U.S. patent application number 15/885671 was filed with the patent office on 2018-08-02 for cereblon ligands and bifunctional compounds comprising the same.
The applicant listed for this patent is Arvinas, Inc.. Invention is credited to Michael Berlin, Andrew P. Crew, Hanqing Dong, Keith R. Homberger, Yimin Qian, Lawrence B. Snyder, Jing Wang, Kurt Zimmermann.
Application Number | 20180215731 15/885671 |
Document ID | / |
Family ID | 62977181 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180215731 |
Kind Code |
A1 |
Crew; Andrew P. ; et
al. |
August 2, 2018 |
CEREBLON LIGANDS AND BIFUNCTIONAL COMPOUNDS COMPRISING THE SAME
Abstract
The description relates to cereblon E3 ligase binding compounds,
including bifunctional compounds comprising the same, which find
utility as modulators of targeted ubiquitination, especially
inhibitors of a variety of polypeptides and other proteins which
are degraded and/or otherwise inhibited by bifunctional compounds
according to the present disclosure. In particular, the description
provides compounds, which contain on one end a ligand which binds
to the cereblon E3 ubiquitin ligase and on the other end a moiety
which binds a target protein such that the target protein is placed
in proximity to the ubiquitin ligase to effect degradation (and
inhibition) of that protein. Compounds can be synthesized that
exhibit a broad range of pharmacological activities consistent with
the degradation/inhibition of targeted polypeptides of nearly any
type.
Inventors: |
Crew; Andrew P.; (Guilford,
CT) ; Berlin; Michael; (Flemington, NJ) ;
Dong; Hanqing; (Madison, CT) ; Homberger; Keith
R.; (Southbury, CT) ; Qian; Yimin;
(Plainsboro, NJ) ; Snyder; Lawrence B.;
(Killingworth, CT) ; Wang; Jing; (Milford, CT)
; Zimmermann; Kurt; (Durham, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arvinas, Inc. |
New Haven |
CT |
US |
|
|
Family ID: |
62977181 |
Appl. No.: |
15/885671 |
Filed: |
January 31, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62452972 |
Jan 31, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 401/14 20130101;
C07D 417/14 20130101; C07D 498/04 20130101; A61P 35/02 20180101;
C07D 495/14 20130101; C07D 471/04 20130101; C07D 487/04 20130101;
C07D 471/10 20130101 |
International
Class: |
C07D 401/14 20060101
C07D401/14; C07D 417/14 20060101 C07D417/14; C07D 471/04 20060101
C07D471/04; C07D 471/10 20060101 C07D471/10; C07D 487/04 20060101
C07D487/04; C07D 495/14 20060101 C07D495/14; C07D 498/04 20060101
C07D498/04; A61P 35/02 20060101 A61P035/02 |
Claims
1. A cereblon E3 ubiquitin ligase binding compound having a
chemical structure selected from: ##STR00783## ##STR00784##
##STR00785## ##STR00786## ##STR00787## ##STR00788## ##STR00789##
wherein: W is independently selected from CH.sub.2, CHR, C.dbd.O,
SO.sub.2, NH, and N-alkyl; Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, Qs
are each independently represent a carbon C or N substituted with a
group independently selected from R', N or N-oxide; R.sup.1 is
selected from absent, H, OH, CN, C1-C3 alkyl, C.dbd.O; R.sup.2 is
selected from the group absent, H, OH, CN, C1-C3 alkyl, CHF.sub.2,
CF.sub.3, CHO, C(.dbd.O)NH.sub.2; R.sup.3 is selected from absent,
H, alkyl (e.g., C1-C6 or C1-C3 alkyl), substituted alkyl (e.g.,
substituted C1-C6 or C1-C3 alkyl), alkoxy (e.g., C1-C6 or C1-C3
alkoxyl), substituted alkoxy (e.g., substituted C1-C6 or C1-C3
alkoxyl); R.sup.4 is selected from H, alkyl, substituted alkyl;
R.sup.5 and R.sup.6 are each independently H, halogen, C(.dbd.O)R';
CN, OH, CF.sub.3; X is C, CH, C.dbd.O, or N; X.sub.1 is C.dbd.O, N,
CH, or CH.sub.2; R' is selected from H, halogen, amine, alkyl
(e.g., C1-C3 alkyl), substituted alkyl (e.g., substituted C1-C3
alkyl), alkoxy (e.g., C1-C3 alkoxyl), substituted alkoxy (e.g.,
substituted C1-C3 alkoxyl), NR.sup.2R.sup.3, C(.dbd.O)OR.sup.2,
optionally substituted phenyl; n is 0-4; and is a single or double
bond.
2. A bifunctional compound having the chemical structure:
CLM-L-PTM, or a pharmaceutically acceptable salt, enantiomer,
stereoisomer, solvate, polymorph or prodrug thereof, wherein: the
PTM is a small molecule comprising a protein targeting moiety; the
L is a bond or a chemical linking moiety covalently coupling the
CLM and the PTM; and the CLM is a small molecule cereblon E3
ubiquitin ligase binding moiety that binds or targets an cereblon
E3 ubiquitin ligase and has a chemical structure selected from the
group consisting of: ##STR00790## ##STR00791## ##STR00792##
##STR00793## ##STR00794## ##STR00795## ##STR00796## wherein: W is
independently selected from CH.sub.2, CHR, C.dbd.O, SO.sub.2, NH,
and N-alkyl; Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, Qs are each
independently represent a carbon C or N substituted with a group
independently selected from R', N or N-oxide; R.sup.1 is selected
from absent, H, OH, CN, C1-C3 alkyl, C.dbd.O; R.sup.2 is selected
from the group absent, H, OH, CN, C1-3 alkyl, CHF.sub.2, CF.sub.3,
CHO, C(.dbd.O)NH.sub.2; R.sup.3 is selected from absent, H, alkyl
(e.g., C1-C6 or C1-C3 alkyl), substituted alkyl (e.g., substituted
C1-C6 or C1-C3 alkyl), alkoxy (e.g., C1-C6 or C1-C3 alkoxyl),
substituted alkoxy (e.g., substituted C1-C6 or C1-C3 alkoxyl);
R.sup.4 is selected from H, alkyl, substituted alkyl; R.sup.5 and
R.sup.6 are each independently H, halogen, C(.dbd.O)R'; CN, OH,
CF.sub.3; X is C, CH, C.dbd.O, or N; X.sub.1 is C.dbd.O, N, CH, or
CH.sub.2; R' is selected from H, halogen, amine, alkyl (e.g., C1-C3
alkyl), substituted alkyl (e.g., substituted C1-C3 alkyl), alkoxy
(e.g., C1-C3 alkoxyl), substituted alkoxy (e.g., substituted C1-C3
alkoxyl), NR.sup.2R.sup.3, C(.dbd.O)OR.sup.2, optionally
substituted phenyl; n is 0-4; and is a single or double bond
3. The bifunctional compound of claim 2, wherein the CLM is linked
to the PTM, the chemical linker group (L), or a combination thereof
via W, X, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R', Q.sub.1, Q.sub.2,
Q.sub.3, Q.sub.4, and Q.sub.5.
4. The bifunctional compound of claim 2, wherein the PTM is a
moiety that binds Brd4, Tau Protein, Estrogen Receptor (ER), or
Androgen Receptor (AR).
5. The bifunctional compound of claim 2, wherein the compound
further comprises a second E3 ubiquitin ligase binding moiety
coupled through a linker group.
6. The bifunctional compound of claim 5, wherein the second E3
ubiquitin ligase binding moiety binds or targets an E3 ubiquitin
ligase selected from the group consisting of Von Hippel-Lindau
(VLM), cereblon (CLM), mouse double-minute homolog2 (MLM), and
inhibitors of apoptosis proteins (ILM).
7. The bifunctional compound of claim 2, wherein the CLM is
represented by a chemical structure selected from the group
consisting of: ##STR00797## ##STR00798## ##STR00799## wherein Rn
comprises a functional group or an atom.
8. The bifunctional compound of claim 2, wherein the CLM is
represented by a chemical structure selected by: ##STR00800##
##STR00801## ##STR00802##
9. The compound of claim 2, wherein the linker (L) comprises a
chemical structural unit represented by the formula: -(A.sup.L)q-
wherein: (A.sup.L).sub.q is a group which is connected to at least
one of the CLM, the PTM, or a combination thereof; q is an integer
greater than or equal to 1; each A.sup.L is independently selected
from the group consisting of, a bond, CR.sup.L1R.sup.L2, O, S, SO,
SO.sub.2, NR.sup.L3, SO.sub.2NR.sup.L3, SONR.sup.L3, CONR.sup.L3,
NR.sup.L3CONR.sup.L4, NR.sup.L3SO.sub.2NR.sup.L4, CO,
CR.sup.L1.dbd.CR.sup.L2, C.ident.C, SiR.sup.L1R.sup.L2,
P(O)R.sup.L1, P(O)OR.sup.L1, NR.sup.L3C(.dbd.NCN)NR.sup.L4,
NR.sup.L3C(.dbd.NCN), NR.sup.L3C(.dbd.CNO.sub.2)NR.sup.L4,
C.sub.3-11cycloalkyl optionally substituted with 0-6 R.sup.L1
and/or R.sup.L2 groups, C.sub.3-11heterocyclyl optionally
substituted with 0-6 R.sup.L1 and/or R.sup.L2 groups, aryl
optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2 groups,
heteroaryl optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2
groups, where R.sup.L1 or R.sup.L2, each independently are
optionally linked to other groups to form cycloalkyl and/or
heterocyclyl moiety, optionally substituted with 0-4 R.sup.L5
groups; and R.sup.L1, R.sup.L2, R.sup.L3, R.sup.L4 and R.sup.L5
are, each independently, H, halo, C.sub.1-8alkyl, OC.sub.1-8alkyl,
SC.sub.1-8alkyl, NHC.sub.1-8alkyl, N(C.sub.1-8alkyl).sub.2,
C.sub.3-11cycloalkyl, aryl, heteroaryl, C.sub.3-11heterocyclyl,
OC.sub.1-8cycloalkyl, SC.sub.1-8cycloalkyl, NHC.sub.1-8cycloalkyl,
N(C.sub.1-8cycloalkyl).sub.2,
N(C.sub.1-8cycloalkyl)(C.sub.1-8alkyl), OH, NH.sub.2, SH,
SO.sub.2C.sub.1-8alkyl, P(O)(OC.sub.1-8alkyl)(C.sub.1-8alkyl),
P(O)(OC.sub.1-8alkyl).sub.2, CC--C.sub.1-8alkyl, C.ident.CH,
CH.dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.C(C.sub.1-8alkyl).sub.2, Si(OH).sub.3,
Si(C.sub.1-8alkyl).sub.3, Si(OH)(C.sub.1-8alkyl).sub.2,
COC.sub.1-8alkyl, CO.sub.2H, halogen, CN, CF.sub.3, CHF.sub.2,
CH.sub.2F, NO.sub.2, SF.sub.5, SO.sub.2NHC.sub.1-8alkyl,
SO.sub.2N(C.sub.1-8alkyl).sub.2, SONHC.sub.1-8alkyl,
SON(C.sub.1-8alkyl).sub.2, CONHC.sub.1-8alkyl,
CON(C.sub.1-8alkyl).sub.2, N(C.sub.1-8alkyl)CONH(C.sub.1-8alkyl),
N(C.sub.1-8alkyl)CON(C.sub.1-8alkyl).sub.2, NHCONH(C.sub.1-8alkyl),
NHCON(C.sub.1-8alkyl).sub.2, NHCONH.sub.2,
N(C.sub.1-8alkyl)SO.sub.2NH(C.sub.1-8alkyl), N(C.sub.1-8alkyl)
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH(C.sub.1-8alkyl), NH
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH.sub.2.
10. The bifunctional compound of claim 9, wherein L is selected
from the group consisting of:
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub-
.q--O(CH2).sub.r-OCH2-,
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r-OCH2-,
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r--O--;
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub-
.q--O(CH2).sub.r--O--;
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--O(-
CH2).sub.r--O--;
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--O(-
CH2).sub.r-OCH2-; ##STR00803## ##STR00804## ##STR00805## wherein m,
n, o, p, q, and r of the linker are independently 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; when the
number is zero, there is no N--O or O--O bond R of the linker is H,
methyl and ethyl; X of the linker is H and F ##STR00806## where m
of the linker can be 2, 3, 4, 5 ##STR00807## ##STR00808##
##STR00809## ##STR00810## ##STR00811## ##STR00812## ##STR00813##
##STR00814## ##STR00815## ##STR00816## ##STR00817## where each n
and m of the linker can independently be 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
11. The bifunctional compound of 9, wherein L is selected from the
group consisting of: ##STR00818## ##STR00819## ##STR00820##
##STR00821## ##STR00822## ##STR00823## ##STR00824## wherein each m
and n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
12. The bifunctional compound of claim 9, wherein the linker (L) is
selected from the group consisting of: ##STR00825## ##STR00826##
##STR00827## ##STR00828## ##STR00829## ##STR00830## ##STR00831##
##STR00832## ##STR00833## ##STR00834## ##STR00835## ##STR00836##
##STR00837## ##STR00838## ##STR00839## ##STR00840## ##STR00841##
##STR00842## ##STR00843## ##STR00844## ##STR00845## ##STR00846##
##STR00847## ##STR00848## ##STR00849## ##STR00850## ##STR00851##
##STR00852## ##STR00853## ##STR00854## ##STR00855## ##STR00856##
##STR00857## ##STR00858## wherein each m, n, o, p, q, and r is
independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20.
13. The bifunctional compound of claim 9, wherein the linker (L) is
selected from the group consisting of: ##STR00859## ##STR00860##
##STR00861## ##STR00862## ##STR00863## ##STR00864## ##STR00865##
##STR00866## ##STR00867## ##STR00868## ##STR00869## ##STR00870##
##STR00871## ##STR00872## ##STR00873## ##STR00874## ##STR00875##
##STR00876## ##STR00877## ##STR00878## ##STR00879## ##STR00880##
##STR00881## ##STR00882## ##STR00883## ##STR00884## ##STR00885##
##STR00886## ##STR00887##
14. The bifunctional compound of claim 9, wherein the linker (L) is
selected from: ##STR00888## ##STR00889## ##STR00890## ##STR00891##
##STR00892## ##STR00893## ##STR00894## ##STR00895## ##STR00896##
wherein: "X" in above structures can be linear chain with atoms
ranging from 2 to 14, and the mentioned chain can contain
heteroatoms such as oxygen; and "Y" in above structures can be O,
N, S(O).sub.n (n=0, 1, 2).
15. The bifunctional compound of claim 2, wherein the linker (L)
comprises a structure selected from: ##STR00897## wherein: W.sup.L1
and W.sup.L2 are each independently a 4-8 membered ring with 0-4
heteroatoms, optionally substituted with R.sup.Q, each R.sup.Q is
independently a H, halo, OH, CN, CF.sub.3, C.sub.1-C.sub.6 alkyl
(linear, branched, optionally substituted), C.sub.1-C.sub.6 alkoxy
(linear, branched, optionally substituted), or 2 R.sup.Q groups
taken together with the atom they are attached to, form a 4-8
membered ring system containing 0-4 heteroatoms; Y.sup.L1 is each
independently a bond, C.sub.1-C.sub.6 alkyl (linear, branched,
optionally substituted) and optionally one or more C atoms are
replaced with O; or C.sub.1-C.sub.6 alkoxy (linear, branched,
optionally substituted); n is 0-10; and a dashed line indicates the
attachment point to the PTM or CLM moieties.
16. The bifunctional compound of claim 2, wherein the linker
comprises a structure selected from: ##STR00898## wherein: W.sup.L1
and W.sup.L2 are each independently aryl, heteroaryl, cyclic,
heterocyclic, C.sub.1-6 alkyl, bicyclic, biaryl, biheteroaryl, or
biheterocyclic, each optionally substituted with R.sup.Q, each
R.sup.Q is independently a H, halo, OH, CN, CF.sub.3, hydroxyl,
nitro, C.ident.CH, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted),
C.sub.1-C.sub.6 alkoxy (linear, branched, optionally substituted),
OC.sub.1-3alkyl (optionally substituted by 1 or more --F), OH,
NH.sub.2, NR.sup.Y1R.sup.Y2, CN, or 2 R.sup.Q groups taken together
with the atom they are attached to, form a 4-8 membered ring system
containing 0-4 heteroatoms; Y.sup.L1 is each independently a bond,
NR.sup.YL1, O, S, NR.sup.YL2, CR.sup.YL1R.sup.YL2, C.dbd.O,
C.dbd.S, SO, SO.sub.2, C.sub.1-C.sub.6 alkyl (linear, branched,
optionally substituted) and optionally one or more C atoms are
replaced with O; C.sub.1-C.sub.6 alkoxy (linear, branched,
optionally substituted); Q.sup.L is a 3-6 membered alicyclic or
aromatic ring with 0-4 heteroatoms, optionally bridged, optionally
substituted with 0-6 R.sup.Q, each R.sup.Q is independently H,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), or 2 R.sup.Q groups taken together
with the atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); R.sup.YL1, R.sup.YL2 are each
independently H, OH, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), or R.sup.1,
R.sup.2 together with the atom they are attached to, form a 3-8
membered ring system containing 0-2 heteroatoms); n is 0-10; and a
dashed line indicates the attachment point to the PTM or CLM
moieties.
17. The bifunctional compound of claim 9, wherein the linker (L) is
a polyethylenoxy group optionally substituted with aryl or phenyl
comprising from 1 to 10 ethylene glycol units.
18. The bifunctional compound of claim 2, wherein the PTM a
chemical structure that includes at least one of (A), (B), (C),
(D), (E), or a combination thereof: (A) an estrogen receptor
binding moiety (EBM) comprising PTM-I or PTM-II: ##STR00899##
wherein: X.sub.PTM is O or C.dbd.O; each of X.sub.PTM1 and
X.sub.PTM2 is independently selected from N or CH; R.sub.PTM1 is
independently selected from OH, O(CO)R.sub.PTM, O-lower alkyl,
wherein R.sub.PTM is an alkyl or aryl group in the ester;
R.sub.PTM2 and R.sub.PTM4 are independently selected from H, OH,
halogen, CN, CF.sub.3, SO.sub.2-alkyl, O-lower alkyl; R.sub.PTM3
and R.sub.PTM5 are independently selected from H, halogen; PTM-I
has at least one R.sub.PTM2 and at least one R.sub.PTM3 on each
respective rings; and the ##STR00900## indicates the site of
attachment of at least one of the linker, the CLM, a CLM', or a
combination thereof; (B) an estrogen receptor protein targeting
moiety represented by the chemical structure: ##STR00901## wherein:
each X.sub.PTM is independently CH, N; indicates the site of
attachment of at least one of the linker, the CLM, a CLM', or a
combination thereof; each R.sub.PTM1 is independently OH, halogen,
alkoxy, methoxy, ethoxy, O(CO)R.sub.PTM, wherein the substitution
can be a mono-, di- or tri-substitution and the R.sub.PTM is alkyl
or cycloalkyl group with 1 to 6 carbons or aryl groups; each
R.sub.PTM2 is independently H, halogen, CN, CF.sub.3, liner or
branched alkyl, alkoxy, methoxy, ethoxy, wherein the substitution
can be mono- or di-substitution; each R.sub.PTM3 is independently
H, halogen, wherein the substitution can be mono- or
di-substitution; and R.sub.PTM4 is a H, alkyl, methyl, ethyl. (C)
an androgen receptor (AR) binding moiety (ABM) comprises a
structure selected from the group consisting of: ##STR00902##
wherein: W.sup.1 is aryl, heteroaryl, bicyclic, or biheterocyclic,
each independently substituted by 1 or more H, halo, hydroxyl,
nitro, CN, C.ident.CH, C.sub.1-6 alkyl (linear, branched,
optionally substituted; for example, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), C.sub.1-6 alkoxyl (linear, branched,
optionally substituted; for example, optionally substituted by 1 or
more halo), C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or CF.sub.3;
Y.sup.1, Y.sup.2 are each independently NR.sup.Y1, O, S, SO2,
heteroaryl, or aryl; Y.sup.3, Y.sup.4, Y.sup.5 are each
independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2, C.dbd.O,
C.dbd.S, SO, SO.sub.2, heteroaryl, or aryl; Q is a 3-6 membered
ring with 0-4 heteroatoms, optionally substituted with 0-6 R.sup.Q,
each R.sup.Q, is independently H, C.sub.1-6 alkyl (linear,
branched, optionally substituted, for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, or 2 R.sup.Q groups taken together with the atom
they are attached to, form a 3-8 membered ring system containing
0-2 heteroatoms); R.sup.1, R.sup.2, R.sup.a, R.sup.b, R.sup.Y1,
R.sup.Y2 are each independently H, C.sub.1-6 alkyl (linear,
branched, optionally substituted; for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, cyclic, heterocyclic or R.sup.1, R.sup.2 together
with the atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); W.sup.2 is a bond, C.sub.1-6 alkyl,
C.sub.1-6 heteroalkyl, O, aryl, heteroaryl, alicyclic,
heterocyclic, biheterocyclic, biaryl, or biheteroaryl, each
optionally substituted by 1-10 R.sup.W2; each R.sup.W2 is
independently H, halo, C.sub.1-6 alkyl (linear or branched
optionally substituted; for example, optionally substituted by 1 or
more F), --OR.sup.W2A, C.sub.3-6 cycloalkyl, C.sub.4-6
cycloheteroalkyl, C.sub.1-6 alkyl (optionally substituted),
heterocyclic (optionally substituted), aryl (optionally
substituted), or heteroaryl (optionally substituted), bicyclic
heteroaryl or aryl, OC.sub.1-3alkyl (optionally substituted; for
example, optionally substituted by 1 or more --F), OH, NH.sub.2,
NR.sup.Y1R.sup.Y2, CN; R.sup.W2A is H, C.sub.1-6 alkyl (linear,
branched), or C.sub.1-6 heteroalkyl (linear, branched), each
optionally substituted by a cycloalkyl, cycloheteroalkyl, aryl,
heterocyclic, heteroaryl, halo, or OC.sub.1-3alkyl; and the dashed
line indicates the site of attachment of at least one of the
linker, the CLM, a CLM', or a combination thereof; (D) a Tau
protein targeting moiety that is represented by at least one of
Formula I-XI: ##STR00903## wherein: A, B, C, D, E, and F are
independently selected from an optionally substituted 5- or
6-membered aryl or heteroaryl ring, an optionally substituted 4- to
7-membered cycloalkyl or a heterocycloalkyl, where contact between
circles indicates ring fusion; L.sub.PTM is selected from a bond,
an alkyl, an alkenyl or an alkynyl, optionally interrupted by one
or more rings (i.e., cycloalkyl, heterocycloalkyl, aryl or
heteroaryl), or one or more functional groups selected from the
groups --O--, --S--, --NR.sup.1.sub.PTM--, --N.dbd.N--, --S(O)--,
--SO.sub.2--, --C(O)--, --NHC(O)--, --C(O)NH--, --NHSO.sub.2--,
--NHC(O)NH--, --NHC(O)O--, or --OC(O)NH--, wherein the said
functional group is optionally located at either end of the linker;
and R.sup.1.sub.PTM is selected from H or alkyl. (E) a tricyclic
diazepine or azepine BET/BRD4 binding ligand comprising a group
according to the chemical structure PTM-a: ##STR00904## wherein:
Y.sub.1, Y.sub.2 and Y.sub.3 are independently selected from the
group of carbon, nitrogen or oxygen and together with the atoms to
form an aromatic fused ring. A and B are independently selected
from the group of a 5-membered aromatic ring, a 6-membered aromatic
ring, a heteroaromatic ring, a carbocyclic, a thiophene a pyrrole
ring, a pyridine, a pyrimidine, a pyrazine, a pyrazole ring each
optionally substituted with alkyl, alkoxy, halogen, an aromatic and
a heteroaromatic ring; wherein ring A is fused to the central
azepine (Y1=C) or diazepine (Y1=N) moiety; and Z1 is selected from
the group of methyl or analkyl group, and wherein the dashed line
indicates the site of attachment of at least one of the linker, the
CLM, a CLM', or a combination thereof.
19. The bifunctional compound of claim 2, wherein the PTM has a
structure selected from the group consisting of: ##STR00905##
##STR00906## ##STR00907## ##STR00908## ##STR00909## wherein R or
Linker is a bond or a chemical linker moiety coupling the CLM to
the PTM, including pharmaceutically acceptable salt forms
thereof.
20. The bifunctional compound of claim 2, wherein the compound is
selected from the group consisting of PROTAC-1 through
PROTAC-112.
21. A composition comprising an effective amount of a bifunctional
compound of any of claim 2, and a pharmaceutically acceptable
carrier.
22. The composition of claim 21, wherein the composition further
comprises at least one of additional bioactive agent or another
bifunctional compound of claim 2.
23. The composition of claim 22, wherein the additional bioactive
agent is anti-cancer agent, an anti-neurodegenerative agent, an
antimicrobial agent, an antiviral agent, an anti-HIV agent, or an
antifungal agent.
24. A method of treating a disease or disorder in a subject, the
method comprising administering a composition comprising an
effective amount of at least one compound of claim 2 and a
pharmaceutically acceptable carrier, additive, and/or excipient to
a subject in need thereof, wherein the compound is effective in
treating or ameliorating at least one symptom of the disease or
disorder.
25. The method of claim 24, wherein the disease or disorder is
associated with the accumulation and/or aggregation of the target
protein.
26. The method of claim 24, wherein the disease or disorder is
selected from the group consisting of asthma, autoimmune diseases
such as multiple sclerosis, various cancers, ciliopathies, cleft
palate, diabetes, heart disease, hypertension, inflammatory bowel
disease, mental retardation, mood disorder, obesity, refractive
error, infertility, Angelman syndrome, Canavan disease, Coeliac
disease, Charcot-Marie-Tooth disease, Cystic fibrosis, Duchenne
muscular dystrophy, Haemochromatosis, Haemophilia, Klinefelter's
syndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney
disease, (PKD1) or 4 (PKD2) Prader-Willi syndrome, Sickle-cell
disease, Tay-Sachs disease, Turner syndrome.
27. The method of claim 24, wherein the disease or disorder is
selected from the group consisting of Alzheimer's disease,
Amyotrophic lateral sclerosis (Lou Gehrig's disease), Anorexia
nervosa, Anxiety disorder, Atherosclerosis, Attention deficit
hyperactivity disorder, Autism, Bipolar disorder, Chronic fatigue
syndrome, Chronic obstructive pulmonary disease, Crohn's disease,
Coronary heart disease, Dementia, Depression, Diabetes mellitus
type 1, Diabetes mellitus type 2, Epilepsy, Guillain-Barre
syndrome, Irritable bowel syndrome, Lupus, Metabolic syndrome,
Multiple sclerosis, Myocardial infarction, Obesity,
Obsessive-compulsive disorder, Panic disorder, Parkinson's disease,
Psoriasis, Rheumatoid arthritis, Sarcoidosis, Schizophrenia,
Stroke, Thromboangiitis obliterans, Tourette syndrome,
Vasculitis.
28. The method of claim 24, wherein the disease or disorder is
selected from the group consisting of aceruloplasminemia,
Achondrogenesis type II, achondroplasia, Acrocephaly, Gaucher
disease type 2, acute intermittent porphyria, Canavan disease,
Adenomatous Polyposis Coli, ALA dehydratase deficiency,
adenylosuccinate lyase deficiency, Adrenogenital syndrome,
Adrenoleukodystrophy, ALA-D porphyria, ALA dehydratase deficiency,
Alkaptonuria, Alexander disease, Alkaptonuric ochronosis, alpha
1-antitrypsin deficiency, alpha-1 proteinase inhibitor, emphysema,
amyotrophic lateral sclerosis Alstrom syndrome, Alexander disease,
Amelogenesis imperfecta, ALA dehydratase deficiency, Anderson-Fabry
disease, androgen insensitivity syndrome, Anemia Angiokeratoma
Corporis Diffusum, Angiomatosis retinae (von Hippel-Lindau disease)
Apert syndrome, Arachnodactyly (Marfan syndrome), Stickler
syndrome, Arthrochalasis multiplex congenital (Ehlers-Danlos
syndrome#arthrochalasia type) ataxia telangiectasia, Rett syndrome,
primary pulmonary hypertension, Sandhoff disease, neurofibromatosis
type II, Beare-Stevenson cutis gyrata syndrome, Mediterranean
fever, familial, Benjamin syndrome, beta-thalassemia, Bilateral
Acoustic Neurofibromatosis (neurofibromatosis type II), factor V
Leiden thrombophilia, Bloch-Sulzberger syndrome (incontinentia
pigmenti), Bloom syndrome, X-linked sideroblastic anemia,
Bonnevie-Ullrich syndrome (Turner syndrome), Bourneville disease
(tuberous sclerosis), prion disease, Birt-Hogg-Dube syndrome,
Brittle bone disease (osteogenesis imperfecta), Broad Thumb-Hallux
syndrome (Rubinstein-Taybi syndrome), Bronze Diabetes/Bronzed
Cirrhosis (hemochromatosis), Bulbospinal muscular atrophy
(Kennedy's disease), Burger-Grutz syndrome (lipoprotein lipase
deficiency), CGD Chronic granulomatous disorder, Campomelic
dysplasia, biotinidase deficiency, Cardiomyopathy (Noonan
syndrome), Cri du chat, CAVD (congenital absence of the vas
deferens), Caylor cardiofacial syndrome (CBAVD), CEP (congenital
erythropoietic porphyria), cystic fibrosis, congenital
hypothyroidism, Chondrodystrophy syndrome (achondroplasia),
otospondylomegaepiphyseal dysplasia, Lesch-Nyhan syndrome,
galactosemia, Ehlers-Danlos syndrome, Thanatophoric dysplasia,
Coffin-Lowry syndrome, Cockayne syndrome, (familial adenomatous
polyposis), Congenital erythropoietic porphyria, Congenital heart
disease, Methemoglobinemia/Congenital methaemoglobinaemia,
achondroplasia, X-linked sideroblastic anemia, Connective tissue
disease, Conotruncal anomaly face syndrome, Cooley's Anemia
(beta-thalassemia), Copper storage disease (Wilson's disease),
Copper transport disease (Menkes disease), hereditary
coproporphyria, Cowden syndrome, Craniofacial dysarthrosis (Crouzon
syndrome), Creutzfeldt-Jakob disease (prion disease), Cockayne
syndrome, Cowden syndrome, Curschmann-Batten-Steinert syndrome
(myotonic dystrophy), Beare-Stevenson cutis gyrata syndrome,
primary hyperoxaluria, spondyloepimetaphyseal dysplasia (Strudwick
type), muscular dystrophy, Duchenne and Becker types (DBMD), Usher
syndrome, Degenerative nerve diseases including de Grouchy syndrome
and Dejerine-Sottas syndrome, developmental disabilities, distal
spinal muscular atrophy, type V, androgen insensitivity syndrome,
Diffuse Globoid Body Sclerosis (Krabbe disease), Di George's
syndrome, Dihydrotestosterone receptor deficiency, androgen
insensitivity syndrome, Down syndrome, Dwarfism, erythropoietic
protoporphyria Erythroid 5-aminolevulinate synthetase deficiency,
Erythropoietic porphyria, erythropoietic protoporphyria,
erythropoietic uroporphyria, Friedreich's ataxia, familial
paroxysmal polyserositis, porphyria cutanea tarda, familial
pressure sensitive neuropathy, primary pulmonary hypertension
(PPH), Fibrocystic disease of the pancreas, fragile X syndrome,
galactosemia, genetic brain disorders, Giant cell hepatitis
(Neonatal hemochromatosis), Gronblad-Strandberg syndrome
(pseudoxanthoma elasticum), Gunther disease (congenital
erythropoietic porphyria), haemochromatosis, Hallgren syndrome,
sickle cell anemia, hemophilia, hepatoerythropoietic porphyria
(HEP), Hippel-Lindau disease (von Hippel-Lindau disease),
Huntington's disease, Hutchinson-Gilford progeria syndrome
(progeria), Hyperandrogenism, Hypochondroplasia, Hypochromic
anemia, Immune system disorders, including X-linked severe combined
immunodeficiency, Insley-Astley syndrome, Kennedy's syndrome,
Jackson-Weiss syndrome, Joubert syndrome, Lesch-Nyhan syndrome,
Jackson-Weiss syndrome, Kidney diseases, including hyperoxaluria,
Klinefelter's syndrome, Kniest dysplasia, Lacunar dementia,
Langer-Saldino achondrogenesis, ataxia telangiectasia, Lynch
syndrome, Lysyl-hydroxylase deficiency, Machado-Joseph disease,
Metabolic disorders, including Kniest dysplasia, Marfan syndrome,
Movement disorders, Mowat-Wilson syndrome, cystic fibrosis, Muenke
syndrome, Multiple neurofibromatosis, Nance-Insley syndrome,
Nance-Sweeney chondrodysplasia, Niemann-Pick disease, Noack
syndrome (Pfeiffer syndrome), Osler-Weber-Rendu disease,
Peutz-Jeghers syndrome, Polycystic kidney disease, polyostotic
fibrous dysplasia (McCune-Albright syndrome), Peutz-Jeghers
syndrome, Prader-Labhart-Willi syndrome, hemochromatosis, primary
hyperuricemia syndrome (Lesch-Nyhan syndrome), primary pulmonary
hypertension, primary senile degenerative dementia, prion disease,
progeria (Hutchinson Gilford Progeria Syndrome), progressive
chorea, chronic hereditary (Huntington) (Huntington's disease),
progressive muscular atrophy, spinal muscular atrophy, propionic
acidemia, protoporphyria, proximal myotonic dystrophy, pulmonary
arterial hypertension, PXE (pseudoxanthoma elasticum), Rb
(retinoblastoma), Recklinghausen disease (neurofibromatosis type
I), Recurrent polyserositis, Retinal disorders, Retinoblastoma,
Rett syndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome,
Roussy-Levy syndrome, severe achondroplasia with developmental
delay and acanthosis nigricans (SADDAN), Li-Fraumeni syndrome,
sarcoma, breast, leukemia, and adrenal gland (SBLA) syndrome,
sclerosis tuberose (tuberous sclerosis), SDAT, SED congenital
(spondyloepiphyseal dysplasia congenita), SED Strudwick
(spondyloepimetaphyseal dysplasia, Strudwick type), SEDc
(spondyloepiphyseal dysplasia congenita) SEMD, Strudwick type
(spondyloepimetaphyseal dysplasia, Strudwick type), Shprintzen
syndrome, Skin pigmentation disorders, Smith-Lemli-Opitz syndrome,
South-African genetic porphyria (variegate porphyria),
infantile-onset ascending hereditary spastic paralysis, Speech and
communication disorders, sphingolipidosis, Tay-Sachs disease,
spinocerebellar ataxia, Stickler syndrome, stroke, androgen
insensitivity syndrome, tetrahydrobiopterin deficiency,
beta-thalassemia, Thyroid disease, Tomaculous neuropathy
(hereditary neuropathy with liability to pressure palsies),
Treacher Collins syndrome, Triplo X syndrome (triple X syndrome),
Trisomy 21 (Down syndrome), Trisomy X, VHL syndrome (von
Hippel-Lindau disease), Vision impairment and blindness (Alstrom
syndrome), Vrolik disease, Waardenburg syndrome, Warburg Sjo
Fledelius Syndrome, Weissenbacher-Zweymuller syndrome,
Wolf-Hirschhorn syndrome, Wolff Periodic disease,
Weissenbacher-Zweymuller syndrome and Xeroderma pigmentosum.
29. The method of claim 24, further comprising an additional
bioactive agent.
30. The method of claim 29, wherein the additional bioactive agent
is at least one of an anti-cancer agent, an anti-neurodegenerative
agent, an antimicrobial agent, an antiviral agent, an anti-HIV
agent, an antifungal agent, or a combination thereof.
31. The method of claim 30, wherein said anticancer agent is
selected from the group consisting of everolimus, trabectedin,
abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744,
ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364,
AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054,
PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor,
an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1
modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor,
a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK
inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT
inhibitor, an mTORC1/2 inhibitor, a JAK/STAT inhibitor, a
checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a
Map kinase kinase (mek) inhibitor, a VEGF trap antibody,
pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,
panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,
batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine,
rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab,
gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,
cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR.sub.1
KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx
102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380,
sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine,
doxorubicin, liposomal doxorubicin, 5'-deoxy-5-fluorouridine,
vincristine, temozolomide, ZK-304709, seliciclib; PD0325901,
AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258,);
3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone,
vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,
Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu
t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sub.4O.sub.2).sub.X
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin,
bleomycin, buserelin, busulfan, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clodronate, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin,
diethylstilbestrol, epirubicin, fludarabine, fludrocortisone,
fluoxymesterone, flutamide, gleevac, gemcitabine, hydroxyurea,
idarubicin, ifosfamide, imatinib, leuprolide, levamisole,
lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin,
porfimer, procarbazine, raltitrexed, rituximab, streptozocin,
teniposide, testosterone, thalidomide, thioguanine, thiotepa,
tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard,
uracil mustard, estramustine, altretamine, floxuridine,
5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine,
deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine,
vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat,
BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974,
interleukin-12, IM862, angiostatin, vitaxin, droloxifene,
idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,
denileukin diftitox, gefitinib, bortezimib, paclitaxel,
cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550,
BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,
ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene,
idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa, darbepoetin alfa and mixtures
thereof.
32. A method for inducing degradation of a target protein in a cell
comprising administering an effective amount of a compound of claim
2 to the cell, wherein the compound effectuates degradation of the
target protein.
33. A method for treating cancer, said method comprising
administering a composition comprising an effective amount of a
compound of claim 2 to a patient in need thereof, wherein the
composition is effectuates for the treatment or alleviation of at
least one symptom of cancer in the patient.
34. The method of claim 33, wherein the cancer is squamous-cell
carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular
carcinomas, and renal cell carcinomas, cancer of the bladder,
bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung,
neck, ovary, pancreas, prostate, and stomach; leukemias; benign and
malignant lymphomas, particularly Burkitt's lymphoma and
Non-Hodgkin's lymphoma; benign and malignant melanomas;
myeloproliferative diseases; multiple myeloma, sarcomas, including
Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,
myosarcomas, peripheral neuroepithelioma, synovial sarcoma,
gliomas, astrocytomas, oligodendrogliomas, ependymomas,
gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas,
medulloblastomas, pineal cell tumors, meningiomas, meningeal
sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast
cancer, prostate cancer, cervical cancer, uterine cancer, lung
cancer, ovarian cancer, testicular cancer, thyroid cancer,
astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer,
liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's
disease, Wilms' tumor or teratocarcinomas, T-lineage Acute
lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma
(T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B
ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma,
B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia
chromosome positive CML.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present disclosure claims priority to U.S. Provisional
Application No. 62/452,972, filed 31 Jan. 2017, which is
incorporated herein by reference in its entirety.
INCORPORATION BY REFERENCE
[0002] U.S. patent application Ser. No. 15/230,354, filed on Aug.
5, 2016, published as U.S. Patent Application Publication No.
2017/0065719; and U.S. patent application Ser. No. 15/801,243,
filed on 1 Nov. 2017; and U.S. patent application Ser. No.
15/206,497 filed 11 Jul. 2016; and U.S. patent application Ser. No.
15/209,648 filed 13 Jul. 2016; and U.S. patent application Ser. No.
15/730,728, filed on Oct. 11, 2017; U.S. patent application Ser.
No. 15/829,541, filed on Dec. 1, 2017; U.S. patent application Ser.
No. 15/881,318, filed on Jan. 26, 2018; and U.S. patent application
Ser. No. 14/686,640, filed on Apr. 14, 2015, published as U.S.
Patent Application Publication No. 2015/0291562; and U.S. patent
application Ser. No. 14/792,414, filed on Jul. 6, 2015, published
as U.S. Patent Application Publication No. 2016/0058872; and U.S.
patent application Ser. No. 14/371,956, filed on Jul. 11, 2014,
published as U.S. Patent Application Publication No. 2014/0356322;
and U.S. patent application Ser. No. 15/074,820, filed on Mar. 18,
2016, published as U.S. Patent Application Publication No.
2016/0272639, are incorporated herein by reference in their
entirety. Furthermore, all references cited herein are incorporated
by reference herein in their entirety.
FIELD OF THE INVENTION
[0003] The description provides imide-based compounds, including
bifunctional compounds comprising the same, and associated methods
of use. The bifunctional compounds are useful as modulators of
targeted ubiquitination, especially with respect to a variety of
polypeptides and other proteins, which are degraded and/or
otherwise inhibited by bifunctional compounds according to the
present disclosure.
BACKGROUND
[0004] Most small molecule drugs bind enzymes or receptors in tight
and well-defined pockets. On the other hand, protein-protein
interactions are notoriously difficult to target using small
molecules due to their large contact surfaces and the shallow
grooves or flat interfaces involved. E3 ubiquitin ligases (of which
hundreds are known in humans) confer substrate specificity for
ubiquitination, and therefore, are more attractive therapeutic
targets than general proteasome inhibitors due to their specificity
for certain protein substrates. The development of ligands of E3
ligases has proven challenging, in part due to the fact that they
must disrupt protein-protein interactions. However, recent
developments have provided specific ligands which bind to these
ligases. For example, since the discovery of nutlins, the first
small molecule E3 ligase inhibitors, additional compounds have been
reported that target E3 ligases but the field remains
underdeveloped.
[0005] One E3 ligase with therapeutic potential is the von
Hippel-Lindau (VHL) tumor suppressor. VHL comprises the substrate
recognition subunit/E3 ligase complex VCB, which includes elongins
B and C, and a complex including Cullin-2 and Rbx1. The primary
substrate of VHL is Hypoxia Inducible Factor 1.alpha.
(HIF-1.alpha.), a transcription factor that upregulates genes such
as the pro-angiogenic growth factor VEGF and the red blood cell
inducing cytokine erythropoietin in response to low oxygen levels.
We generated the first small molecule ligands of Von Hippel Lindau
(VHL) to the substrate recognition subunit of the E3 ligase, VCB,
an important target in cancer, chronic anemia and ischemia, and
obtained crystal structures confirming that the compound mimics the
binding mode of the transcription factor HIF-1.alpha., the major
substrate of VHL.
[0006] Cereblon is a protein that in humans is encoded by the CRBN
gene. CRBN orthologs are highly conserved from plants to humans,
which underscores its physiological importance. Cereblon forms an
E3 ubiquitin ligase complex with damaged DNA binding protein 1
(DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). This
complex ubiquitinates a number of other proteins. Through a
mechanism which has not been completely elucidated, cereblon
ubquitination of target proteins results in increased levels of
fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10
(FGF10). FGF8 in turn regulates a number of developmental
processes, such as limb and auditory vesicle formation. The net
result is that this ubiquitin ligase complex is important for limb
outgrowth in embryos. In the absence of cereblon, DDB1 forms a
complex with DDB2 that functions as a DNA damage-binding
protein.
[0007] Thalidomide, which has been approved for the treatment of a
number of immunological indications, has also been approved for the
treatment of certain neoplastic diseases, including multiple
myeloma. In addition to multiple myeloma, thalidomide and several
of its analogs are also currently under investigation for use in
treating a variety of other types of cancer. While the precise
mechanism of thalidomide's anti-tumor activity is still emerging,
it is known to inhibit angiogenesis. Recent literature discussing
the biology of the imides includes Lu et al Science 343, 305 (2014)
and Kronke et al Science 343, 301 (2014).
[0008] Significantly, thalidomide and its analogs e.g.
pomolinamiode and lenalinomide, are known to bind cereblon. These
agents bind to cereblon, altering the specificity of the complex to
induce the ubiquitination and degradation of Ikaros (IKZF1) and
Aiolos (IKZF3), transcription factors essential for multiple
myeloma growth. Indeed, higher expression of cereblon has been
linked to an increase in efficacy of imide drugs in the treatment
of multiple myeloma.
[0009] BRD4 has captured considerable attention from academia and
Pharmaceutical industry alike due to its great potential as a novel
target in multiple disease settings, particularly in cancer. BRD4
belongs to the bromodomain and extra-terminal domain (BET) family,
which is characterized by two bromodomains (BD domain) at the
N-terminus and an extraterminal domain (ET domain) at the
C-terminus (J. Shi, et al. Molecular cell, 54 (2014) 728-736 and A.
C. Belkina, et al., Nat. Rev. Cancer, 12 (2012) 465-477). The two
BD domains recognize and interact with acetylated-lysine residues
at the N-terminal tail of histone protein; the ET domain is not yet
fully characterized, and is largely considered to serve a
scaffolding function in recruiting diverse transcriptional
regulators. Thus, BRD4 plays a key role in regulating gene
expression by recruiting relevant transcription modulators to
specific genomic loci. Several studies have establish that BRD4 is
preferentially located at super-enhancer regions, which often
reside upstream of important oncogenes, such as c-MYC, Bcl-xL and
BCL-6, and play a key role in regulating their expressions (J.
Loven, et al., Cell, 153 (2013) 320-334 and B. Chapuy, et al.,
Cancer Cell, 24 (2013) 777-790.). Owing to its pivotal role in
modulating the expression of essential oncogenes, BRD4 emerges as a
promising therapeutic target in multiple cancer types, including
midline carcinoma, AML, MM, BL, and prostate cancer (J. Loven, et
al., Cell, 153 (2013) 320-334; J. Zuber, et al., Nature, 478 (2011)
524-528; J. E. Delmore, et al., Cell, 146 (2011) 904-917; J. A.
Mertz, et al., PNAS, 108 (2011) 16669-16674; A. Wyce, et al.,
Oncotarget, 4 (2013) 2419-2429; I. A. Asangani, et al., Nature, 510
(2014) 278-282; and C. A. French, et al., Oncogene, 27 (2008)
2237-2242). BRD4's distinct high occupancy of genomic loci proximal
to specific oncogenes provide a potential therapeutic window that
will allow specific targeting of tumor cells while sparing normal
tissues. Particularly, BRD4 may serve as an alternative strategy of
targeting c-MYC, which contributes to the development and
maintenance of a majority of human cancers but has remained
undruggable (J. E. Delmore, et al., Cell, 146 (2011) 904-917; J. A.
Mertz, et al., PNAS, 108 (2011) 16669-16674; M. G. Baratta, et al.,
PNAS, 112 (2015) 232-237; and M. Gabay, et al., Cold Spring Harb
Perspect Med. (2014) 4:a014241).
[0010] The development of small molecule BRD4 inhibitors, such as
JQ1, iBET and OTX15, has demonstrated promising therapeutic
potential in preclinical models of various cancers, including BL
(J. Loven, et al., Cell, 153 (2013) 320-334; B. Chapuy, et al.,
Cancer Cell, 24 (2013) 777-790; J. E. Delmore, et al., Cell, 146
(2011) 904-917; J. A. Mertz, et al., PNAS, 108 (2011) 16669-16674;
I. A. Asangani, et al., Nature, 510 (2014) 278-282; M. G. Baratta,
et al., PNAS, 112 (2015) 232-237; M. Boi, et al., Clin. Cancer
Res., (2015) 21(7):1628-38; and A. Puissant, et al., Cancer
discovery, 3 (2013) 308-323). Indeed, BRD4 inhibitors have shown
various anti-tumor activities with good tolerability in different
mouse tumor models and, not surprisingly, high sensitivity to BRD4
inhibitors such as JQ1, has been associated with high level of
either c-MYC and N-MYC in different tumor types, including c-MYC
driven BL. Almost all BL cases contain c-myc gene translocation
that places it under control of a super-enhancer located upstream
of IgH, thus driving an abnormally high level of c-MYC expression,
tumor development and maintenance (K. Klapproth, et al., British
journal of haematology, 149 (2010) 484-497).
[0011] Currently, four BET Bromodomain inhibitors are in phase I
clinical trial with focus largely on midline carcinoma and
hematological malignancies (CPI-0610, NCT01949883; GSK525762,
NCT01587703; OTX015, NCT01713582; TEN-010, NCT01987362).
Preclinical studies with BRD4 inhibitors demonstrate their value in
suppressing c-MYC and proliferation in BL cell lines, albeit with
IC.sub.50 values often in the range of 100 nM to 1 uM (J. A. Mertz,
et al., PNAS, 108 (2011) 16669-16674 and M. Ceribelli, et al.,
PNAS, 111 (2014) 11365-11370). Thus, despite the rapid progress of
BRD4 inhibitors, the effect of BRD4 inhibition has been
encouraging, but less than ideal, as the effect is mostly
cytostatic and requires relatively high concentration of
inhibitors.
[0012] An ongoing need exists in the art for effective treatments
for disease, especially hyperplasias and cancers, such as multiple
myeloma. However, non-specific effects, and the inability to target
and modulate certain classes of proteins altogether, such as
transcription factors, remain as obstacles to the development of
effective anti-cancer agents. As such, small molecule therapeutic
agents that leverage or potentiate cereblon's substrate specificity
and, at the same time, are "tunable" such that a wide range of
protein classes can be targeted and modulated with specificity
would be very useful as a therapeutic.
BRIEF SUMMARY OF THE INVENTION
[0013] The present disclosure describes bifunctional compounds
which function to recruit endogenous proteins to an E3 Ubiquitin
Ligase for degradation, and methods of using the same. In
particular, the present disclosure provides bifunctional or
proteolysis targeting chimeric (PROTAC) compounds, which find
utility as modulators of targeted ubiquitination of a variety of
polypeptides and other proteins, which are then degraded and/or
otherwise inhibited by the bifunctional compounds as described
herein. An advantage of the compounds provided herein is that a
broad range of pharmacological activities is possible, consistent
with the degradation/inhibition of targeted polypeptides from
virtually any protein class or family. In addition, the description
provides methods of using an effective amount of the compounds as
described herein for the treatment or amelioration of a disease
condition, such as cancer, e.g., multiple myeloma.
[0014] As such, in one aspect the disclosure provides novel
imide-based compounds as described herein.
[0015] In an additional aspect, the disclosure provides
bifunctional or PROTAC compounds, which comprise an E3 Ubiquitin
Ligase binding moiety (i.e., a ligand for an E3 Ubiquitin Ligase or
"ULM" group), and a moiety that binds a target protein (i.e., a
protein/polypeptide targeting ligand or "PTM" group) such that the
target protein/polypeptide is placed in proximity to the ubiquitin
ligase to effect degradation (and inhibition) of that protein. In a
preferred embodiment, the ULM is a cereblon E3 Ubiquitin Ligase
binding moiety (i.e., a "CLM"). For example, the structure of the
bifunctional compound can be depicted as:
##STR00001##
[0016] The respective positions of the PTM and CLM moieties as well
as their number as illustrated herein is provided by way of example
only and is not intended to limit the compounds in any way. As
would be understood by the skilled artisan, the bifunctional
compounds as described herein can be synthesized such that the
number and position of the respective functional moieties can be
varied as desired.
[0017] In certain embodiments, the bifunctional compound further
comprises a chemical linker ("L"). In this example, the structure
of the bifunctional compound can be depicted as:
##STR00002##
where PTM is a protein/polypeptide targeting moiety, L is a linker,
and CLM is a cereblon E3 ubiquitin ligase binding moiety.
[0018] In certain preferred embodiments, the E3 Ubiquitin Ligase is
cereblon. As such, in certain additional embodiments, the CLM of
the bifunctional compound comprises chemistries such as imide,
amide, thioamide, thioimide derived moieties. In additional
embodiments, the CLM comprises a phthalimido group or an analog or
derivative thereof. In still additional embodiments, the CLM
comprises a phthalimido-glutarimide group or an analog or
derivative thereof. In still other embodiments, the CLM comprises a
member of the group consisting of thalidomide, lenalidomide,
pomalidomide, and analogs or derivatives thereof.
[0019] In certain embodiments, the compounds as described herein
comprise multiple CLMs, multiple PTMs, multiple chemical linkers or
a combination thereof.
[0020] In any aspect or embodiment described herein, the ULM
(ubiquitination ligase modulator) can be Von Hippel-Lindau E3
ubiquitin ligase (VHL) binding moiety (VLM), or a cereblon E3
ubiquitin ligase binding moiety (CLM), or a mouse double minute 2
homolog (MDM2) E3 ubiquitin ligase binding moiety (MLM), or an IAP
E3 ubiquitin ligase binding moiety (i.e., a "ILM"). In any aspect
or embodiments described herein, the bifunctional compound includes
at least one additional E3 ligase binding moiety selected from the
group consisting of VLM, VLM', CLM, CLM', MLM, MLM', ILM, ILM', or
a combination thereof. For example, there can be at least 1, 2, 3,
4, or 5 additional E3 ligase binding moieties.
[0021] In an additional aspect, the description provides
therapeutic compositions comprising an effective amount of a
compound as described herein or salt form thereof, and a
pharmaceutically acceptable carrier. The therapeutic compositions
modulate protein degradation in a patient or subject, for example,
an animal such as a human, and can be used for treating or
ameliorating disease states or conditions which are modulated
through the degraded protein. In certain embodiments, the
therapeutic compositions as described herein may be used to
effectuate the degradation of proteins of interest for the
treatment or amelioration of a disease, e.g., cancer. In yet
another aspect, the present disclosure provides a method of
ubiquitinating/degrading a target protein in a cell. In certain
embodiments, the method comprises administering a bifunctional
compound as described herein comprising an CLM and a PTM,
preferably linked through a linker moiety, as otherwise described
herein, wherein the CLM is coupled to the PTM and wherein the CLM
recognizes a ubiquitin pathway protein (e.g., an ubiquitin ligase,
preferably an E3 ubiquitin ligase such as, e.g., cereblon) and the
PTM recognizes the target protein such that degradation of the
target protein will occur when the target protein is placed in
proximity to the ubiquitin ligase, thus resulting in
degradation/inhibition of the effects of the target protein and the
control of protein levels. The control of protein levels afforded
by the present disclosure provides treatment of a disease state or
condition, which is modulated through the target protein by
lowering the level of that protein in the cells of a patient.
[0022] In an additional aspect, the description provides a method
for assessing (i.e., determining and/or measuring) a CLM's binding
affinity. In certain embodiments, the method comprises providing a
test agent or compound of interest, for example, an agent or
compound having an imide moiety, e.g., a phthalimido group,
phthalimido-glutarimide group, derivatized thalidomide, derivatized
lenalidomide or derivatized pomalidomide, and comparing the
cereblon binding affinity and/or inhibitory activity of the test
agent or compound as compared to an agent or compound known to bind
and/or inhibit the activity of cereblon.
[0023] In still another aspect, the description provides methods
for treating or emeliorating a disease, disorder or symptom thereof
in a subject or a patient, e.g., an animal such as a human,
comprising administering to a subject in need thereof a composition
comprising an effective amount, e.g., a therapeutically effective
amount, of a compound as described herein or salt form thereof, and
a pharmaceutically acceptable carrier, wherein the composition is
effective for treating or ameliorating the disease or disorder or
symptom thereof in the subject.
[0024] In another aspect, the description provides methods for
identifying the effects of the degradation of proteins of interest
in a biological system using compounds according to the present
disclosure.
[0025] The preceding general areas of utility are given by way of
example only and are not intended to be limiting on the scope of
the present disclosure and appended claims. Additional objects and
advantages associated with the compositions, methods, and processes
of the present disclosure will be appreciated by one of ordinary
skill in the art in light of the instant claims, description, and
examples. For example, the various aspects and embodiments of the
invention may be utilized in numerous combinations, all of which
are expressly contemplated by the present description. These
additional advantages objects and embodiments are expressly
included within the scope of the present disclosure. The
publications and other materials used herein to illuminate the
background of the invention, and in particular cases, to provide
additional details respecting the practice, are incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate several embodiments of
the present disclosure and, together with the description, serve to
explain the principles of the invention. The drawings are only for
the purpose of illustrating an embodiment of the invention and are
not to be construed as limiting the invention. Further objects,
features and advantages of the invention will become apparent from
the following detailed description taken in conjunction with the
accompanying figures showing illustrative embodiments of the
invention, in which:
[0027] FIGS. 1A and 1B. Illustration of general principle for
PROTAC function. (A) Exemplary PROTACs comprise a protein targeting
moiety (PTM; darkly shaded rectangle), a ubiquitin ligase binding
moiety (ULM; lightly shaded triangle), and optionally a linker
moiety (L; black line) coupling or tethering the PTM to the ULM.
(B) Illustrates the functional use of the PROTACs as described
herein. Briefly, the ULM recognizes and binds to a specific E3
Ubiquitin Ligase, and the PTM binds and recruits a target protein
bringing it into close proximity to the E3 Ubiquitin Ligase.
Typically, the E3 Ubiquitin Ligase is complexed with an E2
ubiquitin-conjugating protein, and either alone or via the E2
protein catalyzes attachment of ubiquitin (dark circles) to a
lysine on the target protein via an isopeptide bond. The
poly-ubiquitinated protein (far right) is then targeted for
degration by the proteosomal machinery of the cell.
DETAILED DESCRIPTION
[0028] The following is a detailed description provided to aid
those skilled in the art in practicing the present disclosure.
Those of ordinary skill in the art may make modifications and
variations in the embodiments described herein without departing
from the spirit or scope of the present disclosure. All
publications, patent applications, patents, figures and other
references mentioned herein are expressly incorporated by reference
in their entirety.
[0029] Presently described are compositions and methods that relate
to the surprising and unexpected discovery that an E3 Ubiquitin
Ligase protein, e.g., cereblon, ubiquitinates a target protein once
it and the target protein are placed in proximity by a bifunctional
or chimeric construct that binds the E3 Ubiquitin Ligase protein
and the target protein. Accordingly the present disclosure provides
such compounds and compositions comprising an E3 Ubiquintin Ligase
binding moiety ("ULM") coupled to a protein target binding moiety
("PTM"), which result in the ubiquitination of a chosen target
protein, which leads to degradation of the target protein by the
proteasome (see FIGS. 1A and 1B). The present disclosure also
provides a library of compositions and the use thereof.
[0030] In certain aspects, the present disclosure provides
compounds which comprise a ligand, e.g., a small molecule ligand
(i.e., having a molecular weight of below 2,000, 1,000, 500, or 200
Daltons), which is capable of binding to a ubiquitin ligase, such
as IAP, VHL, MDM2, or cereblon. The compounds also comprise a
moiety that is capable of binding to target protein, in such a way
that the target protein is placed in proximity to the ubiquitin
ligase to effect degradation (and/or inhibition) of that protein.
Small molecule can mean, in addition to the above, that the
molecule is non-peptidyl, that is, it is not generally considered a
peptide, e.g., comprises fewer than 4, 3, or 2 amino acids. In
accordance with the present description, the PTM, ULM or PROTAC
molecule can be a small molecule.
[0031] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description is for describing particular
embodiments only and is not intended to be limiting of the
invention.
[0032] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise (such as in the case
of a group containing a number of carbon atoms in which case each
carbon atom number falling within the range is provided), between
the upper and lower limit of that range and any other stated or
intervening value in that stated range is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
[0033] The following terms are used to describe the present
invention. In instances where a term is not specifically defined
herein, that term is given an art-recognized meaning by those of
ordinary skill applying that term in context to its use in
describing the present invention.
[0034] The articles "a" and "an" as used herein and in the appended
claims are used herein to refer to one or to more than one (i.e.,
to at least one) of the grammatical object of the article unless
the context clearly indicates otherwise. By way of example, "an
element" means one element or more than one element.
[0035] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0036] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of."
[0037] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0038] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from anyone or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
nonlimiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0039] It should also be understood that, in certain methods
described herein that include more than one step or act, the order
of the steps or acts of the method is not necessarily limited to
the order in which the steps or acts of the method are recited
unless the context indicates otherwise.
[0040] The terms "co-administration" and "co-administering" or
"combination therapy" refer to both concurrent administration
(administration of two or more therapeutic agents at the same time)
and time varied administration (administration of one or more
therapeutic agents at a time different from that of the
administration of an additional therapeutic agent or agents), as
long as the therapeutic agents are present in the patient to some
extent, preferably at effective amounts, at the same time. In
certain preferred aspects, one or more of the present compounds
described herein, are coadministered in combination with at least
one additional bioactive agent, especially including an anticancer
agent. In particularly preferred aspects, the co-administration of
compounds results in synergistic activity and/or therapy, including
anticancer activity.
[0041] The term "compound", as used herein, unless otherwise
indicated, refers to any specific chemical compound disclosed
herein and includes tautomers, regioisomers, geometric isomers, and
where applicable, stereoisomers, including optical isomers
(enantiomers) and other stereoisomers (diastereomers) thereof, as
well as pharmaceutically acceptable salts and derivatives,
including prodrug and/or deuterated forms thereof where applicable,
in context. Deuterated small molecules contemplated are those in
which one or more of the hydrogen atoms contained in the drug
molecule have been replaced by deuterium.
[0042] Within its use in context, the term compound generally
refers to a single compound, but also may include other compounds
such as stereoisomers, regioisomers and/or optical isomers
(including racemic mixtures) as well as specific enantiomers or
enantiomerically enriched mixtures of disclosed compounds. The term
also refers, in context to prodrug forms of compounds which have
been modified to facilitate the administration and delivery of
compounds to a site of activity. It is noted that in describing the
present compounds, numerous substituents and variables associated
with same, among others, are described. It is understood by those
of ordinary skill that molecules which are described herein are
stable compounds as generally described hereunder. When the bond is
shown, both a double bond and single bond are represented or
understood within the context of the compound shown and well-known
rules for valence interactions.
[0043] The term "Ubiquitin Ligase" refers to a family of proteins
that facilitate the transfer of ubiquitin to a specific substrate
protein, targeting the substrate protein for degradation. For
example, cereblon is an E3 Ubiquitin Ligase protein that alone or
in combination with an E2 ubiquitin-conjugating enzyme causes the
attachment of ubiquitin to a lysine on a target protein, and
subsequently targets the specific protein substrates for
degradation by the proteasome. Thus, E3 ubiquitin ligase alone or
in complex with an E2 ubiquitin conjugating enzyme is responsible
for the transfer of ubiquitin to targeted proteins. In general, the
ubiquitin ligase is involved in polyubiquitination such that a
second ubiquitin is attached to the first; a third is attached to
the second, and so forth. Polyubiquitination marks proteins for
degradation by the proteasome. However, there are some
ubiquitination events that are limited to mono-ubiquitination, in
which only a single ubiquitin is added by the ubiquitin ligase to a
substrate molecule. Mono-ubiquitinated proteins are not targeted to
the proteasome for degradation, but may instead be altered in their
cellular location or function, for example, via binding other
proteins that have domains capable of binding ubiquitin. Further
complicating matters, different lysines on ubiquitin can be
targeted by an E3 to make chains. The most common lysine is Lys48
on the ubiquitin chain. This is the lysine used to make
polyubiquitin, which is recognized by the proteasome.
[0044] The term "patient" or "subject" is used throughout the
specification to describe an animal, preferably a human or a
domesticated animal, to whom treatment, including prophylactic
treatment, with the compositions according to the present
disclosure is provided. For treatment of those infections,
conditions or disease states which are specific for a specific
animal such as a human patient, the term patient refers to that
specific animal, including a domesticated animal such as a dog or
cat or a farm animal such as a horse, cow, sheep, etc. In general,
in the present disclosure, the term patient refers to a human
patient unless otherwise stated or implied from the context of the
use of the term.
[0045] The term "effective" is used to describe an amount of a
compound, composition or component which, when used within the
context of its intended use, effects an intended result. The term
effective subsumes all other effective amount or effective
concentration terms, which are otherwise described or used in the
present application.
Compounds and Compositions
[0046] In one aspect, the description provides compounds comprising
an E3 Ubiquitin Ligase binding moiety ("ULM") that is a cereblon E3
Ubiquitin Ligase binding moiety ("CLM"). In one embodiment, the CLM
is coupled to a chemical linker (L) according to the structure:
L-CLM (I)
wherein L is a chemical linker group and CLM is a cereblon E3
Ubiquitin Ligase binding moiety. The number and/or relative
positions of the moieties in the compounds illustrated herein is
provided by way of example only. As would be understood by the
skilled artisan, compounds as described herein can be synthesized
with any desired number and/or relative position of the respective
functional moieties.
[0047] The terms ULM and CLM are used in their inclusive sense
unless the context indicates otherwise. For example, the term ULM
is inclusive of all ULMs, including those that bind cereblon (i.e.,
CLMs). Further, the term CLM is inclusive of all possible cereblon
E3 Ubiquitin Ligase binding moieties.
[0048] In another aspect, the present disclosure provides
bifunctional or multifunctional PROTAC compounds useful for
regulating protein activity by inducing the degradation of a target
protein. In certain embodiments, the compound comprises a CLM
coupled, e.g., linked covalently, directly or indirectly, to a
moiety that binds a target protein (i.e., protein targeting moiety
or "PTM"). In certain embodiments, the CLM and PTM are joined or
coupled via a chemical linker (L). The CLM recognizes the cereblon
E3 ubiquitin ligase and the PTM recognizes a target protein and the
interaction of the respective moieties with their targets
facilitates the degradation of the target protein by placing the
target protein in proximity to the ubiquitin ligase protein. An
exemplary bifunctional compound can be depicted as:
PTM-CLM (II)
[0049] In certain embodiments, the bifunctional compound further
comprises a chemical linker ("L"). For example, the bifunctional
compound can be depicted as:
PTM-L-CLM (III)
wherein PTM is a protein/polypeptide targeting moiety, L is a
linker, and CLM is a cereblon E3 ligase binding moiety.
[0050] In certain embodiments, the compounds as described herein
comprise multiple PTMs (targeting the same or different protein
targets), multiple CLMs, one or more ULMs (i.e., moieties that bind
specifically to another E3 Ubiquitin Ligase, e.g., VHL) or a
combination thereof. In any of the aspects of embodiments described
herein, the PTMs, CLMs, and ULMs can be coupled directly or via one
or more chemical linkers or a combination thereof. In additional
embodiments, where a compound has multiple ULMs, the ULMs can be
for the same E3 Ubiquintin Ligase or each respective ULM can bind
specifically to a different E3 Ubiquitin Ligase. In still further
embodiments, where a compound has multiple PTMs, the PTMs can bind
the same target protein or each respective PTM can bind
specifically to a different target protein.
[0051] In another embodiment, the description provides a compound
which comprises a plurality of CLMs coupled directly or via a
chemical linker moiety (L). For example, a compound having two CLMs
can be depicted as:
CLM-CLM or (IV)
CLM-L-CLM (V)
[0052] In certain embodiments, where the compound comprises
multiple CLMs, the CLMs are identical. In additional embodiments,
the compound comprising a plurality of CLMs further comprises at
least one PTM coupled to a CLM directly or via a chemical linker
(L) or both. In certain additional embodiments, the compound
comprising a plurality of CLMs further comprises multiple PTMs. In
still additional embodiments, the PTMs are the same or, optionally,
different. In still further embodiments, wherein the PTMs are
different the respective PTMs may bind the same protein target or
bind specifically to a different protein target.
[0053] In additional embodiments, the description provides a
compound comprising at least two different CLMs coupled directly or
via a chemical linker (L) or both. For example, such a compound
having two different CLMs can be depicted as:
CLM-CLM' or (VI)
CLM-L-CLM' (VII)
wherein CLM' indicates a cereblon E3 Ubiquitin Ligase binding
moiety that is structurally different from CLM. In certain
embodiments, the compound may comprise a plurality of CLMs and/or a
plurality of CLM's. In further embodiments, the compound comprising
at least two different CLMs, a plurality of CLMs, and/or a
plurality of CLM's further comprises at least one PTM coupled to a
CLM or a CLM' directly or via a chemical linker or both. In any of
the embodiments described herein, a compound comprising at least
two different CLMs can further comprise multiple PTMs. In still
additional embodiments, the PTMs are the same or, optionally,
different. In still further embodiments, wherein the PTMs are
different the respective PTMs may bind the same protein target or
bind specifically to a different protein target. In still further
embodiments, the PTM itself is a ULM or CLM (or ULM' or CLM').
[0054] In a preferred embodiment, the CLM comprises a moiety that
is a ligand of the cereblon E3 Ubiquitin Ligase (CRBN). In certain
embodiments, the CLM comprises a chemotype from the "imide" class
of molecules. In certain additional embodiments, the CLM comprises
a phthalimido group or an analog or derivative thereof. In still
additional embodiments, the CLM comprises a phthalimido-glutarimide
group or an analog or derivative thereof. In still other
embodiments, the CLM comprises a member of the group consisting of
thalidomide, lenalidomide, pomalidomide, and analogs or derivatives
thereof.
[0055] In additional embodiments, the description provides the
compounds as described herein including their enantiomers,
diastereomers, solvates and polymorphs, including pharmaceutically
acceptable salt forms thereof, e.g., acid and base salt forms.
[0056] Exemplary Cereblon Binding and/or Inhibiting Compounds
[0057] In one aspect the description provides compounds useful for
binding and/or inhibiting cereblon E3 Ubiquitin Ligase binding
moiety. In certain embodiments, the compound has a chemical
structure that includes at least one of (e.g., the compound has a
chemical structure selected from the group consisting of):
##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008## ##STR00009##
wherein: [0058] W is independently selected from CH.sub.2, CHR,
C.dbd.O, SO.sub.2, NH, and N-alkyl; [0059] Q.sub.1, Q.sub.2,
Q.sub.3, Q.sub.4, Q.sub.5 are each independently represent a carbon
C or N substituted with a group independently selected from R', N
or N-oxide; [0060] R.sup.1 is selected from absent, H, OH, CN,
C1-C3 alkyl, C.dbd.O; [0061] R.sup.2 is selected from the group
absent, H, OH, CN, C1-C3 alkyl, CHF.sub.2, CF.sub.3, CHO,
C(.dbd.O)NH.sub.2; [0062] R.sup.3 is selected from absent, H, alkyl
(e.g., C1-C6 or C1-C3 alkyl), substituted alkyl (e.g., substituted
C1-C6 or C1-C3 alkyl), alkoxy (e.g., C1-C6 or C1-C3 alkoxyl),
substituted alkoxy (e.g., substituted C1-C6 or C1-C3 alkoxyl);
[0063] R.sup.4 is selected from H, alkyl, substituted alkyl; [0064]
R.sup.5 and R.sup.6 are each independently H, halogen, C(.dbd.O)R';
CN, OH, CF.sub.3 [0065] X is C, CH, C.dbd.O, or N; [0066] X.sub.1
is C.dbd.O, N, CH, or CH.sub.2; [0067] R' is selected from H,
halogen, amine, alkyl (e.g., C1-C3 alkyl), substituted alkyl (e.g.,
substituted C1-C3 alkyl), alkoxy (e.g., C1-C3 alkoxyl), substituted
alkoxy (e.g., substituted C1-C3 alkoxyl), NR.sup.2R.sup.3,
C(.dbd.O)OR.sup.2, optionally substituted phenyl; [0068] n is 0-4;
and [0069] is a single or double bond.
[0070] Exemplary CLMs
[0071] In any of the compounds described herein, the CLM comprises
a chemical structure selected from the group:
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016##
wherein: [0072] W is independently selected from CH.sub.2, CHR,
C.dbd.O, SO.sub.2, NH, and N-alkyl; [0073] Q.sub.1, Q.sub.2,
Q.sub.3, Q.sub.4, Q.sub.5 are each independently represent a carbon
C or N substituted with a group independently selected from R', N
or N-oxide; [0074] R.sup.1 is selected from absent H, OH, CN, C1-C3
alkyl, C.dbd.O; [0075] R.sup.2 is selected from the group absent H,
OH, CN, C1-C3 alkyl, CHF.sub.2, CF.sub.3, CHO, C(.dbd.O)NH.sub.2;
[0076] R.sup.3 is selected from H, alkyl (e.g., C1-C6 or C1-C3
alkyl), substituted alkyl (e.g., substituted C1-C6 or C1-C3 alkyl),
alkoxy (e.g., C1-C6 or C1-C3 alkoxyl), substituted alkoxy (e.g.,
substituted C1-C6 or C1-C3 alkoxyl); [0077] R.sup.4 is selected
from H, alkyl, substituted alkyl; [0078] R.sup.5 and R.sup.6 are
each independently H, halogen, C(.dbd.O)R', CN, OH, CF.sub.3;
[0079] X is C, CH, C.dbd.O, or N; [0080] X.sub.1 is C.dbd.O, N, CH,
or CH.sub.2; [0081] R' is selected from H, halogen, amine, alkyl
(e.g., C1-C3 alkyl), substituted alkyl (e.g., substituted C1-C3
alkyl), alkoxy (e.g., C1-C3 alkoxyl), substituted alkoxy (e.g.,
substituted C1-C3 alkoxyl), NR.sup.2R.sup.3, C(.dbd.O)OR.sup.2,
optionally substituted phenyl; [0082] n is 0-4; [0083] is a single
or double bond; and [0084] the CLM is covalently joined to a PTM, a
chemical linker group (L), a ULM, CLM (or CLM') or combination
thereof.
[0085] In any aspect or embodiment described herein, the CLM or
CLM' is covalently joined to a PTM, a chemical linker group (L), a
ULM, a CLM, a CLM', or a combination thereof via an R group (such
as, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R'), W, X, or a Q
group (such as, Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, or
Q.sub.5).
[0086] In any of the embodiments described herein, the CLM or CLM'
is covalently joined to a PTM, a chemical linker group (L), a ULM,
a CLM, a CLM', or a combination thereof via W, X, R, R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R', Q.sub.1, Q.sub.2, Q.sub.3,
Q.sub.4, and Q.sub.5.
[0087] In any of the embodiments described herein, the W, X,
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R', Q.sub.1, Q.sub.2, Q.sub.3,
Q.sub.4, and Q.sub.5 can independently be covalently coupled to a
linker and/or a linker to which is attached to one or more PTM,
ULM, ULM', CLM or CLM' groups.
[0088] The term "independently" is used herein to indicate that the
variable, which is independently applied, varies independently from
application to application.
[0089] The term "alkyl" shall mean within its context a linear,
branch-chained or cyclic fully saturated hydrocarbon radical or
alkyl group, preferably a C.sub.1-C.sub.10, more preferably a
C.sub.1-C.sub.6, alternatively a C.sub.1-C.sub.3 alkyl group, which
may be optionally substituted. Examples of alkyl groups are methyl,
ethyl, n-butyl, sec-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl,
n-decyl, isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl,
cyclobutyl, cyclopentyl, cyclopentylethyl, cyclohexylethyl and
cyclohexyl, among others. In certain embodiments, the alkyl group
is end-capped with a halogen group (At, Br, Cl, F, or I). In
certain preferred embodiments, compounds according to the present
disclosure which may be used to covalently bind to dehalogenase
enzymes. These compounds generally contain a side chain (often
linked through a polyethylene glycol group) which terminates in an
alkyl group which has a halogen substituent (often chlorine or
bromine) on its distal end which results in covalent binding of the
compound containing such a moiety to the protein.
[0090] The term "Alkoxy" refers to an alkyl group singularly bonded
to oxygen.
[0091] The term "Alkenyl" refers to linear, branch-chained or
cyclic C.sub.2-C.sub.10 (preferably C.sub.2-C.sub.6) hydrocarbon
radicals containing at least one C.dbd.C bond.
[0092] The term "Alkynyl" refers to linear, branch-chained or
cyclic C.sub.2-C.sub.10 (preferably C.sub.2-C.sub.6) hydrocarbon
radicals containing at least one C.ident.C bond.
[0093] The term "alkylene" when used, refers to a
--(CH.sub.2).sub.n-- group (n is an integer generally from 0-6),
which may be optionally substituted. When substituted, the alkylene
group preferably is substituted on one or more of the methylene
groups with a C.sub.1-C.sub.6 alkyl group (including a cyclopropyl
group or a t-butyl group), but may also be substituted with one or
more halo groups, preferably from 1 to 3 halo groups or one or two
hydroxyl groups, O--(C.sub.1-C.sub.6 alkyl) groups or amino acid
sidechains as otherwise disclosed herein. In certain embodiments,
an alkylene group may be substituted with a urethane or alkoxy
group (or other group) which is further substituted with a
polyethylene glycol chain (of from 1 to 10, preferably 1 to 6,
often 1 to 4 ethylene glycol units) to which is substituted
(preferably, but not exclusively on the distal end of the
polyethylene glycol chain) an alkyl chain substituted with a single
halogen group, preferably a chlorine group. In still other
embodiments, the alkylene (often, a methylene) group, may be
substituted with an amino acid sidechain group such as a sidechain
group of a natural or unnatural amino acid, for example, alanine,
.beta.-alanine, arginine, asparagine, aspartic acid, cysteine,
cystine, glutamic acid, glutamine, glycine, phenylalanine,
histidine, isoleucine, lysine, leucine, methionine, proline,
serine, threonine, valine, tryptophan or tyrosine.
[0094] The term "unsubstituted" shall mean substituted only with
hydrogen atoms. A range of carbon atoms which includes C.sub.0
means that carbon is absent and is replaced with H. Thus, a range
of carbon atoms which is C.sub.0-C.sub.6 includes carbons atoms of
1, 2, 3, 4, 5 and 6 and for C.sub.0, H stands in place of
carbon.
[0095] The term "substituted" or "optionally substituted" shall
mean independently (i.e., where more than substituent occurs, each
substituent is independent of another substituent) one or more
substituents (independently up to five substitutents, preferably up
to three substituents, often 1 or 2 substituents on a moiety in a
compound according to the present disclosure and may include
substituents which themselves may be further substituted) at a
carbon (or nitrogen) position anywhere on a molecule within
context, and includes as substituents hydroxyl, thiol, carboxyl,
cyano (C.ident.N), nitro (NO.sub.2), halogen (preferably, 1, 2 or 3
halogens, especially on an alkyl, especially a methyl group such as
a trifluoromethyl), an alkyl group (preferably, C.sub.1-C.sub.10,
more preferably, C.sub.1-C.sub.6), aryl (especially phenyl and
substituted phenyl for example benzyl or benzoyl), alkoxy group
(preferably, C.sub.1-C.sub.6 alkyl or aryl, including phenyl and
substituted phenyl), thioether (C.sub.1-C.sub.6 alkyl or aryl),
acyl (preferably, C.sub.1-C.sub.6 acyl), ester or thioester
(preferably, C.sub.1-C.sub.6 alkyl or aryl) including alkylene
ester (such that attachment is on the alkylene group, rather than
at the ester function which is preferably substituted with a
C.sub.1-C.sub.6 alkyl or aryl group), preferably, C.sub.1-C.sub.6
alkyl or aryl, halogen (preferably, F or Cl), amine (including a
five- or six-membered cyclic alkylene amine, further including a
C.sub.1-C.sub.6 alkyl amine or a C.sub.1-C.sub.6 dialkyl amine
which alkyl groups may be substituted with one or two hydroxyl
groups) or an optionally substituted --N(C.sub.0-C.sub.6
alkyl)C(O)(O--C.sub.1-C.sub.6 alkyl) group (which may be optionally
substituted with a polyethylene glycol chain to which is further
bound an alkyl group containing a single halogen, preferably
chlorine substituent), hydrazine, amido, which is preferably
substituted with one or two C.sub.1-C.sub.6 alkyl groups (including
a carboxamide which is optionally substituted with one or two
C.sub.1-C.sub.6 alkyl groups), alkanol (preferably, C.sub.1-C.sub.6
alkyl or aryl), or alkanoic acid (preferably, C.sub.1-C.sub.6 alkyl
or aryl). Substituents according to the present disclosure may
include, for example --SiR.sub.1subR.sub.2subR.sub.3sub groups
where each of R.sub.1sub and R.sub.2sub is as otherwise described
herein and R.sub.3sub is H or a C.sub.1-C.sub.6 alkyl group,
preferably R.sub.1sub, R.sub.2sub, R.sub.3sub in this context is a
C.sub.1-C.sub.3 alkyl group (including an isopropyl or t-butyl
group). Each of the above-described groups may be linked directly
to the substituted moiety or alternatively, the substituent may be
linked to the substituted moiety (preferably in the case of an aryl
or heteroaryl moiety) through an optionally substituted
--(CH.sub.2).sub.m-- or alternatively an optionally substituted
--(OCH.sub.2).sub.m--, --(OCH.sub.2CH.sub.2).sub.m-- or
--(CH.sub.2CH.sub.2O).sub.m-- group, which may be substituted with
any one or more of the above-described substituents. Alkylene
groups --(CH.sub.2).sub.m-- or --(CH.sub.2).sub.n-- groups or other
chains such as ethylene glycol chains, as identified above, may be
substituted anywhere on the chain. Preferred substitutents on
alkylene groups include halogen or C.sub.1-C.sub.6 (preferably
C.sub.1-C.sub.3) alkyl groups, which may be optionally substituted
with one or two hydroxyl groups, one or two ether groups
(O--C.sub.1-C.sub.6 groups), up to three halo groups (preferably
F), or a sidechain of an amino acid as otherwise described herein
and optionally substituted amide (preferably carboxamide
substituted as described above) or urethane groups (often with one
or two C.sub.0-C.sub.6 alkyl substitutents, which group(s) may be
further substituted). In certain embodiments, the alkylene group
(often a single methylene group) is substituted with one or two
optionally substituted C.sub.1-C.sub.6 alkyl groups, preferably
C.sub.1-C.sub.4 alkyl group, most often methyl or O-methyl groups
or a sidechain of an amino acid as otherwise described herein. In
the present disclosure, a moiety in a molecule may be optionally
substituted with up to five substituents, preferably up to three
substituents. Most often, in the present disclosure, moieties which
are substituted are substituted with one or two substituents.
[0096] The term "substituted" (each substituent being independent
of any other substituent) shall also mean within its context of use
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, amido,
carboxamido, sulfone, including sulfonamide, keto, carboxy,
C.sub.1-C.sub.6 ester (oxyester or carbonylester), C.sub.1-C.sub.6
keto, urethane --O--C(O)--NR.sub.1subR.sub.2sub or
--N(R.sub.1sub)--C(O)--O--R.sub.1sub, nitro, cyano and amine
(especially including a C.sub.1-C.sub.6
alkylene-NR.sub.1subR.sub.2sub, a mono- or di-C.sub.1-C.sub.6 alkyl
substituted amines which may be optionally substituted with one or
two hydroxyl groups). Each of these groups contain unless otherwise
indicated, within context, between 1 and 6 carbon atoms. In certain
embodiments, preferred substituents will include for example,
--NH--, --NHC(O)--, --O--, .dbd.O, --(CH.sub.2).sub.m-- (here, m
and n are in context, 1, 2, 3, 4, 5 or 6), --S--, --S(O)--,
SO.sub.2-- or --NH--C(O)--NH--, --(CH.sub.2).sub.nOH,
--(CH.sub.2).sub.nSH, --(CH.sub.2).sub.nCOOH, C.sub.1-C.sub.6
alkyl, --(CH.sub.2).sub.nO--(C.sub.1-C.sub.6 alkyl),
--(CH.sub.2).sub.n--C(O)--(C.sub.1-C.sub.6 alkyl),
--(CH.sub.2).sub.nOC(O)--(C.sub.1-C.sub.6 alkyl),
--(CH.sub.2).sub.nC(O)O--(C.sub.1-C.sub.6 alkyl),
--(CH.sub.2).sub.nNHC(O)--R.sub.1sub,
--(CH.sub.2).sub.nC(O)--NR.sub.1subR.sub.2sub,
--(OCH.sub.2).sub.nOH, --(CH.sub.2O).sub.nCOOH, C.sub.1-C.sub.6
alkyl, --(OCH.sub.2).sub.nO--(C.sub.1-C.sub.6 alkyl),
--(CH.sub.2O).sub.nC(O)--(C.sub.1-C.sub.6 alkyl),
--(OCH.sub.2).sub.nNHC(O)--R.sub.1sub,
--(CH.sub.2O).sub.nC(O)--NR.sub.1subR.sub.2sub,
--S(O).sub.2--R.sub.S, --S(O)--R.sub.S (R.sub.S is C.sub.1-C.sub.6
alkyl or a --(CH.sub.2).sub.m--NR.sub.1subR.sub.2sub group),
NO.sub.2, CN or halogen (F, Cl, Br, I, preferably F or Cl),
depending on the context of the use of the substituent. R.sub.1sub
and R.sub.2sub are each, within context, H or a C.sub.1-C.sub.6
alkyl group (which may be optionally substituted with one or two
hydroxyl groups or up to three halogen groups, preferably
fluorine). The term "substituted" shall also mean, within the
chemical context of the compound defined and substituent used, an
optionally substituted aryl or heteroaryl group or an optionally
substituted heterocyclic group as otherwise described herein.
