U.S. patent application number 13/841349 was filed with the patent office on 2014-03-20 for processes for preparing tubulysin derivatives and conjugates thereof.
This patent application is currently assigned to ENDOCYTE, INC.. The applicant listed for this patent is ENDOCYTE, INC.. Invention is credited to Paul J. KLEINDL, Christopher P. LEAMON, Hari Krishna R. SANTHAPURAM, Iontcho R. VLAHOV, Fei YOU.
Application Number | 20140080175 13/841349 |
Document ID | / |
Family ID | 49261307 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140080175 |
Kind Code |
A1 |
VLAHOV; Iontcho R. ; et
al. |
March 20, 2014 |
PROCESSES FOR PREPARING TUBULYSIN DERIVATIVES AND CONJUGATES
THEREOF
Abstract
The invention described herein pertains to processes for
preparing tubulysin derivatives, conjugates of tubulysins, and
intermediates therefore.
Inventors: |
VLAHOV; Iontcho R.; (West
Lafayette, IN) ; SANTHAPURAM; Hari Krishna R.; (West
Lafayette, IN) ; KLEINDL; Paul J.; (Lebanon, IN)
; LEAMON; Christopher P.; (West Lafayette, IN) ;
YOU; Fei; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENDOCYTE, INC.; |
|
|
US |
|
|
Assignee: |
ENDOCYTE, INC.
West Lafayette
IN
|
Family ID: |
49261307 |
Appl. No.: |
13/841349 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61617386 |
Mar 29, 2012 |
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61684450 |
Aug 17, 2012 |
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61771451 |
Mar 1, 2013 |
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61794720 |
Mar 15, 2013 |
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Current U.S.
Class: |
435/68.1 ;
530/323; 530/331 |
Current CPC
Class: |
C07K 7/02 20130101; C07F
7/188 20130101; C07D 277/56 20130101; C07F 7/1892 20130101; C07K
5/02 20130101; C07D 417/12 20130101; C07D 213/76 20130101; C07D
417/14 20130101; C07D 213/71 20130101; C07C 241/02 20130101 |
Class at
Publication: |
435/68.1 ;
530/323; 530/331 |
International
Class: |
C07K 7/02 20060101
C07K007/02; C07K 5/02 20060101 C07K005/02 |
Claims
1. A process for preparing a compound of the formula ##STR00187##
or a salt or solvate thereof, wherein Ar.sub.1 is optionally
substituted aryl or optionally substituted heteroaryl; Ar.sub.2 is
optionally substituted aryl or optionally substituted heteroaryl; L
is selected from the group consisting of
--(CR.sup.2).sub.pCR.sup.aR.sup.b--, ##STR00188## where p is an
integer from about 1 to about 3, m is an integer from about 1 to
about 4, and * indicates the points of attachment; R.sup.a,
R.sup.b, and R are each independently selected in each instance
from the group consisting of hydrogen and alkyl; or any two of
R.sup.a, R.sup.b, and R are taken together with the attached carbon
atom(s) to form a carbocyclic ring; R.sub.Ar represents 0 to 4
substituents selected from the group consisting of amino, or
derivatives thereof, hydroxy or derivatives thereof, halo, thio or
derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof; Y is acyloxy or
R.sub.12O; R.sub.3 is optionally substituted alkyl; R.sub.4 is
optionally substituted alkyl or optionally substituted cycloalkyl;
R.sub.5 and R.sub.6 are each independently selected from the group
consisting of optionally substituted alkyl and optionally
substituted cycloalkyl; R.sub.7 is optionally substituted alkyl;
R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and n is 1, 2, 3, or 4; wherein the
process comprises the step of treating a compound of formula A with
triethylsilyl chloride and imidazole in an aprotic solvent, where
R.sub.8 is C1-C6 unbranched alkyl ##STR00189## or the step of
treating a compound of formula B with a base and a compound of the
formula ClCH.sub.2OC(O)R.sub.2 in an aprotic solvent at a
temperature from about -78.degree. C. to about 0.degree. C.;
wherein the molar ratio of the compound of the formula
ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from about 1 to
about 1.5, where R.sub.8 is C1-C6 unbranched alkyl ##STR00190## or
the steps of a) preparing a compound of formula (E1) from a
compound of formula (E), where X.sub.1 is a leaving group
##STR00191## and b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1, where
R.sub.8 is C1-C6 unbranched alkyl ##STR00192## or the step of
treating compound of formula D with a hydrolase enzyme or with a
trialkyltin hydroxide, where R.sub.8 is C1-C6 unbranched alkyl
##STR00193## or the step of treating a compound of formula F1 with
a non-basic fluoride reagent ##STR00194## or the step of treating a
compound of formula G1 with an acylating agent of formula
R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving group ##STR00195##
or the steps of c) forming an active ester intermediate from a
compound of formula H1 ##STR00196## and d) reacting the active
ester intermediate with a compound of the formula I ##STR00197## or
combinations thereof.
