U.S. patent application number 16/529228 was filed with the patent office on 2020-03-05 for bcl-2 inhibitors.
The applicant listed for this patent is Newave Pharmaceutical Inc.. Invention is credited to Yi Chen, Yan Lou.
Application Number | 20200071329 16/529228 |
Document ID | / |
Family ID | 59399138 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200071329 |
Kind Code |
A1 |
Chen; Yi ; et al. |
March 5, 2020 |
BCL-2 INHIBITORS
Abstract
The disclosure includes compounds of Formula (I) ##STR00001##
wherein W, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, m,
n, L, and Z.sub.1, are defined herein. Also disclosed is a method
for treating a neoplastic disease and autoimmune disease with these
compounds.
Inventors: |
Chen; Yi; (Pleasanton,
CA) ; Lou; Yan; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Newave Pharmaceutical Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
59399138 |
Appl. No.: |
16/529228 |
Filed: |
August 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16045736 |
Jul 26, 2018 |
10377755 |
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16529228 |
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PCT/US2017/015281 |
Jan 27, 2017 |
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16045736 |
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62289209 |
Jan 30, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 471/14 20130101; A61P 35/02 20180101 |
International
Class: |
C07D 471/14 20060101
C07D471/14; A61P 35/02 20060101 A61P035/02; A61P 35/00 20060101
A61P035/00 |
Claims
1. A compound of Formula (I), or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug of said compound of
Formula (I) or N-oxide thereof: ##STR00066## wherein Z.sub.1 is
absent, (CH.sub.2).sub.p, N(H), O, S, C(O), S(O).sub.2, OC(O),
C(O)O, OS(O).sub.2, S(O).sub.2O, C(O)S, SC(O), C(O)C(O), C(O)N(H),
N(H)C(O), S(O).sub.2N(H), N(H)S(O).sub.2, OC(O)O, OC(O)S,
OC(O)N(H), N(H)C(O)O, N(H)C(O)S, N(H)C(O)N(H),
(CH.sub.2).sub.pN(H)(CH.sub.2).sub.q,
(CH.sub.2).sub.pN(H)C(O)(CH.sub.2).sub.q,
(CH.sub.2).sub.pC(O)N(H)(CH.sub.2).sub.q,
OC(O)N(H)(CH.sub.2).sub.p+1N(H)(CH.sub.2).sub.q, a bivalent alkenyl
group, or a bivalent alkynyl group; W is CH or N, and the tricylic
##STR00067## is optionally substituted with one or more R.sub.7;
each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7, independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,
heteroaryl, halo, nitro, oxo, cyano, OR.sub.a, SR.sub.a,
alkyl-R.sub.a, NH(CH.sub.2).sub.pR.sub.a, C(O)R.sub.a, S(O)R.sub.a,
SO.sub.2R.sub.a, C(O)OR.sub.a, OC(O)R.sub.a, NR.sub.bR.sub.c,
P(O)R.sub.bR.sub.c, C(O)N(R.sub.b)R.sub.c, N(R.sub.b)C(O)R.sub.c,
SO.sub.2N(R.sub.b)R.sub.c, or N(R.sub.b)SO.sub.2R.sub.c, in which
said cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, heteroaryl is optionally substituted with
one or more R.sub.d; optionally, two of R.sub.2 group, taken
together with the atom to which they are attached, may optionally
form a cycloalkyl or heterocycloalkyl optionally substituted with
one or more R.sub.d; R.sub.a, R.sub.b, R.sub.c and R.sub.d,
independently, is H, alkyl, alkenyl, alkynyl, halo, cyano, amine,
nitro, hydroxy, C(O)NHOH, alkoxy, alkoxyalkyl, haloalkyl,
hydroxyalkyl, aminoalkyl, alkylcarbonyl, alkoxycarbonyl,
alkylcarbonylamino, alkylamino, oxo, halo-alkylamino, cycloalkyl,
cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or
heteroaryl; L is absent, alkylene, alkenylene, alkynylene,
cycloalkylene, cycloalkenylene, heterocycloalkylene,
heterocycloalkenylene, wherein said alkylene, alkenylene,
alkynylene, cycloalkylene, cycloalkenylene, heterocycloalkylene, or
heterocycloalkenylene is optionally substituted with one or more
R.sub.d; and each of m, n, p, and q, independently, is 0, 1, 2, 3,
or 4.
2. The compound according to claim 1 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein the
compound is represented by Formula (II) ##STR00068## wherein the
tricylic ##STR00069## is unsubstituted.
3. The compound according to claim 2 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein the
compound is represented by Formula (III) ##STR00070##
4. The compound according to claim 3 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein
R.sub.1 is Cl.
5. The compound according to claim 4 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein
R.sub.5 independently, is nitro, halo, or SO.sub.2R.sub.a.
6. The compound according to claim 5 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein
Z.sub.1 is absent, NH, O, or S.
7. The compound according to claim 6 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein L is
absent or C.sub.1-3alkylene.
8. The compound according to claim 7 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein
R.sub.6 is H, 4-6 membered cycloalkyl or 4-6 membered heterocyclyl,
wherein the 4-6 membered cycloalkyl or 4-6 membered heterocyclyl is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-4alkyl, CN, --OH,
C.sub.1-4alkoxy, --S(O).sub.2CH.sub.3, --COCH.sub.3, 3-6 membered
cycloalkyl, and 3-6 membered heterocyclyl,
9. The compound according to claim 1 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein the
compound is ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077##
10. The compound according to claim 1 or an N-oxide thereof, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug thereof, wherein the
compound is ##STR00078## ##STR00079## ##STR00080## ##STR00081##
##STR00082## ##STR00083## ##STR00084## ##STR00085##
11. A pharmaceutical composition comprising a compound of Formula
(I) or an N-oxide thereof as defined in claim 1, or a
pharmaceutically acceptable salt, solvate, polymorph, tautomer,
stereoisomer, an isotopic form, or a prodrug of said compound of
Formula (I) or an N-oxide thereof, and a pharmaceutically
acceptable diluent or carrier.
12. A method of treating a neoplastic disease or autoimmune
disease, comprising administering to a subject in need thereof an
effective amount of a compound of Formula (I) or an N-oxide thereof
as defined in claim 1, or a pharmaceutically acceptable salt,
solvate, polymorph, tautomer, stereoisomer, an isotopic form, or a
prodrug of said compound of Formula (I) or an N-oxide thereof.
13. The method according to claim 12, wherein the neoplastic
disease is leukemia, lymphoma, or multiple myeloma.
14. A compound or an N-oxide thereof, or a pharmaceutically
acceptable salt, solvate, polymorph, tautomer, stereoisomer, an
isotopic form, or a prodrug of said compound or N-oxide thereof,
wherein the compound is: ##STR00086##
15. A pharmaceutical composition comprising a compound or an
N-oxide thereof as defined in claim 14, or a pharmaceutically
acceptable salt, solvate, polymorph, tautomer, stereoisomer, an
isotopic form, or a prodrug of said compound or an N-oxide thereof,
and a pharmaceutically acceptable diluent or carrier.
16. A method of treating a neoplastic disease or autoimmune
disease, comprising administering to a subject in need thereof an
effective amount of a compound or an N-oxide thereof as defined in
claim 14, or a pharmaceutically acceptable salt, solvate,
polymorph, tautomer, stereoisomer, an isotopic form, or a prodrug
of said compound or an N-oxide thereof.
17. The method according to claim 16, wherein the neoplastic
disease is leukemia, lymphoma, or multiple myeloma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/045,736, filed on Jul. 26, 2018, which is a
continuation of International Patent Application No.
PCT/US2017/015281, filed on Jan. 27, 2017, which claims the benefit
of the filing date of U.S. Provisional Application No. 62/289,209,
filed on Jan. 30, 2016. The entire contents of each of the
aforementioned applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Apoptosis, or programmed cell death, is a conserved and
regulated process that is the primary mechanism for the removal of
aged, damaged and unnecessary cells. The ability to block apoptotic
signaling is a key hallmark of cancer and is thus important for
oncogenesis, tumor maintenance and chemoresistance Manahan, D.
& Weinberg, R. A. The hallmarks of cancer. Cell 100, 57-70
(2000)1. Dynamic binding interactions between prodeath (for
example, BCL-2associated X protein (BAX), BCL-2 antagonist/killer 1
(BAK), BCL-2associated agonist of cell death (BAD), BCL-2like 11
(BIM), NOXA and BCL-2 binding component 3 (PUMA)) and prosurvival
(BCL-2, BCL-XL, BCL-2like 2 (BCL-W), myeloid cell leukemia sequence
1 (MCL-1) and BCL-2related protein Al (BFL-1)) proteins in the
BCL-2 family control commitment to programmed cell death. Altering
the balance among these opposing factions provides one means by
which cancer cells undermine normal apoptosis and gain a survival
advantage [Youle, R. J. & Strasser, A. The BCL-2 protein family
opposing activities that mediate cell death. Nat. Rev. Mol. Cell
Biol. 9, 47-59 (2008)].
[0003] BCL-2, the first identified apoptotic regulator, was
originally cloned from the breakpoint of a t(14;18) translocation
present in human B cell lymphomas[Tsujimoto, Y., et al. Science
228, 1440-1443 (1985); Cleary, M. L., et al Cell 47, 19-28 (1986);
Boise, L. H. et al. Cell 74, 597-608 (1993)]. This protein has
since been shown to have a dominant role in the survival of
multiple lymphoid malignancies [Vaux, D. L., et al pre-B cells.
Nature 335, 440-442 (1988)]. Overexpression of Bcl-2 proteins
correlates with resistance to chemotherapy, clinical outcome,
disease progression, overall prognosis or a combination thereof in
various cancers and disorders of the immune system. Involvement of
Bcl-2 proteins in bladder cancer, brain cancer, breast cancer, bone
marrow cancer, cervical cancer, chronic lymphocytic leukemia,
colorectal cancer, esophageal cancer, hepatocellular cancer,
lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies
of T-cell or B-cell origin, melanoma, myelogenous leukemia,
myeloma, oral cancer, ovarian cancer, non-small cell lung cancer,
prostate cancer, small cell lung cancer, spleen cancer, and the
like is described in commonly-owned PCT/US2004/36770, published as
WO 2005/049593, and PCT/US/2004/37911, published as WO 2005/024636.
Involvement of Bcl-2 proteins in immune and autoimmune diseases is
described in Current Allergy and Asthma Reports 2003, 3, 378-384;
British Journal of Hematology 2000, 110(3), 584-90; Blood 2000,
95(4), 1283-92; and New England Journal of Medicine 2004, 351(14),
1409-1418. Involvement of Bcl-2 proteins in arthritis is disclosed
in commonly-owned U.S. Provisional Patent Application Ser. No.
60/988,479. Involvement of Bcl-2 proteins in bone marrow transplant
rejection is disclosed in commonly-owned U.S. patent application
Ser. No. 11/941,196.
[0004] In the last decade, several Bcl-2 inhibitors such as
ABT-737, ABT-263, and ABT-199 as shown below have been identified
and entered human clinical trials for cancers treatment.
##STR00002##
[0005] ABT-737 is discovered by nuclear magnetic resonance
(NMR)-based screening, parallel synthesis and structure based
fragment drug design [Tilman Oltersdorf, et al, Nature, Vol 435,
2005, p 677]. ABT-737 a small-molecule inhibitor of the
anti-apoptotic proteins Bcl-2, Bcl-XL and Bcl-w, with an affinity
two to three orders of magnitude more potent than previously
reported compounds. Mechanistic studies reveal that ABT-737 does
not directly initiate the apoptotic process, but enhances the
effects of death signals, displaying synergistic cytotoxicity with
chemotherapeutics and radiation. ABT-737 exhibits
single-agent-mechanism-based killing of cells from lymphoma and
small-cell lung carcinoma lines, as well as primary patient-derived
cells, and in animal models, ABT-737 improves survival, causes
regression of established tumors, and produces cures in a high
percentage of the mice. Unfortunately, ABT-737 is not orally
bioavailable and its low aqueous solubility makes formulation for
intravenous delivery extremely difficult.
[0006] After extensive MedChem effort, an orally bioavailable Bcl-2
inhibitor ABT-263 (Navitoclax) has been developed [Cheol-Min Park,
et al J. Med. Chem. 2008, 51, 6902-6915]. ABT-263 is a potent
inhibitor of Bcl-xL, Bcl-2 and Bcl-w with Ki of .ltoreq.0.5 nM,
.ltoreq.nM and .ltoreq.1 nM. ABT-263 has a IC.sub.50 of 110 nM
against SCLC H146 cell line, When ABT-263 is administered at 100
mg/kg/day in the H345 xenograft model, significant antitumor
efficacy is observed with 80% TGI and 20% of treated tumors
indicating at least a 50% reduction in tumor volume. Oral
administration of ABT-263 alone causes complete tumor regressions
in xenograft models of small-cell lung cancer and acute
lymphoblastic leukemia [Tse C, et al. Cancer Res. 2008, 68(9),
3421-3428]. Unfortunately, in the clinical trial, the inhibition of
BCL-XL by ABT-263 (navitoclax) induces a rapid,
concentration-dependent decrease in the number of circulating
platelets. This mechanism-based thrombocytopenia is the
dose-limiting toxicity of single-agent navitoclax treatment in
patients and limits the ability to drive drug concentrations into a
highly efficacious range.
[0007] Thus, a BCL-2 selective (BCL-XL sparing) inhibitor would
culminate in substantially reduced thrombocytopenia while
maintaining efficacy in lymphoid malignancies. The resulting
increase in the therapeutic window should allow for greater BCL-2
suppression and clinical efficacy in BCL-2dependent tumor types,
After extensive MedChem, ABT-199 (GDC-0199) has been successfully
developed[Andrew J Souers, et al, Nature Medicine, Volume 19, 22,
p202, 2013]. ABT-199 is a Bcl-2-selective inhibitor with Ki of
<0.01 nM, >4800-fold more selective versus Bcl-xL and Bcl-w,
and no activity to Mc1-1. ABT-199 potently inhibits RS4; 11 cells
with EC.sub.50 of 8 nM. In addition, ABT-199 induces a rapid
apoptosis in RS4; 11 cells with cytochrome c release, caspase
activation, and the accumulation of sub-G0/G1 DNA. Quantitative
immunoblotting reveals that sensitivity to ABT-199 correlated
strongly with the expression of Bcl-2, including NHL, DLBCL, MCL,
AML and ALL cell lines. ABT-199 also induces apoptosis in CLL with
an average EC.sub.50 of 3.0 nM. A single dose of 100 mg/kg of
ABT-199 causes a maximal tumor growth inhibition of 95% and tumor
growth delay of 152% in RS4; 11 xenografts. ABT-199 also inhibits
xenograft growth (DoHH2, Granta-519) as a single agent or in
combination with Bendamustine and other agents. Human Phase I and
II data showed that ABT-199 is highly efficacious for CLL, however,
ABT.sup.-199 causes lethal tumour lysis syndrome in which the
debris of dying cancer cells overwhelms the kidney and other
organs.
[0008] Although the Bcl-2 inhibitors, particularly the Bcl-2
selective inhibitor ABT-199 have made a significant contribution to
the art, there is a continuing search in this field of art for
improved pharmaceuticals.
SUMMARY OF THE INVENTION
[0009] In a first embodiment, this invention provides compounds of
the Formula (I) or an N-oxide thereof, or a pharmaceutically
acceptable salt, solvate, polymorph, tautomer, stereoisomer, an
isotopic form, or a prodrug of said compound of Formula (I) or
N-oxide thereof:
##STR00003##
wherein
[0010] Z.sub.1 is absent, (CH.sub.2).sub.p, N(H), O, S, C(O),
S(O).sub.2, OC(O), C(O)O, OS(O).sub.2, S(O).sub.2O, C(O)S, SC(O),
C(O)C(O), C(O)N(H), N(H)C(O), S(O).sub.2N(H), N(H)S(O).sub.2,
OC(O)O, OC(O)S, OC(O)N(H), N(H)C(O)O, N(H)C(O)S, N(H)C(O)N(H),
(CH.sub.2).sub.pN(H)(CH.sub.2).sub.q,
(CH.sub.2).sub.pN(H)C(O)(CH.sub.2).sub.q,
(CH.sub.2).sub.pC(O)N(H)(CH.sub.2).sub.q,
OC(O)N(H)(CH.sub.2).sub.p+1N(H)(CH.sub.2).sub.q, a bivalent alkenyl
group, or a bivalent alkynyl group;
[0011] W is CH or N, and the tricylic
##STR00004##
is optionally substituted with one or more R.sub.7;
[0012] each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7, independently, is H, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,
aryl, heteroaryl, halo, nitro, oxo, cyano, OR.sub.a, SR.sub.a,
alkyl-R.sub.a, NH(CH.sub.2).sub.pR.sub.a, C(O)R.sub.a, S(O)R.sub.a,
SO.sub.2R.sub.a, C(O)OR.sub.a, OC(O)R.sub.a, NR.sub.bR.sub.c,
P(O)R.sub.bR.sub.c, C(O)N(R.sub.b)R.sub.c, N(R.sub.b)C(O)R.sub.c,
SO.sub.2N(R.sub.b)R.sub.c, or N(R.sub.b)SO.sub.2R.sub.c, in which
said cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, heteroaryl is optionally substituted with
one or more R.sub.d;
[0013] optionally, two of R.sub.2 group, taken together with the
atom to which they are attached, may optionally form a cycloalkyl
or heterocycloalkyl optionally substituted with one or more
R.sub.d;
[0014] R.sub.a, R.sub.b, R.sub.c and R.sub.d, independently, is H,
alkyl, alkenyl, alkynyl, halo, cyano, amine, nitro, hydroxy,
C(O)NHOH, alkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, aminoalkyl,
alkylcarbonyl, alkoxycarbonyl, alkylcarbonylamino, alkylamino, oxo,
halo-alkylamino, cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, or heteroaryl;
[0015] each of R.sub.1, R.sub.2, independently, is H or alkyl; or
two of R.sub.2 group, taken together with the atom to which they
are attached, may optionally form a cycloalkyl;
[0016] L is absent, alkylene, alkenylene, alkynylene,
cycloalkylene, cycloalkenylene, heterocycloalkylene,
heterocycloalkenylene, wherein said alkylene, alkenylene,
alkynylene, cycloalkylene, cycloalkenylene, heterocycloalkylene, or
heterocycloalkenylene is optionally substituted with one or more
R.sub.d; and
[0017] each of m, p, and q, independently, is 0, 1, 2, 3, or 4.
[0018] In a second embodiment, the invention provides the compounds
according to the previous embodiment, wherein the compound is
represented by Formula (II):
##STR00005##
wherein the tricylic
##STR00006##
is unsubstituted, wherein the reminder of the variables are as
defined in the first embodiment.
[0019] In a third embodiment, the invention provides the compounds
according to the previous embodiments, wherein the compound is
represented by Formula (III)
##STR00007##
wherein the reminder of the variables are as defined in the first
embodiment.
[0020] In a fourth embodiment, the invention provides the compounds
according to the previous embodiments, wherein R.sub.1 is halo
(e.g., Cl), wherein the reminder of the variables are as defined in
the first, second or third embodiment.
[0021] In a fifth embodiment, the invention provides the compounds
according to the previous embodiments, wherein R.sub.5
independently, is nitro, halo, or SO.sub.2R.sub.a, wherein the
reminder of the variables are as defined in the first, second,
third or fourth embodiment.
[0022] In a sixth embodiment, the invention provides the compounds
according to the previous embodiments, wherein Z.sub.1 is absent,
NH, O, or S, wherein the reminder of the variables are as defined
in the first, second, third, fourth, or fifth embodiment. In a
seventh embodiment, the invention provides the compounds according
to the previous embodiments, wherein L is absent or
C.sub.1-3alkylene, wherein the reminder of the variables are as
defined in the first, second, third, fourth, fifth, or sixth
embodiment.
[0023] In an eighth embodiment, the invention provides the
compounds according to the previous embodiments, wherein R.sub.6 is
H, 4-6 membered cycloalkyl or 4-6 membered heterocyclyl, wherein
the 4-6 membered cycloalkyl or 4-6 membered heterocyclyl is
optionally substituted with one or more substituents selected from
the group consisting of halo, C.sub.1-4alkyl, CN, --OH,
C.sub.1-4alkoxy, --S(O).sub.2CH.sub.3, --COCH.sub.3, 3-6 membered
cycloalkyl, and 3-6 membered heterocyclyl, wherein the reminder of
the variables are as defined in the first, second, third, fourth,
fifth, sixth, or seventh embodiment
[0024] Compounds of the invention may contain one or more
asymmetric carbon atoms. Accordingly, the compounds may exist as
diastereomers, enantiomers, or mixtures thereof. Each of the
asymmetric carbon atoms may be in the R or S configuration, and
both of these configurations are within the scope of the
invention.