Alkylene groups may also be substituted as otherwise disclosed
herein, preferably with optionally substituted C.sub.1-C.sub.6
alkyl groups (methyl, ethyl or hydroxymethyl or hydroxyethyl is
preferred, thus providing a chiral center), a sidechain of an amino
acid group as otherwise described herein, an amido group as
described hereinabove, or a urethane group
O--C(O)--NR.sub.1subR.sub.2sub group where R.sub.1sub and
R.sub.2sub are as otherwise described herein, although numerous
other groups may also be used as substituents. Various optionally
substituted moieties may be substituted with 3 or more
substituents, preferably no more than 3 substituents and preferably
with 1 or 2 substituents. It is noted that in instances where, in a
compound at a particular position of the molecule substitution is
required (principally, because of valency), but no substitution is
indicated, then that substituent is construed or understood to be
H, unless the context of the substitution suggests otherwise.
[0097] The term "aryl" or "aromatic", in context, refers to a
substituted (as otherwise described herein) or unsubstituted
monovalent aromatic radical having a single ring (e.g., benzene,
phenyl, benzyl) or condensed rings (e.g., naphthyl, anthracenyl,
phenanthrenyl, etc.) and can be bound to the compound according to
the present disclosure at any available stable position on the
ring(s) or as otherwise indicated in the chemical structure
presented. Other examples of aryl groups, in context, may include
heterocyclic aromatic ring systems, "heteroaryl" groups having one
or more nitrogen, oxygen, or sulfur atoms in the ring (moncyclic)
such as imidazole, furyl, pyrrole, furanyl, thiene, thiazole,
pyridine, pyrimidine, pyrazine, triazole, oxazole or fused ring
systems such as indole, quinoline, indolizine, azaindolizine,
benzofurazan, etc., among others, which may be optionally
substituted as described above. Among the heteroaryl groups which
may be mentioned include nitrogen-containing heteroaryl groups such
as pyrrole, pyridine, pyridone, pyridazine, pyrimidine, pyrazine,
pyrazole, imidazole, triazole, triazine, tetrazole, indole,
isoindole, indolizine, azaindolizine, purine, indazole, quinoline,
dihydroquinoline, tetrahydroquinoline, isoquinoline,
dihydroisoquinoline, tetrahydroisoquinoline, quinolizine,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, imidazopyridine, imidazotriazine, pyrazinopyridazine,
acridine, phenanthridine, carbazole, carbazoline, pyrimidine,
phenanthroline, phenacene, oxadiazole, benzimidazole,
pyrrolopyridine, pyrrolopyrimidine and pyridopyrimidine;
sulfur-containing aromatic heterocycles such as thiophene and
benzothiophene; oxygen-containing aromatic heterocycles such as
furan, pyran, cyclopentapyran, benzofuran and isobenzofuran; and
aromatic heterocycles comprising 2 or more hetero atoms selected
from among nitrogen, sulfur and oxygen, such as thiazole,
thiadizole, isothiazole, benzoxazole, benzothiazole,
benzothiadiazole, phenothiazine, isoxazole, furazan, phenoxazine,
pyrazoloxazole, imidazothiazole, thienofuran, furopyrrole,
pyridoxazine, furopyridine, furopyrimidine, thienopyrimidine and
oxazole, among others, all of which may be optionally
substituted.
[0098] The term "substituted aryl" refers to an aromatic
carbocyclic group comprised of at least one aromatic ring or of
multiple condensed rings at least one of which being aromatic,
wherein the ring(s) are substituted with one or more substituents.
For example, an aryl group can comprise a substituent(s) selected
from: --(CH.sub.2).sub.nOH,
--(CH.sub.2).sub.n--O--(C.sub.1-C.sub.6)alkyl,
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.n--(C.sub.1-C.sub.6)alkyl,
--(CH.sub.2).sub.n--C(O)(C.sub.0-C.sub.6) alkyl,
--(CH.sub.2).sub.n--C(O)O(C.sub.0-C.sub.6)alkyl,
--(CH.sub.2).sub.n--OC(O)(C.sub.0-C.sub.6)alkyl, amine, mono- or
di-(C.sub.1-C.sub.6 alkyl) amine wherein the alkyl group on the
amine is optionally substituted with 1 or 2 hydroxyl groups or up
to three halo (preferably F, Cl) groups, OH, COOH, C.sub.1-C.sub.6
alkyl, preferably CH.sub.3, CF.sub.3, OMe, OCF.sub.3, NO.sub.2, or
CN group (each of which may be substituted in ortho-, meta- and/or
para-positions of the phenyl ring, preferably para-), an optionally
substituted phenyl group (the phenyl group itself is preferably
substituted with a linker group attached to a PTM group, including
a ULM group), and/or at least one of F, Cl, OH, COOH, CH.sub.3,
CF.sub.3, OMe, OCF.sub.3, NO.sub.2, or CN group (in ortho-, meta-
and/or para-positions of the phenyl ring, preferably para-), a
naphthyl group, which may be optionally substituted, an optionally
substituted heteroaryl, preferably an optionally substituted
isoxazole including a methylsubstituted isoxazole, an optionally
substituted oxazole including a methylsubstituted oxazole, an
optionally substituted thiazole including a methyl substituted
thiazole, an optionally substituted isothiazole including a methyl
substituted isothiazole, an optionally substituted pyrrole
including a methylsubstituted pyrrole, an optionally substituted
imidazole including a methylimidazole, an optionally substituted
benzimidazole or methoxybenzylimidazole, an optionally substituted
oximidazole or methyloximidazole, an optionally substituted diazole
group, including a methyldiazole group, an optionally substituted
triazole group, including a methylsubstituted triazole group, an
optionally substituted pyridine group, including a
halo-(preferably, F) or methylsubstitutedpyridine group or an
oxapyridine group (where the pyridine group is linked to the phenyl
group by an oxygen), an optionally substituted furan, an optionally
substituted benzofuran, an optionally substituted
dihydrobenzofuran, an optionally substituted indole, indolizine or
azaindolizine (2, 3, or 4-azaindolizine), an optionally substituted
quinoline, and combinations thereof.
[0099] "Carboxyl" denotes the group --C(O)OR, where R is hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or
substituted heteroaryl, whereas these generic substituents have
meanings which are identical with definitions of the corresponding
groups defined herein.
[0100] The term "heteroaryl" or "hetaryl" can mean but is in no way
limited to an optionally substituted quinoline (which may be
attached to the pharmacophore or substituted on any carbon atom
within the quinoline ring), an optionally substituted indole
(including dihydroindole), an optionally substituted indolizine, an
optionally substituted azaindolizine (2, 3 or 4-azaindolizine) an
optionally substituted benzimidazole, benzodiazole, benzoxofuran,
an optionally substituted imidazole, an optionally substituted
isoxazole, an optionally substituted oxazole (preferably methyl
substituted), an optionally substituted diazole, an optionally
substituted triazole, a tetrazole, an optionally substituted
benzofuran, an optionally substituted thiophene, an optionally
substituted thiazole (preferably methyl and/or thiol substituted),
an optionally substituted isothiazole, an optionally substituted
triazole (preferably a 1,2,3-triazole substituted with a methyl
group, a triisopropylsilyl group, an optionally substituted
--(CH.sub.2).sub.m--O--C.sub.1-C.sub.6 alkyl group or an optionally
substituted --(CH.sub.2).sub.m--C(O)--O--C.sub.1-C.sub.6 alkyl
group), an optionally substituted pyridine (2-, 3, or 4-pyridine)
or a group according to the chemical structure:
##STR00017##
wherein [0101] S.sup.c is CHR.sup.SS, NR.sup.URE, or O; [0102]
R.sup.HET is H, CN, NO.sub.2, halo (preferably Cl or F), optionally
substituted C.sub.1-C.sub.6 alkyl (preferably substituted with one
or two hydroxyl groups or up to three halo groups (e.g. CF.sub.3),
optionally substituted O(C.sub.1-C.sub.6 alkyl) (preferably
substituted with one or two hydroxyl groups or up to three halo
groups) or an optionally substituted acetylenic group
--C.ident.C--R.sub.a where R.sub.a is H or a C.sub.1-C.sub.6 alkyl
group (preferably C.sub.1-C.sub.3 alkyl); [0103] R.sup.SS is H, CN,
NO.sub.2, halo (preferably F or Cl), optionally substituted
C.sub.1-C.sub.6 alkyl (preferably substituted with one or two
hydroxyl groups or up to three halo groups), optionally substituted
O--(C.sub.1-C.sub.6 alkyl) (preferably substituted with one or two
hydroxyl groups or up to three halo groups) or an optionally
substituted --C(O)(C.sub.1-C.sub.6 alkyl) (preferably substituted
with one or two hydroxyl groups or up to three halo groups); [0104]
R.sup.URE is H, a C.sub.1-C.sub.6 alkyl (preferably H or
C.sub.1-C.sub.3 alkyl) or a --C(O)(C.sub.1-C.sub.6 alkyl), each of
which groups is optionally substituted with one or two hydroxyl
groups or up to three halogen, preferably fluorine groups, or an
optionally substituted heterocycle, for example piperidine,
morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene,
piperidine, piperazine, each of which is optionally substituted,
and [0105] Y.sup.C is N or C--R.sup.YC, where R.sup.YC is H, OH,
CN, NO.sub.2, halo (preferably Cl or F), optionally substituted
C.sub.1-C.sub.6 alkyl (preferably substituted with one or two
hydroxyl groups or up to three halo groups (e.g. CF.sub.3),
optionally substituted O(C.sub.1-C.sub.6 alkyl) (preferably
substituted with one or two hydroxyl groups or up to three halo
groups) or an optionally substituted acetylenic group
--C.ident.C--R.sub.a where R.sub.a is H or a C.sub.1-C.sub.6 alkyl
group (preferably C.sub.1-C.sub.3 alkyl).
[0106] The term "Heterocycle" refers to a cyclic group which
contains at least one heteroatom, e.g., N, O or S, and may be
aromatic (heteroaryl) or non-aromatic. Thus, the heteroaryl
moieties are subsumed under the definition of heterocycle,
depending on the context of its use. Exemplary heteroaryl groups
are described hereinabove.
[0107] Exemplary heterocyclics include: azetidinyl, benzimidazolyl,
1,4-benzodioxanyl, 1,3-benzodioxolyl, benzoxazolyl, benzothiazolyl,
benzothienyl, dihydroimidazolyl, dihydropyranyl, dihydrofuranyl,
dioxanyl, dioxolanyl, ethyleneurea, 1,3-dioxolane, 1,3-dioxane,
1,4-dioxane, furyl, homopiperidinyl, imidazolyl, imidazolinyl,
imidazolidinyl, indolinyl, indolyl, isoquinolinyl,
isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl,
morpholinyl, naphthyridinyl, oxazolidinyl, oxazolyl, pyridone,
2-pyrrolidone, pyridine, piperazinyl, N-methylpiperazinyl,
piperidinyl, phthalimide, succinimide, pyrazinyl, pyrazolinyl,
pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl,
quinolinyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydroquinoline, thiazolidinyl, thiazolyl, thienyl,
tetrahydrothiophene, oxane, oxetanyl, oxathiolanyl, thiane among
others.
[0108] Heterocyclic groups can be optionally substituted with a
member selected from the group consisting of alkoxy, substituted
alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,
cyano, halogen, hydroxyl, keto, thioketo, carboxy, carboxyalkyl,
thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,
heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino,
alkoxyamino, nitro, --SO-alkyl, --SO-substituted alkyl, --SOaryl,
--SO-heteroaryl, --SO2-alkyl, --SO2-substituted alkyl, --SO2-aryl,
oxo (.dbd.O), and --SO2-heteroaryl. Such heterocyclic groups can
have a single ring or multiple condensed rings. Examples of
nitrogen heterocycles and heteroaryls include, but are not limited
to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,
pyridazine, indolizine, isoindole, indole, indazole, purine,
quinolizine, isoquinoline, quinoline, phthalazine,
naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,
carbazole, carboline, phenanthridine, acridine, phenanthroline,
isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,
imidazolidine, imidazoline, piperidine, piperazine, indoline,
morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as
N-alkoxy-nitrogen containing heterocycles. The term "heterocyclic"
also includes bicyclic groups in which any of the heterocyclic
rings is fused to a benzene ring or a cyclohexane ring or another
heterocyclic ring (for example, indolyl, quinolyl, isoquinolyl,
tetrahydroquinolyl, and the like).
[0109] The term "cycloalkyl" can mean but is in no way limited to
univalent groups derived from monocyclic or polycyclic alkyl groups
or cycloalkanes, as defined herein, e.g., saturated monocyclic
hydrocarbon groups having from three to twenty carbon atoms in the
ring, including, but not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and the like. The term
"substituted cycloalkyl" can mean but is in no way limited to a
monocyclic or polycyclic alkyl group and being substituted by one
or more substituents, for example, amino, halogen, alkyl,
substituted alkyl, carbyloxy, carbylmercapto, aryl, nitro, mercapto
or sulfo, whereas these generic substituent groups have meanings
which are identical with definitions of the corresponding groups as
defined in this legend.
[0110] The term "hydrocarbyl" shall mean a compound which contains
carbon and hydrogen and which may be fully saturated, partially
unsaturated or aromatic and includes aryl groups, alkyl groups,
alkenyl groups and alkynyl groups.
[0111] The term "lower alkyl" refers to methyl, ethyl or propyl
[0112] The term "lower alkoxy" refers to methoxy, ethoxy or
propoxy.
[0113] More specifically, non-limiting examples of CLMs include
those shown below as well as "hybrid" molecules or compounds that
arise from combining 1 or more features of the following
compounds:
##STR00018## ##STR00019## ##STR00020##
wherein: [0114] W is independently selected from the group
CH.sub.2, CHR, C.dbd.O, SO.sub.2, NH, and N-alkyl; [0115] R.sup.1
is selected from the group absent, H, CH, CN, C1-C3 alkyl; [0116]
R.sup.2 is H or a C1-C3 alkyl; [0117] R.sup.3 is selected from H,
alkyl, substituted alkyl, alkoxy, substituted alkoxy; [0118]
R.sup.4 is methyl or ethyl; [0119] R.sup.5 is H or halo; [0120]
R.sup.6 is H or halo; [0121] R of the CLM is H; [0122] R' is H or
an attachment point for a PTM, a PTM', a chemical linker group (L),
a ULM, a CLM, a CLM', [0123] Q1 and Q2 are each independently C or
N substituted with a group independently selected from H or C1-C3
alkyl; [0124] is a single or double bond; and [0125] Rn comprises a
functional group or an atom.
[0126] In any of the embodiments described herein, the W, R.sup.1,
R.sup.2, Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, and Rn can
independently be covalently coupled to a linker and/or a linker to
which is attached one or more PTM, ULM, ULM', CLM or CLM'
groups.
[0127] In any of the embodiments described herein, the R.sup.1,
R.sup.2, Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, and Rn can
independently be covalently coupled to a linker and/or a linker to
which is attached one or more PTM, ULM, ULM', CLM or CLM'
groups.
[0128] In any of the embodiments described herein, the Q.sub.1,
Q.sub.2, Q.sub.3, Q.sub.4, and Rn can independently be covalently
coupled to a linker and/or a linker to which is attached one or
more PTM, ULM, ULM', CLM or CLM' groups.
[0129] In any aspect or embodiment described herein, R.sub.n is
modified to be covalently joined to the linker group (L), a PTM, a
ULM, a second CLM having the same chemical structure as the CLM, a
CLM', a second linker, or any multiple or combination thereof.
[0130] In any aspect or embodiment described herein, the CLM is
selected from:
##STR00021## ##STR00022## ##STR00023##
wherein R' is a halogen and R.sup.1 is as described in any aspect
or embodiment described herein.
[0131] In certain cases, "CLM" can be imides that bind to cereblon
E3 ligase. These imides and linker attachment point can be but not
limited to the following structures:
##STR00024##
[0132] Exemplary Linkers
[0133] In certain embodiments, the compounds as described herein
include one or more CLMs chemically linked or coupled to one or
more PTMs (e.g., PTM and/or PTM'), ULMs (e.g., ULM, ULM', and/or
CLM') via a chemical linker (L). In certain embodiments, the linker
group L is a group comprising one or more covalently connected
structural units (e.g., -A.sup.L.sub.1 . . . (A.sup.L).sub.q- or
-(A.sup.L).sub.q-), wherein A.sub.1 is a group coupled to PTM, and
Aq is a group coupled to at least one of a ULM, a ULM', a CLM, a
CLM', or a combination thereof. In certain embodiments,
A.sup.L.sub.1 links a CLM or CLM' directly to another ULM, PTM, or
combination thereof. In other embodiments, A.sup.L.sub.1 links a
CLM or CLM' indirectly to another ULM, PTM, or combination thereof
through A.sub.q.
[0134] In certain embodiments, the linker group is
-(A.sup.L).sub.q-, wherein [0135] (A.sup.L).sub.q is a group which
is connected to at least one of a ULM moiety, a PTM moiety, or a
combination thereof; [0136] q of the linker is an integer greater
than or equal to 1; [0137] each A.sup.L is independently selected
from the group consisting of a bond, CR.sup.L1R.sup.L2, O, S, SO,
SO.sub.2, NR.sup.L3, SO.sub.2NR.sup.L3, SONR.sup.L3, CONR.sup.L3,
NR.sup.L3CONR.sup.L4, NR.sup.L3SO.sub.2NR.sup.L4, CO,
CR.sup.L1.dbd.CR.sup.L2, C.ident.C, SiR.sup.L1R.sup.L2,
P(O)R.sup.L1, P(O)OR.sup.L1, NR.sup.L3C(.dbd.NCN)NR.sup.L4,
NR.sup.L3C(.dbd.NCN), NR.sup.L3C(.dbd.CNO.sub.2)NR.sup.L4,
C.sub.3-11cycloalkyl optionally substituted with 0-6 R.sup.L1
and/or R.sup.L2 groups, C.sub.5-13 spirocycloalkyl optionally
substituted with 0-9 R.sup.L1 and/or R.sup.L2 groups,
C.sub.3-11heterocyclyl optionally substituted with 0-6 R.sup.L1
and/or R.sup.L2 groups, C.sub.5-13 spiroheterocycloalkyl optionally
substituted with 0-8 R.sup.L1 and/or R.sup.L2 groups, aryl
optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2 groups,
heteroaryl optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2
groups, where R.sup.L1 or R.sup.L2, each independently are
optionally linked to other groups to form cycloalkyl and/or
heterocyclyl moiety, optionally substituted with 0-4 R.sup.L5
groups; and [0138] R.sup.L1, R.sup.L2, R.sup.L3, R.sup.L4 and
R.sup.L5 are, each independently, H, halo, C.sub.1-8alkyl,
OC.sub.1-8alkyl, NHC.sub.1-8alkyl, N(C.sub.1-8alkyl).sub.2,
C.sub.3-11cycloalkyl, aryl, heteroaryl, C.sub.3-11heterocyclyl,
OC.sub.1-8cycloalkyl, SC.sub.1-8cycloalkyl, NHC.sub.1-8cycloalkyl,
N(C.sub.1-8cycloalkyl).sub.2,
N(C.sub.1-8cycloalkyl)(C.sub.1-8alkyl), OH, NH.sub.2, SH,
SO.sub.2C.sub.1-8alkyl, P(O)(OC.sub.1-8alkyl)(C.sub.1-8alkyl),
P(O)(OC.sub.1-8alkyl).sub.2, CC--C.sub.1-8alkyl, CCH,
CH.dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.C(C.sub.1-8alkyl).sub.2, Si(OH).sub.3,
Si(C.sub.1-8alkyl).sub.3, Si(OH)(C.sub.1-8alkyl).sub.2, CO.sub.2H,
halogen, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NO.sub.2, SF.sub.5,
SO.sub.2NHC.sub.1-8alkyl, SO.sub.2N(C.sub.1-8alkyl).sub.2,
SONHC.sub.1-8alkyl, SON(C.sub.1-8alkyl).sub.2, CONHC.sub.1-8alkyl,
CON(C.sub.1-8alkyl).sub.2, N(C.sub.1-8alkyl)CONH(C.sub.1-8alkyl),
N(C.sub.1-8alkyl)CON(C.sub.1-8alkyl).sub.2, NHCONH(C.sub.1-8alkyl),
NHCON(C.sub.1-8alkyl).sub.2, NHCONH.sub.2,
N(C.sub.1-8alkyl)SO.sub.2NH(C.sub.1-8alkyl), N(C.sub.1-8alkyl)
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH(C.sub.1-8alkyl), NH
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH.sub.2.
[0139] In certain embodiments, q of the linker is an integer
greater than or equal to 0. In certain embodiments, q is an integer
greater than or equal to 1.
[0140] In certain embodiments, e.g., where q is greater than 2,
A.sup.L.sub.q is a group which is connected to a ULM or ULM' moiety
(such as CLM or CLM'), and A.sup.L.sub.1 and A.sup.L.sub.q are
connected via structural units of the linker (L).
[0141] In certain embodiments, e.g., where q of the linker is 2,
A.sup.L.sub.q is a group which is connected to A.sup.L.sub.1 and to
a ULM or a ULM' moiety (such as CLM or CLM').
[0142] In certain embodiments, e.g., where q of the linker is 1,
the structure of the linker group L is -A.sup.L.sub.1-, and
A.sup.L.sub.1 is a group which is connected to a ULM or ULM' moiety
(such as CLM or CLM') and a PTM moiety.
[0143] In certain embodiments, the linker (L) comprises a group
represented by a general structure selected from the group
consisting of: [0144] NR(CH.sub.2).sub.n-(lower alkyl)-,
--NR(CH.sub.2).sub.n-(lower alkoxyl)-, --NR(CH.sub.2).sub.n-(lower
alkoxyl)-OCH.sub.2--, --NR(CH.sub.2).sub.n-(lower alkoxyl)-(lower
alkyl)-OCH.sub.2--, --NR(CH.sub.2).sub.n-(cycloalkyl)-(lower
alkyl)-OCH.sub.2--, --NR(CH.sub.2).sub.n-(hetero cycloalkyl)-,
--NR(CH.sub.2CH.sub.2O).sub.n-(lower alkyl)-O--CH.sub.2--,
--NR(CH.sub.2CH.sub.2O).sub.n-(hetero cycloalkyl)-O--CH.sub.2--,
--NR(CH.sub.2CH.sub.2O).sub.n-Aryl-O--CH.sub.2--,
--NR(CH.sub.2CH.sub.2O).sub.n-(hetero aryl)-O--CH.sub.2--,
--NR(CH.sub.2CH.sub.2O).sub.n-(cyclo alkyl)-O-(hetero
aryl)-O--CH.sub.2--, --NR(CH.sub.2CH.sub.2O).sub.n-(cyclo
alkyl)-O-Aryl-O--CH.sub.2--, --NR(CH.sub.2CH.sub.2O).sub.n-(lower
alkyl)-NH-Aryl-O--CH.sub.2--, --NR(CH.sub.2CH.sub.2O).sub.n-(lower
alkyl)-O-Aryl-CH.sub.2,
--NR(CH.sub.2CH.sub.2O).sub.n-cycloalkyl-O-Aryl-,
--NR(CH.sub.2CH.sub.2O).sub.n-cycloalkyl-O-(heteroaryl)l-,
--NR(CH.sub.2CH.sub.2).sub.n-(cycloalkyl)-O-(heterocycle)-CH.sub.2,
--NR(CH.sub.2CH.sub.2).sub.n-(heterocycle)-(heterocycle)-CH.sub.2,
--N(R1R2)-(heterocycle)-CH.sub.2; where [0145] n of the linker can
be 0 to 10; [0146] R of the linker can be H, lower alkyl; [0147] R1
and R2 of the linker can form a ring with the connecting N.
[0148] In certain embodiments, the linker (L) comprises a group
represented by a general structure selected from the group
consisting of: [0149]
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O-
(CH2).sub.q--O(CH2).sub.r--OCH2-, [0150]
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r--OCH2-, [0151]
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r--O--; [0152]
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub-
.q--O(CH2).sub.r--O--; [0153]
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--O(-
CH2).sub.r--O--; [0154]
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--O(-
CH2).sub.r--OCH2-;
##STR00025## ##STR00026## ##STR00027##
[0154] wherein [0155] m, n, o, p, q, and r of the linker are
independently 0, 1, 2, 3, 4, 5, 6; 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20; [0156] when the number is zero, there is no
N--O or O--O bond [0157] R of the linker is H, methyl and ethyl;
[0158] X of the linker is H and F
[0158] ##STR00028## [0159] where m of the linker can be 2, 3, 4,
5
[0159] ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## [0160] where each n and m of
the linker can independently be 0, 1, 2, 3, 4, 5, 6.
[0161] In any aspect or embodiment described herein, the linker (L)
is selected from the group consisting of:
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049##
wherein each m and n is independently selected from 0, 1, 2, 3, 4,
5, or 6.
[0162] In any aspect or embodiment described herein, the linker (L)
is selected from the group consisting of:
##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059##
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069##
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088##
wherein each m, n, o, p, q, and r is independently 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
[0163] In any aspect or embodiment described herein, L is selected
from the group consisting of:
##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093##
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108##
##STR00109## ##STR00110## ##STR00111##
[0164] In additional embodiments, the linker (L) comprises a
structure selected from, but not limited to the structure shown
below, where a dashed line indicates the attachment point to the
PTM or ULM moieties:
##STR00112##
wherein: [0165] W.sup.L1 and W.sup.L2 are each independently a 4-8
membered ring with 0-4 heteroatoms, optionally substituted with
R.sup.Q, each R.sup.Q is independently a H, halo, OH, CN, CF.sub.3,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted),
C.sub.1-C.sub.6 alkoxy (linear, branched, optionally substituted),
or 2 R.sup.Q groups taken together with the atom they are attached
to, form a 4-8 membered ring system containing 0-4 heteroatoms;
[0166] Y.sup.L1 is each independently a bond, C.sub.1-C.sub.6 alkyl
(linear, branched, optionally substituted) and optionally one or
more C atoms are replaced with O; or C.sub.1-C.sub.6 alkoxy
(linear, branched, optionally substituted); [0167] n is 0-10; and
[0168] a dashed line indicates the attachment point to the PTM or
ULM moieties.
[0169] In additional embodiments, the linker (L) comprises a
structure selected from, but not limited to the structure shown
below, where a dashed line indicates the attachment point to the
PTM or ULM moieties:
##STR00113##
wherein: [0170] W.sup.L1 and W.sup.L2 are each independently aryl,
heteroaryl, cyclic, heterocyclic, C.sub.1-6 alkyl, bicyclic,
biaryl, biheteroaryl, or biheterocyclic, each optionally
substituted with R.sup.Q, each R.sup.Q is independently a H, halo,
OH, CN, CF.sub.3, hydroxyl, nitro, C.ident.CH, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-C.sub.6 alkyl (linear, branched,
optionally substituted), C.sub.1-C.sub.6 alkoxy (linear, branched,
optionally substituted), OC.sub.1-3alkyl (optionally substituted by
1 or more --F), OH, NH.sub.2, NR.sup.Y1R.sup.Y2, CN, or 2 R.sup.Q
groups taken together with the atom they are attached to, form a
4-8 membered ring system containing 0-4 heteroatoms; [0171]
Y.sup.L1 is each independently a bond, NR.sup.YL1, O, S,
NR.sup.YL2, CR.sup.YL1R.sup.YL2, C.dbd.O, C.dbd.S, SO, SO.sub.2,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted)
and optionally one or more C atoms are replaced with O;
C.sub.1-C.sub.6 alkoxy (linear, branched, optionally substituted);
[0172] Q.sup.L is a 3-6 membered alicyclic or aromatic ring with
0-4 heteroatoms, optionally bridged, optionally substituted with
0-6 R.sup.Q, each R.sup.Q is independently H, C.sub.1-6 alkyl
(linear, branched, optionally substituted by 1 or more halo,
C.sub.1-6 alkoxyl), or 2 R.sup.Q groups taken together with the
atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); [0173] R.sup.YL1, R.sup.YL2 are each
independently H, OH, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), or R.sup.1,
R.sup.2 together with the atom they are attached to, form a 3-8
membered ring system containing 0-2 heteroatoms); [0174] n is 0-10;
and [0175] a dashed line indicates the attachment point to the PTM
or ULM moieties.
[0176] In additional embodiments, the linker group is optionally
substituted (poly)ethyleneglycol having between 1 and about 100
ethylene glycol units, between about 1 and about 50 ethylene glycol
units, between 1 and about 25 ethylene glycol units, between about
1 and 10 ethylene glycol units, between 1 and about 8 ethylene
glycol units and 1 and 6 ethylene glycol units, between 2 and 4
ethylene glycol units, or optionally substituted alkyl groups
interdispersed with optionally substituted, O, N, S, P or Si atoms.
In certain embodiments, the linker is substituted with an aryl,
phenyl, benzyl, alkyl, alkylene, or heterocycle group. In certain
embodiments, the linker may be asymmetric or symmetrical.
[0177] In any of the embodiments of the compounds described herein,
the linker group may be any suitable moiety as described herein. In
one embodiment, the linker is a substituted or unsubstituted
polyethylene glycol group ranging in size from about 1 to about 12
ethylene glycol units, between 1 and about 10 ethylene glycol
units, about 2 about 6 ethylene glycol units, between about 2 and 5
ethylene glycol units, between about 2 and 4 ethylene glycol
units.
[0178] In another embodiment, the present disclosure is directed to
a compound which comprises a PTM group, which binds to a target
protein or polypeptide, which is ubiquitinated by an ubiquitin
ligase and is chemically linked directly to the ULM group (such as
CLM) or through a linker moiety L, or PTM is alternatively a ULM'
group (such as CLM') which is also a ubiquitin ligase binding
moiety, which may be the same or different than the ULM group as
described above and is linked directly to the ULM group directly or
through the linker moiety; and L is a linker moiety as described
above which may be present or absent and which chemically
(covalently) links ULM to PTM, or a pharmaceutically acceptable
salt, enantiomer, stereoisomer, solvate or polymorph thereof.
[0179] In certain embodiments, the linker group L is a group
comprising one or more covalently connected structural units
independently selected from the group consisting of:
##STR00114##
The X is selected from the group consisting of O, N, S, S(O) and
SO.sub.2; n is integer from 1 to 5; R.sup.L1 is hydrogen or
alkyl,
##STR00115##
is a mono- or bicyclic aryl or heteroaryl optionally substituted
with 1-3 substituents selected from alkyl, halogen, haloalkyl,
hydroxy, alkoxy or cyano;
##STR00116##
is a mono- or bicyclic cycloalkyl or a heterocycloalkyl optionally
substituted with 1-3 substituents selected from alkyl, halogen,
haloalkyl, hydroxy, alkoxy or cyano; and the phenyl ring fragment
can be optionally substituted with 1, 2 or 3 substituents selected
from the group consisting of alkyl, halogen, haloalkyl, hydroxy,
alkoxy and cyano. In an embodiment, the linker group L comprises up
to 10 covalently connected structural units, as described
above.
[0180] Although the ULM group and PTM group may be covalently
linked to the linker group through any group which is appropriate
and stable to the chemistry of the linker, in preferred aspects of
the present disclosure, the linker is independently covalently
bonded to the ULM group and the PTM group preferably through an
amide, ester, thioester, keto group, carbamate (urethane), carbon
or ether, each of which groups may be inserted anywhere on the ULM
group and PTM group to provide maximum binding of the ULM group on
the ubiquitin ligase and the PTM group on the target protein to be
degraded. (It is noted that in certain aspects where the PTM group
is a ULM group, the target protein for degradation may be the
ubiquitin ligase itself). In certain preferred aspects, the linker
may be linked to an optionally substituted alkyl, alkylene, alkene
or alkyne group, an aryl group or a heterocyclic group on the ULM
and/or PTM groups.
[0181] In additional embodiments, q is an integer from 1 to 100, 1
to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to
20, or 1 to 10.
[0182] In certain embodiments, the linker (L) is selected from the
group consisting of:
##STR00117## ##STR00118## ##STR00119## ##STR00120##
[0183] In additional embodiments, the linker group is optionally
substituted (poly)ethyleneglycol having between 1 and about 100
ethylene glycol units, between about 1 and about 50 ethylene glycol
units, between 1 and about 25 ethylene glycol units, between about
1 and 10 ethylene glycol units, between 1 and about 8 ethylene
glycol units and 1 and 6 ethylene glycol units, between 2 and 4
ethylene glycol units, or optionally substituted alkyl groups
interdispersed with optionally substituted, O, N, S, P or Si atoms.
In certain embodiments, the linker is substituted with an aryl,
phenyl, benzyl, alkyl, alkylene, or heterocycle group. In certain
embodiments, the linker may be asymmetric or symmetrical.
[0184] In any of the embodiments of the compounds described herein,
the linker group may be any suitable moiety as described herein. In
one embodiment, the linker is a substituted or unsubstituted
polyethylene glycol group ranging in size from about 1 to about 12
ethylene glycol units, between 1 and about 10 ethylene glycol
units, about 2 about 6 ethylene glycol units, between about 2 and 5
ethylene glycol units, between about 2 and 4 ethylene glycol
units.
[0185] Although the CLM (or ULM) group and PTM group may be
covalently linked to the linker group through any group which is
appropriate and stable to the chemistry of the linker, in preferred
aspects of the present disclosure, the linker is independently
covalently bonded to the CLM group and the PTM group preferably
through an amide, ester, thioester, keto group, carbamate
(urethane), carbon or ether, each of which groups may be inserted
anywhere on the CLM group and PTM group to provide maximum binding
of the CLM group on the ubiquitin ligase and the PTM group on the
target protein to be degraded. (It is noted that in certain aspects
where the PTM group is a ULM group, the target protein for
degradation may be the ubiquitin ligase itself). In certain
preferred aspects, the linker may be linked to an optionally
substituted alkyl, alkylene, alkene or alkyne group, an aryl group
or a heterocyclic group on the CLM and/or PTM groups.
[0186] In certain embodiments, "L" can be linear chains with linear
atoms from 4 to 24, the carbon atom in the linear chain can be
substituted with oxygen, nitrogen, amide, fluorinated carbon, etc.,
such as the following:
##STR00121## ##STR00122## ##STR00123##
[0187] In certain embodiments, "L" can be nonlinear chains, and can
be aliphatic or aromatic or heteroaromatic cyclic moieties, some
examples of "L" include but not be limited to the following:
##STR00124## ##STR00125##
wherein: [0188] "X" in above structures can be linear chain with
atoms ranging from 2 to 14, and the mentioned chain can contain
heteroatoms such as oxygen; and [0189] "Y" in above structures can
be O, N, S(O).sub.n (n=0, 1, 2).
[0190] Exemplary PTMs
[0191] In preferred aspects of the present disclosure, the PTM
group is a group, which binds to target proteins. Targets of the
PTM group are numerous in kind and are selected from proteins that
are expressed in a cell such that at least a portion of the
sequences is found in the cell and may bind to a PTM group. The
term "protein" includes oligopeptides and polypeptide sequences of
sufficient length that they can bind to a PTM group according to
the present disclosure. Any protein in a eukaryotic system or a
microbial system, including a virus, bacteria or fungus, as
otherwise described herein, are targets for ubiquitination mediated
by the compounds according to the present disclosure. Preferably,
the target protein is a eukaryotic protein. In certain aspects, the
protein binding moiety is a haloalkane (preferably a
C.sub.1-C.sub.10 alkyl group which is substituted with at least one
halo group, preferably a halo group at the distal end of the alkyl
group, i.e., away from the linker or CLM group), which may
covalently bind to a dehalogenase enzyme in a patient or subject or
in a diagnostic assay.
[0192] PTM groups according to the present disclosure include, for
example, include any moiety which binds to a protein specifically
(binds to a target protein) and includes the following non-limiting
examples of small molecule target protein moieties: Hsp90
inhibitors, kinase inhibitors, androgen receptor inhibitors, HDM2
& MDM2 inhibitors, compounds targeting Human BET
Bromodomain-containing proteins, HDAC inhibitors, human lysine
methyltransferase inhibitors, angiogenesis inhibitors, nuclear
hormone receptor compounds, immunosuppressive compounds, and
compounds targeting the aryl hydrocarbon receptor (AHR), among
numerous others. The compositions described below exemplify some of
the members of these nine types of small molecule target protein
binding moieties. Such small molecule target protein binding
moieties also include pharmaceutically acceptable salts,
enantiomers, solvates and polymorphs of these compositions, as well
as other small molecules that may target a protein of interest.
These binding moieties are linked to the ubiquitin ligase binding
moiety preferably through a linker in order to present a target
protein (to which the protein target moiety is bound) in proximity
to the ubiquitin ligase for ubiquitination and degradation.
[0193] Any protein, which can bind to a protein target moiety or
PTM group and acted on or degraded by a ubiquitin ligase is a
target protein according to the present disclosure. In general,
target proteins may include, for example, structural proteins,
receptors, enzymes, cell surface proteins, proteins pertinent to
the integrated function of a cell, including proteins involved in
catalytic activity, aromatase activity, motor activity, helicase
activity, metabolic processes (anabolism and catabolism),
antioxidant activity, proteolysis, biosynthesis, proteins with
kinase activity, oxidoreductase activity, transferase activity,
hydrolase activity, lyase activity, isomerase activity, ligase
activity, enzyme regulator activity, signal transducer activity,
structural molecule activity, binding activity (protein, lipid
carbohydrate), receptor activity, cell motility, membrane fusion,
cell communication, regulation of biological processes,
development, cell differentiation, response to stimulus, behavioral
proteins, cell adhesion proteins, proteins involved in cell death,
proteins involved in transport (including protein transporter
activity, nuclear transport, ion transporter activity, channel
transporter activity, carrier activity, permease activity,
secretion activity, electron transporter activity, pathogenesis,
chaperone regulator activity, nucleic acid binding activity,
transcription regulator activity, extracellular organization and
biogenesis activity, translation regulator activity. Proteins of
interest can include proteins from eurkaryotes and prokaryotes
including humans as targets for drug therapy, other animals,
including domesticated animals, microbials for the determination of
targets for antibiotics and other antimicrobials and plants, and
even viruses, among numerous others.
[0194] In still other embodiments, the PTM group is a haloalkyl
group, wherein said alkyl group generally ranges in size from about
1 or 2 carbons to about 12 carbons in length, often about 2 to 10
carbons in length, often about 3 carbons to about 8 carbons in
length, more often about 4 carbons to about 6 carbons in length.
The haloalkyl groups are generally linear alkyl groups (although
branched-chain alkyl groups may also be used) and are end-capped
with at least one halogen group, preferably a single halogen group,
often a single chloride group. Haloalkyl PT, groups for use in the
present disclosure are preferably represented by the chemical
structure --(CH.sub.2).sub.v-Halo where v is any integer from 2 to
about 12, often about 3 to about 8, more often about 4 to about 6.
Halo may be any halogen, but is preferably Cl or Br, more often
Cl.
[0195] In another embodiment, the present disclosure provides a
library of compounds. The library comprises more than one compound
wherein each composition has a formula of A-B, wherein A is a
ubiquitin pathway protein binding moiety (preferably, an E3
ubiquitin ligase moiety as otherwise disclosed herein) and B is a
protein binding member of a molecular library, wherein A is coupled
(preferably, through a linker moiety) to B, and wherein the
ubiquitin pathway protein binding moiety recognizes an ubiquitin
pathway protein, in particular, an E3 ubiquitin ligase, such as
cereblon. In a particular embodiment, the library contains a
specific cereblon E3 ubiquitin ligase binding moiety bound to
random target protein binding elements (e.g., a chemical compound
library). As such, the target protein is not determined in advance
and the method can be used to determine the activity of a putative
protein binding element and its pharmacological value as a target
upon degradation by ubiquitin ligase.
[0196] The present disclosure may be used to treat a number of
disease states and/or conditions, including any disease state
and/or condition in which proteins are dysregulated and where a
patient would benefit from the degradation of proteins.
[0197] In an additional aspect, the description provides
therapeutic compositions comprising an effective amount of a
compound as described herein or salt form thereof, and a
pharmaceutically acceptable carrier, additive or excipient, and
optionally an additional bioactive agent. The therapeutic
compositions modulate protein degradation in a patient or subject,
for example, an animal such as a human, and can be used for
treating or ameliorating disease states or conditions which are
modulated through the degraded protein. In certain embodiments, the
therapeutic compositions as described herein may be used to
effectuate the degradation of proteins of interest for the
treatment or amelioration of a disease, e.g., cancer (such as
prostate cancer) and Kennedy's Disease. In certain additional
embodiments, the disease is prostate cancer.
[0198] In alternative aspects, the present disclosure relates to a
method for treating a disease state or ameliorating the symptoms of
a disease or condition in a subject in need thereof by degrading a
protein or polypeptide through which a disease state or condition
is modulated comprising administering to said patient or subject an
effective amount, e.g., a therapeutically effective amount, of at
least one compound as described hereinabove, optionally in
combination with a pharmaceutically acceptable carrier, additive or
excipient, and optionally an additional bioactive agent, wherein
the composition is effective for treating or ameliorating the
disease or disorder or symptom thereof in the subject. The method
according to the present disclosure may be used to treat a large
number of disease states or conditions including cancer, by virtue
of the administration of effective amounts of at least one compound
described herein. The disease state or condition may be a disease
caused by a microbial agent or other exogenous agent such as a
virus, bacteria, fungus, protozoa or other microbe or may be a
disease state, which is caused by overexpression of a protein,
which leads to a disease state and/or condition.
[0199] In another aspect, the description provides methods for
identifying the effects of the degradation of proteins of interest
in a biological system using compounds according to the present
disclosure.
[0200] The term "target protein" is used to describe a protein or
polypeptide, which is a target for binding to a compound according
to the present disclosure and degradation by ubiquitin ligase
hereunder. Such small molecule target protein binding moieties also
include pharmaceutically acceptable salts, enantiomers, solvates
and polymorphs of these compositions, as well as other small
molecules that may target a protein of interest. These binding
moieties are linked to CLM or ULM groups through linker groups
L.
[0201] Target proteins which may be bound to the protein target
moiety and degraded by the ligase to which the ubiquitin ligase
binding moiety is bound include any protein or peptide, including
fragments thereof, analogues thereof, and/or homologues thereof.
Target proteins include proteins and peptides having any biological
function or activity including structural, regulatory, hormonal,
enzymatic, genetic, immunological, contractile, storage,
transportation, and signal transduction. In certain embodiments,
the target proteins include structural proteins, receptors,
enzymes, cell surface proteins, proteins pertinent to the
integrated function of a cell, including proteins involved in
catalytic activity, aromatase activity, motor activity, helicase
activity, metabolic processes (anabolism and catabolism),
antioxidant activity, proteolysis, biosynthesis, proteins with
kinase activity, oxidoreductase activity, transferase activity,
hydrolase activity, lyase activity, isomerase activity, ligase
activity, enzyme regulator activity, signal transducer activity,
structural molecule activity, binding activity (protein, lipid
carbohydrate), receptor activity, cell motility, membrane fusion,
cell communication, regulation of biological processes,
development, cell differentiation, response to stimulus, behavioral
proteins, cell adhesion proteins, proteins involved in cell death,
proteins involved in transport (including protein transporter
activity, nuclear transport, ion transporter activity, channel
transporter activity, carrier activity, permease activity,
secretion activity, electron transporter activity, pathogenesis,
chaperone regulator activity, nucleic acid binding activity,
transcription regulator activity, extracellular organization and
biogenesis activity, translation regulator activity. Proteins of
interest can include proteins from eukaryotes and prokaryotes,
including microbes, viruses, fungi and parasites, including humans,
microbes, viruses, fungi and parasites, among numerous others, as
targets for drug therapy, other animals, including domesticated
animals, microbials for the determination of targets for
antibiotics and other antimicrobials and plants, and even viruses,
among numerous others.
[0202] More specifically, a number of drug targets for human
therapeutics represent protein targets to which protein target
moiety may be bound and incorporated into compounds according to
the present disclosure. These include proteins which may be used to
restore function in numerous polygenic diseases, including for
example B7.1 and B7, TINFR1m, TNFR2, NADPH oxidase, BclIBax and
other partners in the apotosis pathway, C5a receptor, HMG-CoA
reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase
type 4, PDE I, PDEII, PDEIII, squalene cyclase inhibitor, CXCR1,
CXCR2, nitric oxide (NO) synthase, cyclo-oxygenase 1,
cyclo-oxygenase 2, 5HT receptors, dopamine receptors, G Proteins,
i.e., Gq, histamine receptors, 5-lipoxygenase, tryptase serine
protease, thymidylate synthase, purine nucleoside phosphorylase,
GAPDH trypanosomal, glycogen phosphorylase, Carbonic anhydrase,
chemokine receptors, JAW STAT, RXR and similar, HIV 1 protease, HIV
1 integrase, influenza, neuramimidase, hepatitis B reverse
transcriptase, sodium channel, multi drug resistance (MDR), protein
P-glycoprotein (and MRP), tyrosine kinases, CD23, CD124, tyrosine
kinase p56 lck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR,
ICAM1, Cat+ channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L,
newokinins and receptors, inosine monophosphate dehydrogenase, p38
MAP Kinase, Ras1Raf1MEWERK pathway, interleukin-1 converting
enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase,
glycinamide ribonucleotide formyl transferase, rhinovirus 3C
protease, herpes simplex virus-1 (HSV-I), protease, cytomegalovirus
(CMV) protease, poly (ADP-ribose) polymerase, cyclin dependent
kinases, vascular endothelial growth factor, oxytocin receptor,
microsomal transfer protein inhibitor, bile acid transport
inhibitor, 5 alpha reductase inhibitors, angiotensin 11, glycine
receptor, noradrenaline reuptake receptor, endothelin receptors,
neuropeptide Y and receptor, estrogen receptors, androgen receptors
(AR), adenosine receptors, adenosine kinase and AMP deaminase,
purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7),
farnesyltransferases, geranylgeranyl transferase, TrkA a receptor
for NGF, beta-amyloid, tyrosine kinase Flk-IIKDR, vitronectin
receptor, integrin receptor, Her-21 neu, telomerase inhibition,
cytosolic phospholipaseA2 and EGF receptor tyrosine kinase.
Additional protein targets include, for example, ecdysone
20-monooxygenase, ion channel of the GABA gated chloride channel,
acetylcholinesterase, voltage-sensitive sodium channel protein,
calcium release channel, and chloride channels. Still further
target proteins include Acetyl-CoA carboxylase, adenylosuccinate
synthetase, protoporphyrinogen oxidase, and
enolpyruvylshikimate-phosphate synthase.
[0203] Haloalkane dehalogenase enzymes are another target of
specific compounds according to the present disclosure. Compounds
according to the present disclosure which contain chloroalkane
peptide binding moieties (C.sub.1-C.sub.12 often about
C.sub.2-C.sub.10 alkyl halo groups) may be used to inhibit and/or
degrade haloalkane dehalogenase enzymes which are used in fusion
proteins or related dioagnostic proteins as described in
PCT/US2012/063401 filed Dec. 6, 2011 and published as WO
2012/078559 on Jun. 14, 2012, the contents of which is incorporated
by reference herein.
[0204] These various protein targets may be used in screens that
identify compound moieties which bind to the protein and by
incorporation of the moiety into compounds according to the present
disclosure, the level of activity of the protein may be altered for
therapeutic end result.
[0205] The term "protein target moiety" or PTM is used to describe
a small molecule which binds to a target protein or other protein
or polypeptide of interest and places/presents that protein or
polypeptide in proximity to an ubiquitin ligase such that
degradation of the protein or polypeptide by ubiquitin ligase may
occur. Non-limiting examples of small molecule target protein
binding moieties include Hsp90 inhibitors, kinase inhibitors, MDM2
inhibitors, compounds targeting Human BET Bromodomain-containing
proteins, HDAC inhibitors, human lysine methyltransferase
inhibitors, angiogenesis inhibitors, immunosuppressive compounds,
and compounds targeting the aryl hydrocarbon receptor (AHR), among
numerous others. The compositions described below exemplify some of
the members of these nine types of small molecule target
protein.
[0206] Exemplary protein target moieties according to the present
disclosure include, haloalkane halogenase inhibitors, Hsp90
inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting
Human BET Bromodomain-containing proteins, HDAC inhibitors, human
lysine methyltransferase inhibitors, angiogenesis inhibitors,
immunosuppressive compounds, and compounds targeting the aryl
hydrocarbon receptor (AHR).
[0207] The compositions described below exemplify some of the
members of these types of small molecule target protein binding
moieties. Such small molecule target protein binding moieties also
include pharmaceutically acceptable salts, enantiomers, solvates
and polymorphs of these compositions, as well as other small
molecules that may target a protein of interest. References which
are cited hereinbelow are incorporated by reference herein in their
entirety.
[0208] I. Heat Shock Protein 90 (HSP90) Inhibitors:
[0209] HSP90 inhibitors as used herein include, but are not limited
to:
[0210] 1. The HSP90 inhibitors identified in Vallee, et al.,
"Tricyclic Series of Heat Shock Protein 90 (HSP90) Inhibitors Part
I: Discovery of Tricyclic Imidazo[4,5-C]Pyridines as Potent
Inhibitors of the HSP90 Molecular Chaperone (2011) J. Med. Chem.
54: 7206, including YKB
(N-[4-(3H-imidazo[4,5-C]Pyridin-2-yl)-9H-Fluoren-9-yl]-succinamide):
##STR00126##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal amide group;
[0211] 2. The HSP90 inhibitor p54 (modified)
(8-[(2,4-dimethylphenyl)sulfanyl]-3]pent-4-yn-1-yl-3H-purin-6-amine):
##STR00127##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal acetylene group;
[0212] 3. The HSP90 inhibitors (modified) identified in Brough, et
al., "4,5-Diarylisoxazole HSP90 Chaperone Inhibitors: Potential
Therapeutic Agents for the Treatment of Cancer", J. MED. CHEM. vol:
51, pag: 196 (2008), including the compound 2GJ
(5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-n-ethyl-4-[4-(morpholin-4-ylme-
thyl)phenyl]isoxazole-3-carboxamide) having the structure:
##STR00128##
derivatized, where a linker group L or a -(L-CLM) group is
attached, for example, via the amide group (at the amine or at the
alkyl group on the amine);
[0213] 4. The HSP90 inhibitors (modified) identified in Wright, et
al., Structure-Activity Relationships in Purine-Based Inhibitor
Binding to HSP90 Isoforms, Chem Biol. 2004 June; 11(6):775-85,
including the HSP90 inhibitor PU3 having the structure:
##STR00129##
derivatized where a linker group L or -(L-CLM) is attached, for
example, via the butyl group; and
[0214] 5. The HSP90 inhibitor geldanamycin
((4E,6Z,8S,9S,10E,12S,13R,14S,16R)-13-hydroxy-8,14,19-trimethoxy-4,10,12,-
16-tetramethyl-3,20,22-trioxo-2-azabicyclo[16.3.1] (derivatized) or
any of its derivatives (e.g.
17-alkylamino-17-desmethoxygeldanamycin ("17-AAG") or
17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin
("17-DMAG")) (derivatized, where a linker group L or a -(L-CLM)
group is attached, for example, via the amide group).
[0215] II. Kinase and Phosphatase Inhibitors:
[0216] Kinase inhibitors as used herein include, but are not
limited to:
[0217] 1. Erlotinib Derivative Tyrosine Kinase Inhibitor:
##STR00130##
where R is a linker group L or a -(L-CLM) group attached, for
example, via the ether group;
[0218] 2. The kinase inhibitor sunitinib (derivatized):
##STR00131##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the pyrrole moiety;
[0219] 3. Kinase Inhibitor sorafenib (derivatized):
##STR00132##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the amide moiety;
[0220] 4. The kinase inhibitor desatinib (derivatized):
##STR00133##
derivatized where R is a linker group L or a -(L-CLM) attached, for
example, to the pyrimidine;
[0221] 5. The kinase inhibitor lapatinib (derivatized):
##STR00134##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal methyl of the sulfonyl methyl
group;
[0222] 6. The kinase inhibitor U09-CX-5279 (derivatized):
##STR00135##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the amine (aniline), carboxylic acid or amine
alpha to cyclopropyl group, or cyclopropyl group;
[0223] 7. The kinase inhibitors identified in Millan, et al.,
Design and Synthesis of Inhaled P38 Inhibitors for the Treatment of
Chronic Obstructive Pulmonary Disease, J. MED. CHEM. vol: 54, pag:
7797 (2011), including the kinase inhibitors Y1W and Y1X
(Derivatized) having the structures:
##STR00136##
[0224]
YIX(1-ethyl-3-(2-{[3-(1-methylethyl)[1,2,4]triazolo[4,3-a]pyridine--
6-yl]sulfanyl}benzyl)urea, derivatized where a linker group L or a
-(L-CLM) group is attached, for example, via the .sup.ipropyl
group;
##STR00137##
[0225] YIW
[0226]
1-(3-ten-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(2-{[3-(1-methylethyl)[1-
,2,4]triazolo[4,3-a]pyridin-6-yl]sulfanyl}benzyl)urea
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, preferably via either the i-propyl group or the
t-butyl group;
[0227] 8. The kinase inhibitors identified in Schenkel, et al.,
Discovery of Potent and Highly Selective Thienopyridine Janus
Kinase 2 Inhibitors J. Med. Chem., 2011, 54 (24), pp 8440-8450,
including the compounds 6TP and 0TP (Derivatized) having the
structures:
##STR00138##
[0228] 6TP
[0229]
4-amino-2-[4-(tert-butylsulfamoyl)phenyl]-N-methylthieno[3,2-c]pyri-
dine-7-carboxamide
[0230] Thienopyridine 19
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal methyl group bound to amide
moiety;
##STR00139##
[0231] 0TP
[0232]
4-amino-N-methyl-2-[4-(morpholin-4-yl)phenyl]thieno[3,2-c]pyridine--
7-carboxamide
[0233] Thienopyridine 8
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal methyl group bound to the amide
moiety;
[0234] 9. The kinase inhibitors identified in Van Eis, et al.,
"2,6-Naphthyridines as potent and selective inhibitors of the novel
protein kinase C isozymes", Biorg. Med. Chem. Lett. 2011 Dec. 15;
21(24):7367-72, including the kinase inhibitor 07U having the
structure:
##STR00140##
[0235] 07U
[0236]
2-methyl-N.about.1.about.-[3-(pyridin-4-yl)-2,6-naphthyridin-1-yl]p-
ropane-1,2-diamine
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the secondary amine or terminal amino group;
[0237] 10. The kinase inhibitors identified in Lountos, et al.,
"Structural Characterization of Inhibitor Complexes with Checkpoint
Kinase 2 (Chk2), a Drug Target for Cancer Therapy", J. STRUCT.
BIOL. vol: 176, pag: 292 (2011), including the kinase inhibitor YCF
having the structure:
##STR00141##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via either of the terminal hydroxyl groups;
[0238] 11. The kinase inhibitors identified in Lountos, et al.,
"Structural Characterization of Inhibitor Complexes with Checkpoint
Kinase 2 (Chk2), a Drug Target for Cancer Therapy", J. STRUCT.
BIOL. vol: 176, pag: 292 (2011), including the kinase inhibitors
XK9 and NXP (derivatized) having the structures:
##STR00142##
[0239] XK9
[0240]
N-{4-[(1E)-N--(N-hydroxycarbamimidoyl)ethanehydrazonoyl]phenyl}-7-n-
itro-1H-indole-2-carboxamide;
##STR00143##
[0241] NXP
[0242]
N-{4-[(1E)-N-CARBAMIMIDOYLETHANEHYDRAZONOYL]PHENYL}-1H-INDOLE-3-CAR-
BOXAMIDE
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via the terminal hydroxyl group (XK9) or the hydrazone
group (NXP);
[0243] 12. The kinase inhibitor afatinib (derivatized)
(N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-
-6-quinazolinyl]-4(dimethylamino)-2-butenamide) (Derivatized where
a linker group L or a -(L-CLM) group is attached, for example, via
the aliphatic amine group);
[0244] 13. The kinase inhibitor fostamatinib (derivatized)
([6-({5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]pyrimidin-4-yl}amino)-2,2-
-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b]-1,4-oxazin-4-yl]methyl
disodium phosphate hexahydrate) (Derivatized where a linker group L
or a -(L-CLM) group is attached, for example, via a methoxy
group);
[0245] 14. The kinase inhibitor gefitinib (derivatized)
(N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinaz-
olin-4-amine):
##STR00144##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, via a methoxy or ether group;
[0246] 15. The kinase inhibitor lenvatinib (derivatized)
(4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxy-quinoline-6--
carboxamide) (derivatized where a linker group L or a -(L-CLM)
group is attached, for example, via the cyclopropyl group);
[0247] 16. The kinase inhibitor vandetanib (derivatized)
(N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]-
quinazolin-4-amine) (derivatized where a linker group L or a
-(L-CLM) group is attached, for example, via the methoxy or
hydroxyl group);
[0248] 17. The kinase inhibitor vemurafenib (derivatized)
(propane-1-sulfonic acid
{3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-
-phenyl}-amide), derivatized where a linker group L or a -(L-CLM)
group is attached, for example, via the sulfonyl propyl group;
[0249] 18. The kinase inhibitor Gleevec (derivatized):
##STR00145##
derivatized where R as a linker group L or a -(L-CLM) group is
attached, for example, via the amide group or via the aniline amine
group;
[0250] 19. The kinase inhibitor pazopanib (derivatized) (VEGFR3
inhibitor):
##STR00146##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety or via the aniline
amine group;
[0251] 20. The kinase inhibitor AT-9283 (Derivatized) Aurora Kinase
Inhibitor
##STR00147##
where R is a linker group L or a -(L-CLM) group attached, for
example, to the phenyl moiety);
[0252] 21. The kinase inhibitor TAE684 (derivatized) ALK
inhibitor
##STR00148##
where R is a linker group L or a -(L-CLM) group attached, for
example, to the phenyl moiety);
[0253] 22. The kinase inhibitor nilotanib (derivatized) Abl
inhibitor:
##STR00149##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety or the aniline amine
group;
[0254] 23. Kinase Inhibitor NVP-BSK805 (derivatized) JAK2
Inhibitor
##STR00150##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety or the diazole
group;
[0255] 24. Kinase Inhibitor crizotinib Derivatized Alk
Inhibitor
##STR00151##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety or the diazole
group;
[0256] 25. Kinase Inhibitor JNJ FMS (derivatized) Inhibitor
##STR00152##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety;
[0257] 26. The kinase inhibitor foretinib (derivatized) Met
Inhibitor
##STR00153##
derivatized where R is a linker group L or a -(L-CLM) group
attached, for example, to the phenyl moiety or a hydroxyl or ether
group on the quinoline moiety;
[0258] 27. The allosteric Protein Tyrosine Phosphatase Inhibitor
PTP1B (derivatized):
##STR00154##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R, as indicated;
[0259] 28. The inhibitor of SHP-2 Domain of Tyrosine Phosphatase
(derivatized):
##STR00155##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R;
[0260] 29. The inhibitor (derivatized) of BRAF
(BRAF.sup.V600E)/MEK:
##STR00156##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R;
[0261] 30. Inhibitor (derivatized) of Tyrosine Kinase ABL
##STR00157##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R;
[0262] 31. The kinase inhibitor OSI-027 (derivatized) mTORC1/2
inhibitor
##STR00158##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R;
[0263] 32. The kinase inhibitor OSI-930 (derivatized) c-Kit/KDR
inhibitor
##STR00159##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R; and
[0264] 33. The kinase inhibitor OSI-906 (derivatized) IGF1R/IR
inhibitor
##STR00160##
derivatized where a linker group L or a -(L-CLM) group is attached,
for example, at R.
[0265] Wherein, in any of the embodiments described in sections
I-XVII, "R" designates a site for attachment of a linker group L or
a -(L-CLM) group on the piperazine moiety.
[0266] III. HDM2/MDM2 Inhibitors:
[0267] HDM2/MDM2 inhibitors as used herein include, but are not
limited to:
[0268] 1. The HDM2/MDM2 inhibitors identified in Vassilev, et al.,
In vivo activation of the p53 pathway by small-molecule antagonists
of MDM2, SCIENCE vol: 303, pag: 844-848 (2004), and Schneekloth, et
al., Targeted intracellular protein degradation induced by a small
molecule: En route to chemical proteomics, Bioorg. Med. Chem. Lett.
18 (2008) 5904-5908, including (or additionally) the compounds
nutlin-3, nutlin-2, and nutlin-1 (derivatized) as described below,
as well as all derivatives and analogs thereof:
##STR00161##
(derivatized where a linker group L or a -(L-CLM) group is
attached, for example, at the methoxy group or as a hydroxyl
group);
##STR00162##
(derivatized where a linker group L or a -(L-CLM) group is
attached, for example, at the methoxy group or hydroxyl group);
##STR00163##
(derivatized where a linker group L or a -(L-CLM) group is
attached, for example, via the methoxy group or as a hydroxyl
group); and
[0269] 2. Trans-4-Iodo-4'-Boranyl-Chalcone
##STR00164##
[0270] (derivatized where a linker group L or a a linker group L or
a -(L-CLM) group is attached, for example, via a hydroxy
group).
[0271] IV. Compounds Targeting Human BET Bromodomain-Containing
Proteins:
[0272] In certain embodiments, "PTM" can be ligands binding to
Bromo- and Extra-terminal (BET) proteins BRD2, BRD3 and BRD4.
Compounds targeting Human BET Bromodomain-containing proteins
include, but are not limited to the compounds associated with the
targets as described below, where "R" or "linker" designates a site
for linker group L or a -(L-CLM) group attachment, for example:
[0273] 1. JQ1, Filippakopoulos et al. Selective inhibition of BET
bromodomains. Nature (2010):
##STR00165## ##STR00166##
[0274] 2. I-BET, Nicodeme et al. Supression of Inflammation by a
Synthetic Histone Mimic. Nature (2010). Chung et al. Discovery and
Characterization of Small Molecule Inhibitors of the BET Family
Bromodomains. J. Med Chem. (2011):
##STR00167##
[0275] 3. Compounds described in Hewings et al.
3,5-Dimethylisoxazoles Act as Acetyl-lysine Bromodomain Ligands. J.
Med. Chem. (2011) 54 6761-6770.
##STR00168##
[0276] 4. I-BET151, Dawson et al. Inhibition of BET Recruitment to
Chromatin as an Effective Treatment for MLL-fusion Leukemia. Nature
(2011):
##STR00169##
[0277] 5. Carbazole type (US 2015/0256700)
##STR00170##
[0278] 6. Pyrrolopyridone type (US 2015/0148342)
##STR00171##
[0279] 7. Tetrahydroquinoline type (WO 2015/074064)
##STR00172##
[0280] 8. Triazolopyrazine type (WO 2015/067770)
##STR00173##
[0281] 9. Pyridone type (WO 2015/022332)
##STR00174##
[0282] 10. Quinazolinone type (WO 2015/015318)
##STR00175##
[0283] 11. Dihydropyridopyrazinone type (WO 2015/011084)
##STR00176##
[0284] (Where R or L or linker, in each instance, designates a site
for attachment, for example, of a linker group L or a -(L-CLM)
group).
[0285] In any aspect or embodiment described herein, the claimed
structure the PTM may be composed of tricyclic diazepine or
tricyclic azepine as BET/BRD4 ligand (PTM-a), where the dashed
lines indicate the linker connection trajectory and three sites are
defined to which linkers may be attached:
##STR00177##
wherein: [0286] A and B are independently an aromatic ring, a
heteroaromatic ring, a 5-membered carbocyclic, a 6-membered
carbocyclic, a 5-membered heterocyclic, a 6-membered heterocyclic,
a thiophene, a pyrrole, a pyrazole, a pyridine, a pyrimidine, a
pyrazine, optionally substituted by alkyl, aloxy, halogen, nitrile
or another aromatic or heteroaromatic ring, where A is fused to the
central azepine (Y1=C) or diazepine (Y1=N) moiety; [0287] Y1, Y2,
and Y3 and Y4 can be carbon, nitrogen or oxygen for to form a fused
5-membered aromatic ring as triazole or isoxazole; and [0288] Z1 is
methyl, or lower alkyl group.
[0289] The fragment of PTM-a as BET/BRD4 ligand is described in the
literature (WO 2016/069578; WO2014/001356; WO2016/050821; WO
2015/195863; WO 2014/128111).
[0290] In any aspect or embodiment described herein comprising the
structure CLM-L-PTM-a, PTM-a can be represented by the following
general structures, where dashed line indicates a possible linker
connection point. In structure PTM-aa through PTM-ai, the
substitution pattern of X and Y can be mono- or
bis-substitution.
##STR00178##
X=Cl, F, Br, H, CN, methyl, acetylene, methoxy
##STR00179##
X=Cl, F, Br, H, CN, methyl, acetylene, methoxy
##STR00180##
X=Cl, F, Br, H, CN, methyl, methoxy, acetylene Y: mono- or
di-substitution, Y=Me, OMe, N-methylpyrazole/imidazole
##STR00181##
X=Cl, F, Br, H, CN, methyl, methoxy, acetylene Y: mono- or
di-substitution, Y=Me, OMe, N-methylpyrazole/imidazole
##STR00182##
X=Cl, F, Br, H, CN, methyl, methoxy, acetylene R=lower alkyl, aryl,
substituted aryl
##STR00183##
R=lower alkyl, aryl, substituted aryl
##STR00184##
Y: mono- or di-substitution, Y=Me, OMe, N-methylpyrazole/imidazole
R=lower alkyl, aryl, substituted aryl
##STR00185##
X=Cl, F, Br, H, CN, methyl, acetylene, methoxy
##STR00186##
X=Cl, F, Br, H, CN, methyl, acetylene, methoxy
[0291] In any aspect or embodiment described herein, the structures
of PTM-a as the BET/BRD4 ligand includes, wherein the dashed line
indicates the connection point between BET/BRD4 ligand and the
linkers:
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## ##STR00193##
[0292] In certain embodiments, the description provides, but not
limited to, the following exemplary BET PROTAC (compounds 1 or 2),
including salts, prodrugs, polymorphs, analogs, derivatives, and
deuterated forms thereof:
##STR00194##
[0293] V. HDAC Inhibitors:
[0294] HDAC Inhibitors (derivatized) include, but are not limited
to:
[0295] 1. Finnin, M. S. et al. Structures of Histone Deacetylase
Homologue Bound to the TSA and SAHA Inhibitors. Nature 40, 188-193
(1999).
##STR00195##
(Derivatized where "R" designates a site for attachment, for
example, of a linker group L or a -(L-CLM) group); and
[0296] 2. Compounds as defined by formula (I) of PCT WO0222577
("DEACETYLASE INHIBITORS") (Derivatized where a linker group L or a
-(L-CLM) group is attached, for example, via the hydroxyl
group);
[0297] VI. Human Lysine Methyltransferase Inhibitors:
[0298] Human Lysine Methyltransferase inhibitors include, but are
not limited to:
[0299] 1. Chang et al. Structural Basis for G9a-Like protein Lysine
Methyltransferase Inhibition by BIX-1294. Nat. Struct. Biol. (2009)
16(3) 312.
##STR00196##
(Derivatized where "R" designates a site for attachment, for
example, of a linker group L or a -(L-CLM) group);
[0300] 2. Liu, F. et al Discovery of a
2,4-Diamino-7-aminoalkoxyquinazoline as a Potent and Selective
Inhibitor of Histone Methyltransferase G9a. J. Med. Chem. (2009)
52(24) 7950.
##STR00197##
[0301] (Derivatized where "R" designates a potential site for
attachment, for example, of a linker group L or a -(L-CLM)
group);
[0302] 3. Azacitidine (derivatized)
(4-amino-1-.beta.-D-ribofuranosyl-1,3,5-triazin-2(1H)-one)
(Derivatized where a linker group L or a -(L-CLM) group is
attached, for example, via the hydroxy or amino groups); and
[0303] 4. Decitabine (derivatized)
(4-amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1, 3,
5-triazin-2(1H)-one) (Derivatized where a linker group L or a
-(L-CLM) group is attached, for example, via either of the hydroxy
groups or at the amino group).
[0304] VII. Angiogenesis Inhibitors:
[0305] Angiogenesis inhibitors include, but are not limited to:
[0306] 1. GA-1 (derivatized) and derivatives and analogs thereof,
having the structure(s) and binding to linkers as described in
Sakamoto, et al., Development of Protacs to target cancer-promoting
proteins for ubiquitination and degradation, Mol Cell Proteomics
2003 Dec.; 2(12):1350-8;
[0307] 2. Estradiol (derivatized), which may be bound to a linker
group L or a -(L-CLM) group as is generally described in
Rodriguez-Gonzalez, et al., Targeting steroid hormone receptors for
ubiquitination and degradation in breast and prostate cancer,
Oncogene (2008) 27, 7201-7211;
[0308] 3. Estradiol, testosterone (derivatized) and related
derivatives, including but not limited to DHT and derivatives and
analogs thereof, having the structure(s) and binding to a linker
group L or a -(L-CLM) group as generally described in Sakamoto, et
al., Development of Protacs to target cancer-promoting proteins for
ubiquitination and degradation, Mol Cell Proteomics 2003 Dec.;
2(12):1350-8; and
[0309] 4. Ovalicin, fumagillin (derivatized), and derivatives and
analogs thereof, having the structure(s) and binding to a linker
group L or a -(L-CLM) group as is generally described in Sakamoto,
et al., Protacs: chimeric molecules that target proteins to the
Skp1-Cullin-F box complex for ubiquitination and degradation Proc
Natl Acad Sci USA. 2001 Jul. 17; 98(15):8554-9 and U.S. Pat. No.
7,208,157.
[0310] VIII. Immunosuppressive Compounds:
[0311] Immunosuppressive compounds include, but are not limited
to:
[0312] 1. AP21998 (derivatized), having the structure(s) and
binding to a linker group L or a -(L-CLM) group as is generally
described in Schneekloth, et al., Chemical Genetic Control of
Protein Levels: Selective in Vivo Targeted Degradation, J. AM.
CHEM. SOC. 2004, 126, 3748-3754;
[0313] 2. Glucocorticoids (e.g., hydrocortisone, prednisone,
prednisolone, and methylprednisolone) (Derivatized where a linker
group L or a -(L-CLM) group is to bound, e.g. to any of the
hydroxyls) and beclometasone dipropionate (Derivatized where a
linker group or a -(L-CLM) is bound, e.g. to a proprionate);
[0314] 3. Methotrexate (Derivatized where a linker group or a
-(L-CLM) group can be bound, e.g. to either of the terminal
hydroxyls);
[0315] 4. Ciclosporin (Derivatized where a linker group or a
-(L-CLM) group can be bound, e.g. at any of the butyl groups);
[0316] 5. Tacrolimus (FK-506) and rapamycin (Derivatized where a
linker group L or a -(L-CLM) group can be bound, e.g. at one of the
methoxy groups); and
[0317] 6. Actinomycins (Derivatized where a linker group L or a
-(L-CLM) group can be bound, e.g. at one of the isopropyl
groups).
[0318] IX. Compounds Targeting the Aryl Hydrocarbon Receptor
(AHR):
[0319] Compounds targeting the aryl hydrocarbon receptor (AHR)
include, but are not limited to:
[0320] 1. Apigenin (Derivatized in a way which binds to a linker
group L or a -(L-CLM) group as is generally illustrated in Lee, et
al., Targeted Degradation of the Aryl Hydrocarbon Receptor by the
PROTAC Approach: A Useful Chemical Genetic Tool, ChemBioChem Volume
8, Issue 17, pages 2058-2062, Nov. 23, 2007); and
[0321] 2. SR1 and LGC006 (derivatized such that a linker group L or
a -(L-CLM) is bound), as described in Boitano, et al., Aryl
Hydrocarbon Receptor Antagonists Promote the Expansion of Human
Hematopoietic Stem Cells, Science 10 Sep. 2010: Vol. 329 no. 5997
pp. 1345-1348.
[0322] X. Compounds Targeting RAF Receptor (Kinase):
##STR00198##
[0323] PLX4032
[0324] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment, for example).
[0325] XI. Compounds Targeting FKBP:
##STR00199##
[0326] (Derivatized where "R" designates a site for a linker group
L or a -(L-CLM) group attachment, for example).
[0327] XII. Compounds Targeting Androgen Receptor (AR)
[0328] 1. RU59063 Ligand (derivatized) of Androgen Receptor
##STR00200##
[0329] (Derivatized where "R" designates a site for a linker group
L or a -(L-CLM) group attachment, for example).
[0330] 2. SARM Ligand (derivatized) of Androgen Receptor
##STR00201##
[0331] (Derivatized where "R" designates a site for a linker group
L or a -(L-CLM) group attachment, for example).
[0332] 3. Androgen Receptor Ligand DHT (derivatized)
##STR00202##
[0333] (Derivatized where "R" designates a site for a linker group
L or -(L-CLM) group attachment, for example).
[0334] 4. MDV3100 Ligand (derivatized)
##STR00203##
[0335] 5. ARN-509 Ligand (derivatized)
##STR00204##
[0336] 6. Hexahydrobenzisoxazoles
##STR00205##
[0337] 7. Tetramethylcyclobutanes
##STR00206##
[0338] 8. In any aspect or embodiment described herein, the PTM is
a chemical moiety that binds to the androgen receptor (AR) (ABM).
Various androgen receptor binding compounds have been described in
literature, including various androgen derivatives such as
testosterone, dihydrotestosterone, and metribolone (also known as
methyltrienolone or R1881), and non-steroidal compounds such as
bicalutamide, enzalutamide, some of which are described above.
Those of ordinary skill in the art would appreciate that these
androgen receptor binding compounds could be potentially used as an
ABM moiety in a PROTAC compound. Such literature includes, but not
limited to, G. F. Allan et. al, Nuclear Receptor Signaling, 2003,
1, e009; R. H. Bradbury et. al, Bioorganic & Medicinal
Chemistry Letters, 2011 5442-5445; C. Guo et. al, Bioorganic &
Medicinal Chemistry Letters, 2012 2572-2578; P. K. Poutiainen et.
al, J. Med. Chem. 2012, 55, 6316-6327 A. Pepe et. al, J. Med. Chem.
2013, 56, 8280-8297; M. E. Jung et al, J. Med. Chem. 2010, 53,
2779-2796, which are incorporated by reference herein
[0339] In any aspect or embodiment described herein, the ABM
comprises a structure selected from, but not limited to the
structures shown below, wherein a dashed line indicates the
attachment point of a linker moiety or a ULM, such as a CLM:
##STR00207##
wherein: [0340] W.sup.1 is aryl, heteroaryl, bicyclic, or
biheterocyclic, each independently substituted by 1 or more H,
halo, hydroxyl, nitro, CN, C.ident.CH, C.sub.1-6 alkyl (linear,
branched, optionally substituted; for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), C.sub.1-6
alkoxyl (linear, branched, optionally substituted; for example,
optionally substituted by 1 or more halo), C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or CF.sub.3; [0341] Y.sup.1, Y.sup.2 are each
independently NR.sup.Y1, 0, S; [0342] Y.sup.3, Y.sup.4, Y.sup.5 are
each independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2,
C.dbd.O, C.dbd.S, SO, SO.sub.2, heteroaryl, or aryl; [0343] Q is a
3-6 membered ring with 0-4 heteroatoms, optionally substituted with
0-6 R.sup.Q, each R.sup.Q, is independently H, C.sub.1-6 alkyl
(linear, branched, optionally substituted; for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, or 2 R.sup.Q groups taken together with the atom
they are attached to, form a 3-8 membered ring system containing
0-2 heteroatoms); [0344] R.sup.1, R.sup.2, R.sup.a, R.sup.b,
R.sup.Y1, R.sup.Y2 are each independently H, C.sub.1-6 alkyl
(linear, branched, optionally substituted; for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, cyclic, heterocyclic, or R.sup.1, R.sup.2
together with the atom they are attached to, form a 3-8 membered
ring system containing 0-2 heteroatoms); [0345] W.sup.2 is a bond,
C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, O, aryl, heteroaryl,
alicyclic, heterocyclic, biheterocyclic, biaryl, or biheteroaryl,
each optionally substituted by 1-10 R.sup.W2; [0346] each R.sup.W2
is independently H, halo, C.sub.1-6 alkyl (linear, branched,
optionally substituted; for example, optionally substituted by 1 or
more F), --OR.sup.W2A, C.sub.3-6 cycloalkyl, C.sub.4-6
cycloheteroalkyl, C.sub.1-6 alicyclic (optionally substituted),
heterocyclic (optionally substituted), aryl (optionally
substituted), or heteroaryl (optionally substituted), bicyclic
heteroaryl or aryl, OC.sub.1-3alkyl (optionally substituted), OH,
NH.sub.2, NR.sup.Y1R.sup.Y2, CN; and [0347] R.sup.W2A is H,
C.sub.1-6 alkyl (linear, branched), or C.sub.1-6 heteroalkyl
(linear, branched), each optionally substituted by a cycloalkyl,
cycloheteroalkyl, aryl, heterocyclic, heteroaryl, halo, or
OC.sub.1-3alkyl.
[0348] In any aspect or embodiment described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0349] In any aspect or embodiment described herein, W.sup.1 is
##STR00208##
wherein each R.sub.22 is independently halo, H, optionally
substituted alkyl, haloalkyl, cyano, or nitro; and each R.sub.23 is
independently H, halo, CF.sub.3, optionally substituted alkyl,
alkoxy, haloalkyl, cyano, or nitro.
[0350] In any aspect or embodiment described herein, W.sup.1 is
selected from the group consisting of:
##STR00209##
[0351] In any aspect or embodiment described herein, the ABM
comprises a structure selected from the following structures shown
below, where a indicates the attachment point of a linker or a
ULM:
##STR00210##
wherein: [0352] R.sup.Q2 is a H, halogen, CH.sub.3 or CF.sub.3;
[0353] R.sup.Q3 is H, halo, hydroxyl, nitro, CN, C.ident.CH,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), C.sub.1-6 alkoxyl (linear, branched,
optionally substituted by 1 or more halo), C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or CF.sub.3; [0354] Y.sup.3, Y.sup.4, Y.sup.5
are each independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2,
C.dbd.O, heteroaryl, or aryl, [0355] R.sup.Y1, R.sup.Y2 are each
independently H, or C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl, cyclic, or
heterocyclic); and [0356] R.sup.Q each independently is H,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted by
1 or more halo, or C.sub.1-6 alkoxyl), or two R.sup.Q together with
the atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms.
[0357] In any aspect or embodiment described herein, each R.sup.Q
is independently H or CH.sub.3. In another embodiment R.sup.Q3 is
CN.
[0358] In any aspect or embodiment described herein, the ABM
comprises a structure selected from the following structures shown
below, where a indicates the attachment point of a linker or a
ULM:
##STR00211##
wherein: [0359] R.sup.Q2 is a H, halogen, CN, CH.sub.3 or CF.sub.3;
and [0360] R.sup.Q3 is H, halo, hydroxyl, nitro, CN, C.ident.CH,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), C.sub.1-6 alkoxyl (linear, branched,
optionally substituted by 1 or more halo), C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or CF.sub.3; [0361] Y.sup.3, Y.sup.4, Y.sup.5
are each independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2,
C.dbd.O, heteroaryl, or aryl; and [0362] R.sup.Y1, R.sup.Y2 are
each independently H or C.sub.1-6 alkyl (linear, branched,
optionally substituted by 1 or more halo, C.sub.1-6 alkoxyl,
cyclic, or heterocyclic); and [0363] X is N or C.
[0364] In any aspect or embodiment described herein, R.sup.Q3 is a
CN.
[0365] In any aspect or embodiment described herein, the ABM
comprises a structure shown below, where a dashed line indicates
the attachment point of a linker moiety or a ULM or a CLM:
##STR00212##
wherein: [0366] W.sup.1 is
[0366] ##STR00213## [0367] each R.sub.22 is independently H or
--CN; [0368] each R.sub.23 is independently H, halo,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted),
C.sub.1-C.sub.6 alkoxy, or --CF.sub.3; [0369] Y.sup.3 is a bond or
O; [0370] Y.sup.4 is a bond or NH; [0371] Y.sup.5 is a bond,
C.dbd.O, C.sub.1-C.sub.6 heteroaryl, or C.sub.1-C.sub.6 aryl;
[0372] R.sup.1, R.sup.2, are each independently H, or
C.sub.1-C.sub.6 alkyl (linear or branched, optionally substituted;
for example, optionally substituted by 1 or more halo, or C.sub.1-6
alkoxyl); [0373] W.sup.2 is a bond, C.sub.1-6 aryl, C.sub.1-6
heteroaryl, C.sub.1-6 alicyclic, or C.sub.1-6 heterocyclic,
biheterocyclic, biaryl, or biheteroaryl, each optionally
substituted by 1-10 R.sup.W2; and [0374] each R.sup.W2 is
independently H, or halo; and represents a bond that may be
stereospecific ((R) or (S)) or non-stereospecific.
[0375] In any aspect or embodiment described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0376] In any aspect or embodiment described herein, W.sup.1 is
selected from the group consisting of:
##STR00214##
[0377] In any aspect or embodiment described herein, W.sup.2 is
selected from the group consisting of:
##STR00215##
[0378] In any aspect or embodiment described herein, the ABM
comprises a structure selected from, but not limited to the
structures shown below, where a dashed line indicates the
attachment point of a linker moiety or a ULM:
##STR00216##
wherein: [0379] W.sup.1 is
[0379] ##STR00217## [0380] each R.sub.22 is independently H or
--CN; [0381] each R.sub.23 is independently H, halo, or --CF.sub.3;
[0382] Y.sup.1, Y.sup.2 are each independently O or S; [0383]
R.sup.1, R.sup.2, are each independently H or a methyl group;
[0384] W.sup.2 is a bond, C.sub.1-6 aryl, or heteroaryl, each
optionally substituted by 1, 2 or 3 R.sup.W2; and [0385] each
R.sup.W2 is independently H, halo, C.sub.1-6 alkyl (optionally
substituted by 1 or more F), OC.sub.1-3alkyl (optionally
substituted by 1 or more --F).