2. The process of claim 1 comprising the step of treating a
compound of formula A with triethylsilyl chloride and imidazole in
an aprotic solvent, where R.sub.8 is C1-C6 unbranched alkyl
##STR00198##
3. The process of claim 1 comprising the step of treating a
compound of formula B with a base and a compound of the formula
ClCH.sub.2OC(O)R.sub.2 in an aprotic solvent at a temperature from
about -78.degree. C. to about 0.degree. C.; wherein the molar ratio
of the compound of the formula ClCH.sub.2OC(O)R.sub.2 to the
compound of formula B from about 1 to about 1.5, where R.sub.8 is
C1-C6 unbranched alkyl ##STR00199##
4. The process of claim 1 comprising the steps of a) preparing a
compound of formula (E1) from a compound of formula E, where
X.sub.1 is a leaving group ##STR00200## and b) treating a compound
of formula C under reducing conditions in the presence of the
compound of formula E1, where R.sub.8 is C1-C6 unbranched alkyl
##STR00201##
5. The process of claim 1 comprising the step of treating a
compound of formula D with a hydrolase enzyme or a trialkyltin
hydroxide, where R.sub.8 is C1-C6 unbranched alkyl ##STR00202##
6. The process of claim 1 comprising the step of treating a
compound of formula G1 with an acylating agent of formula
R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving group
##STR00203##
7. The process of claim 1 comprising the steps of c) forming an
active ester intermediate from a compound of formula H1
##STR00204## and d) reacting the active ester intermediate with a
compound of the formula I ##STR00205##
8. The process of claim 1 wherein L is ##STR00206##
9. The process of claim 1 wherein O-L-S is ##STR00207##
10. The process of claim 1 wherein O-L-S is ##STR00208##
11. The process of claim 1 wherein Y is acyloxy.
12. The process of claim 12 wherein R.sub.2 is C1-C8 alkyl or C3-C8
cycloalkyl.
13. The process of claim 1 wherein R.sub.2 is
CH.sub.2CH(CH.sub.3).sub.2, CH.sub.2CH.sub.2CH.sub.3,
CH.sub.2CH.sub.3, CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
14. The process of claim 1 wherein R.sub.3 is C1-C4 alkyl.
15. The process of claim 1 wherein Ar.sub.1 is optionally
substituted aryl.
16. The process of claim 1 wherein R.sub.4 is C1-C8 alkyl or C3-C8
cycloalkyl.
17. The process of claim 1 wherein R.sub.5 is branched C3-C6 or
C3-C8 cycloalkyl.
18. The process of claim 1 wherein R.sub.6 is branched C3-C6 or
C3-C8 cycloalkyl.
19. The process of claim 1 wherein R.sub.7 is C1-C6 alkyl.
20. The process of claim 1 wherein Ar.sub.2 is optionally
substituted heteroaryl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit, under 35 U.S.C.
.sctn.119(e), of U.S. Provisional Application No. 61/617,386, filed
Mar. 29, 2012, Provisional Application No. 61/684,450, filed Aug.