[0025] A modified compound of any one of such compounds including a
modification having an improved (e.g., enhanced, greater)
pharmaceutical solubility, stability, bioavailability, and/or
therapeutic index as compared to the unmodified compound is also
contemplated. Exemplary modifications include (but are not limited
to) applicable prodrug derivatives, and deuterium-enriched
compounds.
[0026] It should be recognized that the compounds of the present
invention may be present and optionally administered in the form of
salts or solvates. The invention encompasses any pharmaceutically
acceptable salts and solvates of any one of the above-described
compounds and modifications thereof.
[0027] Also within the scope of this invention is a pharmaceutical
composition containing one or more of the compounds, modifications,
and/or salts and thereof described above for use in treating a
neoplastic disease, therapeutic uses thereof, and use of the
compounds for the manufacture of a medicament for treating the
disease/disorder.
[0028] This invention also relates to a method of treating a
neoplastic disease or an autoimmune disease by administering to a
subject in need thereof an effective amount of one or more of the
compounds, modifications, and/or salts, and compositions thereof
described above. The neoplastic disease or the autoimmune disease
can be ameliorated by inhibition of bcl-2. In one embodiment, the
neoplastic disease is leukemia, lymphoma, or multiple myeloma.
[0029] Also provided herein is the use of one or more of the
disclosed compounds, or a pharmaceutically acceptable salt thereof,
or the pharmaceutical composition thereof, for the preparation of a
medicament for the treatment of a neoplastic disease or an
autoimmune disease.
[0030] In another embodiment provided herein, the disclosed
compounds, or a pharmaceutically acceptable salt thereof, or the
pharmaceutical composition thereof are for use in treating a
neoplastic disease or an autoimmune disease.
[0031] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and from the claims. It should be understood that all
embodiments/features of the invention (compounds, pharmaceutical
compositions, methods of make/use, etc) described herein, including
any specific features described in the examples and original
claims, can combine with one another unless not applicable or
explicitly disclaimed.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Exemplary compounds described herein include, but are not
limited to, the following:
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050##
[0033] Compounds of the invention may contain one or more
asymmetric carbon atoms. Accordingly, the compounds may exist as
diastereomers, enantiomers or mixtures thereof. The syntheses of
the compounds may employ racemates, diastereomers or enantiomers as
starting materials or as intermediates. Diastereomeric compounds
may be separated by chromatographic or crystallization methods.
Similarly, enantiomeric mixtures may be separated using the same
techniques or others known in the art. Each of the asymmetric
carbon atoms may be in the R or S configuration and both of these
configurations are within the scope of the invention.
[0034] A modified compound of any one of such compounds including a
modification having an improved (e.g., enhanced, greater)
pharmaceutical solubility, stability, bioavailability and/or
therapeutic index as compared to the unmodified compound is also
contemplated. The examples of modifications include but not limited
to the prodrug derivatives, and the deuterium-enriched compounds.
For example: [0035] Prodrug derivatives: prodrugs, upon
administration to a subject, will converted in vivo into active
compounds of the present invention [Nature Reviews of Drug
Discovery, 2008, Volume 7, p255]. It is noted that in many
instances, the prodrugs themselves also fall within the scope of
the range of compounds according to the present invention. The
prodrugs of the compounds of the present invention can be prepared
by standard organic reaction, for example, by reacting with a
carbamylating agent (e.g., 1,1-acyloxyalkylcarbonochloridate,
para-nitrophenyl carbonate, or the like) or an acylating agent.
Further examples of methods and strategies of making prodrugs are
described in Bioorganic and Medicinal Chemistry Letters, 1994, Vol.
4, p. 1985. [0036] Deuterium-enriched compounds: deuterium (D or
.sup.2H) is H) a stable, non-radioactive isotope of hydrogen and
has an atomic weight of 2.0144. Hydrogen naturally occurs as a
mixture of the isotopes .sup.XH (hydrogen or protium), D (.sup.2H
or deuterium), and T (.sup.3H or tritium). The natural abundance of
deuterium is 0.015%. One of ordinary skill in the art recognizes
that in all chemical compounds with a H atom, the H atom actually
represents a mixture of H and D, with about 0.015% being D. Thus,
compounds with a level of deuterium that has been enriched to be
greater than its natural abundance of 0.015%, should be considered
unnatural and, as a result, novel over their non-enriched
counterparts.
[0037] It should be recognized that the compounds of the present
invention may be present and optionally administered in the form of
salts, and solvates. For example, it is within the scope of the
present invention to convert the compounds of the present invention
into and use them in the form of their pharmaceutically acceptable
salts derived from various organic and inorganic acids and bases in
accordance with procedures well known in the art.
[0038] When the compounds of the present invention possess a free
base form, the compounds can be prepared as a pharmaceutically
acceptable acid addition salt by reacting the free base form of the
compound with a pharmaceutically acceptable inorganic or organic
acid, e.g., hydrohalides such as hydrochloride, hydrobromide,
hydroiodide; other mineral acids such as sulfate, nitrate,
phosphate, etc.; and alkyl and monoarylsulfonates such as
ethanesulfonate, toluenesulfonate and benzenesulfonate; and other
organic acids and their corresponding salts such as acetate,
tartrate, maleate, succinate, citrate, benzoate, salicylate and
ascorbate. Further acid addition salts of the present invention
include, but are not limited to: adipate, alginate, arginate,
aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate,
camphorsulfonate, caprylate, chloride, chlorobenzoate,
cyclopentanepropionate, digluconate, dihydrogenphosphate,
dinitrobenzoate, dodecylsulfate, fumarate, galacterate (from mucic
acid), galacturonate, glucoheptaoate, gluconate, glutamate,
glycerophosphate, hemisuccinate, hemisulfate, heptanoate,
hexanoate, hippurate, 2-hydroxyethanesulfonate, iodide,
isethionate, iso-butyrate, lactate, lactobionate, malonate,
mandelate, metaphosphate, methanesulfonate, methylbenzoate,
monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, oxalate,
oleate, pamoate, pectinate, persulfate, phenylacetate,
3-phenylpropionate, phosphonate and phthalate. It should be
recognized that the free base forms will typically differ from
their respective salt forms somewhat in physical properties such as
solubility in polar solvents, but otherwise the salts are
equivalent to their respective free base forms for the purposes of
the present invention.
[0039] When the compounds of the present invention possess a free
acid form, a pharmaceutically acceptable base addition salt can be
prepared by reacting the free acid form of the compound with a
pharmaceutically acceptable inorganic or organic base. Examples of
such bases are alkali metal hydroxides including potassium, sodium
and lithium hydroxides; alkaline earth metal hydroxides such as
barium and calcium hydroxides; alkali metal alkoxides, e.g.,
potassium ethanolate and sodium propanolate; and various organic
bases such as ammonium hydroxide, piperidine, diethanolamine and
N-methylglutamine Also included are the aluminum salts of the
compounds of the present invention. Further base salts of the
present invention include, but are not limited to: copper, ferric,
ferrous, lithium, magnesium, manganic, manganous, potassium, sodium
and zinc salts. Organic base salts include, but are not limited to,
salts of primary, secondary and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion exchange resins, e.g., arginine, betaine, caffeine,
chloroprocaine, choline, N,N'-dibenzylethylenediamine (benzathine),
dicyclohexylamine, diethanolamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, iso-propylamine, lidocaine, lysine,
meglumine, N-methyl-D-glucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethanolamine, triethylamine, trimethylamine, tripropylamine and
tris-(hydroxymethyl)-methylamine (tromethamine) It should be
recognized that the free acid forms will typically differ from
their respective salt forms somewhat in physical properties such as
solubility in polar solvents, but otherwise the salts are
equivalent to their respective free acid forms for the purposes of
the present invention.
[0040] In one aspect, a pharmaceutically acceptable salt is a
hydrochloride salt, hydrobromide salt, methanesulfonate,
toluenesulfonate, acetate, fumarate, sulfate, bisulfate, succinate,
citrate, phosphate, maleate, nitrate, tartrate, benzoate,
biocarbonate, carbonate, sodium hydroxide salt, calcium hydroxide
salt, potassium hydroxide salt, tromethamine salt, or mixtures
thereof.
[0041] Compounds of the present invention that comprise tertiary
nitrogen-containing groups may be quaternized with such agents as
(C.sub.1-4) alkyl halides, e.g., methyl, ethyl, iso-propyl and
tert-butyl chlorides, bromides and iodides; di-(C.sub.1-.sub.4)
alkyl sulfates, e.g., dimethyl, diethyl and diamyl sulfates; alkyl
halides, e.g., decyl, dodecyl, lauryl, myristyl and stearyl
chlorides, bromides and iodides; and aryl (C.sub.1-.sub.4) alkyl
halides, e.g., benzyl chloride and phenethyl bromide. Such salts
permit the preparation of both water- and oil-soluble compounds of
the invention.
[0042] Amine oxides, also known as amine-N-oxide and N-oxide, of
anti-cancer agents with tertiary nitrogen atoms have been developed
as prodrugs [Mol Cancer Therapy. 2004 March; 3(3):233-44].
Compounds of the present invention that comprise tertiary nitrogen
atoms may be oxidized by such agents as hydrogen peroxide
(H.sub.2O.sub.2), Caro's acid or peracids like
meta-Chloroperoxybenzoic acid (mCPBA) to from amine oxide.
[0043] The invention encompasses pharmaceutical compositions
comprising the compound of the present invention and pharmaceutical
excipients, as well as other conventional pharmaceutically inactive
agents. Any inert excipient that is commonly used as a carrier or
diluent may be used in compositions of the present invention, such
as sugars, polyalcohols, soluble polymers, salts and lipids. Sugars
and polyalcohols which may be employed include, without limitation,
lactose, sucrose, mannitol, and sorbitol. Illustrative of the
soluble polymers which may be employed are polyoxyethylene,
poloxamers, polyvinylpyrrolidone, and dextran. Useful salts
include, without limitation, sodium chloride, magnesium chloride,
and calcium chloride. Lipids which may be employed include, without
limitation, fatty acids, glycerol fatty acid esters, glycolipids,
and phospholipids.
[0044] In addition, the pharmaceutical compositions may further
comprise binders (e.g., acacia, cornstarch, gelatin, carbomer,
ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, povidone), disintegrating agents (e.g.,
cornstarch, potato starch, alginic acid, silicon dioxide,
croscarmellose sodium, crospovidone, guar gum, sodium starch
glycolate, Primogel), buffers (e.g., tris-HCL, acetate, phosphate)
of various pH and ionic strength, additives such as albumin or
gelatin to prevent absorption to surfaces, detergents (e.g., Tween
20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,
surfactants (e.g., sodium lauryl sulfate), permeation enhancers,
solubilizing agents (e.g., glycerol, polyethylene glycerol,
cyclodextrins), a glidant (e.g., colloidal silicon dioxide),
anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated
hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose,
hydroxypropylmethyl cellulose), viscosity increasing agents (e.g.,
carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),
sweeteners (e.g., sucrose, aspartame, citric acid), flavoring
agents (e.g., peppermint, methyl salicylate, or orange flavoring),
preservatives (e.g., Thimerosal, benzyl alcohol, parabens),
lubricants (e.g., stearic acid, magnesium stearate, polyethylene
glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon
dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate),
emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl
sulfate, methyl cellulose, hydroxyethyl cellulose,
carboxymethylcellulose sodium), polymer coatings (e.g., poloxamers
or poloxamines), coating and film forming agents (e.g., ethyl
cellulose, acrylates, polymethacrylates) and/or adjuvants.
[0045] In one embodiment, the pharmaceutical compositions 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. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0046] Additionally, the invention encompasses pharmaceutical
compositions comprising any solid or liquid physical form of the
compound of the invention. For example, the compounds can be in a
crystalline form, in amorphous form, and have any particle size.
The particles may be micronized, or may be agglomerated,
particulate granules, powders, oils, oily suspensions or any other
form of solid or liquid physical form.
[0047] When compounds according to the present invention exhibit
insufficient solubility, methods for solubilizing the compounds may
be used. Such methods are known to those of skill in this art, and
include, but are not limited to, pH adjustment and salt formation,
using co-solvents, such as ethanol, propylene glycol, polyethylene
glycol (PEG) 300, PEG 400, DMA (10-30%), DMSO (10-20%), NMP
(10-20%), using surfactants, such as polysorbate 80, polysorbate 20
(1-10%), cremophor EL, Cremophor RH40, Cremophor RH60 (5-10%),
Pluronic F68/Poloxamer 188 (20-50%), Solutol HS15 (20-50%), Vitamin
E TPGS, and d-.alpha.-tocopheryl PEG 1000 succinate (20-50%), using
complexation such as HPPCD and SBEr3CD (10-40%), and using advanced
approaches such as micelle, addition of a polymer, nanoparticle
suspensions, and liposome formation.
[0048] A wide variety of administration methods may be used in
conjunction with the compounds of the present invention. Compounds
of the present invention may be administered or coadministered
orally, parenterally, intraperitoneally, intravenously,
intraarterially, transdermally, sublingually, intramuscularly,
rectally, transbuccally, intranasally, liposomally, via inhalation,
vaginally, intraoccularly, via local delivery (for example by
catheter or stent), subcutaneously, intraadiposally,
intraarticularly, or intrathecally. The compounds according to the
invention may also be administered or coadministered in slow
release dosage forms. Compounds may be in gaseous, liquid,
semi-liquid or solid form, formulated in a manner suitable for the
route of administration to be used. For oral administration,
suitable solid oral formulations include tablets, capsules, pills,
granules, pellets, sachets and effervescent, powders, and the like.
Suitable liquid oral formulations include solutions, suspensions,
dispersions, emulsions, oils and the like. For parenteral
administration, reconstitution of a lyophilized powder is typically
used.
[0049] As used herein, "acyl" means a carbonyl containing
substituent represented by the formula --C(O)--R in which R is H,
alkyl, a carbocycle, a heterocycle, carbocycle-substituted alkyl or
heterocycle-substituted alkyl wherein the alkyl, alkoxy, carbocycle
and heterocycle are as defined herein. Acyl groups include alkanoyl
(e.g. acetyl), aroyl (e.g. benzoyl), and heteroaroyl.
[0050] "Aliphatic" means a moiety characterized by a straight or
branched chain arrangement of constituent carbon atoms and may be
saturated or partially unsaturated with one or more double or
triple bonds.
[0051] The term "alkyl" refers to a straight or branched
hydrocarbon containing 1-20 carbon atoms (e.g., C.sub.1-C.sub.10).
Examples of alkyl include, but are not limited to, methyl,
methylene, ethyl, ethylene, n-propyl, i-propyl, n-butyl, i-butyl,
and t-butyl. Preferably, the alkyl group has one to ten carbon
atoms. More preferably, the alkyl group has one to four carbon
atoms.
[0052] The term "alkenyl" refers to a straight or branched
hydrocarbon containing 2-20 carbon atoms (e.g., C.sub.2-C.sub.10)
and one or more double bonds. Examples of alkenyl include, but are
not limited to, ethenyl, propenyl, and allyl. Preferably, the
alkylene group has two to ten carbon atoms. More preferably, the
alkylene group has two to four carbon atoms.
[0053] The term "alkynyl" refers to a straight or branched
hydrocarbon containing 2-20 carbon atoms (e.g., C.sub.2-C.sub.10)
and one or more triple bonds. Examples of alkynyl include, but are
not limited to, ethynyl, 1-propynyl, 1- and 2-butynyl, and
1-methyl-2-butynyl. Preferably, the alkynyl group has two to ten
carbon atoms. More preferably, the alkynyl group has two to four
carbon atoms.
[0054] The term "alkylamino" refers to an N(R)-alkyl in which R can
be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl.
[0055] "Alkoxy" means an oxygen moiety having a further alkyl
substituent.
[0056] "Alkoxycarbonyl" means an alkoxy group attached to a
carbonyl group.
[0057] "Oxoalkyl" means an alkyl, further substituted with a
carbonyl group. The carbonyl group may be an aldehyde, ketone,
ester, amide, acid or acid chloride.
[0058] The term "cycloalkyl" refers to a saturated hydrocarbon ring
system having 3 to 30 carbon atoms (e.g., C.sub.3-C.sub.12,
C.sub.3-C.sub.8, C.sub.3-C.sub.6). Examples of cycloalkyl include,
but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, and cyclooctyl. The term "cycloalkenyl"
refers to a non-aromatic hydrocarbon ring system having 3 to 30
carbons (e.g., C.sub.3-C.sub.12) and one or more double bonds.
Examples include cyclopentenyl, cyclohexenyl, and
cycloheptenyl.
[0059] The term "heterocycloalkyl" refers to a nonaromatic 5-8
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having one or more heteroatoms (such as O, N,
S, B, P, Si, or Se). Examples of heterocycloalkyl groups include,
but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl,
morpholinyl, and tetrahydrofuranyl.
[0060] The term "heterocycloalkenyl" refers to a nonaromatic 5-8
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having one or more heteroatoms (such as O, N,
S, P, B, Si, or Se) and one or more double bonds.
[0061] The term "aryl" refers to a 6-carbon monocyclic, 10-carbon
bicyclic, 14-carbon tricyclic aromatic ring system. Examples of
aryl groups include, but are not limited to, phenyl, naphthyl, and
anthracenyl. The term "heteroaryl" refers to an aromatic 5-8
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having one or more heteroatoms (such as O, N,
S, P, or Se). Examples of heteroaryl groups include pyridyl, furyl,
imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl,
indolyl, and thiazolyl.
[0062] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, heterocycloalkenyl, alkylamino, aryl, and heteroaryl
mentioned above include both substituted and unsubstituted
moieties. Possible substituents on alkylamino, cycloalkyl,
heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, and
heteroaryl include, but are not limited to, C.sub.1-C.sub.10 alkyl,
C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
C.sub.3-C.sub.20 cycloalkyl, C.sub.3-C.sub.20 cycloalkenyl,
C.sub.1-C.sub.20 heterocycloalkyl, C.sub.1-C.sub.20
heterocycloalkenyl, C.sub.1-C.sub.10 alkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, amino, C.sub.1-C.sub.10 alkylamino,
arylamino, hydroxy, halo, oxo (0.dbd.), thioxo (S.dbd.), thio,
silyl, C.sub.1-C.sub.10 alkylthio, arylthio, C.sub.1-C.sub.10
alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl,
amidino, mercapto, amido, thioureido, thiocyanato, sulfonamido,
guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy,
carbamido, carbamyl, carboxyl, and carboxylic ester. On the other
hand, possible substituents on alkyl, alkenyl, or alkynyl include
all of the above-recited substituents except C.sub.1-C.sub.10
alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, and heteroaryl can also be fused with
each other.
[0063] "Amino" means a nitrogen moiety having two further
substituents where each substituent has a hydrogen or carbon atom
alpha bonded to the nitrogen. Unless indicated otherwise, the
compounds of the invention containing amino moieties may include
protected derivatives thereof. Suitable protecting groups for amino
moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl,
and the like.
[0064] "Aromatic" means a moiety wherein the constituent atoms make
up an unsaturated ring system, all atoms in the ring system are sp2
hybridized and the total number of pi electrons is equal to 4n+2.
An aromatic ring may be such that the ring atoms are only carbon
atoms or may include carbon and non-carbon atoms (see
Heteroaryl).
[0065] "Carbamoyl" means the radical --OC(O)NR.sub.aR.sub.b where
R.sub.a and R.sub.b are each independently two further substituents
where a hydrogen or carbon atom is alpha to the nitrogen. It is
noted that carbamoyl moieties may include protected derivatives
thereof. Examples of suitable protecting groups for carbamoyl
moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl,
and the like. It is noted that both the unprotected and protected
derivatives fall within the scope of the invention.
[0066] "Carbonyl" means the radical --C(O)--. It is noted that the
carbonyl radical may be further substituted with a variety of
substituents to form different carbonyl groups including acids,
acid halides, amides, esters, and ketones.
[0067] "Carboxy" means the radical --C(O)O--. It is noted that
compounds of the invention containing carboxy moieties may include
protected derivatives thereof, i.e. , where the oxygen is
substituted with a protecting group. Suitable protecting groups for
carboxy moieties include benzyl, tert-butyl, and the like.
[0068] "Cyano" means the radical --CN.
[0069] "Formyl" means the radical CH.dbd.O.
[0070] "Formimino" means the radical HC.dbd.NH.
[0071] "Halo" means fluoro, chloro, bromo or iodo.
[0072] "Halo-substituted alkyl", as an isolated group or part of a
larger group, means "alkyl" substituted by one or more "halo"
atoms, as such terms are defined in this Application.
Halo-substituted alkyl includes haloalkyl, dihaloalkyl,
trihaloalkyl, perhaloalkyl and the like.
[0073] "Hydroxy" means the radical --OH.