[0386] In any of the embodiments described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0387] In certain additional embodiments, W.sup.1 is selected from
the group consisting of:
##STR00218##
[0388] In any aspect or embodiment described herein, W2 is selected
from the group consisting of:
##STR00219##
[0389] In any aspect or embodiment described herein, ABM is
selected from the group consisting of:
##STR00220## ##STR00221## ##STR00222## ##STR00223## ##STR00224##
##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229##
##STR00230## ##STR00231## ##STR00232## ##STR00233##
[0390] In any aspect or embodiment described herein, the ABM
comprises the structure:
##STR00234##
wherein: [0391] W.sup.1 is aryl, or heteroaryl, each independently
substituted by 1 or more H, halo, hydroxyl, nitro, CN, C.ident.CH,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), C.sub.1-6 alkoxyl (linear, branched,
optionally substituted by 1 or more halo), C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, or CF.sub.3; [0392] Y.sup.3, Y.sup.4, Y.sup.5
are each independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2,
C.dbd.O, C.dbd.S, SO, SO.sub.2, heteroaryl, or aryl; [0393] Q is a
4 membered alicyclic ring with 0-2 heteroatoms, optionally
substituted with 0-6 R.sup.Q, each R.sup.Q is independently H,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more halo, C.sub.1-6 alkoxyl), or 2 R.sup.Q groups taken together
with the atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); [0394] R.sup.Y1, R.sup.Y2 are each
independently H, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl); [0395] W.sup.2
is a bond, C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, O, C.sub.1-6
alicyclic, heterocyclic, aryl, biheterocyclic, biaryl, or
biheteroaryl, or heteroaryl, each optionally substituted by 1, 2 or
3 R.sup.W2; and [0396] each R.sup.W2 is independently H, halo,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more F), C.sub.1-6 heteroalkyl (linear, branched, optionally
substituted), --OR.sup.W2A OC.sub.1-3alkyl (optionally substituted
by 1 or more --F), C.sub.3-6 cycloalkyl, C.sub.4-6 cycloheteroalkyl
(optionally substituted), C.sub.1-6 alkyl (optionally substituted),
C.sub.1-6 alicyclic (optionally substituted), heterocyclic
(optionally substituted), aryl (optionally substituted), heteroaryl
(optionally substituted), bicyclic heretoaryl (optionally
substituted), bicyclic aryl, OH, NH.sub.2, NR.sup.Y1R.sup.Y2, or
CN; and [0397] R.sup.W2A is H, C.sub.1-6 alkyl (linear, branched),
or C.sub.1-6 heteroalkyl (linear, branched), each optionally
substituted by a cycloalkyl, cycloheteroalkyl, aryl, heterocyclic,
heteroaryl, halo, or OC.sub.1-3alkyl.
[0398] In any aspect or embodiment described herein, the
description provides an androgen receptor binding compound
comprising a structure of:
##STR00235##
wherein: [0399] W.sup.1 is aryl, heteroaryl, bicyclic, or
biheterocyclic, each independently substituted by 1 or more H,
halo, hydroxyl, nitro, CN, C.ident.CH, C.sub.1-6 alkyl (linear,
branched, optionally substituted by 1 or more halo, C.sub.1-6
alkoxyl), C.sub.1-6 alkoxyl (linear, branched, optionally
substituted by 1 or more halo), C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, or CF.sub.3; [0400] Y.sup.1, Y.sup.2 are each
independently NR.sup.Y1, O, or S; [0401] Y.sup.3, Y.sup.4, Y.sup.5
are each independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2,
C.dbd.O, C.dbd.S, SO, SO.sub.2, heteroaryl, or aryl; [0402] Q is a
3-6 membered alicyclic or aromatic ring with 0-4 heteroatoms,
optionally substituted with 0-6 R.sup.Q, each R.sup.Q, is
independently H, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), or 2 R.sup.Q
groups taken together with the atom they are attached to, form a
3-8 membered ring system containing 0-2 heteroatoms); [0403]
R.sup.1, R.sup.2, R.sup.a, R.sup.b, R.sup.Y1, R.sup.Y2 are each
independently H, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), or R.sup.1,
R.sup.2 together with the atom they are attached to, form a 3-8
membered ring system containing 0-2 heteroatoms); [0404] W.sup.2 is
a bond, C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, O, C.sub.1-6
alicyclic, heterocyclic, aryl, biheterocyclic, biaryl, or
biheteroaryl, or heteroaryl, each optionally substituted by 1, 2 or
3 R.sup.W2; [0405] each R.sup.W2 is independently H, halo,
C.sub.1-6 alkyl (linear, branched, optionally substituted by 1 or
more F), C.sub.1-6 heteroalkyl (linear, branched, optionally
substituted), --OR.sup.W2A, OC.sub.1-3alkyl (optionally substituted
by 1 or more --F), C.sub.3-6 cycloalkyl, C.sub.4-6
cycloheteroalkyl, C.sub.1-6 alkyl (optionally substituted),
C.sub.1-6 alicyclic (optionally substituted), heterocyclic
(optionally substituted), aryl (optionally substituted), or
heteroaryl (optionally substituted), bicyclic heteroaryl or aryl,
OH, NH.sub.2, NR.sup.Y1R.sup.Y2, CN; and [0406] R.sup.W2A is H,
C.sub.1-6 alkyl (linear, branched), or C.sub.1-6 heteroalkyl
(linear, branched), each optionally substituted by a cycloalkyl,
cycloheteroalkyl, aryl, heterocyclic, heteroaryl, halo, or
OC.sub.1-3alkyl.
[0407] In any aspect or embodiment described herein, an androgen
receptor binding moiety has a structure of:
##STR00236##
wherein: [0408] W.sup.1 is
[0408] ##STR00237## [0409] each R.sub.22 is independently H or
--CN; [0410] each R.sub.23 is independently H, halo, or --CF.sub.3;
[0411] Y.sup.3 is a bond or O; [0412] Q is a 4 member ring,
optionally substituted with 0-4 R.sup.Q, each R.sup.Q is
independently H or methyl; [0413] Y4 is a bond or NH; [0414] Y5 is
a bond, a C.dbd.O, or a C.dbd.S; and [0415] each W.sup.2 is
independently a bond, C1-6 aryl or heteroaryl, each optionally
substituted by 1, 2 or 3 R.sup.W2, each R.sup.W2 is independently
H, halo, a 6 member alicyclic ring with 1 or 2 heteroatoms or a 5
member aromatic ring with 1 or 2 or 3 heteroatoms.
[0416] In any aspect or embodiment described herein, W.sup.2 is
selected from the group consisting of:
##STR00238##
[0417] In any aspect or embodiment described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0418] In any aspect or embodiment described herein, W.sup.1 is
selected from the group consisting of:
##STR00239##
[0419] In any aspect or embodiment described herein, an androgen
binding moiety has a structure of:
##STR00240##
wherein: [0420] W.sup.1 is aryl, independently substituted by 1 or
more halo, CN; [0421] Y.sup.3 are each independently a bond,
NR.sup.Y2, CR.sup.Y1R.sup.Y2, C.dbd.O; [0422] Q is a 5 membered
aromatic ring with 1 or 2 heteroatoms; [0423] R.sup.Y1, R.sup.Y2
are each independently H, C.sub.1-6 alkyl (linear, branched);
[0424] W.sup.2 is a bond, aryl, or heteroaryl, each optionally
substituted by 1, 2 or 3 R.sup.W2; and [0425] each R.sup.W2 is
independently H, halo, C.sub.1-6 alkyl (optionally substituted by 1
or more F), OC.sub.1-3alkyl (optionally substituted by 1 or more
--F).
[0426] In any aspect or embodiment described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0427] In any aspect or embodiment described herein, W.sup.1 is
##STR00241##
wherein each R.sub.22 is independently halo or CN; and each
R.sub.23 is independently H or halo.
[0428] In any aspect or embodiment described herein, W.sup.1 is
selected from the group consisting of:
##STR00242##
[0429] In any aspect or embodiment described herein, Q is
##STR00243##
[0430] In any aspect or embodiment described herein, W.sup.2 is
##STR00244##
[0431] In any aspect or embodiment described herein,
(Y.sup.3).sub.0-5 is
##STR00245##
[0432] In any aspect or embodiment described herein, the ABM
comprises a structure selected from, but not limited to the
structures shown below, where a dashed line indicates the
attachment point of a linker moiety or a ULM, such as a CLM:
##STR00246##
wherein: [0433] W.sup.1 is
[0433] ##STR00247## [0434] each R.sub.22 is independently H or
--CN; [0435] each R.sub.23 is independently H, halo, or --CF.sub.3;
[0436] Y.sup.1, Y.sup.2 are each independently O or S; [0437]
Y.sup.3, Y.sup.4, Y.sup.5 are each independently a bond, O,
NR.sup.Y2, CR.sup.Y1R.sup.Y2, C.dbd.O, C.dbd.S, SO, or
SO.sub.2;
[0438] R.sup.1, R.sup.2, are each independently H or a methyl
group; [0439] W.sup.2 is a bond, C.sub.1-6 aryl, or heteroaryl,
each optionally substituted by 1, 2 or 3 R.sup.W2; and [0440] each
R.sup.W2 is independently H, halo, C.sub.1-6 alkyl (optionally
substituted by 1 or more F), C.sub.3-6 cycloalkyl, C.sub.4-6
cycloheteroalkyl, OC.sub.1-3alkyl (optionally substituted by 1 or
more --F).
[0441] In any aspect or embodiment described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0442] In any aspect or embodiment described herein, W.sup.1 is
selected from the group consisting of:
##STR00248##
[0443] In any aspect or embodiment described herein, W2 is selected
from the group consisting of:
##STR00249##
[0444] In any aspect or embodiment described herein, the ABM
comprises a structure shown below, where a dashed line indicates
the attachment point of a linker moiety or a ULM or a CLM:
##STR00250##
wherein: [0445] W.sup.1 is
[0445] ##STR00251## [0446] each R.sub.22 is independently H or
--CN; [0447] each R.sub.23 is independently H, halo, or --CF.sub.3;
[0448] Y.sup.3 is a bond or O; [0449] Y.sup.4 is a bond or NH;
[0450] Y.sup.5 is a bond, C.dbd.O, C.sub.1-C.sub.6 heteroaryl, or
C.sub.1-C.sub.6 aryl; [0451] R.sup.1, R.sup.2, are each
independently H, or C.sub.1-C.sub.6 alkyl (linear or branched,
optionally substituted by 1 or more halo, or C.sub.1-6 alkoxyl);
[0452] W.sup.2 is a bond, C.sub.1-6 aryl, C.sub.1-6 heteroaryl,
C.sub.1-6 alicyclic, or C.sub.1-6 heterocyclic, each optionally
substituted by 1-10 R.sup.W2; and [0453] each R.sup.W2 is
independently H, or halo; and
[0454] represents a bond that may be stereospecific ((R) or (S)) or
non-stereospecific.
[0455] In any of the embodiments described herein, the W.sup.2 is
covalently coupled to one or more ULM or CLM groups, or a linker to
which is attached one or more ULM or CLM groups as described
herein.
[0456] In certain additional embodiments, W.sup.1 is selected from
the group consisting of:
##STR00252##
[0457] In certain additional embodiments, W.sup.2 is selected from
the group consisting of:
##STR00253## ##STR00254##
[0458] In certain embodiments, the androgen receptor binding
compound of ABM is selected from the group consisting of: [0459]
trans-2-Chloro-4-[3-amino-2,2,4,4-tetramethylcyclobutoxy]benzonitrile;
[0460]
cis-2-Chloro-4-[3-amino-2,2,4,4-tetramethylcyclobutoxy]benzonitril-
e; [0461] trans
6-Amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyri-
dazine-3-carboxamide; [0462] trans tert-Butyl
N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]carbamate;
[0463] trans
4-Amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benz-
amide; [0464] trans
5-Amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyra-
zine-2-carboxamide; [0465] trans
2-Amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyri-
midine-5-carboxamide; [0466]
4-Methoxy-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclo-
butyl]benzamide; [0467] trans
1-(2-Hydroxyethyl)-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcycl-
obutyl]-1H-pyrazole-4-carboxamide; [0468] trans
6-Amino-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyri-
dine-3-carboxamide; [0469] trans
4-[(5-Hydroxypentyl)amino]-N-[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetrame-
thylcyclobutyl]benzamide; and [0470] trans tert-Butyl
2-({5-[(4-{[3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]car-
bamoyl}phenyl)aminopentyl}oxy)acetate; and [0471]
N-((1r,3r)-3-(4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-methylbenz-
amide.
[0472] In certain embodiments, the description provides, but is not
limited to, the following exemplary androgen receptor PROTAC
molecules (PROTAC 3 through PROTAC-30), including salts, prodrugs,
polymorphs, analogs, derivatives, and deuterated forms thereof:
##STR00255## ##STR00256## ##STR00257## ##STR00258## ##STR00259##
##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264##
##STR00265##
[0473] XIII. Compounds Targeting Estrogen Receptor (ER)
ICI-182780
[0474] 1. Estrogen Receptor Ligand
##STR00266##
[0475] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment).
[0476] In any embodiment or aspect described herein, the PTM may be
represented by the Formula PTM-I:
##STR00267##
wherein: [0477] X.sub.PTM is O or C.dbd.O; [0478] each of
X.sub.PTM1 and X.sub.PTM2 is independently selected from N or CH;
[0479] R.sub.PTM1 is independently selected from OH,
O(CO)R.sub.PTM, O-lower alkyl, wherein R.sub.PTM is an alkyl or
aryl group in the ester; [0480] at least one R.sub.PTM2, each
independently selected from H, OH, halogen, CN, CF.sub.3,
SO.sub.2-alkyl, O-lower alkyl; [0481] at least one R.sub.PTM3, each
independently selected from H, halogen; and [0482] the dashed line
indicates the site of attachment of at least one linker, CLM, CLM',
PTM, PTM', or a combination thereof.
[0483] In any embodiment or aspect described herein, the PTM may be
represented by the Formula PTM-I:
##STR00268##
wherein: [0484] X.sub.PTM is O or C.dbd.O; [0485] each of
X.sub.PTM1 and X.sub.PTM2 is independently selected from N or CH;
[0486] R.sub.PTM1 is independently selected from OH,
O(CO)R.sub.PTM, O-lower alkyl, wherein R.sub.PTM is an alkyl or
aryl group in the ester; [0487] each R.sub.PTM2 is independently
selected from H, OH, halogen, CN, CF.sub.3, SO.sub.2-alkyl, O-lower
alkyl; [0488] each R.sub.PTM3 is independently selected from H,
halogen; [0489] the PTM-I comprises as least one R.sub.PTM2, at
least one R.sub.PTM3, or a combination thereof on the respective
rings; and [0490] the dashed line indicates the site of attachment
of at least one linker, CLM, CLM', PTM, PTM', or a combination
thereof.
[0491] In any embodiment or aspect described herein, PTM-I has at
least one of: two R.sub.PTM2, two R.sub.PTM3, or a combination
thereof.
[0492] In any embodiment or aspect described herein, the PTM may be
represented by the Formula PTM-II:
##STR00269##
wherein: [0493] X.sub.PTM is O or C.dbd.O; [0494] each of
X.sub.PTM1 and X.sub.PTM2 is independently selected from N or CH;
[0495] R.sub.PTM1 is independently selected from OH,
O(CO)R.sub.PTM, O-lower alkyl, wherein R.sub.PTM is an alkyl or
aryl group in the ester; [0496] R.sub.PTM2 and R.sub.PTM4 are
independently selected from H, OH, halogen, CN, CF.sub.3,
SO.sub.2-alkyl, O-lower alkyl; [0497] R.sub.PTM3 and R.sub.PTM5 are
independently selected from H, halogen; and [0498] the dashed line
indicates the site of attachment of at least one linker, CLM, CLM',
PTM, PTM', or a combination thereof.
[0499] In aspect or embodiment described herein, O(CO)R.sub.PTM
functions as a prodrug of the corresponding phenol in Formula PTM-I
or PTM-II.
[0500] In any embodiment or aspect described herein, the O-lower
alkyl of PTM-I or PTM-II an alkyl chain with carbon number 1 to
3.
[0501] In aspect or embodiment described herein, the present
disclosure provides a compound or PTM of Formula (I.sub.PTM):
##STR00270##
wherein: [0502] each X.sub.PTM is independently CH, N; [0503]
indicates the site of attachment of at least one linker, CLM, CLM',
PTM, PTM', or a combination thereof; [0504] each R.sub.PTM1 is
independently OH, halogen, O(CO)R.sub.PTM, where R.sub.PTM is alkyl
or cycloalkyl group with 1 to 6 carbons or aryl groups,
substitution can be mono-, di- or tri-substituted; [0505] each
R.sub.PTM2 is independently H, halogen, CN, CF.sub.3, alkoxy,
substitution can be mono- or di-substitution; and [0506] each
R.sub.PTM3 is independently H, halogen, substitution can be mono-
or di-substitution.
[0507] In any aspect or embodiment described herein, the PTM is
represented by the Formula (II.sub.PTM):
##STR00271##
wherein: [0508] X.sub.PTM is CH, N; [0509] indicates the site of
attachment of at least one linker, CLM, CLM', PTM, PTM', or a
combination thereof; [0510] each R.sub.PTM1 is independently OH,
halogen (e.g., F); [0511] each R.sub.PTM2 is independently H,
halogen (e.g., F), CF.sub.3, substitution can be mono- or
di-substitution; and [0512] each R.sub.PTM3 is independently
halogen (e.g. F), substitution can be mono- or di-substitution.
[0513] In certain embodiments, at least one of: [0514] X.sub.PTM of
Formula (II.sub.PTM) is CH; [0515] R.sub.PTM1 of Formula
(II.sub.PTM) is OH; [0516] R.sub.PTM2 of Formula (II.sub.PTM) is H;
[0517] each R.sub.PTM3 of Formula (II.sub.PTM) is independently H
or F; or [0518] a combination thereof.
[0519] XIV. Compounds Targeting Thyroid Hormone Receptor (TR)
[0520] 1. Thyroid Hormone Receptor Ligand (derivatized)
##STR00272##
[0521] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment and MOMO indicates a methoxymethoxy
group).
[0522] XV. Compounds Targeting HIV Protease
[0523] 1. Inhibitor of HIV Protease (derivatized)
##STR00273##
[0524] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment). See, J. Med. Chem. 2010, 53,
521-538.
[0525] 2. Inhibitor of HIV Protease
##STR00274##
[0526] (Derivatized where "R" designates a potential site for
linker group L or -(L-CLM) group attachment). See, J. Med. Chem.
2010, 53, 521-538.
[0527] XVI. Compounds Targeting HIV Integrase
[0528] 1. Inhibitor of HIV Integrase (derivatized)
##STR00275##
[0529] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment). See, J. Med. Chem. 2010, 53,
6466.
[0530] 2. Inhibitor of HIV Integrase (derivatized)
##STR00276##
[0531] 3. Inhibitor of HIV integrase Isetntress (derivatized)
##STR00277##
[0532] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment). See, J. Med. Chem. 2010, 53,
6466.
[0533] XVII. Compounds Targeting HCV Protease
[0534] 1. Inhibitors of HCV Protease (derivatized)
##STR00278##
[0535] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment).
[0536] XVIII. Compounds Targeting Acyl-Protein Thioesterase-1 and
-2 (APT1 and APT2)
[0537] 1. Inhibitor of APT1 and APT2 (derivatized)
##STR00279##
[0538] (Derivatized where "R" designates a site for linker group L
or -(L-CLM) group attachment). See, Angew. Chem. Int. Ed. 2011, 50,
9838-9842, where L is a linker group as otherwise described herein
and said CLM group is as otherwise described herein such that
-(L-CLM) binds the CLM group to a PTMgroup as otherwise described
herein.
[0539] VIV. Compound Targeting Tau Protein
[0540] In any aspect or embodiment described herein, the PTM may
include a Tau protein binding moieties. For example, the PTM may be
represented by Formula I, Formula II, Formula III, Formula IV,
Formula V, Formula VI, Formula, VII, Formula, VIII, Formula IX,
Formula X, or Formula XI:
##STR00280##
wherein: [0541] A, B, C, D, E, and F are independently selected
from an optionally substituted 5- or 6-membered aryl or heteroaryl
ring, an optionally substituted 4- to 7-membered cycloalkyl or a
heterocycloalkyl, where contact between circles indicates ring
fusion; and [0542] L.sub.PTM is selected from a bond, an alkyl, an
alkenyl or an alkynyl, optionally interrupted by one or more rings
(i.e., cycloalkyl, heterocycloalkyl, aryl or heteroaryl), or one or
more functional groups selected from the groups --O--, --S--,
--NR.sup.1.sub.PTM-- (where R.sup.1.sub.PTM is selected from H or
alkyl), --N.dbd.N--, --S(O)--, --SO.sub.2--, --C(O)--, --NHC(O)--,
--C(O)NH--, --NHSO.sub.2--, --NHC(O)NH--, --NHC(O)O--, or
--OC(O)NH--, wherein the said functional group are optionally
located at either end of the linker.
[0543] In any aspect or embodiment described herein, aryl and
heteroaryl rings of A, B, C, D, E, and F of PTM are optionally
substituted with 1-3 substituents each independently selected from
alkyl, alkenyl, haloalkyl, halogen, hydroxyl, alkoxy, fluoroalkoxy,
amino, alkylamino, dialkylamino, acylamino, trifluoromethyl, and
cyano, wherein the said alkyl and alkenyl groups are further
optionally substituted.
[0544] In any aspect or embodiment described herein, the rings of
at least one of A, B, C, F, or a combination thereof is selected
from optionally substituted 5- or 6-membered aryl or heteroaryl
rings;
[0545] In any aspect or embodiment described herein, the PTM has
the chemical structure of Formula I, wherein: [0546] A, B and C
rings are independently 5- or 6-membered fused aryl or heteroaryl
rings; [0547] L.sub.PTM is selected from a bond or an alkyl, and
[0548] D is selected from a 6-membered aryl, heteroaryl or
heterocycloalkyl, [0549] wherein A, B, C and D are optionally
substituted with alkyl, haloalkyl, halogen, hydroxyl, alkoxy,
amino, alkylamino, dialkylamino or cyano.
[0550] In any aspect or embodiment described herein, The PTM has
the chemical structure of Formula I, wherein: [0551] A and C are a
phenyl or a 6-membered heteroaryl ring; [0552] B is a 5-membered
heteroaryl ring; [0553] L.sub.PTM is a bond; and [0554] D is a
6-membered heteroaryl or a 6-membered heterocycloalkyl ring; [0555]
wherein each A, B, C and D is optionally independently substituted
with alkyl, haloalkyl, halogen, hydroxyl, alkoxy, amino,
dialkylamino or cyano, and wherein a nitrogen atom of any of the A,
B, C and D rings is not directly connected to a heteroatom or to a
carbon atom, to which another heteroatom is directly attached.
[0556] In any aspect or embodiment described herein, the PTM has
the chemical structure of Formula III or IV, wherein A, B and C are
5- or 6-membered fused aryl or heteroaryl rings, L.sub.PTM is
selected from a bond or an alkyl, and D and E are 5- or 6-membered
fused aryl or heteroaryl rings, wherein A, B, C, D and E are
optionally substituted with alkyl, haloalkyl, halogen, hydroxyl,
alkoxy, amino, alkylamino, dialkylamino or cyano.
[0557] In any aspect or embodiment described herein, the PTM is
represented by following chemical structure:
##STR00281## ##STR00282## ##STR00283##
wherein: [0558] R.sup.1, R.sup.2 and R.sup.3 are independently
selected from H, methyl, ethyl, 2-fluoroethyl and
2,2,2-trifluoroethyl; [0559] R.sup.4 and R.sup.5 are independently
selected from H, methyl, ethyl and halogen; and [0560] R.sup.6 is 1
to 2 substituents independently selected from H, methyl, ethyl and
halogen, wherein the PTM is coupled to a ULM via L.
[0561] In any of the aspects or embodiments described herein, the
PTM is covalently coupled to one or more ULM (VLM or CLM) groups,
or a linker to which is attached one or more ULM (VLM or CLM)
groups as described herein.
[0562] In any aspect or embodiment described herein, PTM is
represented by chemical structure:
##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288##
##STR00289##
wherein: [0563] R.sup.1, R.sup.2 and R.sup.3 are independently
selected from H, optionally substituted alkyl, methyl, ethyl,
2-fluoroethyl and 2,2,2-trifluoroethyl; and [0564] R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are 1 to 8 substituents independently
selected from H, optionally substituted alkyl, haloalkyl, halogen,
hydroxyl, alkoxy, amino, dialkylamino, aceylamino, trifluoromethyl
or cyano, and wherein the PTM is coupled to a ULM (VLM or CLM) via
L.
[0565] In any aspect or embodiment described herein, PTM is
represented by chemical
##STR00290## ##STR00291## ##STR00292## ##STR00293## ##STR00294##
##STR00295## ##STR00296##
[0566] In any aspect or embodiment described herein, the linker
attachment point to PTM is as indicated by the dotted line:
##STR00297##
[0567] Therapeutic Compositions
[0568] Pharmaceutical compositions comprising combinations of an
effective amount of at least one bifunctional compound as described
herein, and one or more of the compounds otherwise described
herein, all in effective amounts, in combination with a
pharmaceutically effective amount of a carrier, additive or
excipient, represents a further aspect of the present
disclosure.
[0569] The present disclosure includes, where applicable, the
compositions comprising the pharmaceutically acceptable salts, in
particular, acid or base addition salts of compounds as described
herein. The acids which are used to prepare the pharmaceutically
acceptable acid addition salts of the aforementioned base compounds
useful according to this aspect are those which form non-toxic acid
addition salts, i.e., salts containing pharmacologically acceptable
anions, such as the hydrochloride, hydrobromide, hydroiodide,
nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,
lactate, citrate, acid citrate, tartrate, bitartrate, succinate,
maleate, fumarate, gluconate, saccharate, benzoate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate and pamoate [i.e.,
1,1'-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous
others.
[0570] Pharmaceutically acceptable base addition salts may also be
used to produce pharmaceutically acceptable salt forms of the
compounds or derivatives according to the present disclosure. The
chemical bases that may be used as reagents to prepare
pharmaceutically acceptable base salts of the present compounds
that are acidic in nature are those that form non-toxic base salts
with such compounds. Such non-toxic base salts include, but are not
limited to those derived from such pharmacologically acceptable
cations such as alkali metal cations (eg., potassium and sodium)
and alkaline earth metal cations (eg, calcium, zinc and magnesium),
ammonium or water-soluble amine addition salts such as
N-methylglucamine-(meglumine), and the lower alkanolammonium and
other base salts of pharmaceutically acceptable organic amines,
among others.
[0571] The compounds as described herein may, in accordance with
the disclosure, be administered in single or divided doses by the
oral, parenteral or topical routes. Administration of the active
compound may range from continuous (intravenous drip) to several
oral administrations per day (for example, Q.I.D.) and may include
oral, topical, parenteral, intramuscular, intravenous,
sub-cutaneous, transdermal (which may include a penetration
enhancement agent), buccal, sublingual and suppository
administration, among other routes of administration. Enteric
coated oral tablets may also be used to enhance bioavailability of
the compounds from an oral route of administration. The most
effective dosage form will depend upon the pharmacokinetics of the
particular agent chosen as well as the severity of disease in the
patient. Administration of compounds according to the present
disclosure as sprays, mists, or aerosols for intra-nasal,
intra-tracheal or pulmonary administration may also be used. The
present disclosure therefore also is directed to pharmaceutical
compositions comprising an effective amount of compound as
described herein, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient. Compounds according to
the present disclosure ion may be administered in immediate
release, intermediate release or sustained or controlled release
forms. Sustained or controlled release forms are preferably
administered orally, but also in suppository and transdermal or
other topical forms. Intramuscular injections in liposomal form may
also be used to control or sustain the release of compound at an
injection site.
[0572] The compositions as described herein may be formulated in a
conventional manner using one or more pharmaceutically acceptable
carriers and may also be administered in controlled-release
formulations. Pharmaceutically acceptable carriers that may be used
in these pharmaceutical compositions include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as prolamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat.
[0573] The compositions as described herein may be administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intravenous, intramuscular, intra-articular, intra-synovial,
intrasternal, intrathecal, intrahepatic, intralesional and
intracranial injection or infusion techniques. Preferably, the
compositions are administered orally, intraperitoneally or
intravenously.
[0574] Sterile injectable forms of the compositions as described
herein may be aqueous or oleaginous suspension. These suspensions
may be formulated according to techniques known in the art using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile injectable
solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent, for example as a solution in 1, 3-butanediol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose, any bland fixed oil
may be employed including synthetic mono- or di-glycerides. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as Ph. Helv or similar alcohol.
[0575] The pharmaceutical compositions as described herein may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0576] Alternatively, the pharmaceutical compositions as described
herein may be administered in the form of suppositories for rectal
administration. These can be prepared by mixing the agent with a
suitable non-irritating excipient, which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0577] The pharmaceutical compositions as described herein may also
be administered topically. Suitable topical formulations are
readily prepared for each of these areas or organs. Topical
application for the lower intestinal tract can be effected in a
rectal suppository formulation (see above) or in a suitable enema
formulation. Topically-acceptable transdermal patches may also be
used.
[0578] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water. In certain
preferred aspects of the invention, the compounds may be coated
onto a stent which is to be surgically implanted into a patient in
order to inhibit or reduce the likelihood of occlusion occurring in
the stent in the patient.
[0579] Alternatively, the pharmaceutical compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0580] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0581] The pharmaceutical compositions as described herein may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0582] The amount of compound in a pharmaceutical composition as
described herein that may be combined with the carrier materials to
produce a single dosage form will vary depending upon the host and
disease treated, the particular mode of administration. Preferably,
the compositions should be formulated to contain between about 0.05
milligram to about 750 milligrams or more, more preferably about 1
milligram to about 600 milligrams, and even more preferably about
10 milligrams to about 500 milligrams of active ingredient, alone
or in combination with at least one other compound according to the
present disclosure.
[0583] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease or condition being treated.
[0584] A patient or subject in need of therapy using compounds
according to the methods described herein can be treated by
administering to the patient (subject) an effective amount of the
compound according to the present disclosure including
pharmaceutically acceptable salts, solvates or polymorphs, thereof
optionally in a pharmaceutically acceptable carrier or diluent,
either alone, or in combination with other known erythopoiesis
stimulating agents as otherwise identified herein.
[0585] These compounds can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, or topically, including
transdermally, in liquid, cream, gel, or solid form, or by aerosol
form.
[0586] The active compound is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount for the desired
indication, without causing serious toxic effects in the patient
treated. A preferred dose of the active compound for all of the
herein-mentioned conditions is in the range from about 10 ng/kg to
300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5
to about 25 mg per kilogram body weight of the recipient/patient
per day. A typical topical dosage will range from 0.01-5% wt/wt in
a suitable carrier.
[0587] The compound is conveniently administered in any suitable
unit dosage form, including but not limited to one containing less
than 1 mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active
ingredient per unit dosage form. An oral dosage of about 25-250 mg
is often convenient.
[0588] The active ingredient is preferably administered to achieve
peak plasma concentrations of the active compound of about
0.00001-30 mM, preferably about 0.1-30 .mu.M. This may be achieved,
for example, by the intravenous injection of a solution or
formulation of the active ingredient, optionally in saline, or an
aqueous medium or administered as a bolus of the active ingredient.
Oral administration is also appropriate to generate effective
plasma concentrations of active agent.
[0589] The concentration of active compound in the drug composition
will depend on absorption, distribution, inactivation, and
excretion rates of the drug as well as other factors known to those
of skill in the art. It is to be noted that dosage values will also
vary with the severity of the condition to be alleviated. It is to
be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at varying intervals of time.
[0590] Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound or its prodrug derivative can
be incorporated with excipients and used in the form of tablets,
troches, or capsules. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition.
[0591] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a dispersing
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring. When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a liquid
carrier such as a fatty oil. In addition, dosage unit forms can
contain various other materials which modify the physical form of
the dosage unit, for example, coatings of sugar, shellac, or
enteric agents.
[0592] The active compound or pharmaceutically acceptable salt
thereof can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0593] The active compound or pharmaceutically acceptable salts
thereof can also be mixed with other active materials that do not
impair the desired action, or with materials that supplement the
desired action, such as erythropoietin stimulating agents,
including EPO and darbapoietin alfa, among others. In certain
preferred aspects of the invention, one or more compounds according
to the present disclosure are coadministered with another bioactive
agent, such as an erythropoietin stimulating agent or a would
healing agent, including an antibiotic, as otherwise described
herein.
[0594] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0595] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0596] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0597] Liposomal suspensions may also be pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 (which is incorporated herein by reference
in its entirety). For example, liposome formulations may be
prepared by dissolving appropriate lipid(s) (such as stearoyl
phosphatidyl ethanolamine, stearoyl phosphatidyl choline,
arachadoyl phosphatidyl choline, and cholesterol) in an inorganic
solvent that is then evaporated, leaving behind a thin film of
dried lipid on the surface of the container. An aqueous solution of
the active compound are then introduced into the container. The
container is then swirled by hand to free lipid material from the
sides of the container and to disperse lipid aggregates, thereby
forming the liposomal suspension.
[0598] Therapeutic Methods
[0599] In an additional aspect, the description provides
therapeutic compositions comprising an effective amount of a
compound as described herein or salt form thereof, and a
pharmaceutically acceptable carrier. The therapeutic compositions
modulate protein degradation in a patient or subject, for example,
an animal such as a human, and can be used for treating or
ameliorating disease states or conditions which are modulated
through the degraded protein.
[0600] The terms "treat", "treating", and "treatment", etc., as
used herein, refer to any action providing a benefit to a patient
for which the present compounds may be administered, including the
treatment of any disease state or condition which is modulated
through the protein to which the present compounds bind. Disease
states or conditions, including cancer, which may be treated using
compounds according to the present disclosure are set forth
hereinabove.
[0601] The description provides therapeutic compositions as
described herein for effectuating the degradation of proteins of
interest for the treatment or amelioration of a disease, e.g.,
cancer. In certain additional embodiments, the disease is multiple
myeloma. As such, in another aspect, the description provides a
method of ubiquitinating/degrading a target protein in a cell. In
certain embodiments, the method comprises administering a
bifunctional compound as described herein comprising, e.g., a CLM
and a PTM, preferably linked through a linker moiety, as otherwise
described herein, wherein the CLM is coupled to the PTM and wherein
the CLM recognizes a ubiquitin pathway protein (e.g., an ubiquitin
ligase, preferably an E3 ubiquitin ligase such as, e.g., cereblon)
and the PTM recognizes the target protein such that degradation of
the target protein will occur when the target protein is placed in
proximity to the ubiquitin ligase, thus resulting in
degradation/inhibition of the effects of the target protein and the
control of protein levels. The control of protein levels afforded
by the present disclosure provides treatment of a disease state or
condition, which is modulated through the target protein by
lowering the level of that protein in the cell, e.g., cell of a
patient. In certain embodiments, the method comprises administering
an effective amount of a compound as described herein, optionally
including a pharamaceutically acceptable excipient, carrier,
adjuvant, another bioactive agent or combination thereof.
[0602] In additional embodiments, the description provides methods
for treating or emeliorating a disease, disorder or symptom thereof
in a subject or a patient, e.g., an animal such as a human,
comprising administering to a subject in need thereof a composition
comprising an effective amount, e.g., a therapeutically effective
amount, of a compound as described herein or salt form thereof, and
a pharmaceutically acceptable excipient, carrier, adjuvant, another
bioactive agent or combination thereof, wherein the composition is
effective for treating or ameliorating the disease or disorder or
symptom thereof in the subject.
[0603] In another aspect, the description provides methods for
identifying the effects of the degradation of proteins of interest
in a biological system using compounds according to the present
disclosure.
[0604] In another embodiment, the present disclosure is directed to
a method of treating a human patient in need for a disease state or
condition modulated through a protein where the degradation of that
protein will produce a therapeutic effect in that patient, the
method comprising administering to a patient in need an effective
amount of a compound according to the present disclosure,
optionally in combination with another bioactive agent. The disease
state or condition may be a disease caused by a microbial agent or
other exogenous agent such as a virus, bacteria, fungus, protozoa
or other microbe or may be a disease state, which is caused by
overexpression of a protein, which leads to a disease state and/or
condition
[0605] The term "disease state or condition" is used to describe
any disease state or condition wherein protein dysregulation (i.e.,
the amount of protein expressed in a patient is elevated) occurs
and where degradation of one or more proteins in a patient may
provide beneficial therapy or relief of symptoms to a patient in
need thereof. In certain instances, the disease state or condition
may be cured.
[0606] Disease states of conditions which may be treated using
compounds according to the present disclosure include, for example,
asthma, autoimmune diseases such as multiple sclerosis, various
cancers, ciliopathies, cleft palate, diabetes, heart disease,
hypertension, inflammatory bowel disease, mental retardation, mood
disorder, obesity, refractive error, infertility, Angelman
syndrome, Canavan disease, Coeliac disease, Charcot-Marie-Tooth
disease, Cystic fibrosis, Duchenne muscular dystrophy,
Haemochromatosis, Haemophilia, Klinefelter's syndrome,
Neurofibromatosis, Phenylketonuria, Polycystic kidney disease,
(PKD1) or 4 (PKD2) Prader-Willi syndrome, Sickle-cell disease,
Tay-Sachs disease, Turner syndrome.
[0607] Further disease states or conditions which may be treated by
compounds according to the present disclosure include Alzheimer's
disease, Amyotrophic lateral sclerosis (Lou Gehrig's disease),
Anorexia nervosa, Anxiety disorder, Atherosclerosis, Attention
deficit hyperactivity disorder, Autism, Bipolar disorder, Chronic
fatigue syndrome, Chronic obstructive pulmonary disease, Crohn's
disease, Coronary heart disease, Dementia, Depression, Diabetes
mellitus type 1, Diabetes mellitus type 2, Epilepsy, Guillain-Barre
syndrome, Irritable bowel syndrome, Lupus, Metabolic syndrome,
Multiple sclerosis, Myocardial infarction, Obesity,
Obsessive-compulsive disorder, Panic disorder, Parkinson's disease,
Psoriasis, Rheumatoid arthritis, Sarcoidosis, Schizophrenia,
Stroke, Thromboangiitis obliterans, Tourette syndrome,
Vasculitis.
[0608] Still additional disease states or conditions which can be
treated by compounds according to the present disclosure include
aceruloplasminemia, Achondrogenesis type II, achondroplasia,
Acrocephaly, Gaucher disease type 2, acute intermittent porphyria,
Canavan disease, Adenomatous Polyposis Coli, ALA dehydratase
deficiency, adenylosuccinate lyase deficiency, Adrenogenital
syndrome, Adrenoleukodystrophy, ALA-D porphyria, ALA dehydratase
deficiency, Alkaptonuria, Alexander disease, Alkaptonuric
ochronosis, alpha 1-antitrypsin deficiency, alpha-1 proteinase
inhibitor, emphysema, amyotrophic lateral sclerosis Alstrom
syndrome, Alexander disease, Amelogenesis imperfecta, ALA
dehydratase deficiency, Anderson-Fabry disease, androgen
insensitivity syndrome, Anemia Angiokeratoma Corporis Diffusum,
Angiomatosis retinae (von Hippel-Lindau disease) Apert syndrome,
Arachnodactyly (Marfan syndrome), Stickler syndrome, Arthrochalasis
multiplex congenital (Ehlers-Danlos syndrome#arthrochalasia type)
ataxia telangiectasia, Rett syndrome, primary pulmonary
hypertension, Sandhoff disease, neurofibromatosis type II,
Beare-Stevenson cutis gyrata syndrome, Mediterranean fever,
familial, Benjamin syndrome, beta-thalassemia, Bilateral Acoustic
Neurofibromatosis (neurofibromatosis type II), factor V Leiden
thrombophilia, Bloch-Sulzberger syndrome (incontinentia pigmenti),
Bloom syndrome, X-linked sideroblastic anemia, Bonnevie-Ullrich
syndrome (Turner syndrome), Bourneville disease (tuberous
sclerosis), prion disease, Birt-Hogg-Dube syndrome, Brittle bone
disease (osteogenesis imperfecta), Broad Thumb-Hallux syndrome
(Rubinstein-Taybi syndrome), Bronze Diabetes/Bronzed Cirrhosis
(hemochromatosis), Bulbospinal muscular atrophy (Kennedy's
disease), Burger-Grutz syndrome (lipoprotein lipase deficiency),
CGD Chronic granulomatous disorder, Campomelic dysplasia,
biotinidase deficiency, Cardiomyopathy (Noonan syndrome), Cri du
chat, CAVD (congenital absence of the vas deferens), Caylor
cardiofacial syndrome (CBAVD), CEP (congenital erythropoietic
porphyria), cystic fibrosis, congenital hypothyroidism,
Chondrodystrophy syndrome (achondroplasia),
otospondylomegaepiphyseal dysplasia, Lesch-Nyhan syndrome,
galactosemia, Ehlers-Danlos syndrome, Thanatophoric dysplasia,
Coffin-Lowry syndrome, Cockayne syndrome, (familial adenomatous
polyposis), Congenital erythropoietic porphyria, Congenital heart
disease, Methemoglobinemia/Congenital methaemoglobinaemia,
achondroplasia, X-linked sideroblastic anemia, Connective tissue
disease, Conotruncal anomaly face syndrome, Cooley's Anemia
(beta-thalassemia), Copper storage disease (Wilson's disease),
Copper transport disease (Menkes disease), hereditary
coproporphyria, Cowden syndrome, Craniofacial dysarthrosis (Crouzon
syndrome), Creutzfeldt-Jakob disease (prion disease), Cockayne
syndrome, Cowden syndrome, Curschmann-Batten-Steinert syndrome
(myotonic dystrophy), Beare-Stevenson cutis gyrata syndrome,
primary hyperoxaluria, spondyloepimetaphyseal dysplasia (Strudwick
type), muscular dystrophy, Duchenne and Becker types (DBMD), Usher
syndrome, Degenerative nerve diseases including de Grouchy syndrome
and Dejerine-Sottas syndrome, developmental disabilities, distal
spinal muscular atrophy, type V, androgen insensitivity syndrome,
Diffuse Globoid Body Sclerosis (Krabbe disease), Di George's
syndrome, Dihydrotestosterone receptor deficiency, androgen
insensitivity syndrome, Down syndrome, Dwarfism, erythropoietic
protoporphyria Erythroid 5-aminolevulinate synthetase deficiency,
Erythropoietic porphyria, erythropoietic protoporphyria,
erythropoietic uroporphyria, Friedreich's ataxia, familial
paroxysmal polyserositis, porphyria cutanea tarda, familial
pressure sensitive neuropathy, primary pulmonary hypertension
(PPH), Fibrocystic disease of the pancreas, fragile X syndrome,
galactosemia, genetic brain disorders, Giant cell hepatitis
(Neonatal hemochromatosis), Gronblad-Strandberg syndrome
(pseudoxanthoma elasticum), Gunther disease (congenital
erythropoietic porphyria), haemochromatosis, Hallgren syndrome,
sickle cell anemia, hemophilia, hepatoerythropoietic porphyria
(HEP), Hippel-Lindau disease (von Hippel-Lindau disease),
Huntington's disease, Hutchinson-Gilford progeria syndrome
(progeria), Hyperandrogenism, Hypochondroplasia, Hypochromic
anemia, Immune system disorders, including X-linked severe combined
immunodeficiency, Insley-Astley syndrome, Kennedy's syndrome,
Jackson-Weiss syndrome, Joubert syndrome, Lesch-Nyhan syndrome,
Jackson-Weiss syndrome, Kidney diseases, including hyperoxaluria,
Klinefelter's syndrome, Kniest dysplasia, Lacunar dementia,
Langer-Saldino achondrogenesis, ataxia telangiectasia, Lynch
syndrome, Lysyl-hydroxylase deficiency, Machado-Joseph disease,
Metabolic disorders, including Kniest dysplasia, Marfan syndrome,
Movement disorders, Mowat-Wilson syndrome, cystic fibrosis, Muenke
syndrome, Multiple neurofibromatosis, Nance-Insley syndrome,
Nance-Sweeney chondrodysplasia, Niemann-Pick disease, Noack
syndrome (Pfeiffer syndrome), Osler-Weber-Rendu disease,
Peutz-Jeghers syndrome, Polycystic kidney disease, polyostotic
fibrous dysplasia (McCune-Albright syndrome), Peutz-Jeghers
syndrome, Prader-Labhart-Willi syndrome, hemochromatosis, primary
hyperuricemia syndrome (Lesch-Nyhan syndrome), primary pulmonary
hypertension, primary senile degenerative dementia, prion disease,
progeria (Hutchinson Gilford Progeria Syndrome), progressive
chorea, chronic hereditary (Huntington) (Huntington's disease),
progressive muscular atrophy, spinal muscular atrophy, propionic
acidemia, protoporphyria, proximal myotonic dystrophy, pulmonary
arterial hypertension, PXE (pseudoxanthoma elasticum), Rb
(retinoblastoma), Recklinghausen disease (neurofibromatosis type
I), Recurrent polyserositis, Retinal disorders, Retinoblastoma,
Rett syndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome,
Roussy-Levy syndrome, severe achondroplasia with developmental
delay and acanthosis nigricans (SADDAN), Li-Fraumeni syndrome,
sarcoma, breast, leukemia, and adrenal gland (SBLA) syndrome,
sclerosis tuberose (tuberous sclerosis), SDAT, SED congenital
(spondyloepiphyseal dysplasia congenita), SED Strudwick
(spondyloepimetaphyseal dysplasia, Strudwick type), SEDc
(spondyloepiphyseal dysplasia congenita) SEMD, Strudwick type
(spondyloepimetaphyseal dysplasia, Strudwick type), Shprintzen
syndrome, Skin pigmentation disorders, Smith-Lemli-Opitz syndrome,
South-African genetic porphyria (variegate porphyria),
infantile-onset ascending hereditary spastic paralysis, Speech and
communication disorders, sphingolipidosis, Tay-Sachs disease,
spinocerebellar ataxia, Stickler syndrome, stroke, androgen
insensitivity syndrome, tetrahydrobiopterin deficiency,
beta-thalassemia, Thyroid disease, Tomaculous neuropathy
(hereditary neuropathy with liability to pressure palsies),
Treacher Collins syndrome, Triplo X syndrome (triple X syndrome),
Trisomy 21 (Down syndrome), Trisomy X, VHL syndrome (von
Hippel-Lindau disease), Vision impairment and blindness (Alstrom
syndrome), Vrolik disease, Waardenburg syndrome, Warburg Sjo
Fledelius Syndrome, Weissenbacher-Zweymuller syndrome,
Wolf-Hirschhorn syndrome, Wolff Periodic disease,
Weissenbacher-Zweymuller syndrome and Xeroderma pigmentosum, among
others.
[0609] The term "neoplasia" or "cancer" is used throughout the
specification to refer to the pathological process that results in
the formation and growth of a cancerous or malignant neoplasm,
i.e., abnormal tissue that grows by cellular proliferation, often
more rapidly than normal and continues to grow after the stimuli
that initiated the new growth cease. Malignant neoplasms show
partial or complete lack of structural organization and functional
coordination with the normal tissue and most invade surrounding
tissues, metastasize to several sites, and are likely to recur
after attempted removal and to cause the death of the patient
unless adequately treated. As used herein, the term neoplasia is
used to describe all cancerous disease states and embraces or
encompasses the pathological process associated with malignant
hematogenous, ascitic and solid tumors. Exemplary cancers which may
be treated by the present compounds either alone or in combination
with at least one additional anti-cancer agent include
squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma,
hepatocellular carcinomas, and renal cell carcinomas, cancer of the
bladder, bowel, breast, cervix, colon, esophagus, head, kidney,
liver, lung, neck, ovary, pancreas, prostate, and stomach;
leukemias; benign and malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; benign and malignant
melanomas; myeloproliferative diseases; sarcomas, including Ewing's
sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,
myosarcomas, peripheral neuroepithelioma, synovial sarcoma,
gliomas, astrocytomas, oligodendrogliomas, ependymomas,
gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas,
medulloblastomas, pineal cell tumors, meningiomas, meningeal
sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast
cancer, prostate cancer, cervical cancer, uterine cancer, lung
cancer, ovarian cancer, testicular cancer, thyroid cancer,
astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer,
liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's
disease, Wilms' tumor and teratocarcinomas. Additional cancers
which may be treated using compounds according to the present
disclosure include, for example, T-lineage Acute lymphoblastic
Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL),
Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B
Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL,
Philadelphia chromosome positive ALL and Philadelphia chromosome
positive CML.
[0610] The term "bioactive agent" is used to describe an agent,
other than a compound according to the present disclosure, which is
used in combination with the present compounds as an agent with
biological activity to assist in effecting an intended therapy,
inhibition and/or prevention/prophylaxis for which the present
compounds are used. Preferred bioactive agents for use herein
include those agents which have pharmacological activity similar to
that for which the present compounds are used or administered and
include for example, anti-cancer agents, antiviral agents,
especially including anti-HIV agents and anti-HCV agents,
antimicrobial agents, antifungal agents, etc.
[0611] The term "additional anti-cancer agent" is used to describe
an anti-cancer agent, which may be combined with compounds
according to the present disclosure to treat cancer. These agents
include, for example, everolimus, trabectedin, abraxane, TLK 286,
AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244
(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,
vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263,
a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an
aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an
HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk
inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF
antibody, a PI3 kinase inhibitor, an AKT inhibitor, an mTORC1/2
inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a
focal adhesion kinase inhibitor, a Map kinase kinase (mek)
inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,
nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,
nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab,
edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen,
ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110,
BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001,
IPdR.sub.1 KRX-0402, lucanthone, LY317615, neuradiab, vitespan, Rta
744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin,
ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide,
gemcitabine, doxorubicin, liposomal doxorubicin,
5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,
seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d[pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258);
3-[5-(methylsulfonylpiperadinemethyl)-indolyl-quinolone, vatalanib,
AG-013736, AVE-0005, goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin,
bleomycin, buserelin, busulfan, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clodronate, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin,
diethylstilbestrol, epirubicin, fludarabine, fludrocortisone,
fluoxymesterone, flutamide, gleevec, gemcitabine, hydroxyurea,
idarubicin, ifosfamide, imatinib, leuprolide, levamisole,
lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesa,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin,
porfimer, procarbazine, raltitrexed, rituximab, streptozocin,
teniposide, testosterone, thalidomide, thioguanine, thiotepa,
tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard,
uracil mustard, estramustine, altretamine, floxuridine,
5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine,
deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine,
vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat,
BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974,
interleukin-12, IM862, angiostatin, vitaxin, droloxifene,
idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,
denileukin diftitox, gefitinib, bortezimib, paclitaxel,
cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550,
BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,
ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene,
idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonist,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa, darbepoetin alfa and mixtures
thereof.
[0612] The term "anti-HIV agent" or "additional anti-HIV agent"
includes, for example, nucleoside reverse transcriptase inhibitors
(NRTI), other non-nucleoside reverse transcriptase inhibitors
(i.e., those which are not representative of the present
disclosure), protease inhibitors, fusion inhibitors, among others,
exemplary compounds of which may include, for example, 3TC
(Lamivudine), AZT (Zidovudine), (-)-FTC, ddI (Didanosine), ddC
(zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset),
D4T (Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV
(Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV
(Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV (Nelfinavir),
APV (Amprenavir), LPV (Lopinavir), fusion inhibitors such as T20,
among others, fuseon and mixtures thereof, including anti-HIV
compounds presently in clinical trials or in development.
[0613] Other anti-HIV agents which may be used in coadministration
with compounds according to the present disclosure include, for
example, other NNRTI's (i.e., other than the NNRTI's according to
the present disclosure) may be selected from the group consisting
of nevirapine (BI-R6-587), delavirdine (U-901525/T), efavirenz
(DMP-266), UC-781
(N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide-
), etravirine (TMC125), Trovirdine (Ly300046.HCl), MKC-442
(emivirine, coactinon), HI-236, HI-240, HI-280, HI-281, rilpivirine
(TMC-278), MSC-127, HBY 097, DMP266, Baicalin (TJN-151) ADAM-II
(Methyl
3',3'-dichloro-4',4''-dimethoxy-5',5''-bis(methoxycarbonyl)-6,6-diphenylh-
exenoate), Methyl
3-Bromo-5-(1-5-bromo-4-methoxy-3-(methoxycarbonyl)phenyl)hept-1-enyl)-2-m-
ethoxybenzoate (Alkenyldiarylmethane analog, Adam analog),
(5-chloro-3-(phenylsulfinyl)-2'-indolecarboxamide), AAP-BHAP
(U-104489 or PNU-104489), Capravirine (AG-1549, S-1153), atevirdine
(U-87201E), aurin tricarboxylic acid (SD-095345),
1-[(6-cyano-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperaz-
ine,
1-[5-[[N-(methyl)methylsulfonylamino]-2-indolylcarbonyl-4-[3-(isoprop-
ylamino)-2-pyridinyl]piperazine,
1-[3-(Ethylamino)-2-[pyridinyl]-4-[(5-hydroxy-2-indolyl)carbonyl]piperazi-
ne,
1-[(6-Formyl-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]pip-
erazine,
1-[[5-(Methylsulfonyloxy)-2-indoyly)carbonyl]-4-[3-(isopropylamin-
o)-2-pyridinyl]piperazine, U88204E, Bis(2-nitrophenyl)sulfone (NSC
633001), Calanolide A (NSC675451), Calanolide B,
6-Benzyl-5-methyl-2-(cyclohexyloxy)pyrimidin-4-one (DABO-546), DPC
961, E-EBU, E-EBU-dm, E-EPSeU, E-EPU, Foscarnet (Foscavir), HEPT
(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine), HEPT-M
(1-[(2-Hydroxyethoxy)methyl]-6-(3-methylphenyl)thio)thymine),
HEPT-S (1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine),
Inophyllum P, L-737,126, Michellamine A (NSC650898), Michellamine B
(NSC649324), Michellamine F,
6-(3,5-Dimethylbenzyl)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil,
6-(3,5-Dimethylbenzyl)-1-(ethyoxymethyl)-5-isopropyluracil, NPPS,
E-BPTU (NSC 648400), Oltipraz
(4-Methyl-5-(pyrazinyl)-3H-1,2-dithiole-3-thione),
N-{2-(2-Chloro-6-fluorophenethyl]-N'-(2-thiazolyl)thiourea (PETT
Cl, F derivative),
N-{2-(2,6-Difluorophenethyl]-N'-[2-(5-bromopyridyl)]thiourea {PETT
derivative),
N-{2-(2,6-Difluorophenethyl]-N'-[2-(5-methylpyridyl)]thiourea {PETT
Pyridyl derivative),
N-[2-(3-Fluorofuranyl)ethyl]-N'-[2-(5-chloropyridyl)]thiourea,
N-[2-(2-Fluoro-6-ethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]thiourea,
N-(2-Phenethyl)-N'-(2-thiazolyl)thiourea (LY-73497), L-697,639,
L-697,593, L-697,661,
342-(4,7-Difluorobenzoxazol-2-yl)ethyl}-5-ethyl-6-methyl(pypridin-2(1H)-t-
hione (2-Pyridinone Derivative),
3-[[(2-Methoxy-5,6-dimethyl-3-pyridyl)methyl]amine]-5-ethyl-6-methyl(pyri-
din-2(1H)-thione, R82150, R82913, R87232, R88703, R89439
(Loviride), R90385, S-2720, Suramin Sodium, TBZ
(Thiazolobenzimidazole, NSC 625487), Thiazoloisoindol-5-one,
(+)(R)-9b-(3,5-Dimethylphenyl-2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-o-
ne, Tivirapine (R86183), UC -38 and UC-84, among others.
[0614] The term "pharmaceutically acceptable salt" is used
throughout the specification to describe, where applicable, a salt
form of one or more of the compounds described herein which are
presented to increase the solubility of the compound in the gastic
juices of the patient's gastrointestinal tract in order to promote
dissolution and the bioavailability of the compounds.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids,
where applicable. Suitable salts include those derived from alkali
metals such as potassium and sodium, alkaline earth metals such as
calcium, magnesium and ammonium salts, among numerous other acids
and bases well known in the pharmaceutical art. Sodium and
potassium salts are particularly preferred as neutralization salts
of the phosphates according to the present disclosure.
[0615] The term "pharmaceutically acceptable derivative" is used
throughout the specification to describe any pharmaceutically
acceptable prodrug form (such as an ester, amide other prodrug
group), which, upon administration to a patient, provides directly
or indirectly the present compound or an active metabolite of the
present compound.
[0616] General Synthetic Approach
[0617] The synthetic realization and optimization of the
bifunctional molecules as described herein may be approached in a
step-wise or modular fashion. For example, identification of
compounds that bind to the target molecules can involve high or
medium throughput screening campaigns if no suitable ligands are
immediately available. It is not unusual for initial ligands to
require iterative design and optimization cycles to improve
suboptimal aspects as identified by data from suitable in vitro and
pharmacological and/or ADMET assays. Part of the optimization/SAR
campaign would be to probe positions of the ligand that are
tolerant of substitution and that might be suitable places on which
to attach the linker chemistry previously referred to herein. Where
crystallographic or NMR structural data are available, these can be
used to focus such a synthetic effort.
[0618] In a very analogous way one can identify and optimize
ligands for an E3 Ligase, i.e. ULMs/CLMs.
[0619] With PTMs and ULMs (e.g. CLMs) in hand one skilled in the
art can use known synthetic methods for their combination with or
without a linker moiety. Linker moieties can be synthesized with a
range of compositions, lengths and flexibility and functionalized
such that the PTM and ULM groups can be attached sequentially to
distal ends of the linker. Thus a library of bifunctional molecules
can be realized and profiled in in vitro and in vivo
pharmacological and ADMET/PK studies. As with the PTM and ULM
groups, the final bifunctional molecules can be subject to
iterative design and optimization cycles in order to identify
molecules with desirable properties.
[0620] Exemplary compounds described in this application can be
synthesized by connecting the right hand key fragment prepared
according to Schemes 2-30, 2-31, 2-40, 2-41, 2-45, and 2-46. The
detailed preparation of representative compounds claimed in this
application are further described in Schemes 3-10, 3-56, 3-58, and
3-72.
[0621] A. Exemplary Cereblon Ligand General Synthetic Schemes
[0622] Synthetic schemes 2-30, 2-31, 2-40, 2-41, 2-45, and 2-46
describe the routes used in the preparation of CRBN ligands, as
well as CRBN ligands with partial linker moieties connected.
[0623] General Synthetic Scheme 2-30 to Prepare Intermediate.
##STR00298##
[0624] General Synthetic Scheme 2-31 to Prepare Intermediate.
##STR00299##
[0625] General Synthetic Scheme 2-40 to Prepare Intermediate.
##STR00300##
[0626] General Synthetic Scheme 2-41 to Prepare Intermediate.
##STR00301##
[0627] General Synthetic Scheme 2-45 to Prepare Intermediate.
##STR00302## ##STR00303##
[0628] General Synthetic Scheme 2-46 to Prepare Intermediate.
##STR00304##
[0629] B. Exemplary PROTAC General Synthetic Schemes
[0630] Synthetic schemes 3-10, 3-56, 3-58, and 3-72 describe the
routes used in the preparation of representative chimeric compounds
claimed in this application.
[0631] General Synthetic Scheme 3-10 to Prepare Claimed
Compounds.
##STR00305##
[0632] General Synthetic Scheme 3-56 to Prepare Claimed
Compounds.
##STR00306##
[0633] General Synthetic Scheme 3-58 to Prepare Claimed
Compounds.
##STR00307##
[0634] General Synthetic Scheme 3-72 to Prepare Claimed
Compounds.
##STR00308##
[0635] Synthesis of Exemplary PROTAC 1
##STR00309## [0636]
2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-
-a][1,4]diazepin-6-yl)-N-(3-(3-((3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-y-
l)quinolin-6-yl)oxy)propoxy)propyl)acetamide
[0637] Synthetic Scheme:
##STR00310##
[0638] To a solution of
(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo-
[4,3-a][1,4]diazepin-6-yl)acetic acid (20.6 mg, 0.051 mmol) and
1-(5-(3-(3-aminopropoxy)propoxy)quinolin-3-yl)pyrimidine-2,4(1H,3H)-dione
hydrochloride (21.6 mg, 0.053 mmol) in DCM (1 mL) was added
diisopropylethylamine (0.022 mL, 0.128 mmol), HATU (20.1 mg, 0.053
mmol) was added and it was stirred at room temperature for 2 hours.
The reaction was washed by NaHCO3 solution and the organic layer
was separated and dried. The product was purified by column
chromatography on silica (10% MeOH/DCM) to give
2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-
-a][1,4]diazepin-6-yl)-N-(3-(3-((3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-y-
l)quinolin-6-yl)oxy)propoxy)propyl)acetamide (25 mg, 65%)
[0639] LCMS (m/e+)=753.35 [M+H].sup.+ and m/e+=377.17
[M+2H].sup.2+
[0640] Synthesis of Exemplary PROTAC 29
##STR00311##
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(6-((1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)oxy)hex-
yl)piperazin-1-yl)nicotinamide
Synthetic Scheme Part 1--Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide
##STR00312##
[0641] Step 1: Synthesis of
6-(4-(tert-butoxycarbonyl)piperazin-1-yl)nicotinic acid
##STR00313##
[0643] 6-Chloronicotinic acid (1.6 g, 10.0 mmol) was dissolved in
N,N-dimethylacetamide (15 mL), and tert-butyl
piperazine-1-carboxylate (1.9 g, 10.0 mmol) and
ethyldiisopropylamine (2.6 g, 20 mmol) were added thereto, followed
by stirring at 130.degree. C. overnight. The reaction mixture was
concentrated under reduced pressure, and to the obtained residue
was added a 1 M aqueous NaOH solution (10 mL), followed by washing
with CHCl.sub.3 (50 mL). The pH of the aqueous layer was adjusted
to around 6 to 7 by the addition of 1 M hydrochloric acid, followed
by extraction with CHCl.sub.3 (50 mL.times.3). The organic layer
was dried over anhydrous sodium sulfate and the solvent was
concentrated under reduced pressure. The obtained residue was
purified by silica gel column chromatography
(CH.sub.2Cl.sub.2/MeOH=10/1) to give
6-(4-(tert-butoxycarbonyl)piperazin-1-yl)nicotinic acid (2.0 g, 65%
yield) as a white solid.
[0644] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 83.17%, Rt=1.312 min; MS Calcd.:
307.15; MS Found: 308.2 [M+H].sup.+.
[0645] Chemical Formula: C.sub.15H.sub.21N.sub.3O.sub.4, Molecular
Weight: 307.34
Step 2: Synthesis of tert-butyl
4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylca-
rbamoyl)pyridin-2-yl)piperazine-1-carboxylate
##STR00314##
[0647] A mixture of
6-(4-(tert-butoxycarbonyl)piperazin-1-yl)nicotinic acid (614 mg,
2.0 mmol),
4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile
hydrochloride (630 mg, 2.0 mmol),
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (1.1 g, 3.0 mmol) and ethyldiisopropylamine
(516 mg, 4.0 mmol) in dichloromethane (20 mL) was stirred at room
temperature overnight. Water (50 mL) was added and extracted with
dichloromethane (50 mL.times.3). Combined organic layers were
washed by brine (50 mL.times.2), dried over anhydrous sodium
sulfate. The solvent was concentrated to give the residue, which
was purified by column chromatography on silica gel (petroleum
ether/ethyl acetate=1/1) to give tert-butyl
4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylca-
rbamoyl)pyridin-2-yl)piperazine-1-carboxylate (977 mg, 86% yield)
as a white solid.
[0648] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 88.26%, Rt=2.161 min; MS Calcd.:
567.26; MS Found: 568.3 [M+H].sup.+.
[0649] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.1.12 (6H, s),
1.22 (6H, s), 1.43 (9H, s), 3.42-3.44 (4H, m), 3.60-3.63 (4H, m),
4.02-4.07 (1H, m), 4.31 (1H, s), 6.88 (1H, d, J=8.8 Hz), 7.00 (1H,
dd, J=8.4, 2.4 Hz), 7.21 (1H, d, J=2.4 Hz), 7.65 (1H, d, J=9.2 Hz),
7.91 (1H, d, J=8.8 Hz), 7.99 (1H, dd, J=8.8, 2.4 Hz), 8.64 (1H, d,
J=2.4 Hz).
[0650] Chemical Formula: C.sub.30H.sub.38ClN.sub.5O.sub.4,
Molecular Weight: 568.11
[0651] Total H count from HNMR data: 38.
Step 3: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride
##STR00315##
[0653] A mixture of tert-butyl
4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl
carbamoyl)pyridin-2-yl)piperazine-1-carboxylate (405 mg, 0.7 mmol)
in HCl/1,4-dioxane (10 mL) was stirred at room temperature for 4 h.
The solvent was removed in vacuum to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride (353 mg, 100% yield) as a
white solid.
[0654] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.791 min; MS Calcd.: 467.21; MS Found:
468.3 [M+H].sup.+.
[0655] Chemical Formula: C.sub.25H.sub.31Cl.sub.2N.sub.5O.sub.2,
Molecular Weight: 504.45
[0656] Synthetic Scheme Part 2
##STR00316##
Step 4: Synthesis of
4,5-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one
##STR00317##
[0658] The mixture of 4,5-dichloropyridazin-3(2H)-one (5.0 g, 30.5
mmol), 1-(chloromethyl)-4-methoxybenzene (7.1 g, 45.7 mmol) and
potassium carbonate (12.6 g, 91.5 mmol) in N',N'-Dimethylformamide
(100 mL) was stirred at room temperature for 12 hours. The mixture
was poured into water and extracted with ethyl acetate (100
mL.times.3). The combined organic phase was concentrated in vacuo
and the residue was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate=3/1) to give
4,5-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one (6.3 g, 73%
yield) as a white solid.
[0659] LC-MS (Agilent LCMS 1200-6110, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 95%
[water+0.05% TFA] and 5% [CH.sub.3CN+0.05% TFA] to 0% [water+0.05%
TFA] and 100% [CH.sub.3CN+0.05% TFA] in 1.5 min, then under this
condition for 0.5 min, finally changed to 95% [water+0.05% TFA] and
5% [CH.sub.3CN+0.05% TFA] in 0.1 min and under this condition for
0.1 min). Rt=1.220 min; MS Calcd.: 284.0; MS Found: 285.1
[M+H].sup.+.
[0660] Chemical Formula: C.sub.12H.sub.10Cl.sub.2N.sub.2O.sub.2,
Molecular Weight: 285.13
Step 5: Synthesis of
5-(6-(benzyloxy)hexyloxy)-4-chloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one
##STR00318##
[0662] To a solution of 6-(benzyloxy)hexan-1-ol (1.04 g, 50 mmol)
in dried THF (100 mL) was added 60% NaH (240 mg, 60 mmol) at
0.degree. C., then it was stirred for 30 minutes,
4,5-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one (1.42 g, 50
mmol) was added, the resulting mixture was refluxed overnight.
After cooling to room temperature, the mixture was quenched by
aqueous NH.sub.4Cl and then extracted with ethyl acetate (50
mL.times.2). The combined organic phase was washed with brine (50
mL), dried over Na.sub.2SO.sub.4, filtered, concentrated in vacuo
and the residue was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate=10/1) to give
5-(6-(benzyloxy)hexyloxy)-4-chloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one
(1.59 g, 70% yield) as a colorless gel.
[0663] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.40-1.47 (4H, m),
1.59-1.65 (2H, m), 1.69-1.75 (2H, m), 3.46 (2H, t, J=6.4 Hz), 3.78
(3H, s), 4.50 (2H, s), 4.56 (2H, t, J=6.4 Hz), 5.21 (2H, s), 6.85
(2H, d, J=8.4 Hz), 7.26-7.29 (1H, m), 7.33-7.38 (6H, m), 7.69 (1H,
s).
[0664] Total H count from HNMR data: 29.
Step 6: Synthesis of
5-(6-(benzyloxy)hexyloxy)-4-chloropyridazin-3(2H)-one
##STR00319##
[0666] To a solution of
5-(6-(benzyloxy)hexyloxy)-4-chloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one
(450 mg, 1 mmol) in CH.sub.3CN (30 mL) at 0.degree. C., was added a
solution of CAN (1.37 g, 2.5 mmol) in H.sub.2O (10 mL), and the
solution was allowed to warm to room temperature and stirred
overnight. At this point the mixture was partitioned between ethyl
acetate (30 mL) and half saturated brine (20 mL). The phases were
separated, and the aqueous phase was extracted with ethyl acetate
(30 mL), then with CH.sub.2Cl.sub.2 (30 mL). The combined organic
phases were dried (Na.sub.2SO.sub.4), filtered, and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel (petroleum ether/ethyl acetate=10/1) to afford
5-(6-(benzyloxy)hexyloxy)-4-chloropyridazin-3(2H)-one (250 mg, 74%
yield) as a yellow gel. LC-MS (Agilent LCMS 1200-6110, Column:
Waters X-Bridge C18 (50 mm.times.4.6 mm.times.3.5 .mu.m); Column
Temperature: 40.degree. C.; Flow Rate: 1.5 mL/min; Mobile Phase:
from 95% [water+0.05% TFA] and 5% [CH.sub.3CN+0.05% TFA] to 0%
[water+0.05% TFA] and 100% [CH.sub.3CN+0.05% TFA] in 1.5 min, then
under this condition for 0.5 min, finally changed to 95%
[water+0.05% TFA] and 5% [CH.sub.3CN+0.05% TFA] in 0.1 min and
under this condition for 0.1 min). Rt=1.346 min; MS Calcd.: 336.1;
MS Found: 337.3 [M+H].sup.+.
[0667] Chemical Formula: C.sub.17H.sub.21ClN.sub.2O.sub.3,
Molecular Weight: 336.81.
Step 7: Synthesis of
3-(4-(6-(benzyloxy)hexyloxy)-5-chloro-6-oxopyridazin-1(6H)-yl)piperidine--
2,6-dione
##STR00320##
[0669] The mixture of
5-(6-(benzyloxy)hexyloxy)-4-chloropyridazin-3(2H)-one (250 mg, 0.74
mmol), 3-bromopiperidine-2,6-dione (143 mg, 0.74 mmol) and
potassium carbonate (205 mg, 1.48 mmol) in acetonitrile (40 mL) was
stirred at room temperature for 3 days, and then filtrated. The
filtrate was concentrated and purified by column chromatography on
silica gel (petroleum ether/ethyl acetate=3/2) to give
3-(4-(6-(benzyloxy)hexyloxy)-5-chloro-6-oxopyridazin-1(6H)-yl)piperidine--
2,6-dione (180 mg, 54% yield) as a light yellow gel.
[0670] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.40-1.49 (4H, m),
1.61-1.66 (2H, m), 1.72-1.78 (2H, m), 2.20-2.24 (1H, m), 2.65-2.79
(2H, m), 2.86-2.90 (1H, m), 3.47 (2H, t, J=6.4 Hz), 4.50 (2H, s),
4.55-4.61 (2H, m), 5.65 (1H, dd, J=10.8, 5.6 Hz), 7.26-7.34 (5H,
m), 7.76 (1H, s), 8.46 (1H, s).
[0671] Total H count from HNMR data: 26.
Step 8: Synthesis of
3-(4-(6-hydroxyhexyloxy)-6-oxopyridazin-1(6H)-yl)piperidine-2,6-dione
##STR00321##
[0673] The mixture of
3-(4-(6-(benzyloxy)hexyloxy)-5-chloro-6-oxopyridazin-1(6H)-yl)piperidine--
2,6-dione (180 mg, 0.4 mmol) and 10% palladium on activated carbon
(100 mg) in MeOH (20 mL) was stirred under 1 atm. hydrogen
atmosphere at room temperature for 2 h. It was filtered to remove
the solid, the filtrate was concentrated to give
3-(4-(6-hydroxyhexyloxy)-6-oxopyridazin-1(6H)-yl)piperidine-2,6-dione
(118 mg, 90% yield) as a light yellow solid.
[0674] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.34-1.45 (6H,
m), 1.71-1.77 (2H, m), 2.04-2.08 (1H, m), 2.46-2.60 (2H, m),
2.84-2.90 (1H, m), 3.39 (2H, t, J=6.4 Hz), 4.02 (2H, t, J=6.4 Hz),
4.72 (1H, brs), 5.69 (1H, dd, J=12.4, 5.2 Hz), 6.77 (1H, d, J=5.2
Hz), 7.82 (1H, d, J=4.8 Hz), 11.03 (1H, s).
[0675] Total H count from HNMR data: 21.
Step 9: Synthesis of
6-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yloxy)hexanal
##STR00322##
[0677] A solution of
3-(4-(6-hydroxyhexyloxy)-6-oxopyridazin-1(6H)-yl)piperidine-2,6-dione
(64 mg, 0.2 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added Dess-Martin
reagent (127 mg, 0.6 mmol), and the mixture was stirred at room
temperature overnight. After removal of undissolved solid by
suction, the filtrate was concentrated at room temperature to give
crude
6-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yloxy)hexanal
(64 mg, 99% yield) as a white semi-solid, it was directly used to
the next step without further purification.
[0678] LC-MS (Agilent LCMS 1200-6110, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 95%
[water+0.05% TFA] and 5% [CH.sub.3CN+0.05% TFA] to 0% [water+0.05%
TFA] and 100% [CH.sub.3CN+0.05% TFA] in 1.5 min, then under this
condition for 0.5 min, finally changed to 95% [water+0.05% TFA] and
5% [CH.sub.3CN+0.05% TFA] in 0.1 min and under this condition for
0.1 min). Rt=0.721 min; MS Calcd.: 321.1; MS Found: 322.3
[M+H].sup.+.
[0679] Chemical Formula: C.sub.15H.sub.19N.sub.3O.sub.5, Molecular
Weight: 321.33.
Step 10: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(6-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yloxy)hexyl-
)piperazin-1-yl)nicotinamide
##STR00323##
[0681] To a solution of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride (100 mg, 0.2 mmol) in
MeOH (5 mL) was added a solution of
6-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yloxy)hexanal
(64 mg, 0.2 mmol) in CH.sub.2Cl.sub.2 (5 mL), then NaBH.sub.3CN (40
mg, 0.6 mmol) was added, the resulting mixture was stirred at room
temperature overnight. The reaction mixture was concentrated,
diluted with water (10 mL) and extracted with CH.sub.2Cl.sub.2 (20
mL.times.2). The organic extract was washed with brine (20 mL),
dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by
Prep-TLC and then Prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(6-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yloxy)hexyl-
)piperazin-1-yl)nicotinamide (20 mg, 13% yield) as a white
solid.
[0682] LC-MS (Agilent LCMS 1200-6120, Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 94.07%, Rt=2.741 min; MS Calcd.:
772.4; MS Found: 773.3 [M+H].sup.+.
[0683] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 93.35%, Rt=9.681 min.
[0684] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25
(6H, s), 1.39-1.44 (2H, m), 1.49-1.62 (4H, m), 1.87-1.93 (2H, m),
2.24-2.28 (1H, m), 2.36-2.43 (2H, m), 2.56 (4H, s), 2.70-2.81 (2H,
m), 2.87-2.92 (1H, m), 3.66-3.69 (4H, m), 4.00-4.04 (3H, m), 4.14
(1H, d, J=8.0 Hz), 5.74 (1H, dd, J=11.2, 5.6 Hz), 6.07 (1H, d,
J=8.4 Hz), 6.40 (1H, d, J=4.8 Hz), 6.66 (1H, d, J=8.8 Hz), 6.80
(1H, dd, J=8.8, 2.4 Hz), 6.96 (1H, d, J=2.4 Hz), 7.57 (1H, d, J=8.8
Hz), 7.71 (1H, d, J=4.8 Hz), 7.93 (1H, dd, J=8.8, 2.4 Hz), 8.16
(1H, brs), 8.58 (1H, d, J=2.4 Hz).
[0685] Chemical Formula: C.sub.40H.sub.49ClN.sub.8O.sub.6,
Molecular Weight: 773.32
[0686] Total H count from HNMR data: 49.
[0687] Synthesis of Exemplary PROTAC 30
##STR00324## [0688]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-8--
yl)oxy)pentyl)piperazin-1-yl)nicotinamide
[0689] Synthetic Scheme
##STR00325##
Step 1: Synthesis of
3-(8-hydroxy-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione
##STR00326##
[0691] To a stirred mixture of 2-amino-3-hydroxybenzoic acid (2.0
g, 13.1 mmol) and imidazole (2.0 g, 29.4 mmol) in acetonitrile (30
mL), was added acetyl chloride (2.0 mL, 28.7 mmol) at room
temperature. The mixture was stirred at room temperature for 2
days. To the mixture, was added 3-amino-piperidine-2, 6-dione
hydrogen chloride (2.2 g, 13.1 mmol), imidazole (2.0 g, 29.4 mmol)
and triphenyl phosphite (4.11 mL, 15.7 mmol) and heated to reflux
for 3 days. To the mixture, was added water (60 mL) and conc HCl
until pH=1. The solvent was removed in vacuo. To the residue, was
added water (50 mL). The aqueous layer was extracted with ethyl
acetate (2.times.50 mL). To the aqueous layer, was added sodium
hydrogen carbonate (1.8 g) to pH=7-8, and the mixture was stirred
at room temperature to give a suspension. The suspension was
filtered and dried to give
3-(8-hydroxy-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-di-
one (230 mg, 6% yield) as a gray solid.
[0692] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.14-2.19 (1H,
m), 2.57-2.69 (5H, m), 2.80-2.87 (1H, m), 5.26 (1H, dd, J=11.6, 5.6
Hz), 7.19 (1H, dd, J=8.0, 1.6 Hz), 7.30 (1H, t, J=8.0 Hz), 7.45
(1H, dd, J=8.0, 1.6 Hz), 9.66 (1H, s), 11.03 (1H, s).
[0693] Total H count from HNMR data: 13.
Step 2: Synthesis of
3-(8-(5-chloropentyloxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-
-dione
##STR00327##
[0695] To a solution of
3-(8-hydroxy-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione
(91 mg, 0.32 mmol) and 5-chloropentyl 4-methylbenzenesulfonate (88
mg, 0.32 mmol) in DMF (10 mL) was added K.sub.2CO.sub.3 (88 mg,
0.64 mmol) at room temperature, then it was heated to 40.degree. C.
and stirred for 2 days. The mixture was purified by reverse phase
HPLC to give
3-(8-(5-chloropentyloxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-
-dione (19 mg, 15% yield) as a white solid.
[0696] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.65-1.73 (2H, m),
1.87-2.02 (4H, m), 2.13-2.17 (1H, m), 2.66-2.74 (4H, m), 2.89-3.02
(2H, m), 3.60 (2H, t, J=6.4 Hz), 4.19 (2H, t, J=6.4 Hz), 4.77 (1H,
dd, J=11.6, 6.4 Hz), 7.21 (1H, d, J=8.0 Hz), 7.38 (1H, t, J=8.0
Hz), 7.76 (1H, d, J=7.2 Hz).
[0697] Total H count from HNMR data: 21.
Step 3: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-8-y-
loxy)pentyl)piperazin-1-yl)nicotinamide
##STR00328##
[0699] A mixture of
3-(8-(5-chloropentyloxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-
-dione (15 mg, 0.038 mmol), DIEA (25 mg, 0.19 mmol), KI (6 mg,
0.038 mmol) and
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-
-6-(piperazin-1-yl)nicotinamide (20 mg, 0.038 mmol) in CH.sub.3CN
(10 mL) was stirred at 100.degree. C. overnight. Then it was
evaporated, to the residue was added DIEA (25 mg, 0.19 mmol) and
EtCN (10 mL), and the solution was stirred at 100.degree. C.
overnight. At this point the mixture was diluted with water (10 mL)
and extracted by ethyl acetate (20 mL.times.2). The organic extract
was washed with brine (10 mL), dried (Na.sub.2SO.sub.4), filtered,
and concentrated in vacuo. The crude product was purified by
prep-HPLC to afford
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-8-y-
loxy)pentyl)piperazin-1-yl)nicotinamide (5.5 mg, 17% yield) as a
white solid.
[0700] LC-MS (Agilent LCMS 1200-6120, Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 93.89%, Rt=1.987 min; MS Calcd.:
822.4; MS Found: 823.4 [M+H].sup.+.
[0701] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 93.92%, Rt=9.851 min.
[0702] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25
(6H, s), 1.59-1.62 (4H, m), 1.90-2.00 (2H, m), 2.14-2.17 (1H, m),
2.70-2.79 (5H, m), 2.86-2.96 (6H, m), 3.15 (1H, dd, J=14.8, 7.2
Hz), 3.88 (4H, s), 4.05 (1H, s), 4.13-4.20 (3H, m), 4.82 (1H, dd,
J=11.2, 5.6 Hz), 6.14 (1H, d, J=8.4 Hz), 6.68 (1H, d, J=9.2 Hz),
6.80 (1H, dd, J=8.8, 2.4 Hz), 6.96 (1H, d, J=2.4 Hz), 7.20 (1H, d,
J=8.0 Hz), 7.38 (1H, t, J=8.0 Hz), 7.57 (1H, d, J=8.8 Hz), 7.74
(1H, d, J=8.0 Hz), 7.94 (1H, dd, J=8.8, 2.0 Hz), 8.30 (1H, brs),
8.57 (1H, d, J=2.0 Hz).
[0703] Chemical Formula: C.sub.44H.sub.51ClN.sub.8O.sub.6,
Molecular Weight: 823.38
[0704] Total H count from HNMR data: 51.
[0705] Synthesis of Exemplary PROTAC 33
##STR00329##
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]is-
othiazol-6-yl)oxy)pentyl)piperazin-1-yl)nicotinamide
[0706] Synthetic Scheme
##STR00330##
Step 1: Synthesis of
6-((5-hydroxypentyl)oxy)benzo[d]isothiazol-3(2H)-one
1,1-dioxide
##STR00331##
[0708] To a solution of pentane-1,5-diol (1.73 g, 16.7 mmol) in
N,N-dimethylformamide (15.0 mL) was added sodium hydride (266 mg,
6.66 mmol) under nitrogen. The reaction mixture was stirred at room
temperature for 1 h. Then 6-nitrobenzo[d]isothiazol-3(2H)-one
1,1-dioxide (760 mg, 3.33 mmol) was added and stirred at 70.degree.
C. for 12 hours. After cooling to room temperature, the solvent was
removed in vacuo. The residue was extracted with ethyl acetate (30
mL.times.3) and water (30 mL). The organic layer was washed with
brine (5 mL). The combined organic phases were dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The residue
was washed by methanol (3 mL) to give
6-((5-hydroxypentyl)oxy)benzo[d]isothiazol-3(2H)-one 1,1-dioxide
(560 mg, 59%) as a pale yellow solid.
[0709] Agilent LC-MS (Agilent LCMS 1200-6120, Column: Waters
X-Bridge C18 (50 mm*4.6 mm*3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 78.69%, Rt=1.159 min; MS Calcd.:
285.1; MS Found: 284.2 [M-H].sup.+.
Step 2: Synthesis of
5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl
methanesulfonate
##STR00332##
[0711] To a solution of
6-((5-hydroxypentyl)oxy)benzo[d]isothiazol-3(2H)-one (120 mg, 0.421
mmol) in tetrahydrofuran (10.0 mL) was added triethylamine (85.1
mg, 0.841 mmol) and methanesulfonyl chloride (38.5 mg, 0.336 mmol)
under nitrogen. The resulting reaction mixture was stirred at room
temperature for 0.5 hour. The solvent was concentrated in vacuo.
The residue was extracted with dichloromethane (10 mL.times.3) and
water (20 mL). The organic phase was dried and concentrated in
vacuo to give crude
5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl
methanesulfonate as yellow oil, which was used to the next step
without further purification.
[0712] Agilent LC-MS (Agilent LCMS 1200-6120, Column: Waters
X-Bridge C18 (30 mm*4.6 mm*3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5%
[water+10 mM NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min,
then under this condition for 1.5 min, finally changed to 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and
under this condition for 0.5 min). Purity is 77.93%, Rt=0.613 min;
MS Calcd.: 363.0; MS Found: 362.0 [M-H].sup.+.