17, 2012, Provisional Application No. 61/771,451, filed Mar. 1,
2013, and U.S. Provisional Application 61/794,720, filed Mar. 15,
2013, the entirety of each of the disclosures of which are hereby
incorporated herein by reference.
TECHNICAL FIELD
[0002] The invention described herein pertains to processes for
preparing tubulysin derivatives, conjugates of tubulysins, and
intermediates therefore.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] The tubulysins are members of a new class of natural
products isolated from myxobacterial species (F. Sasse, et al., J.
Antibiot. 2000, 53, 879-885). As cytoskeleton interacting agents,
the tubulysins are mitotic poisons that inhibit tubulin
polymerization and lead to cell cycle arrest and apoptosis (H.
Steinmetz, et al., Chem. Int. Ed. 2004, 43, 4888-4892; M. Khalil,
et al., ChemBioChem. 2006, 7, 678-683; G. Kaur, et al., Biochem. J.
2006, 396, 235-242). Tubulysins are extremely potent cytotoxic
molecules, and exceed the cell growth inhibition of many other
clinically relevant traditional chemotherapeutics, including
epothilones, paclitaxel, and vinblastine. Furthermore, they are
potent against multidrug resistant cell lines (A. Domling, et al.,
Mol. Diversity. 2005, 9, 141-147). These compounds show high
cytotoxicity tested against a panel of cancer cell lines with
IC.sub.50 values in the low picomolar range; thus, they are of
interest as potential anticancer therapeutics. However, tubulysins
have been reported to exhibit a narrow, or in some cases
nonexistent, therapeutic window such that disease treatment with
tubulysins is hampered by toxicity and other unwanted side effects.
Accordingly, tubulysins have been conjugated with targeting agents
to improve their therapeutic window. A total synthesis of tubulysin
D possessing C-terminal tubuphenylalanine (R.sub.A=H) (H. Peltier,
et al., J. Am. Chem. Soc. 2006, 128, 16018-16019) has been
reported. Recently, a modified synthetic protocol toward the
synthesis of tubulysin B (R.sub.A=OH) (O. Pando, et at., Org. Lett.
2009, 11, 5567-5569) has been reported. However, attempts to follow
the published procedures to provide larger quantities of tubulysins
were unsuccessful, being hampered in part by low yields, difficult
to remove impurities, the need for expensive chromatographic steps,
and/or the lack of reproducibility of several steps. The interest
in using tubulysins for anticancer therapeutics accents the need
for reliable and efficient processes for preparing tubulysins, and
analogs and derivatives thereof. Therefore, there is a need for
tubulysin derivatives, tubulysin analogs, and other tubulysin
conjugate intermediates that are useful for preparing such targeted
conjugates.
[0004] Tubulysin derivatives useful for preparing vitamin receptor
binding tubulysin conjugates (also referred to herein as tubulysin
linker derivatives) are described herein. Structurally, tubulysin
linker derivatives include linear tetrapeptoid backbones, including
illustrative compounds having the following formula
##STR00001##
or a salt thereof, wherein
[0005] Ar.sub.1 is optionally substituted aryl or optionally
substituted heteroaryl;
[0006] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0007] L is selected from the group consisting of
[0008] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00002##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0009] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring; R.sub.Ar
represents hydrogen, or 1 to 4 substituents each independently
selected from the group consisting of amino or derivatives thereof,
hydroxy or derivatives thereof, halo, thio or derivatives thereof,
nitro, sulfonic acids and derivatives thereof, carboxylic acids and
derivatives thereof, and alkyl, heteroalkyl, aryl, arylalkyl,
arylheteroalkyl, heteroaryl, heteroarylalkyl, and
heteroarylheteroalkyl, each of which is optionally substituted;
[0010] Y is R.sub.2C(O)O or R.sub.12O;
[0011] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0012] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0013] R.sub.3 is optionally substituted alkyl;
[0014] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0015] R.sub.7 is optionally substituted alkyl;
[0016] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted;
[0017] R.sub.Ar represents hydrogen, or 1 to 4 substituents each
independently selected from the group consisting of amino or
derivatives thereof, hydroxy or derivatives thereof, halo, thio or
derivatives thereof, nitro, sulfonic acids and derivatives thereof,
carboxylic acids and derivatives thereof, and alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, and heteroarylheteroalkyl; and n is 1, 2, 3, or
4.