[0074] "Imine derivative" means a derivative comprising the moiety
--C(.dbd.NR)--, wherein R comprises a hydrogen or carbon atom alpha
to the nitrogen.
[0075] "Isomers" mean any compound having identical molecular
formulae but differing in the nature or sequence of bonding of
their atoms or in the arrangement of their atoms in space. Isomers
that differ in the arrangement of their atoms in space are termed
"stereoisomers." Stereoisomers that are not mirror images of one
another are termed "diastereomers" and stereoisomers that are
nonsuperimposable mirror images are termed "enantiomers" or
sometimes "optical isomers." A carbon atom bonded to four
nonidentical substituents is termed a "chiral center." A compound
with one chiral center has two enantiomeric forms of opposite
chirality. A mixture of the two enantiomeric forms is termed a
"racemic mixture."
[0076] "Nitro" means the radical --NO.sub.2.
[0077] "Protected derivatives" means derivatives of compounds in
which a reactive site are blocked with protecting groups. Protected
derivatives are useful in the preparation of pharmaceuticals or in
themselves may be active as inhibitors. A comprehensive list of
suitable protecting groups can be found in T.W.Greene, Protecting
Groups in Organic Synthesis, 3rd edition, Wiley & Sons,
1999.
[0078] The term "substituted" means that an atom or group of atoms
has replaced hydrogen as the substituent attached to another group.
For aryl and heteroaryl groups, the term "substituted" refers to
any level of substitution, namely mono-, di-, tri-, tetra-, or
penta-substitution, where such substitution is permitted. The
substituents are independently selected, and substitution may be at
any chemically accessible position. The term "unsubstituted" means
that a given moiety may consist of only hydrogen substituents
through available valencies (unsubstituted).
[0079] If a functional group is described as being "optionally
substituted," the function group may be either (1) not substituted,
or (2) substituted. If a carbon of a functional group is described
as being optionally substituted with one or more of a list of
substituents, one or more of the hydrogen atoms on the carbon (to
the extent there are any) may separately and/or together be
replaced with an independently selected optional substituent.
[0080] "Sulfide" means --S--R wherein R is H, alkyl, carbocycle,
heterocycle, carbocycloalkyl or heterocycloalkyl. Particular
sulfide groups are mercapto, alkylsulfide, for example
methylsulfide (--S-Me); arylsulfide, e.g., phenylsulfide;
aralkylsulfide, e.g., benzylsulfide.
[0081] "Sulfinyl" means the radical --S(O)--. It is noted that the
sulfinyl radical may be further substituted with a variety of
substituents to form different sulfinyl groups including sulfinic
acids, sulfinamides, sulfinyl esters, and sulfoxides.
[0082] "Sulfonyl" means the radical --S(O)(O)--. It is noted that
the sulfonyl radical may be further substituted with a variety of
substituents to form different sulfonyl groups including sulfonic
acids, sulfonamides, sulfonate esters, and sulfones.
[0083] "Thiocarbonyl" means the radical --C(S)--. It is noted that
the thiocarbonyl radical may be further substituted with a variety
of substituents to form different thiocarbonyl groups including
thioacids, thioamides, thioesters, and thioketones.
[0084] Certain of the compounds described herein may exist in
various stereoisomeric or tautomeric forms. Stereoisomers are
compounds which differ only in their spatial arrangement. When a
disclosed compound is named or depicted by structure without
indicating stereochemistry, it is understood that the name or
structure encompasses all possible stereoisomers, geometric
isomers, including essentially pure stereo or geometric isomers, as
well as combination thereof.
[0085] It is to be understood that when a compound herein is
represented by a structural formula or designated by a chemical
name herein, all other tautomeric forms which may exist for the
compound are encompassed by the structural formula.
[0086] Certain of the disclosed compounds may exist in various
stereoisomeric forms. Stereoisomers are compounds that differ only
in their spatial arrangement. Enantiomers are pairs of
stereoisomers whose mirror images are not superimposable, most
commonly because they contain an asymmetrically substituted carbon
atom that acts as a chiral center. "Enantiomer" means one of a pair
of molecules that are mirror images of each other and are not
superimposable. Diastereomers are stereoisomers that contain two or
more asymmetrically substituted carbon atoms.
[0087] When the stereochemistry of a disclosed compound is named or
depicted by structure, the named or depicted stereoisomer is at
least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to
all of the other stereoisomers. Percent by weight pure relative to
all of the other stereoisomers is the ratio of the weight of one
stereoisomer over the weight of the other stereoisomers. When a
single enantiomer is named or depicted by structure, the depicted
or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by
weight optically pure (also referred to as "enantiomerically
pure"). Percent optical purity by weight is the ratio of the weight
of the enantiomer over the weight of the enantiomer plus the weight
of its optical isomer.
[0088] When the stereochemistry of a disclosed compound is named or
depicted by structure, and the named or depicted structure
encompasses more than one stereoisomer (e.g., as in a
diastereomeric pair), it is to be understood that one of the
encompassed stereoisomers or any mixture of the encompassed
stereoisomers are included. It is to be further understood that the
stereoisomeric purity of the named or depicted stereoisomers at
least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to
all of the other stereoisomers. The stereoisomeric purity in this
case is determined by dividing the total weight in the mixture of
the stereoisomers encompassed by the name or structure by the total
weight in the mixture of all of the stereoisomers.
[0089] When a disclosed compound is named or depicted by structure
without indicating the stereochemistry, and the compound has one
chiral center, it is to be understood that the name or structure
encompasses one enantiomer of compound free from the corresponding
optical isomer, a racemic mixture of the compound and mixtures
enriched in one enantiomer relative to its corresponding optical
isomer.
[0090] When a disclosed compound is named or depicted by structure
without indicating the stereochemistry and e.g., the compound has
at least two chiral centers, it is to be understood that the name
or structure encompasses one stereoisomer free of other
stereoisomers, mixtures of stereoisomers, and mixtures of
stereoisomers in which one or more stereoisomers is enriched
relative to the other stereoisomer(s). For example, the name or
structure may encompass one stereoisomer free of other
diastereomers, mixtures of stereoisomers, and mixtures of
stereoisomers in which one or more diastereomers is enriched
relative to the other diastereomer(s).
[0091] Enantiomeric and diastereomeric mixtures can be resolved
into their component enantiomers or stereoisomers by well-known
methods, such as chiral-phase gas chromatography, chiral-phase high
performance liquid chromatography, crystallizing the compound as a
chiral salt complex, or crystallizing the compound in a chiral
solvent. Enantiomers and diastereomers can also be obtained from
diastereomerically- or enantiomerically-pure intermediates,
reagents, and catalysts by well-known asymmetric synthetic
methods.
[0092] "Animal" includes humans, non-human mammals (e.g., non-human
primates, rodents, mice, rats, hamsters, dogs, cats, rabbits,
cattle, horses, sheep, goats, swine, deer, and the like) and
non-mammals (e.g., birds, and the like).
[0093] "Bioavailability" as used herein is the fraction or
percentage of an administered dose of a drug or pharmaceutical
composition that reaches the systemic circulation intact. In
general, when a medication is administered intravenously, its
bioavailability is 100%. However, when a medication is administered
via other routes (e.g., orally), its bioavailability decreases
(e.g., due to incomplete absorption and first-pass metabolism).
Methods to improve the bioavailability include prodrug approach,
salt synthesis, particle size reduction, complexation, change in
physical form, solid dispersions, spray drying, and hot-melt
extrusion.
[0094] "Disease" specifically includes any unhealthy condition of
an animal or part thereof and includes an unhealthy condition that
may be caused by, or incident to, medical or veterinary therapy
applied to that animal, i.e., the "side effects" of such
therapy.
[0095] "Pharmaceutically acceptable" means that which is useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic and neither biologically nor otherwise undesirable and
includes that which is acceptable for veterinary use as well as
human pharmaceutical use.
[0096] "Pharmaceutically acceptable salts" means organic or
inorganic salts of compounds of the present invention which are
pharmaceutically acceptable, as defined above, and which possess
the desired pharmacological activity. Such salts include acid
addition salts formed with inorganic acids, or with organic acids.
Pharmaceutically acceptable salts also include base addition salts
which may be formed when acidic protons present are capable of
reacting with inorganic or organic bases. Exemplary salts include,
but are not limited, to sulfate, citrate, acetate, oxalate,
chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid
phosphate, isonicotinate, lactate, salicylate, acid citrate,
tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,
succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,
saccharate, formate, benzoate, glutamate, methanesulfonate
"mesylate," ethanesulfonate, benzenesulfonate, p-toluenesulfonate,
pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts,
alkali metal (e.g., sodium and potassium) salts, alkaline earth
metal (e.g., magnesium) salts, and ammonium salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counter ion. The counter ion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counter ion.
[0097] "Pharmaceutically acceptable carrier" means a non-toxic
solvent, dispersant, excipient, adjuvant, or other material which
is mixed with the compounds of the present invention in order to
form a pharmaceutical composition, i.e., a dose form capable of
administration to the patient. Examples of pharmaceutically
acceptable carrier includes suitable polyethylene glycol (e.g.,
PEG400), surfactant (e.g., Cremophor), or cyclopolysaccharide
(e.g., hydroxypropyl-.beta.-cyclodextrin or sulfobutyl ether
.beta.-cyclodextrins), polymer, liposome, micelle, nanosphere,
etc.
[0098] "Pharmacophore," as defined by The International Union of
Pure and Applied Chemistry, is an ensemble of steric and electronic
features that is necessary to ensure the optimal supramolecular
interactions with a specific biological target and to trigger (or
block) its biological response. For example, Camptothecin is the
pharmacophore of the well known drug topotecan and irinotecan.
Mechlorethamine is the pharmacophore of a list of widely used
nitrogen mustard drugs like Melphalan, Cyclophosphamide,
Bendamustine, and so on.
[0099] "Prodrug" means a compound that is convertible in vivo
metabolically into an active pharmaceutical according to the
present invention. For example, an inhibitor comprising a hydroxyl
group may be administered as an ester that is converted by
hydrolysis in vivo to the hydroxyl compound.
[0100] "Stability" in general refers to the length of time a drug
retains its properties without loss of potency. Sometimes this is
referred to as shelf life. Factors affecting drug stability
include, among other things, the chemical structure of the drug,
impurity in the formulation, pH, moisture content, as well as
environmental factors such as temperature, oxidization, light, and
relative humidity. Stability can be improved by providing suitable
chemical and/or crystal modifications (e.g., surface modifications
that can change hydration kinetics; different crystals that can
have different properties), excipients (e.g., anything other than
the active substance in the dosage form), packaging conditions,
storage conditions, etc.
[0101] "Therapeutically effective amount" of a composition
described herein is meant an amount of the composition which
confers a therapeutic effect on the treated subject, at a
reasonable benefit/risk ratio applicable to any medical treatment.
The therapeutic effect may be objective (i.e., measurable by some
test or marker) or subjective (i.e., subject gives an indication of
or feels an effect). An effective amount of the composition
described above may range from about 0.1 mg/kg to about 500 mg/kg,
preferably from about 0.2 to about 50 mg/kg. Effective doses will
also vary depending on route of administration, as well as the
possibility of co-usage with other agents. It will be understood,
however, that the total daily usage of the compositions of the
present invention will be decided by the attending physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular patient will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; the activity of the specific compound
employed; the specific composition employed; the age, body weight,
general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of
the specific compound employed; the duration of the treatment;
drugs used in combination or contemporaneously with the specific
compound employed; and like factors well known in the medical
arts.
[0102] As used herein, the term "treating" refers to administering
a compound to a subject that has a neoplastic or immune disorder,
or has a symptom of or a predisposition toward it, with the purpose
to cure, heal, alleviate, relieve, alter, remedy, ameliorate,
improve, or affect the disorder, the symptoms of or the
predisposition toward the disorder. The term "an effective amount"
refers to the amount of the active agent that is required to confer
the intended therapeutic effect in the subject. Effective amounts
may vary, as recognized by those skilled in the art, depending on
route of administration, excipient usage, and the possibility of
co-usage with other agents.
[0103] A "subject" refers to a human and a non-human animal.
Examples of a non-human animal include all vertebrates, e.g.,
mammals, such as non-human primates (particularly higher primates),
dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals,
such as birds, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model.
[0104] "Combination therapy" includes the administration of the
subject compounds of the present invention in further combination
with other biologically active ingredients (such as, but not
limited to, a second and different antineoplastic agent) and
non-drug therapies (such as, but not limited to, surgery or
radiation treatment). For instance, the compounds of the invention
can be used in combination with other pharmaceutically active
compounds, or non-drug therapies, preferably compounds that are
able to enhance the effect of the compounds of the invention. The
compounds of the invention can be administered simultaneously (as a
single preparation or separate preparation) or sequentially to the
other therapies. In general, a combination therapy envisions
administration of two or more drugs/treatments during a single
cycle or course of therapy.
[0105] In one embodiment, the compounds of the invention are
administered in combination with one or more of traditional
chemotherapeutic agents. The traditional chemotherapeutic agents
encompass a wide range of therapeutic treatments in the field of
oncology. These agents are administered at various stages of the
disease for the purposes of shrinking tumors, destroying remaining
cancer cells left over after surgery, inducing remission,
maintaining remission and/or alleviating symptoms relating to the
cancer or its treatment. Examples of such agents include, but are
not limited to, alkylating agents such as Nitrogen Mustards (e.g.,
Bendamustine, Cyclophosphamide, Melphalan, Chlorambucil,
Isofosfamide), Nitrosureas (e.g., Carmustine, Lomustine and
Streptozocin), ethylenimines (e.g., thiotepa, hexamethylmelanine),
Alkylsulfonates (e.g., Busulfan), Hydrazines and Triazines (e.g.,
Altretamine, Procarbazine, Dacarbazine and Temozolomide), and
platinum based agents (e.g., Carboplatin, Cisplatin, and
Oxaliplatin); plant alkaloids such as Podophyllotoxins (e.g.,
Etoposide and Tenisopide), Taxanes (e.g., Paclitaxel and
Docetaxel), Vinca alkaloids (e.g., Vincristine, Vinblastine and
Vinorelbine); anti-tumor antibiotics such as Chromomycins (e.g.,
Dactinomycin and Plicamycin), Anthracyclines (e.g., Doxorubicin,
Daunorubicin, Epirubicin, Mitoxantrone, and Idarubicin), and
miscellaneous antibiotics such as Mitomycin and Bleomycin;
anti-metabolites such as folic acid antagonists (e.g.,
Methotrexate), pyrimidine antagonists (e.g., 5-Fluorouracil,
Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine
antagonists (e.g., 6-Mercaptopurine and 6-Thioguanine) and
adenosine deaminase inhibitors (e.g., Cladribine, Fludarabine,
Nelarabine and Pentostatin); topoisomerase inhibitors such as
topoisomerase I inhibitors(Topotecan, Irinotecan), topoisomerase II
inhibitors (e.g., Amsacrine, Etoposide, Etoposide phosphate,
Teniposide), and miscellaneous anti-neoplastics such as
ribonucleotide reductase inhibitors (Hydroxyurea), adrenocortical
steroid inhibitor (Mitotane), anti-microtubule agents
(Estramustine), and retinoids (Bexarotene, Isotretinoin, Tretinoin
(ATRA).
[0106] In one aspect of the invention, the compounds may be
administered in combination with one or more targeted anti-cancer
agents that modulate protein kinases involved in various disease
states. Examples of such kinases may include, but are not limited
ABL1, ABL2/ARG, ACK1, AKT1, AKT2, AKT3, ALK, ALK1/ACVRL1,
ALK2/ACVR1, ALK4/ACVR1B, ALK5/TGFBR1, ALK6/BMPR1B, AMPK(A1/B1/G1),
AMPK(A1/B1/G2), AMPK(A1/B1/G3), AMPK(A1/B2/G1), AMPK(A2/B1/G1),
AMPK(A2/B2/G1), AMPK(A2/B2/G2), ARAF, ARK5/NUAK1, ASK1/MAP3K5, ATM,
Aurora A, Aurora B , Aurora C , AXL, BLK, BMPR2, BMX/ETK, BRAF,
BRK, BRSK1, BRSK2, BTK, CAMK1a , CAMK1b, CAMK1d, CAMK1g , CAMKIIa ,
CAMKIIb , CAMKIId , CAMKIIg , CAMK4, CAMKK1, CAMKK2, CDC7-DBF4,
CDK1-cyclin A, CDK1-cyclin B, CDK1-cyclin E, CDK2-cyclin A,
CDK2-cyclin A1, CDK2-cyclin E, CDK3-cyclin E, CDK4-cyclin D1,
CDK4-cyclin D3, CDK5-p25, CDK5-p35, CDK6-cyclin D1, CDK6-cyclin D3,
CDK7-cyclin H, CDK9-cyclin K, CDK9-cyclin Tl, CHK1, CHK2, CK1a1,
CK1d , CK1epsilon , CK1g1 , CK1g2, CK1g3 , CK2a , CK2a2, c-KIT,
CLK1 , CLK2, CLK3, CLK4, c-MER, c-MET, COT1/MAP3K8, CSK, c-SRC,
CSF1R, CTK/MATK, DAPK1, DAPK2, DCAMKL1, DCAMKL2, DDR1, DDR2,
DLK/MAP3K12, DMPK, DMPK2/CDC42BPG, DNA-PK, DRAK1/STK17A,
DYRK1/DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, EEF2K, EGFR, EIF2AK1,
EIF2AK2, EIF2AK3, EIF2AK4/GCN2, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5,
EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, ERBB2/HER2,
ERBB4/HER4, ERK1/MAPK3, ERK2/MAPK1, ERK5/MAPK7, FAK/PTK2, FER,
FES/FPS, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1/VEGFR1, FLT3,
FLT4/VEGFR3, FMS, FRK/PTK5, FYN, GCK/MAP4K2, GRK1, GRK2, GRK3,
GRK4, GRK5, GRK6, GRK7, GSK3a, GSK3b, Haspin, HCK, HGK/MAP4K4,
HIPK1, HIPK2, HIPK3, HIPK4, HPK1/MAP4K1, IGF1R, IKKa/CHUK ,
IKKb/IKBKB, IKKe/IKBKE, IR, IRAK1, IRAK4, IRR/INSRR, ITK, JAK1,
JAK2, JAK3, JNK1 , JNK2 , JNK3, KDR/VEG1-R2, KHS/MAP4K5, LATS1,
LATS2, LCK, LCK2/ICK, LKB1 , LIMK1, LOK/STK10, LRRK2, LYN, LYNB,
MAPKAPK2, MAPKAPK3, MAPKAPK5/PRAK, MARK1, MARK2/PAR-1Ba, MARK3,
MARK4, MEK1, MEK2, MEKK1, MEKK2, MEKK3, MELK, MINK/MINK1, MKK4,
MKK6, MLCK/MYLK, MLCK2/MYLK2, MLK1/MAP3K9, MLK2/MAP3K10,
MLK3/MAP3K11, MNK1, MNK2, MRCKa/, CDC42BPA, MRCKb/, CDC42BPB,
MSK1/RPS6KA5, MSK2/RPS6KA4, MSSK1/STK23, MST1/STK4, MST2/STK3,
MST3/STK24, MST4, mTOR/FRAP1, MUSK, MYLK3, MYO3b, NEK1, NEK2, NEK3,
NEK4, NEK6, NEK7, NEK9, NEK11, NIK/MAP3K14, NLK, OSR1/OXSR1,
P38a/MAPK14, P38b/MAPK11, P38d/MAPK13 , P38g/MAPK12 ,
P70S6K/RPS6KB1, p70S6Kb/, RPS6KB2, PAK1, PAK2, PAK3, PAK4, PAK5,
PAK6, PASK, PBK/TOPK, PDGFRa, PDGFRb, PDK1/PDPK1, PDK1/PDHK1,
PDK2/PDHK2 , PDK3/PDHK3, PDK4/PDHK4, PHKg1 , PHKg2 , PI3Ka,
(p110a/p85a), PI3Kb, (p110b/p85a), PI3Kd, (p110d/p85a),
PI3Kg(p120g), PIM1, PIM2, PIM3, PKA, PKAcb, PKAcg , PKCa , PKCb1 ,
PKCb2 , PKCd , PKCepsilon, PKCeta, PKCg , PKCiota, PKCmu/PRKD1,
PKCnu/PRKD3, PKCtheta, PKCzeta, PKD2/PRKD2, PKG1a , PKG1b ,
PKG2/PRKG2, PKN1/PRK1, PKN2/PRK2, PKN3/PRK3, PLK1, PLK2, PLK3,
PLK4/SAK, PRKX, PYK2, RAF1, RET, RIPK2, RIPK3, RIPK5, ROCK1, ROCK2,
RON/MST1R, ROS/ROS1, RSK1, RSK2, RSK3, RSK4, SGK1, SGK2, SGK3/SGKL,
SIK1, SIK2, SLK/STK2, SNARK/NUAK2, SRMS, SSTK/TSSK6, STK16,
STK22D/TSSK1, STK25/YSK1, STK32b/YANK2, STK32c/YANK3, STK33,
STK38/NDR1, STK38L/NDR2, STK39/STLK3, SRPK1, SRPK2, SYK, TAK1,
TAOK1, TAOK2/TAO1, TAOK3/JIK, TBK1, TEC, TESK1, TGFBR2, TIE2/TEK,
TLK1, TLK2, TNIK, TNK1, TRKA, TRKB, TRKC, TRPM7/CHAK1, TSSK2,
TSSK3/STK22C, TTBK1, TTBK2, TTK, TXK, TYK1/LTK, TYK2, TYRO3/SKY,
ULK1, ULK2, ULK3, VRK1, VRK2, WEE1, WNK1, WNK2, WNK3, YES/YES1,
ZAK/MLTK, ZAP70, ZIPK/DAPK3, KINASE, MUTANTS, ABL1(E255K),
ABL1(F317I), ABL1(G250E), ABL1(H396P), ABL1(M351T), ABL1(Q252H),
ABL1(T315I), ABL1(Y253F), ALK (C1156Y), ALK(L1196M), ALK (F1174L),
ALK (R1275Q), BRAF(V599E), BTK(E41K), CHK2(I157T), c-Kit(A829P),
c-KIT(D816H), c-KIT(D816V), c-Kit(D820E), c-Kit(N822K), C-Kit
(T670I), c-Kit(V559D), c-Kit(V559D/V654A), c-Kit(V559D/T670I),
C-Kit (V560G), c-KIT(V654A), C-MET(D1228H), C-MET(D1228N),
C-MET(F1200I), c-MET(M1250T), C-MET(Y1230A), C-MET(Y1230C),
C-MET(Y1230D), C-MET(Y1230H), c-Src(T341M), EGFR(G719C),
EGFR(G719S), EGFR(L858R), EGFR(L861Q), EGFR(T790M), EGFR,
(L858R,T790M) , EGFR(d746-750/T790M), EGFR(d746-750),
EGFR(d747-749/A750P), EGFR(d747-752/P753S), EGFR(d752-759),
FGFR1(V561M), FGFR2(N549H), FGFR3(G697C), FGFR3(K650E),
FGFR3(K650M), FGFR4(N535K), FGFR4(V550E), FGFR4(V550L),
FLT3(D835Y), FLT3(ITD), JAK2 (V617F), LRRK2 (G2019S), LRRK2
(I2020T), LRRK2 (R1441C), p38a(T106M), PDGFRa(D842V),
PDGFRa(T674I), PDGFRa(V561D), RET(E762Q), RET(G691S), RET(M918T),
RET(R749T), RET(R813Q), RET(V804L), RET(V804M), RET(Y791F),
TIF2(R849W), TIF2(Y897S), and TIF2(Y1108F).