Step 3: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl)pip-
erazin-1-yl)nicotinamide
##STR00333##
[0714] To a solution of
5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl
methanesulfonate (0.421 mmol) in acetonitrile (5 mL) was added
potassium carbonate (291 mg, 2.11 mmol) and
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride (212 mg, 0.421 mmol). The
resulting reaction mixture was stirred at 90.degree. C. for 16
hours. The solvent was concentrated in vacuo. The residue was
extracted with ethyl acetate (20 mL.times.3) and water (20 mL). The
organic phase was dried and concentrated in vacuo. The residue was
purified by prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl)pip-
erazin-1-yl)nicotinamide (34 mg, 11% for two steps) as a pale
yellow solid.
[0715] Agilent LC-MS (Agilent LCMS 1200-6120, Column: Waters
X-Bridge C18 (30 mm*4.6 mm*3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5%
[water+10 mM NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min,
then under this condition for 1.5 min, finally changed to 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and
under this condition for 0.5 min). Purity is 97.67%, Rt=1.037 min;
MS Calcd.: 734.3; MS Found: 735.0 [M+H].sup.+.
Step 4: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]is-
othiazol-6-yl)oxy)pentyl)piperazin-1-yl)nicotinamide
##STR00334##
[0717] To a solution of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)oxy)pentyl)pip-
erazin-1-yl)nicotinamide (30 mg, 0.0408 mmol) in
1,4-dioxane/N,N-dimethylformamide (5 mL/0.5 mL) was added
3-bromopiperidine-2,6-dione (11.8 mg, 0.0612 mmol) and potassium
tert-butoxide (9.16 mg, 0.0816 mmol). The reaction mixture was
stirred at 100.degree. C. for overnight. After cooling to room
temperature, ice-water (2.0 mL) was added, and adjust to
PH=2.about.3 by hydrochloric acid (1N), then extracted with ethyl
acetate (20.0 mL.times.3). The combined organic phase was washed
with brine (5.0 mL), dried over anhydrous sodium sulfate, filtered,
and concentrated in vacuo. The residue was purified by prep-HPLC
and prep-TLC (dichloromethane/methanol=10:1) to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]is-
othiazol-6-yl)oxy)pentyl)piperazin-1-yl)nicotinamide (6.8 mg, 20%)
as a white solid.
[0718] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.). Purity is 99.03%, Rt=3.087 min; MS Calcd.:
845.3; MS Found: 846.3 [M+H].sup.+.
[0719] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow Rate:
1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH.sub.4HCO.sub.3]
and 5% [CH.sub.3CN] to 0% [water+10 mM NH.sub.4HCO.sub.3] and 100%
[CH.sub.3CN] in 10 min, then under this condition for 5 min,
finally changed to 95% [water+10 mM NH.sub.4HCO.sub.3] and 5%
[CH.sub.3CN] in 0.1 min and under this condition for 5 min). Purity
is 96.34%, Rt=10.536 min.
[0720] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.19 (6H, s),
1.22 (6H, s), 1.46-1.55 (4H, m), 1.79-1.80 (2H, m), 2.34-2.40 (3H,
m), 2.45 (4H, s), 2.54-2.92 (3H, m), 3.59 (4H, s), 4.06 (1H, d,
J=9.2 Hz), 4.20-4.25 (2H, m), 4.30 (1H, s), 5.23-5.28 (0.5H, m),
5.98 (0.5H, t, J=9.2 Hz), 6.87 (1H, d, J=9.2 Hz), 6.99-7.02 (1H,
m), 7.21 (1H, d, J=2.0 Hz), 7.35-7.50 (1H, m), 7.63 (1H, d, J=9.2
Hz), 7.81-7.83 (1H, m), 7.90-8.02 (3H, m), 8.62 (1H, d, J=2.0 Hz),
11.19 (1H, t, J=9.6 Hz).
[0721] Chemical Formula: C.sub.42H.sub.48ClN.sub.7O.sub.8S,
Molecular Weight: 846.39
[0722] Total H count from HNMR data: 48.
[0723] Synthesis of Exemplary PROTAC 39
##STR00335## [0724]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)ethyl)piperazin-1-y-
l)nicotinamide
[0725] Synthetic Scheme:
##STR00336##
Step 1: Synthesis of
N-(2,4-difluorophenyl)-3-methoxy-2-methylbenzamide
##STR00337##
[0727] A mixture of 3-methoxy-2-methylbenzoic acid (5 g, 30 mmol)
and oxalyl chloride (5.6 g, 150 mmol) and N,N-dimethylformamide
(0.1 ml) in dichloromethane (20 ml) was stirred at room temperature
for 2 hours. TLC showed the reaction was complete. The volatiles
were evaporated under reduced pressure to afford
3-methoxy-2-methylbenzoyl chloride (crude) as yellow oil which was
used in next step without further purification. A mixture of
3-methoxy-2-methylbenzoyl chloride (crude), 2,4-difluoroaniline
(3.8 g, 30 mmol) and triethylamine (12 g, 120 mmol) in
dichloromethane (20 ml) was stirred at room temperature for 1 hour.
TLC showed the reaction was complete. The reaction mixture was
diluted with dichloromethane (20 ml) and washed with brine (20 ml)
dried over anhydrous sodium sulfate, and concentrated under reduced
pressure to give a crude residue which was purified by silica gel
flash chromatography
N-(2,4-difluorophenyl)-3-methoxy-2-methylbenzamide (5.8 g, yield
69%) as yellow oil.
Step 2: Synthesis of
2-(bromomethyl)-N-(2,4-difluorophenyl)-3-methoxybenzamide
##STR00338##
[0729] A mixture of
N-(2,4-difluorophenyl)-3-methoxy-2-methylbenzamide (5.8 g, 20.9
mmol), N-Bromosuccinimide (3.9 g, 31.4 mmol) and AIBN
(2,2'-Azobis(2-methylpropionitrile)) (342 mg, 2.09 mmol) in carbon
tetrachloride (30 ml) was stirred at 70.degree. C. overnight. The
volatiles were evaporated under reduced pressure which was purified
by silica gel flash column chromatography (eluted with 10-20% ethyl
acetate in hexane) to afford
2-(bromomethyl)-N-(2,4-difluorophenyl)-3-methoxybenzamide (5.9 g,
yield 80%) as white solid.
[0730] LC_MS: (ES.sup.+): m/z 356.0, 357.9 [M+H].sup.+.
t.sub.R=2.907 min.
Step 3: Synthesis of
2-(2,4-difluorophenyl)-4-methoxyisoindolin-1-one
##STR00339##
[0732] To a solution of
2-(bromomethyl)-N-(2,4-difluorophenyl)-3-methoxybenzamide (2.0 g,
5.6 mmol) in anhydrous tetrahydrofuran (20 ml) was added potassium
tert-butanolate (1M in tetrahydrofuran, 8.4 ml, 8.4 mmol) at
0.degree. C., and the resulting mixture was stirred at 0.degree. C.
for 2 hours. TLC showed the reaction was complete. The reaction
mixture was partitioned between water (50 ml) and ethyl acetate (50
ml). The organic layer was collected, washed with brine (20
ml.times.2), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to afford a crude residue which was purified
by silica gel flash chromatography (eluted with 20% ethyl acetate
in hexane) to afford
2-(2,4-difluorophenyl)-4-methoxyisoindolin-1-one (500 mg, yield
33%) as yellow solid.
Step 4: Synthesis of
2-(2,4-difluorophenyl)-4-hydroxyisoindolin-1-one
##STR00340##
[0734] A mixture of
2-(2,4-difluorophenyl)-4-methoxyisoindolin-1-one (200 mg, 0.727
mmol) in hydrogen bromide in acetic acid solution (33%, 3 ml) was
stirred at 100.degree. C. for 2 days. TLC showed the reaction was
complete. The volatiles were evaporated under reduced pressure to
give a crude residue which was purified by silica gel flash
chromatography (eluted with 30%-50 ethyl acetate in hexane) to
afford 2-(2,4-difluorophenyl)-4-hydroxyisoindolin-1-one (180 mg,
yield 95%) as yellow oil.
[0735] LC_MS: (ES+): m/z 262.1 [M+H].sup.+. t.sub.R=2.64 min.
Step 5: Synthesis of
4-(allyloxy)-2-(2,4-difluorophenyl)isoindolin-1-one
##STR00341##
[0737] To a stirred solution of
2-(2,4-difluorophenyl)-4-hydroxyisoindolin-1-one (180 mg, 0.68
mmol), triphenylphosphine (539 mg, 2.06 mmol) and prop-2-en-1-ol
(119 mg, 2.06 mmol) in tetrahydrofuran (5 ml) was added diisopropyl
azodicarboxylate (416 mg, 2.06 mmol) in tetrahydrofuran (2 ml) at
0.degree. C., and the reaction mixture was stirred at 0.degree. C.
for 30 minutes. TLC showed the reaction was complete. The volatiles
were evaporated under reduced pressure to give a crude residue
which was purified by silica gel flash chromatography (eluted with
10-20% ethyl acetate in hexane) to afford
4-(allyloxy)-2-(2,4-difluorophenyl)isoindolin-1-one (180 mg, yield
87%) as colorless oil.
[0738] LC_MS: (ES+): m/z 302.2 [M+H].sup.+. t.sub.R=2.86 min.
Step 6: Synthesis of
2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)acetaldehyde
##STR00342##
[0740] An ozone-enrichen steam of oxygen was bubbled through a
solution of 4-(allyloxy)-2-(2,4-difluorophenyl)isoindolin-1-one
(180 mg, 0.59 mmol) in dichloromethane (20 ml) at -78.degree. C.
until the reaction mixture turned dark blue. The solution was
purged with oxygen at -78.degree. C. for 20 min to remove the
excess ozone. Then to the reaction mixture was added dimethyl
sulfide (1.5 ml, 20.4 mmol) at -78.degree. C., the mixture was
allowed to warm up to room temperature and stirred overnight. TLC
showed the reaction was complete. The reaction mixture was
concentrated under reduced pressure to give
2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)acetaldehyde
(180 mg, 100%) which was used in next step without further
purification.
[0741] .sup.1H NMR (400 MHz, DMSO-d6): .delta. 4.68-4.69 (m, 2H),
4.77-4.79 (m, 2H), 6.86-6.93 (m, 4H), 7.33-7.55 (m, 2H), 9.80 (s,
1H).
[0742] Chemical Formula: C.sub.16H.sub.11F.sub.2NO.sub.3; Molecular
Weight: 303.26;
[0743] Total H count from HNMR data: 11;
Step 7: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)ethyl)piperazin-1-y-
l)nicotinamide
##STR00343##
[0745] To a stirred solution of
2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)acetaldehyde
(160 mg, 0.53 mmol),
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide (300 mg, 0.6 mmol, intermediate in
synthesis of exemplary PROTAC 29) and acetic acid (2 drops) in
methanol (3 ml) was added sodium cyanoborohydride (150 mg, 2.4
mmol) at room temperature. The reaction mixture was stirred at room
temperature overnight. TLC showed the reaction was complete. The
reaction mixture was partitioned between ethyl acetate (40 ml) and
water (20 ml). The organic layer was collected, washed with brine
(20 ml), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to give a crude residue which was purified
by prep-TLC (eluted with 10% methanol in dichloromethane) to afford
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobut-
yl)-6-(4-(2-((2-(2,4-difluorophenyl)-1-oxoisoindolin-4-yl)oxy)ethyl)pipera-
zin-1-yl)nicotinamide (50 mg, yield 12%, 3 steps) as light yellow
solid.
[0746] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.23 (s, 6H),
1.29 (s, 6H), 2.67-2.82 (m, 4H), 2.92-3.01 (m, 2H), 3.72 (s, 4H),
4.15 (s, 1H), 4.29-4.39 (m, 3H), 4.88 (s, 2H), 6.85-6.87 (m, 2H),
7.10-7.34 (m, 4H), 7.47-7.75 (m, 4H), 7.96-7.98 (m, 1H), 8.61 (s,
1H).
[0747] Chemical Formula: C.sub.41H.sub.41ClF.sub.2N.sub.6O.sub.4;
Molecular Weight: 755.25;
[0748] Total H count from HNMR data: 40;
[0749] LC_MS: (ES+): m/z 755.6 [M+H].sup.+. t.sub.R=2.534 min.
[0750] Synthesis of Exemplary PROTAC 41
##STR00344## [0751]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl)piperazi-
n-1-yl)nicotinamide
[0752] Synthetic Scheme:
##STR00345##
Step 1: Synthesis of
2-(2,4-difluorophenyl)-5-hydroxyisoindoline-1,3-dione
##STR00346##
[0754] To a solution of 4-hydroxyphthalic acid (2 g, 10.98 mmol) in
acetonitrile (50 ml) was added 1,1'-carbonyldiimidazole (3.9 g,
24.16 mmol) in portions at room temperature. After stirring for 30
mins, 2,4-difluoroaniline (1.6 g, 12.08 mmol) was added, and the
resulting mixture was stirred at 70.degree. C. for 3 hours. TLC
showed the reaction was complete. The reaction mixture was
partitioned between ethyl acetate (50 ml) and water (50 ml), the
organic layer was washed with brine (50 ml.times.2) and dried over
anhydrous sodium sulfate and concentrated under reduced pressure to
give a crude residue which was purified by silica gel flash column
chromatography (eluted with 25-35% ethyl acetate in hexane) to
afford 2-(2,4-difluorophenyl)-5-hydroxyisoindoline-1,3-dione (2.1
g, yield 70%) as yellow solid.
[0755] LC_MS: (ES.sup.+): m/z 276.1 [M+H].sup.+. t.sub.R=2.462
min.
[0756] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 7.21-7.31 (m,
3H), 7.51-7.56 (m, 1H), 7.60-7.66 (m, 1H), 7.83 (d, J=8.4 Hz, 1H),
11.17 (br, 1H).
[0757] Chemical Formula: C.sub.14H.sub.7F.sub.2NO.sub.3; Molecular
Weight: 275.21;
[0758] Total H count from HNMR data: 7.
Step 2: Synthesis of
2-(2,4-difluorophenyl)-5-((5-hydroxypentyl)oxy)isoindoline-1,3-dione
##STR00347##
[0760] A mixture of
2-(2,4-difluorophenyl)-5-hydroxyisoindoline-1,3-dione (300 mg, 1.09
mmol), 5-hydroxypentyl 4-methylbenzenesulfonate (282 mg, 1.09 mmol)
and potassium carbonate (301 mg, 2.18 mmol) in
N,N-dimethylformamide (5 ml) was stirred at 50.degree. C.
overnight. TLC showed the reaction was complete. The reaction
mixture was partitioned between ethyl acetate (30 ml) and water (30
ml), the organic layer was washed with brine (30 ml.times.2) and
dried over anhydrous sodium sulfate and concentrated under reduced
pressure to give a crude residue which was purified by silica gel
flash column chromatography (eluted with 40-50% ethyl acetate in
hexane) to afford
2-(2,4-difluorophenyl)-5-((5-hydroxypentyl)oxy)isoindoline-1,3-dione
(217 mg, yield 55%) as white solid.
[0761] LC_MS: (ES.sup.+): m/z 362.1 [M+H].sup.+. t.sub.R=2.658
min.
[0762] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.57-1.69 (m,
4H), 1.88-1.91 (m, 2H), 3.70 (t, J=6.2 Hz, 2H), 4.12 (t, J=6.4 Hz,
2H), 6.99-7.05 (m, 2H), 7.22-7.24 (m, 1H), 7.31-7.36 (m, 1H),
7.40-7.41 (m, 1H), 7.85 (d, J=8.4 Hz, 1H).
[0763] Chemical Formula: C.sub.19H.sub.17F.sub.2NO.sub.4; Molecular
Weight: 361.34;
[0764] Total H count from HNMR data: 16.
Step 3: Synthesis of
5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl
4-methylbenzenesulfonate
##STR00348##
[0766] To a solution of
2-(2,4-difluorophenyl)-5-((5-hydroxypentyl)oxy)isoindoline-1,3-dione
(217 mg, 0.60 mmol), triethylamine (122 mg, 1.20 mmol) and
N,N-dimethylpyridin-4-amine (7.3 mg, 0.06 mmol) in dichloromethane
(20 ml) was added 4-toluenesulfonyl chloride (171 mg, 0.90 mmol) at
0.degree. C., the reaction mixture was allowed to warm up to room
temperature and stirred overnight. TLC showed the reaction was
complete. The reaction mixture was diluted with dichloromethane (30
ml), washed with water (50 ml) then brine (50 ml), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure
to give a crude residue which was purified by silica gel flash
chromatography (eluted with 30-50% ethyl acetate in hexane) to
afford
5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl
4-methylbenzenesulfonate (208 mg, yield 67%) as white solid.
[0767] LC_MS: (ES.sup.+): m/z 516.2 [M+H].sup.+. t.sub.R=3.183
min.
[0768] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 1.53-1.58 (m,
2H), 1.74-1.85 (m, 4H), 2.45 (s, 3H), 4.05-4.09 (m, 4H), 7.00-7.04
(m, 2H), 7.20-7.22 (m, 1H), 7.31-7.38 (m, 4H), 7.79-7.86 (m,
3H).
[0769] Chemical Formula: C.sub.26H.sub.23F.sub.2NO.sub.6S;
Molecular Weight: 515.53;
[0770] Total H count from HNMR data: 23.
Step 4: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl)piperazi-
n-1-yl)nicotinamide
##STR00349##
[0772] To a stirred solution of
5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl
4-methylbenzenesulfonate (110 mg, 0.21 mmol),
N-ethyl-N-isopropylpropan-2-amine (55 mg, 0.43 mmol) and potassium
iodide (3 mg, 0.02 mmol) in N,N-dimethylformamide (2 ml) was added
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide (100 mg, 0.21 mmol, intermediate in
synthesis of exemplary PROTAC 29), and the mixture was stirred at
50.degree. C. overnight under nitrogen. TLC showed the reaction was
complete. The reaction mixture was partitioned between ethyl
acetate (50 ml) and water (30 ml), the organic layer collected and
washed with brine (20 ml.times.2), dried over anhydrous sodium
sulfate and concentrated under reduced pressure to give a crude
residue which was purified by silica gel flash column
chromatography (eluted with 2-5% methanol in dichloromethane) to
afford
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(2,4-difluorophenyl)-1,3-dioxoisoindolin-5-yl)oxy)pentyl)piperazi-
n-1-yl)nicotinamide (98.4 mg, yield 57%) as white solid.
[0773] LC_MS: (ES.sup.+): m/z 811.3 [M+H].sup.+. t.sub.R=2.630
min.
[0774] .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.12 (s, 6H),
1.22 (s, 6H), 1.48-1.61 (m, 4H), 1.80-1.83 (m, 2H), 2.35-2.44 (m,
6H), 3.59 (br, 4H), 4.06 (d, J=9.2 Hz, 1H), 4.22 (t, J=6.4 Hz, 2H),
4.31 (s, 1H), 6.88-6.90 (m, 1H), 6.99-7.02 (m, 1H), 7.20-7.21 (m,
1H), 7.28-7.32 (m, 1H), 7.40-7.42 (m, 1H), 7.52-7.55 (m, 2H),
7.63-7.65 (m, 2H), 7.89-7.93 (m, 2H), 7.97-7.99 (m, 1H), 8.64 (br,
1H).
[0775] Chemical Formula: C.sub.44H.sub.45ClF.sub.2N.sub.6O.sub.5;
Molecular Weight: 811.32;
[0776] Total H count from HNMR data: 45.
[0777] Synthesis of Exemplary PROTAC 42
##STR00350## [0778]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((2-(6-cyano-2-oxo-1,2-dihydropyridin-3-yl)-1,3-dioxoisoindolin-5-yl)-
oxy)pentyl)piperazin-1-yl)nicotinamide
[0779] Synthetic Scheme
##STR00351##
Step 1: Synthesis of
5-(5-hydroxy-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitrile
##STR00352##
[0781] The mixture of 5-amino-6-methoxypicolinonitrile (600 mg,
4.02 mmol) and 5-hydroxyisobenzofuran-1,3-dione (660 mg, 4.02 mmol)
in acetic acid glacial (4 mL) was stirred at 100.degree. C.
overnight and then cooled down to room temperature. Water (40 mL)
was added. The mixture was neutralized with saturated sodium
bicarbonate until pH>7. The mixture was extracted with ethyl
acetate (20 mL.times.3). The combined organic layer was washed with
brine (10 mL.times.3), dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was washed with
ether to give
5-(5-hydroxy-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitrile
(650 mg, 55%) as a yellow solid.
[0782] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(30 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5% [water+10 mM
NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min, then under this
condition for 1.5 min, finally changed to 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and under this
condition for 0.5 min). Purity is 69.2%, Rt=0.852 min; MS Calcd.:
295.1; MS Found: 296.0 [M+H].sup.+.
Step 2: Synthesis of
5-(5-(5-chloropentyloxy)-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitri-
le
##STR00353##
[0784] The mixture of
5-(5-hydroxy-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitrile
(200 mg, 0.68 mmol), potassium carbonate (188 mg, 1.36 mmol) and
5-chloropentyl 4-methylbenzenesulfonate (187 mg, 0.68 mmol) in
dimethyl sulfoxide (5 mL) was stirred at 40.degree. C. for 2 hour.
The resulting mixture was allowed to cool down to room temperature.
Water (20 mL) and ethyl acetate (20 mL) was added. The organic
layer was separated, washed with brine (10 mL.times.2), dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo to
give the crude product which was purified by prep-TLC (ethyl
acetate/petroleum ether=1:1) to give
5-(5-(5-chloropentyloxy)-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitri-
le (100 mg, 37%) as a yellow solid
Step 3: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(2-(6-cyano-2-methoxypyridin-3-yl)-1,3-dioxoisoindolin-5-yloxy)pentyl-
)piperazin-1-yl)nicotinamide
##STR00354##
[0786] The mixture of methyl
5-(5-(5-chloropentyloxy)-1,3-dioxoisoindolin-2-yl)-6-methoxypicolinonitri-
le (100 mg, 025 mmol), ethyldiisopropylamine (96.8 mg, 0.75 mmol),
potassium iodide (41.5 mg, 0.25 mmol) and
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide (117 mg, 0.25 mmol) in dimethyl
sulfoxide (3 mL) was stirred at 70.degree. C. overnight. The
resulting mixture was allowed to cool down to room temperature.
Water (20 mL) and ethyl acetate (20 mL) was added. The organic
layer was separated, washed with brine (50 mL.times.2), dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo to
give the crude product which was purified by prep-TLC (ethyl
acetate) to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(2-(6-cyano-2-methoxypyridin-3-yl)-1,3-dioxoisoindolin-5-yloxy)pentyl-
)piperazin-1-yl)nicotinamide (53 mg, 34%) as a yellow solid.
Step 4: Synthesis of
5-(5-(5-(4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyc-
lobutylcarbamoyl)pyridin-2-yl)piperazin-1-yl)pentyloxy)-1,3-dioxoisoindoli-
n-2-yl)-6-hydroxypicolinamide
##STR00355##
[0788] The mixture of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(2-(6-cyano-2-methoxypyridin-3-yl)-1,3-dioxoisoindolin-5-yloxy)pentyl-
)piperazin-1-yl)nicotinamide (70 mg, 0.084 mmol) in Hydrogen
bromide/acetic acid glacial (w/w 48%, 0.5 mL) was stirred at
45.degree. C. for 5 hours. The resulting mixture was allowed to
cool down to room temperature. Water (20 mL) was added. The mixture
was neutralized with saturated sodium bicarbonate until pH>7 and
extracted with ethyl acetate (10 mL.times.2). The combined organic
layers were washed with brine (10 mL.times.2), dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo to give
5-(5-(5-(4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyc-
lobutylcarbamoyl)pyridin-2-yl)piperazin-1-yl)pentyloxy)-1,3-dioxoisoindoli-
n-2-yl)-6-hydroxypicolinamide (50 mg, 71%) as a white solid.
Step 5: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(2-(6-cyano-2-hydroxypyridin-3-yl)-1,3-dioxoisoindolin-5-yloxy)pentyl-
)piperazin-1-yl)nicotinamide
##STR00356##
[0790] To a solution of
5-(5-(5-(4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyc-
lobutylcarbamoyl)pyridin-2-yl)piperazin-1-yl)pentyloxy)-1,3-dioxoisoindoli-
n-2-yl)-6-hydroxypicolinamide (45 mg, 0.053 mmol) and triethylamine
(21.2 mg, 0.21 mmol) in dichloromethane (4 mL) was added
trifluoroacetic anhydride (44.1 mg, 0.21 mmol). The mixture was
stirred for 2 hour. The mixture was poured into ice-water (40 mL).
Dichloromethane (40 mL) was added. The organic layer was separated,
washed with brine (10 mL.times.2), dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The residue was
dissolved in tetrahydrofuran (5 mL) and water (5 mL) and stirred
overnight. Ethyl acetate (10 mL) was added. The organic layer was
separated, washed with brine (10 mL.times.2), dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo to give crude
product which was purified by prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(2-(6-cyano-2-hydroxypyridin-3-yl)-1,3-dioxoisoindolin-5-yloxy)pentyl-
)piperazin-1-yl)nicotinamide (6.8 mg, 16%) as a white solid
[0791] LC-MS (Agilent LCMS 1200-6110, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+0.05% TFA] and 5%
[CH.sub.3CN+0.05% TFA] to 0% [water+0.05% TFA] and 100%
[CH.sub.3CN+0.05% TFA] in 1.6 min, then under this condition for
1.4 min, finally changed to 95% [water+0.05% TFA] and 5%
[CH.sub.3CN+0.05% TFA] in 0.05 min and under this condition for 0.7
min). Purity is 99.5%, Rt=1.842 min; MS Calcd.: 816.3; MS Found: no
mass responded.
[0792] HPLC (Agilent HPLC 1200; Column: L-column2 ODS (150 mm*4.6
mm*5.0 .mu.m); Column Temperature: 40.degree. C.; Flow Rate: 1.0
mL/min; Mobile Phase: from 95% [water+0.1% TFA] and 5%
[CH.sub.3CN+0.1% TFA] to 0% [water+0.1% TFA] and 100%
[CH.sub.3CN+0.1% TFA] in 10 min, then under this condition for 5
min, finally changed to 95% [water+0.1% TFA] and 5%
[CH.sub.3CN+0.1% TFA] in 0.1 min and under this condition for 5
min). Purity is 91.3%, Rt=8.215 min.
[0793] .sup.1H NMR (400 MHz, DMSO-d.sup.6) .delta. 1.12 (6H, s),
1.22 (6H, s), 1.42-1.60 (4H, m), 1.77-1.82 (2H, m), 2.36-2.44 (2H,
m), 3.30-3.35 (4H, m), 3.58-3.66 (4H, m), 4.06 (1H, d, J=9.2 Hz),
4.21 (1H, t, J=6.2 Hz), 4.30 (1H, s), 6.88 (1H, d, J=8.8 Hz),
6.99-7.02 (1H, m), 7.21 (1H, d, J=2.4 Hz), 7.38-7.41 (1H, m),
7.48-7.52 (2H, m), 7.64 (1H, d, J=9.2 Hz), 7.89-7.98 (4H, m), 8.63
(1H, d, J=2.0 Hz).
[0794] Chemical Formula: C.sub.44H.sub.45ClN.sub.8O.sub.6;
Molecular Weight: 817.33
[0795] Total H count from HNMR data: 45
[0796] Synthesis of Exemplary PROTAC 43
##STR00357## [0797]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(1,3-dioxo-2-(6-oxo-1,6-dihydropyridin-3-yl)isoindolin-5-yl)piperazi-
n-1-yl)methyl)piperidin-1-yl)benzamide
[0798] Synthetic Scheme
##STR00358## ##STR00359##
Step 1: Synthesis of 4-[4-(hydroxymethyl)-1-piperidyl]benzoic
acid
##STR00360##
[0800] To a solution of ethyl
4-[4-(hydroxymethyl)-1-piperidyl]benzoate (52 g, 197.47 mmol, 1 eq)
in tetrahydrofuran (250 mL), methanol (250 mL) and water (250 mL)
was added sodium hydroxide (31.6 g, 0.79 mmol, 4 eq). The mixture
was stirred at 30.degree. C. for 12 hours. Thin layer
chromatography (petroleum ether:ethyl acetate=1:1) showed the
reaction was completed. The mixture was adjusted to pH 3.about.4
with hydrochloric acid (2 M) and filtered. The filter cake was
dried in vacuum. The residue was triturated with ethyl acetate (500
mL) to give 4-[4-(hydroxymethyl)-1-piperidyl]benzoic acid (35 g,
148.76 mmol, 75% yield) as a white solid.
[0801] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta.: 12.19 (s, 1H),
7.74 (d, J=8.8 Hz, 2H), 6.93 (d, J=8.8 Hz, 2H), 4.48 (br t, J=5.2
Hz, 1H), 3.90 (d, J=12.8 Hz, 2H), 3.27 (br t, J=5.2 Hz, 2H),
2.86-2.72 (m, 2H), 1.72 (d, J=12.8 Hz, 2H), 1.66-1.51 (m, 1H), 1.17
(dq, J=4.0, 12.0 Hz, 2H)
[0802] Chemical Formula: C.sub.13H.sub.17NO.sub.3, Molecular
Weight: 235.28
[0803] Total H count from HNMR data: 17.
Step 2: Synthesis of
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-[4-(hyd-
roxymethyl)-1-piperidyl]benzamide
##STR00361##
[0805] To a solution of 4-[4-(hydroxymethyl)-1-piperidyl]benzoic
acid (38 g, 161.51 mmol, 1 eq) and
4-(3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chloro-benzonitrile
(50.9 g, 161.51 mmol, 1 eq, hydrochloride) in dimethylformamide
(800 mL) was added diisopropylethylamine (83.5 g, 646.04 mmol, 112
mL, 4 eq). The mixture was stirred at 30.degree. C. for 10 min, and
then o-(7-azabenzotriazol-1-yl)-n,n,n',n'-tetramethyluronium
hexafluorophosphate (64.48 g, 169.59 mmol, 1.05 eq) was added. The
mixture was stirred at 30.degree. C. for 1 hour. LCMS showed the
reaction was completed and desired MS can be detected. The mixture
was poured into water (4 L) and filtered. The filter cake was
concentrated and triturated with methanol (500 mL.times.2) to give
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-[4-(hyd-
roxymethyl)-1-piperidyl]benzamide (72 g, 137.89 mmol, 85% yield,
95% purity) as a white solid.
[0806] LCMS: MS (ESI) m/z: 496.1 [M+1].sup.+
[0807] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta.: 7.90 (d, J=8.8
Hz, 1H), 7.73 (d, J=8.8 Hz, 2H), 7.48 (d, J=9.2 Hz, 1H), 7.20 (d,
J=2.4 Hz, 1H), 7.00 (dd, J=2.4, 8.8 Hz, 1H), 6.95 (d, J=8.8 Hz,
2H), 4.48 (t, J=5.2 Hz, 1H), 4.31 (s, 1H), 4.05 (d, J=9.2 Hz, 1H),
3.86 (d, J=12.8 Hz, 2H), 3.27 (t, J=5.6 Hz, 2H), 2.80-2.70 (m, 2H),
1.73 (d, J=11.2 Hz, 2H), 1.63-1.52 (m, 1H), 1.27-1.15 (m, 8H), 1.12
(s, 6H)
[0808] Chemical Formula: C.sub.28H.sub.34ClN.sub.3O.sub.3,
Molecular Weight: 496.04
[0809] Total H count from HNMR data: 34.
Step 3: Synthesis of
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-(4-form-
yl-1-piperidyl)benzamide
##STR00362##
[0811] To a solution of
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-[4-(hyd-
roxymethyl)-1-piperidyl]benzamide (65 g, 131.04 mmol, 1 eq) in
dichloromethane (700 mL) was added Dess-Martin reagent (76.70 g,
180.83 mmol, 1.38 eq). The mixture was stirred at 30.degree. C. for
2 hours. Thin layer chromatography (dichloromethane:methanol=1:1)
showed the reaction was completed. The reaction was adjusted to pH
8.about.9 with saturated sodium bicarbonate. The mixture was
diluted with water (3 L) and extracted with dichloromethane (1.5
L.times.3). The combined organic phase was washed with saturated
brine (1.5 L.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum. The residue was purified by
silica gel chromatography (dichloromethane:methanol=100:0 to 50:1)
to give
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-(4-form-
yl-1-piperidyl)benzamide (34.6 g, 67.94 mmol, 51% yield, 97%
purity) as a white solid.
[0812] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta.: 9.63 (s, 1H),
7.90 (d, J=8.8 Hz, 1H), 7.74 (d, J=8.8 Hz, 2H), 7.49 (d, J=9.2 Hz,
1H), 7.20 (d, J=2.4 Hz, 1H), 7.03-6.94 (m, 3H), 4.32 (s, 1H), 4.05
(d, J=9.2 Hz, 1H), 3.76 (td, J=3.6, 12.8 Hz, 2H), 3.01-2.92 (m,
2H), 2.62-2.55 (m, 1H), 2.62-2.55 (m, 1H), 1.92 (dd, J=3.6, 12.8
Hz, 2H), 1.62-1.48 (m, 2H), 1.21 (s, 6H), 1.12 (s, 6H)
[0813] Chemical Formula: C.sub.28H.sub.32ClN.sub.3O.sub.3,
Molecular Weight: 494.02
[0814] Total H count from HNMR data: 32.
Step 4: Synthesis of
5-fluoro-2-(6-methoxypyridin-3-yl)isoindoline-1,3-dione
##STR00363##
[0816] A mixture of 5-fluoro-1,3-dihydro-2-benzofuran-1,3-dione
(100.0 mg, 602 .mu.mol), 6-methoxypyridin-3-amine (82.1 mg, 662
.mu.mol), sodium acetate (59.2 mg, 722 .mu.mol), and acetic acid
(499 .mu.L, 8.74 mmol) was heated at 118.degree. C. with stirring
for 2 hours. The reaction was monitored by LCMS (CF-820-1), which
showed a major peak with a mass consistent with the desired
product. The reaction was cooled to 90.degree. C. and quenched with
water (2 mL). The mixture was allowed to cool to room temperature.
The resulting precipitate was filtered and washed with water. The
material was dried to give the desired product as a light purple
solid,
5-fluoro-2-(6-methoxypyridin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione
(149.1 mg, 547 .mu.mol, 91.4% Yield).
[0817] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. 8.26 (dd,
J=0.49, 2.64 Hz, 1H), 7.98 (dd, J=4.50, 8.22 Hz, 1H), 7.65 (d,
J=2.54 Hz, 1H), 7.62-7.64 (m, 1H), 7.48 (dt, J=2.35, 8.51 Hz, 1H),
6.89 (dd, J=0.78, 8.80 Hz, 1H), 4.00 (s, 3H)
[0818] LCMS m/e+=273.16 [M+H].sup.+
Step 5: Synthesis of tert-butyl
4-(2-(6-methoxypyridin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carbox-
ylate
##STR00364##
[0820] A solution of tert-butyl piperazine-1-carboxylate (34.0 mg,
183 .mu.mol) and
5-fluoro-2-(6-methoxypyridin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione
(50.0 mg, 183 .mu.mol) in methylpyrrolidone (1.0 mL) was charged
with N,N-diisopropylethylamine (95.5 .mu.L, 549 .mu.mol). The
reaction mixture was heated at 120.degree. C. for 2 hours. The
reaction was monitored by LCMS, which showed a major peak with a
mass consistent with the desired product and small peak with a mass
consistent with the starting material. The reaction was allowed to
stir at 120.degree. C. for an additional 16 hours. LCMS showed a
major peak with a mass consistent with the desired product. The
reaction mixture was quenched with water (2 mL) and extracted with
EtOAc (2 mL). The organic layer was washed with brine (1 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
crude material was purified by silica gel chromatography on a
Teledyne Combiflash ISCO eluting with DCM/MeOH (gradient 100:0 to
95:5). The fraction containing product was concentrated under
reduced pressure to yield the desired product as a white solid,
tert-butyl
4-[2-(6-methoxypyridin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piper-
azine-1-carboxylate (39.6 mg, 90.3 .mu.mol, 49.3% Yield).
[0821] LCMS m/e+=439.33 [M+H].sup.+
[0822] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. 8.25 (d, J=2.15
Hz, 1H), 7.79 (d, J=8.61 Hz, 1H), 7.64 (dd, J=2.74, 8.80 Hz, 1H),
7.35 (d, J=2.35 Hz, 1H), 7.11 (dd, J=2.45, 8.51 Hz, 1H), 6.87 (dd,
J=0.59, 8.80 Hz, 1H), 3.98 (s, 3H), 3.60-3.66 (m, 4H), 3.42-3.48
(m, 4H), 1.50 (s, 9H)
Step 6: Synthesis of
2-(6-oxo-1,6-dihydropyridin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione
##STR00365##
[0824] A solution of tert-butyl
4-[2-(6-methoxypyridin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]piper-
azine-1-carboxylate (39.6 mg, 90.3 .mu.mol) in 4.0 M Hydrochloric
acid in 1,4-dioxane (1.0 mL, 4.00 mmol) was stirred at 100.degree.
C. for 16 hours. The reaction mixture was
[0825] concentrated under reduced pressure to yield a white solid,
2-(6-oxo-1,6-dihydropyridin-3-yl)-5-(piperazin-1-yl)-2,3-dihydro-1H-isoin-
dole-1,3-dione hydrochloride (32.5 mg, 90.0 .mu.mol, 100% Yield).
The material was used in the next reaction without any further
purification.
[0826] LCMS m/e+=425.22 [M+H].sup.+
[0827] Step 7: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(1,3-dioxo-2-(6-oxo-1,6-dihydropyridin-3-yl)isoindolin-5-yl)piperazi-
n-1-yl)methyl)piperidin-1-yl)benzamide
##STR00366##
[0828] A solution of
4-(4-formylpiperidin-1-yl)-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-
-tetramethylcyclobutyl]benzamide (44.4 mg, 90.0 .mu.mol) and
2-(6-oxo-1,6-dihydropyridin-3-yl)-5-(piperazin-1-yl)-2,3-dihydro-1H-isoin-
dole-1,3-dione hydrochloride (32.5 mg, 90.0 .mu.mol) in ethylene
dichloride (1.0 mL) was charged with triethylamine (37.4 .mu.L, 269
.mu.mol) and sodium triacetoxyborohydride (57.0 mg, 269 .mu.mol).
The reaction mixture was allowed to stir at room temperature for 5
hours. The reaction mixture was monitored by LCMS, which showed a
peak with a mass consistent with the desired product and peaks with
masses consistent with the starting materials. The reaction mixture
was allowed to stir at room temperature for an additional 16 hours.
LMCS showed a major peak with a mass consistent with the desired
product. The reaction mixture was quenched with aq. NaHCO3 (1 mL)
and extracted with DCM (1 mL). The organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
crude material was purified by silica gel chromatography on a
Teledyne Combiflash ISCO eluting with DCM/MeOH (gradient 100:0 to
90:10). The fractions containing product were combined and
concentrated under reduced pressure to yield the desired product
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(1,3-dioxo-2-(6-oxo-1,6-dihydropyridin-3-yl)isoindolin-5-yl)piperazi-
n-1-yl)methyl)piperidin-1-yl)benzamide as a yellow solid.-mg, (30
mg, 37.3 .mu.mol, 41.5% Yield).
[0829] .sup.1H NMR (400 MHz, DMSO-d6): .delta. 7.91 (d, J=8.80 Hz,
1H), 7.72 (t, J=8.41 Hz, 3H), 7.56 (d, J=2.54 Hz, 1H), 7.44-7.53
(m, 2H), 7.38 (d, J=1.96 Hz, 1H), 7.28 (dd, J=2.05, 8.71 Hz, 1H),
7.21 (d, J=2.35 Hz, 1H), 7.00 (dd, J=2.35, 8.80 Hz, 1H), 6.96 (d,
J=9.00 Hz, 2H), 6.41 (d, J=9.78 Hz, 1H), 4.32 (s, 1H), 4.05 (d,
J=9.00 Hz, 1H), 3.86 (d, J=12.52 Hz, 2H), 3.45 (br. s., 4H), 2.79
(t, J=11.74 Hz, 2H), 2.21 (d, J=6.46 Hz, 2H), 1.81 (d, J=11.15 Hz,
3H), 1.21 (s, 6H), 1.12 (s, 6H)
[0830] LCMS m/e+=802.57 [M+
[0831] Synthesis of Exemplary PROTAC 46
##STR00367## [0832]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-yl)me-
thoxy)ethoxy)ethyl)piperazin-1-yl)nicotinamide
[0833] Synthetic Scheme:
##STR00368## ##STR00369##
Step 1: Synthesis of
N-(2,6-dioxopiperidin-3-yl)-2-iodobenzamide
[0834] Into a 100-mL round-bottom flask, was placed 2-iodobenzoic
acid (5.0 g, 20.16 mmol, 1.00 equiv), N,N-dimethylformamide (40
mL), HATU (7.66 g, 20.15 mmol, 1.00 equiv), DIEA (7.80 g, 60.35
mmol, 3.00 equiv), after stirred 10 minutes,
3-aminopiperidine-2,6-dione (3.30 g, 25.76 mmol, 1.00 equiv) was
added. The resulting solution was stirred for 2 hours at room
temperature. The reaction was then quenched by the addition of 500
mL of water/ice. The solids were collected by filtration. The
resulting mixture was concentrated under vacuum. This resulted in
6.48 g (90%) of N-(2,6-dioxopiperidin-3-yl)-2-iodobenzamide as a
off-white solid.
[0835] LC-MS (ES.sup.+): m/z 358.85 [MH.sup.+], t.sub.R=0.56 min,
(1.90 minute run).
Step 2: Synthesis of
([2-[2-(prop-2-yn-1-yloxy)ethoxy]ethoxy]methyl)benzene
[0836] Into a 250-mL 3-necked round-bottom flask purged and
maintained with an inert atmosphere of nitrogen, was placed
2-[2-(benzyloxy)ethoxy]ethan-1-ol (10.0 g, 50.96 mmol, 1.00 equiv),
N,N-dimethylformamide (100 mL). This was followed by the addition
of sodium hydride (2.4 g, 100.00 mmol, 1.20 equiv) in several
batches at 0.degree. C., after stirred 30 minutes. To this was
added a solution of 3-bromoprop-1-yne (7.285 g, 61.24 mmol, 1.20
equiv) in N,N-dimethylformamide (30 mL) dropwise with stirring at
0.degree. C. The resulting solution was stirred overnight at room
temperature. The reaction was then quenched by the addition of 300
mL of water/ice. The resulting solution was extracted with ethyl
acetate (300 mL) and the organic layers combined. The resulting
mixture was washed with brine (300 mL). The mixture was dried over
anhydrous sodium sulfate. The residue was applied onto a silica gel
column with ethyl acetate/petroleum ether (1/4). This resulted in
9.5 g (80%) of
([2-[2-(prop-2-yn-1-yloxy)ethoxy]ethoxy]methyl)benzene as light
yellow oil.
[0837] LC-MS (ES.sup.+): m/z 234.95 [MH.sup.+], t.sub.R=1.15 min,
(2.00 minute run).
Step 3: Synthesis of
2-(3-(2-(2-(benzyloxy)ethoxy)ethoxy)prop-1-ynyl)-N-(2,6-dioxopiperidin-3--
yl)benzamide
[0838] Into a 25-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed
N-(2,6-dioxopiperidin-3-yl)-2-iodobenzamide (1.5 g, 4.1 mmol, 1.00
equiv), N,N-dimethylformamide (20 mL), (PPh.sub.3).sub.2PdCl.sub.2
(293 mg, 0.41 mmol, 0.1 equiv), CuI (79 mg, 0.41 mmol, 0.1 equiv),
triethylamine (1.69 g, 16 mmol, 4.00 equiv),
(2-[2-(prop-2-yn-1-yloxy)ethoxy]ethoxymethyl)benzene (1.17 g, 5.0
mmol, 1.20 equiv). The resulting solution was stirred overnight at
room temperature. The resulting solution was extracted with ethyl
acetate (300 mL) and the organic layers combined. The resulting
mixture was washed with brine (300 mL). The mixture was dried over
anhydrous sodium sulfate. The residue was applied onto a silica gel
column with ethyl acetate/petroleum ether (7/3). This resulted in
1.74 g of
2-(3-(2-(2-(benzyloxy)ethoxy)ethoxy)prop-1-ynyl)-N-(2,6-dioxopiperidin-3--
yl)benzamide as light yellow oil.
[0839] LC-MS (ES.sup.+): m/z 465.10 [MH.sup.+], t.sub.R=0.79 min,
(1.90 minute run).
Step 4: Synthesis of
3-[3-([2-[2-(benzyloxy)ethoxy]ethoxy]methyl)-1-oxo-1,2-dihydroisoquinolin-
-2-yl]piperidine-2,6-dione
[0840] Into a 25-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed a solution of
2-(3-[2-[2-(benzyloxy)ethoxy]ethoxy]prop-1-yn-1-yl)-N-(2,6-dioxopiperidin-
-3-yl)benzamide (1.0 g, 2.15 mmol, 1.00 equiv) in
N,N-dimethylformamide (10 mL), Pd(OAc).sub.2 (24.0 mg, 0.11 mmol,
0.05 equiv), LiCl (90.0 mg, 2.14 mmol, 1.00 equiv), potassium
carbonate (594.0 mg, 4.30 mmol, 2.00 equiv). The resulting solution
was stirred overnight at 100.degree. C. in an oil bath. The solids
were filtered out. The residue was applied onto a silica gel column
with ethyl acetate/petroleum ether (7/3). This resulted in 465.0 mg
(47%) of
3-[3-[2-[2-(benzyloxy)ethoxy]ethoxy]methyl)-1-oxo-1,2-dihydroisoquinolin--
2-yl]piperidine-2,6-dione as light yellow oil.
[0841] LC-MS (ES.sup.+): m/z 465.10 [MH.sup.+], t.sub.R=0.74 min,
(1.90 minute run).
Step 5: Synthesis of
3-(3-[[2-(2-hydroxyethoxy)ethoxy]methyl]-1-oxo-1,2-dihydroisoquinolin-2-y-
l)piperidine-2,6-dione
[0842] Into a 100-mL 3-necked round-bottom flask purged and
maintained with an inert atmosphere of nitrogen, was placed
3-[3-([2-[2-(benzyloxy)ethoxy]ethoxy]methyl)-1-oxo-1,2-dihydroisoquinolin-
-2-yl]piperidine-2,6-dione (420.0 mg, 0.90 mmol, 1.00 equiv),
dichloromethane (10 mL). This was followed by the addition of
BBr.sub.3 (IM in DCM) (3.61 mL, 4.00 equiv) dropwise with stirring
at -78.degree. C. The resulting solution was stirred for 1 hat
-78.degree. C. in a liquid nitrogen bath. The reaction was then
quenched by the addition of 20 mL of sodium bicarbonate at
-78.degree. C. The resulting solution was extracted with
dichloromethane (100 mL) and the organic layers combined and dried
over anhydrous sodium sulfate. The residue was applied onto a
silica gel column with dichloromethane/methanol (10/1). This
resulted in 212.0 mg (63%) of
3-(3-[[2-(2-hydroxyethoxy)ethoxy]methyl]-1-oxo-1,2-dihydroisoquinolin-2-y-
l)piperidine-2,6-dione as light yellow oil.
[0843] LC-MS (ES.sup.+): m/z 374.95 [MH.sup.+], t.sub.R=0.41 min,
(1.90 minute run).
Step 6: Synthesis of
2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-yl]meth-
oxy]ethoxy)ethyl 4-methylbenzene-1-sulfonate
[0844] Into a 50-mL round-bottom flask, was placed
3-(3-[[2-(2-hydroxyethoxy)ethoxy]methyl]-1-oxo-1,2-dihydroisoquinolin-2-y-
l)piperidine-2,6-dione (212.0 mg, 0.57 mmol, 1.00 equiv),
dichloromethane (10.0 mL), TsCl (215.4 mg, 1.13 mmol, 2.00 equiv),
triethylamine (171.0 mg, 1.69 mmol, 3.00 equiv),
4-dimethylaminopyridine (6.98 mg, 0.06 mmol, 0.10 equiv). The
resulting solution was stirred for 3 hours at room temperature. The
resulting solution was extracted with dichloromethane (100 mL) and
the organic layers combined. The resulting mixture was washed with
brine (100 mL). The mixture was dried over anhydrous sodium
sulfate. The residue was applied onto a silica gel column with
ethyl acetate/petroleum ether (4/1). This resulted in 238.0 mg
(80%) of
2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-yl]meth-
oxy]ethoxy)ethyl 4-methylbenzene-1-sulfonate as light yellow
oil.
[0845] LC-MS (ES.sup.+): m/z 529.10 [MH.sup.+], t.sub.R=0.76 min,
(1.90 minute run).
Step 7: Synthesis of
6-[4-[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-y-
l]methoxy]ethoxy)ethyl]piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanopheno-
xy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide
[0846] Into a 20-mL microwave tube purged and maintained with an
inert atmosphere of nitrogen, was placed
6-(piperazin-1-yl)-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetrame-
thylcyclobutyl]pyridine-3-carboxamide (65.0 mg, 0.14 mmol, 1.00
equiv), acetonitrile (5.0 mL), potassium carbonate (71.3 mg, 0.52
mmol, 4.00 equiv),
2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-
-yl]methoxy]ethoxy)ethyl 4-methylbenzene-1-sulfonate (68.0 mg, 0.13
mmol, 1.00 equiv), NaI (19.38 mg, 0.13 mmol, 1.00 equiv). The
resulting solution was stirred for 24 hours at 75.degree. C. in an
oil bath. The solids were filtered out. The resulting mixture was
concentrated under vacuum. Then purified by Prep-HPLC-Column:
XBridge Shield RP18 OBD Column, 5 um, 19*150 mm; Mobile Phase
A:water (10 mmol/L NH.sub.4HCO.sub.3), Mobile Phase B:
acetonitrile; Flow rate: 20 mL/min; Gradient: 61% B to 70% B in 8
min; 254 nm; Rt: 6.7 min This resulted in 50.0 mg (47%) of
6-[4-[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-3-y-
l]methoxy]ethoxy)ethyl]piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanopheno-
xy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide as a white
solid.
[0847] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.81 (s, 1H),
8.58-8.57 (d, J=2.4 Hz, 1H), 8.23-8.21 (d, J=7.6 Hz, 1H), 7.92-7.89
(m, 1H), 7.57-7.48 (m, 2H), 7.38-7.34 (m, 1H), 7.26-7.21 (m, 1H),
6.97-6.96 (d, J=2.0 Hz, 1H), 6.81-6.78 (m, 1H), 6.61-6.59 (d, J=9.2
Hz, 1H), 6.25 (s, 1H), 6.11-6.09 (d, J=8.0 Hz, 1H), 4.82-4.79 (m,
1H), 4.32-4.29 (m, 2H), 4.26-4.23 (m, 1H), 4.15-4.13 (m, 1H), 4.04
(s, 1H), 3.76-3.67 (m, 10H), 2.95-2.90 (m, 1H), 2.70-2.62 (m, 7H),
2.23-2.19 (m, 2H), 1.25 (s, 6H), 1.21 (s, 6H);
[0848] LC-MS (ES.sup.+): m/z 824.75/826.75 [MH.sup.+], t.sub.R=2.43
min, (4.80 minute run).
[0849] Chemical formula: C.sub.44H.sub.50ClN.sub.7O7
[823.35/825.35]
[0850] Total H count from HNMR data: 50
[0851] Synthesis of Exemplary PROTAC 47
##STR00370## [0852]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yl)oxy)pentyl-
)piperazin-1-yl)nicotinamide
[0853] Synthetic Scheme
##STR00371##
Step 1: Synthesis of methyl
5-(3-bromoquinolin-6-yloxy)pentan-1-ol
##STR00372##
[0855] A mixture of 3-bromoquinolin-6-ol (700 mg, 3.1 mmol),
5-bromopentan-1-ol (518 mg, 3.1 mmol) and potassium carbonate (856
mg, 6.2 mmol) in N,N-dimethylformamide (5 mL) was heated at
80.degree. C. for 6 hours. The reaction mixture was cooled to room
temperature. Water (10 mL) was added and extracted with ethyl
acetate (20 mL.times.3). The combined organic layers were washed
with water (20 mL.times.2) and brine (20 mL), dried over anhydrous
sodium sulfate. The solvent was concentrated to give the residue,
which was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate=1/1) to give
5-(3-bromoquinolin-6-yloxy)pentan-1-ol (750 mg, 78% yield) as a
yellow solid.
[0856] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 91.43%, Rt=1.767 min; MS Calcd.:
309.04; MS Found: 310.0 [M+H].sup.+.
Step 2: Synthesis of
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)pyrimidine-2,4(1H,3H)-dione
##STR00373##
[0858] A solution of 5-(3-bromoquinolin-6-yloxy)pentan-1-ol (496
mg, 1.6 mmol), pyrimidine-2,4(1H,3H)-dione (538 mg, 4.8 mmol),
potassium phosphate (1.0 g, 4.8 mmol), cuprous iodide (304 mg, 1.6
mmol), N-(2-cyanophenyl)picolinamide (357 mg, 1.6 mmol) in dimethyl
sulfoxide (10 mL) was heated at 120.degree. C. for 5 hours under
argon atmosphere. The reaction mixture was cooled to room
temperature. Water (10 mL) was added, extracted with ethyl acetate
(20 mL.times.2). Combined organic layers were washed with brine (10
mL.times.2), dried over anhydrous sodium sulfate. The solvent was
removed and the residue was purified by column chromatography on
silica gel (methanol/dichloromethane=1/20) to give
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)pyrimidine-2,4(1H,3H)-dione
(200 mg, 37% yield) as an off-white solid.
[0859] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.325 min; MS Calcd.: 341.14; MS Found:
342.2 [M+H].sup.+.
Step 3: Synthesis of
5-(3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pentanal
##STR00374##
[0861] A mixture of
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)pyrimidine-2,4(1H,3H)-dione
(150 mg, 0.4 mmol) and Dess-Martin periodinane (559 mg, 1.3 mmol)
in dichloromethane (15 mL) was stirred at room temperature
overnight. The reaction mixture was filtered and the filtered cake
was washed with dichloromethane (10 mL.times.2). The filtrate was
concentrated and the residue was purified by Prep-TLC
(dichloromethane/methanol=5/1) to give
5-(3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pentanal
(100 mg, 67% yield) as a yellow solid.
[0862] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.396 min; MS Calcd.: 339.12; MS Found:
340.2 [M+H].sup.+.
Step 4: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pentyl)p-
iperazin-1-yl)nicotinamide
##STR00375##
[0864] A mixture of
5-(3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pentanal
(100 mg, 0.29 mmol),
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride (149 mg, 0.29 mmol),
sodium cyanoborohydride (36 mg, 0.58 mmol) in methanol (5 mL) and
acetic acid glacial (0.5 mL) was stirred at room temperature
overnight. Water (10 mL) was added and extracted with
dichloromethane (20 mL.times.3). Combined organic layers were
washed with brine (10 mL.times.2), dried over anhydrous sodium
sulfate. The solvent was concentrated to give the residue, which
was purified by Prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pentyl)p-
iperazin-1-yl)nicotinamide (23 mg, 10% yield) as a white solid.
[0865] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 94.84%, Rt=2.864 min; MS Calcd.:
790.34; MS Found: 791.30 [M+H].sup.+.
[0866] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 95.31%, Rt=9.913 min.
[0867] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25
(6H, s), 1.58-1.66 (4H, m), 1.90-1.94 (2H, m), 2.43-2.47 (2H, m),
2.56-2.58 (4H, m), 3.67-3.70 (4H, m), 4.04 (1H, s), 4.09-4.15 (3H,
m), 5.93 (1H, d, J=8.0 Hz), 6.07 (1H, d, J=8.0 Hz), 6.66 (1H, d,
J=9.2 Hz), 6.80 (1H, dd, J=8.8, 2.4 Hz), 6.96 (1H, d, J=2.4 Hz),
7.09 (1H, d, J=2.8 Hz), 7.41-7.46 (2H, m), 7.57 (1H, d, J=8.8 Hz),
7.93 (1H, dd, J=9.2, 2.4 Hz), 8.05-8.07 (2H, m), 8.58 (1H, d, J=2.4
Hz), 8.73 (1H, d, J=2.4 Hz).
[0868] Chemical Formula: C.sub.43H.sub.47ClN.sub.8O.sub.5,
Molecular Weight: 791.34
[0869] Total H count from HNMR data: 46.
[0870] Synthesis of Exemplary PROTAC 48
##STR00376## [0871]
rac-N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-
-6-(4-(5-(4-((2,6-dioxopiperidin-3-yl)(ethyl)carbamoyl)phenoxy)pentyl)pipe-
razin-1-yl)nicotinamide
[0872] Synthetic Scheme
##STR00377##
Step 1: Synthesis of methyl 4-(5-hydroxypentyloxy)benzoate
##STR00378##
[0874] A mixture of methyl 4-hydroxybenzoate (3.0 g, 20 mmol),
5-bromopentan-1-ol (3.3 g, 20 mmol), potassium carbonate (5.5 g, 40
mmol) and potassium iodide (0.3 g, 2 mmol) in N,N-dimethylformamide
(20 mL) was heated at 110.degree. C. overnight. The reaction
mixture was cooled to room temperature. Water (50 mL) was added.
Extracted with ethyl acetate (50 mL.times.3) and combined organic
layers were washed with water (30 mL.times.2) and brine (30
mL.times.2), dried over anhydrous sodium sulfate. The solvent was
concentrated to give the residue, which was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate=10/1)
to give methyl 4-(5-hydroxypentyloxy)benzoate (2.2 g, 46% yield) as
a white solid.
[0875] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 98.48%, Rt=1.637 min; MS Calcd.:
238.1; MS Found: 239.2 [M+H].sup.+.
Step 2: Synthesis of 4-(5-hydroxypentyloxy)benzoic acid
##STR00379##
[0877] A mixture of methyl 4-(5-hydroxypentyloxy)benzoate (2.2 g,
9.2 mmol), lithium hydroxide (1.6 g, 36.9 mmol) in methanol (10 mL)
and water (1 mL) was stirred at room temperature overnight. The
solvent was removed in vacuum and water (5 mL) was added. It was
extracted with ethyl acetate and the water phase was adjust pH=5-6
with IN aqueous hydrochloride. Filtered and the solid was
collected, which was dried in vacuum to afford
4-(5-hydroxypentyloxy)benzoic acid (1.9 g, 90% yield) as a white
solid.
[0878] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.073 min; MS Calcd.: 224.1; MS Found:
225.3 [M+H].sup.+.
Step 3: Synthesis of 3-(ethylamino)piperidine-2,6-dione
##STR00380##
[0880] A mixture of 3-aminopiperidine-2,6-dione hydrochloride (3.8
g, 23 mmol), acetaldehyde (1.0 g, 23 mmol), sodium cyanoborohydride
(4.3 g, 69 mmol) in methanol (30 mL) and acetic acid glacial (0.5
mL) was stirred at room temperature overnight. Water (10 mL) was
added and extracted with dichloromethane (50 mL.times.3). Combined
organic layers were washed by brine (30 mL.times.2), dried over
anhydrous sodium sulfate. The solvent was concentrated to give the
residue, which was purified by column chromatography on silica gel
(dichloromethane/methanol=10/1) to give
3-(ethylamino)piperidine-2,6-dione (3.0 g, 33% yield) as a yellow
oil.
[0881] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=0.737 min; MS Calcd.: 156.1; MS Found:
157.2 [M+H].sup.+.
Step 4: Synthesis of
N-(2,6-dioxopiperidin-3-yl)-N-ethyl-4-(5-hydroxypentyloxy)benzamide
##STR00381##
[0883] A mixture of 3-(ethylamino)piperidine-2,6-dione (500 mg, 3.2
mmol), 4-(5-hydroxypentyloxy)benzoic acid (3.3 g, 20 mmol),
ethyldiisopropylamine (826 mg, 6.4 mmol) and
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (1.8 g, 4.8 mmol) in N,N-dimethylformamide (5
mL) was stirred at room temperature overnight. Water (10 mL) was
added. Extracted with ethyl acetate (20 mL.times.3) and combined
organic layers were washed with water (20 mL.times.2) and brine (20
mL.times.2), dried over anhydrous sodium sulfate. The solvent was
concentrated to give the residue, which was purified by column
chromatography on silica gel (dichloromethane/methanol=10/1) to
give N-(2,
6-dioxopiperidin-3-yl)-N-ethyl-4-(5-hydroxypentyloxy)benzamide (108
mg, 9% yield) as a white solid.
[0884] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.377 min; MS Calcd.: 362.2; MS Found:
363.2 [M+H].sup.+.
Step 5: Synthesis of
N-(2,6-dioxopiperidin-3-yl)-N-ethyl-4-(5-oxopentyloxy)
benzamide
##STR00382##
[0886] A mixture of
N-(2,6-dioxopiperidin-3-yl)-N-ethyl-4-(5-hydroxypentyloxy)benzamide
(108 mg, 0.3 mmol) and Dess-Martin periodinane (254 mg, 0.6 mmol)
in dichloromethane (10 mL) was stirred at room temperature for 2
hours. The reaction mixture was filtered and the cake was washed by
dichloromethane (10 mL.times.2). The filtrate was concentrated and
the residue was purified by prep-TLC (dichloromethane/methanol=5/1)
to give
N-(2,6-dioxopiperidin-3-yl)-N-ethyl-4-(5-oxopentyloxy)benzamide (97
mg, 90% yield) as a yellow solid.
[0887] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.465 min; MS Calcd.: 360.2; MS Found:
361.2 [M+H].sup.+.
Step 6: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(4-((2,6-dioxopiperidin-3-yl)(ethyl)carbamoyl)phenoxy)pentyl)piperazi-
n-1-yl)nicotinamide
##STR00383##
[0889] A mixture of
N-(2,6-dioxopiperidin-3-yl)-N-ethyl-4-(5-oxopentyloxy)benzamide (97
mg, 0.27 mmol),
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide hydrochloride (136 mg, 0.27 mmol),
sodium cyanoborohydride (34 mg, 0.54 mmol) in methanol (5 mL) and
acetic acid glacial (0.5 mL) was stirred at room temperature
overnight. Water (10 mL) was added and extracted with
dichloromethane (20 mL.times.3). Combined organic layers were
washed by brine (10 mL.times.2), dried over anhydrous sodium
sulfate. The solvent was concentrated to give the residue, which
was purified by prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(4-((2,6-dioxopiperidin-3-yl)(ethyl)carbamoyl)phenoxy)pentyl)piperazi-
n-1-yl)nicotinamide (55 mg, 25% yield) as an off-white solid.
[0890] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 98.20%, Rt=2.918 min; MS Calcd.:
811.38; MS Found: 812.30 [M+H].sup.+.
[0891] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 99.92%, Rt=10.259 min.
[0892] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.1.10-1.13 (9H,
m), 1.21 (6H, s), 1.44-1.53 (4H, m), 1.74-1.77 (2H, m), 1.99-2.08
(1H, m), 2.31-2.34 (3H, m), 2.42-2.45 (5H, m), 2.67-2.68 (1H, m),
3.29-3.34 (3H, m), 3.58-3.59 (4H, m), 4.00-4.07 (3H, m), 4.30 (1H,
s), 6.86 (1H, d, J=8.8 Hz), 6.98-7.02 (3H, m), 7.22 (1H, d, J=2.4
Hz), 7.31 (2H, d, J=8.0 Hz), 7.63 (1H, d, J=9.2 Hz), 7.91 (1H, d,
J=8.8 Hz), 7.95 (1H, dd, J=8.8, 2.4 Hz), 8.62 (1H, d, J=2.0 Hz),
10.78 (1H, s).
[0893] Chemical Formula: C.sub.44H.sub.54ClN.sub.7O.sub.6,
Molecular Weight: 812.40
[0894] Total H count from HNMR data: 54.
[0895] Synthesis of Exemplary PROTAC 50
##STR00384## [0896]
5-(3-(4-(5-(((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclo-
butyl)carbamoyl)pyridin-2-yl)piperazin-1-yl)propoxy)-N-(2,6-dioxopiperidin-
-3-yl)picolinamide
[0897] Synthetic Scheme
##STR00385## ##STR00386##
Step 1: Synthesis of methyl 5-(3-hydroxypropoxy)picolinate
##STR00387##
[0899] To a solution of methyl 5-hydroxypicolinate (5.0 g, 32.6
mmol) in N,N-dimethylformamide (60.0 mL) was added
3-bromopropan-1-ol (5.45 g, 39.2 mmol), potassium carbonate (9.03
g, 65.3 mmol). The reaction mixture was stirred at 70.degree. C.
overnight. The solvent was removed in vacuo. The residue was
purified by silica gel chromatography
(dichloromethane/methanol=20:1) to give methyl
5-(3-hydroxypropoxy)picolinate (2.5 g, 36%) as a pale yellow
solid.
[0900] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.90 (2H, t,
J=6.0 Hz), 3.57 (2H, q, J=5.9 Hz), 3.84 (3H, s), 4.20 (2H, t, J=6.4
Hz), 4.62 (1H, t, J=5.2 Hz), 7.52 (1H, dd, J=8.8 Hz, 2.8 Hz), 8.04
(1H, d, J=8.8 Hz), 8.37 (1H, d, J=2.8 Hz).
[0901] Chemical Formula: C.sub.10H.sub.13NO.sub.4, Molecular
Weight: 211.21
[0902] Total H count from HNMR data: 13.
Step 2: Synthesis of 5-(3-hydroxypropoxy)picolinic acid
##STR00388##
[0904] To a solution of methyl 5-(3-hydroxypropoxy)picolinate (2.5
g, 11.8 mmol) in methanol (50 mL) was added lithium hydroxide (1.49
g, 35.5 mmol). The mixture was stirred at room temperature for 3
hours. The solvent was removed and added aq. hydrochloric acid (0.5
M) adjust to PH=2.about.3. The water was removed in vacuo and the
residue was washed with dichloromethane/methanol (10:1), filtered
and concentrated in vacuo to give crude
5-(3-hydroxypropoxy)picolinic acid as a pale yellow solid, which
was used for the next step without further purification.
Step 3: Synthesis of
N-(2,6-dioxopiperidin-3-yl)-5-(3-hydroxypropoxy)picolinamide
##STR00389##
[0906] 5-(3-hydroxypropoxy)picolinic acid (crude, 11.8 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(3.39 g, 17.7 mmol), 1-hydroxybenzotriazole hydrate (HOBt) (2.40 g,
17.7 mmol) and ethyldiisopropylamine (4.58 g, 35.4 mmol) in N,
N-dimethylformamide (DMF) (30 mL) was stirred for 30 minutes, and
then 3-aminopiperidine-2,6-dione (2.14 g, 13.0 mmol) was added. The
mixture was stirred at room temperature overnight and water (100
mL) was added. The aqueous layer was extracted by ethyl acetate
(100 mL.times.3). The combined organic layer was washed by brine
(20 mL.times.4), dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
(dichloromethane/methanol=20:1) to give
N-(2,6-dioxopiperidin-3-yl)-5-(3-hydroxypropoxy)picolinamide (2.1
g, 58% for two steps) as a pale yellow solid.
Step 4: Synthesis of
3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl
methanesulfonate
##STR00390##
[0908] To a solution of
N-(2,6-dioxopiperidin-3-yl)-5-(3-hydroxypropoxy)picolinamide (500
mg, 1.63 mmol) in dichloromethane (50.0 mL) was added triethylamine
(329 mg, 3.25 mmol) and methanesulfonyl chloride (224 mg, 1.95
mmol) under nitrogen. The resulting reaction mixture was stirred at
0.degree. C. for 1 hour. Then water (20.0 mL) was added and
extracted with dichloromethane (20 mL.times.3), washed by brine,
dried and concentrated in vacuo to give crude
3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl
methanesulfonate as pale yellow oil, which was used for the next
step without further purification.
Step 5: Synthesis of tert-butyl 6-chloronicotinate
##STR00391##
[0910] A solution of 6-chloronicotinic acid (31.6 g, 200 mmol) and
4-dimethylaminopyridine (2.4 g, 20 mmol) in THF (250 mL) was
refluxed for 3 hours. Then di-tert-butyl dicarbonate (65.0 g, 300
mmol) was added dropwise. After addition, the reaction mixture was
refluxed for 3 hours. Upon reaction completion, the reaction
mixture was cooled to room temperature. The solvent was removed and
the residue was purified by column chromatography on silica gel
(ethyl acetate/petroleum ether=0.about.1/10) to give tert-butyl
6-chloronicotinate (40 g, 94% yield) as a white solid.
[0911] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 100%, Rt=1.984 min; MS Calcd.:
213.06; MS Found: 214.2 [M+H].sup.+.
[0912] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.56 (9H, s),
7.67 (1H, d, J=8.4 Hz), 8.26 (1H, dd, J=8.0, 2.4 Hz), 8.86 (1H, d,
J=2.4 Hz).
[0913] Chemical Formula: C.sub.10H.sub.12ClNO.sub.2, Molecular
Weight: 213.66
[0914] Total H count from HNMR data: 12.
Step 6: Synthesis of tert-butyl 6-(piperazin-1-yl)nicotinate
##STR00392##
[0916] A mixture of tert-butyl 6-chloronicotinate (20.0 g, 94 mmol)
and piperazine (8.9 g, 103 mmol) in N,N-dimethylacetamide (100 mL)
was stirred at 140.degree. C. overnight. The reaction mixture was
cooled to room temperature and saturated aqueous potassium
carbonate solution (200 mL) was added portionwise. The mixture was
filtered and the filtrate was extracted with ethyl acetate (600
mL.times.2). The combined organic layers were washed with water
(600 mL.times.4) and brine (600 mL), dried over anhydrous sodium
sulfate. The solvent was concentrated under reduced pressure and
the residue was purified by column chromatography on silica gel
(dichloride/methanol=10/1) to give tert-butyl
6-(piperazin-1-yl)nicotinate (6.5 g, 26% yield) as a yellow
solid.
[0917] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 100%, Rt=2.068 min; MS Calcd.:
263.16; MS Found: 264.3 [M+H].sup.+.
[0918] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 97.11%, Rt=7.311 min.
[0919] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.51 (9H, s),
2.75 (4H, t, J=4.8 Hz), 3.30 (1H, brs), 3.54 (4H, t, J=4.8 Hz),
6.80 (1H, d, J=9.2 Hz), 7.86 (1H, dd, J=8.8, 2.4 Hz), 8.57 (1H, d,
J=2.4 Hz).
[0920] Chemical Formula: C.sub.14H.sub.21N.sub.3O.sub.2, Molecular
Weight: 263.34
[0921] Total H count from HNMR data: 21.
Step 7: Synthesis of tert-butyl
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinate
##STR00393##
[0923] To a solution of
3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl
methanesulfonate (crude, 1.63 mmol) in dimethyl sulfoxide (5.0 mL)
was added tert-butyl 6-(piperazin-1-yl)nicotinate (472 mg, 1.79
mmol), ethyldiisopropylamine (632 mg, 4.89 mmol) and potassium
iodide (27.1 mg, 0.163 mmol). The reaction mixture was stirred at
45.degree. C. overnight. Then water (20 mL) was added and extracted
with ethyl acetate (20 mL.times.3), washed with brine (5
mL.times.4). The combined organic phases were dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The residue
was purified by prep-TLC (dichloromethane/methanol=10:1) to give
tert-butyl
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinate (250 mg, 28% for two steps) as a pale yellow
solid.
[0924] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.57 (9H, s),
1.70-1.72 (2H, m), 1.99-2.08 (2H, m), 2.54-2.64 (6H, m), 2.79-2.85
(2H, m), 3.69 (4H, t, J=4.8 Hz), 4.17 (2H, t, J=6.4 Hz), 4.76-4.82
(1H, m), 6.58 (1H, d, J=9.2 Hz), 7.31 (1H, dd, J=8.8 Hz, 3.2 Hz),
7.98 (1H, dd, J=8.8 Hz, 2.4 Hz), 8.13 (1H, d, J=8.8 Hz), 8.16 (1H,
brs), 8.25 (1H, d, J=2.8 Hz), 8.51 (1H, d, J=6.8 Hz), 8.76 (1H, d,
J=2.0 Hz).
[0925] Chemical Formula: C.sub.28H.sub.36N.sub.6O.sub.6, Molecular
Weight: 552.62
[0926] Total H count from HNMR data: 36.
Step 8: Synthesis of
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinic acid
##STR00394##
[0928] To a solution of tert-butyl
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinate (250 mg, 0.452 mmol) in dichloromethane (3.0
mL) was added trifluoroacetic acid (1 mL). The reaction mixture was
stirred at room temperature for 2 hours. Then solvent was removed
in vacuo to give
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinic acid (crude) as pale yellow oil, which was used
for the next step without further purification.
Step 9: Synthesis of
5-(3-(4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclob-
utylcarbamoyl)pyridin-2-yl)piperazin-1-yl)propoxy)-N-(2,6-dioxopiperidin-3-
-yl)picolinamide
##STR00395##
[0930] To a solution of
6-(4-(3-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yloxy)propyl)piper-
azin-1-yl)nicotinic acid (crude, 0.452 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(130 mg, 0.678 mmol), 1-hydroxybenzotriazole hydrate (HOBt) (91.9
mg, 0.678 mmol) and ethyldiisopropylamine (175 mg, 1.36 mmol) in N,
N-dimethylformamide (DMF) (15 mL) was stirred for 30 minutes, and
then
4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile
(139 mg, 0.497 mmol) was added. The mixture was stirred at room
temperature overnight and water (20 mL) was added. The aqueous
layer was extracted by ethyl acetate (20 mL.times.3). The combined
organic layer was washed by brine (5 mL.times.4), dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified by prep-TLC (dichloromethane/methanol=10:1)
and prep-HPLC to give
5-(3-(4-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclob-
utylcarbamoyl)pyridin-2-yl)piperazin-1-yl)propoxy)-N-(2,6-dioxopiperidin-3-
-yl)picolinamide (57.7 mg, 17% for two steps) as a white solid.
[0931] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.). Purity is 94.69%, Rt=2.803 min; MS Calcd.:
756.3; MS Found: 757.3 [M+H].sup.+.
[0932] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow Rate:
1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH.sub.4HCO.sub.3]
and 5% [CH.sub.3CN] to 0% [water+10 mM NH.sub.4HCO.sub.3] and 100%
[CH.sub.3CN] in 10 min, then under this condition for 5 min,
finally changed to 95% [water+10 mM NH.sub.4HCO.sub.3] and 5%
[CH.sub.3CN] in 0.1 min and under this condition for 5 min). Purity
is 85.08%, Rt=9.741 min.
[0933] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s),
1.22 (6H, s), 1.94-2.01 (3H, m), 2.18-2.22 (1H, m), 2.49-2.50 (6H,
m), 2.75-2.83 (1H, m), 2.99 (1H, d, J=4.8 Hz), 3.61 (4H, s), 4.06
(1H, d, J=9.2 Hz), 4.19-4.23 (2H, m), 4.31 (1H, s), 4.74-4.80 (1H,
m), 6.88 (1H, d, J=9.2 Hz), 7.01 (1H, dd, J=8.8 Hz, 2.4 Hz), 7.21
(1H, d, J=2.4 Hz), 7.58 (1H, dd, J=8.8 Hz, 2.4 Hz), 7.63 (1H, d,
J=9.2 Hz), 7.90 (1H, d, J=8.4 Hz), 7.96 (1H, dd, J=8.8 Hz, 2.4 Hz),
8.02 (1H, d, J=8.8 Hz), 8.34 (1H, d, J=2.8 Hz), 8.63 (1H, d, J=2.4
Hz), 8.89 (1H, d, J=8.4 Hz), 10.87 (1H, s).
[0934] Chemical Formula: C.sub.39H.sub.45ClN.sub.8O.sub.6,
Molecular Weight: 757.28
[0935] Total H count from HNMR data: 45.
[0936] Synthesis of Exemplary PROTAC 53
##STR00396## [0937]
5-(4-((1-(5-(((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcycl-
obutyl)carbamoyl)pyridin-2-yl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(2,6-
-dioxopiperidin-3-yl)picolinamide
[0938] Synthetic Scheme:
##STR00397## ##STR00398##
Step 1: Synthesis of tert-butyl
4-(6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate
##STR00399##
[0940] To a solution of methyl 5-bromopicolinate (14.8 g, 68.5
mmol) and tert-butyl piperazine-1-carboxylate (15.3 g, 82.2 mmol)
in toluene (150 mL) was added cesium carbonate (55.8 g, 171.3
mmol), tris(dibenzylideneacetone)dipalladium(O) (3.15 g, 3.44 mmol)
and (+/-)-2,2'-Bis(diphenylphosphino)-1,1'-binaphthyl (4.62 g, 7.42
mmol), then it was stirred at 100.degree. C. under nitrogen
overnight. After cooling, it was quenched by water (100 mL) and
extracted with ethyl acetate (100 mL.times.3). The combined organic
layers were washed by brine (200 mL), dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (petroleum
ether/ethyl acetate=5/1) to give tert-butyl
4-(6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate (12.0
g, 55% yield) as a brown solid.
[0941] LC-MS: (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(30 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5%
[water+10 mM NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 1.0 min,
then under this condition for 1.0 min). Purity is 82.48%, Rt=0.991
min; MS Calcd.: 321.17; MS Found: 322.2 [M+H].sup.+.
[0942] Chemical Formula: C.sub.16H.sub.23N.sub.3O.sub.4, Molecular
Weight: 321.37.
Step 2: Synthesis of methyl 5-(piperazin-1-yl)picolinate
##STR00400##
[0944] A mixture of tert-butyl
4-(6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate (12.0
g, 37.4 mmol) in a solution of HCl gas in 1,4-dioxane (100 mL, 4.0
M) was stirred at 30.degree. C. for 1 h. The reaction mixture was
concentrated in vacuo to give methyl 5-(piperazin-1-yl)picolinate
(7.6 g, 93% yield) as a brown solid.
[0945] Chemical Formula: C.sub.11H.sub.15N.sub.3O.sub.2, Molecular
Weight: 221.26.
Step 3: Synthesis of tert-butyl
6-(4-formylpiperidin-1-yl)nicotinate
##STR00401##
[0947] A mixture of tert-butyl
6-(4-(hydroxymethyl)piperidin-1-yl)nicotinate (5.0 g, 17.1 mmol)
and Dess-Martin periodinane (21.8 g, 51.4 mmol) in DCM (200 mL) was
stirred at room temperature for 4 hours. The mixture was filtered
and the filtrate was concentrated in vacuo give tert-butyl
6-(4-formylpiperidin-1-yl)nicotinate (3.5 g, 70% yield) as yellow
gel.
[0948] Chemical Formula: C.sub.16H.sub.22N.sub.2O.sub.3, Molecular
Weight: 290.36
Step 4: Synthesis of methyl
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)methyl)piper-
azin-1-yl)picolinate
##STR00402##
[0950] To a solution of tert-butyl
6-(4-formylpiperidin-1-yl)nicotinate (3.5 g, 12.1 mmol) and methyl
5-(piperazin-1-yl)picolinate (2.67 g, 12.1 mmol) in MeOH (50 mL)
was added NaBH.sub.3CN (1.52 g, 18.0 mmol) and AcOH (2 mL), then it
was stirred at room temperature overnight. It was diluted with
water (50 mL), extracted with DCM (50 mL.times.3). The combined
organic layers were washed by brine (50 mL), dried over anhydrous
sodium sulfate, filtered and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (DCM/MeOH=20/1) to
give methyl
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)methyl)piper-
azin-1-yl)picolinate (1.6 g, 27% yield) as a brown solid.
[0951] LC-MS: (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.987 min; MS Calcd.: 495.28 MS Found:
496.3 [M+H].sup.+.
[0952] Chemical Formula: C.sub.27H.sub.37N.sub.5O.sub.4, Molecular
Weight: 495.61.
Step 5: Synthesis of
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)methyl)piper-
azin-1-yl)picolinic acid
##STR00403##
[0954] To a solution of methyl
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)methyl)piper-
azin-1-yl)picolinate (1.6 g, 2.35 mmol) in THF (60 mL) was added 1
mol/L aqueous NaOH (30 mL), then it was stirred at 30.degree. C.
for 2 hours. It was quenched with water (100 mL) and extracted with
DCM (50 mL.times.3). The combined organic layers were washed by
brine (100 mL), dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo to give
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)meth-
yl)piperazin-1-yl)picolinic acid (1.5 g, 96% yield) as a brown
solid.
[0955] LC-MS: (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.557 min; MS Calcd.: 481.27, MS Found:
482.3 [M+H].sup.+.
[0956] Chemical Formula: C.sub.26H.sub.35N.sub.5O.sub.4, Molecular
Weight: 481.59.
Step 6: Synthesis of tert-butyl
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinate
##STR00404##
[0958] A mixture of
5-(4-((1-(5-(tert-butoxycarbonyl)pyridin-2-yl)piperidin-4-yl)methyl)piper-
azin-1-yl)picolinic acid (1.5 g, 3.1 mmol),
3-aminopiperidine-2,6-dione (0.56 g, 3.4 mmol), HATU (1.77 g, 4.65
mmol) and DIEA (0.8 g, 6.2 mmol) in DMF (50 mL) was stirred at room
temperature for 1 hour. The mixture was poured into water (30 mL)
and extracted with DCM (30 mL.times.3). The combined organic phase
was concentrated and the residue was purified by Prep-HPLC to give
tert-butyl
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinate (1.0 g, 54% yield) as a white
solid.
[0959] LC-MS: (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.879 min; MS Calcd.: 591.32; MS Found:
592.3 [M+H].sup.+.
[0960] Chemical Formula: C.sub.31H.sub.41N.sub.7O.sub.5, Molecular
Weight: 591.70.
Step 7: Synthesis of
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinic acid
##STR00405##
[0962] To a solution of tert-butyl
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinate (500 mg, 0.85 mmol) in DCM (10
mL) was added TFA (5 mL), then it was stirred at room temperature
for 2 hours. The reaction mixture was concentrated in vacuo to give
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinic acid (400 mg, 88% yield) as a
white solid.
[0963] LC-MS: (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Rt=1.215 min; MS Calcd.: 535.25; MS Found:
536.3 [M+H].sup.+.
[0964] Chemical Formula: C.sub.27H.sub.33N.sub.7O.sub.5, Molecular
Weight: 535.59.
Step 8: Synthesis of
5-(4-((1-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclo-
butylcarbamoyl)pyridin-2-yl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(2,6-d-
ioxopiperidin-3-yl)picolinamide
##STR00406##
[0966] A mixture of
6-(4-((4-(6-(2,6-dioxopiperidin-3-ylcarbamoyl)pyridin-3-yl)piperazin-1-yl-
)methyl)piperidin-1-yl)nicotinic acid (400 mg, 0.75 mmol),
4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile
(207.7 mg, 0.75 mmol), EDCI (158.4 mg, 0.825 mmol), HOBt (153 mg,
1.125 mmol) and DIEA (290.25 mg, 2.25 mmol) in DMF (10 mL) were
stirred at room temperature overnight. Then the reaction mixture
was quenched by water (20 mL) and extracted by DCM (20 mL.times.3).
The combined organic layers were washed by brine (30 mL), dried
over anhydrous sodium sulfate, filtered and concentrated in vacuo.