[0018] In another embodiment, the tubulysin linker derivative has
formula T1
##STR00003##
or a salt thereof. In another embodiment, the tubulysin linker
derivative has formula T2
##STR00004##
or a salt thereof.
[0019] In another embodiment, in any of the embodiments described
herein Ar.sup.2 is optionally substituted aryl.
[0020] In another embodiment, in any of the embodiments described
herein Ar.sup.2 is optionally substituted heteroaryl.
[0021] Another illustrative group of tubulysins described herein
are more particularly comprised of one or more non-naturally
occurring or hydrophobic amino acid segments, such as N-methyl
pipecolic acid (Mep), isoleucine (Ile),
##STR00005##
and analogs and derivative of each of the foregoing. Derivatives
and analogs of tubuvaline include compounds of the following
formula,
##STR00006##
wherein R.sub.4, R.sub.5 and R.sub.6 are as described in any of the
embodiments described herein. Derivatives and analogs of
tubutyrosine or tubuphenylalanine include compounds having
formula,
##STR00007##
wherein R.sub.3 and Ar.sub.1 are as described in any of the
embodiment described herein. A common feature in the molecular
architecture of potent natural occurring tubulysins is the acid
and/or base sensitive N-acyloxymethyl substituent (or a N,O-acetal
of formaldehyde) represented by R.sub.2CO.sub.2CH.sub.2 in the
formula (T1).
[0022] In another embodiment, the compounds described herein are
NHNH--C(O)O-L-SS-Ar.sub.2 derivatives of naturally occurring
tubulysins. An illustrative group of tubulysin derivatives
described herein are those having formula 1.
##STR00008##
[0023] Formula 1, Structures of several tubulysin derivatives
TABLE-US-00001 Tubulysin R.sub.A R.sub.2 A OH
CH.sub.2CH(CH.sub.3).sub.2 B OH CH.sub.2CH.sub.2CH.sub.3 C OH
CH.sub.2CH.sub.3 D H CH.sub.2CH(CH.sub.3).sub.2 E H
CH.sub.2CH.sub.2CH.sub.3 F H CH.sub.2CH.sub.3 G OH
CH.dbd.C(CH.sub.3).sub.2 H H CH.sub.3 I OH CH.sub.3
[0024] Processes for preparing tubulysins, and analogs and
derivatives thereof, are also described in WO 2012/019123, the
disclosure of which is incorporated herein by reference in its
entirety.
[0025] The formation of tubulysins conjugated to vitamin receptor
binding moieties for targeted and/or selective delivery to cell
populations expressing, overexpressing or selectively expressing
cell surface vitamin receptors necessitates further modification of
the highly toxic tubulysins. Described herein are improved
processes for making natural tubulysins analogs or derivatives,
which are useful for preparing vitamin receptor binding tubulysin
conjugates including compounds of formula (T) and formula (I).
Vitamin receptor binding conjugates of tubulysins are described in
U.S. Patent Publication 2010/0048490, the disclosure of which is
incorporated herein by reference in its entirety.
[0026] In one illustrative embodiment of the invention, processes
for derivatives or analogs of natural tubulysins including
compounds of formula (T). In another embodiment, vitamin receptor
binding conjugates of tubulysins are described. The processes
include one or more steps described herein. In another embodiment,
a process is described for preparing a compound of formula B,
wherein R.sub.5 and R.sub.6 are as described in the various
embodiments herein, such as each being independently selected from
optionally substituted alkyl or optionally substituted cycloalkyl;
and R.sub.8 is C1-C6 n-alkyl; wherein the process comprises the
step of treating a compound of formula A with a silylating agent,
such as triethylsilyl chloride, and a base, such as imidazole in an
aprotic solvent.