[0107] In another aspect of the invention, the subject compounds
may be administered in combination with one or more targeted
anti-cancer agents that modulate non-kinase biological targets,
pathway, or processes. Such targets pathways, or processes include
but not limited to heat shock proteins (e.g. HSP90), poly-ADP
(adenosine diphosphate)-ribose polymerase (PARP), hypoxia-inducible
factors(HIF), proteasome, Wnt/Hedgehog/Notch signaling proteins,
TNF-alpha, matrix metalloproteinase, farnesyl transferase,
apoptosis pathway (e.g Bcl-xL, Bcl-2, Bcl-w), histone deacetylases
(HDAC), histone acetyltransferases (HAT), and methyltransferase
(e.g histone lysine methyltransferases, histone arginine
methyltransferase, DNA methyltransferase, etc), and other
immunotherapies(e.g anti-PD1, anti-PDL1, anti-CTLA4, A2A antagonist
etc).
[0108] In another aspect of the invention, the compounds of the
invention are administered in combination with one or more of other
anti-cancer agents that include, but are not limited to, gene
therapy, RNAi cancer therapy, chemoprotective agents (e.g.,
amfostine, mesna, and dexrazoxane), antibody conjugate(e.g
brentuximab vedotin, ibritumomab tioxetan), cancer immunotherapy
such as Interleukin-2, cancer vaccines(e.g., sipuleucel-T) or
monoclonal antibodies (e.g., Bevacizumab, Alemtuzumab, Rituximab,
Trastuzumab, etc).
[0109] In another aspect of the invention, the subject compounds
are administered in combination with radiation therapy or
surgeries. Radiation is commonly delivered internally (implantation
of radioactive material near cancer site) or externally from a
machine that employs photon (x-ray or gamma-ray) or particle
radiation. Where the combination therapy further comprises
radiation treatment, the radiation treatment may be conducted at
any suitable time so long as a beneficial effect from the co-action
of the combination of the therapeutic agents and radiation
treatment is achieved. For example, in appropriate cases, the
beneficial effect is still achieved when the radiation treatment is
temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0110] In certain embodiments, the compounds of the invention are
administered in combination with one or more of radiation therapy,
surgery, or anti-cancer agents that include, but are not limited
to, DNA damaging agents, anti-metabolites, topoisomerase
inhibitors, anti-microtubule agents, kinase inhibitors, epigenetic
agents, HSP90 inhibitors, PARP inhibitors, and antibodies targeting
VEGF, HER2, EGFR, CD50, CD20, CD30, CD33, etc.
[0111] In certain embodiments, the compounds of the invention are
administered in combination with one or more of abarelix,
abiraterone acetate, aldesleukin, alemtuzumab, altretamine,
anastrozole, asparaginase, bendamustine, bevacizumab, bexarotene,
bicalutamide, bleomycin, bortezombi, brentuximab vedotin, busulfan,
capecitabine, carboplatin, carmustine, cetuximab, chlorambucil,
cisplatin, cladribine, clofarabine, clomifene, crizotinib,
cyclophosphamide, dasatinib, daunorubicin liposomal, decitabine,
degarelix, denileukin diftitox, denileukin diftitox, denosumab,
docetaxel, doxorubicin, doxorubicin liposomal, epirubicin, eribulin
mesylate, erlotinib, estramustine, etoposide phosphate, everolimus,
exemestane, fludarabine, fluorouracil, fotemustine, fulvestrant,
gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate,
histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin,
ifosfamide, imatinib mesylate, interferon alfa 2a, ipilimumab,
ixabepilone, lapatinib ditosylate, lenalidomide, letrozole,
leucovorin, leuprolide acetate, levamisole, lomustine,
mechlorethamine, melphalan, methotrexate, mitomycin C,
mitoxantrone, nelarabine, nilotinib, oxaliplatin, paclitaxel,
paclitaxel protein-bound particle, pamidronate, panitumumab,
pegaspargase, peginterferon alfa-2b, pemetrexed disodium,
pentostatin, raloxifene, rituximab, sorafenib, streptozocin,
sunitinib maleate, tamoxifen, temsirolimus, teniposide,
thalidomide, toremifene, tositumomab, trastuzumab, tretinoin,
uramustine, vandetanib, vemurafenib, vinorelbine, zoledronate,
radiation therapy, or surgery.
[0112] The invention further provides methods for the prevention or
treatment of a neoplastic disease or autoimmune disease. In one
embodiment, the invention relates to a method of treating a
neoplastic disease or autoimmune disease, in a subject in need of
treatment comprising administering to said subject a
therapeutically effective amount of a compound of the invention. In
one embodiment, the invention further provides for the use of a
compound of the invention in the manufacture of a medicament for
halting or decreasing a neoplastic disease or autoimmune
disease.
[0113] In certain embodiments, the neoplastic disease is a lung
cancer, head and neck cancer, central nervous system cancer,
prostate cancer, testicular cancer, colorectal cancer, pancreatic
cancer, liver cancer, stomach cancer, biliary tract cancer,
esophageal cancer, gastrointestinal stromal tumor, breast cancer,
cervical cancer, ovarian cancer, uterine cancer, leukemia,
lymphomas, multiple myeloma, melanoma, basal cell carcinoma,
squamous cell carcinoma, bladder cancer, renal cancer, sarcoma,
mesothelioma, thymoma, myelodysplastic syndrome, or
myeloproliferative disease.
[0114] The autoimmune diseases that can be affected using compounds
and compositions according to the invention include, but are not
limited to allergy, Alzheimer's disease, acute disseminated
encephalomyelitis, Addison's disease, ankylosing spondylitis,
antiphospholipid antibody syndrome, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune hemolytic and
thrombocytopenic states, autoimmune hepatitis, autoimmune inner ear
disease, bullous pemphigoid, coeliac disease, chagas disease,
chronic obstructive pulmonary disease, chronic Idiopathic
thrombocytopenic purpura (ITP), churg-strauss syndrome, Crohn's
disease, dermatomyositis, diabetes mellitus type 1, endometriosis,
Goodpasture's syndrome (and associated glomerulonephritis and
pulmonary hemorrhage), graves' disease, guillain-bane syndrome,
hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, irritable bowel
syndrome, lupus erythematosus, morphea, multiple sclerosis,
myasthenia gravis, narcolepsy, neuromyotonia, Parkinson's disease,
pemphigus vulgaris, pernicious anaemia, polymyositis, primary
biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid
arthritis, schizophrenia, septic shock, scleroderma, Sjogren's
disease, systemic lupus erythematosus (and associated
glomerulonephritis), temporal arteritis, tissue graft rejection and
hyperacute rejection of transplanted organs, vasculitis
(ANCA-associated and other vasculitides), vitiligo, and wegener's
granulomatosis.
[0115] It should be understood that the invention is not limited to
the particular embodiments shown and described herein, but that
various changes and modifications may be made without departing
from the spirit and scope of the invention as defined by the
claims.
[0116] The compounds according to the present invention may be
synthesized according to a variety of schemes. Necessary starting
materials may be obtained by standard procedures of organic
chemistry. The compounds and processes of the present invention
will be better understood in connection with the following
representative synthetic schemes and examples, which are intended
as an illustration only and not limiting of the scope of the
invention. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art and such
changes and modifications including, without limitation, those
relating to the chemical structures, substituents, derivatives,
and/or methods of the invention may be made without departing from
the spirit of the invention and the scope of the appended
claims.
[0117] A typical approach to synthesize of the intermediate
##STR00051##
in which W is N is described in Scheme 1. The intermedediate with N
is CH can be made similarly.
##STR00052##
[0118] In Scheme 1, the starting material 1-1 is protected with SEM
group to give intermediate 1-2, which is followed by Buckwald
coupling to yield intermediate 1-3. Hydrolysis of imine 1-3 gives
aniline intermediate 1-4. Condensation of 1-4 with acetic acid
anhydride results in SEM-protected tricyclic intermediate 1-5.
[0119] A typical approach to synthesize of the intermediate
##STR00053##
is described in Scheme 2:
##STR00054##
[0120] In Scheme 2, the appropriate ketone starting material 2-1
can react with tribromophosphine to form the aldehyde intermediate
2-2, which can couple with Boc-protected piperazine to form the
intermediate 2-3. After that, 2-3 will couple with appropriate
phenylboronic acid via a Suzuki reaction to form intermediate 2-4,
followed by a de-boc process to yield key intermediate 2-5.
[0121] A typical approach to synthesize of the intermediate
##STR00055##
is described in Scheme 3:
##STR00056##
[0122] In Scheme 3, the staring intermediate 1-5 reacts with ethyl
4-Br-2-F-benzoic ester under basic conditions to give 3-2. Buckwald
coupling or nucleophilic substitution reactions between 3-2 and 5-5
produces intermediate 3-3. Hydrolysis of 3-3 gives intermediate
3-4.
[0123] A typical approach to synthesize of the intermediate
##STR00057##
in which R.sub.5 is NO.sub.2 is described in Scheme 4:
##STR00058##
[0124] In Scheme 4, the staring intermediate 4-1 reacts with
appropriate alcohol or amine will yield 4-2.
[0125] A typical approach to synthesize of the Formula (I)
compounds with Z.sub.1=NH or 0 is described in Scheme A:
##STR00059##
[0126] The intermediate 3-4 can undergo an amide coupling with
ammonia gives intermediate A-2. Sequential substitution reaction of
chlorobenzene sulfonyl halides (X is Cl or F) with appropriate
amine NH.sub.2LR.sub.4 or alcohol HO-L-R.sub.4 and coupling
reaction with intermediate A-2 followed by deprotection of SEM
group leads to Formula (I).
[0127] Similarly, a typical approach to synthesize of the Forumula
(I) compounds is described in Scheme B:
##STR00060##
[0128] In Scheme B, The intermediate 3-4 can undergo an amide
coupling with ammonia gives intermediate A-2. After that, the
substitution reaction of appropriate sulfonyl chloride followed by
deprotection of SEM group leads to final product Formula (I).
[0129] Similarly, a typical approach to synthesize of the Forumula
(I) compounds is described in Scheme C:
##STR00061##
[0130] In Scheme C, the amide coupling of 3-4 and 4-2 leads to
final product Formula (I).
[0131] The compounds and processes of the present invention will be
better understood in connection with the following examples, which
are intended as an illustration only and not limiting of the scope
of the invention. Various changes and modifications to the
disclosed embodiments will be apparent to those skilled in the art
and such changes and modifications including, without limitation,
those relating to the chemical structures, substituents,
derivatives, formulations and/or methods of the invention may be
made without departing from the spirit of the invention and the
scope of the appended claims.
[0132] Where NMR data are presented, .sup.1H spectra were obtained
on XL400 (400 MHz) and are reported as ppm down field from
Me.sub.4Si with number of protons, multiplicities, and coupling
constants in Hertz indicated parenthetically. Where HPLC data are
presented, analyses were performed using an Agilent 1100 system.
Where LC/MS data are presented, analyses were performed using an
Applied Biosystems API-100 mass spectrometer and Shimadzu SCL-10A
LC column:
EXAMPLE 1
Preparation of Key Intermediate IM-14-1
##STR00062## ##STR00063##
[0134] Synthesis of 2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde
2: A solution of anhydrous chloroform (57 ml) and anhydrous
N,N-dimethylformamide (9 mL) was cooled to .about.3.degree. C.
(internal temperature) under nitrogen before phosphorus tribromide
(10 mL, 0.1 mol) was introduced dropwise at a rate so that the
reaction was maintained at .about.3.degree. C. After the addition
was complete the reaction was allowed to warm slowly to
.about.10.degree. C. and then the temperature was raised to
70.degree. C. where it was maintained for 30 min. The reaction was
cooled to rt and 3,3-dimethylcyclohexanone 1 (5 g, 0.04 mol) was
added slowly over 20 min. After the addition was complete the
reaction was warmed to 70.degree. C. and it was stirred for 1.5 h.
The mixture was then cooled to 0.degree. C. and a solution of 4M
sodium acetate (53 ml) was added slowly. The pH of the resulting
solution was adjusted to .about.7 using a solution of 5M NaOH and
the mixture was then extracted with heptanes (100 mL.times.3). The
combined organic fractions were dried (Na.sub.2SO.sub.4), filtered
and concentrated under reduced pressure to give
2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde 2 (4 g, 49%)as a
yellow oil.
[0135] Synthesis of
2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde 3: To a
degassed solution of 2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde
2 (5 g, 0.023 mol) and 4-chlorophenyl boronic acid (3.6 g, 0.023
mol) in 1,4-dioxane (50 mL) at rt was added a solution of 2M
Na.sub.2CO.sub.3 (20.4 ml). Nitrogen was bubbled through the
mixture for 2 min and then PdCl.sub.2(dppf) (0.5 g) was added. The
reaction flask was heated to 120.degree. C. where it was maintained
for 3 h. After this time the suspension was cooled to rt and
filtered through Celite. The collected solids were washed with
additional dichloromethane and the combined filtrate and washings
were concentrated under reduced pressure. Purification by column
chromatography on silica with PE: EA=20:1 gave
2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde 3 (3 g,
53%) as a white solid. MS: 249[M+H].sup.+
[0136] Synthesis of
(2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4: A
solution of
2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde 3 (20 g,
80.6 mmol) in MeOH (100 mL) was cooled to 0.degree. C., NaBH.sub.4
(3.1g, 80.6 mmol) was added portionwise to the reaction at a rate
so that the reaction was maintained at 0.about.5.degree. C. After
added, the mixture was stirred for 1 h at 0.degree. C. Water was
added slowly to the mixture and extracted with EA (200 mL.times.3),
the organic layer was washed with brine and dried Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure to give
(2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4 (15 g,
75%) as a white solid. MS: 233[M+H-H.sub.2O].sup.+
[0137] Synthesis of
1-(2-(bromomethyl)-5,5-dimethylcyclohex-1-enyl)-4-chlorobenzene 5:
A solution of
(2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4 (15 g,
0.060 mol) and in Et.sub.2O (300 ml) was cooled to 0.degree. C.
before phosphorus tribromide (7.5 mL) was added dropwise to the
mixture, after added, the mixture was stirred for 1 h at 0.degree.
C. for 90 minutes. The reaction mixture was added H.sub.2O before
being extracted with EA. The organic layer was washed with a
saturated NaHCO.sub.3 solution and brine and dried
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure
to give
1-(2-(bromomethyl)-5,5-dimethylcyclohex-1-enyl)-4-chlorobenzene 5
(18 g, 96%) as a colorless oil.
[0138] Synthesis of tert-butyl
4-4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1--
carboxylate--To a solution of
1-bromo-2-(bromomethyl)-5,5-dimethylcyclohex-1-ene 5 (21 g, 0.067
mol) and tert-butyl piperazine-1-carboxylate (12.4 g, 0.067 mol) in
dichloromethane (200 ml) at rt was added TEA (12.2 g, 0.12 mol).
The reaction was stirred for 2 h. The reaction mixture was
concentrated under reduced pressure to give the crude product.
Purification by column chromatography on silica with PE:EA=20:1
provided tert-butyl
4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-ca-
rboxylate 6 (21 g, 75%).
[0139] Synthesis of
1-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine
hydrogen chloride: To a solution of tert-butyl
4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-ca-
rboxylate 6 (30 g, 0.072 mol) in MeOH (20 ml) was added conc. HCl
(50 mL). The reaction was stirred for 24 hours and then
concentrated under reduced pressure. A saturated solution of
Na.sub.2CO.sub.3 was added to adjust the pH to .about.8-9 and the
mixture was extracted with dichloromethane (x2). The combined
extracts were washed with brine, dried (Na.sub.2SO.sub.4), filtered
and concentrated under reduced pressure. The oil product was
treated with MeOH/HCl(g) (3M, 500 mL) and stirred for 1 hour, then
concentrated under reduced pressure to get the product
1-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enylnnethyl)piperazine
hydrogen chloride IM-14-1 (23 g, 83%). MS: 319[M+H].sup.+ 1H NMR
(400 MHz, DMSO) .delta. 11.51 (s, 1H), 9.60 (s, 1H), 9.18 (s, 1H),
7.45 (d, J=8.2 Hz, 2H), 7.15 (d, J=8.0 Hz, 2H), 3.43 (s, 8H), 2.84
(s, 2H), 2.39 (s, 2H), 2.03 (s, 2H), 1.45 (t, J=6.0 Hz, 2H), 0.96
(s, 6H).
EXAMPLE 2
Preparation of Key Intermediate IM-14-3
##STR00064##
[0141] Step 1: To a solution of compound 1 (200 g, 1.3 mol) in a
solution of sulfuric acid (314 mL) and water (2.5 L) was added
potassium iodide (217 g, 1.3 mol) and KIO.sub.3 (140 g, 655 mmol)
at 20.degree. C. The mixture was stirred at 80.degree. C. for 4 h.
TLC (petroleum ether:ethyl acetate=1:1, R.sub.f=0.43) showed
reactant was consumed completely, and one major new spot with
larger polarity was detected. The five reactions were combined for
workup. The mixture was cooled to RT and adjusted to pH 9 with the
careful addition of NaOH (5N). Sodium sulfite saturated solution
was added with vigorous stirring until the iodine color had been
disappeared. A significant amount of brown solid remained out of
solution, which was collected by filtration, washed with water
(2000 mL) to give compound 2 (1.5 kg, 5.4 mol, 82.1% yield) as a
brown solid. .sup.1H NMR: (400 MHz, DMSO-d.sub.6) .delta. 8.50 (s,
1H), 2.58 (s, 3H).
[0142] Step 2: To a solution of compound 2 (387 g, 1.4 mol) in THF
(2.4 L) was added TEA (422 g, 4.2 mol) and CuI (26.47 g, 139 mmol).