The residue was purified by Prep-HPLC to give
5-(4-((1-(5-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramet-
hylcyclobutylcarbamoyl)pyridin-2-yl)piperidin-4-yl)methyl)piperazin-1-yl)--
N-(2,6-dioxopiperidin-3-yl)picolinamide (215 mg, 36% yield) as a
white solid.
[0967] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 90.80%, Rt=3.023 min; MS Calcd.:
795.36; MS Found: 796.3 [M+H].sup.+.
[0968] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 90.34%, Rt=10.276 min.
[0969] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.76-0.81 (1H, m),
1.15-1.21 (16H, m), 1.80-2.23 (5H, m), 2.53-2.59 (4H, m), 2.71-2.78
(2H, m), 2.85-2.91 (2H, m), 3.29 (3H, brs), 3.97 (1H, s), 4.07 (1H,
d, J=8 Hz), 4.38 (2H, d, J=12.8 Hz), 4.69-4.75 (1H, m), 5.98 (1H,
d, J=8.4 Hz), 6.60 (1H, d, J=8.8 Hz), 6.73 (1H, dd, J=8.8, 2.4 Hz),
6.89 (1H, d, J=2.4 Hz), 7.14-7.17 (1H, m), 7.50 (1H, d, J=8.8 Hz),
7.84 (1H, dd, J=8.8, 2.4 Hz), 7.92 (1H, s), 7.97 (1H, d, J=8.8 Hz),
8.15 (1H, d, J=2.4 Hz), 8.38 (1H, d, J=6.8 Hz), 8.50 (1H, d, J=2.4
Hz).
[0970] Chemical Formula: C.sub.42H.sub.50ClN.sub.9O.sub.5,
Molecular Weight: 796.36.
[0971] Total H count from HNMR data: 50.
[0972] Synthesis of Exemplary PROTAC 61
##STR00407## [0973]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(4-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)piperazi-
n-1-yl)butyl)nicotinamide
[0974] Synthetic Scheme Part 1
##STR00408##
[0975] Synthetic Scheme Part 2
##STR00409##
Step 1: Synthesis of tert-butyl
6-(4-hydroxybut-1-ynyl)nicotinate
##STR00410##
[0977] tert-butyl 6-chloronicotinate (2.0 g, 9.39 mmol) is
dissolved in dimethoxyethane (50 ml) and added in succession with
water (30 mL), potassium carbonate (5.18 g, 37.6 mmol), copper(I)
iodide (0.1 g, 0.5 mmol), triphenylphosphine (0.26 g, 1 mmol) and
10 percent (w/w) palladium on carbon (0.3 g). The reaction mixture
is stirred for 30 minutes at room temperature, then added with
2-methyl-3-butyn-2-ol (5 ml, 50 mmol), heated at 80.degree. C. for
5 hours, then cooled, filtered through Celite, diluted with water
(150 mL) and extracted with ethyl acetate (100 mL.times.2). The
organic phase is washed with water, dried over sodium sulfate,
filtered and concentrated evaporated in vacuo. The resulting
reaction crude is purified by column and flash chromatography on
silica gel to give tert-butyl 6-(4-hydroxybut-1-ynyl)nicotinate
(1.7 g, 73%) as colorless oil.
Step 2: Synthesis of tert-butyl 6-(4-hydroxybutyl)nicotinate
##STR00411##
[0979] A solution of tert-butyl 6-(4-hydroxybut-1-ynyl)nicotinate
(500 mg, 2.0 mmol), Pd/C (50 mg) in tert-butanol (10 mL) was
stirred at room temperature overnight under an atmosphere of
hydrogen (g). The mixture was filtered through a pad of celite to
remove the palladium. The solvent was evaporated in vacuo to give
tert-butyl 6-(4-hydroxybutyl)nicotinate (450 mg, 88% yield) as a
yellow oil. The residue was used to next step without further
purification.
Step 3: Synthesis of 6-(4-hydroxybutyl)nicotinic acid
##STR00412##
[0981] To a solution of 6-(4-hydroxybutyl)nicotinate (200 mg, 0.79
mmol) in dichloromethane (5 mL) was added TFA (5 mL), then it was
stirred at room temperature for 2 hours. It was concentrated in
vacuo to give crude 6-(4-hydroxybutyl)nicotinic acid (130 mg, 84%
yield) as yellow oil, which was directly used to the next step
without further purification.
Step 4: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-hydroxybutyl)nicotinamide
##STR00413##
[0983] To a solution of 6-(4-hydroxybutyl)nicotinic acid (570 mg,
crude, 2.9 mmol),
4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile
(400 mg, 1.4 mmol), EDCI (472 mg, 2.4 mmol) and HOBt (332 mg, 2.4
mmol) in DMF (10 mL) was added DIEA (800 mg, 6.2 mmol), then it was
stirred at room temperature for two days. It was diluted by water
(20 ml) and extracted by ethyl acetate (20 mL.times.2). The organic
extract was washed by water (40 mL.times.3) and brine (40 mL),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in
vacuo. The residue was purified by Prep-TLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-hydroxybutyl)nicotinamide (262 mg, 20% yield) as pale yellow
solid.
[0984] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.6 mm.times.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%[(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] to 10% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 90% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 1.6 min, then under
this condition for 2.4 min, finally changed to 90% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 0.1 min and under
this condition for 0.7 min). Rt=1.832 min; MS Calcd.: 455.98 MS
Found: 456.2[M+H].sup.+.
[0985] Chemical Formula: C.sub.25H.sub.30ClN.sub.3O.sub.3,
Molecular Weight: 455.98
Step 5: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-oxobutyl)nicotinamide
##STR00414##
[0987] A mixture of
N-(2,6-dioxopiperidin-3-yl)-5-(5-hydroxypentyloxy) picolinamide
(240 mg, 0.53 mmol) and Dess-Martin periodinane (269 mg, 0.64 mmol)
in dichloromethane (10 mL) was stirred at room temperature 1.5
hours. The reaction mixture was filtered, and the filter cake was
washed by dichloromethane (10 mL.times.3). The filtrate was
concentrated and purified by Prep-TLC (DCM/MeOH=100/5) to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-oxobutyl)nicotinamide (100 mg, 42% yield) as a yellow solid.
[0988] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 90% [(total 10 mM AcONH.sub.4)
water/CH.sub.3CN=900/100 (v/v)] and 10% [(total 10 mM AcONH.sub.4)
water/CH.sub.3CN=100/900 (v/v)] to 10% [(total 10 mM AcONH.sub.4)
water/CH.sub.3CN=900/100 (v/v)] and 90% [(total 10 mM AcONH.sub.4)
water/CH.sub.3CN=100/900 (v/v)] in 1.6 min, then under this
condition for 2.4 min, finally changed to 90% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10% [(total 10 mM
AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 0.1 min and under
this condition for 0.7 min). Purity is 52.80, Rt=1.977 min; MS
Calcd.: 453.96; MS Found: 454.2 [M+H].sup.+.
Step 6: Synthesis of tert-butyl
4-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)piperazine-1-carboxylate
##STR00415##
[0990] To a solution of 4,5-dichloropyridazin-3(2H)-one (10 g, 60.6
mmol), in N,N-dimethylformamide (40 mL) was added tert-butyl
piperazine-1-carboxylate (22.5 g, 121.2 mmol) and DIEA (25 g, 182
mmol). The mixture was stirred at 80.degree. C. overnight. After
cooling to room temperature, the mixture was filtered, and the
residue was washed with ethyl acetate (100 mL.times.3) and DCM (100
mL.times.3) to give compound tert-butyl
4-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)piperazine-1-carboxylate
(8 g, 42% yield) as pale yellow solid.
[0991] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(30 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5% [water+10 mM
NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min, then under this
condition for 1.5 min, finally changed to 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and under this
condition for 0.5 min.) Purity is 87.88%. Rt=0.903 min; MS Calcd.:
314.77 MS Found: 315.2 [M+H].sup.+.
[0992] Chemical Formula: C.sub.13H.sub.19ClN.sub.4O.sub.3,
Molecular Weight: 314.77
Step 7: Synthesis of tert-butyl
4-(5-chloro-1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)p-
iperazine-1-carboxylate
##STR00416##
[0994] To a solution of 1-bromo-4-(5-bromopentyloxy)benzene (4 g,
12.7 mmol) in DMSO (20 mL) was added 3-bromopiperidine-2,6-dione
(4.8 mg, 25.4 mmol) and potassium carbonate (5.3 g, 38.1 mmol). The
mixture was stirred at 40.degree. C. for two days. After cooling to
room temperature, the mixture was filtered, and the residue was
washed with ethyl acetate (20 mL.times.3) and DCM (20 mL.times.3).
The combined organic phases were dried over anhydrous sodium
sulfate and concentrated in vacuo and purified by column
chromatography on silica gel (petroether/ethyl acetate=1:4) to give
tert-butyl
4-(5-chloro-1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)p-
iperazine-1-carboxylate (3.7 g, 67% yield) as pale yellow
solid.
Step 8: Synthesis of tert-butyl
4-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)piperazine-
-1-carboxylate
##STR00417##
[0996] A mixture of tert-butyl
4-(5-chloro-1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)p-
iperazine-1-carboxylate (300 mg, 0.7 mmol) and 10% palladium on
activated carbon (90 mg) in MeOH (30 mL) was stirred under 1 atm
hydrogen atmosphere at 37.degree. C. overnight. It was filtered to
remove the solid, the filtrate was concentrated in vacuo to give
3-(4-(3-hydroxypropoxy)-6-oxopyridazin-1(6H)-yl)piperidine-2,6-dione
(190 mg, 93% yield) as a yellow solid.
[0997] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(30 mm.times.6 mm.times.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5% [water+10 mM
NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min, then under this
condition for 1.5 min, finally changed to 90% [water+10 mM
NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and under this
condition for 0.5 min.). Purity is 77.70%. Rt=0.873 min; MS Calcd.:
391.42. MS Found: 392.2 [M+H].sup.+.
[0998] Chemical Formula: C.sub.18H.sub.25N.sub.5O.sub.5, Molecular
Weight: 391.42
Step 9: Synthesis of
3-(6-oxo-4-(piperazin-1-yl)pyridazin-1(6H)-yl)piperidine-2,6-dione
##STR00418##
[1000] To a solution of
3-(4-(3-hydroxypropoxy)-6-oxopyridazin-1(6H)-yl)piperidine-2,6-dione
(50 mg, 0.10 mmol) in DCM (3 mL) and trifluoroacetic acid (3 mL)
was stirred at rt for 3 h. Then the solvent was directly removed to
give
3-(6-oxo-4-(piperazin-1-yl)pyridazin-1(6H)-yl)piperidine-2,6-dione
(124 mg, crude, 88% yield) which was directly used to the next step
without further purification.
Step 10: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(4-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)piperazi-
n-1-yl)butyl)nicotinamide
##STR00419##
[1002] To a solution of
3-(6-oxo-4-(piperazin-1-yl)pyridazin-1(6H)-yl)piperidine-2,6-dione
(100 mg, 0.34 mmol),
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-oxobutyl)nicotinamide (130 mg, 0.29 mmol) in MeOH (6 mL) was
added acetic acid (3 drops), then NaBH.sub.3CN (23 mg, 0.35 mmol)
was added in 7 portions during 6 hours at room temperature. The
resulting mixture was stirred at room temperature for another 1
hour. The reaction mixture was concentrated, diluted with brine (15
mL) and extracted with CH.sub.2Cl.sub.2/MeOH (10/1, 20 mL.times.2).
The organic was dried over Na.sub.2SO.sub.4, filtered, concentrated
and purified by Prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl-
)-6-(4-(4-(1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl)pip-
erazin-1-yl)butyl)nicotinamide (56 mg, 27% yield) as a pale yellow
solid.
[1003] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.). Purity is 99.06%, Rt=2.650 min; MS Calcd.:
728.3; MS Found: 729.4 [M+H].sup.+.
[1004] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 96.51%, Rt=9.185 min.
[1005] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.16 (7H, s), 1.21
(7H, s), 1.71-1.75 (2H, m), 2.13-2.16 (1H, m), 2.34-2.37 (2H, m),
2.45-2.47 (4H, m), 2.55-2.71 (2H, m), 2.77-2.84 (3H, m), 3.25-3.28
(4H, m), 3.99 (1H, s), 4.09 (1H, d, J=8.4 Hz), 5.63-5.68 (1H, m),
5.82 (1H, d, J=2.8 Hz), 6.11 (1H, d, J=8.0 Hz), 6.74 (1H, dd,
J=8.8, 2.4 Hz), 6.90 (1H, d, J=2.0 Hz), 7.21 (1H, s), 7.50 (1H, d,
J=8.8 Hz), 7.64 (1H, d, J=3.2 Hz), 7.91 (1H, brs), 7.96 (1H, dd,
J=8.0, 2.0 Hz), 8.83 (1H, d, J=1.6 Hz).
[1006] Chemical Formula: C.sub.38H.sub.45ClN.sub.8O.sub.5,
Molecular Weight: 729.27
[1007] Total H count from HNMR data: 45.
[1008] Synthesis of Exemplary PROTAC 70
##STR00420## [1009]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,2,4--
triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide
[1010] Synthetic Scheme
##STR00421## ##STR00422##
Step 1: Synthesis of tert-butyl
4-(4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate
##STR00423##
[1012] The mixture of methyl 4-fluorobenzoate (3.1 g, 20.0 mmol)
tert-butyl piperazine-1-carboxylate (3.7 g, 20.0 mmol) and
potassium carbonate (2.7 g, 40.0 mmol) in dimethyl sulfoxide (30
mL) was heaed at 120.degree. C. for 24 hours. The mixture was
poured into water (100 mL) and extracted with ethyl acetate (50
mL.times.3). The combined organic phase was concentrated in vacuo
to give tert-butyl
4-(4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate (5.1 g, 80%
yield) as a white solid.
[1013] Chemical Formula: C.sub.17H.sub.24NO.sub.2, Molecular
Weight: 320.38
Step 2: Synthesis of tert-butyl
4-(4-(hydrazinecarbonyl)phenyl)piperazine-1-carboxylate
##STR00424##
[1015] The mixture of tert-butyl
4-(4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate (3.2 g, 10.0
mmol) and hydrazine hydrate (1.0 g, 20.0 mmol) in ethanol (30 mL)
was refluxed overnight. The mixture was concentrated to give
tert-butyl 4-(4-(hydrazinecarbonyl)phenyl)piperazine-1-carboxylate
(2.6 g, 80% yield) as a white solid used directly.
[1016] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.) Purity is 78.9%, Rt=1.609 min. MS Calcd.:
320.1; MS Found: 321.3 [M+H].sup.+.
[1017] Chemical Formula: C.sub.16H.sub.24N.sub.4O.sub.3, Molecular
Weight: 320.39
Step 3: Synthesis of tert-butyl
4-(4-(2-(methylcarbamoyl)hydrazinecarbonyl)
phenyl)piperazine-1-carboxylate
##STR00425##
[1019] A mixture of tert-butyl
4-(4-(hydrazinecarbonyl)phenyl)piperazine-1-carboxylate (2.0 g, 6.3
mmol) and 2,5-dioxopyrrolidin-1-yl methylcarbamate (1.1 g, 6.3
mmol) in acetonitrile (30 mL) was stirred at room temperature
overnight. The mixture was poured into water (30 mL) and filtered
to give tert-butyl
4-(4-(2-(methylcarbamoyl)hydrazinecarbonyl)phenyl)piperazine-1-carboxylat-
e (1.7 g, 70%) as a white solid.
[1020] Chemical Formula: C.sub.18H.sub.27N.sub.5O.sub.4, Molecular
Weight: 377.44
Step 4: Synthesis of tert-butyl
4-(4-(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)piperazine--
1-carboxylate
##STR00426##
[1022] The mixture of tert-butyl
4-(4-(2-(methylcarbamoyl)hydrazinecarbonyl)phenyl)piperazine-1-carboxylat-
e (1.7 g, 4.5 mmol) and sodium hydroxide (360 mg, 9.0 mmol) in
water (15 mL) was refluxed for 3 hours. The mixture was cooled to
room temperature and the pH value of the mixture was adjusted to
5-6 by hydrochloride acid (1.0 N). The mixture was extracted with
dichloromethane (30 mL.times.3) and the combined organic phase was
concentrated in vacuo to give tert-butyl
4-(4-(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)piperazine--
1-carboxylate (1.2 g, 75% yield) as a white solid.
[1023] Chemical Formula: C.sub.18H.sub.25N.sub.5O.sub.3, Molecular
Weight: 359.42
Step 5: Synthesis of tert-butyl
4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-tri-
azol-3-yl)phenyl)piperazine-1-carboxylate
##STR00427##
[1025] The mixture of tert-butyl
4-(4-(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl)piperazine--
1-carboxylate (1.2 g, 3.3 mmol), 3-bromopiperidine-2,6-dione (1.3
g, 6.6 mmol) and potassium tert-butoxide (1.1 g, 9.9 mmol) in
acetonitrile (20 mL) was refluxed overnight. The mixture was poured
into saturated ammonium chloride solution (30 mL) and extracted
with dichloromethane (30 mL.times.3). The combined organic phase
was concentrated in vacuo and the residue was purified by Prep-HPLC
to give tert-butyl
4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-tri-
azol-3-yl)phenyl)piperazine-1-carboxylate (465 mg, 30% yield) as a
white solid.
[1026] Chemical Formula: C.sub.23H.sub.30N.sub.6O.sub.5, Molecular
Weight: 470.52
Step 6: Synthesis of
3-(4-methyl-5-oxo-3-(4-(piperazin-1-yl)phenyl)-4,5-dihydro-1H-1,2,4-triaz-
ol-1-yl)piperidine-2,6-dione
##STR00428##
[1028] A solution of tert-butyl
4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-tri-
azol-3-yl)phenyl)piperazine-1-carboxylate (465 mg, 0.99 mmol) in
dry hydrochloride/1,4-dioxane (20 mL, 4.0 N.) was stirred at room
temperature for 4 h. The mixture was concentrated in vacuo to give
3-(4-methyl-5-oxo-3-(4-(piperazin-1-yl)phenyl)-4,5-dihydro-1H-1,2,4-triaz-
ol-1-yl)piperidine-2,6-dione (293 mg, 80% yield) as a white
solid.
[1029] Chemical Formula: C.sub.18H.sub.22N.sub.6O.sub.3, Molecular
Weight: 370.41
Step 7: Synthesis of tert-butyl
4-(4-(hydroxymethyl)piperidin-1-yl)benzoate
##STR00429##
[1031] To a solution of tert-butyl 4-fluorobenzoate (23 g, 0.12
mmol) in DMSO (100 mL) was added piperidin-4-ylmethanol (40.5 g,
0.35 mmol). The mixture was heated to 120.degree. C. overnight
under nitrogen. After cooling to room temperature, water (50 mL)
was added to the reaction mixture, and extracted with ethyl acetate
(20 mL.times.3). The organic layer was washed with brine (15
mL.times.3). The combined organic phases were dried over anhydrous
sodium sulfate and concentrated in vacuo, and purified by CC
(PE/EA=10:1) to give compound tert-butyl
4-(4-(hydroxymethyl)piperidin-1-yl)benzoate (31 g, 91.2%) as a
white solid.
[1032] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] to 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 1.6
min, then under this condition for 2.4 min, finally changed to 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 0.1
min and under this condition for 0.7 min). Purity is 99.57%,
Rt=2.035 min.; MS Calcd.: 291.2; MS Found: 292.2 [M+H]+.
[1033] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 93.27%, Rt=9.542 min.
[1034] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.29-1.40 (2H, m),
1.49 (1H, d, J=5.4 Hz), 1.57 (9H, s), 1.70-1.75 (1H, m), 1.82 (2H,
d, J=12.8 Hz), 2.80-2.87 (2H, m), 3.53 (2H, t, J=5.8 Hz), 3.87-3.90
(2H, m), 6.85 (2H, d, J=9.2 Hz), 7.84 (2H, d, J=9.2 Hz).
[1035] Chemical Formula: C.sub.17H.sub.25NO.sub.3, Molecular
Weight: 291.39
[1036] Total H count from HNMR data: 25.
Step 8: Synthesis of tert-butyl
4-(4-formylpiperidin-1-yl)benzoate
##STR00430##
[1038] To a solution of tert-butyl
4-(4-(hydroxymethyl)piperidin-1-yl)benzoate (300 mg, 1.03 mmol) in
dichloromethane (20 mL) was added Dess-Martin periodinane (1.31 g,
3.09 mmol) slowly at 0.degree. C. The reaction mixture was stirred
at room temperature for 1 hour. Then filtered, and concentrated in
vacuo to give compound tert-butyl
4-(4-formylpiperidin-1-yl)benzoate (240 mg, 81%) as a pale yellow
solid.
Step 9: Synthesis of tert-butyl
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoate
##STR00431##
[1040] The mixture of
3-(4-methyl-5-oxo-3-(4-(piperazin-1-yl)phenyl)-4,5-dihydro-1H-1,2,4-triaz-
ol-1-yl)piperidine-2,6-dione (200 mg, 0.54 mmol), tert-butyl
4-(4-formylpiperidin-1-yl)benzoate (156 mg, 0.54 mmol), sodium
cyanoborohydride (100 mg, 1.6 mmol) and acetic acid (0.5 mL) in
methanol (10 mL) was stirring at room temperature overnight. The
mixture was poured into water (20 mL) and extracted with
dichloromethane (20 mL.times.3). The combined organic phase was
purified by column chromatography on silica gel
(dichloromethane/methanol=20/1) to give tert-butyl
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoate
(173 mg, 50% yield) as a brown solid.
[1041] Chemical Formula: C.sub.35H.sub.45N.sub.7O.sub.5, Molecular
Weight: 643.78
Step 10: Synthesis of
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoic
acid
##STR00432##
[1043] The mixture of tert-butyl
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoate
(150 mg, 0.23 mmol) and trifluoroacetic acid (265 mg, 2.3 mmol) in
1,2-dichloroethane (10 mL) was stirred for 2 h. The mixture was
concentrated in vacuo to give
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoic
acid (95 mg, 70% yield) as a brown solid, which was directly used
to next step without further purification.
[1044] Chemical Formula: C.sub.31H.sub.37N.sub.7O.sub.5, Molecular
Weight: 587.67
Step 11: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,2,4--
triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide
##STR00433##
[1046] The mixture of
4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1H-1,-
2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzoic
acid (95 mg, 0.16 mmol),
4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile
(45 mg, 0.16 mmol),
2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (91 mg, 0.24 mmol) and ethyldiisopropylamine
(62 mg, 0.48 mmol) in N,N-dimethylformamide (5 mL) was stirring at
room temperature overnight. The mixture was poured into water (10
mL) and extracted with dichloromethane (10 mL.times.3). The
combined organic phase was concentrated in vacuo and the residue
was purified by Prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobu-
tyl)-4-(4-((4-(4-(1-(2,6-dioxopiperidin-3-yl)-4-methyl-5-oxo-4,5-dihydro-1-
H-1,2,4-triazol-3-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide
(54 mg, 40% yield) as a white solid.
[1047] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95%
[water+10 mM NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10
mM NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 99.4%, Rt=3.160 min; MS Calcd.:
847.3; MS Found: 848.4 [M+H].sup.+.
[1048] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 10 min, then under this
condition for 5 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 5 min). Purity is 94.0%, Rt=10.750 min.
[1049] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.12 (7H, brs),
1.21 (8H, brs), 1.79-1.82 (3H, m), 2.08-2.12 (1H, m), 2.20-2.22
(2H, m), 2.41-2.45 (3H, m), 2.59-2.63 (1H, m), 2.76-2.87 (3H, m),
3.26-3.27 (5H, m), 3.30 (3H, s), 3.84-3.87 (2H, m), 4.04-4.06 (1H,
m), 4.32 (1H, s), 5.18 (1H, dd, J=5.6, 12.8 Hz), 6.94-7.05 (5H, m),
7.20 (1H, d, J=2.4 Hz), 7.47-7.53 (3H, m), 7.73 (1H, d, J=8.8 Hz),
7.90 (1H, d, J=8.8 Hz), 11.0 (1H, s).
[1050] Chemical Formula: C.sub.46H.sub.54ClN.sub.9O.sub.5,
Molecular Weight: 848.43
[1051] Total H count from HNMR data: 54
[1052] Synthesis of Exemplary PROTAC 79
##STR00434## [1053]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yl)oxy)pen-
tyl)piperazin-1-yl)nicotinamide
[1054] Synthetic Scheme
##STR00435##
Step 1: Synthesis of 7-bromoisoquinoline
##STR00436##
[1056] To a solution of 3-bromobenzaldehyde (50.0 g, 0.27 mol) in
toluene (250 mL) was added aminoacetaldehyde dimethyl acetal (31.1
g, 0.30 mol) was stirred at room temperature for few minutes, then
heated at 100.degree. C. overnight. The reaction solvent was
evaporated to afford 3-bromobenzalaminoacetal (70 g, 95%) as yellow
oil which was directly used to next step without further
purification.
[1057] To a solution of phosphorus pentoxide (140 g, 2v) in
concentrated sulphuric acid (70 mL, 1v) stirred at room temperature
for few minutes, then stirred at 0.degree. C.,
3-bromobenzalaminoacetal (70 g, 0.26 mol) was added slowly to the
mixture prepared above. Then the mixture was heated to 160.degree.
C. for 30 minutes. After cooling to room temperature, the reaction
mixture was carefully poured into ice water (100 mL) while
vigorously stirred, then filtered, the pH was further increased to
9 using saturated sodium hydroxide and extracted with
dichloromethane (100 mL.times.3), the combined organic phases were
dried over anhydrous sodium sulfate, filtered, and concentrated in
vacuo, and purified by silica gel (petroleum ether/ethyl
acetate=6:1) to give mixture of 7-bromoisoquinoline and
5-bromoisoquinoline (15.0 g, 28%) as a yellow solid.
Step 2: Synthesis of 4,7-dibromoisoquinoline
##STR00437##
[1059] To a solution of a mixture of 7-bromoisoquinoline and
5-bromoisoquinoline (15.0 g, 0.072 mol) in acetic acid (30 mL) was
added N-bromosuccinimide (19.3 g, 0.11 mol). The mixture was heated
to 100.degree. C. overnight under nitrogen. After cooling to room
temperature, water (10 mL) was added to the reaction mixture and
neutralized by saturated sodium hydroxide then extracted with ethyl
acetate (10 mL.times.3). The combined organic phases were dried
over anhydrous sodium sulfate and concentrated in vacuo, and
purified by silica gel (petroleum ether/ethyl acetate=15:1) to give
compound 4,7-dibromoisoquinoline (6.0 g, 29%) as a yellow
solid.
[1060] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.87-7.90 (1H, m),
8.05 (1H, d, J=8.8 Hz), 8.15 (1H, m), 8.75 (1H, s), 9.01 (1H,
s).
[1061] Chemical Formula: C.sub.9H.sub.5Br.sub.2N, Molecular Weight:
286.95
[1062] Total H count from HNMR data: 5.
Step 3: Synthesis of 4-bromo-7-methoxyisoquinoline
##STR00438##
[1064] To a solution of 4,7-dibromoisoquinoline (1.0 g, 3.5 mmol)
in dimethyl sulfoxide/methanol (4:3) (10 mL) was added sodium
methanolate (0.3 g, 5.6 mmol). The mixture was heated in a
microwave reactor at 140.degree. C. for 1 hour. Water (5 mL) was
added to the mixture and extracted with ethyl acetate (5
mL.times.3). The combined organic layer was washed with brine (5
mL.times.2), dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo and purified by silica gel (petroleum
ether/ethyl acetate=10:1) to give 4-bromo-7-methoxyisoquinoline
(180 mg, 22%) as a yellow solid.
[1065] .sup.1H NMR (400 MHz, DMSO-d.sup.6) 6 3.95 (3H, s),
7.57-7.60 (1H, m), 7.63 (1H, d, J=2.4 Hz), 7.99 (1H, d, J=8.8 Hz),
8.59 (1H, s), 9.21 (1H, s).
[1066] Chemical Formula: C.sub.10H.sub.8BrNO, Molecular Weight:
238.08
[1067] Total H count from HNMR data: 8.
Step 4: Synthesis of 4-bromoisoquinolin-7-ol
##STR00439##
[1069] To a solution of 4-bromo-7-methoxyisoquinoline (110 mg, 0.46
mmol) in dichloromethane (2 mL) was added BBr.sub.3 (1.0M) in
dichloromethane (4.6 mL, 4.6 mmol) at -20.degree. C., then stirred
at room temperature for 12 hours. The reaction mixture was poured
in cold water and neutralized with saturated sodium bicarbonate,
then extracted with dichloromethane (5 mL.times.3). The combined
organic layer was dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo and purified by prep-TLC (petroether/ethyl
acetate=3:1) to give 4-bromoisoquinolin-7-ol (60 mg, 58%) as light
oil.
[1070] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(30 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] to 5%
[water+10 mM NH.sub.4HCO.sub.3] and 95% [CH.sub.3CN] in 0.5 min,
then under this condition for 1.5 min, finally changed to 90%
[water+10 mM NH.sub.4HCO.sub.3] and 10% [CH.sub.3CN] in 0.1 min and
under this condition for 0.5 min). Purity is 90.50%, Rt=1.078 min;
MS Calcd.: 223.7; MS Found: 224.7 [M+H].sup.+.
Step 5: Synthesis of 5-(4-bromoisoquinolin-7-yloxy)pentan-1-ol
##STR00440##
[1072] To a solution of compound 4-bromoisoquinolin-7-ol (0.90 g,
4.02 mmol) in DMF (10 mL) was added 5-bromopentan-1-ol (0.66 g,
4.02 mmol) and potassium carbonate (0.74 g, 8.04 mmol), then
stirred at 70.degree. C. for 8 hours. The reaction mixture was
poured in cold water and extracted with dichloromethane/methanol
(10 mL.times.3). The combined organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated in vacuo and
purified by prep-TLC (dichloromethane/methanol=15:1) to give
5-(4-bromoisoquinolin-7-yloxy)pentan-1-ol (1.0 g, 81%) as a yellow
solid.
[1073] .sup.1H NMR (400 MHz, DMSO-d.sup.6) .delta. 1.49-1.51 (4H,
m), 1.82 (2H, t, J=6.8 Hz), 3.43-3.44 (2H, m), 4.16 (2H, t, J=6.4
Hz), 4.41 (1H, t, J=5.2 Hz), 7.58-7.64 (2H, m), 8.00 (1H, d, J=9.2
Hz), 8.59 (1H, s), 9.19 (1H, s).
[1074] Chemical Formula: C.sub.14H.sub.16BrNO.sub.2, Molecular
Weight: 310.19
[1075] Total H count from HNMR data: 16.
Step 6: Synthesis of
1-(7-(5-hydroxypentyloxy)isoquinolin-4-yl)pyrimidine-2,4(1H,3H)-dione
##STR00441##
[1077] A solution of 5-(4-bromoisoquinolin-7-yloxy)pentan-1-ol (100
mg, 0.32 mmol), pyrimidine-2,4(1H,3H)-dione (48 mg, 0.38 mmol),
K.sub.3PO.sub.4 (200 mg, 0.96 mmol), CuI (30 mg, 0.16 mmol),
N-(2-cyanophenyl)picolinamide (22 mg, 0.16 mmol) in DMSO (6 mL) was
heated at 120.degree. C. for 2 hours under argon atmosphere. The
reaction mixture was cooled to room temperature poured in cold
water and extracted with dichloromethane/methanol (10 mL.times.3).
The combined organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated in vacuo and purified by prep-TLC
(dichloromethane/methanol=12:1) to give
1-(7-(5-hydroxypentyloxy)isoquinolin-4-yl)pyrimidine-2,4(1H,3H)-dione
(21 mg, 19%) as a yellow solid.
[1078] .sup.1H NMR (400 MHz, DMSO-d.sup.6) .delta. 1.49-1.51 (4H,
m), 1.80-1.83 (2H, m), 3.42-3.44 (2H, m), 4.16 (2H, t, J=6.4 Hz),
4.41 (1H, t, J=5.2 Hz), 5.75-5.78 (1H, m), 7.50 (1H, dd, J=9.2, 2.8
Hz), 7.69-7.77 (3H, m), 8.44 (1H, s), 9.31 (1H, s), 11.61 (1H,
s).
[1079] Chemical Formula: C.sub.18H.sub.19N.sub.3O.sub.4, Molecular
Weight: 341.36
[1080] Total H count from HNMR data: 19.
Step 7: Synthesis of
5-(4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yloxy)pentana-
l
##STR00442##
[1082] To a solution of
1-(7-(5-hydroxypentyloxy)isoquinolin-4-yl)pyrimidine-2,4(1H,3H)-dione
(30 mg, 0.088 mmol) in dichloromethane (10 mL) was added
Dess-Martin periodinane (112 mg, 0.26 mmol). The mixture was
stirred at room temperature for 2 hours. The mixture was added to
water (10.0 mL) and extracted with dichloromethane (10.0
mL.times.2). The combined organic layer was washed with brine (20
mL), dried over anhydrous sodium sulfate, filtered and concentrated
in vacuo and purified by prep-TLC (dichloromethane/methanol=12:1)
to give
5-(4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yloxy)pentana-
l (20 mg, 67%) as a yellow solid.
Step 8: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yloxy)penty-
l)piperazin-1-yl)nicotinamide
##STR00443##
[1084] To a solution of
5-(4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yloxy)pentana-
l (20 mg, 0.058 mmol) in dry methanol/1,2-dichloroethane/HOAc (5
mL/3 mL/0.1 mL) was added
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide (27 mg, 0.058 mmol). The mixture was
left to stir for 30 minutes under N.sub.2 gas. Then sodium
cyanoborohydride (7 mg, 0.116 mmol) was added and the reaction
mixture was left to stir overnight. The solvent was removed and the
residue partitioned between dichloromethane and water, washed with
brine, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give crude product. The residue was
purified by prep-HPLC to give compound
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoquinolin-7-yloxy)penty-
l)piperazin-1-yl)nicotinamide (6.0 mg, 13%) as a yellow solid.
[1085] LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min). Purity is 93.61%, Rt=2.885 min.; MS Calcd.:
790.3; MS Found: 791.3 [M+H].sup.+.
[1086] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow Rate:
1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH.sub.4HCO.sub.3]
and 5% [CH.sub.3CN] to 0% [water+10 mM NH.sub.4HCO.sub.3] and 100%
[CH.sub.3CN] in 10 min, then under this condition for 5 min,
finally changed to 95% [water+10 mM NH.sub.4HCO.sub.3] and 5%
[CH.sub.3CN] in 0.1 min and under this condition for 5 min). Purity
is 92.34%, Rt=9.952 min.
[1087] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s),
1.21 (6H, s), 1.49-1.57 (4H, m), 1.83-1.86 (2H, m), 2.31-2.40 (5H,
m), 2.67-2.68 (1H, m), 3.58-3.60 (4H, m), 4.05 (1H, d, J=9.2 Hz),
4.17-4.20 (2H, m), 4.30 (1H, s), 5.76 (1H, d, J=8.4 Hz), 6.86 (1H,
d, J=8.8 Hz), 6.99-7.02 (1H, m), 7.21 (1H, d, J=2.0 Hz), 7.50-7.52
(1H, m), 7.63 (1H, d, J=9.6 Hz), 7.70-7.76 (3H, m), 7.90-7.97 (2H,
m), 8.44 (1H, s), 8.62 (1H, d, J=1.6 Hz), 9.31 (1H, s).
[1088] Chemical Formula: C.sub.43H.sub.47ClN.sub.8O.sub.5,
Molecular Weight: 791.34
[1089] Total H count from HNMR data: 47.
[1090] Synthesis of Exemplary PROTAC 80
##STR00444## [1091]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-((3-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yl)ox-
y)pentyl)piperazin-1-yl)nicotinamide
[1092] Synthetic Scheme
##STR00445##
Step 1: Synthesis of 5-(3-aminoquinolin-6-yloxy)pentan-1-ol
##STR00446##
[1094] To a solution of 5-(3-bromoquinolin-6-yloxy)pentan-1-ol (1.1
g, 3.6 mmol), benzophenone imine (684 mg, 3.8 mmol) and sodium
tert-butoxide (691 mg, 7.2 mmol) in toluene (20 mL) was added
(+/-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (448 mg, 0.7
mmol) and tris(dibenzylideneacetone)dipalladium (207 mg, 0.36 mmol)
under nitrogen atmosphere, and the mixture was refluxed for 2
hours. When it was cooled to room temperature, water (20 mL) was
added. The resulted mixture was extracted by ethyl acetate (10
mL.times.3), washed by brine (20 mL.times.3), dried over anhydrous
sodium sulfate and filtered. Then 4N HCl (5 mL) was added to the
filtrate, the mixture was stirred for an hour. The layers were
separated and the organic layer was extracted by water (10
mL.times.3). Then the combined water phase was adjusted to pH=9
with sat. NaHCO.sub.3, extracted by ethyl acetate (10 mL.times.3),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by column chromatography on silica gel
(dichloromethane/methanol=8/1) to give
5-(3-aminoquinolin-6-yloxy)pentan-1-ol (600 mg, 69% yield) as a
white solid.
[1095] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm x 3.5 .mu.m); Column Temperature: 40.degree.
C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 1.6 min, then under
this condition for 1.4 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.) Purity is 97.35%, Rt=1.361 min. MS Calcd.:
246.14; MS Found: 247.3 [M+H].sup.+.
[1096] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.45-1.49 (4H,
m), 1.76 (2H, t, J=6.8 Hz), 3.42 (2H, dd, J=11.2, 6.0 Hz), 4.03
(2H, t, J=6.4 Hz), 4.40 (1H, t, J=5.2 Hz), 5.60 (2H, s), 6.93 (1H,
dd, J=8.8, 2.4 Hz), 6.97 (1H, d, J=2.4 Hz), 7.02 (1H, d, J=2.4 Hz),
7.62 (1H, d, J=8.8 Hz), 8.23 (1H, d, J=2.8 Hz).
[1097] Chemical Formula: C.sub.14H.sub.18N.sub.2O.sub.2, Molecular
Weight: 246.30.
[1098] Total H count from HNMR data: 18.
Step 2: Synthesis of
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrim-
idine-5-carbonitrile
##STR00447##
[1100] The solution of 5-(3-aminoquinolin-6-yloxy)pentan-1-ol (600
mg, 2.44 mmol), N-carbamoyl-2-cyanoacetamide (1.2 g, 9.76 mmol) and
trimethoxymethane (1.0 g, 9.76 mmol) in dimethyl sulfoxide (10 mL)
was stirred at 80.degree. C. overnight, and the reaction mixture
continued to stir at 120.degree. C. for 2 hours. When it was cooled
to room temperature, water (30 mL) was added to the mixture and a
white solid resulted. The resulted mixture was filtered and the
solid was purified by Prep-HPLC to give
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrim-
idine-5-carbonitrile (110 mg, 12% yield) as a white solid.
[1101] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.49-1.51 (4H,
m), 1.81 (2H, t, J=6.4 Hz), 3.43 (2H, d, J=5.2 Hz), 4.13 (2H, t,
J=6.4 Hz), 4.41 (1H, t, J=5.2 Hz), 7.44 (1H, d, J=2.4 Hz), 7.49
(1H, dd, J=9.2, 2.8 Hz), 7.99 (1H, d, J=9.2 Hz), 8.36 (1H, d, J=2.4
Hz), 8.77 (1H, d, J=2.4 Hz), 8.95 (1H, s), 12.31 (1H, brs).
[1102] Chemical Formula: C.sub.19H.sub.18N.sub.4O.sub.4, Molecular
Weight: 366.37.
[1103] Total H count from HNMR data: 18.
Step 3: Synthesis of
5-(3-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pe-
ntyl methanesulfonate
##STR00448##
[1105] To a solution of
1-(6-(5-hydroxypentyloxy)quinolin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrim-
idine-5-carbonitrile (110 mg, 0.30 mmol) and triethylamine (98 mg,
0.90 mmol) in dichloromethane (4 mL) was added methanesulfonyl
chloride (51 mg, 0.45 mmol) at 0.degree. C., and the mixture was
stirred at room temperature for 30 minutes. Then water (5 mL) was
added to the mixture, and the resulted mixture was extracted by
dichloromethane (5 mL.times.3), washed by brine (5 mL.times.3),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated.
The crude product (150 mg) was directly used to the next step
without further purification.
Step 4: Synthesis of
1-(6-(5-iodopentyloxy)quinolin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidi-
ne-5-carbonitrile
##STR00449##
[1107] To a solution of
5-(3-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)pe-
ntyl methanesulfonate (150 mg) in acetonitrile (3 mL) was added
potassium iodide (50 mg, 0.3 mmol), and the mixture was stirred at
90.degree. C. for 4 hours. When it was cooled to room temperature,
water (5 mL) was added to the mixture, and the resulted mixture was
extracted by dichloromethane (5 mL.times.3), washed by brine (5
mL.times.3), dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified by Prep-TLC
(dichloromethane/methanol=10/1) to give the desired product (40 mg,
28% yield over two steps).
[1108] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm.times.4.6 mm.times.3.5 .mu.m); Column Temperature:
40.degree. C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] to 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 1.6
min, then under this condition for 2.4 min, finally changed to 90%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=900/100 (v/v)] and 10%
[(total 10 mM AcONH.sub.4) water/CH.sub.3CN=100/900 (v/v)] in 0.1
min and under this condition for 0.7 min.) Purity is 66.97%,
Rt=2.066 min. MS Calcd.: 476.03; MS Found: 477.0 [M+H].sup.+.
Step 5: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)-
pentyl)piperazin-1-yl)nicotinamide
##STR00450##
[1110] A solution of
1-(6-(5-iodopentyloxy)quinolin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidi-
ne-5-carbonitrile (40 mg, 0.08 mmol),
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
piperazin-1-yl)nicotinamide (39 mg, 0.08 mmol), and
ethyldiisopropylamine (30 mg, 0.25 mmol) in acetonitrile (2 mL) was
stirred at 90.degree. C. overnight. When it was cooled to room
temperature, water (5 mL) was added and the mixture was extracted
by ethyl acetate (2 mL.times.3), washed by brine (5 mL.times.3),
dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated.
The residue was purified by Prep-HPLC to give
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-6-(-
4-(5-(3-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)quinolin-6-yloxy)-
pentyl)piperazin-1-yl)nicotinamide (12 mg, 18% yield) as a white
solid.
[1111] LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18
(50 mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow
Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] to 0% [water+10 mM
NH.sub.4HCO.sub.3] and 100% [CH.sub.3CN] in 3.0 min, then under
this condition for 1.0 min, finally changed to 95% [water+10 mM
NH.sub.4HCO.sub.3] and 5% [CH.sub.3CN] in 0.1 min and under this
condition for 0.7 min.) Purity is 94.03%, Rt=2.703 min; MS Calcd.:
815.33; MS Found: 816.3 [M+H].sup.+.
[1112] HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150
mm*4.6 mm*3.5 .mu.m); Column Temperature: 40.degree. C.; Flow Rate:
1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH.sub.4HCO.sub.3]
and 5% [CH.sub.3CN] to 0% [water+10 mM NH.sub.4HCO.sub.3] and 100%
[CH.sub.3CN] in 10 min, then under this condition for 5 min,
finally changed to 95% [water+10 mM NH.sub.4HCO.sub.3] and 5%
[CH.sub.3CN] in 0.1 min and under this condition for 5 min.) Purity
is 96.02%, Rt=9.232 min.
[1113] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.11 (6H, s),
1.21 (6H, s), 1.50-1.57 (4H, m), 1.81-1.86 (2H, m), 2.33-2.37 (2H,
m), 2.45-2.50 (4H, m), 3.59 (4H, s), 4.05 (1H, d, J=9.2 Hz), 4.15
(2H, t, J=6.4 Hz), 4.30 (1H, s), 6.86 (1H, d, J=9.2 Hz), 7.00 (1H,
dd, J=8.8, 2.0 Hz), 7.21 (1H, d, J=2.4 Hz), 7.45 (1H, d, J=2.4 Hz),
7.50 (1H, dd, J=5.2, 2.4 Hz), 7.63 (1H, d, J=9.2 Hz), 7.91 (1H, d,
J=8.8 Hz), 7.95 (1H, dd, J=8.8, 2.0 Hz), 8.00 (1H, d, J=9.2 Hz),
8.37 (1H, d, J=2.0 Hz), 8.62 (1H, d, J=2.0 Hz), 8.78 (1H, d, J=2.4
Hz), 8.96 (1H, s), 12.28 (1H, brs).
[1114] Chemical Formula: C.sub.44H.sub.46ClN.sub.9O.sub.5,
Molecular Weight: 816.35.
[1115] Total H count from HNMR data: 46.
[1116] Synthesis of Exemplary PROTAC 81
##STR00451## [1117]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(2-(2,6-dioxopiperidin-3-yl)-4-methylene-1-oxo-1,2,3,4-tetrahydroiso-
quinolin-6-yl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide
[1118] Reaction Scheme:
##STR00452## ##STR00453##
Step 1: Synthesis of methyl 4-bromo-2-iodobenzoate
[1119] Into a 1000-mL 3-necked round-bottom flask, was placed
methyl 2-amino-4-bromobenzoate (5.0 g, 21.73 mmol, 1.00 equiv), a
solution of sulfuric acid (20%) (20 mL) in water (100 mL). This was
followed by the addition of a solution of NaNO.sub.2 (1.8 g, 26.09
mmol, 1.20 equiv) in water (20 mL) dropwise with stirring at
0.degree. C., after stirred 1 hour at 0.degree. C. To this was
added a solution of iodopotassium (7.21 g, 43.43 mmol, 2.00 equiv)
in water (30 mL) dropwise with stirring at 0.degree. C. The
resulting solution was stirred for 1 hour at 0.degree. C. in a
water/ice bath. The reaction was then quenched by the addition of
200 mL of water/ice. The resulting solution was extracted with
ethyl acetate (100 mL.times.3) and the organic layers combined. The
resulting mixture was washed with brine (100 mL.times.1). The
residue was applied onto a silica gel column with ethyl
acetate/petroleum ether (1/5). This resulted in 5.97 g (81%) of
methyl 4-bromo-2-iodobenzoate as light yellow oil.
Step 2: Synthesis of methyl 4-bromo-2-cyanobenzoate
[1120] Into a 250-mL round-bottom flask, was placed methyl
4-bromo-2-iodobenzoate (5.8 g, 17.01 mmol, 1.00 equiv), NMP (60
mL), CuCN (1.82 g, 20.45 mmol, 1.20 equiv). The resulting solution
was stirred for 2 hours at 60.degree. C. in an oil bath. The
resulting solution was extracted with ethyl acetate (50 mL.times.2)
and the organic layers combined. The resulting mixture was washed
with FeSO4 (aq.) (50 mL.times.2). The mixture was dried over
anhydrous sodium sulfate. The residue was applied onto a silica gel
column with ethyl acetate/petroleum ether (1/3). This resulted in
3.68 g (90%) of methyl 4-bromo-2-cyanobenzoate as a white
solid.
Step 3: Synthesis of
6'-bromospiro[cyclopropane-1,1'-isoindolin]-3'-one
[1121] Into a 100-mL 3-necked round-bottom flask purged and
maintained with an inert atmosphere of nitrogen, was placed methyl
4-bromo-2-cyanobenzoate (2.0 g, 8.33 mmol, 1.00 equiv), ether (40
mL), 2-(propan-2-yloxy)propane propan-2-ol propan-2-yltitanium
dihydrate (2.75 mL, 1.10 equiv). This was followed by the addition
of EtMgBr (3M) (5.5 mL, 2.00 equiv) dropwise with stirring at
0.degree. C. The resulting solution was stirred for 3 hour at room
temperature. The reaction was then quenched by the addition of 20
mL of hydrogen chloride (1M). The resulting solution was extracted
with ethyl acetate (50 mL.times.2) and the organic layers combined
and dried over anhydrous sodium sulfate. The residue was applied
onto a silica gel column with ethyl acetate/petroleum ether (7/3).
This resulted in 409 mg (21%) of
6'-bromospiro[cyclopropane-1,1'-isoindolin]-3'-one as a yellow
solid.
[1122] LC-MS (ES.sup.+): m/z 238.00, 240.00 [MH.sup.+],
t.sub.R=0.79 min, (1.90 minute run).
Step 4: Synthesis of dimethyl
2-(6'-bromo-3'-oxospiro[cyclopropane-1,1'-isoindolin]-2'-yl)pentanedioate
[1123] Into a 100-mL round-bottom flask, was placed
6'-bromospiro[cyclopropane-1,1'-isoindolin]-3'-one (895.0 mg, 3.76
mmol, 1.00 equiv), N,N-dimethylformamide (15.0 mL),
Cs.sub.2CO.sub.3 (2.44 g, 7.49 mmol, 2.00 equiv), 1,5-dimethyl
2-bromopentanedioate (2.69 g, 11.25 mmol, 3.00 equiv). The
resulting solution was stirred overnight at 100.degree. C. in an
oil bath. The resulting solution was extracted with ethyl acetate
(50 mL.times.2) and the organic layers combined. The resulting
mixture was washed with brine (50 mL.times.2). The mixture was
dried over anhydrous sodium sulfate. The residue was applied onto a
silica gel column with ethyl acetate/petroleum ether (3/7). This
resulted in 740.0 mg (50%) of dimethyl
2-(6'-bromo-3'-oxospiro[cyclopropane-1,1'-isoindolin]-2'-yl)pentanedioate
as light yellow oil.
[1124] LC-MS (ES.sup.+): m/z 395.85, 397.85 [MH.sup.+],
t.sub.R=1.01 min, (1.90 minute run).
Step 5: Synthesis of dimethyl
2-(6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-4-methylene-1-oxo-3,4-dihydr-
oisoquinolin-2(1H)-yl)pentanedioate
[1125] Into a 20-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed dimethyl
2-(6'-bromo-3'-oxospiro[cyclopropane-1,1'-isoindolin]-2'-yl)pentanedioate
(740.0 mg, 1.87 mmol, 1.00 equiv), toluene (10 mL), tert-butyl
piperazine-1-carboxylate (418.0 mg, 2.24 mmol, 1.20 equiv),
Cs.sub.2CO.sub.3 (1.217 g, 3.74 mmol, 2.00 equiv), RuphosPd (140.5
mg, 0.17 mmol, 0.10 equiv). The resulting solution was stirred for
8 hours at 100.degree. C. in an oil bath. The residue was applied
onto a silica gel column with ethyl acetate/petroleum ether (1/1).
This resulted in 303.0 mg (32%) of dimethyl
2-(6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-4-methylene-1-oxo-3,4-dihydr-
oisoquinolin-2(1H)-yl)pentanedioate as light yellow oil.
[1126] LC-MS (ES.sup.+): m/z 502.20 [MH.sup.+], t.sub.R=0.96 min,
(1.90 minute run).
Step 6: Synthesis of tert-butyl
4-[2-(1-carbamoyl-4-methoxy-4-oxobutyl)-4-methylidene-1-oxo-1,2,3,4-tetra-
hydroisoquinolin-6-yl]piperazine-1-carboxylate
[1127] Into a 100 mL round-bottom flask, was placed 1,5-dimethyl
2-(6-[4-[(tert-butoxy)carbonyl]piperazin-1-yl]-4-methylidene-1-oxo-1,2,3,-
4-tetrahydroisoquinolin-2-yl)pentanedioate (400 mg, 0.80 mmol, 1
equiv), MeOH (50 mL), NH.sub.3. The resulting solution was stirred
for 5 hours at room temperature. The residue was applied onto a
silica gel column with dichloromethane/methanol (20:1). This
resulted in 100 mg (25.77%) of tert-butyl
4-[2-(1-carbamoyl-4-methoxy-4-oxobutyl)-4-methylidene-1-oxo-1,2,3,4-tetra-
hydroisoquinolin-6-yl]piperazine-1-carboxylate (and/or it's
regioisomere as shown in the scheme above) as a yellow solid.
Step 7: Synthesis of tert-butyl
4-[2-(2,6-dioxopiperidin-3-yl)-4-methylidene-1-oxo-1,2,3,4-tetrahydroisoq-
uinolin-6-yl]piperazine-1-carboxylate
[1128] Into a 50 mL round-bottom flask, was placed tert-butyl
4-[2-(1-carbamoyl-4-methoxy-4-oxobutyl)-4-methylidene-1-oxo-1,2,3,4-tetra-
hydroisoquinolin-6-yl]piperazine-1-carboxylate (188 mg, 0.39 mmol,
1 equiv), acetonitrile (20 mL), Cs.sub.2CO.sub.3 (629.5 mg, 1.93
mmol, 5 equiv). The resulting solution was stirred for 3 hours at
80.degree. C. in an oil bath. The solids were filtered out. The
residue was applied onto a silica gel column with
dichloromethane/methanol (20:1). The collected fractions were
combined and concentrated under vacuum. This resulted in 100 mg
(56.94%) of tert-butyl
4-[2-(2,6-dioxopiperidin-3-yl)-4-methylidene-1-oxo-1,2,3,4-tetrahydroisoq-
uinolin-6-yl]piperazine-1-carboxylate as a yellow solid.
Step 8: Synthesis of
3-[4-methylidene-1-oxo-6-(piperazin-1-yl)-1,2,3,4-tetrahydroisoquinolin-2-
-yl]piperidine-2,6-dione (Trifluoroacetate salt)
[1129] Into a 50 mL round-bottom flask, was placed tert-butyl
4-[2-(2,6-dioxopiperidin-3-yl)-4-methylidene-1-oxo-1,2,3,4-tetrahydroisoq-
uinolin-6-yl]piperazine-1-carboxylate (120 mg, 0.26 mmol, 1 equiv),
dichloromethane (20 mL), TFA (1.5 mL). The resulting solution was
stirred for 2 hours at room temperature. The resulting mixture was
concentrated under vacuum. This resulted in 93 mg (77.86%) of
3-[4-methylidene-1-oxo-6-(piperazin-1-yl)-1,2,3,4-tetrahydroisoquinolin-2-
-yl]piperidine-2,6-dione (TFA salt) as a yellow solid.
Step 9: Synthesis of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(2-(2,6-dioxopiperidin-3-yl)-4-methylene-1-oxo-1,2,3,4-tetrahydroiso-
quinolin-6-yl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide
[1130] Into a 50 mL round-bottom flask, was placed
4-(4-formylpiperidin-1-yl)-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-
-tetramethylcyclobutyl]benzamide (83 mg, 0.17 mmol, 1 equiv),
dichloromethane (20 mL),
3-[4-methylidene-1-oxo-6-(piperazin-1-yl)-1,2,3,4-tetrahydroisoquinolin-2-
-yl]piperidine-2,6-dione (TFA salt) (91.2 mg, 0.20 mmol, 1.2
equiv), NaBH(OAc).sub.3 (106.8 mg, 0.50 mmol, 3 equiv). The
resulting solution was stirred for 1 overnight at room temperature.
The reaction was then quenched by the addition of water. The
resulting solution was extracted with dichloromethane The resulting
mixture was washed with brine. The mixture was dried over anhydrous
sodium sulfate. The crude product was purified by Prep-HPLC with
the following conditions: Column, XBridge Prep C18 OBD Column, 19
150 mm Sum; mobile phase, water (10 mmol/L NH.sub.4HCO.sub.3) and
acetonitrile (58.0% acetonitrile up to 78.0% in 8 min); Detector,
UV 254 nm. The product was obtained and concentrated under vacuum,
and lyophiliation. This resulted in 80.3 mg (57.42%) of
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(2-(2,6-dioxopiperidin-3-yl)-4-methylene-1-oxo-1,2,3,4-tetrahydroiso-
quinolin-6-yl)piperazin-1-yl)methyl)piperidin-1-yl)benzamide as a
white solid.
[1131] .sup.1H NMR (400 MHz, DMSO) .delta. 10.88 (s, 1H), 7.91-7.89
(m, 1H), 7.78-7.72 (m, 3H), 7.50-7.47 (d, J=9.2 Hz, 1H), 7.21 (s,
1H), 7.09-6.94 (m, 5H), 5.75 (s, 1H), 5.29 (s, 1H), 5.15-4.95 (m,
1H), 4.32 (s, 1H), 4.21-4.04 (m, 3H), 3.87-3.84 (m, 2H), 3.32-3.30
(m, 7H), 2.84-2.76 (m, 3H), 2.65-2.56 (m, 1H), 2.48-2.37 (m, 1H),
2.22-2.18 (m, 2H), 1.90-1.79 (m, 4H), 1.40-1.16 (m, 9H), 1.16-1.09
(m, 6H);
[1132] LC-MS (ES.sup.+): m/z 832.35[MH.sup.+], t.sub.R=1.53 min,
(3.00 minute run).
[1133] Chemical formula: C.sub.47H.sub.54ClN.sub.7O.sub.5
[831.39]
[1134] Total H count from HNMR data: 54
[1135] Synthesis of Exemplary PROTAC 82
##STR00454## [1136]
N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)-4-(-
4-((4-(2-(2,6-dioxopiperidin-3-yl)-1-oxo-1,2-dihydroisoquinolin-6-yl)piper-
azin-1-yl)methyl)piperidin-1-yl)benzamide
[1137] Synthetic Scheme
##STR00455## ##STR00456##
Step 1: Synthesis of tert-butyl
4-(1-oxo-2H-isoquinolin-6-yl)piperazine-1-carboxylate
##STR00457##
[1139] A mixture of 6-bromo-2H-isoquinolin-1-one (2 g, 8.93 mmol, 1
eq), tert-butyl piperazine-1-carboxylate (2.49 g, 13.39 mmol, 1.5
eq), sodium tert-butoxide (2 M, 13.4 mL, 3 eq) and
[2-(2-aminophenyl)phenyl]-chloro-palladium;
dicyclohexyl-[2-(2,6-diisopropoxyphenyl)phenyl]phosphane (693 mg,
0.89 mmol, 0.1 eq) in tert-amyl alcohol (30 mL) was degassed and
purged with nitrogen for 3 times, and then the mixture was stirred
at 100.degree. C. for 12 hours under nitrogen atmosphere. LCMS
showed the reaction was completed and desired MS can be detected.
The reaction mixture was diluted with water (100 mL) and extracted
with ethyl acetate (50 mL.times.3). The combined organic phase was
washed with saturated brine (50 mL.times.2), dried with anhydrous
sodium sulfate, filtered and concentrated in vacuum. The residue
was purified by silica gel chromatography (petroleum ether:ethyl
acetate=20:1 to 3:1) to give tert-butyl
4-(1-oxo-2H-isoquinolin-6-yl)piperazine-1-carboxylate (2.3 g, 6.98
mmol, 78% yield) as a white solid
[1140] LCMS: MS (ESI) m/z: 330.1 [M+1].sup.+
[1141] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta.: 10.73 (s, 1H),
8.27 (d, J=8.8 Hz, 1H), 7.13-7.05 (m, 2H), 6.81 (d, J=2.4 Hz, 1H),
6.42 (d, J=7.2 Hz, 1H), 3.65-3.59 (m, 4H), 3.39-3.34 (m, 4H), 1.50
(s, 9H)
[1142] Chemical Formula: C.sub.18H.sub.23N.sub.3O.sub.3, Molecular
Weight: 329.39
[1143] Total H count from HNMR data: 23.
Step 2: Synthesis of dimethyl
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]pentanedio-
ate
##STR00458##
[1145] To a solution of tert-butyl
4-(1-oxo-2H-isoquinolin-6-yl)piperazine-1-carboxylate (800 mg, 2.43
mmol, 1 eq) in dimethylformamide (16 mL) was added cesium carbonate
(2.37 g, 7.29 mmol, 3 eq) and dimethyl 2-bromopentanedioate (696
mg, 2.91 mmol, 1.2 eq). The mixture was stirred at 100.degree. C.
for 12 hours. LCMS showed the reaction was completed and desired MS
can be detected. The reaction mixture was adjusted to pH 4.about.5
with hydrochloric acid (1 M). The reaction was diluted with water
(60 mL) and extracted with ethyl acetate (30 mL.times.3). The
combined organic phase was washed with saturated brine (30
mL.times.2), dried with anhydrous sodium sulfate, filtered and
concentrated in vacuum to give the crude product dimethyl
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]pentanedio-
ate (700 mg, crude) as a light yellow oil was used into the next
step without further purification.
[1146] LCMS: MS (ESI) m/z: 474.1 [M+1].sup.+
[1147] Chemical Formula: C.sub.25H.sub.33N.sub.3O.sub.7, Molecular
Weight: 487.55
Step 3: Synthesis of
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]pentanedio-
ic acid
##STR00459##
[1149] To a solution of dimethyl
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]
pentanedioate (800 mg, 1.64 mmol, 1 eq) in tetrahydrofuran (5 mL),
methanol (5 mL) and water (5 mL) was added lithium hydroxide
monohydrate (413 mg, 9.85 mmol, 6 eq). The mixture was stirred at
30.degree. C. for 12 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction was adjusted to pH 4-5
with hydrochloric acid (1 M) and diluted with water (25 mL). The
reaction was extracted with ethyl acetate (15 mL.times.3). The
combined organic phase was dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum to give the crude product
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]pentanedio-
ic acid (800 mg, crude) as a yellow solid was used into the next
step without further purification.
[1150] LCMS: MS (ESI) m/z: 460.1 [M+1].sup.+
[1151] Chemical Formula: C.sub.23H.sub.29N.sub.3O.sub.7, Molecular
Weight: 459.49
Step 4: Synthesis of tert-butyl
4-[2-(2,6-dioxo-3-piperidyl)-1-oxo-6-isoquinolyl]piperazine-1-carboxylate
##STR00460##
[1153] To a solution of
2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-1-oxo-2-isoquinolyl]pentanedio-
ic acid (800 mg, 1.74 mmol, 1 eq) in N-methyl-2-pyrrolidone (10 mL)
was added urea (522 mg, 8.71 mmol, 5 eq). The mixture was stirred
at 160.degree. C. for 2 hours. LCMS showed the reaction was
completed and desired MS can be detected. The reaction mixture was
diluted with water (50 mL) and extracted with ethyl acetate (25
mL.times.3). The combined organic phase was washed with saturated
brine (30 mL.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum. The residue was purified by
Semi-preparative reverse phase HPLC (column: Phenomenex Synergi
Max-RP 250*50 mm*10 um; mobile phase: [water(0.225% FA)-ACN]; B %:
30ACN %-60ACN %,30 min; 50% min). Tert-butyl
4-[2-(2,6-dioxo-3-piperidyl)-1-oxo-6-isoquinolyl]piperazine-1-carboxylate
(100 mg, 0.22 mmol, 13% yield) was obtained as a white solid.
[1154] LCMS: MS (ESI) m/z: 441.1 [M+1].sup.+
[1155] Chemical Formula: C.sub.23H.sub.28N.sub.4O.sub.5, Molecular
Weight: 440.49
Step 5: Synthesis of
3-(1-oxo-6-piperazin-1-yl-2-isoquinolyl)piperidine-2,6-dione
##STR00461##
[1157] To a solution of tert-butyl
4-[2-(2,6-dioxo-3-piperidyl)-1-oxo-6-isoquinolyl]piperazine-1-carboxylate
(100 mg, 0.22 mmol, 1 eq) in dichloromethane (3 mL) was added 4 M
hydrochloric acid in dioxane (3 mL, 52.86 eq). The mixture was
stirred at 25.degree. C. for 4 hours. LCMS showed 14% of the
starting material was remained and the reaction was stirred another
1 hour. Thin layer chromatography (dichloromethane:methanol=10:1)
showed the reaction was completed. The reaction mixture was
concentrated under reduced pressure to remove dichloromethane and
dioxane, hydrochloric acid to give the crude product
3-(1-oxo-6-piperazin-1-yl-2-isoquinolyl)piperidine-2,6-dione (85
mg, crude, hydrochloride) as a light yellow solid.
[1158] LCMS: MS (ESI) m/z: 341.0 [M+1].sup.+.
[1159] Chemical Formula: C.sub.18H.sub.20N.sub.4O.sub.3, Molecular
Weight: 340.38
Step 6: Synthesis of
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-[4-[[4--
[2-(2,6-dioxo-3-piperidyl)-1-oxo-6-isoquinolyl]piperazin-1-yl]methyl]-1-pi-
peridyl]benzamide
##STR00462##
[1161] To a solution of
3-(1-oxo-6-piperazin-1-yl-2-isoquinolyl)piperidine-2,6-dione (85
mg, 0.22 mmol, 1 eq, hydrochloride) in 1,2-dichloroethane (4 mL)
was added triethylamine (0.9 mmol, 0.12 mL, 4 eq) and
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-(4-form-
yl-1-piperidyl)benzamide (111 mg, 0.22 mmol, 1 eq). The mixture was
stirred at 20.degree. C. for 0.5 hour. Sodium triacetoxyborohydride
(95 mg, 0.45 mmol, 2 eq) was added and the mixture was stirred at
20.degree. C. for 12 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture was
concentrated under reduced pressure to remove 1,2-dichloroethane.
The residue was dissolved into dimethylformamide (3 mL) and
filtered. The filter was purified by Semi-preparative reverse phase
HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase:
[water(0.05% HCl)-ACN]; B %: 23%-53%,10 min)
N-[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]-4-[4-[[4--
[2-(2,6-dioxo-3-piperidyl)-1-oxo-6-isoquinolyl]piperazin-1-yl]methyl]-1-pi-
peridyl]benzamide (50.9 mg, 0.05 mmol, 25% yield, 95.8% purity,
hydrochloride) as a white solid.
[1162] LCMS: MS (ESI) m/z: 818.4 [M+1].sup.+.
[1163] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta.: 11.07-10.90
(m, 1H), 10.57 (s, 1H), 8.10-8.01 (m, 1H), 7.91 (d, J=8.8 Hz, 1H),
7.80 (d, J=8.8 Hz, 2H), 7.58 (br d, J=9.2 Hz, 1H), 7.33 (d, J=7.6
Hz, 1H), 7.29-7.23 (m, 1H), 7.21 (d, J=2.4 Hz, 1H), 7.16-7.05 (m,
3H), 7.01 (dd, J=2.4, 8.8 Hz, 1H), 6.56-6.37 (m, 1H), 6.56-6.37 (m,
1H), 4.34 (s, 1H), 4.06 (d, J=9.2 Hz, 3H), 3.87 (br d, J=12.8 Hz,
2H), 3.68-3.60 (m, 1H), 3.22-3.08 (m, 4H), 3.00-2.76 (m, 3H),
2.65-2.55 (m, 1H), 2.54-2.52 (m, 2H), 2.47-2.43 (m, 1H), 2.23-2.11
(m, 1H), 2.05-1.90 (m, 3H), 1.55-1.30 (m, 2H), 1.23 (s, 6H), 1.14
(s, 6H)
[1164] Chemical Formula: C.sub.46H.sub.52ClN.sub.7O.sub.5,
Molecular Weight: 818.40
[1165] Total H count from HNMR data: 53.
[1166] Synthesis of Exemplary PROTAC 89
##STR00463## [1167]
3-[3-[4-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-pip-
eridyl]methyl]piperazin-1-yl]phenyl]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-d-
ione
Step 1: Preparation of 6-tert-butoxytetralin-1-one
##STR00464##
[1169] To a stirred solution of 6-hydroxytetralin-1-one (50 g,
308.29 mmol, 1 eq) in anhydrous dichloromethane (2000 mL) at
0.degree. C. was added tert-butyl 2,2,2-trichloroethanimidate
(67.36 g, 308.29 mmol, 55 mL, 1 eq) and pyridinium
para-toluenesulfonate (7.75 g, 30.83 mmol, 0.1 eq). The reaction
mixture was stirred at 10.degree. C. for 3 hours. Additional
portion of tert-butyl 2,2,2-trichloroethanimidate (67.36 g, 308.29
mmol, 55 mL, 1 eq) and pyridinium para-toluenesulfonate (7.75 g,
30.83 mmol, 0.1 eq) was added and the reaction mixture was stirred
at 10.degree. C. for 15 hours. This process was repeated three
times. Thin layer chromatography (petroleum ether:ethyl
acetate=3:1, R.sub.f=0.8) showed the most of reactant was still
remained, the reaction mixture was stirred at 10.degree. C. for 72
hours. The reaction mixture was quenched by addition a solution of
sodium hydrogen carbonate (1500 mL) at 15.degree. C., and then
extracted with dichloromethane (300 mL.times.3). The combined
organic layers were washed with brine (300 mL.times.2), dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(petroleum ether:ethyl acetate=100:1 to 50:1) to get
6-tert-butoxytetralin-1-one (21 g, 96.20 mmol, 31% yield) as a
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.97 (d,
J=8.8 Hz, 1H), 6.91 (dd, J=2.4, 8.8 Hz, 1H), 6.82 (d, J=2.0 Hz,
1H), 2.93-3.90 (t, J=6.0 Hz, 2H), 2.63-2.60 (m, t, J=6.0 Hz, 2H),
2.13 (m, 2H), 1.43 (s, 9H)
Step 2: Preparation of
(6-tert-butoxy-3,4-dihydronaphthalen-1-yl)trifluoromethanesulfonate
##STR00465##
[1171] To a solution of 6-tert-butoxytetralin-1-one (40 g, 183.24
mmol, 1 eq) in tetrahydrofuran (500 mL) was added lithium
diiso-propylamide (2 M, 137 mL, 1.5 eq) at -70.degree. C. The
mixture was stirred at -70.degree. C. for 1 hour, then
1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)
methanesulfonamide (72.01 g, 201.56 mmol, 1.1 eq) in
tetrahydrofuran (200 mL) was added dropwise to the mixture. The
reaction mixture was stirred at 20.degree. C. for 2 hours. Thin
layer chromatography (petroleum ether:ethyl acetate=5:1) showed the
reaction was completed. Saturated ammonium chloride (300 mL) was
added to the mixture, the organic phase was separated. Ethyl
acetate (500 mL.times.3) was added to the mixture, the resulting
mixture was washed with brine (1000 mL.times.2). The combined
organic phase was dried over sodium sulfate, filtered and
concentrated in vacuum. The residue was purified by silica gel
chromatography (petroleum ether:ethyl acetate=1:0 to 50:1) to give
(6-tert-butoxy-3,4-dihydronaphthalen-1-yl)
trifluoromethanesulfonate (52 g, 144.64 mmol, 78% yield, 97%
purity) as a yellow oil. LC-MS (ESI) m/z: 294.9 [M+1-56].sup.+.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 7.30 (d, J=6.4 Hz, 1H),
6.91 (d, J=8.4 Hz, 1H), 6.84 (s, 1H), 5.95 (s, 1H), 2.93-2.78 (m,
2H), 2.59-2.46 (m, 2H), 1.42 (s, 9H).
Step 3: Preparation of
4-(6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol
##STR00466##
[1173] To a solution of (6-tert-butoxy-3,4-dihydronaphthalen-1-yl)
trifluoromethanesulfonate (52 g, 148.42 mmol, 1 eq),
(4-hydroxyphenyl)boronic acid (24.57 g, 178.11 mmol, 1.2 eq) in
dioxane (800 mL) and water (150 mL) was added potassium carbonate
(41.03 g, 296.84 mmol, 2 eq) and
(1,1'-Bis(diphenylphosphino)ferrocene)palladium(II) dichloride
(10.86 g, 14.84 mmol, 0.1 eq) under nitrogen. The reaction mixture
was stirred at 100.degree. C. for 10 hours. Thin layer
chromatography (petroleum ether:ethyl acetate=5:1) showed the
reaction was completed. The residue was diluted with water (500 mL)
and extracted with ethyl acetate (500 mL.times.2). The combined
organic layers were washed with brine (1000 mL.times.2), dried with
anhydrous sodium sulfate, filtered and concentrated in vacuum. The
residue was purified by silica gel chromatography (petroleum
ether:tetrahydrofuran=50:1 to 20:1) to give
4-(6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol (43 g, 131.46
mmol, 88% yield, 90% purity) as a yellow oil. LCMS (ESI) m/z: 239.1
[M+1-56].sup.+; .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.23 (d,
J=7.6 Hz, 2H), 6.91 (d, J=8.0 Hz, 1H), 6.87-6.79 (m, 3H), 6.73 (d,
J=8.4 Hz, 1H), 5.95 (s, 1H), 4.83-4.75 (m, 1H), 2.87-2.73 (m, 2H),
2.44-2.31 (m, 2H), 1.37 (s, 9H)
Step 4: Preparation of
4-(2-bromo-6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol
##STR00467##
[1175] To a solution of
4-(6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol (1 g, 3.06 mmol,
1 eq) in acetonitrile (20 mL) was added N-bromosuccinimide (489 mg,
2.75 mmol, 0.9 eq) in three portions. The reaction mixture was
stirred at 20.degree. C. for 1.5 hours. LC-MS showed the reaction
was completed. The residue was diluted with water (20 mL) and
extracted with ethyl acetate (20 mL.times.2). The combined organic
layers were washed with brine (20 mL.times.2), dried with anhydrous
sodium sulfate, filtered and concentrated in vacuum. The residue
was purified by silica gel chromatography (petroleum ether:ethyl
acetate=1:0 to 20:1) to give
4-(2-bromo-6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol (1 g,
2.46 mmol, 80% yield, 91% purity) as a yellow oil. LC-MS (ESI) m/z:
316.9 [M+1-56].sup.+; .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.12 (d, J=8.4 Hz, 2H), 6.90 (d, J=8.0 Hz, 2H), 6.77 (s, 1H),
6.69-6.62 (m, 1H), 6.60-6.53 (m, 1H), 4.86 (s, 1H), 2.96 (s, 4H),
1.35 (s, 9H).
Step 5: Preparation of
4-(6-tert-butoxy-2-phenyl-3,4-dihydronaphthalen-1-yl)phenol
##STR00468##
[1177] To a solution of
4-(2-bromo-6-tert-butoxy-3,4-dihydronaphthalen-1-yl)phenol (1 g,
2.46 mmol, 1 eq), phenylboronic acid (314 mg, 2.58 mmol, 1.05 eq)
in dioxane (10 mL) and water (2 mL) was added potassium carbonate
(678 mg, 4.91 mmol, 2 eq) and
(1,1'-bis(diphenylphosphino)ferrocene)palladium(II) dichloride (179
mg, 0.24 mmol, 0.1 eq) under nitrogen. The reaction mixture was
stirred at 100.degree. C. for 12 hours. LC-MS showed the reaction
was completed. The residue was diluted with water (20 mL) and
extracted with ethyl acetate (20 mL.times.2). The combined organic
layers were washed with brine (20 mL.times.3), dried with anhydrous
sodium sulfate, filtered and concentrated in vacuum. The residue
was purified by silica gel chromatography (petroleum ether:ethyl
acetate=1:0 to 10:1) to get
4-(6-tert-butoxy-2-phenyl-3,4-dihydronaphthalen-1-yl)phenol (930
mg, 2.35 mmol, 95% yield, 93% purity) as a orange oil. LCMS (ESI)
m/z: 314.1 [M+1-56].sup.+; .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 7.16-7.09 (m, 2H), 7.08-6.99 (m, 3H), 6.97-6.89 (m, 2H),
6.86-6.82 (m, 1H), 6.74-6.66 (m, 4H), 4.70 (s, 1H), 2.99-2.89 (m,
2H), 2.84-2.75 (m, 2H), 1.37 (s, 9H)
Step 6: Preparation of
4-(6-tert-butoxy-2-phenyl-tetralin-1-yl)phenol
##STR00469##
[1179] To a solution of
4-(6-tert-butoxy-2-phenyl-3,4-dihydronaphthalen-1-yl)phenol (930
mg, 2.35 mmol, 1 eq) in tetrahydrofuran (20 mL) and methanol (4 mL)
was added palladium on activated carbon catalyst (100 mg, 10%
purity) under nitrogen. The suspension was degassed under vacuum
and purged with hydrogen three times. The mixture was stirred under
hydrogen (50 psi) at 30.degree. C. for 36 hours. LC-MS showed the
reaction was completed. The reaction mixture was filtered and the
solution was concentrated. The resulting material was directly used
into the next step without further purification to afford
cis-4-(6-tert-butoxy-2-phenyl-tetralin-1-yl)phenol (870 mg, 2.14
mmol, 91% yield, 91% purity) as a white solid. LC-MS (ESI) m/z:
317.0 [M+1-56].sup.+; .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.22-7.12 (m, 3H), 6.89-6.78 (m, 4H), 6.74 (dd, J=2.0, 8.4 Hz, 1H),
6.45 (d, J=8.4 Hz, 2H), 6.27 (d, J=8.4 Hz, 2H), 4.51 (s, 1H), 4.25
(d, J=4.8 Hz, 1H), 3.38 (dd, J=3.2, 12.8 Hz, 1H), 3.08-2.99 (m,
2H), 2.27-2.08 (m, 1H), 1.87-1.76 (m, 1H), 1.37 (s, 9H)
Step 7: Preparation of WX-ARV-HD-012-E1,
4-[(1S,2R)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenol
##STR00470##
[1181] 4-(6-tert-butoxy-2-phenyl-tetralin-1-yl)phenol (870 mg, 2.13
mmol, 1 eq) was subjected to supercritical fluid chromatography for
chiral separation (column: AD, 250 mm.times.30 mm, 5 um; mobile
phase: 0.1% ammonium hydroxide in methanol, 20%-20%, 4.2 min for
each run) to get 4-[(1S,
2R)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenol (420 mg, 1.04 mmol,
97% yield, 92% purity) as the first fraction and 4-[(1R,
2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenol (420 mg, 1.04 mmol,
97% yield, 92% purity) as a second fraction. Fraction 1:
[.quadrature.].sub.D=+336.9 (C=0.50 g/100 mL in ethyl acetate),
LC-MS (ESI) m/z: 395.1 [M+23].sup.+; .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 9.02 (s, 1H), 7.20-7.07 (m, 3H), 6.87-6.79
(m, 3H), 6.79-6.72 (m, 1H), 6.71-6.64 (m, 1H), 6.36 (d, J=8.4 Hz,
2H), 6.15 (d, J=8.4 Hz, 2H), 4.19 (d, J=4.8 Hz, 1H), 3.31-3.26 (m,
1H), 3.09-2.89 (m, 2H), 2.17-2.04 (m, 1H), 1.79-1.65 (m, 1H), 1.29
(s, 9H).
[1182] Fraction 2: [.alpha.].sub.D=-334.1 (C=0.50 g/100 mL in ethyl
acetate), LC-MS (ESI) m/z: 395.2 [M+23].sup.+; .sup.1H-NMR (400
MHz, DMSO-d.sub.6) .delta.: 9.02 (s, 1H), 7.21-7.06 (m, 3H),
6.88-6.78 (m, 3H), 6.78-6.72 (m, 1H), 6.71-6.64 (m, 1H), 6.36 (d,
J=8.4 Hz, 2H), 6.15 (d, J=8.4 Hz, 2H), 4.19 (d, J=4.8 Hz, 1H),
3.30-3.27 (m, 1H), 3.08-2.90 (m, 2H), 2.16-2.04 (m, 1H), 1.79-1.65
(m, 1H), 1.29 (s, 9H).
Step 8: Preparation of
4-(6-benzyloxy-2-phenyl-3,4-dihydronaphthalen-1-yl)phenyl]
1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonate
##STR00471##
[1184] To a solution of
4-[(1R,2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenol (1 g, 2.68
mmol, 1 eq) and 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonyl
fluoride (811 mg, 2.68 mmol, 1 eq) in tetrahydrofuran (5 mL) and
acetonitrile (5 mL) was added potassium carbonate (557 mg, 4.03
mmol, 1.5 eq). The reaction mixture was stirred at 25.degree. C.
for 16 hours. TLC (petroleum ether:ethyl acetate=10:1) indicated
the starting material was consumed completely and one new spot
formed. The reaction mixture was concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(petroleum ether:ethyl acetate=1:0 to 50:1). The desired compound
[4-[(1R,2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenyl]1,1,2,2,-
3,3,4,4,4-nonafluorobutane-1-sulfonate (1.6 g, 2.44 mmol, 91%
yield) was obtained as a colorless oil. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.21-7.11 (m, 3H), 6.94-6.86 (m, 3H), 6.84-6.73
(m, 4H), 6.46 (d, J=8.8 Hz, 2H), 4.33 (d, J=5.2 Hz, 1H), 3.50-3.40
(m, 1H), 3.16-2.95 (m, 2H), 2.20-2.02 (m, 1H), 1.91-1.79 (m, 1H),
1.38 (s, 9H)
Step 9: Preparation of 1-[4-(6-benzyloxy-2-phenyl-3,
4-dihydronaphth alen-1-yl)
phenyl]-4-(dimethoxymethyl)piperidine
##STR00472##
[1186] A mixture of
[4-[(1R,2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenyl]
1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonate (1.6 g, 2.44 mmol, 1
eq), 4-(dimethoxymethyl)piperidine (584 mg, 3.67 mmol, 1.5 eq),
sodium tert-butoxide (705 mg, 7.33 mmol, 3 eq), palladium acetate
(82 mg, 0.37 mmol, 0.15 eq) and
dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (233 mg, 0.49
mmol, 0.2 eq) in toluene (30 mL) was degassed and purged with
nitrogen 3 times, and then the mixture was stirred at 90.degree. C.
for 16 hours under nitrogen atmosphere. LC-MS showed one main peak
with desired MS was detected. TLC (petroleum ether:ethyl
acetate=10:1) indicated the starting material was consumed
completely and one new spot formed. The mixture was cooled, diluted
with ethyl acetate (50 mL), filtered on a plug of celite, the
filter cake was washed with ethyl acetate (30 mL). The filtrate was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether:ethyl acetate=100:1 to 10:1). The desired compound
1-[4-[(1R,2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenyl]-4-(dimethoxyme-
thyl)piperidine (1.1 g, 2.14 mmol, 87% yield) was obtained as a
white solid. LCMS (ESI) m/z: 514.3 [M+1].sup.+; .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.21-7.11 (m, 3H), 6.88-6.78 (m, 4H), 6.73
(dd, J=2.4, 8.0 Hz, 1H), 6.57 (d, J=8.4 Hz, 2H), 6.27 (d, J=8.8 Hz,
2H), 4.23 (d, J=4.8 Hz, 1H), 4.06 (d, J=7.2 Hz, 1H), 3.63-3.52 (m,
2H), 3.41-3.30 (m, 7H), 3.13-2.96 (m, 2H), 2.54 (d, J=2.0, 12.0 Hz,
2H), 2.28-2.10 (m, 1H), 1.85-1.63 (m, 4H), 1.49-1.31 (m, 11H).