##STR00009##
It is to be understood that R.sub.5 and R.sub.6 may each include
conventional protection groups on the optional substituents.
[0027] In another embodiment, a process is described for preparing
a compound of formula C, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as each being
independently selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.8 is C1-C6 n-alkyl; and
R.sub.2 is as described in the various embodiments herein, such as
being selected from optionally substituted alkyl or optionally
substituted cycloalkyl; wherein the process comprises the step of
treating a compound of formula B with a base and a compound of the
formula ClCH.sub.2OC(O)R.sub.2 in an aprotic solvent at a
temperature below ambient temperature, such as in the range from
about -78.degree. C. to about 0.degree. C.; wherein the molar ratio
of the compound of the formula ClCH.sub.2OC(O)R.sub.2 to the
compound of formula B is from about 1 to about 1.5.
##STR00010##
It is to be understood that R.sub.2, R.sub.5 and R.sub.6 may each
include conventional protection groups on the optional
substituents.
[0028] In another embodiment, a process is described for preparing
a compound of formula D, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.8 is C1-C6 n-alkyl; R.sub.2 is as described in
the various embodiments herein, such as being selected from
optionally substituted alkyl or optionally substituted cycloalkyl;
and R.sub.7 is optionally substituted alkyl; wherein the process
comprises the steps of
[0029] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E; and
[0030] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1.
##STR00011##
It is to be understood that R.sub.2, R.sub.5, R.sub.6, and R.sub.7
may each include conventional protection groups on the optional
substituents.
[0031] In another embodiment, a process is described for preparing
a compound of formula F, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating compound D with a hydrolase enzyme.
##STR00012##
[0032] In another embodiment, a process is described for preparing
a compound of formula F, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating compound D with a trialkyltin hydroxide (e.g. trimethyltin
hydroxide). It is to be understood that R.sub.2, R.sub.5, R.sub.6,
and R.sub.7 may each include conventional protection groups on the
optional substituents.
[0033] In another embodiment, a process is described for preparing
a compound of formula AF, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
contacting compound D with an alcohol, R.sub.12OH, where R.sub.12
is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and a transesterification catalyst. In
one embodiment the transesterification catalyst is trifluoroacetic
acid (TFA). In another embodiment, the transesterification catalyst
is selected from the group consisting of
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the transesterification catalyst
is(R.sub.13).sub.2SnO. Illustrative examples of R.sub.13 include
methyl, n-butyl, n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl,
phenethyl, and benzyl.
##STR00013##
It is to be understood that R.sub.5, R.sub.6, R.sub.12, and R.sub.7
may each include conventional protection groups on the optional
substituents.
[0034] In another embodiment, a process is described for preparing
a compound of formula G, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating the silyl ether of compound F with a non-basic fluoride
containing reagent.
##STR00014##
It is to be understood that R.sub.2, R.sub.5, R.sub.6, and R.sub.7
may each include conventional protection groups on the optional
substituents.
[0035] In another embodiment, a process is described for preparing
a compound of formula AG, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
contacting compound F with an alcohol, R.sub.12OH, where R.sub.12
is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and a transesterification catalyst. In
one embodiment the transesterification catalyst is trifluoroacetic
acid (TFA). In another embodiment, the transesterification catalyst
is selected from the group consisting of
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the transesterification catalyst is
(R.sub.13).sub.2SnO. Illustrative examples of R.sub.13 are methyl,
n-butyl, n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl,
and benzyl.
##STR00015##
It is to be understood that R.sub.2, R.sub.5, R.sub.6, R.sub.7, and
R.sub.12 may each include conventional protection groups on the
optional substituents.