The mixture was degassed and purged with nitrogen atmosphere for 3
times, Pd(PPh.sub.3).sub.2Cl.sub.2 (29.3 g, 42 mmol) was added and
the mixture was degassed and purged with nitrogen atmosphere for 3
times again. Then ethynyl(trimethyl)silane (164 g, 1.7 mol) was
added drop-wise. During which the temperature was maintained below
30.degree. C. The mixture was stirred at 25.degree. C. for 3 h
under nitrogen atmosphere. TLC (petroleum ether:ethyl acetate=1:1,
R.sub.f=0.43) show reactant was consumed completely, and one major
new spot with larger polarity was detected. The four reactions were
combined for workup. The mixture was filtered and concentrated
under reduced pressure to give a residue. The resulting residue was
dissolved in water (10 L) and extracted with ethyl acetate
(4.times.1 L). The organic layer was washed with water and then
separated out. The organic layer dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure at
50.degree. C. The solid was added to petroleum ether (300 mL) and
stirred for 30 min Then the suspension was filtered and the solid
was dried in air to give compound 3 (1.3 kg, crude) as a brown
solid. The solid was added to the solution of DCM (5 L) and stirred
at 25.degree. C. for 3 h. Then filtered and concentrated to give
compound 3 (700 g) as yellow solid. .sup.1H NMR: (400 MHz,
DMSO-d.sub.6) .delta. 8.15 (s, 1H), 2.63 (s, 3H), 0.24 (s, 9H).
[0143] Step 3: To a solution of t-BuOK (126 g, 1.1 mol, 2.0 eq) in
NMP (500 mL) was added a solution of compound 3 (140 g, 561 mmol,
1.0 eq) in NMP (400 mL) in dropwise at 140.degree. C. The mixture
was stirred at 140.degree. C. for 3 h. LCMS showed the reaction was
completed. The mixture was cooled to 20.degree. C. and combined
poured into saturated NaCl solution (5 L) and extracted with ethyl
acetate (5 L*3). The combined organic phase was washed with brine
(3 L), dried with anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuum to give compound 4 (140 g, 790.3 mmol, 35.2%
yield) as yellow solid. .sup.1H NMR: (400 MHz, DMSO-d.sub.6)
.delta. 12.20 (s, 1H), 8.68 (s, 1H), 7.61 (t, J=2.1 Hz, 1H), 6.59
(s, 1H), 2.75 (s, 3H),
[0144] Step 4: To a solution of compound 4 (45 g, 254 mmol, 1.0 eq)
in DMF (250 mL) was added NaH (12.2 g, 305 mmol, 60% purity, 1.2
eq) at 0.degree. C. in portion. The mixture was stirred at
0.degree. C. for 1 h. Then the mixture was warmed to 25.degree. C.
and SEM-Cl (50.8 g, 304.8 mmol, 54 mL, 1.2 eq) was added in
dropwise within 1 h. The mixture was stirred at 25.degree. C. for 3
h. LCMS showed the reaction was completed. All the mixture was
poured into ice-water and combined extracted with ethyl acetate (1
L*2). The combined organic phase was washed with brine (500 mL*N),
dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in
vacuum. The mixture was purified by silica gel (Petroleum
ether:Ethyl acetate=10:1 to 5:1, Rf=0.32) to give compound 5 (95 g,
309 mmol, 40.6% yield) as yellow solid. .sup.1H NMR: (400 MHz,
CDCl.sub.3) .delta. 8.70 (s, 1H), 7.49 (d, J=3.2 Hz, 1H), 6.68 (d,
J=4.0 Hz, 1H), 5.74 (s, 2H), 3.61 (t, J=8.0 Hz, 2H), 2.99 (s, 3H),
0.99 (t, J=8.4 Hz, 2H), 0.00 (s, 9H)
[0145] Step 5: To a solution of compound 5 (32 g, 104 mmol, 1.0 eq)
and Pd/C (3.2 g) in THF (300 mL) was added AcOH (31.3 g, 520.5
mmol, 30 mL, 5.0 eq), the suspension was degassed under vacuum and
purged with H.sub.2 three times. The mixture was stirred under
H.sub.2 (50 psi) at 30.degree. C. for 12 hours. LCMS showed the
reaction was completed. The mixture was filtered and concentrated
in vacuum. The residue was purified by silica gel (Petroleum
ether:Ethyl acetate=10:1 to 2:1, Rf=0.2) to give compound 6 (53 g,
191 mmol, 61.2% yield) as yellow solid. .sup.1H NMR: (400 MHz,
CDCl.sub.3) .delta. 7.25 (d, J=4.0 Hz, 1H), 6.37 (d, J=2.8 Hz, 1H),
5.67 (d, J=4.4 Hz, 2H), 3.61 (t, J=8.4 Hz, 2H), 3.48 (s, 2H), 2.58
(s, 3H), 0.99 (t, J=8.4 Hz, 2H), 0.00 (s, 9H)
[0146] Step 6: To a solution of compound 6 (14.3 g, 51.5 mmol, 1.0
eq) in toluene (250 mL) was added Ac.sub.2O (20 g, 195.9 mmol, 18
mL, 3.8 eq) and AcOK (20.2 g, 206.2 mmol, 4.0 eq), the mixture was
stirred at 60.degree. C. for 3 h. Then isopentyl nitrate (17.2 g,
129 mmol, 2.5 eq) was added to the mixture in dropwise, the mixture
was heated to 110.degree. C. and stirred for 9 h. TLC (Petroleum
ether:Ethyl acetate=2:1, Rf=0.1) showed the reaction was completed.
The two mixtures was cooled to 25.degree. C. and poured into water
(500 mL). The aqueous phase was extracted with ethyl acetate (200
mL*2). The combined organic phase was washed with brine (200 mL),
dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in
vacuum. The residue was purified by silica gel (Petroleum
ether:Ethyl acetate=10:1 to 2:1) to give compound 7 (20 g, 60.5
mmol, 58.7% yield) as yellow solid. .sup.1H NMR: (400 MHz,
CDCl.sub.3) .delta. 9.01 (s, 1H), 8.44 (s, 1H), 7.64 (d, J=3.6 Hz,
1H), 6.77 (d, J=3.9 Hz, 1H), 5.82 (d, J=6.4 Hz, 2H), 3.65 (t, J=8.4
Hz, 3H), 2.90 (s, 2H), 1.01 (t, J=8.4 Hz, 2H), 0.00 (s, 9H).
[0147] Step 7: To a solution of compound 7 (20 g, 60.5 mmol, 1.0
eq) in MeOH (250 mL) was added K.sub.2CO.sub.3 (16.7 g, 121.0 mmol,
2.0 eq), the mixture was stirred at 20.degree. C. for 5 h. LCMS
showed the reaction was completed. The mixture was concentrated in
vacuo and water (100 mL) was added. The aqueous phase was extracted
with ethyl acetate (100 mL*3). The combined organic phase was
washed with brine (100 mL*), dried with anhydrous Na.sub.2SO.sub.4,
filtered and concentrated in vacuum. The residue was purified by
silica gel (Petroleum ether:Ethyl acetate=10:1 to 2:1, Rf=0.1) to
give compound IM-14-7 (17 g, 58.9 mmol, 97.4% yield) as yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.62 (s, 1H),
8.43 (s, 1H), 8.06 (s, 1H), 7.60 (d, J=3.6 Hz, 1H), 6.66 (d, J=3.6
Hz, 1H), 5.82 (s, 21H), 3.65-3.69 (m, 2H), 1.09 (s, 2H), 0.00 (s,
9H).
EXAMPLE 3
Alternative Preparation of IM-14-3
##STR00065##
[0149] General procedure for preparation of compound 2: To a
solution of 5-bromo-6-methyl-1H-pyrrolo[2,3-b]pyridine (10 g, 47.4
mmol, 1.00 eq) in DMF (150.00 mL), the mixture was cooled to
0.degree. C. and NaH (2.84 g, 71 mmol, 60% purity, 1.50 eq) was
added to the mixture. The reaction mixture was stirred at 0.degree.
C. for 1 hr, 2-(chloromethoxy)ethyl-trimethyl-silane (10.3 g, 61.6
mmol, 11 mL, 1.30 eq) was added dropwise to the mixture. The
mixture was stirred at 25.degree. C. for 3hr. TLC indicated 0% of
Reactant 1 was remained, and one major new spot with larger
polarity (Petroleum ether:Ethyl acetate=5:1, Rf=0.51) was detected.
The mixture was poured into NH.sub.4Cl solution (40 ml),
concentrated, then extracted with EA (30 ml.times.3), combine the
organic layers, washed with brine (20 ml), and concentrated to give
crude product. The residue was purified by column chromatography
(SiO2, Petroleum ether/Ethyl acetate=100/1 to 5:1). Compound 2
(10.00 g, 29.30 mmol, 61.84% yield) was obtained. .sup.1H NMR:
ET6225-38-P1A 400 MHz CDCl.sub.3.delta. 8.01 (s, 1H), 7.26 (d,
J=3.2 Hz, 1H), 6.40 (d, J=3.2 Hz, 1H), 5.62 (s, 2H), 3.55 (t, J=16
Hz, 2H), 2.75 (s, 3H), 0.91 (t, J=16 Hz, 2H), 0.07 (s, 9H).
[0150] General procedure for preparation of compound 3: A mixture
of compound 2 (5.50 g, 16.1 mmol, 1.00 eq), diphenylmethanimine
(3.50 g, 19.3 mmol, 1.20 eq), Xantphos (1.86 g, 3.22 mmol, 0.20
eq), Cs.sub.2CO.sub.3 (10.5 g, 32.2 mmol, 2.00 eq) in dioxane (150
mL) was degassed and purged with N.sub.2 for 3 times, and then
Pd.sub.2(dba).sub.3 (885 mg, 966 umol, 0.06 eq) was added to the
mixture. Then the mixture was stirred at 100.degree. C. for 15 hour
under N.sub.2 atmosphere. TLC (Petroleum ether/Ethyl acetate=5:1)
showed many spot. The desired compound was detected by LCMS
(RT=1.363). The reaction mixture was concentrated under reduced
pressure to give a residue. The crude product 3 was used into the
next step without further purification
[0151] General procedure for preparation of compound 4: A mixture
of crude compound 3 (8.00 g, 11.8 mmol, 1.00 eq), 0.5 N HCl (100
mL), in THF (100 mL) was degassed and purged with N.sub.2 for 3
times, and then the mixture was stirred at 25.degree. C. for 14
hour under N.sub.2 atmosphere. TLC (Petroleum ether/Ethyl
acetate=3:1) showed the desired product was detected. The mixture
was quenched by aq. NaHCO.sub.3 (200 ml) to adjust pH=8, then
extracted with Ethyl acetate (100 ml.times.3), combine the organic
layers, washed with brine(100 ml), dried over sodium sulfate,
concentrated to give crude product. The residue was purified by
column chromatography (SiO2, Petroleum ether/Ethyl acetate=100/1 to
5:1) Compound 4 (3 g, 8.65 mmol, 73.5% yield, 80% purity) was
obtained as a solid. .sup.1H NMR: ET6225-46-P1A 400 MHz CDCl.sub.3
.delta. 7.25 (s, 2H), 6.42 (s, 1H), 5.67 (s, 2H), 3.60 (t, J=8 Hz,
2H), 2.62 (s, 3H), 0.98 (t, J=8 Hz, 2H), 0.04 (s, 9H).
[0152] General procedure for preparation of compound 5: A mixture
of compound 4 (2.00 g, 7.21 mmol, 1.00 eq) and Ac.sub.2O (1.6 g,
15.6 mmol, 2.2 eq) in CHCl.sub.3 (50.00 mL) was added AcOK (7.08 g,
72.10 mmol, 10 eq), and then the mixture was stirred at 25.degree.
C. for 3 hr, under N.sub.2 atmosphere. then isoamyl nitrate (3.38
g, 28.8 mmol, 4 eq) was added, heated to 60.degree. C. for 14 hr,
TLC(Petroleum ether/Ethyl acetate=3:1, Rf (product=0.43) showed the
starting material was consumed completely. The mixture was quenched
by aq. NaHCO.sub.3 (200 ml), then extracted with DCM (100
ml.times.3),combine the organic layers, washed with brine (100 ml),
dried over sodium sulfate, concentrated to give crude product. The
residue was purified by column chromatography (SiO2, Petroleum
ether/Ethyl acetate=100/1 to 5:1). Compound 5_Ac (600 mg, 21.36%
yield, 85% purity) and
N-[6-methyl-1-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-b]pyridin-5-yl]ac-
etamide (1.50 g, 4.70 mmol, 65.12% yield) were obtained.
[0153] A mixture of Compound 5_Ac (600 mg, 1.82 mmol, 1.00 eq) in
MeOH (5 mL) was added K.sub.2CO.sub.3 (753 mg, 5.45 mmol, 3.00 eq),
and then the mixture was stirred at 25.degree. C. for 2 hr, under
N.sub.2 atmosphere. TLC showed none of Compound 5 was remained. The
mixture was poured into water (200 ml), and then extracted with DCM
(100 ml.times.3), combine the organic layers, washed with brine
(100 ml), and dried over sodium sulfate, concentrated to give crude
product. The residue was purified by column chromatography (SiO2,
Petroleum ether/Ethyl acetate=100/1 to 5:1). Compound 5 (500 mg,
95.25% yield) was obtained as a yellow solid. .sup.1H NMR:
ET6225-47-P1A 400 MHz CDCl.sub.36 9.05 (s, 1H), 8.45 (s, 1H), 7.64
(s, 1H), 6.77(s, 1H), 5.82(s, 2H), 3.65 (t, J=8 Hz, 2H), 2.90(s,
3H), 1.01 (t, J=8 Hz, 2H), 0.01 (s, 9H). .sup.1H NMR: ET6225-51-P1A
400 MHz CDCl.sub.3 .delta. 8.05 (s, 1H), 7.60 (s, 1H), 6.66(s, 1H),
5.82(s, 2H), 3.67 (t, J=16 Hz, 2H), 1.01 (t, J=16 Hz, 2H), 0.04 (s,
9H).
EXAMPLE 4
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino-
)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzami-
de
[0154] A mixture of compound IM-14-3 (500 mg, 1.73 mmol, 1.00 eq),
ethyl 4-bromo-2-fluoro-benzoate (641 mg, 2.60 mmol, 1.50 eq),
Cs.sub.2CO.sub.3 (1.69 g, 5.19 mmol, 3.00 eq) in DMF (10 mL) was
degassed and purged with N.sub.2 for 3 times, and then the mixture
was stirred at 125.degree. C. for 1 hour under N.sub.2 atmosphere
under microwave condition. TLC indicated the desired compound (Rf:
0.24). The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by silica gel column chromatography (Petroleum
ether/Ethyl acetate=100:1 to 3:1). ethyl
4-bromo-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-
-e]pyridin-1(5H)-yl)benzoate (400 mg, 659 umol, 38.1% yield, 85%
purity) was obtained as a yellow oil. .sup.1H NMR: ET6225-63-P1A
400 MHz CDCl.sub.3 .delta.8.50 (s, 1 H) 7.90-7.96 (m, 3 H) 7.72 (d,
J=8.38 1 H) 7.60 (d, J=3.53 Hz, 1 H) 6.60 (d, J=4 Hz, 1 H) 5.81 (s,
2 H) 3.97-4.07 (m, 2 H) 3.62-3.70 (m, 2 H) 0.97-1.05 (m, 2 H) 0.83
(t, J=7.28 Hz, 3 H) 0.01 (s, 9 H).
[0155] A mixture of ethyl
4-bromo-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-
-e]pyridin-1(5H)-yl)benzoate (800 mg, 1.55 mmol, 1.00 eq), compound
7 (594 mg, 1.86 mmol, 1.20 eq), Xantphos (359 mg, 621 umol, 0.40
eq), Cs.sub.2CO.sub.3 (1.52 g, 4.66 mmol, 3.00 eq) in dioxane (20
mL) was degassed and purged with N.sub.2 for 3 times, and then
Pd.sub.2(dba).sub.3 (284 mg, 310 umol, 0.20 eq) was added to the
mixture, the mixture was stirred at 100.degree. C. for 15 hour
under N.sub.2 atmosphere. TLC showed the reaction was completed.
The desired compound (Rf: 0.24) was detected. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. The residue was purified by silica gel
chromatography (100-200 mesh silica gel, Petroleum ether/Ethyl
acetate=100/1-3/1). Ethyl
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate (600 mg, 637 umol, 41.1%
yield, 80% purity) was obtained as a yellow solid. .sup.1H NMR:
ET6225-84-p1a 400 MHz CDCl.sub.3 .delta. 8.46 (s, 1 H) 8.04 (d,
J=8.8 Hz, 1 H) 7.73-7.86 (m, 1 H) 7.55 (d, J=3.5 Hz, 1 H) 6.95-7.05
(m, 4 H) 6.57 (d, J=3.5 Hz, 1 H) 5.80 (s, 2 H) 3.88 (q, J=7 Hz, 2
H) 3.61-3.70 (m, 2 H) 3.37 (d, J=4.8 Hz, 4 H) 2.89 (s, 2 H) 2.42
(d, J=4.8 Hz, 4 H) 2.29 (s, 2 H) 2.07 (s, 2 H) 1.63 (s, 7 H) 1.52
(t, J=6.39 Hz, 2 H) 0.85-1.08 (m, 16 H) 0.68 (t, J=7.2 Hz, 3 H)
0.00 (s, 9 H).
[0156] A mixture of ethyl
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate (500 mg, 664 umol, 1.00
eq), NaOH (79.6 mg, 1.99 mmol, 3.00 eq) in MeOH (10 mL), THF (10
mL), H.sub.2O (5 mL) was stirred at 0-25.degree. C. for 2 hour
under N.sub.2 atmosphere. The desired compound was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. this residue was dissolved in water, then was adjusted to
pH=4-5 with 0.5 M HCl and extracted with Ethyl acetate (20
ml.times.3) combine the organic layers, washed with brine, dried
over sodium sulfate, concentrated to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoic acid (400 mg, 74.8% yield,
90% purity) was obtained as a white solid. .sup.1H NMR:
ET6225-85-p1a 400 MHz CDCl.sub.3 .delta.8.45 (s, 1 H) 8.07 (d,
J=9.2 Hz, 1 H) 7.85 (s, 1 H) 7.53 (d, J=3.9 Hz, 1 H) 7.30-7.34 (m,
3 H) 7.00 (d, J=8.4 Hz, 2 H) 6.84-6.93 (m, 2 H) 6.52-6.57 (m, 1 H)
5.76 (s, 2 H) 3.61-3.69 (m, 2 H) 3.35 (s, 3 H) 2.47 (s, 2 H) 2.28
(s, 2 H) 2.06 (s, 2 H) 1.49 (t, J=5.9 Hz, 2 H) 1.27-1.36 (m, 1 H)
0.96-1.05 (m, 9 H), 0.01 (s, 9 H).
[0157] A mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoic acid (400 mg, 551 umol, 1.00
eq), and HATU (629 mg, 1.65 mmol, 3.00 eq) NH.sub.4Cl (147 mg, 2.76
mmol, 5.00 eq) Et.sub.3N (558 mg, 5.51 mmol, 10.00 eq) in DMF (3
mL) was stirred at 25.degree. C. for 15 hr under N.sub.2
atmosphere. The desired compound was detected by TLC. The reaction
mixture was filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO2, DCM: MeOH=200/1 to 20:1).
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (300 mg, 75% yield) was
obtained as a yellow solid. .sup.1H NMR: ET6225-86-p1a 400 MHz
CDCl.sub.3 .delta.8.44 (s, 1 H) 7.88-7.94 (m, 1 H) 7.83 (d, J=8.8
Hz, 1 H) 7.55-7.60 (m, 1 H) 7.39 (d, J=7.9 Hz, 2 H) 7.05 (d, J=7.9
Hz, 2 H) 6.87 (s, 1 H) 6.65 (d, J=3.5 Hz, 1 H) 5.74-5.80 (s, 2 H)
3.66 (m, 4 H) 3.53 (br. s., 2 H) 3.21-3.34 (m, 11 H) 2.09-2.32 (m,
3 H) 1.41 (m, 17 H) 0.01 (s, 9 H).
[0158] A mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (300 mg, 414 umol, 1.00
eq) in THF (8 mL), then NaH (49.7 mg, 1.24 mmol, 60% purity, 3.00
eq) was added at 0.degree. C., stirred for 0.5 hr, then
4-chloro-3-nitro-benzenesulfonyl chloride (117 mg, 456 umol, 1.10
eq) in THF (2 mL) was added, the mixture was stirred at 25.degree.
C. for 2 hr under N.sub.2 atmosphere. The desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-TLC
(SiO2, DCM/MeOH=10/1).