Step 10: Preparation of 1-[4-[4-(dimethoxymethyl)-1-piperidyl]
phenyl]-2-phenyl-tetralin-6-ol
##STR00473##
[1188] To a solution of
1-[4-[(1R,2S)-6-tert-butoxy-2-phenyl-tetralin-1-yl]phenyl]-4-(dimethoxyme-
thyl)piperidine (1.1 g, 2.14 mmol, 1 eq) in tetrahydrofuran (45 mL)
was added sulfuric acid (2 M, 43 mL, 40 eq). The reaction mixture
was stirred at 70.degree. C. for 1 hour. Thin-Layer Chromatography
(petroleum ether:ethyl acetate=3:1) indicated the starting material
was consumed completely and one new spot formed. The reaction
mixture was quenched by addition saturated sodium bicarbonate
solution to pH=7.about.8, and extracted with ethyl acetate (20
mL.times.2). The combined organic layers were washed with brine (20
mL), dried over sodium sulfate, filtered and concentrated under
reduced pressure. The residue was used into next step without
further purification. The desired compound
1-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]piperidine-4-carbal-
dehyde (900 mg, 2.14 mmol, 99% yield, 97% purity) was obtained as
light yellow solid. LCMS MS (ESI) m/z: 412.1 [M+1].sup.+
Step 11: Preparation of ethyl (Z)-3-(4-bromophenyl)but-2-enoate
##STR00474##
[1190] To a suspension of sodium hydride (2.41 g, 60.29 mmol, 60%
purity, 1.2 eq) in tetrahydrofuran (100 mL) cooled to 0.degree. C.
was slowly added ethyl 2-diethoxyphosphorylacetate (13.52 g, 60.29
mmol, 12 mL, 1.2 eq) and the reaction mixture was stirred at
25.degree. C. for 1 hour. A solution of 1-(4-bromophenyl)ethanone
(10 g, 50.24 mmol, 1 eq) in tetrahydrofuran (100 mL) was added
dropwise and the mixture was stirred at 25.degree. C. for 12 hours.
To this mixture was added saturated aqueous ammonium chloride (50
mL). The mixture was extracted with ethyl acetate (100 mL.times.3).
The organic layer was dried over anhydrous sodium sulfate and
concentrated. The residue was purified with prep-HPLC
(acetonitrile:water=50:1 to 5:1). Ethyl
(Z)-3-(4-bromophenyl)but-2-enoate (6.6 g, 24.52 mmol, 48.9% yield)
was obtained as a yellow oil and ethyl
(E)-3-(4-bromophenyl)but-2-enoate (2.6 g, 9.66 mmol, 19.3% yield)
was also obtained as a yellow oil. LC/MS (ESI) m/z: 270.0
[M+1].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48 (d,
J=8.4 Hz, 2H), 7.09 (d, J=8.4 Hz, 2H), 5.93 (s, 1H), 4.02 (q, J=7.2
Hz, 2H), 2.16 (s, 3H), 1.13 (t, J=7.2 Hz, 3H); .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.58 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.8 Hz,
2H), 6.05 (s, 1H), 4.02 (q, J=14.4 Hz, 2H), 2.52 (s, 3H), 1.13 (q,
J=14.4 Hz, 3H).
Step 12: Preparation of tert-butyl
4-[4-[(Z)-3-ethoxy-1-methyl-3-oxo-prop-1-enyl]phenyl]piperazine-1-carboxy-
late
##STR00475##
[1192] A mixture of ethyl (Z)-3-(4-bromophenyl)but-2-enoate (2.0 g,
7.43 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (2.08 g,
11.15 mmol, 1.5 eq), cesium carbonate (4.84 g, 14.86 mmol, 2 eq),
palladium acetate (334 mg, 1.49 mmol, 0.2 eq) and XPhos (708 mg,
1.49 mmol, 0.2 eq) in toluene (30 mL) was degassed and purged with
nitrogen three times. The mixture was stirred at 100.degree. C. for
12 hours under nitrogen atmosphere. The resulting mixture were
filtered and concentrated under reduced pressure. The residue was
washed with saturated brine (30 mL.times.2) and extracted with
ethyl acetate (30 mL.times.2). The combined organic layers were
dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by
semi-preparative reverse phase HPLC (column: Phenomenex Synergi
Max-RP 250.times.50 mm, 10 um; mobile phase: [water(0.225% formic
acid)-acetonitrile]; B %: 50% acetonitrile-80% acetonitrile, 30
min). Tert-butyl
4-[4-[(Z)-3-ethoxy-1-methyl-3-oxo-prop-1-enyl]phenyl]piperazine-1-carboxy-
late (2.24 g, 5.83 mmol, 78% yield, 97% purity) was obtained as a
white solid. LC/MS (ESI) m/z: 375.1 [M].sup.+.
Step 13: Preparation of tert-butyl
4-[4-[(E)-1-(bromomethyl)-3-ethoxy-3-oxo-prop-1-enyl]phenyl]piperazine-1--
carboxylate
##STR00476##
[1194] To a solution of tert-butyl
4-[4-[(Z)-3-ethoxy-1-methyl-3-oxo-prop-1-enyl]phenyl]
piperazine-1-carboxylate (1.0 g, 2.60 mmol, 1 eq) and
1-bromopyrrolidine-2,5-dione (462.93 mg, 2.60 mmol, 1 eq) in
dichloroethane (10 mL) was added benzoyl peroxide (189 mg, 0.78
mmol, 0.3 eq). The mixture was degassed and purged with nitrogen 3
times. The mixture was stirred at 70.degree. C. for 12 hours under
nitrogen atmosphere. LC-MS showed .about.24% of desired compound
was detected. The reaction mixture was washed with saturated
aqueous brine (25 mL.times.2) and extracted with dichloromethane
(40 mL.times.2). The combined organic layers were dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(petroleum ether/ethyl acetate=50/1 to 25:1). Tert-butyl
4-[4-[(E)-1-(bromomethyl)-3-ethoxy-3-oxo-prop-1-enyl]phenyl]piperazine-1--
carboxylate (0.3 g, 0.43 mmol, 16% yield, 65% purity) was obtained
as a yellow oil. LC/MS (ESI) m/z: 453.0 [M+1].sup.+.
Step 14: Preparation of tert-butyl
4-[4-[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]
phenyl]piperazine-1-carboxylate
##STR00477##
[1196] To a mixture of 3-aminopiperidine-2,6-dione (84.95 mg, 0.52
mmol, 1.2 eq, HCl salt) in dimethyl formamide (3 mL) was added
N,N-diisopropylethylamine (556 mg, 4.30 mmol, 0.7 mL, 10 eq). The
mixture was stirred at 20.degree. C. for 1 hour. Then tert-butyl
4-[4-[(E)-1-(bromomethyl)-3-ethoxy-3-oxo-prop-1-enyl] phenyl]
piperazine-1-carboxylate (0.3 g, 0.43 mmol, 1 eq) was added to the
reaction and the mixture was stirred at 50.degree. C. for 0.5 hour.
The resulting mixture was further heated up to 120.degree. C. and
stirred for 12 hours. LC-MS showed desired compound was detected.
The reaction mixture was cooled, diluted with ethyl acetate, washed
with saturated aqueous brine (25 mL.times.2) and extracted with
ethyl acetate (30 mL.times.2). The combined organic layers were
dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by triturated with
methyl tert-butyl ether (15 mL). The product tert-butyl
4-[4-[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]
phenyl]piperazine-1-carboxylate (175 mg, 0.23 mmol, 52% yield, 58%
purity) was obtained as a brown solid. LC/MS (ESI) m/z: 455.1
[M+1].sup.+.
Step 15: Preparation of
3-[5-oxo-3-(4-piperazin-1-ylphenyl)-2H-pyrrol-1-yl]piperidine-2,6-dione
##STR00478##
[1198] To a solution of tert-butyl
4-[4-[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]
phenyl]piperazine-1-carboxylate (175 mg, 0.22 mmol, 1 eq) was added
HCl in dioxane (4 M, 5 mL). The mixture was stirred at 20.degree.
C. for 1 hour. The reaction mixture was concentrated under vacuum
to give a residue.
3-[5-Oxo-3-(4-piperazin-1-ylphenyl)-2H-pyrrol-1-yl]
piperidine-2,6-dione (260 mg, crude, HCl salt) was obtained as a
brown solid. LC/MS (ESI) m/z: 355.1 [M+1].sup.+.
Step 16: Preparation of
3-[3-[4-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-pip-
eridyl]methyl]piperazin-1-yl]phenyl]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-d-
ione (Exemplary PROTAC 89)
##STR00479##
[1200] To a solution of
3-[5-oxo-3-(4-piperazin-1-ylphenyl)-2H-pyrrol-1-yl]piperidine-2,6-dione
(260 mg, 0.66 mmol, 1 eq, HCl salt) in dichloroethane (3 mL) was
added triethylamine (202 mg, 2.00 mmol, 0.3 mL, 3 eq) and
1-[4-[(1R,
2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]piperidine-4-carbaldehyde
(109 mg, 0.26 mmol, 0.4 eq). The mixture was stirred at 25.degree.
C. for 15 minutes, and then sodium borohydride acetate (282 mg,
1.33 mmol, 2 eq) was added. The mixture was stirred at 25.degree.
C. for another 11.5 hours. LC-MS showed .about.74% of desired
compound was detected. The reaction mixture was diluted with
dichloromethane, washed with saturated brine (20 mL.times.2) and
extracted with dichloromethane (30 mL.times.2). The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure to give a residue. The
residue was purified by prep-HPLC (column: Phenomenex Synergi C18
150.times.25 mm, 10 um; mobile phase: [water (0.225% formic
acid)-acetonitrile]; B %: 22%-43% in 10 min). The product
3-[3-[4-[4-[[1-[4-[(1R,
2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-piperidyl]methyl]piperazin-
-1-yl]phenyl]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione (38.7 mg,
0.04 mmol, 7% yield, 95% purity, formate salt) was obtained as a
brown solid.
[1201] LC/MS (ESI) m/z: 750.3 [M+1].sup.+;
[1202] .sup.1H-NMR (400 MHz, DMSO-d6) .delta. 10.95 (s, 1H), 8.19
(s, 1H), 7.50 (d, J=8.8 Hz, 2H), 7.21-7.06 (m, 3H), 6.96 (d, J=8.8
Hz, 2H), 6.83 (d, J=6.4 Hz, 2H), 6.64 (d, J=8.4 Hz, 1H), 6.59 (d,
J=2.4 Hz, 1H), 6.53 (d, J=8.8 Hz, 2H), 6.47 (dd, J=2.4, 8.4 Hz,
1H), 6.40 (s, 1H), 6.19 (d, J=8.8 Hz, 2H), 4.91 (dd, J=5.2, 13.2
Hz, 1H), 4.45-4.33 (m, 1H), 4.29-4.19 (m, 1H), 4.12 (d, J=4.8 Hz,
1H), 3.52 (s, 1H), 3.49-3.48 (m, 1H), 3.30 (s, 2H), 3.24 (s, 3H),
3.04-2.79 (m, 3H), 2.60 (s, 1H), 2.52 (d, J=2.0 Hz, 2H), 2.47 (b s,
4H), 2.32-2.23 (m, 1H), 2.18 (d, J=6.8 Hz, 2H), 2.13-2.03 (m, 1H),
1.99-1.88 (m, 1H), 1.80-1.59 (m, 4H), 1.22-1.06 (m, 2H).
[1203] Synthesis of Exemplary PROTAC 102
##STR00480## [1204]
3-[4-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-piperi-
dyl]methyl]piperazin-1-yl]-6-oxo-pyridazin-1-yl]piperidine-2,6-dione
Step 1: Preparation of tert-butyl
4-(5-chloro-6-oxo-1H-pyridazin-4-yl) piperazine-1-carboxylate
##STR00481##
[1206] To a solution of 4,5-dichloro-1H-pyridazin-6-one (5 g, 30.31
mmol, 1 eq) in dimethylsulfoxide (100 mL) was added
diisopropylethylamin (11.75 g, 90.92 mmol, 3 eq) and tert-butyl
piperazine-1-carboxylate hydrochloride (6.75 g, 30.31 mmol, 1 eq).
The mixture was stirred at 120.degree. C. for 3 hours. The
resulting mixture was cooled to room temperature, filtered and
quenched by addition of water (500 mL), then extracted with ethyl
acetate (100 mL.times.3). The combined organic phase was washed
with brine (100 mL), dried over anhydrous sodium sulfate, filtered
and concentrated under reduce pressure. The residue was purified by
silica gel chromatography (dichloromethane: methyl alcohol=200:1 to
100:1). Tert-butyl
4-(5-chloro-6-oxo-1H-pyridazin-4-yl)piperazine-1-carboxylate (8.18
g, 24.95 mmol, 82% yield, 96% purity) was obtained as a yellow
solid. LC/MS (ESI) m/z: 315.1 [M+1].sup.+; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 11.95 (s, 1H), 7.66 (s, 1H), 3.64-3.57 (m, 4H),
3.44-3.36 (m, 4H), 1.49 (s, 9H).
Step 2: Preparation of tert-butyl 4-(6-oxo-1H-pyridazin-4-yl)
piperazine-1-carboxylate
##STR00482##
[1208] To a solution of tert-butyl
4-(5-chloro-6-oxo-1H-pyridazin-4-yl)piperazine-1-carboxylate (1 g,
3.18 mmol, 1 eq) in tetrahydrofuran (1 mL) and methanol (9 mL) was
added palladium/active carbon catalyst (200 mg, 10% purity) under
nitrogen. The suspension was degassed under vacuum and purged with
hydrogen several times. The mixture was stirred under hydrogen (45
psi) at 25.degree. C. for 0.5 hour. The reaction was basified with
triethylamine, and then filtered and the filtrate was concentrated.
The residue was used for next step without further purification.
Tert-butyl 4-(6-oxo-1H-pyridazin-4-yl) piperazine-1-carboxylate (1
g, crude) was obtained as a white solid. LC/MS (ESI) m/z: 281.1
[M+1].sup.+; .sup.1H NMR (400 MHz, DMSO) .delta. 12.22 (br s, 1H),
10.38-10.03 (m, 1H), 7.91 (d, J=2.8 Hz, 1H), 3.46-3.37 (m, 4H),
3.04 (br d, J=7.2 Hz, 4H), 1.41 (s, 9H).
Step 3: Preparation of tert-butyl
4-[1-(2,6-dioxo-3-piperidyl)-6-oxo-pyridazin-4-yl]piperazine-1-carboxylat-
e
##STR00483##
[1210] To a solution of tert-butyl
4-(6-oxo-1H-pyridazin-4-yl)piperazine-1-carboxylate (950 mg, 3.39
mmol, 1 eq) in dimethylsulfoxide (15 mL) was added sodium hydride
(271 mg, 6.78 mmol, 60% purity, 2 eq) at 25.degree. C. followed by
the addition of 3-bromopiperidine-2,6-dione (650 mg, 3.39 mmol, 1
eq). The mixture was stirred at 25.degree. C. for 12 hours. The
resulting mixture was filtered and quenched by addition of water
(200 mL), and extracted with ethyl acetate (50 mL.times.3). The
combined organic phase was washed with brine (50 mL.times.3), dried
over anhydrous sodium sulfate, filtered and concentrated under
reduce pressure. The residue was purified by semi-preparative
reverse phase HPLC (column: Phenomenex luna C18 250.times.50 mm, 10
um; mobile phase: [water(0.225% formic acid)-ACN]; B %: 16%-46% in
30 min). Tert-butyl
4-[1-(2,6-dioxo-3-piperidyl)-6-oxo-pyridazin-4-yl]piperazine-1-carboxylat-
e (190 mg, 0.48 mmol, 14% yield) was obtained as a white solid.
LC/MS (ESI) m/z: 392.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO)
.delta. 8.02 (s, 1H), 7.72 (d, J=2.8 Hz, 1H), 5.74 (dd, J=5.3, 11.6
Hz, 1H), 3.62-3.53 (m, 4H), 3.34 (s, 4H), 2.95-2.83 (m, 1H),
2.82-2.58 (m, 2H), 2.27-2.17 (m, 1H), 1.49 (s, 9H).
Step 4: Preparation of 3-(6-oxo-4-piperazin-1-yl-pyridazin-1-yl)
piperidine-2,6-dione
##STR00484##
[1212] To a solution of tert-butyl
4-[1-(2,6-dioxo-3-piperidyl)-6-oxo-pyridazin-4-yl]piperazine-1-carboxylat-
e (190 mg, 0.48 mmol, 1 eq) in dichloromethane (2 mL) was added
hydrochloride in dioxane (4 M, 10 mL, 78 eq). The mixture was
stirred at 25.degree. C. for 0.5 hour. The resulting mixture was
concentrated under reduced pressure to remove dioxane. The crude
product was used into the next step without further purification.
Compound 3-(6-oxo-4-piperazin-1-yl-pyridazin-1-yl)
piperidine-2,6-dione (120 mg, 0.36 mmol, 75% yield, hydrochloride)
was obtained as a light yellow solid. LC/MS (ESI) m/z: 292.0
[M+1].sup.+.
Step 5: Preparation of
3-[4-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-piperi-
dyl]methyl]piperazin-1-yl]-6-oxo-pyridazin-1-yl]piperidine-2,6-dione
(Exemplary PROTAC 102)
##STR00485##
[1214] To a solution of
3-(6-oxo-4-piperazin-1-yl-pyridazin-1-yl)piperidine-2,6-dione (57
mg, 0.17 mmol, 1.2 eq, hydrochloride salt) and 1-[4-[(1R,
2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]
piperidine-4-carbaldehyde (60 mg, 0.14 mmol, 1 eq, see step 10,
synthesisof exemplary PROTAC 89) in 1,2-dichloroethane (3 mL) was
added triethylamine (30 mg, 0.29 mmol, 2 eq) and the mixture was
stirred at 25.degree. C. for 0.5 hour. Then sodium
triacetoxyborohydride (93 mg, 0.43 mmol, 3 eq) was added. The
mixture was further stirred at 25.degree. C. for 0.5 hour. The
reaction mixture was concentrated under reduced pressure to remove
1,2-dichloroethane. The residue was purified by prep-HPLC (column:
Luna C18 150.times.25 mm, 5 um; mobile phase: [water(0.225% formic
acid)-ACN]; B %: 18%-38% in 7.8 min). Compound 3-[4-[4-[[1-[4-[(1R,
2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenyl]-4-piperidyl]methyl]piperazin-
-1-yl]-6-oxo-pyridazin-1-yl]piperidine-2,6-dione (33 mg, 0.04 mmol,
30% yield, 99% purity, formate salt) was obtained as a white solid.
LC/MS (ESI) m/z: 687.3 [M+1].sup.+; .sup.1H-NMR (400 MHz, DMSO-d6)
.delta. 10.96 (s, 1H), 8.22 (s, 1H), 8.04 (d, J=2.4 Hz, 1H),
7.18-7.10 (m, 3H), 6.83 (d, J=6.4 Hz, 2H), 6.64 (d, J=8.4 Hz, 1H),
6.59 (d, J=2.4 Hz, 1H), 6.52 (d, J=8.8 Hz, 2H), 6.47 (dd, J=2.4,
8.4 Hz, 1H), 6.19 (d, J=8.8 Hz, 2H), 5.84 (d, J=2.8 Hz, 1H), 5.58
(dd, J=5.2, 12.4 Hz, 1H), 4.12 (d, J=4.4 Hz, 1H), 3.27 (s, 4H),
3.02-2.79 (m, 3H), 2.57 (d, J=4.0 Hz, 1H), 2.52 (d, J=2.0 Hz, 4H),
2.46 (s, 1H), 2.42 (d, J=4.8 Hz, 5H), 2.20-2.06 (m, 3H), 2.02-1.93
(m, 1H), 1.73 (d, J=14.0 Hz, 3H), 1.61 (s, 1H), 1.19-1.07 (m,
2H).
[1215] Synthesis of Exemplary PROTAC 106
##STR00486## [1216]
3-(4-(3-(1-(3-(4-((1R,2S)-6-hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-
-1-yl)phenoxy)propyl)piperidin-4-yl)phenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-
-yl)piperidine-2,6-dione
[1217] Synthetic Scheme Part 1
##STR00487##
Step 1: Preparation of tert-butyl
4-(3-hydroxyphenyl)-3,6-dihydro-2H-pyridine-1-carboxylate
##STR00488##
[1219] To a mixture of tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-
-carboxylate (7.00 g, 22.64 mmol, 1.00 eq) and 3-iodophenol (4.98
g, 22.64 mmol, 1.00 eq) in dioxane (100 mL) and water (10 mL) was
added potassium carbonate (6.26 g, 45.28 mmol, 2.00 eq) and
cyclopentyl(diphenyl)phosphane; dichloropalladium; iron (1.66 g,
2.26 mmol, 0.10 eq) under nitrogen. The mixture stirred at
90.degree. C. for 4 hours under nitrogen. LC-MS showed the starting
material was consumed completely and one main peak with desired MS
was detected. The reaction mixture was poured into water (500 mL)
and filtered, the filtrated diluted with ethyl acetate (200 mL) and
extracted with ethyl acetate (300 mL*3), the combined organic phase
washed with saturation brine (150 mL), dried over anhydrous sodium
sulfate, filtered and concentrated under reduce pressure. The
residue was purified by silica gel column chromatography (Petroleum
ether/Ethyl acetate=10/1 to 8/1). Tert-butyl
4-(3-hydroxyphenyl)-3,6-dihydro-2H-pyridine-1-carboxylate (4.00 g,
14.53 mmol, 64% yield) was obtained as a white solid
[1220] LCMS: MS (ESI) m/z: 298.1 [M+23].sup.+
[1221] Chemical Formula: C.sub.16H.sub.21NO.sub.3, Molecular
Weight: 275.34
Step 2: Preparation of tert-butyl
4-(3-hydroxyphenyl)piperidine-1-carboxylate
##STR00489##
[1223] To a solution of tert-butyl
4-(3-hydroxyphenyl)-3,6-dihydro-2H-pyridine-1-carboxylate (4.00 g,
14.53 mmol, 1.00 eq) in methanol (4 mL) was added palladium on
activated carbon catalyst (1.00 g, 10% purity) under nitrogen. The
suspension was degassed under vacuum and purged with hydrogen
several times. The mixture was stirred under hydrogen (40 psi) at
30.degree. C. for 4 hours. LC-MS showed that the starting material
was consumed completely and one main peak with desired MS was
detected. The reaction mixture was filtered and concentrated under
reduce pressure. The residue was used for next step without further
purification. Crude tert-butyl
4-(3-hydroxyphenyl)piperidine-1-carboxylate (4.00 g, crude) as an
off-white solid was obtained
[1224] LCMS: MS (ESI) m/z: 300 [M+23].sup.+
[1225] Chemical Formula: C.sub.16H.sub.23NO.sub.3, Molecular
Weight: 277.36
Step 3: Preparation of tert-butyl
4-[3-[(E)-3-methoxy-1-methyl-3-oxo-prop-1-enoxy]phenyl]piperidine-1-carbo-
xylate
##STR00490##
[1227] To a solution of tert-butyl
4-(3-hydroxyphenyl)piperidine-1-carboxylate (2.00 g, 7.21 mmol,
1.00 eq) and methyl but-2-ynoate (1.06 g, 10.82 mmol, 1.50 eq) in
isopropanol (20 mL) was added 1,4-diazabicyclo[2.2.2]octane (808
mg, 7.21 mmol, 1.00 eq). The mixture was stirred at 15.degree. C.
for 12 hours. LC-MS showed that the starting material was consumed
completely and one main peak with desired MS was detected. The
reaction mixture was quenched by water 20 mL at 15.degree. C., and
extracted with ethyl acetate (20 mL.times.3). The combined organic
layers were washed with saturation brine (20 mL.times.2), dried
over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (petroleum ether:ethyl acetate=20:1 to 10:1).
Tert-butyl
4-[3-[(E)-3-methoxy-1-methyl-3-oxo-prop-1-enoxy]phenyl]piperidine-1-carbo-
xylate (1.72 g, 4.58 mmol, 63% yield) was obtained as a white
solid.
[1228] LCMS: MS (ESI) m/z: 398.1 [M+23].sup.+
[1229] Chemical Formula: C.sub.21H.sub.29NO.sub.5, Molecular
Weight: 375.46
Step 4: Preparation of tert-butyl
(E)-4-(3-((1-bromo-4-methoxy-4-oxobut-2-en-2-yl)oxy)phenyl)piperidine-1-c-
arboxylate
##STR00491##
[1231] To a mixture of
tert-butyl4-[3-[(E)-3-methoxy-1-methyl-3-oxo-prop-1-enoxy]phenyl]piperidi-
ne-1-carboxylate (1.2 g, 3.20 mmol, 1.00 eq) in dichloroethane (50
mL) was added a solution of N-bromosuccinimide (853 mg, 4.79 mmol,
1.5 eq) and benzoyl peroxide (232 mg, 0.96 mmol, 0.3 eq). The
mixture stirred at 70.degree. C. for 12 hours. LC-MS showed that
the starting material was consumed completely and one main peak
with desired MS was detected. The mixture was quenched by addition
of water (200 mL), diluted with ethyl acetate (20 mL) and extracted
with ethyl acetate (30 mL.times.3), the combined organic phase
washed with saturation brine (30 mL), dried over anhydrous sodium
sulfate, filtered and concentrated under reduce pressure. The
residue was purified by silica gel column chromatography (petroleum
ether:ethyl acetate=100:1.about.40:1).
Tert-butyl4-[3-[(E)-1-(bromomethyl)-3-methoxy-3-oxo-prop-1-enoxy]phenyl]p-
iperidine-1-carboxylate (960 mg, crude) was obtained as a yellow
oil.
[1232] LCMS: MS (ESI) m/z: 477.9 [M+23].sup.+
[1233] Chemical Formula: C.sub.21H.sub.28BrNO.sub.5, Molecular
Weight: 454.35
Step 5: Preparation of tert-butyl
4-[3-[[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]oxy]phenyl]piperidi-
ne-1-carboxylate
##STR00492##
[1235] To a mixture of 3-aminopiperidine-2,6-dione (1.56 g, 9.46
mmol, 5 eq, hydrochloride) in dimethylformamide (20 mL) was added
diisopropylethylamine (2.45 g, 18.93 mmol, 10 eq). The mixture
stirred at 14.degree. C. for 1 hr. The tert-butyl
4-[3-[(E)-1-(bromomethyl)-3-methoxy-3-oxo-prop-1-enoxy]phenyl]piperidine--
1-carboxylate (860 mg, 1.89 mmol, 1 eq) was added to the reaction.
And then the mixture stirred at 50.degree. C. for 0.5 hour. Then
the mixture was heated up to 100.degree. C. for 12 hours. LC-MS
showed that the starting material bromide was consumed completely
and one main peak with desired MS was detected. The mixture was
quenched by addition of water (200 mL), diluted with ethyl acetate
(50 mL) and extracted with ethyl acetate (50 mL.times.3), the
combined organic phase washed with saturation brine (50
mL.times.3), dried over anhydrous sodium sulfate, filtered and
concentrated under reduce pressure. The residue was purified by
triturated with methyl tert-butyl ether (30 mL). Tert-butyl
4-[3-[[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]oxy]phenyl]piperidi-
ne-1-carboxylate (376 mg, 0.80 mmol, 42% yield) as a brown solid
was obtained
[1236] LCMS: MS (ESI) m/z: 492.2 [M+23].sup.+
[1237] Chemical Formula: C.sub.25H.sub.31N.sub.3O.sub.6, Molecular
Weight: 469.53
Step 6: Preparation of
3-[5-oxo-3-[3-(4-piperidyl)phenoxy]-2H-pyrrol-1-yl]piperidine-2,6-dione
##STR00493##
[1239] To a mixture of tert-butyl
4-[3-[[1-(2,6-dioxo-3-piperidyl)-5-oxo-2H-pyrrol-3-yl]oxy]phenyl]piperidi-
ne-1-carboxylate (420 mg, 0.89 mmol, 1 eq) in dichloromethane (10
mL) was added hydrogen chloride/dioxane (4 M, 4 mL, 20 eq). The
mixture stirred at 14.degree. C. for 0.5 hour. LC-MS showed that
the starting material was consumed completely and one main peak
with desired MS was detected. The reaction was concentrated under
reduce pressure. The residue was used for next step without further
purification. Crude
3-[5-oxo-3-[3-(4-piperidyl)phenoxy]-2H-pyrrol-1-yl]piperidine-2,6-dione
(400 mg, crude, hydrochloride) as a brown solid was obtained
[1240] LCMS: MS (ESI) m/z: 370 [M+1].sup.+
[1241] Chemical Formula: C.sub.20H.sub.23N.sub.3O.sub.4, Molecular
Weight: 369.41
[1242] Synthetic Scheme Part 2
##STR00494##
Step 7: Preparation of
(cis)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
##STR00495##
[1244] To a solution of
4-[(1R,2S)-6-benzyloxy-2-phenyl-tetralin-1-yl]phenol (1.00 g, 2.46
mmol, 1.00 eq) in acetone (20 mL) was added potassium carbonate
(1.02 g, 7.38 mmol, 3.00 eq) and 1,3-dibromopropane (2.48 g, 12.30
mmol, 1.3 mL, 5.00 eq). The mixture was stirred at 70.degree. C.
for 12 hours. LC-MS showed the starting material was consumed
completely and one main peak with desired MS was detected. The
reaction mixture was quenched by addition water (40 mL) at
15.degree. C., and extracted with ethyl acetate (20 mL.times.3).
The combined organic layers were washed with ethyl acetate (20
mL.times.2), dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure. The residue was purified by
prep-HPLC (column: Phenomenex Synergi Max-RP 250*50 mm*10 um;
mobile phase: [water(0.225% FA)-ACN]; B %: 70%-100%,30; 52% min).
(cis)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
(850 mg, 1.61 mmol, 65% yield, 99% purity) was obtained as a white
solid.
[1245] LCMS: MS (ESI) m/z: 527.2 [M+1].sup.+
[1246] Chemical Formula: C.sub.32H.sub.31BrO.sub.2, Molecular
Weight: 527.49
Step 8: Preparation of
(1S,2R)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
and
(1R,2S)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
##STR00496##
[1248] The enantiomers of (cis)
6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin (850 mg,
1.61 mmol, 1.00 eq) were separated using Supercritical Fluid
Chromatography. The residue was separated by Supercritical Fluid
Chromatography (column: OJ(250 mm*30 mm, 10 um); mobile phase:
[0.1% NH3H2O MEOH]; B %:60%-60%,20.9 min; 300minmin) Flow rate: 2
mL/min Wavelength: 220 nm.
[1249]
(1S,2R)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
(350 mg, 0.65 mmol, 81% yield, 97% purity) was obtained as a white
solid.
[1250]
(1R,2S)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
(350 mg, 0.66 mmol, 82% yield, 99% purity) was obtained as a white
solid.
[1251] Chemical Formula: C.sub.32H.sub.31BrO.sub.2, Molecular
Weight: 527.49
Step 9: Preparation of
3-[3-[3-[1-[3-[4-[(1R,2S)-6-benzyloxy-2-phenyl-tetralin-1-yl]phenoxy]prop-
yl]-4-piperidyl]phenoxy]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione
##STR00497##
[1253] To a mixture of
(1R,2S)-6-benzyloxy-1-[4-(3-bromopropoxy)phenyl]-2-phenyl-tetralin
(164 mg, 0.31 mmol, 1.1 eq) and
3-[5-oxo-3-[3-(4-piperidyl)phenoxy]-2H-pyrrol-1-yl]piperidine-2,6-dione
(115 mg, 0.28 mmol, 1 eq, hydrochloride) in acetonitrile (5 mL) was
added diisopropylethylamine (110 mg, 0.85 mmol, 3 eq) and potassium
iodide (47 mg, 0.28 mmol, 1 eq). The mixture stirred at 100.degree.
C. for 1.5 hours. LC-MS showed that the amine starting material was
consumed completely and one main peak with desired MS was detected.
The mixture was quenched by addition water (100 mL), diluted with
ethyl acetate (15 mL), extracted with ethyl acetate (20
mL.times.4), the combined organic phase washed with saturation
brine (20 mL), dried over anhydrous sodium sulfate, filtered and
concentrated under reduce pressure. The residue was purified by
prep-TLC (dichloromethane:methanol=10:1).
3-[3-[3-[1-[3-[4-[(1R,2S)-6-benzyloxy-2-phenyl-tetralin-1-yl]phenoxy]prop-
yl]-4-piperidyl]phenoxy]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione
(100 mg, 0.12 mmol, 43% yield) as a brown solid was obtained.
[1254] LCMS: MS (ESI) m/z: 816.4 [M+1].sup.+
[1255] Chemical Formula: C.sub.52H.sub.53N.sub.3O.sub.6, Molecular
Weight: 815.99
Step 10: Preparation of
3-[3-[3-[1-[3-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenoxy]propyl-
]-4-piperidyl]phenoxy]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione
##STR00498##
[1257] To a mixture of
3-[3-[3-[1-[3-[4-[(1R,2S)-6-benzyloxy-2-phenyl-tetralin-1-yl]phenoxy]prop-
yl]-4-piperidyl]phenoxy]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione
(100 mg, 0.12 mmol, 1 eq) in dichloromethane (5 mL) was added boron
tribromide (92 mg, 0.37 mmol, 3 eq) at -68.degree. C. The mixture
stirred at -68.degree. C. for 30 minutes. LC-MS showed the starting
material was consumed completely and one main peak with desired MS
was detected. The residue was diluted with water (20 mL) and
extracted with ethyl acetate (20 mL.times.2). The combined organic
layers were washed with saturated brines (20 mL.times.3), dried
with anhydrous sodium sulfate, filtered and concentrated in vacuum.
The residue was purified by prep-HPLC (column: Boston Green ODS
150*30 5u; mobile phase: [water(0.225% FA)-ACN]; B %: 34%-55%,10
min).
3-[3-[3-[1-[3-[4-[(1R,2S)-6-hydroxy-2-phenyl-tetralin-1-yl]phenoxy]propyl-
]-4-piperidyl]phenoxy]-5-oxo-2H-pyrrol-1-yl]piperidine-2,6-dione
(16 mg, 0.02 mmol, 16% yield, 97% purity, formate) was obtained as
a white solid
[1258] LCMS: MS (ESI) m/z: 726.3 [M+1].sup.+
[1259] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1260] .delta.=10.92 (s, 1H), 9.48-8.87 (m, 1H), 8.21 (s, 1H),
7.41-7.34 (m, 1H), 7.23-7.06 (m, 6H), 6.82 (d, J=6.8 Hz, 2H),
6.66-6.57 (m, 2H), 6.55-6.44 (m, 3H), 6.25 (d, J=8.8 Hz, 2H),
4.91-4.82 (m, 2H), 4.18-3.97 (m, 3H), 3.84 (t, J=6.4 Hz, 2H),
3.30-3.27 (m, 2H), 3.02-2.82 (m, 5H), 2.55-2.52 (m, 3H), 2.39 (t,
J=6.9 Hz, 2H), 2.26 (dd, J=4.8, 13.6 Hz, 1H), 2.11-1.58 (m,
11H)
[1261] Chemical Formula: C.sub.45H.sub.47N.sub.3O.sub.6, Molecular
Weight: 725.87
[1262] Synthesis of Exemplary PROTAC 107
##STR00499## [1263]
3-(8-((2-(4-(2-(4-((2-(4-bromophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)-
phenoxy)ethyl)piperazin-1-yl)ethyl)amino)-2-methyl-4-oxoquinazolin-3
(4H)-yl)piperidine-2,6-dione
[1264] Synthetic Scheme Part 1:
##STR00500## ##STR00501##
[1265] Synthetic Scheme Part 2:
##STR00502## ##STR00503##
Step 1: Synthesis of
tert-butyl4-[2-(4-benzyloxyphenoxy)ethyl]piperazine-1-carboxylate
##STR00504##
[1267] To a solution of tert-butyl
4-(2-chloroethyl)piperazine-1-carboxylate (1.00 g, 4.02 mmol, 1.00
eq), 4-benzyloxyphenol (965 mg, 4.82 mmol, 1.20 eq) in
N,N-dimethylformamide (20 mL) was added cesium carbonate (1.57 g,
4.82 mmol, 1.20 eq) and potassium iodide (66 mg, 0.4 mmol, 0.10 eq)
under nitrogen. The reaction was stirred at 80.degree. C. for 10
hours. TLC (Petroleum ether/Ethyl acetate=3/1) and LCMS showed most
of the starting material was consumed. Water (100 mL) was added to
the mixture, the resulting mixture was extracted with Ethyl acetate
(50 mL.times.3). The combined organic phase was washed with brine
(80 mL), dried over sodium sulfate, filtered and concentrated in
vacuum. The residue was purified by silica gel chromatography
(Petroleum ether/Ethyl acetate=50/1 to 3/1). tert-butyl
4-[2-(4-benzyloxyphenoxy)ethyl]piperazine-1-carboxylate (1.4 g,
3.39 mmol, 84% yield) was obtained as a colorless oil.
[1268] Chemical Formula: C24H32N2O4, Molecular Weight: 412.5
[1269] Total H count from HNMR data: 32
[1270] .sup.1H NMR: (400 MHz, CHLOROFORM-d)
[1271] .delta.: 7.46-7.29 (m, 5H), 6.95-6.88 (m, 2H), 6.88-6.81 (m,
2H), 5.02 (s, 2H), 4.07 (t, J=5.8 Hz, 2H), 3.51-3.42 (m, 4H), 2.80
(t, J=5.8 Hz, 2H), 2.56-2.48 (m, 4H), 1.47 (s, 9H)
Step 2: Synthesis of tert-butyl
4-[2-(4-hydroxyphenoxy)ethyl]piperazine-1-carboxylate
##STR00505##
[1273] To a solution of tert-butyl
4-[2-(4-benzyloxyphenoxy)ethyl]piperazine-1-carboxylate (1.40 g,
3.39 mmol, 1.00 eq) in methanol (20 mL) was added palladium on
carbon (200 mg, 10% purity) under nitrogen. The suspension was
degassed under vacuum and purged with hydrogen several times. The
mixture was stirred under hydrogen (50 psi) at 20.degree. C. for 4
hours. TLC (Petroleum ether/Ethyl acetate=1/1) showed most of the
starting material was consumed. The reaction mixture was filtered
and the filter was concentrated in vacuum. Tert-butyl
4-[2-(4-hydroxyphenoxy)ethyl]piperazine-1-carboxylate (1 g, 3.07
mmol, 90% yield, 99% purity) was obtained as a light yellow
solid.
[1274] Chemical Formula: C17H26N2O4, Molecular Weight: 322.4
[1275] Total H count from HNMR data: 26
[1276] .sup.1H NMR: (400 MHz, CHLOROFORM-d)
[1277] .delta.: 6.74 (s, 4H), 4.04 (t, J=5.6 Hz, 2H), 3.54-3.38 (m,
5H), 2.79 (t, J=5.6 Hz, 2H), 2.53 (s, 4H), 1.46 (s, 9H)
Step 3: Synthesis of tert-butyl
4-(2-(4-((2-(4-bromophenyl)-6-methoxy-1-oxidobenzo[b]thiophen-3-yl)oxy)ph-
enoxy)ethyl)piperazine-1-carboxylate
##STR00506##
[1279] To a solution of tert-butyl
4-[2-(4-hydroxyphenoxy)ethyl]piperazine-1-carboxylate (234 mg, 0.72
mmol, 1.00 eq) in N,N-dimethylformamide (5 mL) was added NaH (29
mg, 0.72 mmol, 60% mineral oil, 1.00 eq) at 0.degree. C. The
mixture was stirred at 20.degree. C. for 0.5 hour.
3-bromo-2-(4-bromophenyl)-6-methoxy-1-oxido-benzothiophen-1-ium
(300 mg, 0.72 mmol, 1.00 eq) was added, and then the mixture was
stirred at 20.degree. C. for 1 hour. LCMS showed the reaction was
completed and desired MS can be detected. The reaction mixture was
quenched with water (10 mL) and extracted with ethyl acetate (10
mL.times.3). The combined organic phase was washed with saturated
brine (10 mL.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum to afford tert-butyl
4-[2-[4-[2-(4-bromophenyl)-6-methoxy-1-oxido-benzothiophen-1-ium-3-yl]oxy-
phenoxy]ethyl]piperazine-1-carboxylate (430 mg, 0.66 mmol, 90%
yield) as a yellow solid, which was directly used for next step
without further purification.
[1280] LCMS: MS (ESI) m/z: 657.0 [M+1].sup.+
[1281] .sup.1H NMR: (400 MHz, CDCl.sub.3)
[1282] .delta.: 7.65 (d, J=8.4 Hz, 2H), 7.52-7.46 (m, 3H),
7.05-6.89 (m, 4H), 6.81 (d, J=8.4 Hz, 2H), 4.05 (t, J=5.6 Hz, 2H),
3.89 (s, 3H), 3.50-3.42 (m, 4H), 2.81 (t, J=5.6 Hz, 2H), 2.52 (s,
4H), 1.47 (s, 9H)
[1283] Chemical Formula: C.sub.32H.sub.35BrN.sub.2O.sub.6S,
Molecular Weight: 655.60
[1284] Total H count from HNMR data: 35.
Step 4: Synthesis of tert-butyl
4-(2-(4-((2-(4-bromophenyl)-6-methoxybenzo[b]thiophen-3-yl)oxy)phenoxy)et-
hyl)piperazine-1-carboxylate
##STR00507##
[1286] To a solution of tert-butyl
4-[2-[4-[2-(4-bromophenyl)-6-methoxy-1-oxido-benzothiophen-1-ium-3-yl]oxy-
phenoxy]ethyl]piperazine-1-carboxylate (370 mg, 0.56 mmol, 1.00 eq)
in acetonitrile (6 mL) was added sodium iodide (254 mg, 1.69 mmol,
3.00 eq) and trimethylchlorosilane (123 mg, 1.13 mmol, 2.00 eq).
The mixture was stirred at 20.degree. C. for 1 hour. LCMS showed
the reaction was completed and desired MS can be detected. The
reaction mixture was quenched with saturated sodium sulfite (2 mL),
diluted with water (15 mL) and extracted with ethyl acetate (10
mL.times.2). The combined organic phase was washed with saturated
brine (10 mL.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum to give the crude product
tert-butyl
4-[2-[4-[2-(4-bromophenyl)-6-methoxy-benzothiophen-3-yl]oxyphenoxy]ethyl]
piperazine-1-carboxylate (350 mg, crude) as a yellow oil, which was
directly used for next step without further purification.
[1287] LCMS: MS (ESI) m/z: 639.0 [M+1].sup.+.
[1288] Chemical Formula: C.sub.32H.sub.35BrN.sub.2O.sub.5S,
Molecular Weight: 639.60
Step 5: Synthesis of
2-(4-bromophenyl)-3-(4-(2-(piperazin-1-yl)ethoxy)phenoxy)benzo[b]thiophen-
-6-ol
##STR00508##
[1290] To a solution of tert-butyl
4-[2-[4-[2-(4-bromophenyl)-6-methoxy-benzothiophen-3-yl]oxyphenoxy]ethyl]-
piperazine-1-carboxylate (350 mg, 0.55 mmol, 1.00 eq) in
dichloromethane (6 mL) was added boron tribromide (410 mg, 1.64
mmol, 0.16 mL, 3.00 eq) at 0.degree. C. The mixture was stirred at
20.degree. C. for 1 hour. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture was quenched
with saturated sodium bicarbonate (5 mL) at 0.degree. C., and
diluted with water (10 mL) and extracted with dichloromethane (10
mL.times.3). The combined organic phase was washed with saturated
brine (5 mL.times.2), dried with anhydrous sodium sulfate, filtered
and concentrated in vacuum to give
2-(4-bromophenyl)-3-[4-(2-piperazin-1-ylethoxy)phenoxy]benzothiophen-6-ol
(250 mg, crude) as a yellow solid, which was directly used for next
step without further purification.
[1291] LCMS: MS (ESI) m/z: 527.0 [M+1].sup.+.
[1292] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1293] .delta.: 7.65-7.56 (m, 4H), 7.31 (d, J=2.0 Hz, 1H), 7.14 (d,
J=8.4 Hz, 1H), 6.86 (s, 4H), 6.83 (dd, J=2.0, 8.4 Hz, 1H), 5.75 (s,
1H), 3.97 (t, J=5.6 Hz, 2H), 2.78-2.66 (m, 4H), 2.61 (t, J=5.6 Hz,
2H), 2.40 (s, 4H), 2.45-2.34 (m, 1H)
[1294] Chemical Formula: C.sub.26H.sub.25BrN.sub.2O.sub.3S,
Molecular Weight: 525.46
[1295] Total H count from HNMR data: 25.
Step 6: Synthesis of
2-methyl-8-nitro-4H-benzo[d][1,3]oxazin-4-one
##STR00509##
[1297] A mixture of 2-amino-3-nitro-benzoic acid (2 g, 10.98 mmol,
1.00 eq) in acetic anhydride (10 mL) was stirred at 120.degree. C.
for additional 16 hours. TLC (Petroleum ether: Ethyl acetate)
indicated a new spot was formed. The reaction mixture was
concentrated to remove the solvent. The residue was triturated with
petroleum ether:ethyl acetate=2:1 (30 mL), then filtrated. The
filtrate cake was obtained as the desired product
2-methyl-8-nitro-3,1-benzoxazin-4-one (600 mg, 2.91 mmol, 26%
yield).
[1298] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1299] .delta.: 8.42-8.31 (m, 2H), 7.72 (t, J=8.0 Hz, 1H), 3.42 (s,
3H).
[1300] Chemical Formula: C.sub.9H.sub.6N.sub.2O.sub.4, Molecular
Weight: 206.15
[1301] Total H count from HNMR data: 6.
Step 7: Synthesis of
3-(2-methyl-8-nitro-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione
##STR00510##
[1303] To a solution of 2-methyl-8-nitro-3,1-benzoxazin-4-one (1 g,
4.85 mmol, 1.00 eq) and 3-aminopiperidine-2,6-dione (956 mg, 5.82
mmol, 1.20 eq, hydrochloride) in N,N-dimethylformamide (15 mL) was
added triphenyl phosphite (2.26 g, 7.27 mmol, 1.9 mL, 1.50 eq). The
mixture was stirred at 100.degree. C. for 14 hours. LCMS showed the
reaction was completed and desired MS can be detected. The reaction
mixture was diluted with water (40 mL) extracted with ethyl acetate
(30 mL.times.2). The combined organic phase was washed with brine
(30 mL.times.3), dried with anhydrous sodium sulfate, filtered and
concentrated in vacuum to give the crude product
3-(2-methyl-8-nitro-4-oxo-quinazolin-3-yl)piperidine-2,6-dione (450
mg, crude) was used into the next step without further
purification.
[1304] LCMS: MS (ESI) m/z: 316.9 [M+1].sup.+.
[1305] Chemical Formula: C.sub.14H.sub.12N.sub.4O.sub.5, Molecular
Weight: 316.27
Step 8: Synthesis of
3-(8-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione
##STR00511##
[1307] To a solution of
3-(2-methyl-8-nitro-4-oxo-quinazolin-3-yl)piperidine-2,6-dione (450
mg, 1.42 mmol, 1.00 eq) in tetrahydrofuran (50 mL) was added
Palladium/C catalyst (100 mg, 0.14 mmol, 10% purity) under nitrogen
atmosphere. The suspension was degassed and purged with hydrogen
for 3 times. The mixture was stirred under hydrogen (15 Psi) at
20.degree. C. for 16 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture was filtered
and the filter was concentrated to give the crude product
3-(8-amino-2-methyl-4-oxo-quinazolin-3-yl)piperidine-2,6-dione (380
mg, 1.33 mmol, 94% yield) was used into the next step without
further purification.
[1308] LCMS: MS (ESI) m/z: 287.1 [M+1].sup.+.
[1309] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1310] .delta.: 11.01 (s, 1H), 7.20-7.10 (m, 2H), 6.97 (dd, J=2.0,
7.2 Hz, 1H), 5.67 (s, 2H), 5.27-5.18 (m, 1H), 2.91-2.79 (m, 1H),
2.70-2.58 (m, 5H), 2.21-2.10 (m, 1H)
[1311] Chemical Formula: C.sub.14H.sub.14N.sub.4O.sub.3, Molecular
Weight: 286.29
[1312] Total H count from HNMR data: 14.
Step 9: Synthesis of
2-(4-bromophenyl)-3-(4-(2-(4-(2,2-dimethoxyethyl)piperazin-1-yl)ethoxy)ph-
enoxy)benzo[b]thiophen-6-ol
##STR00512##
[1314]
2-(4-bromophenyl)-3-[4-(2-piperazin-1-ylethoxy)phenoxy]benzothiophe-
n-6-ol (250 mg, 0.33 mmol, 1.00 eq, hydrobromide),
diisopropylethylamine (213 mg, 1.65 mmol, 0.3 mL, 5.00 eq) and
2-bromo-1,1-dimethoxy-ethane (112 mg, 0.66 mmol, 0.1 mL, 2.00 eq)
were taken up into a microwave tube in N-methyl-2-pyrrolidone (3.00
mL). The sealed tube was heated at 150.degree. C. for 1 hour under
microwave. TLC (dichloromethane:methanol=10:1, R.sub.f=0.52) the
reaction was completed and a new spot formed. The reaction mixture
was diluted with water (10 mL) and extracted with ethyl acetate (5
mL.times.3). The combined organic phase was washed with saturated
brine (5 mL.times.2), dried with anhydrous sodium sulfate, filtered
and concentrated in vacuum. The residue was purified by prep-TLC
(dichloromethane:methanol=10:1) to give
2-(4-bromophenyl)-3-[4-[2-[4-(2,2-dimethoxyethyl)piperazin-1-yl]ethoxy]ph-
enoxy]benzothiophen-6-ol (120 mg, 0.2 mmol, 59% yield) as a yellow
solid.
[1315] LCMS: MS (ESI) m/z: 615.0 [M+1].sup.+.
[1316] Chemical Formula: C.sub.30H.sub.33BrN.sub.2O.sub.5S,
Molecular Weight: 613.56
Step 10: Synthesis of
2-(4-(2-(4-((2-(4-bromophenyl)-6-hydroxybenzo[b]
thiophen-3-yl)oxy)phenoxy)ethyl)piperazin-1-yl)acetaldehyde
##STR00513##
[1318] To a solution of
2-(4-bromophenyl)-3-[4-[2-[4-(2,2-dimethoxyethyl)piperazin-1-yl]ethoxy]ph-
enoxy]benzothiophen-6-ol (120 mg, 0.20 mmol, 1.00 eq) in dioxane (2
mL) was added hydrochloric acid (2 M, 2 mL, 20.45 eq). The mixture
was stirred at 50.degree. C. for 2 hours. LCMS showed the reaction
was completed and desired MS can be detected. The reaction mixture
was concentrated under reduced pressure to remove dioxane and water
to give the crude product
2-[4-[2-[4-[2-(4-bromophenyl)-6-hydroxy-benzothiophen-3-yl]oxyphenoxy]
ethyl]piperazin-1-yl]acetaldehyde (100 mg, crude) was used into the
next step without further purification.
[1319] LCMS: MS (ESI) m/z: 585.0 [M+18].sup.+.
[1320] Chemical Formula: C.sub.28H.sub.27BrN.sub.2O.sub.4S,
Molecular Weight: 567.49
Step 11: Synthesis of
3-(8-((2-(4-(2-(4-((2-(4-bromophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)-
phenoxy)ethyl)piperazin-1-yl)ethyl)amino)-2-methyl-4-oxoquinazolin-3(4H)-y-
l)piperidine-2,6-dione
##STR00514##
[1322] To a solution of
2-[4-[2-[4-[2-(4-bromophenyl)-6-hydroxy-benzothiophen-3-yl]oxyphenoxy]eth-
yl]piperazin-1-yl]acetaldehyde (1000 mg, 0.18 mmol, 1.00 eq) in
methanol (2 mL) was added acetic acid (0.2 mL) and
3-(8-amino-2-methyl-4-oxo-quinazolin-3-yl) piperidine-2,6-dione (50
mg, 0.18 mmol, 1.00 eq). The mixture was stirred at 20.degree. C.
for 0.5 hour. Borane; 2-methylpyridine (38 mg, 0.35 mmol, 2.00 eq)
was added, then the mixture was stirred at 20.degree. C. for 2
hours. LCMS showed the reaction was completed and desired MS can be
detected. The reaction mixture was purified by prep-HPLC (column:
Boston Green ODS 150*30 5u; mobile phase: [water (0.225% FA)-ACN];
B %: 25%-55%, 10 min). Then the collected fraction was concentrated
to remove most of acetonitrile and hydrochloric acid (1 M, 2 mL)
was added. The solution was lyophilized to
3-[8-[2-[4-[2-[4-[2-(4-bromophenyl)-6-hydroxy-benzothiophen-3-yl]oxypheno-
xy]ethyl]piperazin-1-yl]ethylamino]-2-methyl-4-oxo-quinazolin-3-yl]piperid-
ine-2,6-dione (10 mg, 0.01 mmol, 7% yield, hydrochloride) as a
yellow solid.
[1323] LCMS: MS (ESI) m/z: 839.0 [M+1].sup.+.
[1324] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1325] .delta.: 11.01 (s, 1H), 10.04 (s, 1H), 7.62 (s, 4H), 7.33
(d, J=2.0 Hz, 1H), 7.31-7.23 (m, 1H), 7.21-7.11 (m, 2H), 7.01 (br
d, J=8.0 Hz, 1H), 6.92 (q, J=8.8 Hz, 4H), 6.85 (dd, J=2.0, 8.8 Hz,
1H), 5.25 (dd, J=5.2, 13.2 Hz, 1H), 4.29 (s, 2H), 3.68-3.45 (m,
14H), 2.87-2.79 (m, 1H), 2.69-2.61 (m, 5H), 2.19-2.10 (m, 1H)
[1326] Chemical Formula: C.sub.42H.sub.41BrN.sub.6O.sub.6S,
Molecular Weight: 837.78
[1327] Total H count from HNMR data: 40.
[1328] Synthesis of Exemplary PROTAC 108
##STR00515## [1329]
3-(8-(2-(4-(2-(4-((2-(4-bromophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)p-
henoxy)ethyl)piperazin-1-yl)ethoxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)pipe-
ridine-2,6-dione
[1330] Synthetic Scheme:
##STR00516## ##STR00517##
Step 1: Synthesis of allyl 3-(allyloxy)-2-nitrobenzoate
##STR00518##
[1332] To a solution of 3-hydroxy-2-nitro-benzoic acid (1 g, 5.46
mmol, 1.00 eq) in N,N-dimethylformamide (15 mL) was added potassium
carbonate (3 g, 21.84 mmol, 4.00 eq) and 3-bromoprop-1-ene (2.64 g,
21.84 mmol, 4.00 eq). The mixture was stirred at 20.degree. C. for
15 hours. LCMS showed the reaction was completed and desired MS can
be detected. The residue was diluted with water (100 mL) and
extracted with ethyl acetate (30 mL.times.3). The combined organic
phase was washed with brine (30 mL.times.2), dried with anhydrous
sodium sulfate, filtered and concentrated in vacuum to give allyl
3-allyloxy-2-nitro-benzoate (1.30 g, crude) as a yellow oil.
[1333] LCMS: MS (ESI) m/z: 286.0 [M+23].sup.+.
[1334] Chemical Formula: C.sub.13H.sub.13NO.sub.5, Molecular
Weight: 263.25
Step 2: Synthesis of 3-(allyloxy)-2-nitrobenzoic acid
##STR00519##
[1336] To a solution of allyl 3-allyloxy-2-nitro-benzoate (1.44 g,
5.47 mmol, 1.00 eq) in tetrahydrofuran (40 mL) was added lithium
hydroxide monohydrate (2 M, 11 mL, 4.00 eq). The mixture was
stirred at 20.degree. C. for 12 hours. LCMS showed the reaction was
completed and desired MS can be detected. The reaction mixture was
adjusted to pH=(4-5) by hydrochloric acid (2 M, 10 mL) and diluted
with water (50 mL) and extracted with ethyl acetate (30
mL.times.3). The combined organic phase was washed with saturated
brine (40 mL.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum to give
3-allyloxy-2-nitro-benzoic acid (1.20 g, crude) was used into the
next step without further purification.
[1337] LCMS: MS (ESI) m/z: 246.0 [M+23].sup.+.
[1338] .sup.1H NMR: (400 MHz, CDCl.sub.3)
[1339] .delta.: 7.70 (d, J=8.0 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H),
7.32 (d, J=8.0 Hz, 1H), 6.07-5.93 (m, 1H), 5.47-5.29 (m, 2H), 4.70
(d, J=5.2 Hz, 2H)
[1340] Chemical Formula: C.sub.10H.sub.9NO.sub.5, Molecular Weight:
223.18
[1341] Total H count from HNMR data: 8.
Step 3: Synthesis of 3-(allyloxy)-2-aminobenzoic acid
##STR00520##
[1343] To a solution of 3-allyloxy-2-nitro-benzoic acid (1.2 g,
5.38 mmol, 1.00 eq) in methanol (20 mL) and water (5 mL) was slowly
added iron (1.2 g, 21.52 mmol, 4.00 eq), ammonium chloride (1.44 g,
26.90 mmol, 5.00 eq) at 20.degree. C. The mixture was stirred at
80.degree. C. for 2 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture was filtered
and the filtrate was concentrated to give
3-allyloxy-2-amino-benzoic acid (850 mg, crude) used in the next
step without further purification.
[1344] LCMS: MS (ESI) m/z: 194.1 [M+1].sup.+.
[1345] .sup.1H NMR: (400 MHz, CDCl.sub.3)
[1346] .delta.: 7.54 (s, 1H), 6.99-6.44 (m, 2H), 6.07 (s, 2H), 5.39
(s, 2H), 4.59 (s, 3H), 4.76-4.40 (m, 1H)
[1347] Chemical Formula: C.sub.10H.sub.11NO.sub.3, Molecular
Weight: 193.20
[1348] Total H count from HNMR data: 11.
Step 4: Synthesis of 2-acetamido-3-(allyloxy)benzoic acid
##STR00521##
[1350] To a solution of 3-allyloxy-2-amino-benzoic acid (800 mg,
4.14 mmol, 1.00 eq) in acetonitrile (10 mL) was added imidazole
(282 mg, 4.14 mmol, 1.00 eq) and acetyl chloride (650 mg, 8.28
mmol, 2.00 eq). The mixture was stirred at 20.degree. C. for 12
hours. LCMS showed the reaction was completed and desired MS can be
detected. The reaction was diluted with water (30 mL) and extracted
with ethyl acetate (15 mL.times.3). The combined organic phase was
washed with saturated brine (20 mL.times.2), dried with anhydrous
sodium sulfate, filtered and concentrated in vacuum to give
2-acetamido-3-allyloxy-benzoic acid (900 mg, crude) as a yellow
solid, which was directly used for next step without further
purification.
[1351] LCMS: MS (ESI) m/z: 236.1 [M+1].sup.+.
[1352] Chemical Formula: C.sub.12H.sub.13NO.sub.4, Molecular
Weight: 235.24
Step 5: Synthesis of
3-(8-(allyloxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione
##STR00522##
[1354] To a solution of 2-acetamido-3-allyloxy-benzoic acid (800
mg, 3.40 mmol, 1.00 eq) and 3-aminopiperidine-2,6-dione (672 mg,
4.08 mmol, 1.20 eq, hydrochloride) in N,N-dimethylformamide (15 mL)
was added triphenyl phosphite (1.58 g, 5.10 mmol, 1.50 eq) and
imidazole (232 mg, 92.60 mmol, 27.23 eq). The mixture was stirred
at 100.degree. C. for 16 hours. LCMS showed the reaction was
completed and desired MS can be detected. The reaction mixture was
diluted with water (40 mL) and extracted with ethyl acetate (20
mL.times.2). The combined organic phase was washed with saturated
brine (20 mL.times.2), dried with anhydrous sodium sulfate,
filtered and concentrated in vacuum. The residue was purified by
silica gel column chromatography (dichloromethane:methanol=100:1 to
20:1) to give
3-(8-allyloxy-2-methyl-4-oxo-quinazolin-3-yl)piperidine-2,6-dione
(420 mg, 1.28 mmol, 38% yield) as a light yellow solid.
[1355] LCMS: MS (ESI) m/z: 328.2 [M+1].sup.+.
[1356] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1357] .delta.: 11.03 (s, 1H), 7.58 (dd, J=1.6, 7.6 Hz, 1H),
7.43-7.32 (m, 2H), 6.17-6.01 (m, 1H), 5.45 (dd, J=1.6, 17.2 Hz,
1H), 5.34-5.25 (m, 2H), 4.74 (d, J=4.8 Hz, 2H), 2.88-2.79 (m, 1H),
2.70-2.55 (m, 5H), 2.20-2.12 (m, 1H)
[1358] Chemical Formula: C.sub.17H.sub.17N.sub.3O.sub.4, Molecular
Weight: 327.33
[1359] Total H count from HNMR data: 17.
Step 6: Synthesis of
2-((3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-8-yl-
)oxy)acetaldehyde
##STR00523##
[1361] Ozone was bubbled into a solution of
3-(8-allyloxy-2-methyl-4-oxo-quinazolin-3-yl)piperidine-2,6-dione
(200 mg, 0.61 mmol, 1.00 eq) in dichloromethane (8 mL) and methanol
(2 mL) at -70.degree. C. for 30 minutes. After excess ozone was
purged by nitrogen, and dimethylsulfide (380 mg, 6.11 mmol, 10.00
eq) was added at -70.degree. C. The mixture was stirred at
20.degree. C. for 16 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture was
concentrated under reduced pressure to remove methanol,
dichloromethane and dimethylsulfide to give the
2-[3-(2,6-dioxo-3-piperidyl)-2-methyl-4-oxo-quinazolin-8-yl]oxyacetaldehy-
de (220 mg, crude) as a brown solid.
[1362] LCMS: MS (ESI) m/z: 362.0 [M+23].sup.+.
[1363] Chemical Formula: C.sub.16H.sub.15N.sub.3O.sub.5, Molecular
Weight: 329.31
Step 7: Synthesis of
3-(8-(2-(4-(2-(4-((2-(4-bromophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)p-
henoxy)ethyl)piperazin-1-yl)ethoxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)pipe-
ridine-2,6-dione
##STR00524##
[1365] To a solution of
2-[3-(2,6-dioxo-3-piperidyl)-2-methyl-4-oxo-quinazolin-8-yl]oxyacetaldehy-
de (120 mg, 0.36 mmol, 1.00 eq) in methanol (4 mL) was added
2-(4-bromophenyl)-3-[4-(2-piperazin-1-ylethoxy)phenoxy]benzothiophen-6-ol
(110 mg, 0.18 mmol, 0.50 eq, hydrobromide, intermediate from
synthesis of exemplary PROTAC 107, see above) and acetic acid (44
mg, 0.72 mmol, 2.00 eq). The mixture was stirred at 20.degree. C.
for 0.5 hour. Sodium cyanoborohydride (44 mg, 0.73 mmol, 2.00 eq)
was added at 20.degree. C., and then the mixture was stirred at
20.degree. C. for 2 hours. LCMS showed the reaction was completed
and desired MS can be detected. The reaction mixture concentrated
under reduced pressure to remove methanol. The residue was purified
by prep-HPLC (column: Phenomenex Synergi C18 150*30 mm*4 um; mobile
phase: [water (0.225% FA)-ACN]; B %: 25%-55%, 12 min). Then the
collected fraction was concentrated to remove most of acetonitrile
and hydrochloric acid (1 M, 2 mL) was added. The solution was
lyophilized to give
3-[8-[2-[4-[2-[4-[2-(4-bromophenyl)-6-hydroxy-benzothiophen-3-yl]oxypheno-
xy]ethyl]
piperazin-1-yl]ethoxy]-2-methyl-4-oxo-quinazolin-3-yl]piperidine-
-2,6-dione (18 mg, 0.02 mmol, 5% yield, 91% purity, hydrochloride)
as a white solid.
[1366] LCMS: MS (ESI) m/z: 840.2 [M+1].sup.+.
[1367] .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
[1368] .delta.: 11.06 (s, 1H), 9.99 (s, 1H), 7.66 (d, J=7.2 Hz,
1H), 7.63 (s, 4H), 7.54-7.42 (m, 1H), 7.52-7.42 (m, 1H), 7.33 (d,
J=2.0 Hz, 1H), 7.15 (d, J=8.8 Hz, 1H), 6.97-6.91 (m, 4H), 6.84 (dd,
J=2.0, 8.8 Hz, 1H), 5.28 (dd, J=5.2, 13.2 Hz, 1H), 4.54 (s, 2H),
4.27 (s, 4H), 3.56-3.49 (m, 10H), 2.82-2.80 (m, 1H), 2.65-2.59 (m,
5H), 2.21-2.14 (m, 1H)
[1369] Chemical Formula: C.sub.42H.sub.40BrN.sub.5O.sub.7S,
Molecular Weight: 838.77
[1370] Total H count from HNMR data: 40.
[1371] Synthesis of Exemplary PROTAC 112
##STR00525## [1372]
2-(2,6-dioxopiperidin-3-yl)-8-(14-((5-(5-methyl-5H-pyrido[4,3-b]indol-7-y-
l)pyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-2,8-diazaspiro[4.5]decane-
-1,3-dione
[1373] Reaction Scheme:
##STR00526## ##STR00527##
Step 1:--Preparation of 1-tert-butyl 4-methyl
4-(2-ethoxy-2-oxoethyl)-piperidine-1,4-dicarboxylate
##STR00528##
[1375] To a solution of ethyl 2-bromoacetate (8.65 g, 51.80 mmol,
5.7 mL, 1 eq) in tetrahydrofuran (1000 mL) was added lithium
diiso-propylamide (2 M, 39 mL, 1.5 eq) at -78.degree. C.
[1376] The mixture was stirred at -78.degree. C. for 1 hour. Then
O1-tert-butyl O4-methyl piperidine-1,4-dicarboxylate (20 g, 82.2
mmol, 1.59 eq) was added and the mixture was stirred at this
temperature for 1 h. After this, the mixture was stirred at
15.degree. C. for another 24 hours. Thin-Layer Chromatography
(petroleum ether:ethyl acetate=5:1) indicated 50% of Reactant 1 was
remained, and one major new spot (R.sub.f=0.46) with lower polarity
was detected. The reaction mixture was quenched by addition of
aqueous ammonium chloride 500 mL, and then extracted with ethyl
acetate 1500 mL (500 mL.times.3). The combined organic layers were
washed with brine 1500 mL (500 mL.times.3), dried over sodium
sulfate, filtered and concentrated under reduced pressure. The
residue was purified by flash silica gel chromatography.
O1-tert-butyl O4-methyl 4-(2-ethoxy-2-oxo-ethyl) piperidine-1,
4-dicarboxylate (3.8 g, 11.5 mmol, 22% yield) was obtained as a
brown oil.
[1377] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta. 4.07-3.95 (m,
2H), 3.73-3.50 (m, 5H), 3.06 (br s, 2H), 2.50 (br s, 2H), 1.99 (d,
J=13.6 Hz, 2H), 1.47-1.38 (m, 2H), 1.38-1.33 (m, 9H), 1.21-1.10 (m,
3H).
[1378] Chemical Formula: C.sub.16H.sub.27NO.sub.6, Molecular
Weight: 329.39
2. Step: Preparation of 1-(tert-butoxycarbonyl)-4-(carboxymethyl)
piperidine-4-carboxylic acid
##STR00529##
[1380] To a solution of 01-tert-butyl 04-methyl
4-(2-ethoxy-2-oxo-ethyl) piperidine-1, 4-dicarboxylate (3.8 g,
11.50 mmol, 1 eq) in tetrahydrofuran (20 mL), water (15 mL) was
added sodium hydroxide (2.3 g, 57.7 mmol, 5 eq) and methanol (10
mL). The mixture was stirred at 25.degree. C. for 36 h. High
performance liquid chromatography-mass spectrometry showed Reactant
1 was consumed completely. The reaction mixture was diluted with
water 20 mL and concentrated under reduced pressure to remove
tetrahydrofuran and methanol. The water layer was washed with
petroleum ether (30 mL.times.2), then acided by hydrochloric acid
solution to pH-5, extracted with ethyl acetate (30 mL.times.3). The
combined organic layers were washed with brine 60 mL, dried over
sodium sulfate, filtered and concentrated under reduced pressure.
1-tert-butoxycarbonyl-4-(carboxymethyl) piperidine-4-carboxylic
acid (2.9 g, 10 mmol, 87% yield) was obtained as a brown solid.
[1381] LCMS: MS (ESI) m/z: 286.
[1382] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta. 3.69 (br s, 2H),
3.36-3.23 (m, 2H), 2.72 (s, 2H), 2.19-2.12 (m, 2H), 1.56 (br t,
J=9.7 Hz, 1H), 1.48 (s, 10H)
[1383] Chemical Formula: C.sub.13H.sub.21NO.sub.6, Molecular
Weight: 287.31
3. Step: Preparation of tert-butyl 2-(2, 6-dioxopiperidin-3-yl)-1,
3-dioxo-2, 8-diazaspiro [4.5] decane-8-carboxylate
##STR00530##
[1385] A mixture of
1-tert-butoxycarbonyl-4-(carboxymethyl)piperidine-4-carboxylic acid
(1.9 g, 6.61 mmol, 1 eq) and acetic anhydride (21.80 g, 213.54
mmol, 20 mL, 32.29 eq) was degassed and purged with nitrogen for 3
times, and then the mixture was stirred at 120.degree. C. for 0.5
houe under nitrogen atmosphere. The reaction mixture was
concentrated under reduced pressure to remove acetic anhydride. The
residue was diluted with pyridine (20 mL) and added
3-aminopiperidine-2,6-dione (1.31 g, 7.94 mmol, 1.2 eq,
hydrochloride). The mixture was stirred at 140.degree. C. under
nitrogen atmosphere for 12 h. High performance liquid
chromatography-mass spectrometry showed Reactant 1 was consumed
completely and one main peak with desired mass was detected. The
reaction mixture was concentrated under reduced pressure. The
residue was washed with water (10 mL.times.3) to give the product.
Tert-butyl 2-(2, 6-dioxo-3-piperidyl)-1, 3-dioxo-2, 8-diazaspiro
[4.5] decane-8-carboxylate (1.2 g, 3.2 mmol, 47% yield) was
obtained as a grey solid.
[1386] LCMS: MS (ESI) m/z: 402 [M+23].sup.+
[1387] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta. 7.91 (s, 1H),
4.74 (dd, J=5.3, 12.3 Hz, 1H), 3.94 s, 2H), 2.97 (t, J=11.7 Hz,
2H), 2.80 (d, J=15.4 Hz, 1H), 2.75-2.55 (m, 4H), 2.00-1.88 (m, 3H),
1.50 (s, 2H), 1.40 (s, 9H)
[1388] Chemical Formula: C.sub.18H.sub.25N.sub.3O.sub.6, Molecular
Weight: 379.41
4. Step: Preparation of 2-(2, 6-dioxopiperidin-3-yl)-2,
8-diazaspiro [4.5] decane-1, 3-dione
##STR00531##
[1390] To a solution of tert-butyl 2-(2, 6-dioxo-3-piperidyl)-1,
3-dioxo-2, 8-diazaspiro [4.5] decane-8-carboxylate (1.2 g, 3.16
mmol, 1 eq) in dioxane (15 mL) was added hydrochloric acid solution
(4 M in dioxane, 20 mL, 25.3 eq). The mixture was stirred at
15.degree. C. for 3 hour. The reaction mixture was concentrated
under reduced pressure.
2-(2,6-dioxo-3-piperidyl)-2,8-diazaspiro[4.5]decane-1,3-dione (1.2
g, hydrochloride) was obtained as a grey solid.
[1391] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. 11.08 (s, 1H),
8.93 (s, 1H), 8.64 (s, 1H), 4.95 (dd, J=5.4, 12.8 Hz, 1H), 3.29 (s,
2H), 3.07-2.93 (m, 2H), 2.92-2.87 (m, 2H), 2.86-2.78 (m, 1H), 2.58
(s, 1H), 2.47-2.36 (m, 1H), 2.09-1.87 (m, 3H), 1.80 (d, J=14.1 Hz,
2H)
[1392] Chemical Formula: C.sub.13H.sub.17N.sub.3O.sub.4, Molecular
Weight: 279.29
5. Step: Preparation of
2-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethoxy]e-
thanol
##STR00532##
[1394] To a solution of
2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethanol (2 g, 8.40
mmol, 1 eq) in dichloromethane (20 mL) was added imidazole (1.92 g,
12.6 mmol, 1.9 mL, 1.5 eq) and tert-butyl-chloro-diphenyl-silane
(2.42 g, 8.8 mmol, 2.3 mL, 1.05 eq). The mixture was stirred at
15.degree. C. for 3 hours. Thin-Layer Chromatography (Ethyl
Acetate) indicated 10% of Reactant 1 was remained, and one major
new spot (R.sub.f=0.32) with lower polarity was detected. High
performance liquid chromatography-mass spectrometry showed desired
MS was detected. The reaction mixture was concentrated under
reduced pressure. The residue was purified by silica gel
chromatography (petroleum ether/ethyl acetate=1/1 to 0:1).
2-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethoxy]e-
thanol (1.77 g, 3.7 mmol, 44% yield) was obtained as a colorless
oil.
[1395] LCMS: MS (ESI) m/z: 494 [M+18].sup.+
[1396] HNMR: (400 MHz, CDCl.sub.3) .delta. 7.75-7.66 (m, 4H),
7.48-7.36 (m, 6H), 3.83 (t, J=5.4 Hz, 2H), 3.77-3.58 (m, 18H), 2.51
(s, 1H), 1.07 (s, 9H)
[1397] Chemical Formula: C.sub.26H.sub.40O.sub.6Si, Molecular
Weight: 476.68
6. Step: Preparation of
2-[2-[2-[2-[2-[[5-(5-methylpyrido[4,3-b]indol-7-yl)-2-pyridyl]oxy]ethoxy]-
ethoxy]ethoxy]ethoxy]ethanol
##STR00533##
[1399] To a solution of 2-[2-[2-[2-[2-[tert-butyl (diphenyl) silyl]
oxyethoxy] ethoxy] ethoxy] ethoxy] ethanol (258 mg, 0.54 mmol, 1.5
eq) in N,N-dimethylformamide (5 mL) was added sodium hydride (29
mg, 0.72 mmol, 60% purity in mineral oil, 2 eq) at 0.degree. C. The
mixture was stirred at 15.degree. C. for 1 hour. Then
7-(6-fluoro-3-pyridyl)-5-methyl-pyrido [4, 3-b]indole (0.1 g, 361
umol, 1 eq) was added. The mixture was stirred at 15.degree. C. for
12 hours. High performance liquid chromatography-mass spectrometry
showed Reactant 1 was consumed completely and one main peak with
desired MS was detected. The reaction mixture was quenched by the
addition od water (15 mL) at 0.degree. C., and then extracted with
ethyl acetate 45 mL (15 mL*3). The combined organic layers were
dried over sodium sulfate, filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (dichloromethane:methanol=20:1, R.sub.f=0.21).
2-[2-[2-[2-[2-[[5-(5-methylpyrido[4,3-b] indol-7-yl)-2-pyridyl]
oxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (0.09 g, 0.14 mmol, 39%
yield, 78% purity) was obtained as brown oil.
[1400] LCMS: MS (ESI) m/z: 496.0 [M+1].sup.+
[1401] HNMR: (400 MHz, CDCl.sub.3) .delta. 9.27 (s, 1H), 8.51 (s,
1H), 8.41 (d, J=2.2 Hz, 1H), 8.15 (d, J=8.2 Hz, 1H), 7.88-7.82 (m,
1H), 7.53 (s, 1H), 7.48 (dd, J=1.3, 8.1 Hz, 1H), 7.38-7.33 (m, 1H),
6.87 (d, J=8.7 Hz, 1H), 4.51-4.47 (m, 2H), 3.89 (s, 3H), 3.85-3.82
(m, 2H), 3.70-3.63 (m, 12H)
[1402] Chemical Formula: C.sub.27H.sub.33N.sub.3O.sub.6, Molecular
Weight: 495.57
7. Step: Preparation of
2-[2-[2-[2-[2-[[5-(5-methylpyrido[4,3-b]indol-7-yl)-2-pyridyl]oxy]ethoxy]-
ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate
##STR00534##
[1404] To a solution of 2-[2-[2-[2-[2-[[5-(5-methylpyrido[4,
3-b]indol-7-yl)-2-pyridyl] oxy] ethoxy]
ethoxy]ethoxy]ethoxy]ethanol (90 mg, 0.18 mmol, 1 eq) in
dichloromethane (5 mL) was added triethylamine (37 mg, 0.36 mmol, 2
eq), then p-toluensulfonyl chloride (139 mg, 0.73 mmol, 4 eq) was
added. The mixture was stirred at 15.degree. C. for 12 hours. LCMS
showed Reactant 1 was consumed completely and one main peak with
desired mass was detected. The reaction mixture was concentrated
under reduced pressure. The residue was purified by prep-Thin-Layer
Chromatography (dichloromethane:methanol=10:1, the product
R.sub.f=0.27).
2-[2-[2-[2-[2-[[5-(5-methylpyrido[4,3-b]indol-7-yl)-2-pyridyl]oxy]ethoxy]-
ethoxy]ethoxy] ethoxy]ethyl 4-methylbenzenesulfonate (0.05 g, 0.07
mmol, 36% yield, 86% purity) was obtained as a yellow oil.
[1405] LCMS: MS (ESI) m/z: 650[M+1].sup.+
[1406] Chemical Formula: C.sub.34H.sub.39N.sub.3O.sub.8S, Molecular
Weight: 649.75
8. Step: Preparation of
2-(2,6-dioxopiperidin-3-yl)-8-(14-((5-(5-methyl-5H-pyrido[4,3-b]indol-7-y-
l)pyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-2,8-diazaspiro[4.5]decane-
-1,3-dione
##STR00535##
[1408] A mixture of 2-[2-[2-[2-[2-[[5-(5-methylpyrido[4, 3-b]
indol-7-yl)-2-pyridyl] oxy] ethoxy]
ethoxy]ethoxy]ethoxy]ethyl4-methylbenzenesulfonate (50 mg, 0.07
mmol, 1 eq), 2-(2, 6-dioxo-3-piperidyl)-2,
8-diazaspiro[4.5]decane-1,3-dione (32 mg, 0.10 mmol, 1.33 eq,
hydrochloride), potassium iodide (19 mg, 0.12 mmol, 1.5 eq),
N,N-diisopropylethylamine (30 mg, 0.23 mmol, 3 eq) in acetonitrile
(5 mL) was degassed and purged with nitrogen for 3 times, and then
the mixture was stirred at 100.degree. C. for 12 hours under
nitrogen atmosphere. LCMS showed Reactant 1 was consumed completely
and one main peak with desired MS was detected. The reaction
mixture was concentrated under reduced pressure. The residue was
purified by semi-preparative reverse phase HPLC (column: Phenomenex
Synergi C18 150*25*10 um; mobile phase: [water (0.05% HCl)-ACN]; B
%: 0%-30%, 10 min). The purity of residue was 90%. The residue was
purified by semi-preparative reverse phase HPLC (column: Phenomenex
Synergi C18 150*30 mm*4 um; mobile phase: [water(0.225% FA)-ACN]; B
%: 0%-26%,10.5 min; FlowRate(ml/min): 25).
2-(2,6-dioxo-3-piperidyl)-8-[2-[2-[2[2-[2-[[5-(5-methylpyrido[4,3-b]indol-
-7-yl)-2-pyridyl]oxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-2,8-diazaspiro[4.5-
]decane-1,3-dione (12.9 mg, 0.01 mmol, 20% yield, 99% purity, bis
formate salt) was obtained as a yellow solid.
[1409] LCMS: MS (ESI) m/z: 757.3 [M+1].sup.+
[1410] HNMR: (400 MHz, DMSO-d.sub.6) .delta.: 11.03 (s, 1H), 9.36
(s, 1H), 8.65 (d, J=2.4 Hz, 1H), 8.50 (d, J=6.4 Hz, 1H), 8.33 (d,
J=8.0 Hz, 1H), 8.23-8.19 (m, 3H), 7.99 (s, 1H), 7.63-7.62 (m, 2H),
6.98 (d, J=8.8 Hz, 1H), 4.90 (dd, J=5.2, 13.2 Hz, 1H), 4.45 (t,
J=4.8 Hz, 2H), 3.96 (s, 3H), 3.79 (t, J=4.8 Hz, 2H), 3.61-3.54 (m,
6H), 3.51-3.47 (m, 7H), 2.84-2.76 (m, 3H), 2.67-2.66 (m, 2H),
2.54-2.53 (m, 1H), 2.47-2.33 (m, 4H), 2.03 (t, J=10.4 Hz, 2H),
1.87-1.75 (m, 3H), 1.52-1.49 (m, 2H).
[1411] Chemical Formula: C.sub.40H.sub.48N.sub.6O.sub.9, Molecular
Weight: 756.84
[1412] Protein Level Control
[1413] This description also provides methods for the control of
protein levels with a cell. This is based on the use of compounds
as described herein, which are known to interact with a specific
target protein such that degradation of a target protein in vivo
will result in the control of the amount of protein in a biological
system, preferably to a particular therapeutic benefit.
[1414] The following examples are used to assist in describing the
present invention, but should not be seen as limiting the present
invention in any way.
Exemplary Embodiments of the Present Disclosure
[1415] The present disclosure encompasses the following specific
embodiments. These following embodiments may include all of the
features recited in a proceeding embodiment, as specified. Where
applicable, the following embodiments may also include the features
recited in any proceeding embodiment inclusively or in the
alternative
[1416] One aspect discloses a bifunctional compound having the
chemical structure: CLM-L-PTM, or a pharmaceutically acceptable
salt, enantiomer, stereoisomer, solvate, polymorph or prodrug
thereof, wherein: the PTM is a small molecule comprising a protein
targeting moiety; the L is a bond or a chemical linking moiety
covalently coupling the CLM and the PTM; and the CLM is a small
molecule cereblon E3 ubiquitin ligase binding moiety that binds or
targets an cereblon E3 ubiquitin ligase and has a chemical
structure selected from the group consisting of:
##STR00536## ##STR00537## ##STR00538## ##STR00539## ##STR00540##
##STR00541##
wherein: [1417] W is independently selected from CH.sub.2, CHR,
C.dbd.O, SO.sub.2, NH, and N-alkyl; [1418] Q.sub.1, Q.sub.2,
Q.sub.3, Q.sub.4, Q.sub.5 are each independently represent a carbon
C or N substituted with a group independently selected from R', N
or N-oxide; [1419] R.sup.1 is selected from absent, H, OH, CN,
C1-C3 alkyl, C.dbd.O; [1420] R.sup.2 is selected from the group
absent, H, OH, CN, C1-C3 alkyl, CHF.sub.2, CF.sub.3, CHO,
C(.dbd.O)NH.sub.2; [1421] R.sup.3 is selected from absent, H, alkyl
(e.g., C1-C6 or C1-C3 alkyl), substituted alkyl (e.g., substituted
C1-C6 or C1-C3 alkyl), alkoxy (e.g., C1-C6 or C1-C3 alkoxyl),
substituted alkoxy (e.g., substituted C1-C6 or C1-C3 alkoxyl);
[1422] R.sup.4 is selected from H, alkyl, substituted alkyl; [1423]
R.sup.5 and R.sup.6 are each independently H, halogen, C(.dbd.O)R',
CN, OH, CF.sub.3; [1424] X is C, CH, C.dbd.O, or N; [1425] X.sub.1
is C.dbd.O, N, CH, or CH.sub.2; [1426] R' is selected from H,
halogen, amine, alkyl (e.g., C1-C3 alkyl), substituted alkyl (e.g.,
substituted C1-C3 alkyl), alkoxy (e.g., C1-C3 alkoxyl), substituted
alkoxy (e.g., substituted C1-C3 alkoxyl), NR.sup.2R.sup.3,
C(.dbd.O)OR.sup.2, optionally substituted phenyl; [1427] n is 0-4;
[1428] and is a single or double bond.
[1429] In any aspect or embodiment described herein, the CLM is
linked to the PTM, the chemical linker group (L), or a combination
thereof via W, X, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R', Q.sub.1,
Q.sub.2, Q.sub.3, Q.sub.4, and Q.sub.5.