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (200.00 mg, 211.86 umol, 51.16% yield) was obtained as yellow
solid. .sup.1H NMR: ET6215-89-p1a 400 MHz CDCl.sub.3 .delta.8.44
(s, 1 H) 7.95 (s, 1 H) 7.76 (s, 1 H) 7.40-7.53 (m, 1 H) 6.95 (d,
J=7.8 Hz, 5 H) 6.75 (s, 1 H) 6.51 (s, 1 H) 5.71 (s, 3 H) 5.30 (s, 2
H) 3.50-3.65 (m, 5 H) 3.26 (s, 3 H) 3.06 (d, J=6.65 Hz, 2 H) 2.85
(s, 1 H) 2.36 (s, 2 H) 2.21 (s, 1 H) 2.00 (s, 4 H) 1.44 (s, 1 H)
1.14-1.31 (m, 8 H) 0.96 (m, 13 H) 0.07 (s, 9 H)
[0159] A mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (200 mg, 212 umol, 1.00 eq) in TFA (3.00 mL) and DCM (3.00 mL)
was stirred at 25.degree. C. for 14 hr under N.sub.2 atmosphere.
The desired compound (Rt=1.146) was detected on LC-MS. The reaction
mixture was concentrated under reduced pressure to give a crude as
a yellow solid. A mixture of the crude (150 mg, 204 umol, 1.00 eq)
and cyclohexylmethanamine (69.5 mg, 614 umol, 3.00 eq) in
CH.sub.3CN (4 mL) in a microwave tube, then DIPEA(79.3 mg, 614
umol, 3.00 eq) was added, the mixture was stirred at 125.degree. C.
for 14 hr under N.sub.2 atmosphere under microwave. The desired
compound was detected by LCMS (Rt=0.573). The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by prep-HPLC (neutral) and the recovered product was
purified by Re-prep-HPLC (TFA). Two batches of target compound were
obtained as yellow solid. The first batch is 11 mg; the second
batch is 23 mg. .sup.1H NMR: ET6225-91-p1c 400 MHz DMSO
.delta.11.50 (br. s., 1 H) 8.64 (s, 1 H) 8.40 (s, 1 H) 7.97 (s, 1
H) 7.89 (s, 1 H) 7.60-7.69 (m, 2 H) 7.56 (m, 1 H) 7.40 (m, 2 H)
6.98-7.16 (m, 5 H) 6.35 (s, 1 H) 3.81-4.03 (m, 3 H) 3.12-3.41 (m, 6
H) 2.24 (m, 2 H) 2.05 (s, 2 H) 1.65 (m, 3 H) 1.44-1.53 (m, 2 H)
1.19-1.38 (m, 3 H) 0.96 (s, 6 H), LCMS: (M+H).sup.+: 892.2.
EXAMPLE 6
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((1-(tetrahydro-2H-pyran-4-yl)piperidin--
4-yl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)--
yl)benzamide
[0160] To a mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5-methyl-3,4,5,6-te-
trahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsi-
lyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
(1.00 g, 1.06 mmol, 1.00 eq) in dichloromethane (10 mL) was added
Trifluoroacetic Acid (15.40 g, 135 mmol, 10.00 mL) in one portion
at 20.degree. C. The mixture was stirred at 20.degree. C. for 14
hrs, LCMS showed the product was detected. The reaction mixture was
concentrated under reduced pressure to remove dichloromethane, and
then diluted with water (100 mL), and extracted with ethyl acetate
(20 mL*3). The combined organic layers were washed with brine (100
mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure to give the crude product Compound
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (800.00
mg, crude) was used into the next step without further
purification.
[0161] A mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (100 mg,
118 umol, 1.00 eq) and 1-tetrahydropyran-4-ylpiperidin-4-amine
(65.5 mg, 355 umol, 3.00 eq) in CH.sub.3CN (1.0 mL) in a microwave
tube, then DIPEA (153 mg, 1.19 mmol, 206 uL, 10 eq) was added, the
mixture was stirred at 125.degree. C. for 14 hr under N.sub.2
atmosphere under microwave. LCMS and HPLC showed the product was
detected. The reaction mixture was concentrated under reduced
pressure to remove CH.sub.3CN, and then diluted with water (10 mL)
and extracted with dichloromethane (5 mL*3). The combined organic
layers were washed with brine (10 mL), dried over Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (prep-HPLC (column: YMC-Actus
Triart C18 150*30 5u; liquid phase: [A-10 mM NH.sub.4HCO.sub.3 in
H.sub.2O; B-ACN]B%: 45%-65%,12 min])) to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((1-(tetrahydro-2H-pyran-4-yl)piperidin--
4-yl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)--
yl)benzamide (2.60 mg, 2% yield) as a white solid. LCMS: (M/2+1):
481.1 .sup.1H NMR: CDCl.sub.3 400 MHz .delta. 8.45 (s, 1 H)
8.22-8.38 (m, 2 H) 7.81 (d, J=8.0 Hz, 1 H) 7.54 (s, 2 H) 7.43 (d,
J=8.0 Hz, 1 H) 6.87 (d, J=8.0 Hz, 4 H) 6.67 (s, 1 H) 6.31 (s, 1 H)
3.98 (d, J=8.0 Hz, 2 H) 3.29-3.35 (m, 3 H) 3.20 (s, 5 H) 2.74 (s, 3
H) 2.25 (s, 5 H) 2.14 (s, 3 H) 1.92 (s, 5 H) 1.18 (s, 2 H) 0.89 (s,
9 H)
EXAMPLE 7
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((3-morpholinopropyl)amino)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0162] A mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1 (5H)-yl)benzamide (100
mg, 118 umol, 1.00 eq) and 3-morpholinopropan-1-amine (51 mg, 355
umol, 3.00 eq) in CH.sub.3CN (1.00 mL) in a microwave tube, then
DIPEA (60 mg, 464 umol, 3.92 eq) was added, the mixture was stirred
at 125.degree. C. for 14 hr under N.sub.2 atmosphere under
microwave. LCMS and HPLC showed the product was detected. The
reaction mixture was concentrated under reduced pressure to remove
CH.sub.3CN, and then diluted with water (10 mL) and extracted with
dichloromethane (5 mL*3). The combined organic layers were washed
with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure to give a residue. The residue
was purified by prep-HPLC (prep-HPLC (column: YMC-Actus Triart C18
150*30 5u; liquid phase: [A-10mM NH.sub.4HCO.sub.3 in H.sub.2O;
B-ACN]B%: 45%-75%,12 min])) to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((3-morpholinopropyl)amino)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (2.0
mg, 2% yield) as a yellow solid. LCMS: (M/2+1): 461.1 .sup.1H NMR:
ET10360-22-P1A1 CDCl.sub.3 400 MHz .delta. 9.06 (s, 1 H) 8.46 (s, 1
H) 8.23-8.39 (m, 2 H) 7.81 (d, J=8.0 Hz, 1 H) 7.50-7.59 (m, 2 H)
7.34 (s, 1 H) 6.80-6.92 (m, 3 H) 6.68 (s, 1 H) 6.46 (d, J=8.0 Hz, 1
H) 6.28 (s, 1 H) 3.64-3.78 (m, 4 H) 3.12-3.29 (m, 6 H) 2.66-2.84
(m, 4 H) 2.44 (s, 7 H) 2.26 (s, 4 H) 2.08-2.17 (m, 3 H) 1.92 (s, 2
H) 1.30-1.43 (m, 4 H) 1.06-1.21 (m, 4 H) 0.89 (s, 7 H)
EXAMPLE 8
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0163]
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl--
3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(t-
rimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-
benzamide (200.00 mg, 211.86 .mu.mol, 1.00 eq) was dissolved in DCM
(3.00 mL), to which TFA (4.62 g, 40.52 mmol, 3.00 mL, 191.25 eq)
was added in one portion. The mixture was then stirred at
25.degree. C. under N.sub.2 atmosphere for 14 h. After removal of
the solvent,
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (180.00
mg, crude) was obtained as a dark yellow solid which was confirmed
by LC-MS and used directly for the next step without further
purification.
[0164] To the mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (170.00
mg, 201.47 .mu.mol, 1.00 eq) and morpholine (52.66 mg, 604.41
.mu.mol, 53.19 .mu.L, 3.00 eq) in CH.sub.3CN (4.00 mL), DIEA (78.12
mg, 604.41 .mu.mol, 105.57 .mu.L, 3.00 eq) was added. The resulting
mixture was taken up into a microwave tube, and heated under
microwave at 80.degree. C. under N.sub.2 atmosphere for 2 h. After
removal of the solvent, the crude
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1 (5H)-yl)benzamide (300.00 mg, crude)
was obtained as a black brown oil which was purified together with
EW5403-10 by Prep-HPLC (HCl as additive) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide HCl salt (41.40 mg)
as a yellow solid. The product was confirmed by .sup.1H NMR and
LC-MS.
EXAMPLE 9
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0165] Synthesis of Tert-butyl 3-morpholinoazetidine-1-carboxylate
A mixture of tert-butyl 3-oxoazetidine-1-carboxylate (3.50 g, 20.45
mmol, 1.00 eq) and morpholine (2.32 g, 26.59 mmol, 2.34 mL, 1.30
eq) in DCE (250.00 mL) was stirred at 30.degree. C. for 1 h, and
then NaBH(OAc).sub.3 (5.63 g, 26.59 mmol, 1.30 eq) was added. The
mixture was stirred at 30.degree. C. for additional 15 h. Sat.
Na.sub.2CO.sub.3 solution (200 mL) was added to the mixture, and
the mixture was stirred at 30.degree. C. for 10 min The organic
layer was separated, washed with H.sub.2O (200 mL) and brine (200
mL), and then concentrated under reduced pressure. The residue was
purified by silica gel chromatography eluted with PE to PE:EA=1:1
to give tert-butyl 3-morpholinoazetidine-1-carboxylate (4.50 g,
18.57 mmol, 90.81% yield) as a light yellow oil which was confirmed
by .sup.1H NMR.
[0166] Synthesis of 4-(azetidin-3-yl)morpholine TFA salt: A mixture
of tert-butyl 3-morpholinoazetidine-1-carboxylate (500.00 mg, 2.06
mmol, 1.00 eq) and TFA (7.70 g, 67.53 mmol, 5.00 mL, 32.78 eq) in
DCM (20.00 mL) was stirred at 30.degree. C. for 15 h. After removal
of the solvent, 4-(azetidin-3-yl)morpholine TFA salt (280.00 mg,
crude) was obtained as a yellow oil which was confirmed by LC-MS
used directly without further purification.
[0167] Synthesis of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (200.00 mg, 211.86 .mu.mol, 1.00 eq) was dissolved in DCM (3.00
mL), to which TFA (4.62 g, 40.52 mmol, 3.00 mL, 191.25 eq) was
added in one portion. The resulting mixture was then stirred at
25.degree. C. under N.sub.2 atmosphere for 14 h. After removal of
the solvent,
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (200.00
mg, crude) was obtained as a dark yellow solid which was confirmed
by LC-MS and used directly for the next step without further
purification.
[0168] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide HCl
salt: A mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (78.37
mg, 551.10 .mu.mol, 3.00 eq) in CH.sub.3CN (4.00 mL) was added DIEA
(71.22 mg, 551.10 .mu.mol, 96.24 .mu.L, 3.00 eq). The resulting
mixture was taken up into a microwave tube and heated using
microwave at 80.degree. C. for 2 h under N.sub.2 atmosphere. After
removal of the solvent,
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-N-[4-(3-morpholinoazetidin-1-yl)-3-nitro-phenyl]sulfonyl-2-(5H-pyrazo-
lo[BLAH]pyrrolo[BLAH]-pyridin-1-yl)benzamide (300.00 mg, crude) was
obtained as a black brown oil which was purified together with
EW5403-7 by Prep-HPLC (HCl) and then by SFC (column: Phenomenex
Gemini 150*25mm*10 um; mobile phase: [water (0.05% ammonia
hydroxide v/v)-ACN]; B%: 35%-65%, 10 min) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfon-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide HCl
salt (2.40 mg, 2.51 .mu.mol, 7.69% yield, HCl) as a yellow solid.
The product was confirmed by LC-MS and .sup.1H NMR.
EXAMPLE 10
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)-
sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0169] Synthesis of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
[0170]
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl--
3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(t-
rimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-
benzamide (100.00 mg, 105.93 .mu.mol, 1.00 eq) was dissolved in DCM
(3.00 mL), to which TFA (2.31 g, 20.26 mmol, 1.50 mL, 191.25 eq)
was added in one portion. The resulting mixture was then stirred at
25.degree. C. under N.sub.2 atmosphere for 14 h. After removal of
the solvent,
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (101.00
mg, crude) was obtained as a dark yellow solid which was confirmed
by LC-MS and used directly for the next step without further
purification.
[0171] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)-
sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide.
[0172]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)p-
henyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
HCl salt: The mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymeth-
yl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (101.00
mg, 119.70 .mu.mol, 1.00 eq) and 7-oxa-2-azaspiro[3.5]nonane (78.00
mg, 359.10 .mu.mol, 3.00 eq, OXALIC ACID salt) were dissolved in
CH.sub.3CN (4.00 mL), to which DIEA (92.82 mg, 718.20 .mu.mol,
125.43 .mu.L, 6.00 eq) was added. The resulting mixture was taken
up into a microwave tube. The tube was heated under N.sub.2
atmosphere at 80.degree. C. for 3 h. After removal of the solvent,
the residue was purified by Prep-HPLC (HCl, column: Phenomenex
Synergi C.sub.18 150*25*10 .mu.m; mobile phase:
[water(0.05%HCl)-ACN]; B%: 35%-55%, 7.8 min) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)-
sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
(6.10 mg, 6.48 .mu.mol, 5.42% yield, HCl) as a yellow solid. The
product was confirmed by LC-MS and .sup.1H NMR.
EXAMPLE 11
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamid-
e
[0173] Synthesis of S-(tetrahydropyran-4-ylmethyl)
ethanethioate:
[0174] A mixture of 4-(bromomethyl)tetrahydropyran (300.00 mg, 1.68
mmol, 1.00 eq) and potassium thioacetate (575.62 mg, 5.04 mmol,
3.00 eq) were dissolved in DMF (10.00 mL). The resulting mixture
was then stirred at 25.degree. C. for 3 h under N.sub.2 atmosphere.
The reacting solution was quenched with sat. aq. NaHCO.sub.3
solution (50 mL) and extracted with DCM (50 mL.times.3). The
combined organic layers were concentrated under reduced pressure to
afford S-(tetrahydropyran-4-ylmethyl) ethanethioate (245.00 mg,
1.41 mmol, 83.69% yield) as black brown oil. The product was
confirmed by LC-MS and used directly for the next step without
further purification.
[0175] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyr-
rolo[3,2-e]pyridin-1(5H)-yl)benzamide:
[0176] The mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (100.00 mg, 105.93 82 mol, 1.00 eq) and
S-(tetrahydropyran-4-ylmethyl) ethanethioate (55.38 mg, 317.79
umol, 3.00 eq) were dissolved in CH.sub.3CN (4.00 mL), to which
t-BuOK (59.43 mg, 529.65 .mu.mol, 5.00 eq) was added in one
portion. The resulting mixture was taken up into a microwave tube
and heated under N.sub.2 atmosphere at 80.degree. C. for 2 h. The
reacting solution was poured onto silica gel chromatography and
eluted with pure PE to EA:MeOH=5:1. The eluent was concentrated
under reduced pressure, and the residue was suspended in MeOH (4
mL) and stirred for 30 min The precipitate was collected to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyr-
rolo[3,2-e]pyridin-1(5H)-yl)benzamide (70.00 mg, crude) as an
off-white solid. The crude product was confirmed by LC-MS and used
directly for the next step without further purification.
[0177] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamid-
e:
[0178]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl-
)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)be-
nzamide HCl salt:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyr-
rolo[3,2-e]pyridin-1(5H)-yl)benzamide (30.00 mg, 28.85 .mu.mol,
1.00 eq) was dissolved in DCM (5.00 mL), to which TFA (13.86 g,
121.53 mmol, 9.00 mL, 4211.98 eq) was added. The resulting mixture
was then stirred at 25.degree. C. for 14 h. After removal of the
solvent, the residue was then dissolved in CH.sub.3CN (5.00 mL), to
which DIEA (111.87 mg, 865.58 .mu.mol, 151.17 .mu.L, 30.00 eq) was
added in one portion. The resulting mixture was then stirred at
80.degree. C. for 3 h, and then the solvent and excess reagent were
removed under reduced pressure to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamid-
e (60.00 mg, crude) as a black brown oil which was purified
together with EW5403-56 by Prep-HPLC (HCl) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)-
phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamid-
e HCl salt (21.40 mg, 22.62 .mu.mol, 29.39% yield, HCl) as a yellow
solid. The product was confirmed by LC-MS and .sup.1H NMR.
EXAMPLE 12
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide
[0179] Synthesis of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile:
[0180] A mixture of
1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine
(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile
(8.44 g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40
eq), Pd(dba).sub.2 (3.24 g, 5.63 mmol, 0.20 eq) and
Cs.sub.2CO.sub.3 (27.51 g, 84.42 mmol, 3.00 eq) in dioxane (300.00
mL) was stirred at 100.degree. C. for 16 h under N.sub.2
atmosphere. After removal of the solvent, the residue was purified
by silica gel chromatography eluted with PE to PE:EA=5:1 to give
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 66.53% yield) as a
yellow oil which was confirmed by LC-MS.
[0181] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile:
[0182] A mixture of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 1.00 eq),
5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,-
2-e]pyridine (5.40 g, 18.72 mmol, 1.00 eq) and Cs.sub.2CO.sub.3
(12.20 g, 37.44 mmol, 2.00 eq) in DMF (100.00 mL) was stirred at
120.degree. C. for 3 h. After cooling to 15.degree. C., the mixture
was filtered. The filtrate was concentrated under reduced pressure.
The residue was purified by silica gel chromatography eluted with
PE to PE:EA=3:1 to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro[1,1'-biphenyl]-2-yl)meth-
yl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]p-
yrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (9.60 g, 13.59 mmol,
72.60% yield) as a black oil which was confirmed by LC-MS.
[0183] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
[0184] To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (2.60 g, 3.68 mmol,
1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH (30.00 mL), KOH
(412.97 mg, 7.36 mmol, 2.00 eq) in H.sub.2O (10.00 mL) was added.
After addition, H.sub.2O.sub.2 (3.34 g, 29.44 mmol, 2.83 mL, 30%
purity, 8.00 eq) was added dropwise. The mixture was stirred at
25.degree. C. for 14 h. H.sub.2O (100 mL) was added to the mixture,
and the mixture was extracted with EA (100 mL.times.3). The
combined organic layers were washed with H.sub.2O (500 mL), and
concentrated under reduced pressure. The residue was purified by
reverse phase column with TFA as additive to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (1.20 g, 1.66 mmol, 45.01%
yield) as a yellow solid which was confirmed by LC-MS.
[0185] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide:
[0186] To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (500.00 mg, 690.22 umol,
1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52 mL, 2.20 eq) in THF
was added at 0.degree. C. The mixture was stirred at 0.degree. C.
for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl chloride
(181.92 mg, 759.24 umol, 1.10 eq) was added. The mixture was
stirred at 25.degree. C. for 14 h. The reacting solution was
quenched with sat. aq. NH.sub.4Cl (50 mL) and extracted with EA (50
mL.times.3). The combined organic layers were concentrated under
reduced pressure. The residue was purified by silica gel
chromatogaphy (pure PE to DCM:MeOH=10:1) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (90.00 mg, 97.03 .mu.mol, 14.06% yield) as a yellow oil. The
product was confirmed by LC-MS and used directly for the next step
without further purification.
[0187] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino-
)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[-
4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
[0188] A mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (20.00 mg, 21.56 .mu.mol, 1.00 eq),
(4-fluorotetrahydropyran-4-yl)-methanamine (3.99 mg, 29.97 .mu.mol,
1.39 eq) and DIEA (27.86 mg, 215.60 .mu.mol, 37.65 .mu.L, 10.00 eq)
in DMF (5.00 mL) was stirred at 50.degree. C. for 14 h. The
reacting solution was diluted with water (20 mL) and extracted with
DCM (50 mL.times.3). The combined organic layers were concentrated
under reduced pressure. The residue was purified by silica gel
chromatography(pure PE to PE:EA=1:2) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl-
]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)me-
thyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl-
)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (40.00 mg,
crude) as a yellow oil. The crude product was confirmed by LC-MS
and used directly for the next step without further
purification.
[0189] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide:
[0190]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl-
)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(-
5H)-yl)benzamide HCl salt:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino-
)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[-
4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (40.00 mg, 38.44
.mu.mol, 1.00 eq) was dissolved in DCM (5.00 mL), to which TFA
(43.83 mg, 384.40 .mu.mol, 28.46 .mu.L, 10.00 eq) was added in one
portion. The resulting mixture was then stirred at 25.degree. C.
for 14 h. After removal of the solvent, crude
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino-
)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e-
]pyridin-1(5H)-yl)benzamide (30.00 mg, crude, TFA) was obtained as
a yellow oil which was re-dissolved in MeCN (10.00 mL). To the
solution, DIEA (36.77 mg, 284.50 .mu.mol, 49.69 .mu.L, 10.00 eq)
was added in one portion. The resulting mixture was then stirred at
80.degree. C. for 2 h. The solvent and excess reagent were removed
under reduced pressure. The residue was purified by Prep-HPLC (HCl)
to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-4(4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-
-3-nitrophenyesulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)b-
enzamide HCl salt (6.90 mg, 7.29 .mu.mol, 25.61% yield, HCl) as a
light yellow solid which was confirmed by LC-MS and .sup.1H
NMR.