[1430] In any aspect or embodiment described herein, the PTM is a
moiety that binds Brd4, Tau Protein, Estrogen Receptor (ER) or
Androgen Receptor (AR).
[1431] In any aspect or embodiment described herein, the compound
further comprises a second E3 ubiquitin ligase binding moiety
coupled through a linker group.
[1432] In any aspect or embodiment described herein, the second E3
ubiquitin ligase binding moiety binds or targets an E3 ubiquitin
ligase selected from the group consisting of Von Hippel-Lindau
(VLM), cereblon (CLM), mouse double-minute homolog2 (MLM), and
inhibitors of apoptosis proteins (ILM).
[1433] In any aspect or embodiment described herein, the CLM is
represented by a chemical structure selected from the group
consisting of:
##STR00542## ##STR00543## ##STR00544##
wherein: [1434] W is independently selected from the group
CH.sub.2, CHR, C.dbd.O, SO.sub.2, NH, and N-alkyl; [1435] R.sup.1
is selected from the group absent, H, CH, CN, C1-C3 alkyl; [1436]
R.sup.2 is H or a C1-C3 alkyl; [1437] R.sup.3 is selected from H,
alkyl, substituted alkyl, alkoxy, substituted alkoxy; [1438]
R.sup.4 is methyl or ethyl; [1439] R.sup.5 is H or halo; [1440]
R.sup.6 is H or halo; [1441] R is H or halogen; [1442] R' is H or
an attachment point for a PTM, a PTM', a chemical linker group (L),
a ULM, a CLM, a CLM', [1443] Q.sub.1 and Q.sub.2 are each
independently C or N substituted with a group independently
selected from H or C1-C3 alkyl; [1444] is a single or double bond;
and Rn comprises a functional group or an ato.
[1445] In any aspect or embodiment described herein, the CLM is
represented by a chemical structure selected by:
##STR00545## ##STR00546## ##STR00547##
wherein R' is a halogen.
[1446] In any aspect or embodiment described herein, the CLM is
represented by a chemical structure selected by:
##STR00548##
[1447] In any aspect or embodiment described herein, the linker (L)
comprises a chemical structural unit represented by the
formula:
-(A.sup.L)q-
wherein: [1448] (A.sup.L).sub.q is a group which is connected to a
CLM or PTM moiety; and [1449] q is an integer greater than or equal
to 1; [1450] each A.sup.L is independently selected from the group
consisting of, a bond, CR.sup.L1R.sup.L2, O, S, SO, SO.sub.2,
NR.sup.L3, SO.sub.2NR.sup.L3, SONR.sup.L3, CONR.sup.L3,
NR.sup.L3CONR.sup.L4, NR.sup.L3SO.sub.2NR.sup.L4, CO,
CR.sup.L1.dbd.CR.sup.L2, C.ident.C, SiR.sup.L1R.sup.L2,
P(O)R.sup.L1, P(O)OR.sup.L1, NR.sup.L3C(.dbd.NCN)NR.sup.L4,
NR.sup.L3C(.dbd.NCN), NR.sup.L3C(.dbd.CNO.sub.2)NR.sup.L4,
C.sub.3-11cycloalkyl optionally substituted with 0-6 R.sup.L1
and/or R.sup.L2groups, C.sub.3-11heterocyclyl optionally
substituted with 0-6 R.sup.L1 and/or R.sup.L2 groups, aryl
optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2 groups,
heteroaryl optionally substituted with 0-6 R.sup.L1 and/or R.sup.L2
groups, where R.sup.L1 or R.sup.L2, each independently are
optionally linked to other groups to form cycloalkyl and/or
heterocyclyl moiety, optionally substituted with 0-4 R.sup.L5
groups; and [1451] R.sup.L1, R.sup.L2, R.sup.L3, R.sup.L4 and
R.sup.L5 are, each independently, H, halo, C.sub.1-8alkyl,
OC.sub.1-8alkyl, SC.sub.1-8alkyl, NHC.sub.1-8alkyl,
N(C.sub.1-8alkyl).sub.2, C.sub.3-11cycloalkyl, aryl, heteroaryl,
C.sub.3-11heterocyclyl, OC.sub.1-8cycloalkyl, SC.sub.1-8cycloalkyl,
NHC.sub.1-8cycloalkyl, N(C.sub.1-8cycloalkyl).sub.2,
N(C.sub.1-8cycloalkyl)(C.sub.1-8alkyl), OH, NH.sub.2, SH,
SO.sub.2C.sub.1-8alkyl, P(O)(OC.sub.1-8alkyl)(C.sub.1-8alkyl),
P(O)(OC.sub.1-8alkyl).sub.2, CC--C.sub.1-8alkyl, CCH,
CH.dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.CH(C.sub.1-8alkyl),
C(C.sub.1-8alkyl).dbd.C(C.sub.1-8alkyl).sub.2, Si(OH).sub.3,
Si(C.sub.1-8alkyl).sub.3, Si(OH)(C.sub.1-8alkyl).sub.2,
COC.sub.1-8alkyl, CO.sub.2H, halogen, CN, CF.sub.3, CHF.sub.2,
CH.sub.2F, NO.sub.2, SF.sub.5, SO.sub.2NHC.sub.1-8alkyl,
SO.sub.2N(C.sub.1-8alkyl).sub.2, SONHC.sub.1-8alkyl,
SON(C.sub.1-8alkyl).sub.2, CONHC.sub.1-8alkyl,
CON(C.sub.1-8alkyl).sub.2, N(C.sub.1-8 alkyl)CONH(C.sub.1-8alkyl),
N(C.sub.1-8alkyl)CON(C.sub.1-8alkyl).sub.2, NHCONH(C.sub.1-8alkyl),
NHCON(C.sub.1-8alkyl).sub.2, NHCONH.sub.2,
N(C.sub.1-8alkyl)SO.sub.2NH(C.sub.1-8alkyl), N(C.sub.1-8alkyl)
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH(C.sub.1-8alkyl), NH
SO.sub.2N(C.sub.1-8alkyl).sub.2, NH SO.sub.2NH.sub.2.
[1452] In any aspect or embodiment described herein, the L is
selected from the group consisting of: [1453]
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub-
.q--O(CH2).sub.r-OCH2-, [1454]
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r-OCH2-, [1455]
--O--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--
-O(CH2).sub.r--O--; [1456]
--N(R)--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub-
.q--O(CH2).sub.r--O--; [1457]
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--O(CH2).sub.q--O(-
CH2).sub.r--O--; [1458]
--(CH2).sub.m--O(CH2).sub.n--O(CH2).sub.o--O(CH2).sub.p--(CH2).sub.q--O(C-
H2).sub.r-OCH2-;
##STR00549## ##STR00550## ##STR00551##
[1458] wherein [1459] m, n, o, p, q, and r of the linker are
independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20; [1460] when the number is zero, there is no
N--O or O--O bond [1461] R of the linker is H, methyl and ethyl;
[1462] X of the linker is H and F
[1462] ##STR00552## [1463] where m of the linker can be 2, 3, 4,
5
[1463] ##STR00553## ##STR00554## ##STR00555## ##STR00556##
##STR00557## ##STR00558## ##STR00559## ##STR00560## ##STR00561##
##STR00562## ##STR00563## ##STR00564## ##STR00565## ##STR00566##
##STR00567## [1464] where n and m of the linker can be 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
[1465] In any aspect or embodiment described herein, the L is
selected from the group consisting of:
##STR00568## ##STR00569## ##STR00570## ##STR00571## ##STR00572##
##STR00573## ##STR00574## ##STR00575## ##STR00576##
wherein each m and n is independently selected from 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
[1466] In any aspect or embodiment described herein, the linker (L)
is selected from the group consisting of:
##STR00577## ##STR00578## ##STR00579## ##STR00580## ##STR00581##
##STR00582## ##STR00583## ##STR00584## ##STR00585## ##STR00586##
##STR00587## ##STR00588## ##STR00589## ##STR00590## ##STR00591##
##STR00592## ##STR00593## ##STR00594## ##STR00595##
##STR00596##
##STR00597## ##STR00598## ##STR00599## ##STR00600## ##STR00601##
##STR00602## ##STR00603## ##STR00604## ##STR00605## ##STR00606##
##STR00607## ##STR00608## ##STR00609## ##STR00610## ##STR00611##
##STR00612## ##STR00613## ##STR00614## ##STR00615## ##STR00616##
##STR00617## ##STR00618## ##STR00619##
wherein each m, n, o, p, q, and r is independently 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
[1467] In any aspect or embodiment described herein, the linker (L)
is selected from the group consisting of:
##STR00620## ##STR00621## ##STR00622## ##STR00623## ##STR00624##
##STR00625## ##STR00626## ##STR00627## ##STR00628## ##STR00629##
##STR00630## ##STR00631## ##STR00632## ##STR00633##
[1468] In any aspect or embodiment described herein, the linker (L)
is selected from:
##STR00634## ##STR00635## ##STR00636## ##STR00637## ##STR00638##
##STR00639## ##STR00640## ##STR00641## ##STR00642##
wherein: [1469] "X" in above structures can be linear chain with
atoms ranging from 2 to 14, and the mentioned chain can contain
heteroatoms such as oxygen; and [1470] "Y" in above structures can
be O, N, S(O).sub.n (n=0, 1, 2).
[1471] In any aspect or embodiment described herein, the linker (L)
comprises a structure selected from:
##STR00643##
wherein: [1472] W.sup.L1 and W.sup.L2 are each independently a 4-8
membered ring with 0-4 heteroatoms, optionally substituted with
R.sup.Q, each R.sup.Q is independently a H, halo, OH, CN, CF.sub.3,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted),
C.sub.1-C.sub.6 alkoxy (linear, branched, optionally substituted),
or 2 R.sup.Q groups taken together with the atom they are attached
to, form a 4-8 membered ring system containing 0-4 heteroatoms;
[1473] Y.sup.L1 is each independently a bond, C.sub.1-C.sub.6 alkyl
(linear, branched, optionally substituted) and optionally one or
more C atoms are replaced with O; or C.sub.1-C.sub.6 alkoxy
(linear, branched, optionally substituted); [1474] n is 0-10; and
[1475] a dashed line indicates the attachment point to the PTM or
CLM moieties.
[1476] In any aspect or embodiment described herein, the linker (L)
comprises a structure selected from:
##STR00644##
wherein: [1477] W.sup.L1 and W.sup.L2 are each independently aryl,
heteroaryl, cyclic, heterocyclic, C.sub.1-6 alkyl, bicyclic,
biaryl, biheteroaryl, or biheterocyclic, each optionally
substituted with R.sup.Q, each R.sup.Q is independently a H, halo,
OH, CN, CF.sub.3, hydroxyl, nitro, C.ident.CH, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-C.sub.6 alkyl (linear, branched,
optionally substituted), C.sub.1-C.sub.6 alkoxy (linear, branched,
optionally substituted), OC.sub.1-3alkyl (optionally substituted by
1 or more --F), OH, NH.sub.2, NR.sup.Y1R.sup.Y2, CN, or 2 R.sup.Q
groups taken together with the atom they are attached to, form a
4-8 membered ring system containing 0-4 heteroatoms; [1478]
Y.sup.L1 is each independently a bond, NR.sup.YL1, O, S,
NR.sup.YL2, CR.sup.YL1R.sup.YL2, C.dbd.O, SO, SO.sub.2,
C.sub.1-C.sub.6 alkyl (linear, branched, optionally substituted)
and optionally one or more C atoms are replaced with O;
C.sub.1-C.sub.6 alkoxy (linear, branched, optionally substituted);
[1479] Q.sup.L is a 3-6 membered alicyclic or aromatic ring with
0-4 heteroatoms, optionally bridged, optionally substituted with
0-6 R.sup.Q, each R.sup.Q is independently H, C.sub.1-6 alkyl
(linear, branched, optionally substituted by 1 or more halo,
C.sub.1-6 alkoxyl), or 2 R.sup.Q groups taken together with the
atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); [1480] R.sup.YL1, R.sup.YL2 are each
independently H, OH, C.sub.1-6 alkyl (linear, branched, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), or R.sup.1,
R.sup.2 together with the atom they are attached to, form a 3-8
membered ring system containing 0-2 heteroatoms); [1481] n is 0-10;
and [1482] a dashed line indicates the attachment point to the PTM
or CLM moieties.
[1483] In any aspect or embodiment described herein, the linker (L)
is a polyethylenoxy group optionally substituted with aryl or
phenyl comprising from 1 to 10 ethylene glycol units.
[1484] In any aspect or embodiment described herein, the compound
comprises multiple ULMs, multiple CLMs, multiple PTMs, multiple
linkers or any combinations thereof.
[1485] In any aspect or embodiment described herein, the PTM has a
chemical structure including at least one of (A), (B), (C), (D),
(E), or a combination thereof: [1486] (A) an estrogen receptor
binding moiety (EBM) comprising PTM-I or PTM-II:
[1486] ##STR00645## [1487] wherein: [1488] X.sub.PTM is O or
C.dbd.O; [1489] each of X.sub.PTM1 and X.sub.PTM2 is independently
selected from N or CH; [1490] R.sub.PTM1 is independently selected
from OH, O(CO)R.sub.PTM, O-lower alkyl, wherein R.sub.PTM is an
alkyl or aryl group in the ester; [1491] R.sub.PTM2 and R.sub.PTM4
are independently selected from H, OH, halogen, CN, CF.sub.3,
SO.sub.2-alkyl, O-lower alkyl; [1492] R.sub.PTM3 and R.sub.PTM5 are
independently selected from H, halogen; [1493] P.sub.TM2 and at
least one R.sub.PTM3 on each respective rings; and the
##STR00646##
[1493] indicates the site of attachment of at least one of the
linker, the CLM, a CLM', or a combination thereof; [1494] (B) an
estrogen receptor protein targeting moiety represented by the
chemical structure:
[1494] ##STR00647## [1495] wherein: [1496] each X.sub.PTM is
independently CH, N; [1497] indicates the site of attachment of at
least one of the linker, the CLM, a CLM', or a combination thereof;
[1498] each R.sub.PTM1 is independently OH, halogen, alkoxy,
methoxy, ethoxy, O(CO)R.sub.PTM, wherein the substitution can be a
mono-, di- or tri-substitution and the R.sub.PTM is alkyl or
cycloalkyl group with 1 to 6 carbons or aryl groups; [1499] each
R.sub.PTM2 is independently H, halogen, CN, CF.sub.3, liner or
branched alkyl, alkoxy, methoxy, ethoxy, wherein the substitution
can be mono- or di-substitution; [1500] each R.sub.PTM3 is
independently H, halogen, wherein the substitution can be mono- or
di-substitution; and [1501] R.sub.PTM4 is a H, alkyl, methyl,
ethyl. [1502] (C) an androgen receptor (AR) binding moiety (ABM)
comprises a structure selected from the group consisting of:
[1502] ##STR00648## [1503] wherein: [1504] W.sup.1 is aryl,
heteroaryl, bicyclic, or biheterocyclic, each independently
substituted by 1 or more H, halo, hydroxyl, nitro, CN, C.ident.CH,
C.sub.1-6 alkyl (linear, branched, optionally substituted; for
example, optionally substituted by 1 or more halo, C.sub.1-6
alkoxyl), C.sub.1-6 alkoxyl (linear, branched, optionally
substituted; for example, optionally substituted by 1 or more
halo), C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or CF.sub.3; [1505]
Y.sup.1, Y.sup.2 are each independently NR.sup.Y1, O, S, SO2,
heteroaryl, or aryl; [1506] Y.sup.3, Y.sup.4, Y.sup.5 are each
independently a bond, O, NR.sup.Y2, CR.sup.Y1R.sup.Y2, C.dbd.O,
C.dbd.S, SO, SO.sub.2, heteroaryl, or aryl; [1507] Q is a 3-6
membered ring with 0-4 heteroatoms, optionally substituted with 0-6
R.sup.Q, each R.sup.Q, is independently H, C.sub.1-6 alkyl (linear,
branched, optionally substituted, for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, or 2 R.sup.Q groups taken together with the atom
they are attached to, form a 3-8 membered ring system containing
0-2 heteroatoms); [1508] R.sup.1, R.sup.2, R.sup.a, R.sup.b,
R.sup.Y1, R.sup.Y2 are each independently H, C.sub.1-6 alkyl
(linear, branched, optionally substituted; for example, optionally
substituted by 1 or more halo, C.sub.1-6 alkoxyl), halogen,
C.sub.1-6 alkoxy, cyclic, heterocyclic or R.sup.1, R.sup.2 together
with the atom they are attached to, form a 3-8 membered ring system
containing 0-2 heteroatoms); [1509] W.sup.2 is a bond, C.sub.1-6
alkyl, C.sub.1-6 heteroalkyl, O, aryl, heteroaryl, alicyclic,
heterocyclic, biheterocyclic, biaryl, or biheteroaryl, each
optionally substituted by 1-10 R.sup.W2; [1510] each R.sup.W2 is
independently H, halo, C.sub.1-6 alkyl (linear or branched
optionally substituted; for example, optionally substituted by 1 or
more F), --OR.sup.W2A, C.sub.3-6 cycloalkyl, C.sub.4-6
cycloheteroalkyl, C.sub.1-6 alkyl (optionally substituted),
heterocyclic (optionally substituted), aryl (optionally
substituted), or heteroaryl (optionally substituted), bicyclic
heteroaryl or aryl, OC.sub.1-3alkyl (optionally substituted; for
example, optionally substituted by 1 or more --F), OH, NH.sub.2,
NR.sup.Y1R.sup.Y2, CN; [1511] R.sup.W2A is H, C.sub.1-6 alkyl
(linear, branched), or C.sub.1-6 heteroalkyl (linear, branched),
each optionally substituted by a cycloalkyl, cycloheteroalkyl,
aryl, heterocyclic, heteroaryl, halo, or OC.sub.1-3alkyl; and
[1512] the dashed line indicates the site of attachment of at least
one of the linker, the CLM, a CLM', or a combination thereof;
[1513] (D) a Tau protein targeting moiety that is represented by at
least one of Formula I-XI:
[1513] ##STR00649## [1514] wherein: [1515] A, B, C, D, E, and F are
independently selected from an optionally substituted 5- or
6-membered aryl or heteroaryl ring, an optionally substituted 4- to
7-membered cycloalkyl or a heterocycloalkyl, where contact between
circles indicates ring fusion; [1516] L.sub.PTM is selected from a
bond, an alkyl, an alkenyl or an alkynyl, optionally interrupted by
one or more rings (i.e., cycloalkyl, heterocycloalkyl, aryl or
heteroaryl), or one or more functional groups selected from the
groups --O--, --S--, --NR.sup.1.sub.PTM--, --N.dbd.N--, --S(O)--,
--SO.sub.2--, --C(O)--, --NHC(O)--, --C(O)NH--, --NHSO.sub.2--,
--NHC(O)NH--, --NHC(O)O--, or --OC(O)NH--, wherein the said
functional group is optionally located at either end of the linker;
and [1517] R.sup.1.sub.PTM is selected from H or alkyl. [1518] (E)
a tricyclic diazepine or azepine BET/BRD4 binding ligand comprising
a group according to the chemical structure PTM-a:
[1518] ##STR00650## [1519] wherein: [1520] Y.sub.1, Y.sub.2 and
Y.sub.3 are independently selected from the group of carbon,
nitrogen or oxygen and together with the atoms to form an aromatic
fused ring. [1521] A and B are independently selected from the
group of a 5-membered aromatic ring, a 6-membered aromatic ring, a
heteroaromatic ring, a carbocyclic, a thiophene a pyrrole ring, a
pyridine, a pyrimidine, a pyrazine, a pyrazole ring each optionally
substituted with alkyl, alkoxy, halogen, an aromatic and a
heteroaromatic ring; wherein ring A is fused to the central azepine
(Y1=C) or diazepine (Y1=N) moiety; and [1522] Z1 is selected from
the group of methyl or an alkyl group, and [1523] wherein the
dashed line indicates the site of attachment of at least one of the
linker, the CLM, a CLM', or a combination thereof.
[1524] In any aspect or embodiment described herein, in the Tau
protein targeting moiety, at least one of: [1525] at least one of
A, B, C, F, or a combination thereof is selected from optionally
substituted 5- or 6-membered aryl or heteroaryl rings; [1526] aryl
and heteroaryl rings of A, B, C, D and E of PTM are optionally
substituted with 1-8 substituents each independently selected from
alkyl, alkenyl, haloalkyl, halogen, hydroxyl, alkoxy, fluoroalkoxy,
amino, alkylamino, dialkylamino, acylamino, trifluoromethyl and
cyano, wherein the said alkyl and alkenyl groups are further
optionally substituted; or [1527] a combination thereof.
[1528] In any aspect or embodiment described herein, the PTM is
Formula I and: [1529] A, B and C rings are independently 5- or
6-membered fused aryl or heteroaryl rings; [1530] L.sub.PTM is
selected from a bond or an alkyl; and [1531] D is selected from a
6-membered aryl, heteroaryl or heterocycloalkyl, [1532] wherein A,
B, C and D are optionally substituted with alkyl, haloalkyl,
halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino,
trifluoromethyl or cyano.
[1533] In any aspect or embodiment described herein, the PTM is
Formula I and: [1534] A and C are a phenyl or a 6-membered
heteroaryl ring; [1535] B is a 5-membered heteroaryl ring; [1536]
L.sub.PTM is a bond; and [1537] D is a 6-membered heteroaryl or a
6-membered heterocycloalkyl ring, [1538] wherein each A, B, C and D
is optionally independently substituted with alkyl, haloalkyl,
halogen, hydroxyl, alkoxy, amino, dialkylamino, trifluoromethyl, or
cyano, and wherein a nitrogen atom of any of the A, B, C and D
rings is not directly connected to a heteroatom or to a carbon
atom, to which another heteroatom is directly attached.
[1539] In any aspect or embodiment described herein, the PTM is
Formula III or IV and: [1540] A, B and C are 5- or 6-membered fused
aryl or heteroaryl rings; [1541] L.sub.PTM is selected from a bond
or an alkyl; and [1542] D and E are 5- or 6-membered fused aryl or
heteroaryl rings; [1543] wherein A, B, C, D and E are optionally
substituted with alkyl, haloalkyl, halogen, hydroxyl, alkoxy,
amino, alkylamino, dialkylamino, trifluoromethyl, or cyano.
[1544] In any aspect or embodiment described herein, the PTM has a
structure selected from the group consisting of:
##STR00651## ##STR00652## ##STR00653## ##STR00654## ##STR00655##
[1545] wherein R or Linker is a bond or a chemical linker moiety
coupling the CLM to the PTM, including pharmaceutically acceptable
salt forms thereof.
[1546] In any aspect or embodiment described herein, the compound
is selected from the group consisting of PROTAC-1 through
PROTAC-112.
[1547] In any aspect or embodiment described herein, the compound
is selected from the group consisting of: [1548]
4-{3-[4-({1-[5-chloro-1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyrida-
zin-4-yl]-1,4,7,10-tetraoxadodecan-12-yl}oxy)phenyl]-4,4-dimethyl-5-oxo-2--
sulfanylideneimidazolidin-1-yl}-2-(trifluoromethyl)benzonitrile;
[1549]
4-{3-[4-(2-{2-[4-(2-{[1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyrida-
zin-4-yl]oxy}ethyl)piperazin-1-yl]ethoxy}ethoxy)phenyl]-4,4-dimethyl-5-oxo-
-2-sulfanylideneimidazolidin-1-yl}-2-(trifluoromethyl)benzonitrile;
[1550]
4-[3-(4-{2-[4-(2-{[5-chloro-1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydro-
pyridazin-4-yl]oxy}ethyl)piperazin-1-yl]ethoxy}phenyl)-4,4-dimethyl-5-oxo--
2-sulfanylideneimidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile;
[1551]
6-{4-[5-({6-[(2,6-dioxopiperidin-3-yl)carbamoyl]pyridin-3-yl}oxy)pentyl]p-
iperazin-1-yl}-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylc-
yclobutyl]pyridine-3-carboxamide; [1552]
6-[4-(5-{[3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-1,2,3,4-tetrahydroqu-
inazolin-8-yl]oxy}pentyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanophe-
noxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide; [1553]
6-[4-(6-{[1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl]oxy-
}hexyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetr-
amethylcyclobutyl]pyridine-3-carboxamide; [1554]
6-[4-(5-{[3-(2,6-dioxopiperidin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazoli-
n-8-yl]oxy}pentyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2-
,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide; [1555]
5-(5-{4-[2-(4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-
-sulfanylideneimidazolidin-1-yl}phenoxy)ethyl]piperazin-1-yl}-1,3-dioxo-2,-
3-dihydro-1H-isoindol-2-yl)-6-oxo-1,6-dihydropyridine-2-carbonitrile;
[1556]
4-[3-(4-{2-[4-({1-[5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)p-
yridin-3-yl]piperidin-4-yl}methyl)piperazin-1-yl]ethoxy}phenyl)-4,4-dimeth-
yl-5-oxo-2-sulfanylideneimidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile-
; [1557]
4-[3-(4-{[3-(3-{[3-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)qu-
inolin-5-yl]oxy}propoxy)propyl]amino}phenyl)-4,4-dimethyl-5-oxo-2-sulfanyl-
ideneimidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile; [1558]
4-[3-(4-{[3-(3-{[3-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)quinolin-5-
-yl]oxy}propoxy)propyl]amino}phenyl)-4,4-dimethyl-5-oxo-2-sulfanylideneimi-
dazolidin-1-yl]-2-(trifluoromethyl)benzonitrile; [1559]
4-[4-(2-{2-[(2-{[2-(2,4-dioxo-1,3-diazinan-1-yl)ethyl]carbamoyl}phenyl)am-
ino]ethoxy}ethyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,-
2,4,4-tetramethylcyclobutyl]benzamide; [1560]
5-(4-{2-[(1,3-dioxo-2-{6-oxo-2-oxa-5-azaspiro[3.5]nonan-9-yl}-2,3-dihydro-
-1H-isoindol-4-yl)amino]ethyl}piperazin-1-yl)-N-[(1r,3r)-3-(3-chloro-4-cya-
nophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-2-carboxamide;
[1561]
4-(4,4-dimethyl-3-{4-[4-(3-{[2-(1-methyl-2,4-dioxo-1,2,3,4-tetrahydropyri-
midin-5-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]oxy}propyl)piperazin-1--
yl]phenyl}-5-oxo-2-sulfanylideneimidazolidin-1-yl)-2-(trifluoromethyl)benz-
onitrile; [1562]
5-[4-(2-{[2-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-3-oxo-octahydroindo-
lizin-6-yl]amino}ethyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanopheno-
xy)-2,2,4,4-tetramethylcyclobutyl]pyridine-2-carboxamide; [1563]
4-[3-(4-{[3-(3-{[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)isoquinoli-
n-7-yl]oxy}propoxy)propyl]amino}phenyl)-4,4-dimethyl-5-oxo-2-sulfanylidene-
imidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile; [1564]
4-[3-(4-{1-[3-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-4-methylquinol-
in-7-yl]-1,4,7-trioxa-10-azadecan-10-yl}phenyl)-4,4-dimethyl-5-oxo-2-sulfa-
nylideneimidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile; [1565]
4-[2-(2-{[3-(2,4-dioxo-1,3-diazinan-1-yl)-4-methylquinolin-7-yl]oxy}ethox-
y)ethoxy]-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclob-
utyl]benzamide; [1566]
5-{3-[4-(1,3-dioxo-2-{6-oxo-2-oxa-5-azaspiro[3.5]nonan-9-yl}-2,3-dihydro--
1H-isoindol-5-yl)piperazin-1-yl]propyl}-N-[(1r,3r)-3-(3-chloro-4-cyanophen-
oxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-2-carboxamide; [1567]
4-{4-[2-(2-{[1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-
amino}ethoxy)ethyl]piperazin-1-yl}-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)--
2,2,4,4-tetramethylcyclobutyl]benzamide; [1568]
4-[4-({1-[5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)pyridin-3-yl]pipe-
ridin-4-yl}methyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-4-cyanophenoxy)-2-
,2,4,4-tetramethylcyclobutyl]benzamide; [1569]
4-(4-{2-[4-(2-{[1-(2,6-dioxopiperidin-3-yl)-6-oxo-1,6-dihydropyridazin-4--
yl]oxy}ethyl)piperazin-1-yl]ethoxy}butoxy)-N-[(1r,3r)-3-(3-chloro-4-cyanop-
henoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide; [1570]
2-[(2-{2-[4-(4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo--
2-sulfanylideneimidazolidin-1-yl}phenyl)piperazin-1-yl]ethoxy}ethyl)amino]-
-N-[2-(2,4-dioxo-1,3-diazinan-1-yl)ethyl]benzamide; [1571]
2-{[2-(2-{[4-(4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-
-2-sulfanylideneimidazolidin-1-yl}phenyl)phenyl]amino}ethoxy)ethyl]amino}--
N-[2-(2,4-dioxo-1,3-diazinan-1-yl)ethyl]benzamide; [1572]
4-{4-[2-({1,3-dioxo-2-[2-oxo-6-(trifluoromethyl)piperidin-3-yl]-2,3-dihyd-
ro-1H-isoindol-4-yl}amino)ethyl]piperazin-1-yl}-N-[(1r,3r)-3-(3-chloro-4-c-
yanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide; [1573]
4-{4-[2-({1,3-dioxo-2-[2-oxo-6-(trifluoromethyl)piperidin-3-yl]-2,3-dihyd-
ro-1H-isoindol-5-yl}oxy)ethyl]piperazin-1-yl}-N-[(1r,3r)-3-(3-chloro-4-cya-
nophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide; [1574]
4-{4-[2-({1,3-dioxo-2-[2-oxo-6-(trifluoromethyl)-1,2-dihydropyridin-3-yl]-
-2,3-dihydro-1H-isoindol-4-yl}amino)ethyl]piperazin-1-yl}-N-[(1r,3r)-3-(3--
chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide;
[1575]
4-{4-[2-({1,3-dioxo-2-[2-oxo-6-(trifluoromethyl)-1,2-dihydropyridin-3-yl]-
-2,3-dihydro-1H-isoindol-5-yl}oxy)ethyl]piperazin-1-yl}-N-[(1r,3r)-3-(3-ch-
loro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]benzamide;
[1576]
4-[3-(4-{2-[4-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1-
.lamda..sup.6,2-benzothiazol-6-yl]amino}ethyl)piperazin-1-yl]ethoxy}phenyl-
)-4,4-dimethyl-5-oxo-2-sulfanylideneimidazolidin-1-yl]-2-(trifluoromethyl)-
benzonitrile; [1577]
4-[3-(4-{2-[4-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1-
.lamda..sup.6,2-benzothiazol-6-yl]oxy}ethyl)piperazin-1-yl]ethoxy}phenyl)--
4,4-dimethyl-5-oxo-2-sulfanylideneimidazolidin-1-yl]-2-(trifluoromethyl)be-
nzonitrile; [1578]
6-[4-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1.lamda..s-
up.6,2-benzothiazol-6-yl]oxy}pentyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chloro-
-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide;
[1579]
6-[4-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1.l-
amda..sup.6,2-benzothiazol-6-yl]amino}pentyl)piperazin-1-yl]-N-[(1r,3r)-3--
(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxa-
mide; [1580]
6-[4-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1.lamda..s-
up.6,2-benzothiazol-7-yl]amino}pentyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-chlo-
ro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide;
[1581]
6-[4-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1.l-
amda..sup.6,2-benzothiazol-7-yl]oxy}pentyl)piperazin-1-yl]-N-[(1r,3r)-3-(3-
-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxami-
de; [1582]
4-[3-(4-{2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-
-dihydro-1.lamda..sup.6,2-benzothiazol-6-yl]oxy}ethoxy)ethoxy]ethoxy
phenyl)-4,4-dimethyl-5-oxo-2-sulfanylideneimidazolidin-1-yl]-2-(trifluoro-
methyl)benzonitrile; and [1583]
6-[3-(3-{[2-(2,6-dioxopiperidin-3-yl)-1,1,3-trioxo-2,3-dihydro-1.lamda..s-
up.6,2-benzothiazol-6-yl]oxy}propoxy)propoxy]-N-[(1r,3r)-3-(3-chloro-4-cya-
nophenoxy)-2,2,4,4-tetramethylcyclobutyl]pyridine-3-carboxamide,
including pharmaceutically acceptable salt forms thereof.
[1584] Another aspect discloses a composition comprising an
effective amount of a bifunctional compound of the present
disclosure, and a pharmaceutically acceptable carrier.
[1585] In any aspect or embodiment described herein, the
composition further comprises at least one of additional bioactive
agent or another bifunctional compound of the present
disclosure.
[1586] In any aspect or embodiment described herein, the additional
bioactive agent is anti-cancer agent, an anti-neurodegenerative
agent, an antimicrobial agent, an antiviral agent, an anti-HIV
agent, or an antifungal agent.
[1587] A further aspect disclosures a composition comprising an
effective amount of at least one compound of the present disclosure
and a pharmaceutically acceptable carrier, additive, and/or
excipient for treating a disease or disorder in a subject, the
method comprising administering the composition to a subject in
need thereof, wherein the compound is effective in treating or
ameliorating at least one symptom of the disease or disorder.
[1588] In any aspect or embodiment described herein, the disease or
disorder is associated with the accumulation and/or aggregation of
the target protein.
[1589] In any aspect or embodiment described herein, the disease or
disorder is selected from the group consisting of asthma,
autoimmune diseases such as multiple sclerosis, various cancers,
ciliopathies, cleft palate, diabetes, heart disease, hypertension,
inflammatory bowel disease, mental retardation, mood disorder,
obesity, refractive error, infertility, Angelman syndrome, Canavan
disease, Coeliac disease, Charcot-Marie-Tooth disease, Cystic
fibrosis, Duchenne muscular dystrophy, Haemochromatosis,
Haemophilia, Klinefelter's syndrome, Neurofibromatosis,
Phenylketonuria, Polycystic kidney disease, (PKD1) or 4 (PKD2)
Prader-Willi syndrome, Sickle-cell disease, Tay-Sachs disease,
Turner syndrome.
[1590] In any aspect or embodiment described herein, the disease or
disorder is selected from the group consisting of Alzheimer's
disease, Amyotrophic lateral sclerosis (Lou Gehrig's disease),
Anorexia nervosa, Anxiety disorder, Atherosclerosis, Attention
deficit hyperactivity disorder, Autism, Bipolar disorder, Chronic
fatigue syndrome, Chronic obstructive pulmonary disease, Crohn's
disease, Coronary heart disease, Dementia, Depression, Diabetes
mellitus type 1, Diabetes mellitus type 2, Epilepsy, Guillain-Barre
syndrome, Irritable bowel syndrome, Lupus, Metabolic syndrome,
Multiple sclerosis, Myocardial infarction, Obesity,
Obsessive-compulsive disorder, Panic disorder, Parkinson's disease,
Psoriasis, Rheumatoid arthritis, Sarcoidosis, Schizophrenia,
Stroke, Thromboangiitis obliterans, Tourette syndrome,
Vasculitis.
[1591] In any aspect or embodiment described herein, the disease or
disorder is selected from the group consisting of
aceruloplasminemia, Achondrogenesis type II, achondroplasia,
Acrocephaly, Gaucher disease type 2, acute intermittent porphyria,
Canavan disease, Adenomatous Polyposis Coli, ALA dehydratase
deficiency, adenylosuccinate lyase deficiency, Adrenogenital
syndrome, Adrenoleukodystrophy, ALA-D porphyria, ALA dehydratase
deficiency, Alkaptonuria, Alexander disease, Alkaptonuric
ochronosis, alpha 1-antitrypsin deficiency, alpha-1 proteinase
inhibitor, emphysema, amyotrophic lateral sclerosis Alstrom
syndrome, Alexander disease, Amelogenesis imperfecta, ALA
dehydratase deficiency, Anderson-Fabry disease, androgen
insensitivity syndrome, Anemia Angiokeratoma Corporis Diffusum,
Angiomatosis retinae (von Hippel-Lindau disease) Apert syndrome,
Arachnodactyly (Marfan syndrome), Stickler syndrome, Arthrochalasis
multiplex congenital (Ehlers-Danlos syndrome#arthrochalasia type)
ataxia telangiectasia, Rett syndrome, primary pulmonary
hypertension, Sandhoff disease, neurofibromatosis type II,
Beare-Stevenson cutis gyrata syndrome, Mediterranean fever,
familial, Benjamin syndrome, beta-thalassemia, Bilateral Acoustic
Neurofibromatosis (neurofibromatosis type II), factor V Leiden
thrombophilia, Bloch-Sulzberger syndrome (incontinentia pigmenti),
Bloom syndrome, X-linked sideroblastic anemia, Bonnevie-Ullrich
syndrome (Turner syndrome), Bourneville disease (tuberous
sclerosis), prion disease, Birt-Hogg-Dube syndrome, Brittle bone
disease (osteogenesis imperfecta), Broad Thumb-Hallux syndrome
(Rubinstein-Taybi syndrome), Bronze Diabetes/Bronzed Cirrhosis
(hemochromatosis), Bulbospinal muscular atrophy (Kennedy's
disease), Burger-Grutz syndrome (lipoprotein lipase deficiency),
CGD Chronic granulomatous disorder, Campomelic dysplasia,
biotinidase deficiency, Cardiomyopathy (Noonan syndrome), Cri du
chat, CAVD (congenital absence of the vas deferens), Caylor
cardiofacial syndrome (CBAVD), CEP (congenital erythropoietic
porphyria), cystic fibrosis, congenital hypothyroidism,
Chondrodystrophy syndrome (achondroplasia),
otospondylomegaepiphyseal dysplasia, Lesch-Nyhan syndrome,
galactosemia, Ehlers-Danlos syndrome, Thanatophoric dysplasia,
Coffin-Lowry syndrome, Cockayne syndrome, (familial adenomatous
polyposis), Congenital erythropoietic porphyria, Congenital heart
disease, Methemoglobinemia/Congenital methaemoglobinaemia,
achondroplasia, X-linked sideroblastic anemia, Connective tissue
disease, Conotruncal anomaly face syndrome, Cooley's Anemia
(beta-thalassemia), Copper storage disease (Wilson's disease),
Copper transport disease (Menkes disease), hereditary
coproporphyria, Cowden syndrome, Craniofacial dysarthrosis (Crouzon
syndrome), Creutzfeldt-Jakob disease (prion disease), Cockayne
syndrome, Cowden syndrome, Curschmann-Batten-Steinert syndrome
(myotonic dystrophy), Beare-Stevenson cutis gyrata syndrome,
primary hyperoxaluria, spondyloepimetaphyseal dysplasia (Strudwick
type), muscular dystrophy, Duchenne and Becker types (DBMD), Usher
syndrome, Degenerative nerve diseases including de Grouchy syndrome
and Dejerine-Sottas syndrome, developmental disabilities, distal
spinal muscular atrophy, type V, androgen insensitivity syndrome,
Diffuse Globoid Body Sclerosis (Krabbe disease), Di George's
syndrome, Dihydrotestosterone receptor deficiency, androgen
insensitivity syndrome, Down syndrome, Dwarfism, erythropoietic
protoporphyria Erythroid 5-aminolevulinate synthetase deficiency,
Erythropoietic porphyria, erythropoietic protoporphyria,
erythropoietic uroporphyria, Friedreich's ataxia, familial
paroxysmal polyserositis, porphyria cutanea tarda, familial
pressure sensitive neuropathy, primary pulmonary hypertension
(PPH), Fibrocystic disease of the pancreas, fragile X syndrome,
galactosemia, genetic brain disorders, Giant cell hepatitis
(Neonatal hemochromatosis), Gronblad-Strandberg syndrome
(pseudoxanthoma elasticum), Gunther disease (congenital
erythropoietic porphyria), haemochromatosis, Hallgren syndrome,
sickle cell anemia, hemophilia, hepatoerythropoietic porphyria
(HEP), Hippel-Lindau disease (von Hippel-Lindau disease),
Huntington's disease, Hutchinson-Gilford progeria syndrome
(progeria), Hyperandrogenism, Hypochondroplasia, Hypochromic
anemia, Immune system disorders, including X-linked severe combined
immunodeficiency, Insley-Astley syndrome, Kennedy's syndrome,
Jackson-Weiss syndrome, Joubert syndrome, Lesch-Nyhan syndrome,
Jackson-Weiss syndrome, Kidney diseases, including hyperoxaluria,
Klinefelter's syndrome, Kniest dysplasia, Lacunar dementia,
Langer-Saldino achondrogenesis, ataxia telangiectasia, Lynch
syndrome, Lysyl-hydroxylase deficiency, Machado-Joseph disease,
Metabolic disorders, including Kniest dysplasia, Marfan syndrome,
Movement disorders, Mowat-Wilson syndrome, cystic fibrosis, Muenke
syndrome, Multiple neurofibromatosis, Nance-Insley syndrome,
Nance-Sweeney chondrodysplasia, Niemann-Pick disease, Noack
syndrome (Pfeiffer syndrome), Osler-Weber-Rendu disease,
Peutz-Jeghers syndrome, Polycystic kidney disease, polyostotic
fibrous dysplasia (McCune-Albright syndrome), Peutz-Jeghers
syndrome, Prader-Labhart-Willi syndrome, hemochromatosis, primary
hyperuricemia syndrome (Lesch-Nyhan syndrome), primary pulmonary
hypertension, primary senile degenerative dementia, prion disease,
progeria (Hutchinson Gilford Progeria Syndrome), progressive
chorea, chronic hereditary (Huntington) (Huntington's disease),
progressive muscular atrophy, spinal muscular atrophy, propionic
acidemia, protoporphyria, proximal myotonic dystrophy, pulmonary
arterial hypertension, PXE (pseudoxanthoma elasticum), Rb
(retinoblastoma), Recklinghausen disease (neurofibromatosis type
I), Recurrent polyserositis, Retinal disorders, Retinoblastoma,
Rett syndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome,
Roussy-Levy syndrome, severe achondroplasia with developmental
delay and acanthosis nigricans (SADDAN), Li-Fraumeni syndrome,
sarcoma, breast, leukemia, and adrenal gland (SBLA) syndrome,
sclerosis tuberose (tuberous sclerosis), SDAT, SED congenital
(spondyloepiphyseal dysplasia congenita), SED Strudwick
(spondyloepimetaphyseal dysplasia, Strudwick type), SEDc
(spondyloepiphyseal dysplasia congenita) SEMD, Strudwick type
(spondyloepimetaphyseal dysplasia, Strudwick type), Shprintzen
syndrome, Skin pigmentation disorders, Smith-Lemli-Opitz syndrome,
South-African genetic porphyria (variegate porphyria),
infantile-onset ascending hereditary spastic paralysis, Speech and
communication disorders, sphingolipidosis, Tay-Sachs disease,
spinocerebellar ataxia, Stickler syndrome, stroke, androgen
insensitivity syndrome, tetrahydrobiopterin deficiency,
beta-thalassemia, Thyroid disease, Tomaculous neuropathy
(hereditary neuropathy with liability to pressure palsies),
Treacher Collins syndrome, Triplo X syndrome (triple X syndrome),
Trisomy 21 (Down syndrome), Trisomy X, VHL syndrome (von
Hippel-Lindau disease), Vision impairment and blindness (Alstrom
syndrome), Vrolik disease, Waardenburg syndrome, Warburg Sjo
Fledelius Syndrome, Weissenbacher-Zweymuller syndrome,
Wolf-Hirschhorn syndrome, Wolff Periodic disease,
Weissenbacher-Zweymuller syndrome and Xeroderma pigmentosum.
[1592] In any aspect or embodiment described herein, wherein the
composition further comprising an additional bioactive agent.
[1593] In any aspect or embodiment described herein, the additional
bioactive agent is at least one of an anti-cancer agent, an
anti-neurodegenerative agent, an antimicrobial agent, an antiviral
agent, an anti-HIV agent, an antifungal agent, or a combination
thereof.
[1594] In any aspect or embodiment described herein, the anticancer
agent is selected from the group consisting of everolimus,
trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib,
GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107,
TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197,
MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a
VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor,
a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET
inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor,
an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase
inhibitors, an AKT inhibitor, an mTORC1/2 inhibitor, a JAK/STAT
inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase
inhibitor, a Map kinase (mek) inhibitor, a VEGF trap antibody,
pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,
panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,
batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine,
rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab,
gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,
cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR.sub.1
KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx
102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380,
sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine,
doxorubicin, liposomal doxorubicin, 5'-deoxy-5-fluorouridine,
vincristine, temozolomide, ZK-304709, seliciclib; PD0325901,
AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258,);
3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone,
vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,
Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu
t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sub.4O.sub.2).sub.x
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin,
bleomycin, buserelin, busulfan, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clodronate, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin,
diethylstilbestrol, epirubicin, fludarabine, fludrocortisone,
fluoxymesterone, flutamide, gleevac, gemcitabine, hydroxyurea,
idarubicin, ifosfamide, imatinib, leuprolide, levamisole,
lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin,
porfimer, procarbazine, raltitrexed, rituximab, streptozocin,
teniposide, testosterone, thalidomide, thioguanine, thiotepa,
tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard,
uracil mustard, estramustine, altretamine, floxuridine,
5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine,
deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine,
vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat,
BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974,
interleukin-12, IM862, angiostatin, vitaxin, droloxifene,
idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,
denileukin diftitox, gefitinib, bortezimib, paclitaxel,
cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550,
BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,
ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene,
idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa, darbepoetin alfa and mixtures
thereof.
[1595] Another aspect discloses a method for inducing degradation
of a target protein in a cell comprising administering an effective
amount of a compound of the present disclosure to the cell, wherein
the compound effectuates degradation of the target protein.
[1596] A yet further aspect discloses a composition comprising an
effective amount of a compound of the present disclosure for use in
a method for treating cancer, said method comprising administering
the composition to a patient in need thereof, wherein the
composition is effectuates for the treatment or alleviation of at
least one symptom of cancer in the patient.
[1597] In any aspect or embodiment described herein, the cancer is
squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma,
hepatocellular carcinomas, and renal cell carcinomas, cancer of the
bladder, bowel, breast, cervix, colon, esophagus, head, kidney,
liver, lung, neck, ovary, pancreas, prostate, and stomach;
leukemias; benign and malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; benign and malignant
melanomas; myeloproliferative diseases; multiple myeloma, sarcomas,
including Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma,
liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial
sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas,
gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas,
medulloblastomas, pineal cell tumors, meningiomas, meningeal
sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast
cancer, prostate cancer, cervical cancer, uterine cancer, lung
cancer, ovarian cancer, testicular cancer, thyroid cancer,
astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer,
liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's
disease, Wilms' tumor or teratocarcinomas, T-lineage Acute
lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma
(T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B
ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma,
B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia
chromosome positive CML.
[1598] In any of the aspects or embodiments described herein, the L
comprises nonlinear chains, aliphatic or aromatic or heteroaromatic
cyclic moieties.
[1599] In any of the aspects or embodiments described herein, the L
is selected from the group consisting of:
##STR00656## ##STR00657## ##STR00658## ##STR00659##
##STR00660##
wherein: [1600] "X" is a linear chain with atoms ranging from 2 to
14 optionally substituted to contain heteroatoms; and [1601] "Y" is
independently selected from the group consisting of O, N,
S(O).sub.n (n=0, 1, 2).
EXAMPLES
Abbreviations
[1601] [1602] ACN: acetonitrile [1603] ADDP:
1,1'-(azodicarbonyl)dipiperidine [1604] BAST:
N,N-bis(2-methoxyethyl)aminosulfur trifluoride [1605] BPO: benzoyl
peroxide [1606] Cbz: Carbonylbezyloxy [1607] DAST:
diethylaminosulfur trifluoride [1608] DBE: 1,2-dibromoethane [1609]
DCM: dichloromethane [1610] DEAD: diethyl azodicarboxylate [1611]
DIAD: diisopropyl azodicarboxylate [1612] DIBAL:
disiobutylaluminium hydride [1613] DIEA or DIPEA:
diisopropylethylamine [1614] DMA: N,N-dimethylacetamide [1615] DMF:
N,N-dimethylformamide [1616] DMP: Dess-Martin periodinane [1617]
EA: ethyl acetate [1618] EDCI:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [1619] HBTU:
N,N,N'N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate [1620] HMDS: bis9trimethylsilyl)amine [1621]
HMPA: hexamethylphosphoramide [1622] LDA: lithium diisopropylamide
[1623] MCPBA: meta-chloroperoxybenzoic acid [1624] MsCl:
methanesulfonyl chloride [1625] M.W: microwave [1626] NBS:
N-bromosuccinimide [1627] NMP: N-methylpyrrolidone [1628] PCC:
pyridinium chlorochromate [1629] Pd-118 or Pd(dtpf)Cl.sub.2:
1,1'-bis(di-tert-butylphosphino)ferrocene dichloropalladium [1630]
Pd(dppf)Cl.sub.2: 1,1'-bis(diphenylphosphino)ferrocene
dichloropalladium [1631] Pd(dba).sub.2:
bis(dibenzylideneacetone)palladium [1632] Pd.sub.2(dba).sub.3:
Tris(dibenzylideneacetone)dipalladium [1633] PPTS: pyridium
p-tolunesulfonate [1634] PTSA: p-toluenesulfonic acid [1635]
RuPhos-Pd-G3: XPhos-Pd-G3:
[(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)-2-(2'-amino-1-
,1'-biphenyl)] palladium(II) methanesulfonate [1636] RuPhos-Pd-G2:
Chloro[(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)-2-(2'-a-
mino-1,1'-biphenyl)] palladium(II) [1637] SFC: supercritical fluid
chromatography [1638] t-BuXPhos-Pd-G3:
[(2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-ami-
no-1,1'-biphenyl)] palladium(II) methanesulfonate [1639] TEA:
trimethylamine [1640] TFA: trifluoroacetic acid [1641] TLC: thin
layer chromatography [1642] TMP: 2,2,6,6-tetramethylpiperidine
[1643] TEMPO: 2,2,6,6-tetramethylpiperidine-N-oxide [1644] TosCl or
TsCl: p-toluenesulfonyl chloride [1645] TsOH: p-toluenesulfonic
acid [1646] XantPhos:
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene [1647] XPhos:
2-dicyclohexylphosphino-2' 4'6'-triisopropylbiphenyl [1648]
XPhos-Pd-G3:
[(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amin-
o-1,1'-biphenyl)] palladium(II) methanesulfonate [1649] 12354-85-7:
bis(pentamethylcyclopentadienylrhodium dichloride)
[1650] A. Cloning, Expression and Purification of Human CRBN and
DDB1.
[1651] The proceedure is standard to one versed in the art, as
typified by the description in Lopez-Girona et al. (Cereblon is a
direct protein target for immunomodulatory and antiproliferative
activities of lenalidomide and pomalidomide, A Lopez-Girona, D
Mendy, T Ito, K Miller, A K Gandhi, J Kang, S Karasawa, G Carmel, P
Jackson, M Abbasian, A Mahmoudi, B Cathers, E Rychak, S Gaidarova,
R Chen, P H Schafer, H Handa, T O Daniel, J F Evans and R Chopra,
Leukemia 26: 2326-2335, 2012).
[1652] The cDNAs for the CRBN and DDB1 genes can be amplified by
PCR using Pfusion (NEB) as the polymerase.
[1653] CRBN can be cloned into pBV-ZZ-HT-LIC, pBV-GST-LIC,
pMA-HT-LIC, and DDB1 into pBV-notag-LIC, using ligation-independent
cloning 26. For cloning into the mammalian vector pMA-HT-LIC, the
CRBN-Flag-Reverse oligo adds a C-terminal FLAG tag for
immunodetection. The DDB1-Rev adds a StrepTag 27. A ZZ-tag 28 is
necessary to achieve high expression of soluble CRBN; without it,
the His-CRBN expressed at low level, while a GST-CRBN results in
aggregated protein. Recombinant baculovirus of ZZ-His-CRBN and
DDB1-StrepTag (ST) are generated and amplified using Bac-to-Bac
baculovirus expression system from Invitrogen in Sf9 insect cells.
ZZ-His-CRBN and DDB1-ST are co-expressed in High Five (Tni) insect
in 10 L wave bags at 27.degree. C. using un-supplemented ESF921
media from Expression Systems. Cells are harvested 48 hours post
infection by centrifugation and paste resuspended in PBS
plus5.times. Protease Inhibitor cocktail (Roche, Indianapolis,
Ind.).
[1654] All subsequent protein purification steps are carried out at
4.degree. C. Frozen cells are thawed, resuspended in 5 volumes of
lysis buffer (50 mM Tris HCl pH 8.0, 0.5 M NaCl, 10% glycerol, 2 mM
DTT) plus 20 mM imidazole and protease inhibitors, lysed and
centrifuged to yield a clear supernatant. The CRBN-DDB1 is purified
on a AKTA-xpress system (GE Healthcare) using a Nickel-Sepharose
and 5200 Sephacryl chromatography. The complex is then further
purified using anion exchange chromatography on an 8 ml MonoQ
column and a second pass on a S-200 gel filtration. CRBN-DDB1 is
identified by SDS-PAGE and the CRBN-DDB1 containing fractions were
pooled and stored at -70.degree. C.
[1655] 2. Fluorescence Thermal Melt Assay to Measure Binding of
Compounds to Recombinant CRBN
[1656] The assay is standard to one versed in the art, as typified
by the description in Lopez-Girona et al. (Cereblon is a direct
protein target for immunomodulatory and antiproliferative
activities of lenalidomide and pomalidomide, A Lopez-Girona, D
Mendy, T Ito, K Miller, A K Gandhi, J Kang, S Karasawa, G Carmel, P
Jackson, M Abbasian, A Mahmoudi, B Cathers, E Rychak, S Gaidarova,
R Chen, P H Schafer, H Handa, T O Daniel, J F Evans and R Chopra,
Leukemia 26: 2326-2335, 2012).
[1657] Thermal stabilities of CRBN-DDB1 in the presence or absence
of test compounds are done in the presence of Sypro Orange in a
microplate format according to Pantoliano et al. (Pantoliano M W,
Petrella E C, Kwasnoski J D, Lobanov V S, Myslik J, Graf E et al.
High-density miniaturized thermal shift assays as a general
strategy for drug discovery. J Biomol Screen 2001; 6: 429-440.) Two
mg of protein in 20 ml of assay buffer (25 mM Tris HCl, pH 8.0, 150
mM NaCl, 2 uM Sypro Orange) are subjected to stepwise increase of
temperature from 20 to 70.degree. C. and the fluorescence read at
every 1.degree. C. on an ABIPrism 7900HT (Applied Biosystems,
Carlsbad, Calif., USA). Compounds are dissolved in DMSO (1% final
in assay) and tested in quadruplicate at a concentration range
between 30 nM to 1000 uM; controls contained 1% DMSO only.
[1658] 3. LCMS Method
[1659] The analysis is conducted on a Poroshell 120 EC C18 column
(50 mm.times.3.0 mm internal diameter 2.7 .mu.m packing diameter)
at 45.degree. C.
[1660] The solvents employed are: [1661] A=0.1% v/v solution of
formic acid in water. [1662] B=0.1% v/v solution of formic acid in
acetonitrile.
[1663] The gradient employed are as follows:
TABLE-US-00001 Time Flow Rate (minutes) (mL/min) % A % B 0 1 95 5
0.5 1 95 5 3.0 1 1 99 3.75 1 1 99 4.0 1 95 5
[1664] The UV detection is an averaged signal from wavelength of
210 nm to 350 nm and mass spectra are recorded on a mass
spectrometer using positive mode electrospray ionization.
[1665] The following illustrates the mobile phases and gradients
used when compounds undergo purification by preparative HPLC.
[1666] 4. Preparative HPLC (Formic Acid Modifier)
[1667] The HPLC analysis is conducted on an X Bridge RP18 OBD
column (150 mm.times.19 mm internal diameter, 5 .mu.m packing
diameter) at ambient temperature.
[1668] The solvents employed are: [1669] A=0.1% v/v solution of
formic acid in water. [1670] B=acetonitrile.
[1671] 5. Preparative HPLC (Ammonium Bicarbonate Modifier)
[1672] The HPLC analysis is conducted on an X Bridge RP18 OBD
column (150 mm.times.19 mm internal diameter, 5 .mu.m packing
diameter) at ambient temperature.
[1673] The solvents employed are: [1674] A=10 mM ammonium
bicarbonate in water. [1675] B=acetonitrile.
[1676] For each of the preparative purifications, irrespective of
the modifier used, the gradient employed is dependent upon the
retention time of the particular compound undergoing purification
as recorded in the analytical LCMS. The flow rate is 20 mL/min.
[1677] The UV detection is a signal from wavelength of 254 nm or
220 nm.
[1678] While preferred embodiments of the invention have been shown
and described herein, it will be understood that such embodiments
are provided by way of example only. Numerous variations, changes
and substitutions will occur to those skilled in the art without
departing from the spirit of the invention. Accordingly, it is
intended that the appended claims cover all such variations as fall
within the spirit and scope of the invention.
[1679] B. Synthesis
[1680] The synthetic details for the examples included below are
representative of the general procedures that inform on the
synthesis of the broader example set.
1.
N-(3-(5-bromo-2-chloropyrimidin-4-ylamino)propyl)-N-methylcyclobutane
carboxamide
##STR00661##
[1681] Step 1: tert-butyl
N-{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}-N-methylcarbamate
##STR00662##
[1683] A mixture of tert-butyl N-(3-aminopropyl)-N-methylcarbamate
(826 mg, 4.40 mmol) and 5-bromo-2,4-dichloropyrimidine (400 mg,
1.76 mmol) in MeOH (10 mL) was stirred at rt for 1 h. The reaction
mixture was then concentrated in vacuo, and the residue was
purified using a Teledyne ISCO Chromatography [0.fwdarw.35%
EtOAc/Heptanes] to afford tert-butyl
N-{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}-N-methylcarbamate
(615 mg, 92% yield). LC-MS (ES.sup.+): m/z=381.05/383.05
[MH.sup.+], t.sub.R=2.55 min.
Step 2:
{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}(methyl)amine
##STR00663##
[1685] To a solution of tert-butyl
N-{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}-N-methylcarbamate
(615 mg, 1.62 mmoL) in DCM (5 mL) was added trifluoroacetic acid
(0.54 mL, 6.5 mmol) at rt. After the mixture was stirred for 1 h,
it was concentrated in vacuo. The residue was purified using a
Teledyne ISCO Chromatography [0.fwdarw.15% methanol in DCM] to
afford
{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}(methyl)amine (371
mg, 82% yield). LC-MS (ES.sup.+): m/z=280.99/282.99 [MH.sup.+],
t.sub.R=1.13 min.
Step 3:
N-{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}-N-methylcyclob-
utanecarboxamide
##STR00664##
[1687] To a solution of
{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}(methyl)amine (371
mg, 1.33 mmol) and cyclobutanecarbonyl chloride (188 mg, 1.60 mmol)
in DCM (10 mL) at rt was added triethyl amine (0.41 mL, 2.92 mmol).
The reaction mixture was left to stir at rt for 16 h, then
concentrated in vacuo. The residue was purified using a Teledyne
ISCO Chromatography [0.fwdarw.100% EtOAc/Heptanes] to afford
N-{3-[(5-bromo-2-chloropyrimidin-4-yl)amino]propyl}-N-methylcyclobutane
carboxamide (268 mg, 56%). LC-MS (ES.sup.+): m/z=363.04/365.04
[MH.sup.+], t.sub.R=2.18 min.
2. (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f]
[1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid
##STR00665##
[1689] The title compound was prepared according to the procedures
described in WO2011/143660
3.
(Z)-4-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-2-methoxyphenoxy)-3-(-
trifluoromethyl)benzonitrile
##STR00666##
[1691] The title compound was prepared according to the procedures
described in Patch, R. J. et al J. Med. Chem. 2011, 54,
788-808.
4.
4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-sulfanylideneimidazolidin-1-
-yl]-2-(trifluoromethyl)benzonitrile
##STR00667##
[1693] The title compound was prepared according to the procedures
described in Jung, M. E. et al J. Med. Chem. 2010, 53,
2779-2796.
5.
2-chloro-4-(trans-3-amino-2,2,4,4-tetramethylcyclobutoxy)benzonitrile
hydrogen chloride salt
##STR00668##
[1695] The title compound was prepared according to the procedures
described in Guo, C. et al J. Med. Chem. 2011, 54, 7693-7704.
[1696] C. Protein Degradation Bioassays:
[1697] The following bioassays evaluate the level of protein
degradation observed in various cell types using representative
compounds disclosed herein.
[1698] In each bioassay, cells were treated with varying amounts of
compounds encompassed by the present disclosure. The degradation of
the following proteins may be evaluated: TANK-binding kinase 1
(TBK1), estrogen receptor .alpha. (ER.alpha.),
bromodomain-containing protein 4 (BRD4), androgen receptor (AR),
c-Myc, and tau protein.
[1699] 1. ERE Luciferase Assay for Compounds in Table 2.
[1700] T47D-KBluc cells (ATCC.RTM. #CRL_2865, T47D human breast
cancer cells stably transfected with estrogen responsive
element/promoter/luciferase reporter gene) were seeded into 96-well
white opaque plates in RPMI growth medium supplemented with 10%
fetal bovine serum (PBS) and allowed to adhere overnight in a
37.degree. C. humidified incubator. The following day, cells were
treated with PROTACs in a 12-point concentration curve (top final
concentration of 300 nM with subsequent concentrations being 3-fold
less with 2 pM being the lowest concentration in the assay). Each
PROTAC was tested independently in two experiments on 96-well
plates. After 24 hours, media was removed and lysis buffer was
added to the wells. Following lysis, Bright-Glo.TM. Luciferase
Assay Substrate (Promega, Madison Wis.) was added and the
luciferase activity was measured using a Cytation 3 plate reader
(BioTek.TM., Winooski, Vt.). Each compound was assayed in duplicate
and the activity was calculated as IC50 using GraphPad Prism
software (San Diego, Calif.).
[1701] 2. Estrogen Receptor-Alpha (ER.alpha.) Degradation Assay in
MCF-7 Cells Using Western Blot Method for Table 5.
[1702] The exemplary novel ER.alpha. degraders were assessed for
their activity in degrading ER.alpha. in MCF-7 cells via western
blot. The assay was carried out in the presence of 10% FBS or high
percentage of human or mouse serum. Protocols of the western blot
assay are described below.
[1703] MCF7 cells were grown in DMEM/F12 with 10% FBS and seeded at
24,000 cells per well in 100 .mu.l into 96-well clear tissue
culture plates. The following day, the cells were treated with
PROTACs in a 7-point concentration curve with 100 nM being the top
concentration and serial dilutions to make the other concentrations
(30 nM, 10 nM, 3 nM, 1 nM, and 0.3 nM). At all concentrations,
0.01% DMSO is the final concentration in the well. The following
day, the plates are aspirated, washed with 50 .mu.l of cold PBS.
The cells are lysed with 50 .mu.l/well 4.degree. C. Cell Lysis
Buffer (Catalog#9803; Cell Signaling Technology, Danvers, Mass.)
(20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM Na.sub.2EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
B-glycerophosphate, 1 mM sodium vanadate, 1 ug/ml leupeptin).
Lysates were clarified at 16,000.times.g for 10 minutes, and 2
.mu.g of protein was subjected to SDS-PAGE analysis and followed by
immunoblotting according to standard protocols. The antibodies used
were ER.alpha. (Cell Signaling Technologies Catalog #8644), and
Tubulin (Sigma Catalog #T9026; St. Louis, Mo.). Detection reagents
were Clarity Western ECL substrate (Bio-Rad Catalog #170-5060;
Hercules, Calif.).
[1704] Alternatively, MCF7 cells were grown in DMEM/F12 with 10%
FBS and seeded at 24,000 cells per well in 500 .mu.l in 24-well
clear tissue culture plates. The following day, the cells were
treated with PROTACs in a 5-point concentration curve (100 nM, 33
nM, 11 nM, 3.7 nM, and 1.2 nM) in the presence of 0.01% DMSO. After
72 hours, the wells are aspirated and washed with 500 .mu.l of PBS.
The cells are lysed with 100 .mu.l/well 4.degree. C. Cell Lysis
Buffer (Catalog#9803; Cell Signaling Technology, Danvers, Mass.)
(20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM Na.sub.2EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
B-glycerophosphate, 1 mM sodium vanadate, 1 ug/ml leupeptin).
Lysates were clarified at 16,000.times.g for 10 minutes, and 2
.mu.g of protein was subjected to SDS-PAGE analysis and followed by
immunoblotting according to standard protocols. The antibodies used
were ER.alpha. (Cell Signaling Technologies Catalog #8644), and
Tubulin (Sigma Catalog #T9026; St. Louis, Mo.). Detection reagents
were Clarity Western ECL substrate (Bio-Rad Catalog #170-5060;
Hercules, Calif.).
[1705] 3. Estrogen Receptor-Alpha (ER.alpha.) Degradation Assay
Using in-Cell Western.TM. Assay for Table 5.
[1706] Degradation of ER.alpha. by claimed compounds were
determined in MCF7 cells using an In-Cell Western.TM. assay.
Briefly, MCF7 cells were plated in 96-well plates (2000 cells per
well in 100 .mu.l media) and incubated at 37.degree. C. under an
atmosphere of 5% CO.sub.2 in a humidified incubator overnight.
One-hundred (100) .mu.l of media containing test compound (at
2.times. concentration) was added to the appropriate wells to
provide 11 serially decreasing concentrations (top final
concentration, 1 .mu.M then 3-fold less for the next 10
concentrations); a vehicle control (DMSO) was also added for each
compound. For each experiment, all compounds were assayed in
duplicate plates. Cells were then incubated for 3 or 5 days in the
above-mentioned environment. The assay was terminated by removal of
media, a single wash with ice-cold PBS and the addition of 50 .mu.l
paraformaldehyde (PFA: 4% in PBS). After 15 minutes in PFA at room
temperature, the cells were permeabilized in
Tris-phosphate-buffered saline with Tween (0.1%) (TBST)
supplemented with Triton X-100 (0.5%) for 15 minutes. Cells were
then blocked in BSA (TBST with BSA, 3%) for one hour. Primary
antibodies for the detection of ER.alpha. (rabbit monoclonal,
1:1000, Cell Signaling Technology Catalog #8644) and tubulin (mouse
monoclonal, 1:5000, Sigma Catalog #T6074) in TBST with BSA (3%)
were added. The cells were incubated overnight at 4.degree. C. The
cells were then washed thrice with TBST at room temperature and
then incubated with anti-rabbit and anti-mouse
fluorescently-labelled secondary antibodies (IRDye.RTM.; LI-COR;
Lincoln, Nebr.) in LI-COR blocking buffer (Catalog #927-50000) for
one hour at room temperature. Following 3 washes with TBST, the
buffer was removed and the plates were read on an Odyssey.RTM.
infrared imaging system (LI-COR.RTM.; Lincoln, Nebr.) at 700 nm and
800 nm. Using commercial software (ImageStudio.TM.; LI-COR,
Lincoln, Nebr.), the staining intensity for ER.alpha. and tubulin
in each well was quantified and exported for analysis. For each
data point, ER.alpha. intensity was normalized to tubulin intensity
and for each compound all normalized intensity values were
normalized to the vehicle control. DC.sub.50 and D.sub.max values
were determined following a 4-parameter IC.sub.50 curve fit using
ACAS dose response module (McNeil & Co Inc.).
[1707] 4. BRD4 Western Protocol
[1708] VCaP cells were purchased from ATCC and cultured in
Dulbecco's Modified Eagle's Medium (ATCC), supplemented with 10%
FBS (ATCC) and Penicillin/Streptomycin (Life Technologies). DMSO
control and compound treatments (0.003 .mu.M, 0.01 .mu.M, 0.03
.mu.M and 0.1 .mu.M) were performed in 12-well plates for 16 h.
Cells were harvested, and lysed in RIPA buffer (50 mM Tris pH8, 150
mM NaCl, 1% Tx-100, 0.1% SDS, 0.5% sodium deoxycholate)
supplemented with protease and phosphatase inhibitors. Lysates were
clarified at 16,000 g for 10 minutes, and protein concentration was
determined. Equal amount of protein (20 .mu.g) was subjected to
SDS-PAGE analysis and followed by immunoblotting according to
standard protocols. The antibodies used were BRD4 (Cell Signaling
#13440), and Actin (Sigma #5441). Detection reagents were Clarity
Western ECL substrate (Bio-rad #170-5060).
[1709] 5. AR ELISA Assay Protocol
[1710] Compounds were evaluated in this assay in LNCaP and/or VCaP
cells utilizing similar protocols. The protocols used with VCaP
cells are described below. The androgen receptor ELISA assay was
performed using PathScan AR Sandwich ELISA (Cell Signaling
Catalog#12850) according to the following assay steps:
[1711] VCaP cells were seeded at 40,000 cells/well at a volume of
100 .mu.L/well in VCaP assay medium [Phenol red free RPMI (Gibco
Cat#11835-030); 5% Charcoal Stripped (Dextran treated) FBS (Omega
Scientific, Cat#FB-04); 1% penstrep (Life Technologies, Gibco Cat#:
10378-016)] in Corning 3904 plates. The cells were incubated for a
minimum of 3 days. Cells were dosed with PROTACs diluted in 0.01%
DMSO and the drug treatment was allowed for 5 hours.
[1712] AR ELISA (Cell Signaling) was performed as follows. 1.times.
Cell Signaling Cell lysis buffer was made (Catalogue #9803; comes
with the kit). Media from the treated wells is aspirated, and 100
.mu.L 1.times. cell lysis buffer/well is added. The cells were
placed on a shaker for 10 minutes at 4.degree. C. Twenty
microliters of lysate was transferred to 100 .mu.l of Diluent in
ELISA plate (0.15 .mu.g/ml-0.075 .mu.g/ml). The lysate-diluent
mixture was shaken for 30 minutes at 37.degree. C. Allow mouse AR
antibody, anti-mouse antibody, TMB, and STOP solution to come to
room temperature. The 1.times.ELISA buffer included in kit was made
and loaded in the reservoir. Media from the plates was discarded,
the ELISA plate tapped hard on paper towel, and washed 4.times.200
.mu.l ELISA wash buffer using a plate washer.
[1713] One-hundred (100) .mu.L/well of mouse AR detection Ab was
added; the plates were covered and shaken at 37.degree. C. for 1
hour; media was discarded from the plates, the plates were tapped
on a paper towel, washed 4.times. with 200 .mu.L ELISA wash buffer
with a plate washer.
[1714] One-hundred (100) .mu.L/well of anti-mouse-HRP conjugated Ab
(comes with the kit) was added; the plates were covered and shaken
at 37.degree. C. for 30 minutes; the TMB reagent was allowed to
come to room temperature; the media was discard from the plate, the
plates were tapped on paper towl, washed 4.times. with 200 .mu.L of
ELISA wash buffer; the plates were tapped the plates on paper towl.
One-hundred (100) .mu.L of TMB was added and the plates shaken for
2 minutes--while watching for color development. One-hundred (100)
.mu.L Stop solution was added when light blue color developed.
Plates were shaken and read at 450 nM.
[1715] Progression of prostate cancer in patients treated with
anti-androgen therapy usually involves one of several mechanisms of
enhanced Androgen Receptor (AR) signaling, including increased
intratumoral androgen synthesis, increased AR expression and AR
mutations. PROTACs (PROteolysis TArgeting Chimera), which use
bi-functional molecules that simultaneously bind a target of choice
and an E3 ligase, cause ubiquitination via induced proximity and
degradation of the targeted, pathological protein. As opposed to
traditional target inhibition, which is a competitive process,
degradation is a progressive process. As such, it is less
susceptible to increases in endogenous ligand, target expression,
or mutations in the target. Thus, this technology appears to be
ideal for addressing the mechanisms of AR resistance in patients
with prostate cancer. Data was analyzed and plotted using GraphPad
Prism software.
[1716] 6. BRD4 Human c-Myc ELISA Assay Protocol
[1717] 22RV-1 cells were seeded at 30,000 cells/well at a volume of
75 .mu.L/well in RPMI with 10% FBS media in 96-well plates and
grown overnight at 37.degree. C. Cells were dosed with compounds at
4.times. concentration diluted in 0.4% DMSO; compounds were
serially diluted 1:3 for 8-point dose curve. Twenty-five (25) ul of
the compounds was added to cells for a final concentration starting
at 300 nM-0.3 nM or 1 uM-1 nM in 0.1% DMSO and incubated for 18
hours. Media was aspirated, cells were washed 1.times. with PBS and
aspirated. Cells were lysed in 50 ul RIPA buffer (50 mM Tris pH8,
150 mM NaCl, 1% Tx-100, 0.1% SDS, 0.5% sodium deoxycholate)
supplemented with protease and phosphatase inhibitors. Plates were
incubated on ice for 15 minutes then centrifuged at 4.degree. C.
for 10 minutes at 4000 rpm. Fifty (50) ul of cleared lysate from a
96-well assay plate was added into 96-well c-myc ELISA plate
(Novex, Life Technologies Catalog #KH02041). c-myc standard was
reconstituted with standard diluent buffer; standard curve range of
333 pg/ml-0 pg/ml was prepared, diluted 1:2 for 8-point dose curve.
The rest of the assay was performed following the protocol from the
c-myc ELISA kit. Data was analyzed and plotted using GraphPad Prism
software. Compounds described in the present disclosure were
assayed and c-myc suppression potency are listed in Table 4.
[1718] 7. BRD4 Immunoblotting
[1719] 22Rv1 and VCaP cell lines were purchased from ATCC. BRD2
(#5848), BRD4 (#13440), PARP (#9532), c-Myc (#5605) antibodies were
purchased from cell signaling. BRD3 (sc-81202) antibody was
purchased from Santa Cruz Biotech. Antibodies used for
immunohistochemistry were c-MYC (abcam #ab32072) and BRD4 (Bethyl
Laboratories #a301-985a50). Actin and Tubulin antibodies were
purchased from Sigma.
[1720] Cells were lysed in RIPA buffer (Thermo Fisher Cat#89900)
supplemented with protease inhibitors (Pierce.TM. Protease
Inhibitor Tablets, EDTA-free Cat#88266). Lysates were centrifuged
at 16,000.times.g and supernatants were used for SDS-PAGE. Western
blotting was carried out following standard protocols.
[1721] 8. BRD4 Cell Proliferation Assay
[1722] 22RV-1 cells were seeded at 5,000 cells/well at a volume of
75 .mu.L/well in RPMI+10% FBS media in 96-well plates and grown
overnight at 37.degree. C. Cells were dosed with compounds at four
concentrations diluted in 0.4% DMSO; compounds were serially
diluted 1:3 for 10-point dose curve. Twenty-twenty (25) ul of
compounds was added to cells for a final concentration starting at
300 nM-0.3 nM in 0.1% DMSO and incubated for 72 hours. In a
separate plate, 100 ul of 5,000 cells/well were plated in 8 wells,
100 ul of CellTiter-Glo (CellTiter-Glo.RTM. Luminescent Cell
Viability Assay, Promega # G7573) was added and incubated for 30
minutes, then read on luminometer to assess initial signal for cell
growth. After 72 hours, 100 ul of CellTiter-Glo.RTM. was added and
incubated for 30 minutes, then read on luminometer. Data was
analyzed and plotted using GraphPad Prism software.
[1723] 9. BRD4 Apoptosis Assay
[1724] 22RV-1 cells were seeded at 5,000 cells/well at a volume of
75 .mu.L/well in RPMI+10% FBS media in 96-well plates and grown
overnight at 37.degree. C. Cells were dosed with compounds at
4.times. concentrations diluted in 0.4% DMSO; compounds were
serially diluted 1:3 for 8-point dose curve. Twenty-five (25) ul of
compounds was added to cells for a final concentration starting at
300 nM-0.3 nM in 0.1% DMSO and incubated for 48 hours. After 48
hours, 100 ul of Caspase-Glo.RTM. 3/7 (Promega Caspase-Glo.RTM. 3/7
Assay #G8093 was added and incubated for 30 minutes, then read on
luminometer. Data was analyzed and plotted using GraphPad Prism
software.
[1725] 10. Tau Protein In Vitro Degradation Assay
[1726] To determine effect of PROTACs on tau protein degradation
SK-N-SH cells were seeded in a 24-well tissue culture-treated plate
for at least 18-hours prior to compound addition. Tau PROTACs were
evaluated for tau degradation by lysing the cells in RIM buffer
with protease inhibitors following a 72-hour incubation with tau
PROTACs. Cell lysates were run on standard SDS-PAGE gels, and tau
levels were detected by Western blotting using Tau-13 antibody from
Abcam (Cambridge, UK) that binds to all forms of human tau. The
data was shown in Tables 6.
[1727] Small molecule inhibitors have been the cornerstone of
oncology drug development and generally work by inhibiting enzyme
activity (such as kinase inhibitors) or by interfering
protein-protein interactions (such as BRD4 inhibitors). Given the
reversible binding of most small molecule inhibitors, large
systemic drug concentrations are often required to ensure
sufficient functional inhibition. Additionally, achieving and
maintaining a high systemic drug level that is required for in vivo
efficacy has proven challenging for many targets.
[1728] BRD4, a member of the bromodomain and extraterminal domain
(BET) family, is a protein characterized by two bromodomains (BD
domain) at the N-terminus and an extraterminal domain (ET domain)
at the C-terminus. The two BD domains recognize and interact with
acetylated-lysine residues at the N-terminal tail of histone
protein. The ET domain is considered to serve a scaffolding
function in recruiting diverse transcriptional regulators, but has
not yet been fully characterized. BRD4 has been shown to be located
at super-enhancer regions, which often reside upstream of important
oncogenes, such as c-MYC, Bcl-xL and BCL-6, and play a key role in
regulating their expressions. Based on its role in regulating gene
expression by recruiting relevant transcription modulators to
specific genomic loci, BRD4 is a candidate drug target for treating
and/or preventing a number of human cancers, such as midline
carcinoma, acute myeloid leukemia (AML), multiple myeloma (MM),
Burkitt lymphoma (BL), and prostate cancer.
[1729] Several small molecule BET bromodomain inhibitors have been
developed, such as JQ1, iBET, and OTX15, which have shown
therapeutic potential in certain preclinical models of various
cancers, including BL. Almost all BL cases contain c-myc gene
translocation that places it under control of a super-enhancer
located upstream of IgH, thus driving an abnormally high level of
c-MYC expression, tumor development and maintenance. Preclinical
studies with BRD4 inhibitors demonstrate their ability to suppress
c-MYC and proliferation in BL cell lines; however, the IC.sub.50
values of these inhibitors is often in the range of 100 nM to 1
.mu.M.
[1730] Materials and Methods
[1731] The details of the experimental design and procedures are
provided below:
[1732] Inhibitors JQ1, OTX-15, and pomalidomide were synthesized
according to methods published.
1. K.sub.D DETERMINATION
[1733] The surface plasmon resonance (SPR) experiments were
conducted on a Biacore3000 (GE Healthcare). Myc-tagged cereblon was
immobilized on a carboxymethylated dextran surface (CMS) amine
coupled to anti-Myc antibody to recognize the Myc tag. His-tagged
cereblon protein was immobilized on a carboxymethylated dextran
surface with nitriloacetic acid (NTA), taking advantage of
NTA/Ni.sup.2+ chelation. The prepared surface was allowed to
equilibrate over three hours in running buffer (10 mM HEPES buffer
@ pH 7.4, 150 mM NaCl, 0.005% P20, 2% DMSO).
[1734] All compounds were prepared in 100% DMSO stock plates with a
top concentration of 5 mM in a 3.times. serial dilution. Compounds
were transferred from the stock plate to the assay plate and
diluted into running buffer containing no DMSO. All compounds were
run as a six-concentration series with a final assay top
concentration of 100 .mu.M.
[1735] Data analysis was performed in Scrubber 2 (BioLogic
software, Campbell, Australia). Blanks were subtracted and data was
corrected for DMSO against a standard DMSO curve. All reported KD
values represent an average of at least N=2 and were obtained by
fitting to a minimum of five concentrations using a 1:1 fitting
algorithm. The data is shown in Table 1 below, wherein "a" is a
K.sub.D of <1 .mu.M, "b" represents a K.sub.D of 1 .mu.M to 10
.mu.M, "c" represents a K.sub.D of >10 .mu.M to 100 .mu.M and
"d" represents a K.sub.D of >100 .mu.M or no response.
TABLE-US-00002 TABLE 1 Suface Plasmon Resonance Data for Examplary
CLMs K.sub.D Binding to immobilized CRBN Example protein in SPR
Number Chemical Structure assay Control-1 (Pomalidomide)
##STR00669## a Control-2 (Lenalidomide) ##STR00670## a 1
##STR00671## b 2 ##STR00672## b 3 ##STR00673## c 4 ##STR00674## c 5
##STR00675## c 6 ##STR00676## d 7 ##STR00677## d 8 ##STR00678## d 9
##STR00679## b 10 ##STR00680## d 11 ##STR00681## d 12 ##STR00682##
d 13 ##STR00683## d 14 ##STR00684## d 15 ##STR00685## d 16
##STR00686## d 17 ##STR00687## d 18 ##STR00688## d 19 ##STR00689##
d 20 ##STR00690## d 21 ##STR00691## d 22 ##STR00692## d 23
##STR00693## c 24 ##STR00694## d 25 ##STR00695## d 26 ##STR00696##
d 27 ##STR00697## d 28 ##STR00698## c 29 ##STR00699## c 30
##STR00700## b 31 ##STR00701## d 32 ##STR00702## b 33 ##STR00703##
d 34 ##STR00704## c 35 ##STR00705## d 36 ##STR00706## d 37
##STR00707## d 38 ##STR00708## d 39 ##STR00709## d 40 ##STR00710##
c 41 ##STR00711## d 42 ##STR00712## d 43 ##STR00713## c 44
##STR00714## c
2. CELLS AND REAGENTS
[1736] NAMALWA, Ramos, CA-46 and DAUDI cells were purchased from
ATCC and maintained as instructed. Antibodies against BRD4
(#E2A7X), c-MYC (#D84C12), PARP (#46D11) were purchased from Cell
Signaling Technology. Actin (#A5441) antibody was purchased from
SigmaAldrich. Secondary antibodies (#7074, #7076) were purchased
from Cell Signaling Technology. MG132 (#M7449) was purchased from
SigmaAldrich. Carfizomib (#S2853) was purchased from Selleck.
2. WESTERN BLOT ANALYSIS
[1737] Cultured cells are collected in lysis buffer containing 40
mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM EDTA, 1% NP-40, 0.1% SDS and
protease inhibitor cocktail. After 10 minutes of centrifugation
(14000 rpm), supernatant is collected for protein concentration
determination by BCA method and subjected for immunoblotting by
standard protocol. Western blot results are visualized using
Bio-Rad Clarity ECL Western Blotting Substrate on Bio-Rad
ChemiDoc.TM. MP imaging system.
3. RT-PCR
[1738] RNA extraction is performed with Aurum.TM. Total RNA Mini
Kit (#732-6820) from Bio-Rad. First-strand cDNA from total RNA is
synthesized with High-Capacity cDNA Reverse Transcription Kit
(#4368813) from Life Technologies according to manufacturer's
instruction. Quantitative PCR is performed using Bio-rad
SsoAdvanced.TM. Universal SYBR.RTM. Green Supermix (#172-5271).
4. PROLIFERATION ASSAY
[1739] To assess the effect of the inhibitors on proliferation,
cells (50,000/1000) are seeded in 96-well tissue culture plates
followed by addition of compound at the indicated concentration.