EXAMPLE 13
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0191] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrr-
olo[3,2-e]pyridin-1(5H)-yl)benzamide:
[0192] The mixture of
N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,-
6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (200.00 mg, 211.86 .mu.mol, 1.00 eq) and
(1-methyl-4-piperidyl)methanamine (81.49 mg, 635.58 .mu.mol, 3.00
eq) were dissolved in CH.sub.3CN (10.00 mL), to which DIEA (82.14
mg, 635.58 .mu.mol, 111.00 .mu.L, 3.00 eq) was added in one
portion. The resulting mixture was taken up into a microwave tube
and heated at 80.degree. C. under N.sub.2 atmosphere for 2 h. The
reacting solution was poured onto silica gel chromatography and
eluted with pure DCM to DCM:MeOH=5:1 to afford crude
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrr-
olo[3,2-e]pyridin-1(5H)-yl)benzamide (50.00 mg, 48.27 .mu.mol,
22.79% yield) as a yellow oil which was confirmed by LC-MS.
[0193] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyrid-
in-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)s-
ulfonyl)benzamide:
[0194]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3--
nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3--
b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (50.00 mg, 48.27
.mu.mol, 1.00 eq) was dissolved in DCM (10.00 mL), to which TFA
(16.51 mg, 144.81 .mu.mol, 10.72 .mu.L, 3.00 eq) was added. The
resulting mixture was then stirred at 25.degree. C. for 1 h. The
solvent and excess reagent was removed under reduced pressure to
afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyrid-
in-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)s-
ulfonyl)benzamide (40.00 mg, crude) as a yellow oil which was used
directly in the next step.
[0195] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide-
:
[0196]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3--
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)ben-
zamide HCl salt: The mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyrid-
in-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)s-
ulfonyl)benzamide (40.00 mg, 42.76 .mu.mol, 1.00 eq) and DIEA
(16.58 mg, 128.28 .mu.mol, 22.41 .mu.L, 3.00 eq) in CH.sub.3CN
(10.00 mL) was stirred at 80.degree. C. for 5 h. After removal of
the solvent, the residue was purified by Prep-HPLC (HCl as
additive) and SFC to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
HCl salt (23.60 mg, 26.06 .mu.mol, 92.19% yield) as a light yellow
solid which was confirmed by LC-MS and .sup.1H NMR.
EXAMPLE 14
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl-
)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(-
5H)-yl)benzamide
[0197] Synthesis of 9H-fluoren-9-ylmethyl
4-[tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate:
The mixture of tert-butyl N-[4-fluoro-4-piperidyl)methyl]carbamate
(150.00 mg, 645.74 .mu.mol, 1.00 eq) and 9H-fluoren-9-ylmethyl
carbonochloridate (250.58 mg, 968.61 .mu.mol, 1.50 eq) were
dissolved in THF (20.00 mL) and H.sub.2O (4.00 mL), to which
NaHCO.sub.3 (162.75 mg, 1.94 mmol, 75.35 .mu.L, 3.00 eq) was added
in one portion. The resulting mixture was then stirred at
15.degree. C. for 14 h. The reacting solution was diluted with
water (50 mL) and extracted with EA (50 mL.times.3). The combined
organic layers were concentrated under reduced pressure. The
residue was purified by silica gel chromatography (pure PE to PE:EA
=5:1) to afford 9H-fluoren-9-ylmethyl
4-[(tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate
(163.00 mg, 358.61 .mu.mol, 55.54% yield) as an off-white solid.
The product was confirmed by LC-MS and used directly for the next
step without further purification.
[0198] Synthesis of
4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate TFA salt:
9H-fluoren-9-ylmethyl
4-[(tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate
(163.00 mg, 358.61 .mu.mol, 1.00 eq) was dissolved in DCM (10.00
mL), to which TFA (204.44 mg, 1.79 mmol, 132.76 .mu.L, 5.00 eq) was
added in one portion. The resulting mixture was then stirred at
15.degree. C. for 14 h. The solvent and excess reagent were removed
under reduced pressure to afford 9H-fluoren-9-ylmethyl
4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate (200.00 mg,
crude, TFA) as a yellow oil. The crude product was used directly
for the next step without further purification.
[0199] Synthesis of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile: A mixture of
1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine
(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile
(8.44 g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40
eq), Pd(dba).sub.2 (3.24 g, 5.63 mmol, 0.20 eq) and
Cs.sub.2CO.sub.3 (27.51 g, 84.42 mmol, 3.00 eq) in dioxane (300.00
mL) was stirred at 100.degree. C. for 16 h under N.sub.2
atmosphere. After removal of the solvent, the residue was purified
by silica gel chromatography eluted with PE to PE:EA=5:1 to give
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 66.53% yield) as a
yellow oil which was confirmed by LC-MS.
[0200] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile: A mixture of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 1.00 eq),
5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,-
2-e]pyridine (5.40 g, 18.72 mmol, 1.00 eq) and Cs.sub.2CO.sub.3
(12.20 g, 37.44 mmol, 2.00 eq) in DMF (100.00 mL) was stirred at
120.degree. C. for 3 h. After cooling to 15.degree. C., the mixture
was filtered. The filtrate was concentrated under reduced pressure.
The residue was purified by silica gel chromatography eluted with
PE to PE:EA=3:1 to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (9.60 g, 13.59 mmol,
72.60% yield) as a black oil which was confirmed by LC-MS.
[0201] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide: To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (2.60 g, 3.68 mmol,
1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH (30.00 mL), KOH
(412.97 mg, 7.36 mmol, 2.00 eq) in H.sub.2O (10.00 mL) was added.
After addition, H.sub.2O.sub.2 (3.34 g, 29.44 mmol, 2.83 mL, 30%
purity, 8.00 eq) was added dropwise. The mixture was stirred at
25.degree. C. for 14 h. H.sub.2O (100 mL) was added to the mixture,
and the mixture was extracted with EA (100 mL.times.3). The
combined organic layers were washed with H.sub.2O (500 mL), and
concentrated under reduced pressure. The residue was purified by
reverse phase column with TFA as additive to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (1.20 g, 1.66 mmol, 45.01%
yield) as a yellow solid which was confirmed by LC-MS.
[0202] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide: To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (500.00 mg, 690.22
.mu.mol, 1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52 mL, 2.20 eq)
in THF was added at 0.degree. C. The mixture was stirred at
0.degree. C. for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl
chloride (181.92 mg, 759.24 .mu.mol, 1.10 eq) was added. The
mixture was stirred at 25.degree. C. for 14 h. The reacting
solution was quenched with sat. aq. NH.sub.4Cl (50 mL) and
extracted with EA (50 mL.times.3). The combined organic layers were
concentrated under reduced pressure. The residue was purified by
silica gel chromatogaphy (pure PE to DCM:MeOH=10:1) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (90.00 mg, 97.03 .mu.mol, 14.06% yield) as a yellow oil. The
product was confirmed by LC-MS and used directly for the next step
without further purification.
[0203] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrr-
olo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)
benzamide (250.00 mg, 269.53 .mu.mol, 1.00 eq) and
9H-fluoren-9-ylmethyl
4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate (143.29 mg,
404.29 .mu.mol, 1.50 eq) were dissolved in DMF (50.00 mL), to which
DIEA (348.34 mg, 2.70 mmol, 470.73 .mu.L, 10.00 eq) was added in
one portion. The resulting mixture was then stirred at 50.degree.
C. for 14 h. The reacting solution was diluted with water (50 mL)
and extracted with DCM (50 mL.times.3). The combined organic layers
were concentrated under reduced pressure to afford crude
(9H-fluoren-9-yl)methyl
4-(((4-(N-(4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-bipheny-
l]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyra-
zolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenye-
aminolmethyl)-4-fluoropiperidine-1-carboxylate (400.00 mg, crude)
as a yellow oil which was re-dissolved in DMF (20.00 mL). To the
above solution, piperidine (134.95 mg, 1.58 mmol, 156.92 .mu.L,
5.00 eq) was added in one portion. The resulting mixture was then
stirred at 15.degree. C. for 14 h. The reacting solution was poured
onto silica gel chromatography and eluted with pure PE to
DCM:MeOH=5:1 to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrr-
olo[3,2-e]pyridin-1(5H)-yl)benzamide (268.00 mg, 257.76 .mu.mol,
81.32% yield) as a yellow oil which was confirmed by LC-MS and used
directly for the next step without further purification.
[0204] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1-
(5H)-yl)benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrr-
olo[3,2-e]pyridin-1(5H)-yl)benzamide (268.00 mg, 257.76 .mu.mol,
1.00 eq) was dissolved in DCM (20.00 mL), to which TFA (146.95 mg,
1.29 mmol, 95.42 .mu.L, 5.00 eq) was added in one portion. The
resulting mixture was then stirred at 15.degree. C. for 14 h. The
solvent and excess reagent were removed under reduced pressure to
afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1-
(5H)-yl)benzamide (240.00 mg, crude) as a yellow oil which was
confirmed by LC-MS and used directly for the next step without
further purification.
[0205] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl-
)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(-
5H)-yl)benzamide:
[0206]
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2--
yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)-
methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyri-
din-1(5H)-yl)benzamide: The mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrop-
henyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1-
(5H)-yl)benzamide (120.00 mg, 127.73 .mu.mol, 1.00 eq) and
oxetan-3-one (27.61 mg, 383.19 .mu.mol, 3.00 eq) were dissolved in
MeOH (10.00 mL), to which NaBH.sub.3CN (40.13 mg, 638.65 .mu.mol,
5.00 eq) was added in one portion. The resulting mixture was then
stirred at 15.degree. C. for 14 h. The reacting solution was
diluted with water (50 mL) and extracted with DCM (50 mL.times.3).
The combined organic layers were concentrated under reduced
pressure to afford crude
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-4(4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)-
amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[-
3,2-e]pyridin-1(5H)-yl)benzamide (120.00 mg, crude) as a yellow oil
which was re-dissolved in CH.sub.3CN (20.00 mL). To the above
solution DIEA (77.89 mg, 602.70 .mu.mol, 105.26 .mu.L, 5.00 eq) was
added in one portion. The resulting mixture was then stirred under
N.sub.2 atmosphere at 80.degree. C. for 14 h. The reacting solution
was diluted with water (50 mL) and extracted with DCM (50
mL.times.3). The combined organic layers were concentrated under
reduced pressure. The residue was purified by Prep-TLC
(EA/MeOH=10:1 and then EA:MeOH=5:1) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl-
)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(-
5H)-yl)benzamide (2.80 mg, 2.90 .mu.mol, 2.41% yield) as a yellow
solid. The product was confirmed by LC-MS and .sup.1H NMR.
EXAMPLE 15
Preparation of
(R)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((3-nitro-4(((4-(oxetan-3-yl)morpholin-2-yl)meth-
yl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide
[0207] Synthesis of 9H-fluoren-9-ylmethyl
(2R)-2-[tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate: A
mixture of tert-butyl N-[[(2S)-morpholin-2-yl]methyl]carbamate
(64.00 mg, 295.91 .mu.mol, 1.00 eq), Fmoc-Cl (114.83 mg, 443.87
.mu.mol, 1.50 eq) and Na.sub.2CO.sub.3 (94.09 mg, 887.74 .mu.mol,
3.00 eq) in a mixture of THF (10.00 mL) and H.sub.2O (2.00 mL) was
stirred at 20.degree. C. for 12 h. The mixture was extracted with
EA (20 mL.times.3), and the combined organic layers were
concentrated under reduced pressure. The residue was purified by
Prep-TLC (PE:EA=3:1) to give 9H-fluoren-9-ylmethyl
(2R)-2-[(tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate
(111.00 mg, 253.13 .mu.mol, 85.54% yield) as a colorless oil which
was confirmed by LC-MS.
[0208] Synthesis of 9H-fluoren-9-ylmethyl
(2R)-2-(aminomethyl)morpholine-4-carboxylate TFA salt: A mixture of
9H-fluoren-9-ylmethyl
(2R)-2-[(tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate
(111.00 mg, 253.12 .mu.mol, 1.00 eq) and TFA (577.21 mg, 5.06 mmol,
374.81 .mu.L, 20.00 eq) in DCM (10.00 mL) was stirred at 15.degree.
C. for 3 h. After removal of the solvent, crude
9H-fluoren-9-ylmethyl (2R)-2-(aminomethyl)morpholine-4-carboxylate
TFA salt (80.00 mg) was obtained as a light yellow oil which was
used directly in the next step.
[0209] Synthesis of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile: A mixture of
1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine
(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile
(8.44 g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40
eq), Pd(dba).sub.2 (3.24 g, 5.63 mmol, 0.20 eq) and
Cs.sub.2CO.sub.3 (27.51 g, 84.42 mmol, 3.00 eq) in dioxane (300.00
mL) was stirred at 100.degree. C. for 16 h under N.sub.2
atmosphere. After removal of the solvent, the residue was purified
by silica gel chromatography eluted with PE to PE:EA=5:1 to give
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 66.53% yield) as a
yellow oil which was confirmed by LC-MS.
[0210] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile: A mixture of
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-fluoro-benzonitrile (8.20 g, 18.72 mmol, 1.00 eq),
5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,-
2-e]pyridine (5.40 g, 18.72 mmol, 1.00 eq) and Cs.sub.2CO.sub.3
(12.20 g, 37.44 mmol, 2.00 eq) in DMF (100.00 mL) was stirred at
120.degree. C. for 3 h. After cooling to 15.degree. C., the mixture
was filtered. The filtrate was concentrated under reduced pressure.
The residue was purified by silica gel chromatography eluted with
PE to PE:EA=3:1 to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (9.60 g, 13.59 mmol,
72.60% yield) as a black oil which was confirmed by LC-MS.
[0211] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide: To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile (2.60 g, 3.68 mmol,
1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH (30.00 mL), KOH
(412.97 mg, 7.36 mmol, 2.00 eq) in H.sub.2O (10.00 mL) was added.
After addition, H.sub.2O.sub.2 (3.34 g, 29.44 mmol, 2.83 mL, 30%
purity, 8.00 eq) was added dropwise. The mixture was stirred at
25.degree. C. for 14 h. H.sub.2O (100 mL) was added to the mixture,
and the mixture was extracted with EA (100 mL.times.3). The
combined organic layers were washed with H.sub.2O (500 mL), and
concentrated under reduced pressure. The residue was purified by
reverse phase column with TFA as additive to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (1.20 g, 1.66 mmol, 45.01%
yield) as a yellow solid which was confirmed by LC-MS.
[0212] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide:
[0213] To a solution of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]-
pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (500.00 mg, 690.22
.mu.mol, 1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52 mL, 2.20 eq)
in THF was added at 0.degree. C. The mixture was stirred at
0.degree. C. for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl
chloride (181.92 mg, 759.24 .mu.mol, 1.10 eq) was added. The
mixture was stirred at 25.degree. C. for 14 h. The reacting
solution was quenched with sat. aq. NH.sub.4Cl (50 mL) and
extracted with EA (50 mL.times.3). The combined organic layers were
concentrated under reduced pressure. The residue was purified by
silica gel chromatogaphy (pure PE to DCM:MeOH=10:1) to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (90.00 mg, 97.03 .mu.mol, 14.06% yield) as a yellow oil. The
product was confirmed by LC-MS and used directly for the next step
without further purification.
[0214] Synthesis of
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyes-
ulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-
-e]pyridin-1(5H)-yl)benzamide: A mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (80.00 mg, 86.25 .mu.mol, 1.00 eq), 9H-fluoren-9-ylmethyl
(2R)-2-(aminomethyl)morpholine-4-carboxylate (67.13 mg, 198.38
.mu.mol, 2.30 eq) and DIEA (55.73 mg, 431.25 .mu.mol, 75.31 .mu.L,
5.00 eq) in DMF (10.00 mL) was stirred at 50.degree. C. under
N.sub.2 atmosphere for 14 h. The reacting solution was diluted with
water (20 mL) and extracted with DCM (50 mL.times.3). The combined
organic layers were concentrated under reduced pressure to afford
(R)-(9H-fluoren-9-yl)methyl
2-(((4-(N-(4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-bipheny-
l]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyra-
zolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenyl-
)amino)methyl)morpholine-4-carboxylate (100.00 mg, crude) as a
yellow oil. Next, (R)-(9H-fluoren-9-yl)methyl
2-(((4-(N-(4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-bipheny-
l]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyra-
zolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenyl-
)amino)methyl)morpholine-4-carboxylate (130.00 mg, 104.34 .mu.mol,
1.00 eq) was dissolved in DMF (20.00 mL), to which piperidine
(88.85 mg, 1.04 mmol, 103.31 .mu.L, 10.00 eq) was added in one
portion. The resulting mixture was then stirred at 15.degree. C.
for 14 h. The reacting solution was poured onto silica gel
chromatography (pure PE to DCM:MeOH=5:1) to afford
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-l[1,1'-biphen-
yl]-2-yl)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitr-
ophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]py-
rrolo[3,2-e]pyridin-1(5H)-yl)benzamide (100.00 mg, crude) as a
yellow oil. The crude product was confirmed by LC-MS and used
directly for the next step without further purification.
[0215] Synthesis of
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]p-
yridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfony-
l)benzamide:
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)-
sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,-
2-e]pyridin-1(5H)-yl)benzamide (100.00 mg, 97.68 .mu.mol, 1.00 eq)
was dissolved in DCM (20.00 mL), to which TFA (111.38 mg, 976.84
.mu.mol, 72.32 .mu.L, 10.00 eq) was added in one portion. The
resulting mixture was then stirred at 15.degree. C. for 14 h. The
solvent and excess reagent were removed under reduced pressure to
afford
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]p-
yridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfony-
l)benzamide (100.00 mg, crude, TFA) as a yellow oil. The crude
product was used directly for the next step without further
purification.
[0216] Synthesis of
(R)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)met-
hyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-y-
l)benzamide:
(R)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)met-
hyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-y-
l)benzamide: The mixture of
(S)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]p-
yridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfony-
l)benzamide (100.00 mg, 108.29 .mu.mol, 1.00 eq) and oxetan-3-one
(23.41 mg, 324.87 .mu.mol, 3.00 eq) were dissolved in MeOH (20.00
mL), to which NaBH.sub.3CN (20.41 mg, 324.87 .mu.mol, 3.00 eq) was
added in one portion. The resulting mixture was then stirred at
15.degree. C. under N.sub.2 atmosphere for 14 h. The reacting
solution was diluted with water (20 mL) and extracted with DCM (50
mL.times.3). The combined organic layers were concentrated under
reduced pressure to afford crude
(R)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]p-
yridin-1(5H)-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)methyl)ami-
no)phenyl)sulfonyl)benzamide (100.00 mg, crude) as a yellow oil
which was then suspended in CH.sub.3CN (20.00 mL). To the above
solution, DIEA (131.94 mg, 1.02 mmol, 178.30 .mu.L, 10.00 eq) was
added in one portion. The resulting mixture was then stirred at
80.degree. C. for 14 h. The solvent and excess reagent were removed
under reduced pressure. The residue was purified by Prep-HPLC
(neutral) to afford
(R)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl-
)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)met-
hyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-y-
l)benzamide (1.10 mg, 1.16 .mu.mol, 1.13% yield) as a yellow solid.
The product was confirmed by LC-MS and .sup.1H NMR. 11 mg of crude
product was also obtained.
EXAMPLE 16
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-
benzamide
[0217] Synthesis of Tert-butyl
((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate: A mixture of
tert-butyl ((4-fluoropiperidin-4-yl)methyl)carbamate (330.00 mg,
1.42 mmol, 1.00 eq) and aq. HCHO (37%, 172.88 mg, 2.13 mmol, 158.61
.mu.L, 1.50 eq) in CH.sub.3CN (10.00 mL) was stirred at 30.degree.
C. for 30 min, and then NaBH.sub.3CN (178.47 mg, 2.84 mmol, 2.00
eq) and AcOH (104.88 mg, 1.75 mmol, 99.89 .mu.L, 1.23 eq) were
added in portions. The mixture was stirred at 15.degree. C. for
13.5 h. The reacting solution was diluted with water (100 mL) and
extracted with EA (100 mL.times.4). The combined organic layers
were concentrated under reduced pressure and purified by silica gel
chromatography (PE:EA =1:1 to EA:MeOH=5:1) to afford tert-butyl
((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate (290.00 mg, 1.18
mmol, 82.91% yield) as a colorless oil, which was confirmed by
LC-MS.
[0218] Synthesis of (4-fluoro-1-methylpiperidin-4-yl)methanamine:
Tert-butyl ((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate
(290.00 mg, 1.18 mmol, 1.00 eq) was dissolved in DCM (10.00 mL), to
which TFA (3.08 g, 27.01 mmol, 2.00 mL, 22.89 eq) was added in
portions. The resulting mixture was then stirred at 15.degree. C.
for 15 h. The solvent and excess reagent were removed under reduced
pressure to afford (4-fluoro-1-methylpiperidin-4-yl)methanamine
(320.00 mg, crude) as a yellow oil.
[0219] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide: The mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimeth-
ylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzam-
ide (140.00 mg, 150.94 .mu.mol, 1.00 eq) and
(4-fluoro-1-methylpiperidin-4-yl)methanamine (66.21 mg, 452.82
.mu.mol, 3.00 eq) were dissolved in DMF (20.00 mL), to which DIEA
(195.07 mg, 1.51 mmol, 263.61 .mu.L, 10.00 eq) was added in one
portion. The resulting mixture was then stirred under N.sub.2
atmosphere at 50.degree. C. for 14 h. The solvent and excess
reagent were removed under reduced pressure. The residue was
purified by silica gel chromatography (pure DCM to DCM:MeOH=3:1) to
afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (141.00 mg, 133.81
.mu.mol, 88.65% yield) as a yellow oil, which was confirmed by
LC-MS. LC-MS: EW5403-312-P1A00 (M+H.sup.+=1054,
M/2+H.sup.+=527).
[0220] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]-
pyridin-1(5H)-yl)benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4-
,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (141.00 mg, 133.81
.mu.mol, 1.00 eq) was dissolved in DCM (10.00 mL), to which TFA
(152.57 mg, 1.34 mmol, 99.07 .mu.L, 10.00 eq) was added in one
portion. The resulting mixture was then stirred at 15.degree. C.
for 14 h. The solvent and excess reagent were removed under reduced
pressure to afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]-
pyridin-1(5H)-yl)benzamide (150.00 mg, crude) as a yellow oil,
which was confirmed by LC-MS. LC-MS: EW5403-317-P1Z
(M+H.sup.+=953).
[0221] Synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-
benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl-
]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)met-
hyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-
-1(5H)-yl)benzamide: The mixture of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]-
pyridin-1(5H)-yl)benzamide (150.00 mg, 157.31 .mu.mol, 1.00 eq) and
DIEA (203.31 mg, 1.57 mmol, 274.74 .mu.L, 10.00 eq) were dissolved
in CH.sub.3CN (20.00 mL). The resulting mixture was then stirred at
80.degree. C. under N.sub.2 atmosphere for 14 h. The solvent and
excess reagent were removed under reduced pressure. The residue was
purified by column: Phenomenex Synergi C,.sub.8 150*25*10 .mu.m;
mobile phase: [water (0.05% HCl)-ACN]; B%: 42%-62%, 7.8 min to
afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-
-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-
benzamide (1.8 mg, 1.95 .mu.mol, 1.24% yield) as a yellow solid,
which was confirmed by LC-MS and .sup.1H NMR. LC-MS: EW5403-318-P1B
(M+H.sup.+=924, M/2+H.sup.+=462). .sup.1H NMR: EW5403-318-P1A0 400
MHz DMSO-d.sub.6, .delta. 11.43 (brs, 1 H), 10.65 (brs, 1 H), 8.54
(s, 1 H), 8.30 (s, 1 H), 8.05 (s, 1 H), 7.77 (s, 1 H), 7.36-7.59
(m, 4 H), 7.07-7.09 (m, 3 H), 6.95-6.97 (m, 2 H), 6.30 (s, 1 H),
3.71-3.77 (m, 2 H), 3.24-3.27 (m, 3 H), 3.03-3.17 (m, 3 H),
2.87-2.89 (m, 2 H), 2.10-2.12 (m, 4 H), 2.21-2.50 (m, 4 H), 1.99
(s, 3 H), 1.41-1.45 (m, 2 H), 1.16-1.23 (m, 6 H), 0.95 (s, 6
H).
EXAMPLE 17
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide
[0222] Step 1: Tert-butyl
4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate:
The mixture of 4-fluoro-3-nitro-benzenesulfonamide (300.00 mg, 1.36
mmol, 1.00 eq) and tert-butyl
4-fluoro-4-(hydroxymethyl)piperidine-1-carboxylate (317.26 mg, 1.36
mmol, 1.00 eq) were dissolved in THF (50.00 mL), to which NaH
(163.20 mg, 4.08 mmol, 60% purity, 3.00 eq) was added at 0.degree.
C. in one portion. The resulting mixture was then stirred at
15.degree. C. for 14 h. The reacting solution was quenched with
EtOH (10 mL), diluted with water (50 mL) and extracted with DCM
(100 mL.times.3). The combined organic layers were concentrated
under reduced pressure and purified together with EW5403-280-P1 by
silica gel chromatography (pure PE to PE:EA =1:1) to afford
tert-butyl
4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate
(560 mg, 1.29 mmol, 85.69% yield) as an off-white solid, which was
confirmed by LC-MS. LC-MS: EW5403-286-P1A0 (M+Na.sup.+=456).
[0223] Step 2:
4-((4-fluoropiperidin-4-yl)methoxy)-3-nitrobenzenesulfonamide
Tert-butyl
4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate
(100.00 mg, 230.71 .mu.mol, 1.00 eq) was dissolved in DCM (20.00
mL), to which TFA (131.53 mg, 1.15 mmol, 85.41 .mu.L, 5.00 eq) was
added in one portion. The resulting mixture was then stirred at
15.degree. C. for 14 h. The solvent and excess reagent were removed
under reduced pressure to afford
4-((4-fluoropiperidin-4-yl)methoxy)-3-nitrobenzenesulfonamide (80
mg, crude) as an off-white solid. LC-MS: EW5403-287-P1Z0
(M+H.sup.+=334).
[0224] Step 3:
4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrobenzenesulfona-
mide: The mixture of
4-[(4-fluoro-4-piperidyl)methoxy]-3-nitro-benzenesulfonamide (70.00
mg, 156.47 .mu.mol, 1.00 eq, TFA) and oxetan-3-one (67.65 mg,
938.82 .mu.mol, 6.00 eq) were dissolved in MeOH (10.00 mL), to
which NaBH.sub.3CN (98.33 mg, 1.56 mmol, 10.00 eq) was added in one
portion. The resulting mixture was then stirred at 15.degree. C.
for 14 h. The solvent and excess reagent were removed under reduced
pressure. The residue was purified together with EW5403-288-P1 by
silica gel chromatography (pure DCM to DCM:MeOH=10:1) to afford
4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrobenzenesulfona-
mide (100 mg, crude) as a light yellow oil, which was confirmed by
LC-MS. LC-MS: EW5403-289-P1A2 (M+H.sup.+=390).
[0225] Step 4: The synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide: The mixture of
4-[[4-fluoro-1-(oxetan-3-yl)-4-piperidyl]methoxy]-3-nitro-benzenesulfonam-
ide (50.00 mg, 128.40 .mu.mol, 1.00 eq),
4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-
-yl]-2-(5H-pyrazolo[BLAH]pyrrolo[BLAH]pyridin-1-yl)benzoic acid
(76.42 mg, 128.40 .mu.mol, 1.00 eq), DMAP (31.37 mg, 256.81
.mu.mol, 2.00 eq) and EDCI (49.23 mg, 256.81 .mu.mol, 2.00 eq) were
dissolved in DCM (20.00 mL). The resulting mixture was then stirred
at 15.degree. C. for 14 h. The reacting solution was poured onto
silica gel chromatography and eluted with DCM to DCM:MeOH=5:1, and
then purified by column: Phenomenex Synergi C18 150*25*10 .mu.m;
mobile phase: [water (0.05% HCl)-ACN]; B%: 42%-62%, 7.8 min to
afford
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy-
)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl-
)benzamide (8.4 mg, 8.69 .mu.mol, 6.8% yield) as a light yellow
solid, which was confirmed by LC-MS and .sup.1H NMR. LC-MS:
EW5403-300-P1C0 (M/2+H.sup.+=483). .sup.1H NMR: EW5403-300-P1A0 400
MHz CD.sub.3OD, .delta. 8.26 (s, 1 H), 8.18 (s, 2 H), 7.99-8.00 (m,
1 H), 7.67-7.70 (m, 1 H), 7.65 (s, 1 H), 7.15-7.37 (m, 7 H), 6.52
(s, 1 H), 4.44-4.49 (m, 2 H), 4.12-4.18 (m, 3 H), 3.95 (s, 2 H),
3.54-3.82 (m, 8 H), 3.35 (m, 2 H), 2.90 (s, 3 H), 2.38-2.49 (m, 6
H), 2.14 (s, 2 H), 1.60 (s, 2 H), 1.29 (s, 1 H), 1.03 (s, 6 H).
EXAMPLE 18
Preparation of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)pheny-
l)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
[0226] Step 1: the synthesis of
3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide: To
a solution of (tetrahydro-2H-pyran-4-yl)methanol (263.68 mg, 2.27
mmol, 1.00 eq) in THF (20.00 mL), NaH (272.40 mg, 6.81 mmol, 60%
purity, 3.00 eq) was added at 0.degree. C. The mixture was stirred
at 0.degree. C. for 30 min, and then
4-fluoro-3-nitrobenzenesulfonamide (500.00 mg, 2.27 mmol, 1.00 eq)
was added. The mixture was stirred at 25.degree. C. for 15 h, and
then the reaction mixture was quenched by MeOH (10 mL). After
removal of the solvent, the residue was purified by silica gel
chromatography eluted with PE:EA=5:1 to 1:1 to give
3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide
(446.00 mg, 1.41 mmol, 62.11% yield) as yellow solid which was
confirmed by LC-MS. LC-MS: EW6259-87-P1A2 (M+H.sup.+=317.0).
[0227] Step 2, the synthesis of
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)pheny-
l)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)pheny-
l)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:
To a mixture of
3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide
(296.00 mg, 935.73 .mu.mol, 1.50 eq) and
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzo-
ic acid (371.25 mg, 623.82 .mu.mol, 1.00 eq) in THF (20.00 mL) was
added EDCI (358.76 mg, 1.87 mmol, 3.00 eq) and DMAP (228.64 mg,
1.87 mmol, 3.00 eq). The mixture was stirred at 25.degree. C. for
12 h. After removal of the solvent, the residue was purified
together with EW6259-90 by Prep-HPLC (column: Phenomenex Synergi
C,.sub.8 150*25*10 .mu.m; mobile phase: [water(0.05% HCl)-ACN]; B%:
38%-58%, 7.8 min) to give
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)met-
hyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)pheny-
l)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
(68.4 mg, 50.81 .mu.mol, 8.15% yield) as yellow solid which was
confirmed by LC-MS and .sup.1H NMR. LC-MS: EW6259-91-P1G
(M+H.sup.+=893.2) 1HNMR: EW6259-91-P1P 400 MHz CD.sub.3OD, .delta.
8.43 (s, 1 H), 8.22 (s, 1 H), 8.17 (d, J=2.0 Hz, 1 H), 8.01-8.04
(m, 1 H), 7.69-7.71 (m, 2 H), 7.33-7.39 (m, 3 H), 7.24 (s, 1 H),
7.13-7.15 (m, 3 H), 6.64-6.65 (m, 1 H), 4.11-4.13 (m, 2 H),
3.75-4.04 (m, 5 H), 3.75 (s, 2 H), 3.54-3.56 (m, 2 H), 3.48-3.51
(m, 2 H), 3.31-3.41 (m, 2 H), 2.39 (s, 2 H), 2.14-2.17 (m, 3 H),
1.80-1.83 (m, 2 H), 1.52-1.59 (m, 5 H), 1.03 (s, 6 H).
[0228] The compounds below are prepared by methods substantially
identical, similar, or analogous to those disclosed in Scheme A-C
and above examples:
TABLE-US-00001 Ex- am- ple m/ No. Chemical Name z(MH.sup.+) 19
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 923
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-
methylpiperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 20
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 910
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
fluoropiperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 21
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 893
biphenyl]-2-yl)methyl)piperazin-1-yl)-
N-((4-((morpholin-2-ylmethyl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-
b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 22
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 907
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-
methylmorpholin-2-yl)methyl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 23
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 920
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-hydroxy-
4-methylcyclohexyl)methyl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 24
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 911
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
fluorotetrahydro-2H-pyran-4-yl)methoxy)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 25
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 921
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-hydroxy-
4-methylcyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 26
N-((3-chloro-4-((4-fluoro-1-(oxetan-3-yl)piperidin-4- 955
yl)methoxy)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-
3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 27
N-((3-chloro-4-((4-fluoro-1-methylpiperidin-4- 913
yl)methoxy)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-
3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-
yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 28
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 961
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
morpholinocyclohexyl)amino)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 29
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 906
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-
hydroxycyclohexyl)methyl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 30
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 920
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-
methoxycyclohexyl)methyl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3 -b]pyrrolo [3,2-
e]pyridin-1(5H)-yl)benzamide 31
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 941
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((1-
(methylsulfonyl)pyrrolidin-3-yl)amino)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3 -b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 32
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 961
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3 -nitro-4-(((1-
(tetrahydro-2H-pyran-4-yl)pyrrolidin-3-
yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-
b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 33
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 962
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
morpholinocyclohexyl)oxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 34
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 907
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
hydroxycyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 35
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 921
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
methoxycyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 36
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 921
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-hydroxy-
4-methylcyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 37
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 942
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((1-
(methylsulfonyl)pyrrolidin-3-yl)oxy)-3-
nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-
e]pyridin-1(5H)-yl)benzamide 38
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 962
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((1-
(tetrahydro-2H-pyran-4-yl)pyrrolidin-3-
yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-
b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 39
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 908
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-
methylmorpholin-2-yl)methoxy)-3-nitrophenyl)sulfonyl)-
2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 40
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 926
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4,4-
difluorocyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-
2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 41
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 944
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4- (((1,4,4-
trifluorocyclohexyl)methyl)amino)phenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 42
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 927
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4,4-
difluorocyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 43
4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'- 945
biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-
((1,4,4-trifluorocyclohexyl)methoxy)phenyl)sulfonyl)-2-
(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide
BIOLOGICAL EXAMPLE 1
Bcl-2 Competition Binding (Fluorescence Polarization) Assay
[0229] The fluorescence-labeled 23 amino acid peptide BH3 was
purchased from CalBiochem (NLWAAQRYGRELRRMSDKFVD). An unbound
Fluorescein labeled BH3 peptide emits random light with respect to
the plane of polarization plane of excited light, resulting in a
lower polarization degree (mP) value. When the peptide is bound to
Bcl-2, the complex tumble slower and the emitted light can have a
higher level of polarization, resulting in a higher mP value. This
binding assay was performed in 96-well plate and with each assay
contained 15 and 30nM of labeled peptide and purified Bcl-2 protein
(purchased from R&D Systems, Inc). The assay buffer contained
20 mM Hepes (pH 7.0), 50 mM KCl, 5 mM MgCl.sub.2, 20 mM
Na.sub.2MoO.sub.4, 0.1 mg/ml Bovine Gamma Globulin and 0.01% NP40.
Compounds were diluted in DMSO and added to the final assay with
concentration range from 20 uM to 2 nM. The polarization degree
(mP) value was determined by BioTek Synergy II with background
subtraction after 3 hours of incubation at room temperature.
IC.sub.50 was calculated using Prism software with sigmoidal
dose-response curve fitting. ABT-737 was used as reference
compound. Such assays, carried out with a range of doses of test
compounds, allow the determination of an approximate IC.sub.50
value. Although the inhibitory properties of the compounds of the
present invention vary with structural change as expected, the
activity generally exhibited by these agents is in the range of
IC.sub.50=0.1-1000 nM. The following table lists the IC.sub.50
values of certain compounds of the invention.
TABLE-US-00002 Example No. BCL-2 ABT-199 <10 nM ABT-263 <10
nM 4 <10 nM 6 <10 nM 7 <10 nM 8 <10 nM 12 <10 nM 13
<10 nM 16 <10 nM 11 <10 nM 14 <10 nM 17 <10 nM 15
<10 nM 19 <10 nM 18 <10 nM
BIOLOGICAL EXAMPLE 2
In Vitro Anti-Proliferation Assay in BCL-2Dependent Acute
Lymphoblastic Leukemia Cell Line RS4; 11
[0230] Cell antiproliferation is assayed by PerkinElmer ATPlite.TM.
Luminescence Assay System. Briefly, the various test cancer cell
lines are plated at a density of about 1.times.10.sup.4 cells per
well in Costar 96-well plates, and are incubated with different
concentrations of compounds for about 72 hours in medium
supplemented with 5% FBS. One lyophilized substrate solution vial
is then reconstituted by adding 5 mL of substrate buffer solution,
and is agitated gently until the solution is homogeneous. About 50
.mu.L of mammalian cell lysis solution is added to 100 .mu.L of
cell suspension per well of a microplate, and the plate is shaken
for about five minutes in an orbital shaker at .about.700 rpm. This
procedure is used to lyse the cells and to stabilize the ATP. Next,
50 .mu.L substrate solution is added to the wells and microplate is
shaken for five minutes in an orbital shaker at .about.700 rpm.
Finally, the luminescence is measured by a PerkinElmer
TopCount.RTM. Microplate Scintillation Counter. Such assays,
carried out with a range of doses of test compounds, allow the
determination of the cellular anti-antiproliferative IC.sub.50 of
the compounds of the present invention. The following table lists
the IC.sub.50 values of certain compounds of the invention.
TABLE-US-00003 Example No. RS4; 11 IC.sub.50 4 <15 nM 12 <15
nM
Biological Example 3 In Vitro Anti-Proliferation Assay in BCL-XL
Dependent Lines Small Cell Lung Cancer Cell Line H146
[0231] Cell antiproliferation is assayed by PerkinElmer ATPlite.TM.
Luminescence Assay System. Briefly, the various test cancer cell
lines are plated at a density of about 1.times.10.sup.4 cells per
well in Costar 96-well plates, and are incubated with different
concentrations of compounds for about 72 hours in medium
supplemented with 5% FBS. One lyophilized substrate solution vial
is then reconstituted by adding 5 mL of substrate buffer solution,
and is agitated gently until the solution is homogeneous. About 50
.mu.L of mammalian cell lysis solution is added to 100 .mu.L of
cell suspension per well of a microplate, and the plate is shaken
for about five minutes in an orbital shaker at .about.700 rpm. This
procedure is used to lyse the cells and to stabilize the ATP. Next,
50 .mu.L substrate solution is added to the wells and microplate is
shaken for five minutes in an orbital shaker at .about.700 rpm.
Finally, the luminescence is measured by a PerkinElmer
TopCount.RTM. Microplate Scintillation Counter. Such assays,
carried out with a range of doses of test compounds, allow the
determination of the cellular anti-antiproliferative IC.sub.50 of
the compounds of the present invention. The following table lists
the IC50 values of certain compounds of the invention in the BCL-XL
depedent cell line H146, indicating that Example 4, like ABT-199,
is a BCL-2 selective inhibitor.
TABLE-US-00004 Example No. H146 IC.sub.50 (nM) ABT-263 60.0 ABT-199
>2000 4 >2000 12 >2000
BIOLOGICAL EXAMPLE 4
In Vivo Xenograft Studies
[0232] Typically, athymic nude mice (CD-1 nu/nu) or SCID mice are
obtained at age 6-8 weeks from vendors and acclimated for a minimum
7-day period. The cancer cells are then implanted into the nude
mice. Depending on the specific tumor type, tumors are typically
detectable about two weeks following implantation. When tumor sizes
reach 100-200 mm.sup.3, the animals with appreciable tumor size and
shape are randomly assigned into groups of 8 mice each, including
one vehicle control group and treatment groups. Dosing varies
depending on the purpose and length of each study, which typically
proceeds for about 3-4 weeks. Tumor sizes and body weight are
typically measured three times per week. In addition to the
determination of tumor size changes, the last tumor measurement is
used to generate the tumor size change ratio (T/C value), a
standard metric developed by the National Cancer Institute for
xenograft tumor evaluation. In most cases, % T/C values are
calculated using the following formula: %
T/C=100.times..DELTA.T/.DELTA.C if .DELTA.T>0. When tumor
regression occurred (.DELTA.T<0), however, the following formula
is used: % T/T0=100.times..DELTA.T/T0. Values of <42% are
considered significant.
Sequence CWU 1
1
1121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu
Arg Arg Met Ser1 5 10 15Asp Lys Phe Val Asp 20
* * * * *