After 72 hours, 100 .mu.L per well of reconstituted CellTiter-Glo
(CTG) reagent (#G7572 from Promega) is added and read on Cytation 3
imaging reader from BioTek. Relative cell growth is determined by
comparing assay readings of treated cells with control DMSO treated
cells.
TABLE-US-00003 TABLE 2 Exemplary PROTACs of the present disclosure
PROTAC-# Chemical Structure Name Synthesis PROTAC- 27 ##STR00715##
rac-5-((5-(4-(5-(((1r,3r)-3-(3- chloro-4-cyanophenoxy)- 2,2,4,4-
tetramethylcyclobutyl) carbamoyl)pyridin-2-yl)
piperazin-1-yl)pentyl)oxy)- N-(2,6-dioxopiperidin-3-
yl)picolinamide Following route described for PROTAC 50 PROTAC- 28
##STR00716## rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (5-((3-(2,6-dioxopiperidin-3-
yl)-2-methyl-4-oxo-1,2,3,4- tetrahydroquinazolin-8-
yl)oxy)pentyl)piperazin-1- yl)nicotinamide Byproduct from synthesis
of PROTAC- 30 PROTAC- 29 ##STR00717## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(6-((1-(2,6-dioxopiperidin- 3-yl)-6-oxo-1,6- dihydropyridazin-4-
yl)oxy)hexyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 30 ##STR00718## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(5-((3-(2,6-dioxopiperidin- 3-yl)-2-methyl-4-oxo-3,4-
dihydroquinazolin-8- yl)oxy)pentyl)piperazin-1- yl)nicotinamide
Described in detail PROTAC- 33 ##STR00719##
rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (5-((2-(2,6-dioxopiperidin-
3-yl)-1,1-dioxido-3-oxo-2,3- dihydrobenzo[d]isothiazol-
6-yl)oxy)pentyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 39 ##STR00720## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(2-((2-(2,4-difluorophenyl)- 1-oxoisoindolin-4-
yl)oxy)ethyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 40 ##STR00721## rac-N-((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(2-(2-((2-(2,4-difluoro- phenyl)-1-oxoisoindolin-4-
yl)oxy)ethoxy)ethyl) piperazin-1-yl)nicotinamide Following route
described for PROTAC 40 PROTAC- 41 ##STR00722##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (5-((2-(2,4-difluorophenyl)-
1,3-dioxoisoindolin-5- yl)oxy)pentyl)piperazin-1- yl)nicotinamide
Described in detail PROTAC- 42 ##STR00723##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (5-((2-(6-cyano-2-oxo-1,2-
dihydropyridin-3-yl)-1,3- dioxoisoindolin-5-
yl)oxy)pentyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 43 ##STR00724## rac-N-((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
((4-(1,3-dioxo-2-(6-oxo-1,6- dihydropyridin-3-
yl)isoindolin-5-yl)piperazin- 1-yl)methyl)piperidin-1- yl)benzamide
Described in detail PROTAC- 44 ##STR00725##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- ((4-(1,3-dioxo-2-(2-oxo-1,2-
dihydropyridin-3- yl)isoindolin-5-yl)piperazin-
1-yl)methyl)piperidin-1- yl)benzamide Following route described for
PROTAC 43 PROTAC- 45 ##STR00726## N-((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
((4-(2-(6-hydroxy-2-oxo-1,2- dihydropyridin-3-yl)-1,3-
dioxoisoindolin-5- yl)piperazin-1- yl)methyl)piperidin-1-
yl)benzamide Following route described for PROTAC 43 (using
HBr/HOAc for demethyl- ation step) PROTAC- 46 ##STR00727##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (2-(2-((2-(2,6-dioxo-
piperidin-3-yl)-1-oxo-1,2- dihydroisoquinolin-3-
yl)methoxy)ethoxy)ethyl) piperazin-1-yl)nicotinamide Described in
detail PROTAC- 47 ##STR00728## rac-N-((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(5-((3-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)- yl)quinolin-6-
yl)oxy)pentyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 48 ##STR00729## rac-N-((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(5-(4-((2,6-dioxopiperidin-3- yl)(ethyl)carbamoyl)phenoxy)
pentyl)piperazin-1- yl)nicotinamide Described in detail PROTAC- 49
##STR00730## rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (5-(4-((2,6-dioxopiperidin-3-
yl)(methyl)carbamoyl) phenoxy)pentyl)piperazin-1- yl)nicotinamide
Following route described for PROTAC 48 PROTAC- 50 ##STR00731##
rac-5-(3-(4-(5-(((1r,3r)-3-(3- chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclo- butyl)carbamoyl)pyridin-
2-yl)piperazin-1- yl)propoxy)-N-(2,6- dioxopiperidin-3-
yl)picolinamide Described in detail PROTAC- 51 ##STR00732##
rac-5-(4-(4-(5-(((1r,3r)-3-(3- chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclo- butyl)carbamoyl)pyridin-
2-yl)piperazin-1- yl)butoxy)-N-(2,6- dioxopiperidin-3-
yl)picolinamide Following route described for PROTAC 50 PROTAC- 52
##STR00733## rac-5-(4-(2-(1-(5-(((1r,3r)- 3-(3-chloro-4-cyano-
phenoxy)-2,2,4,4- tetramethylcyclobutyl) carbamoyl)pyridin-2-yl)
piperidin-4-yl)ethyl) piperazin-1-yl)-N- (2,6-dioxopiperidin-3-
yl)picolinamide Following route described for PROTAC 53 PROTAC- 53
##STR00734## rac-5-(4-((1-(5-(((1r,3r)-3-
(3-chloro-4-cyanophenoxy)- 2,2,4,4-tetramethylcyclo-
butyl)carbamoyl)pyridin- 2-yl)piperidin-4-yl)methyl)
piperazin-1-yl)-N-(2,6- dioxopiperidin-3-yl) picolinamide Described
in detail PROTAC- 54 ##STR00735## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
((4-(6-((2,6-dioxopiperidin- 3-yl)carbamoyl)pyridin-3-
yl)piperazin-1- yl)methyl)piperidin-1- yl)pyridazine-3-carboxamide
Following route described for PROTAC 53 PROTAC- 55 ##STR00736##
rac-5-(4-((1-(4-(((1r,3r)-3- (3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclo- butyl)carbamoyl)phenyl)
piperidin-4-yl)methyl) piperazin-1-yl)-N- (2,6-dioxopiperidin-3-
yl)picolinamide Following route described for PROTAC 53 PROTAC- 56
##STR00737## rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-5-(4- ((4-(6-((2,6-dioxopiperidin-
3-yl)carbamoyl)pyridin-3- yl)piperazin-1- yl)methyl)piperidin-1-
yl)pyrazine-2-carboxamide Following route described for PROTAC 53
PROTAC- 57 ##STR00738## 5-(4-((1-(6-(((1r,4r)-4-(3-
chloro-4-cyanophenoxy) cyclohexyl)carbamoyl)
pyridazin-3-yl)piperidin-4- yl)methyl)piperazin-1-yl)-
2-((2,6-dioxopiperidin-3- yl)carbamoyl)-4- fluorobenzoic acid
Following route described for PROTAC 53 PROTAC- 58 ##STR00739##
4-(4-((1-(6-(((1r,4r)-4-(3- chloro-4-cyanophenoxy)
cyclohexyl)carbamoyl) pyridazin-3-yl)piperidin-4-
yl)methyl)piperazin-1-yl)- 2-((2,6-dioxopiperidin-3-
yl)carbamoyl)-5- fluorobenzoic acid Following route described for
PROTAC 53 PROTAC- 59 ##STR00740## N-((1r,4r)-4-(3-chloro-4-
cyanophenoxy)cyclohexyl)- 6-(4-((4-(4-((2,6-dioxo-
piperidin-3-yl)carbamoyl) phenyl)piperazin-1-yl)
methyl)piperidin-1-yl) pyridazine-3-carboxamide Following route
described for PROTAC 53 PROTAC- 60 ##STR00741##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (3-((1-(2,6-dioxopiperidin-
3-yl)-6-oxo-1,6- dihydropyridazin-4- yl)oxy)propyl)piperazin-1-
yl)nicotinamide Following route described for PROTAC 29 PROTAC- 61
##STR00742## rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6-(4- (4-(1-(2,6-dioxopiperidin-
3-yl)-6-oxo-1,6- dihydropyridazin-4- yl)piperazin-1-yl)
butyl)nicotinamide Described in detail PROTAC- 62 ##STR00743##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- ((4-(1-(2,6-dioxopiperidin-
3-yl)-6-oxo-1,6- dihydropyridazin-4- yl)piperazin-1-
yl)methyl)piperidin-1- yl)benzamide Following route described for
PROTAC 61 PROTAC- 63 ##STR00744## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
(2-(4-(1-(2,6-dioxopiperidin- 3-yl)-6-oxo-1,6- dihydropyridazin-4-
yl)piperazin-1- yl)ethyl)piperidin-1- yl)benzamide Following route
described for PROTAC 61 PROTAC- 66 ##STR00745##
N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- ((4-(2-(2,5-dioxopyrrolidin-
3-yl)-1,3-dioxoisoindolin-5- yl)piperazin-1- yl)methyl)piperidin-1-
yl)benzamide Following route described for PROTAC 43, using 3-
amino- pyrrolidine- 2,5-dione as starting material PROTAC- 67
##STR00746## rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- (5-(3-(1-(2,6-dioxopiperidin-
3-yl)-4-methyl-5-oxo-4,5- dihydro-1H-1,2,4-triazol-3-
yl)phenoxy)pentyl) piperazin-1-yl)benzamide Following route
described for PROTAC 70 PROTAC- 68 ##STR00747##
rac-N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- ((4-(3-(1-(2,6-dioxo-
piperidin-3-yl)-4-methyl- 5-oxo-4,5-dihydro-1H-
1,2,4-triazol-3-yl)phenyl) piperazin-1-yl)methyl)
piperidin-1-yl)benzamide Following route described for PROTAC 70
PROTAC- 69 ##STR00748## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
(5-(4-(1-(2,6-dioxo- piperidin-3-yl)-4-methyl-
5-oxo-4,5-dihydro-1H- 1,2,4-triazol-3-yl)phenoxy)
pentyl)piperazin-1-yl) benzamide Following route described for
PROTAC 70 PROTAC- 70 ##STR00749## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
((4-(4-(1-(2,6-dioxo- piperidin-3-yl)-4-methyl-
5-oxo-4,5-dihydro-1H- 1,2,4-triazol-3-yl)phenyl)
piperazin-1-yl)methyl) piperidin-1-yl)benzamide Described in detail
PROTAC- 75 ##STR00750## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-(4-
((2-(2,6-dioxopiperidin-3- yl)-1,3-dioxo-2,8-
diazaspiro[4.5]decan-8- yl)methyl)piperidin-1- yl)benzamide
Following route described for PROTAC 53 and 112 PROTAC- 76
##STR00751## rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(3- (2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxo-2,8- diazaspiro[4.5]decan-8- yl)propyl)benzamide
Following route described for PROTAC 61 and 112 PROTAC- 77
##STR00752## rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4- ((E)-3-(2-(2,6-dioxo-
piperidin-3-yl)-1,3- dioxo-2,8-diazaspiro [4.5]decan-8-yl)prop-1-
en-1-yl)benzamide Following route described for PROTAC 61 and 112
PROTAC- 78 ##STR00753## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-4-
(4-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,8-
diazaspiro[4.5]decan-8- yl)butyl)benzamide Following route
described for PROTAC 61 and 112 PROTAC- 79 ##STR00754##
rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-6- (4-(5-((4-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)- yl)isoquinolin-7-
yl)oxy)pentyl)piperazin-1- yl)nicotinamide Described in detail
PROTAC- 80 ##STR00755## rac-N-((1r,3r)-3-(3-chloro-
4-cyanophenoxy)-2,2,4,4- tetramethylcyclobutyl)-6-(4-
(5-((3-(5-cyano-2,4-dioxo- 3,4-dihydropyrimidin-
1(2H)-yl)quinolin-6- yl)oxy)pentyl)piperazin-1- yl)nicotinamide
Described in detail PROTAC- 81 ##STR00756##
rac-N-((1r,3r)-3-(3-chloro- 4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4-(4- ((4-(2-(2,6-dioxopiperidin-
3-yl)-4-methylene-1-oxo- 1,2,3,4-tetrahydroiso-
quinolin-6-yl)piperazin-1- yl)methyl)piperidin-1- yl)benzamide
Described in detail PROTAC- 82 ##STR00757##
N-((1r,3r)-3-(3-chloro-4- cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl)-4- (4-((4-(2-(2,6-dioxo-
piperidin-3-yl)-1-oxo-1,2- dihydroisoquinolin-6- yl)piperazin-1-
yl)methyl)piperidin-1- yl)benzamide Described in detail PROTAC- 1
##STR00758## rac-(R)-2-(4-(4-chloro- phenyl)-2,3,9-trimethyl-6H-
thieno[3,2-f][1,2,4] triazolo[4,3-a] [1,4]diazepin-6-yl)-N-(3-(3-
((3-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)- yl)quinolin-6-yl)oxy)
propoxy)propyl)acetamide Described in detail PROTAC- 89
##STR00759## rac-3-(4-(4-(4-((1-(4- ((1R,2S)-6-hydroxy-2-
phenyl-1,2,3,4- tetrahydronaphthalen-1- yl)phenyl)piperidin-4-
yl)methyl)piperazin-1- yl)phenyl)-2-oxo-2,5-
dihydro-1H-pyrrol-1-yl) piperidine-2,6-dione Described in detail;
see also Scheme 2- 30, 3-58 PROTAC- 90 ##STR00760##
rac-N-(2,6-dioxopiperidin- 3-yl)-4-(4-(5-(4-((1R,2S)-
6-hydroxy-2-phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)pentyl) piperazin-1-yl)-N- methylbenzamide Scheme 2- 45,
3-10 PROTAC- 91 ##STR00761## rac-N-(2,6-dioxopiperidin-
3-yl)-5-(4-(5-(4-((1R,2S)- 6-hydroxy-2-phenyl-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)pentyl)
piperazin-1-yl)picolinamide Scheme 2- 45, 3-10 PROTAC- 92
##STR00762## rac-N-(2,6-dioxopiperidin- 3-yl)-N-ethyl-4-(4-(5-(4-
((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)pentyl) piperazin-1-yl)-2- methoxybenzamide Scheme 2-
45, 3-10 PROTAC- 93 ##STR00763## rac-3-(4-((6-(((R)-1-(2-(4-
((1R,2S)-2-(4-fluorophenyl)- 6-hydroxy-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)ethyl)pyrrolidin-
3-yl)oxy)pyridin-3-yl)oxy)- 6-oxopyridazin-1(6H)-
yl)piperidine-2,6-dione Scheme 2- 40, 3-10 PROTAC- 94 ##STR00764##
rac-3-(4-((6-(((R)-1-(2-(4- ((1S,2R)-2-(4-fluorophenyl)-
6-hydroxy-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)ethyl)pyrrolidin- 3-yl)oxy)pyridin-3-yl)oxy)-
6-oxopyridazin-1(6H)- yl)piperidine-2,6-dione Scheme 2- 40, 3-10
PROTAC- 95 ##STR00765## rac-3-(4-((6-(4-(2-(4- ((1R,2S)-2-(4-
fluorophenyl)-6- hydroxy-1,2,3,4- tetrahydronaphthalen-
1-yl)phenoxy)ethyl) piperazin-1-yl)pyridin- 3-yl)oxy)-6-
oxopyridazin-1(6H)- yl)piperidine-2,6-dione Scheme 2- 41, 3-10
PROTAC- 96 ##STR00766## rac-3-(4-((6-(4-(2-(4-
((1R,2S)-2-(4-fluoro- phenyl)-6-hydroxy- 1,2,3,4-tetrahydro-
naphthalen-1-yl) phenoxy)ethyl)piperazin- 1-yl)pyridin-3-yl)oxy)-6-
oxopyridazin-1(6H)- yl)piperidine-2,6-dione Scheme 2- 41, 3-10
PROTAC- 97 ##STR00767## rac-3-(4-((6-(((R)-1-(2-(4-
((1S,2R)-2-(4-fluorophenyl)- 6-hydroxy-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)ethyl) pyrrolidin-3-yl)oxy)
pyridin-3-yl)oxy)-6- oxopyridazin-1(6H)- yl)piperidine-2,6-dione
Scheme 2- 40, 3-10 PROTAC- 98 ##STR00768## rac-3-(4-((6-(4-(4-
((1R,2S)-2-(4- fluorophenyl)-6-hydroxy- 1,2,3,4-tetrahydro-
naphthalen-1-yl)phenoxy) butyl)pyridin-3-yl) oxy)-6-oxopyridazin-
1(6H)-yl)piperidine-2,6- dione Scheme 3- 56 PROTAC- 99 ##STR00769##
rac-3-(4-((6-(5-(4- ((1R,2S)-2-(4- fluorophenyl)-6-hydroxy-
1,2,3,4-tetrahydro- naphthalen-1-yl) phenoxy)pentyl)pyridin-
3-yl)oxy)-6-oxopyridazin- 1(6H)-yl)piperidine-2,6- dione Scheme 3-
56 PROTAC- 100 ##STR00770## rac-3-(4-(4-(5-(4- ((1R,2S)-6-hydroxy-
2-phenyl-1,2,3,4- tetrahydronaphthalen-1- yl)phenoxy)pentyl)
piperazin-1-yl)-6- oxopyridazin-1(6H)- yl)piperidine-2,6-dione
Scheme 2- 31, 3-10 PROTAC- 101 ##STR00771## rac-3-(4-(4-(5-(4-
((1R,2S)-6-hydroxy- 2-phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)pentyl) piperazin-1-yl)-6- oxopyrimidin-1(6H)-
yl)piperidine-2,6-dione Scheme 2- 46, 3-10 PROTAC- 102 ##STR00772##
3-(4-(4-((1-(4-((1R,2S)-6- hydroxy-2-phenyl-1,2,3,4-
tetrahydronaphthalen-1- yl)phenyl)piperidin-4-
yl)methyl)piperazin-1-yl)- 6-oxopyridazin-1(6H)-
yl)piperidine-2,6-dione Described in detail; see also Scheme 2- 31,
3-58 PROTAC- 103 ##STR00773## 3-(4-((6-(((R)-1-(2-(4-
((1R,2S)-2-(4-fluorophenyl)- 6-hydroxy-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)ethyl) pyrrolidin-3-yl)oxy)
pyridin-3-yl)oxy)-6- oxopyridazin-1(6H)- yl)piperidine-2,6-dione
Scheme 2- 40, 3-10 PROTAC- 104 ##STR00774## rac-3-(4-(3-(1-(4-(4-
((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)butyl) piperidin-4-yl)phenoxy)- 2-oxo-2,5-
dihydro-1H-pyrrol-1- yl)piperidine-2,6-dione Following route
described for PROTAC 106 PROTAC- 105 ##STR00775##
rac-3-(4-(3-(1-(3-(4- ((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)propyl) piperidin-4-yl)phenoxy)-
2-oxo-2,5- dihydro-1H-pyrrol-1- yl)piperidine-2,6-dione Following
route described for PROTAC 106 PROTAC- 106 ##STR00776##
rac-3-(4-(3-(1-(3-(4- ((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)propyl) piperidin-4-yl)phenoxy)-
2-oxo-2,5- dihydro-1H-pyrrol-1- yl)piperidine-2,6-dione Described
in detail PROTAC- 107 ##STR00777## 3-(8-((2-(4-(2-(4-((2-(4-
bromophenyl)-6- hydroxybenzo[b]thiophen- 3-yl)oxy)phenoxy)ethyl)
piperazin-1-yl)ethyl) amino)-2-methyl- 4-oxoquinazolin-3(4H)-
yl)piperidine-2,6-dione Described in detail PROTAC- 108
##STR00778## 3-(8-(2-(4-(2-(4-((2-(4- bromophenyl)-6-
hydroxybenzo[b]thiophen- 3-yl)oxy)phenoxy) ethyl)piperazin-1-yl)
ethoxy)-2-methyl-4- oxoquinazolin-3(4H)- yl)piperidine-2,6-dione
Described in detail PROTAC- 109 ##STR00779## rac-3-(4-(3-(1-(4-(4-
((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)butyl) piperidin-4-yl)phenoxy)- 2-oxo-2,5-dihydro-1H-
pyrrol-1-yl)piperidine- 2,6-dione Following route described for
PROTAC 106 PROTAC- 110 ##STR00780## rac-3-(4-(4-(4-(3-(4-
((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4- tetrahydronaphthalen-1-
yl)phenoxy)propyl) piperazin-1-yl)phenyl)- 1H-pyrazol-1-yl)
piperidine-2,6-dione Scheme 3- 72 PROTAC- 111 ##STR00781##
rac-3-(4-(6-(4-(3-(4- ((1R,2S)-6-hydroxy-2- phenyl-1,2,3,4-
tetrahydronaphthalen-1- yl)phenoxy)propyl) piperazin-1-yl)pyridin-
3-yl)-1H-pyrazol-1-yl) piperidine-2,6-dione Scheme 3- 72 PROTAC-
112 ##STR00782## 2-(2,6-dioxopiperidin-3-yl)-
8-(14-((5-(5-methyl-5H- pyrido[4,3-b]indol-7-yl)
pyridin-2-yl)oxy)-3,6,9,12- tetraoxatetradecyl)-2,8-
diazaspiro[4.5]decane- 1,3-dione Synthesis described in detail
TABLE-US-00004 TABLE 3 Characteristics of exemplary androgen
receptor PROTACs MS AR AR signal 1 PROTAC- DC50 Dmax m/e+ # (nM)*
%** [M + H].sup.+ NMR PROTAC- B B 785.06 .sup.1H NMR (400 MHz,
d.sup.6-DMSO) .delta. 1.11 (6H, s), 1.21 (6H, s), 27 1.46-1.54 (4H,
m), 1.77-1.81 (2H, m), 1.96-2.00 (1H, m), 2.14- 2.25 (1H, m), 2.33
(2H, t, J = 6.8), 2.44 (5H, s), 2.75-2.83 (1H, m), 3.59 (1H, s),
4.03 (1H, d, J = 6.0 Hz), 4.15 (2H, t, J = 6.8), 4.30 (1H, s),
4.73-4.80 (1H, m), 6.86 (1H, d, J = 9.2 Hz), 7.00 (1H, dd, J = 2.4,
8.8 Hz), 7.21 (1H, d, J = 2.4 Hz), 7.56 (1H, dd, J = 2.8, 5.6 Hz,),
7.63 (1H, d, J = 9.2 Hz), 7.91 (1H, d, J = 8.8 Hz), 7.95 (1H, dd, J
= 2.4, 6.8 Hz,), 8.01 (1H, d, J = 8.8 Hz), 8.33 (1H, d, J = 2.8
Hz), 8.62 (1H, d, J = 2.4 Hz), 8.89 (1H, d, J = 6.8 Hz), 10.87 (1H,
brs). PROTAC- D C 825.36 28 PROTAC- A B 773.33 .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25 (6H, s), 1.39- 29 1.44
(2H, m), 1.49-1.62 (4H, m), 1.87-1.93 (2H, m), 2.24-2.28 (1H, m),
2.36-2.43 (2H, m), 2.56 (4H, s), 2.70-2.81 (2H, m), 2.87-2.92 (1H,
m), 3.66-3.69 (4H, m), 4.00-4.04 (3H, m), 4.14 (1H, d, J = 8.0 Hz),
5.74 (1H, dd, J = 11.2, 5.6 Hz), 6.07 (1H, d, J = 8.4 Hz), 6.40
(1H, d, J = 4.8 Hz), 6.66 (1H, d, J = 8.8 Hz), 6.80 (1H, dd, J =
8.8, 2.4 Hz), 6.96 (1H, d, J = 2.4 Hz), 7.57 (1H, d, J = 8.8 Hz),
7.71 (1H, d, J = 4.8 Hz), 7.93 (1H, dd, J = 8.8, 2.4 Hz), 8.16 (1H,
brs), 8.58 (1H, d, J = 2.4 Hz), PROTAC- D C 823.34 .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25 (6H, s), 1.59- 30 1.62
(4H, m), 1.90-2.00 (2H, m), 2.14-2.17 (1H, m), 2.70-2.79 (5H, m),
2.86-2.96 (6H, m), 3.15 (1H, dd, J = 14.8, 7.2 Hz), 3.88 (4H, s),
4.05 (1H, s), 4.13-4.20 (3H, m), 4.82 (1H, dd, J = 11.2, 5.6 Hz),
6.14 (1H, d, J = 8.4 Hz), 6.68 (1H, d, J = 9.2 Hz), 6.80 (1H, dd, J
= 8.8, 2.4 Hz), 6.96 (1H, d, J = 2.4 Hz), 7.20 (1H, d, J = 8.0 Hz),
7.38 (1H, t, J = 8.0 Hz), 7.57 (1H, d, J = 8.8 Hz), 7.74 (1H, d, J
= 8.0 Hz), 7.94 (1H, dd, J = 8.8, 2.0 Hz), 8.30 (1H, brs), 8.57
(1H, d, J = 2.0 Hz). PROTAC- B B 846.18 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.19 (6H, s), 1.22 (6H, s), 33 1.46-1.55 (4H,
m), 1.79-1.80 (2H, m), 2.34-2.40 (3H, m), 2.45 (4H, s), 2.54-2.92
(3H, m), 3.59 (4H, s), 4.06 (1H, d, J = 9.2 Hz), 4.20-4.25 (2H, m),
4.30 (1H, s), 5.23-5.28 (0.5H, m), 5.98 (0.5H, t, J = 9.2 Hz), 6.87
(1H, d, J = 9.2 Hz), 6.99-7.02 (1H, m), 7.21 (1H, d, J = 2.0 Hz),
7.35-7.50 (1H, m), 7.63 (1H, d, J = 9.2 Hz), 7.81-7.83 (1H, m),
7.90-8.02 (3H, m), 8.62 (1H, d, J = 2.0 Hz), 11.19 (1H, t, J = 9.6
Hz). PROTAC- D 0C 755.23 .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
1.23 (s, 6H), 1.29 (s, 6H), 39 2.67-2.82 (m, 4H), 2.92-3.01 (m,
2H), 3.72 (s, 4H), 4.15 (s, 1H), 4.29-4.39 (m, 3H), 4.88 (s, 2H),
6.85-6.87 (m, 2H), 7.10- 7.34 (m, 4H), 7.47-7.75 (m, 4H), 7.96-7.98
(m, 1H), 8.61 (s, 1H). PROTAC- D C 799.3 .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 1.21 (s, 6H), 1.27 (s, 6H), 40 2.68-2.73 (m,
6H), 3.61 (br, 4H), 3.75-3.76 (m, 2H), 3.87-.89 (m, 2H), 4.12 (s,
1H), 4.27 (s, 1H), 4.33-4.34 (m, 2H), 6.74 (d, J = 9.2 Hz, 1H),
6.95-6.98 (m, 1H), 7.06-7.16 (m, 3H), 7.29 (d, J = 8.2 Hz, 1H),
7.43 (d, J = 7.2 Hz, 1H), 7.51-7.75 (m, 3H), 7.71 (d, J = 8.8 Hz,
1H), 7.92 (d, J = 8.8 Hz, 1H), 8.55 (s, 1H). PROTAC- D C 811.23
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.12 (s, 6H), 1.22 (s,
6H), 41 1.48-1.61 (m, 4H), 1.80-1.83 (m, 2H), 2.35-2.44 (m, 6H),
3.59 (br, 4H), 4.06 (d, J = 9.2 Hz, 1H), 4.22 (t, J = 6.4 Hz, 2H),
4.31 (s, 1H), 6.88-6.90 (m, 1H), 6.99-7.02 (m, 1H), 7.20-7.21 (m,
1H), 7.28-7.32 (m, 1H), 7.40-7.42 (m, 1H), 7.52-7.55 (m, 2H),
7.63-7.65 (m, 2H), 7.89-7.93 (m, 2H), 7.97-7.99 (m, 1H), 8.64 (br,
1H). PROTAC- 0C C 817.2 .sup.1H NMR (400 MHz, DMSO-d.sup.6) .delta.
1.12 (6H, s), 1.22 (6H, s), 42 1.42-1.60 (4H, m), 1.77-1.82 (2H,
m), 2.36-2.44 (2H, m), 3.30- 3.35 (4H, m), 3.58-3.66 (4H, m), 4.06
(1H, d, J = 9.2 Hz), 4.21 (1H, t, J = 6.2 Hz), 4.30 (1H, s), 6.88
(1H, d, J = 8.8 Hz), 6.99- 7.02 (1H, m), 7.21 (1H, d, J = 2.4 Hz),
7.38-7.41 (1H, m), 7.48-7.52 (2H, m), 7.64 (1H, d, J = 9.2 Hz),
7.89-7.98 (4H, m), 8.63 (1H, d, J = 2.0 Hz). PROTAC- D C 802.57
.sup.1H NMR (400 MHz, DMSO-d6): .delta. 7.91 (d, J = 8.80 Hz, 1H),
43 7.72 (t, J = 8.41 Hz, 3H), 7.56 (d, J = 2.54 Hz, 1H), 7.44-7.53
(m, 2H), 7.38 (d, J = 1.96 Hz, 1H), 7.28 (dd, J = 2.05, 8.71 Hz,
1H), 7.21 (d, J = 2.35 Hz, 1H), 7.00 (dd, J = 2.35, 8.80 Hz, 1H),
6.96 (d, J = 9.00 Hz, 2H), 6.41 (d, J = 9.78 Hz, 1H), 4.32 (s, 1H),
4.05 (d, J = 9.00 Hz, 1H), 3.86 (d, J = 12.52 Hz, 2H), 3.45 (br.
s., 4H), 2.79 (t, J = 11.74 Hz, 2H), 2.21 (d, J = 6.46 Hz, 2H),
1.81 (d, J = 11.15 Hz, 3H), 1.21 (s, 6H), 1.12 (s, 6H) PROTAC- D C
802.57 .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. 7.76 (d, J =
8.41 44 Hz, 1H), 7.69 (d, J = 9.00 Hz, 2H), 7.58 (d, J = 8.80 Hz,
1H), 7.51-7.55 (m, 1H), 7.40 (dd, J = 1.96, 6.65 Hz, 1H), 7.36 (d,
J = 2.15 Hz, 1H), 7.10 (dd, J = 2.05, 8.71 Hz, 1H), 6.97 (d, J =
2.35 Hz, 1H), 6.94 (d, J = 9.00 Hz, 2H), 6.82 (dd, J = 2.45, 8.71
Hz, 1H), 6.37 (t, J = 6.85 Hz, 1H), 6.12 (d, J = 8.02 Hz, 1H), 4.16
(d, J = 8.22 Hz, 1H), 4.05 (s, 1H), 3.83-3.91 (m, 2H), 3.40-3.50
(m, 4H), 2.85 (t, J = 11.44 Hz, 2H), 2.55- 2.66 (m, 4H), 2.30 (d, J
= 7.04 Hz, 2H), 1.92 (d, J = 12.91 Hz, 2H), 1.27 (s, 6H), 1.23 (s,
6H) PROTAC- D C 818.56 45 PROTAC- D C 823.96 .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.81 (s, 1H), 8.58-8.57 (d, 46 J = 2.4 Hz,
1H), 8.23-8.21 (d, J = 7.6 Hz, 1H), 7.92-7.89 (m, 1H), 7.57-7.48
(m, 2H), 7.38-7.34 (m, 1H), 7.26-7.21 (m, 1H), 6.97- 6.96 (d, J =
2.0 Hz, 1H), 6.81-6.78 (m, 1H), 6.61-6.59 (d, J = 9.2 Hz, 1H), 6.25
(s, 1H), 6.11-6.09 (d, J = 8.0 Hz, 1H), 4.82- 4.79 (m, 1H),
4.32-4.29 (m, 2H), 4.26-4.23 (m, 1H), 4.15-4.13 (m, 1H), 4.04 (s,
1H), 3.76-3.67 (m, 10H), 2.95-2.90 (m, 1H), 2.70-2.62 (m, 7H),
2.23-2.19 (m, 2H), 1.25 (s, 6H), 1.21 (s, 6H); PROTAC- B C 791.3
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.21 (6H, s), 1.25 (6H,
s), 1.58- 47 1.66 (4H, m), 1.90-1.94 (2H, m), 2.43-2.47 (2H, m),
2.56-2.58 (4H, m), 3.67-3.70 (4H, m), 4.04 (1H, s), 4.09-4.15 (3H,
m), 5.93 (1H, d, J = 8.0 Hz), 6.07 (1H, d, J = 8.0 Hz), 6.66 (1H,
d, J = 9.2 Hz), 6.80 (1H, dd, J = 8.8, 2.4 Hz), 6.96 (1H, d, J =
2.4 Hz), 7.09 (1H, d, J = 2.8 Hz), 7.41-7.46 (2H, m), 7.57 (1H, d,
J = 8.8 Hz), 7.93 (1H, dd, J = 9.2, 2.4 Hz), 8.05-8.07 (2H, m),
8.58 (1H, d, J = 2.4 Hz), 8.73 (1H, d, J = 2.4 Hz). PROTAC- D C
812.15 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.10-1.13 (9H,
m), 1.21 (6H, 48 s), 1.44-1.53 (4H, m), 1.74-1.77 (2H, m),
1.99-2.08 (1H, m), 2.31-2.34 (3H, m), 2.42-2.45 (5H, m), 2.67-2.68
(1H, m), 3.29- 3.34 (3H, m), 3.58-3.59 (4H, m), 4.00-4.07 (3H, m),
4.30 (1H, s), 6.86 (1H, d, J = 8.8 Hz), 6.98-7.02 (3H, m), 7.22
(1H, d, J = 2.4 Hz), 7.31 (2H, d, J = 8.0 Hz), 7.63 (1H, d, J = 9.2
Hz), 7.91 (1H, d, J = 8.8 Hz), 7.95 (1H, dd, J = 8.8, 2.4 Hz), 8.62
(1H, d, J = 2.0 Hz), 10.78 (1H, s). PROTAC- D C 798.33 .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s), 1.21-1.26 (8H, 49 m),
1.45-1.53 (4H, m), 1.74-1.76 (2H, m), 1.96-1.98 (1H, m), 2.33-2.44
(7H, m), 2.78-2.87 (4H, m), 3.58-3.59 (4H, m), 4.01- 4.07 (3H, m),
4.30 (1H, s), 6.87 (1H, d, J = 9.2 Hz), 6.98-7.02 (3H, m), 7.21
(1H, d, J = 2.8 Hz), 7.41 (2H, d, J = 8.0 Hz), 7.63 (1H, d, J = 9.2
Hz), 7.91 (1H, d, J = 8.8 Hz), 7.96 (1H, dd, J = 8.8, 2.4 Hz), 8.62
(1H, d, J = 2.0 Hz), 10.89 (1H, s). PROTAC- A B 757.54 .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s), 1.22 (6H, s), 50
1.94-2.01 (3H, m), 2.18-2.22 (1H, m), 2.49-2.50 (6H, m), 2.75- 2.83
(1H, m), 2.99 (1H, d, J = 4.8 Hz), 3.61 (4H, s), 4.06 (1H, d, J =
9.2 Hz), 4.19-4.23 (2H, m), 4.31 (1H, s), 4.74-4.80 (1H, m) 6.88
(1H, d, J = 9.2 Hz), 7.01 (1H, dd, J = 8.8 Hz, 2.4 Hz), 7.21 (1H,
d, J = 2.4 Hz), 7.58 (1H, dd, J = 8.8 Hz, 2.4 Hz), 7.63 (1H, d, J =
9.2 Hz), 7.90 (1H, d, J = 8.4 Hz), 7.96 (1H, dd, J = 8.8 Hz, 2.4
Hz), 8.02 (1H, d, J = 8.8 Hz), 8.34 (1H, d, J = 2.8 Hz), 8.63 (1H,
d, J = 2.4 Hz), 8.89 (1H, d, J = 8.4 Hz), 10.87 (1H, s). PROTAC- A
C 771.55 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s),
1.21 (6H, s), 51 1.62-1.66 (2H, m), 1.79-1.83 (2H, m), 1.97-2.01
(1H, m), 2.18- 2.22 (1H, m), 2.37-2.41 (2H, m), 2.46-2.50 (5H, m),
2.77-2.80 (1H, m), 3.60 (4H, s), 4.05 (1H, d, J = 9.2 Hz), 4.18
(2H, t, J = 6.0 Hz), 4.30 (1H, s), 4.74-4.79 (1H, m), 6.87 (1H, d,
J = 9.2 Hz), 7.01 (1H, dd, J = 2.4 Hz, 8.8 Hz), 7.21 (1H, d, J =
2.4 Hz), 7.57 (1H, dd, J = 2.8 Hz, 8.8 Hz), 7.64 (1H, d, J = 9.2
Hz), 7.91 (1H, d, J = 8.8 Hz), 7.96 (1H, dd, J = 2.4 Hz, 8.8 Hz),
8.02 (1H, d, J = 8.8 Hz), 8.34 (1H, d, J = 2.8 Hz), 8.62 (1H, d, J
= 2.4 Hz), 8.90 (1H, d, J = 8.8 Hz), 10.88 (1H, s). PROTAC- A B
810.61 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s),
1.22 (6H, s), 52 1.42-1.43 (2H, m), 1.61-1.63 (1H, m), 1.75-1.78
(2H, m), 1.96- 2.01 (1H, m), 2.14-2.22 (1H, m), 2.33-2.40 (3H, m),
2.45-2.49 (5H, m), 2.51-2.55 (5H, m), 2.85-2.91 (3H, m), 4.05 (1H,
d, J = 9.6 Hz), 4.30 (1H, s), 4.41 (2H, d, J = 13.2 Hz), 4.72-4.76
(1H, m), 6.85 (1H, d, J = 8.8 Hz), 7.01 (1H, dd, J = 8.8, 2.4 Hz),
7.21 (1H, d, J = 2.4 Hz), 7.42 (1H, dd, J = 8.8, 2.4 Hz), 7.60 (1H,
d, J = 9.2 Hz), 7.85-7.94 (3H, m), 8.32 (1H, d, J = 2.8 Hz), 8.61
(1H, d, J = 2.0 Hz), 8.74 (1H, d, J = 8.4 Hz), 10.86 (1H, s).
PROTAC- A A 796.59 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
0.76-0.81 (1H, m), 1.15-1.21 53 (16H, m), 1.80-2.23 (5H, m),
2.53-2.59 (4H, m), 2.71-2.78 (2H, m), 2.85-2.91 (2H, m), 3.29 (3H,
brs), 3.97 (1H, s), 4.07 (1H, d, J = 8 Hz), 4.38 (2H, d, J = 12.8
Hz), 4.69-4.75 (1H, m), 5.98 (1H, d, J = 8.4 Hz), 6.60 (1H, d, J =
8.8 Hz), 6.73 (1H, dd, J = 8.8, 2.4 Hz), 6.89 (1H, d, J = 2.4 Hz),
7.14-7.17 (1H, m), 7.50 (1H, d, J = 8.8 Hz), 7.84 (1H, dd, J = 8.8,
2.4 Hz), 7.92 (1H, s), 7.97 (1H, d, J = 8.8 Hz), 8.15 (1H, d, J =
2.4 Hz), 8.38 (1H, d, J = 6.8 Hz), 8.50 (1H, d, J = 2.4 Hz).
PROTAC- B B 797.59 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
1.07-1.23 (14H, m), 1.84- 54 2.03 (4H, m), 2.16-2.23 (3H, m),
2.51-2.60 (5H, m), 2.75-2.80 (1H, m), 3.02-3.08 (2H, m), 3.36 (4H,
s), 4.01 (1H, d, J = 9.2 Hz), 4.46-4.52 (3H, m), 4.72-4.78 (1H, m),
7.03 (1H, dd, J = 8.8, 2.4 Hz), 7.25 (1H, d, J = 2.0 Hz), 7.36-7.44
(2H, m), 7.81- 7.92 (3H, m), 8.24 (1H, d, J = 9.2 Hz), 8.33 (1H, d,
J = 1.6 Hz), 8.73 (1H, d, J = 8.4 Hz), 10.85 (1H, s). PROTAC- A B
795.59 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.13 (6H, s),
1.15-1.22 (8H, 55 m), 1.80-1.83 (3H, m), 1.99-2.03 (1H, m),
2.16-2.23 (3H, m), 2.50-2.51 (5H, m), 2.76-2.82 (3H, m), 3.36 (4H,
s), 3.86 (2H, d, J = 12.4 Hz), 4.05 (1H, d, J = 9.2 Hz), 4.32 (1H,
s), 4.71- 4.78 (1H, m), 6.95-7.02 (3H, m), 7.21 (1H, d, J = 2.0
Hz), 7.42 (1H, dd, J = 8.8, 2.4 Hz), 7.49 (1H, d, J = 9.2 Hz), 7.74
(2H, d, J = 8.8 Hz), 7.86-7.92 (2H, m), 8.32 (1H, d, J = 2.4 Hz),
8.73 (1H, d, J = 8.4 Hz), 10.85 (1H, s). PROTAC- B B 797.58 .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 1.13 (6H, s), 1.19 (6H, s), 56
1.83-1.86 (2H, m), 1.90-2.02 (2H, m), 2.16-2.23 (3H, m), 2.48- 2.51
(6H, m), 2.76-2.82 (1H, m), 2.99-3.05 (3H, m), 3.35 (4H, s), 3.96
(1H, d, J = 9.2 Hz), 4.43 (1H, s), 4.47-4.50 (2H, m), 4.71-4.78
(1H, m), 7.03 (1H, dd, J = 2.4 Hz, 8.8 Hz), 7.25 (1H, d, J = 2.4
Hz), 7.42 (1H, dd, J = 2.8 Hz, 8.8 Hz), 7.80-7.91 (3H, m), 8.33
(2H, d, J = 4.8 Hz), 8.60 (1H, d, J = 1.2 Hz), 8.72 (1H, d, J = 8.0
Hz), 10.85 (1H, s). PROTAC- B A 830.53 57 PROTAC- 0C B 830.53 58
PROTAC- A A 768.53 59 PROTAC- D C 731.51 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.12 (6H, s), 1.22 (6H, s), 60 1.92-2.08 (3H,
m), 2.41-2.50 (7H, m), 2.55-2.60 (1H, m), 2.83- 2.91 (1H, m), 3.61
(4H, s), 4.05-4.13 (3H, m), 4.30 (1H, s), 5.65 (1H, dd, J = 12.4,
5.2 Hz), 6.36 (1H, d, J = 2.8 Hz), 6.87 (1H, d, J = 9.2 Hz), 7.00
(1H, dd, J = 8.8, 2.4 Hz), 7.21 (1H, d, J = 2.4 Hz), 7.63 (1H, d, J
= 9.2 Hz), 7.82 (1H, d, J = 2.8 Hz), 7.91 (1H, d, J = 8.8 Hz), 7.96
(1H, dd, J = 8.8, 2.8 Hz), 8.63 (1H, d, J = 2.4 Hz), 11.03 (1H, s).
PROTAC- A B 729.53 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.16
(7H, s), 1.21 (7H, s), 1.71- 61 1.75 (2H, m), 2.13-2.16 (1H, m),
2.34-2.37 (2H, m), 2.45-2.47 (4H, m), 2.55-2.71 (2H, m), 2.77-2.84
(3H, m), 3.25-3.28 (4H, m) 3.99 (1H, s), 4.09 (1H, d, J = 8.4 Hz),
5.63-5.68 (1H, m), 5.82 (1H, d, J = 2.8 Hz), 6.11 (1H, d, J = 8.0
Hz), 6.74 (1H, dd, J = 8.8, 2.4 Hz), 6.90 (1H, d, J = 2.0 Hz), 7.21
(1H, s), 7.50 (1H, d, J = 8.8 Hz), 7.64 (1H, d, J = 3.2 Hz), 7.91
(1H, brs), 7.96 (1H, dd, J = 8.0, 2.0 Hz), 8.83 (1H, d, J = 1.6
Hz). PROTAC- A B 769.57 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
1.12 (6H, s), 1.22 (6H, s), 62 1.78-1.81 (3H, m), 1.97-2.01 (1H,
m), 2.19-2.21 (2H, m), 2.44 (6H, brs), 2.75-2.81 (3H, m), 3.35 (6H,
brs), 3.85 (2H, d, J = 11.2 Hz), 4.05 (1H, d, J = 9.2 Hz), 4.32
(1H, s), 5.57-5.61 (1H, m), 5.87 (1H, d, J = 2.8 Hz), 6.95-7.02
(3H, m), 7.21 (1H, d, J = 2.4 Hz), 7.50 (1H, d, J = 9.2 Hz), 7.73
(2H, d, J = 8.8 Hz), 7.91 (1H, d, J = 8.8 Hz), 8.06 (1H, d, J = 2.4
Hz), 10.98 (1H, s). PROTAC- A A 783.59 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.12 (6H, s), 1.22-1.26 (8H, 63 m), 1.39-1.44
(3H, m), 1.74-1.77 (2H, m), 1.98-2.00 (1H, m), 2.33-2.45 (8H, m),
2.72-2.85 (3H, m), 3.32-3.34 (4H, m), 3.83- 3.86 (2H, m), 4.05 (1H,
d, J = 8.8 Hz), 4.32 (1H, s), 5.58-5.61 (1H, m), 5.85-5.86 (1H, m),
6.95 (2H, d, J = 9.2 Hz), 7.00 (1H, dd, J = 9.2, 2.4 Hz), 7.21 (1H,
d, J = 2.4 Hz), 7.50 (1H, d, J = 8.8 Hz), 7.73 (2H, d, J = 8.8 Hz),
7.91 (1H, d, J = 8.8 Hz), 8.05 (1H, d, J = 2.8 Hz), 10.97 (1H, s).
PROTAC- D C 806.57
66 PROTAC- B C 837.61 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
1.13 (6H, s), 1.22 (6H, s), 67 1.46-1.54 (4H, m), 1.75-1.79 (2H,
m), 2.07-2.17 (1H, m), 2.33- 2.41 (3H, m), 2.60-2.70 (1H, m),
2.85-2.94 (1H, m), 3.22-3.28 (4H, m), 3.30-3.36 (7H, m), 4.03-4.07
(3H, m), 4.33 (1H, s), 5.21-5.25 (1H, m), 6.95-7.02 (3H, m),
7.12-7.14 (1H, m), 7.20- 7.26 (3H, m), 7.45 (1H, t, J = 8.0 Hz),
7.52 (1H, d, J = 9.2 Hz), 7.75 (2H, d, J = 8.8 Hz), 7.91 (1H, d, J
= 8.8 Hz), 11.1 (1H, s). PROTAC- A A 848.62 .sup.1H NMR (400 MHz,
D6-DMSO): .delta. 1.13 (s, 6H), 1.18-1.27 (m, 68 2H), 1.22 (s, 6H),
1.76-1.83 (m, 3H), 2.07-2.15 (m, 1H), 2.18- 2.25 (m, 2H), 2.42-2.50
(m, 4H), 2.50-2.56 (m, 3H), 2.59-2.67 (m, 1H), 2.75-2.82 (m, 2H),
2.84-2.93 (m, 1H), 3.16-3.26 (m, 4H), 3.29 (s, 1H), 3.86 (d, J =
12.4 Hz, 2H), 4.05 (d, J = 9.2 Hz, 1H), 4.32 (s, 1H), 5.19-5.24 (m,
1H), 6.96 (d, J = 8.8 Hz, 2H), 7.00 (dd, J = 2.4, 8.8 Hz, 1H), 7.05
(d, J = 7.6 Hz, 1H), 7.11-7.14 (m, 2H), 7.21 (d, J = 2.4 Hz, 1H),
7.37 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 9.2 Hz, 1H), 7.74 (d, J =
8.8 Hz, 2H), 7.90 (d, J = 8.8 Hz, 1H), 11.04 (s, 1H),. PROTAC- A B
837.61 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.13 (6H, s),
1.22 (6H, s), 69 1.37-1.59 (4H, m), 1.74-1.81 (2H, m), 2.09-2.13
(1H, m), 2.31- 2.38 (3H, m), 2.59-2.67 (1H, m), 2.80-2.93 (1H, m),
3.22-3.27 (4H, m), 3.30 (3H, m), 3.30-3.34 (4H, m), 4.02-4.08 (3H,
m), 4.32 (1H, s), 5.18-5.22 (1H, m), 6.95-7.02 (3H, m), 7.06-7.10
(2H, m), 7.21 (1H, d, J = 2.4 Hz), 7.51 (1H, d, J = 9.2 Hz),
7.60-7.64 (2H, m), 7.75 (2H, d, J = 8.8 Hz), 7.91 (1H, d, J = 8.8
Hz), 11.03 (1H, s). PROTAC- A A 848.62 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.12 (7H, brs), 1.21 (8H, 70 brs), 1.79-1.82
(3H, m), 2.08-2.12 (1H, m), 2.20-2.22 (2H, m), 2.41-2.45 (3H, m),
2.59-2.63 (1H, m), 2.76-2.87 (3H, m), 3.26- 3.27 (5H, m), 3.30 (3H,
s), 3.84-3.87 (2H, m), 4.04-4.06 (1H, m), 4.32 (1H, s), 5.18 (1H,
dd, J = 5.6, 12.8 Hz), 6.94-7.05 (5H, m), 7.20 (1H, d, J = 2.4 Hz),
7.47-7.53 (3H, m), 7.73 (1H, d, J = 8.8 Hz), 7.90 (1H, d, J = 8.8
Hz), 11.0 (1H, s). PROTAC- D 6C 757.55 75 PROTAC- D C 702.5 .sup.1H
NMR (400 MHz, METHANOL-d.sub.4) .delta. 7.74 (dd, J = 8.51, 76
12.03 Hz, 3H), 7.35 (d, J = 8.22 Hz, 2H), 7.13 (d, J = 2.54 Hz,
1H), 6.99 (dd, J = 2.45, 8.71 Hz, 1H), 4.95 (dd, J = 5.48, 12.72
Hz, 1H), 4.29 (s, 1H), 4.16 (s, 1H), 2.96 (d, J = 11.93 Hz, 2H),
2.66-2.82 (m, 6H), 2.61 (dd, J = 4.40, 13.40 Hz, 1H), 2.41- 2.48
(m, 2H), 2.14-2.24 (m, 2H), 1.98-2.11 (m, 3H), 1.85- 1.94 (m, 2H),
1.68 (d, J = 12.91 Hz, 2H), 1.29 (s, 6H), 1.23 (s, 6H) PROTAC- D C
700.48 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. 7.81 (d, J =
8.41 Hz, 77 2H), 7.73 (d, J = 8.61 Hz, 1H), 7.57 (d, J = 8.41 Hz,
2H), 7.13 (d, J = 2.54 Hz, 1H), 6.99 (dd, J = 2.45, 8.71 Hz, 1H),
6.77 (d, J = 15.85 Hz, 1H), 6.40-6.50 (m, 1H), 4.97 (dd, J = 5.58,
12.62 Hz, 1H), 4.30 (s, 1H), 4.17 (s, 1H), 3.45 (d, J = 6.26 Hz,
2H), 3.12-3.22 (m, 2H), 2.56-2.86 (m, 6H), 2.52 (br. s., 1H),
2.09-2.21 (m, 2H), 1.98-2.05 (m, 1H), 1.80 (d, J = 13.50 Hz, 2H),
1.29 (s, 6H), 1.24 (s, 6H) PROTAC- D C 716.52 .sup.1H NMR (400 MHz,
METHANOL-d.sub.4) .delta. 7.74 (dd, J = 8.51, 78 11.25 Hz, 3H),
7.33 (d, J = 8.22 Hz, 2H), 7.13 (d, J = 2.35 Hz, 1H), 6.99 (dd, J =
2.35, 8.80 Hz, 1H), 4.95 (dd, J = 5.48, 12.72 Hz, 1H), 4.29 (s,
1H), 4.16 (s, 1H), 2.99 (d, J = 11.54 Hz, 2H), 2.67-2.86 (m, 6H),
2.61 (dd, J = 4.21, 13.21 Hz, 1H), 2.46- 2.54 (m, 2H), 2.25 (t, J =
11.25 Hz, 2H), 1.97-2.12 (m, 3H), 1.65-1.75 (m, 4H), 1.55-1.64 (m,
2H), 1.29 (s, 6H), 1.23 (s, 6H) PROTAC- B C 791.23 .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 1.12 (6H, s), 1.21 (6H, s), 79 1.49-1.57
(4H, m), 1.83-1.86 (2H, m), 2.31-2.40 (5H, m), 2.67- 2.68 (1H, m),
3.58-3.60 (4H, m), 4.05 (1H, d, J = 9.2 Hz), 4.17- 4.20 (2H, m),
4.30 (1H, s), 5.76 (1H, d, J = 8.4 Hz), 6.86 (1H, d, J = 8.8 Hz),
6.99-7.02 (1H, m), 7.21 (1H, d, J = 2.0 Hz), 7.50- 7.52 (1H, m),
7.63 (1H, d, J = 9.6 Hz), 7.70-7.76 (3H, m), 7.90-7.97 (2H, m),
8.44 (1H, s), 8.62 (1H, d, J = 1.6 Hz), 9.31 (1H, s). PROTAC- D C
816.23 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.11 (6H, s),
1.21 (6H, s), 80 1.50-1.57 (4H, m), 1.81-1.86 (2H, m), 2.33-2.37
(2H, m), 2.45- 2.50 (4H, m), 3.59 (4H, s), 4.05 (1H, d, J = 9.2
Hz), 4.15 (2H, t, J = 6.4 Hz), 4.30 (1H, s), 6.86 (1H, d, J = 9.2
Hz), 7.00 (1H, dd, J = 8.8, 2.0 Hz), 7.21 (1H, d, J = 2.4 Hz), 7.45
(1H, d, J = 2.4 Hz), 7.50 (1H, dd, J = 5.2, 2.4 Hz), 7.63 (1H, d, J
= 9.2 Hz), 7.91 (1H, d, J = 8.8 Hz), 7.95 (1H, dd, J = 8.8, 2.0
Hz), 8.00 (1H, d, J = 9.2 Hz), 8.37 (1H, d, J = 2.0 Hz), 8.62 (1H,
d, J = 2.0 Hz), 8.78 (1H, d, J = 2.4 Hz), 8.96 (1H, s), 12.28 (1H,
brs). PROTAC- A A 832.62 .sup.1H NMR (400 MHz, DMSO) .delta. 10.88
(s, 1H), 7.91-7.89 (m, 81 1H), 7.78-7.72 (m, 3H), 7.50-7.47 (d, J =
9.2 Hz, 1H), 7.21 (s, 1H), 7.09-6.94 (m, 5H), 5.75 (s, 1H), 5.29
(s, 1H), 5.15-4.95 (m, 1H), 4.32 (s, 1H), 4.21-4.04 (m, 3H),
3.87-3.84 (m, 2H), 3.32- 3.30 (m, 7H), 2.84-2.76 (m, 3H), 2.65-2.56
(m, 1H), 2.48-2.37 (m, 1H), 2.22-2.18 (m, 2H), 1.90-1.79 (m, 4H),
1.40-1.16 (m, 9H), 1.16-1.09 (m, 6H); PROTAC- A A 818.59 .sup.1H
NMR: (400 MHz, DMSO-d.sub.6) .delta.: 11.07-10.90 (m, 1H), 82 10.57
(s, 1H), 8.10-8.01 (m, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.80 (d, J =
8.8 Hz, 2H), 7.58 (br d, J = 9.2 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H),
7.29-7.23 (m, 1H), 7.21 (d, J = 2.4 Hz, 1H), 7.16-7.05 (m, 3H),
7.01 (dd, J = 2.4, 8.8 Hz, 1H), 6.56-6.37 (m, 1H), 6.56- 6.37 (m,
1H), 4.34 (s, 1H), 4.06 (d, J = 9.2 Hz, 3H), 3.87 (br d, J = 12.8
Hz, 2H), 3.68-3.60 (m, 1H), 3.22-3.08 (m, 4H), 3.00- 2.76 (m, 3H),
2.65-2.55 (m, 1H), 2.54-2.52 (m, 2H), 2.47- 2.43 (m, 1H), 2.23-2.11
(m, 1H), 2.05-1.90 (m, 3H), 1.55- 1.30 (m, 2H), 1.23 (s, 6H), 1.14
(s, 6H) *DC50 (nM) and IC50 (nM): A < 1 1 <= B < 10 10
<= C < 100 D >= 100 **Dmax (% degraded) A > 75 50 <
B <= 75 C <= 50
TABLE-US-00005 TABLE 4 Characteristic of exemplary BDR4 PROTACs MS
signal IC.sub.50 Myc I.sub.Max Myc 1 m/e+ PROTAC-# (nM)* (%)** [M +
H].sup.+ NMR PROTAC-1 >1D 40C 753.35 *DC50 (nM) and IC50 (nM): A
< 1 1 <= B < 10 10 <= C < 100 D >= 100 **Dmax (%
degraded) A > 75 50 < B <= 75 C <= 50
TABLE-US-00006 TABLE 5 Characteristics of exemplary estrogen
receptor PROTACs ER MS ER Target degradation ER signal 1 PROTAC-
Engagement DC.sub.50 Degradation m/e+ #* IC.sub.50 (nM)*
(nM)*.sup.+ D.sub.max (%)** [M + H].sup.+ NMR PROTAC- B B 8A 750.6
.sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta.: 10.95 89 (s, 1H),
8.19 (s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.21-7.06 (m, 3H), 6.96 (d,
J = 8.8 Hz, 2H), 6.83 (d, J = 6.4 Hz, 2H), 6.64 (d, J = 8.4 Hz,
1H), 6.59 (d, J = 2.4 Hz, 1H), 6.53 (d, J = 8.8 Hz, 2H), 6.47 (dd,
J = 2.4, 8.4 Hz, 1H), 6.40 (s, 1H), 6.19 (d, J = 8.8 Hz, 2H), 4.91
(dd, J = 5.2, 13.2 Hz, 1H), 4.45-4.33 (m, 1H), 4.29-4.19 (m, 1H),
4.12 (d, J = 4.8 Hz, 1H), 3.52 (s, 1H), 3.49-3.48 (m, 1H), 3.30 (s,
2H), 3.24 (s, 3H), 3.04-2.79 (m, 3H), 2.60 (s, 1H), 2.52 (d, J =
2.0 Hz, 2H), 2.47 (b s, 4H), 2.32-2.23 (m, 1H), 2.18 (d, J = 6.8
Hz, 2H), 2.13-2.03 (m, 1H), 1.99-1.88 (m, 1H), 1.80-1.59 (m, 4H),
1.22-1.06 (m, 2H). PROTAC- B B A 715.58 .sup.1H NMR: (400 MHz,
DMSO-d.sub.6) .delta. = 10.86 90 (s, 1H), 9.13 (s, 1H), 8.13 (s,
1H), 7.42- 7.21 (m, 2H), 7.18-7.07 (m, 3H), 6.97 (d, J = 7.5 Hz,
2H), 6.82 (d, J = 6.4 Hz, 2H), 6.69- 6.57 (m, 2H), 6.56-6.44 (m,
3H), 6.26 (d, J = 8.4 Hz, 2H), 5.13-4.56 (m, 1H), 4.17 (d, J = 5.2
Hz, 1H), 3.81 (t, J = 6.4 Hz, 2H), 3.53- 3.38 (m, 7H), 3.05-2.70
(m, 9H), 2.55 (d, J = 12.0 Hz, 2H), 2.43-2.35 (m, 1H), 2.18- 1.92
(m, 2H), 1.77-1.61 (m, 3H), 1.55 (s, 2H), 1.44-1.31 (m, 2H) PROTAC-
B B A 702.56 .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. = 10.85
91 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 2.8 Hz, 1H),
8.20 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.40 (dd, J = 2.8, 8.8 Hz,
1H), 7.19- 7.08 (m, 3H), 6.82 (d, J = 6.8 Hz, 2H), 6.64 (d, J = 7.6
Hz, 2H), 6.60 (s, 2H), 6.55-6.46 (m, 3H), 6.25 (d, J = 8.8 Hz, 2H),
4.79-4.65 (m, 1H), 4.17 (d, J = 4.8 Hz, 1H), 3.81 (t, J = 6.4 Hz,
2H), 3.03-2.88 (m, 2H), 2.87- 2.72 (m, 1H), 2.70-2.59 (m, 1H),
2.55- 2.52 (m, 4H), 2.46-2.27 (m, 4H), 2.24- 1.96 (m, 4H),
1.77-1.58 (m, 3H), 1.54- 1.42 (m, 2H), 1.39 (d, J = 6.4 Hz, 2H)
PROTAC- D C 759.62 .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. =
10.69 92 (s, 1H), 9.10 (s, 1H), 8.12 (s, 1H), 7.18- 7.04 (m, 3H),
6.90 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 6.8 Hz, 2H), 6.65-6.56 (m,
2H), 6.54- 6.42 (m, 5H), 6.23 (d, J = 8.8 Hz, 2H), 4.26 (s, 1H),
4.15 (d, J = 4.8 Hz, 1H), 4.38-4.06 (m, 1H), 3.86-3.68 (m, 5H),
3.23-3.04 (m, 6H), 3.00-2.87 (m, 2H), 2.72 (s, 1H), 2.50 (s, 5H),
2.31 (d, J = 1.6 Hz, 2H), 2.15-1.85 (m, 2H), 1.73-1.58 (m, 3H),
1.53-1.42 (m, 1H), 1.36 (d, J = 6.8 Hz, 2H), 1.07 (br s, 1H), 0.97
(t, J = 6.8 Hz, 2H) PROTAC- C C 746.5 .sup.1H NMR: (400 MHz,
DMSO-d6) .delta.: 11.06 (s, 93 1H), 10.93-10.63 (m, 1H), 8.21 (d, J
= 2.0 Hz, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.85- 7.76 (m, 1H),
7.04-6.94 (m, 3H), 6.90- 6.81 (m, 2H), 6.69-6.58 (m, 4H), 6.50-6.48
(m, 1H), 6.33 (d, J = 8.4 Hz, 2H), 5.87 (d, J = 2.8 Hz, 1H),
5.70-5.65 (m, 1H), 5.61- 5.45 (m, 1H), 4.30-4.16 (m, 3H), 3.74 (d,
J = 18.1 Hz, 2H), 3.65-3.56 (m, 3H), 3.40- 3.22 (m, 1H), 3.05-2.82
(m, 3H), 2.65- 2.54 (m, 4H), 2.33-1.90 (m, 4H), 1.71 (d, J = 7.3
Hz, 1H). PROTAC- A C 746.5 .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
.delta.: 11.28- 94 10.91 (m, 2H), 8.21 (d, J = 2.9 Hz, 1H), 8.14
(d, J = 2.7 Hz, 1H), 7.82-7.80 (m, 1H), 7.05- 6.94 (m, 3H),
6.89-6.78 (m, 2H), 6.68- 6.58 (m, 4H), 6.51-6.49 (m, 1H), 6.33 (d,
J = 8.6 Hz, 2H), 5.87 (s, 1H), 5.69-6.57 (m, 1H), 5.61-5.45 (m,
1H), 4.30-4.14 (m, 3H), 3.62-3.49 (m, 3H), 3.45-3.27 (m, 3H),
3.07-2.80 (m, 3H), 2.63-2.54 (m, 4H), 2.28-1.94 (m, 4H), 1.70 (d, J
= 6.6 Hz, 1H). PROTAC- D C 745.52 .sup.1H NMR: (400 MHz, DMSO)
.delta. 11.05 (s, 95 1H), 9.19 (br s, 1H), 8.22 (s, 1H), 8.09 (t, J
= 2.6 Hz, 2H), 7.56 (dd, J = 2.9, 9.2 Hz, 1H), 7.03-6.91 (m, 3H),
6.85 (dd, J = 5.8, 8.5 Hz, 2H), 6.68-6.55 (m, 4H), 6.49 (dd, J =
2.5, 8.3 Hz, 1H), 6.29 (d, J = 8.7 Hz, 2H), 5.81 (d, J = 2.8 Hz,
1H), 5.67 (br dd, J = 5.2, 12.2 Hz, 1H), 4.17 (br d, J = 4.9 Hz,
1H), 3.98 (br t, J = 5.6 Hz, 2H), 3.57 (s, 1H), 3.49 (br s, 3H),
3.33 (br dd, J = 3.2, 12.7 Hz, 1H), 3.17-2.80 (m, 4H), 2.70-2.68
(m, 2H), 2.64-2.61 (m, 1H), 2.58-2.52 (m, 4H), 2.19-1.94 (m, 2H),
1.70 (br d, J = 7.0 Hz, 1H). PROTAC- A C 745.52 .sup.1H NMR: (400
MHz, DMSO) .delta. 11.05 (s, 96 1H), 9.19 (br s, 1H), 8.22 (s, 1H),
8.09 (t, J = 2.6 Hz, 2H), 7.56 (dd, J = 2.9, 9.2 Hz, 1H), 7.03-6.91
(m, 3H), 6.85 (dd, J = 5.8, 8.5 Hz, 2H), 6.68-6.55 (m, 4H), 6.49
(dd, J = 2.5, 8.3 Hz, 1H), 6.29 (d, J = 8.7 Hz, 2H), 5.81 (d, J =
2.8 Hz, 1H), 5.67 (br dd, J = 5.2, 12.2 Hz, 1H), 4.17 (br d, J =
4.9 Hz, 1H), 3.98 (br t, J = 5.6 Hz, 2H), 3.57 (s, 1H), 3.49 (br s,
3H), 3.33 (br dd, J = 3.2, 12.7 Hz, 1H), 3.17-2.80 (m, 4H),
2.70-2.68 (m, 2H), 2.64-2.61 (m, 1H), 2.58-2.52 (m, 4H), 2.19-1.94
(m, 2H), 1.70 (br d, J = 7.0 Hz, 1H). PROTAC- C C 746.5 .sup.1H
NMR: (400 MHz, DMSO-d.sub.6) .delta.: 11.06 (s, 97 1H), 10.95-10.67
(m, 1H), 9.18 (s, 1H), 8.20 (d, J = 2.9 Hz, 1H), 8.14 (d, J = 2.8
Hz, 1H), 7.80 (dd, J = 2.8, 8.9 Hz, 1H), 7.03- 6.92 (m, 3H), 6.85
(dd, J = 5.7, 8.4 Hz, 2H), 6.66-6.60 (m, 4H), 6.49 (dd, J = 2.2,
8.3 Hz, 1H), 6.32 (d, J = 8.7 Hz, 2H), 5.86 (d, J = 2.8 Hz, 1H),
5.68 (dd, J = 5.0, 12.2 Hz, 1H), 5.60- 5.46 (m, 1H), 4.28-4.14 (m,
3H), 4.11- 3.98 (m, 1H), 3.81-3.67 (m, 2H), 3.65- 3.55 (m, 3H),
3.38-3.21 (m, 1H), 2.97- 2.91 (m, 3H), 2.67-2.61 (m, 2H), 2.57-2.5
(m, 1H), 2.33-2.06 (m, 3H), 1.71-1.69 (m, 1H) PROTAC- B C 689.47
.sup.1H NMR: (400 MHz, DMSO-d6) .delta. 11.06 (s, 98 1H), 9.23 (s,
1H), 8.47 (d, J = 2.2 Hz, 1H), 8.14 (d, J = 2.4 Hz, 1H), 7.72 (dd,
J = 2.4, 8.6 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 6.98 (t, J = 8.8
Hz, 2H), 6.88-6.78 (m, 2H), 6.67- 6.60 (m, 2H), 6.56 (d, J = 8.2
Hz, 2H), 6.49 (d, J = 8.2 Hz, 1H), 6.28 (d, J = 8.2 Hz, 2H), 5.88
(d, J = 2.5 Hz, 1H), 5.69 (dd, J = 4.6, 12.0 Hz, 1H), 4.16 (br d, J
= 4.6 Hz, 1H), 3.86 (br t, J = 5.8 Hz, 2H), 3.32-3.26 (m, 1H),
3.05-2.87 (m, 3H), 2.82 (br t, J = 7.6 Hz, 2H), 2.62 (br s, 1H),
2.46-2.39 (m, 1H), 2.06 (br d, J = 5.6 Hz, 2H), 1.80 (br d, J = 7.0
Hz, 2H), 1.70 (br d, J = 6.4 Hz, 3H) PROTAC- C C 703.49 .sup.1H
NMR: (400 MHz, DMSO-d6) .delta. 11.07 (s, 99 1H), 9.18 (s, 1H),
8.51-8.38 (m, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.71 (dd, J = 2.8, 8.4
Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.01- 6.92 (m, 2H), 6.88-6.76
(m, 2H), 6.67- 6.59 (m, 2H), 6.55 (d, J = 8.7 Hz, 2H), 6.49 (br d,
J = 8.3 Hz, 1H), 6.27 (br d, J = 8.4 Hz, 2H), 5.86 (d, J = 2.6 Hz,
1H), 5.69 (br dd, J = 5.2, 12.4 Hz, 1H), 4.16 (d, J = 4.6 Hz, 1H),
3.82 (br t, J = 6.3 Hz, 2H), 2.99- 2.85 (m, 3H), 2.78 (t, J = 7.6
Hz, 2H), 2.59 (d, J = 17.4 Hz, 1H), 2.47-2.40 (m, 1H), 2.08-2.00
(m, 2H), 1.78-1.65 (m, 5H), 1.42 (d, J = 6.4 Hz, 2H) PROTAC- B B B
676.54 .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. = 10.95 100 (s,
1H), 9.12 (s, 1H), 8.13 (s, 1H), 8.02 (d, J = 2.8 Hz, 1H),
7.18-7.08 (m, 3H), 6.81 (d, J = 6.4 Hz, 2H), 6.66-6.58 (m, 2H),
6.57- 6.42 (m, 4H), 6.28-6.22 (m, 2H), 5.83 (d, J = 2.4 Hz, 1H),
5.57 (m, 1H), 4.16 (d, J = 4.8 Hz, 1H), 3.79 (t, J = 6.4 Hz, 2H),
3.02-2.77 (m, 4H), 2.59-2.50 (m, 5H), 2.44-2.39 (m, 4H), 2.33-2.26
(m, 2H), 2.14-1.96 (m, 2H), 1.74-1.61 (m, 3H), 1.50-1.31 (m, 4H)
PROTAC- B B B 676.54 .sup.1H NMR: (400 MHz, DMSO) .delta. 10.93 (s,
101 1H) 9.13 (br s, 1H) 8.22 (d, J = 3.51 Hz, 2H) 7.09-7.19 (m, 3H)
6.83 (br d, J = 6.53 Hz, 2H) 6.65 (d, J = 8.68 Hz, 1H) 6.61 (s, 1H)
6.46-6.57 (m, 3H) 6.26 (d, J = 8.66 Hz, 2H) 6.16 (s, 1H) 5.87-5.94
(m, 1H) 4.18 (d, J = 4.77 Hz, 1H) 3.81 (br t, J = 6.40 Hz, 2H) 3.56
(br s, 3H) 3.30 (br s, 1H) 2.76-3.03 (m, 3H) 2.56-2.58 (m, 1H) 2.58
(br d, J = 17.69 Hz, 1H) 2.35-2.41 (m, 1H) 2.29 (br t, J = 7.03 Hz,
4H) 1.99-2.21 (m, 3H) 1.56-1.77 (m, 3H) 1.43-1.54 (m, 2H) 1.38 (br
d, J = 7.15 Hz, 2H) PROTAC- B B A 687.55 .sup.1H NMR: (400 MHz,
DMSO-d.sub.6) .delta.: 10.96 (s, 102 1H), 8.22 (s, 1H), 8.04 (d, J
= 2.4 Hz, 1H), 7.18-7.10 (m, 3H), 6.83 (d, J = 6.4 Hz, 2H), 6.64
(d, J = 8.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.52 (d, J = 8.8 Hz,
2H), 6.47 (dd, J = 2.4, 8.4 Hz, 1H), 6.19 (d, J = 8.8 Hz, 2H), 5.84
(d, J = 2.8 Hz, 1H), 5.58 (dd, J = 5.2, 12.4 Hz, 1H), 4.12 (d, J =
4.4 Hz, 1H), 3.27 (s, 4H), 3.02-2.79 (m, 3H), 2.57 (d, J = 4.0 Hz,
1H), 2.52 (d, J = 2.0 Hz, 4H), 2.46 (s, 1H), 2.42 (d, J = 4.8 Hz,
5H), 2.20-2.06 (m, 3H), 2.02- 1.93 (m, 1H), 1.73 (d, J = 14.0 Hz,
3H), 1.61 (s, 1H), 1.19-1.07 (m, 2H) PROTAC- A C 746.5 .sup.1H NMR:
(400 MHz, DMSO-d6) .delta.: 11.33- 103 10.84 (m, 2H), 8.20 (t, J =
2.9 Hz, 1H), 8.13 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 2.9, 8.3 Hz,
1H), 7.03-6.92 (m, 3H), 6.85 (dd, J = 5.7, 8.3 Hz, 2H), 6.68-6.58
(m, 4H), 6.49 (dd, J = 2.1, 8.3 Hz, 1H), 6.32 (d, J = 8.6 Hz, 2H),
5.86 (d, J = 2.0 Hz, 1H), 5.72-5.62 (m, 1H), 5.60-5.46 (m, 1H),
4.26-4.12 (m, 3H), 4.10-3.99 (m, 1H), 3.63-3.51 (m, 1H), 3.46-3.21
(m, 4H), 3.06-2.80 (m, 3H), 2.61 (d, J = 3.2 Hz, 3H), 2.61-2.60 (m,
1H), 2.60-2.50 (m, 1H), 2.18-1.96 (m, 2H), 1.69 (d, J = 7.7 Hz, 1H)
PROTAC- 6B D C 740.58 1H NMR: (400 MHz, DMSO-d6) .delta.: 10.93 104
(s, 1H), 8.28 (s, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.24-7.08 (m, 6H),
6.83 (d, J = 7.2 Hz, 2H), 6.67-6.60 (m, 2H), 6.56-6.45 (m, 3H),
6.27 (d, J = 8.4 Hz, 2H), 4.94-4.85 (m, 2H), 4.20-4.00 (m, 3H),
3.84 (d, J = 5.6 Hz, 2H), 3.37-3.23 (m, 2H), 2.96 (d, J = 8.0 Hz,
5H), 2.58 (s, 1H), 2.38-2.22 (m, 4H), 2.15-1.86 (m, 4H), 1.80-1.53
(m, 9H) PROTAC- B D C 726.56 .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
.delta. = 10.93 105 (s, 1H), 9.17 (s, 1H), 8.24 (s, 1H), 7.43- 7.36
(m, 1H), 7.24-7.07 (m, 6H), 6.83 (d, J = 6.4 Hz, 2H), 6.68-6.59 (m,
2H), 6.56- 6.45 (m, 3H), 6.27 (d, J = 8.4 Hz, 2H), 4.94- 4.84 (m,
2H), 4.20-3.99 (m, 3H), 3.86 (t, J = 6.4 Hz, 2H), 3.30 (s, 2H),
3.04-2.84 (m, 5H), 2.59-2.53 (m, 3H), 2.41 (t, J = 7.2 Hz, 2H),
2.28 (dd, J = 4.8, 13.6 Hz, 1H), 2.13- 1.60 (m, 11H) PROTAC- A B C
726.56 .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. = 10.92 106 (s,
1H), 9.48-8.87 (m, 1H), 8.21 (s, 1H), 7.41-7.34 (m, 1H), 7.23-7.06
(m, 6H), 6.82 (d, J = 6.8 Hz, 2H), 6.66-6.57 (m, 2H), 6.55-6.44 (m,
3H), 6.25 (d, J = 8.8 Hz, 2H), 4.91-4.82 (m, 2H), 4.18-3.97 (m,
3H), 3.84 (t, J = 6.4 Hz, 2H), 3.30-3.27 (m, 2H), 3.02-2.82 (m,
5H), 2.55-2.52 (m, 3H), 2.39 (t, J = 6.9 Hz, 2H), 2.26 (dd, J =
4.8, 13.6 Hz, 1H), 2.11-1.58 (m, 11H) PROTAC- C D C 837.14 1H NMR:
(400 MHz, DMSO-d6) .delta.: 11.01 (s, 107 1H), 10.04 (s, 1H), 7.62
(s, 4H), 7.33 (d, J = 2.0 Hz, 1H), 7.31-7.23 (m, 1H), 7.21- 7.11
(m, 2H), 7.01 (br d, J = 8.0 Hz, 1H), 6.92 (q, J = 8.8 Hz, 4H),
6.85 (dd, J = 2.0, 8.8 Hz, 1H), 5.25 (dd, J = 5.2, 13.2 Hz, 1H),
4.29 (s, 2H), 3.68-3.45 (m, 14H), 2.87-2.79
(m, 1H), 2.69-2.61 (m, 5H), 2.19-2.10 (m, 1H) PROTAC- B D C 838.12
1H NMR: (400 MHz, DMSO-d6) .delta.: 11.06 (s, 108 1H), 9.99 (s,
1H), 7.66 (d, J = 7.2 Hz, 1H), 7.63 (s, 4H), 7.54-7.42 (m, 1H),
7.52-7.42 (m, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.15 (d, J = 8.8 Hz,
1H), 6.97-6.91 (m, 4H), 6.84 (dd, J = 2.0, 8.8 Hz, 1H), 5.28 (dd, J
= 5.2, 13.2 Hz, 1H), 4.54 (s, 2H), 4.27 (s, 4H), 3.56-3.49 (m,
10H), 2.82-2.80 (m, 1H), 2.65-2.59 (m, 5H), 2.21-2.14 (m, 1H)
PROTAC- D D C 740.58 1H NMR: (400 MHz, DMSO-d6) .delta.: 10.90 109
(s, 1H), 8.23 (s, 1H), 7.40-7.33 (m, 1H), 7.20-7.05 (m, 6H), 6.80
(d, J = 6.8 Hz, 2H), 6.64-6.55 (m, 2H), 6.53-6.43 (m, 3H), 6.23 (d,
J = 8.4 Hz, 2H), 4.91-4.80 (m, 2H), 4.17-3.97 (m, 3H), 3.83-3.77
(m, 2H), 3.25 (d, J = 3.6 Hz, 1H), 3.01-2.79 (m, 5H), 2.55-2.50 (m,
3H), 2.39-2.23 (m, 4H), 2.11-1.85 (m, 4H), 1.75-1.51 (m, 8H)
PROTAC- A A B 696.54 .sup.1H NMR: (400 MHz, DMSO-d6) .delta.: 11.08
110 (s, 1H), 9.13 (br s, 1H), 8.14 (s, 1H), 8.09 (s, 1H), 7.82 (s,
1H), 7.42 (d, J = 8.7 Hz, 2H), 7.20-7.08 (m, 3H), 6.93 (d, J = 8.9
Hz, 2H), 6.83 (br d, J = 6.7 Hz, 2H), 6.68-6.58 (m, 2H), 6.54 (d, J
= 8.7 Hz, 2H), 6.48 (dd, J = 2.4, 8.3 Hz, 1H), 6.26 (d, J = 8.5 Hz,
2H), 5.35 (dd, J = 5.1, 11.9 Hz, 1H), 4.18 (d, J = 5.3 Hz, 1H),
3.87 (br t, J = 6.3 Hz, 2H), 3.30-3.21 (m, 4H), 3.13 (br s, 4H),
3.04-2.76 (m, 3H), 2.66-2.55 (m, 2H), 2.45 (br s, 3H), 2.30-2.19
(m, 1H), 2.17-2.02 (m, 1H), 1.90-1.77 (m, 2H), 1.76-1.66 (m, 1H)
PROTAC- A C 697.54 .sup.1H NMR: (400 MHz, DMSO-d6) .delta.: 11.08
111 (br s, 1H), 9.40-8.74 (m, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.21
(br s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 7.73 (dd, J = 2.1, 8.8 Hz,
1H), 7.20-7.07 (m, 3H), 6.88-6.77 (m, 3H), 6.68-6.58 (m, 2H),
6.57-6.45 (m, 3H), 6.26 (br d, J = 8.4 Hz, 2H), 5.37 (br dd, J =
5.0, 11.9 Hz, 1H), 4.17 (br d, J = 4.8 Hz, 1H), 3.87 (br t, J = 6.1
Hz, 2H), 3.48-3.46 (m, 4H), 3.32-3.30 (m, 4H), 3.00-2.74 (m, 5H),
2.33-2.19 (m, 4H), 2.16-2.01 (m, 1H), 1.90-1.79 (m, 2H), 1.70 (br
d, J = 7.2 Hz, 1H) .sup.+Exemplary PROTACS 93-97, 103, 107, and 108
were assessed in MCF7 cells with a 3 day incubation; exemplary
PROTACs 89-91, 98-102, 110, and 111 were assessed in MCF7 cells
with a 5 day incubation; and exemplary PROTACs 92, 104-106, and 109
were assessed in T47D with a 5 day incubation. *DC50 (nM) and IC50
(nM): A < 1 1 <= B < 10 10 <= C < 100 D >= 100
**Dmax (% degraded) A > 75 50 < B <= 75 C <= 50
TABLE-US-00007 TABLE 6 Characteristics of an exemplary Tau PROTAC
MS Tau signal 1 degradation % m/e+ PROTAC-# DMax** [M + H].sup.+
NMR PROTAC-112 C 757.57 HNMR: (400 MHz, DMSO-d6) .delta.: 11.03 (s,
1H), 9.36 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 8.50 (d, J = 6.4 Hz,
1H), 8.33 (d, J = 8.0 Hz, 1H), 8.23-8.19 (m, 3H), 7.99 (s, 1H),
7.63-7.62 (m, 2H), 6.98 (d, J = 8.8 Hz, 1H), 4.90 (dd, J = 5.2,
13.2 Hz, 1H), 4.45 (t, J = 4.8 Hz, 2H), 3.96 (s, 3H), 3.79 (t, J =
4.8 Hz, 2H), 3.61-3.54 (m, 6H), 3.51-3.47 (m, 7H), 2.84-2.76 (m,
3H), 2.67-2.66 (m, 2H), 2.54-2.53 (m, 1H), 2.47-2.33 (m, 4H), 2.03
(t, J = 10.4 Hz, 2H), 1.87-1.75 (m, 3H), 1.52-1.49 (m, 2H). **Dmax
(% degraded) A > 75 50 < B <= 75 C <= 50
5. INDUSTRIAL APPLICABILITY
[1740] A novel bifunctional molecule, which contains a BRD4 or an
androgen receptor recruiting moiety and an E3 Ligase Cereblon
recruiting moiety, through PROTAC technology is described. The
bifunctional molecules of the present disclosure actively degrades
BRD4, leading to significant and persistent downstream MYC
suppression and robust cellular proliferation suppression and
apoptosis induction. PROTAC mediated protein degradation provides a
promising strategy in targeting the "undruggable" pathological
proteins by traditional approaches.
[1741] The contents of all references, patents, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference.
[1742] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. It is understood that the detailed examples and
embodiments described herein are given by way of example for
illustrative purposes only, and are in no way considered to be
limiting to the invention. Various modifications or changes in
light thereof will be suggested to persons skilled in the art and
are included within the spirit and purview of this application and
are considered within the scope of the appended claims. For
example, the relative quantities of the ingredients may be varied
to optimize the desired effects, additional ingredients may be
added, and/or similar ingredients may be substituted for one or
more of the ingredients described. Additional advantageous features
and functionalities associated with the systems, methods, and
processes of the present disclosure will be apparent from the
appended claims. Moreover, those skilled in the art will recognize,
or be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed
by the following claims.
* * * * *