[0036] In another embodiment, a process is described for preparing
a compound of formula BG, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.12 is as described in the
various embodiments herein, such as being selected from alkyl,
alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
arylalkyl or heteroarylalkyl, each of which is optionally
substituted; and R.sub.2 is optionally substituted alkyl; wherein
the process comprises the step of contacting compound AF with a
metal hydroxide or carbonate. Illustrative examples of a metal
hydroxide or carbonate include LiOH, Li.sub.2CO.sub.3, NaOH,
Na.sub.2CO.sub.3, KOH, K.sub.2CO.sub.3, Ca(OH).sub.2, CaCO.sub.3,
Mg(OH).sub.2, MgCO.sub.3, and the like.
##STR00016##
It is to be understood that R.sub.5, R.sub.6, R.sub.7, and R.sub.12
may each include conventional protection groups on the optional
substituents.
[0037] In another embodiment, a process is described for preparing
a compound of formula H, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 and R.sub.4 are as described in the various
embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.7
is optionally substituted alkyl; wherein the process comprises the
step of treating a compound of formula G with an acylating agent of
formula R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving group.
##STR00017##
It is to be understood that R.sub.2, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 may each include conventional protection groups on the
optional substituents.
[0038] In another embodiment, a process is described for preparing
a compound of formula AH, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 and R.sub.4 are as described in the various
embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; R.sub.12 is
as described in the various embodiments herein, such as being
selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and R.sub.7 is optionally substituted
alkyl; wherein the process comprises the step of treating a
compound of formula BG with an acylating agent of formula
R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving group.
##STR00018##
It is to be understood that R.sub.4, R.sub.5, R.sub.6, and R.sub.7
may each include conventional protection groups on the optional
substituents.
[0039] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (T1), wherein Ar.sub.1 is
optionally substituted aryl; Ar.sub.2 is optionally substituted
aryl or optionally substituted heteroaryl; L is selected from the
group consisting of
[0040] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00019##
where
[0041] p is an integer from about 1 to about 3, m is an integer
from about 1 to about 4, and * indicates the points of
attachment;
[0042] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0043] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof; R.sub.1 is hydrogen, optionally substituted alkyl,
optionally substituted arylalkyl or a pro drug forming group;
R.sub.5 and R.sub.6 are as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.3 is optionally
substituted alkyl; R.sub.2 and R.sub.4 are as described in the
various embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.2
is optionally substituted alkyl;
[0044] wherein the process comprises the step of forming an active
ester intermediate from a compound of formula H; and reacting the
active ester intermediate with a compound of the formula I to give
a compound of the formula T.
##STR00020##
It is to be understood that Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.2,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 may each include
conventional protection groups on the optional substituents.
[0045] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (T2), wherein Ar.sub.1 is
optionally substituted aryl; Ar.sub.2 is optionally substituted
aryl or optionally substituted heteroaryl; L is selected from the
group consisting of
[0046] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00021##
where
[0047] p is an integer from about 1 to about 3, m is an integer
from about 1 to about 4, and * indicates the points of
attachment;
[0048] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0049] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0050] R.sub.1 is hydrogen, optionally substituted alkyl,
optionally substituted arylalkyl or a pro-drug forming group;
R.sub.5 and R.sub.6 are as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.3 is optionally
substituted alkyl; R.sub.2 and R.sub.4 are as described in the
various embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.7
is optionally substituted alkyl; wherein the process comprises the
step of contacting compound T, with an alcohol, R.sub.12OH, where
R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and a transesterification catalyst.
[0051] In one embodiment the transesterification catalyst is
trifluoroacetic acid (TFA). In another embodiment, the
transesterification catalyst is selected from the group consisting
of (R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sup.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the transesterification catalyst is
(R.sub.13).sub.2SnO. Illustrative examples of R.sub.13 are methyl,
n-butyl, n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl,
and benzyl. It is to be understood that Ar.sub.1, Ar.sub.2,
R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.12
may each include conventional protection groups on the optional
substituents.
##STR00022##
[0052] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (T2), wherein Ar.sub.1 is
optionally substituted aryl; Ar.sub.2 is optionally substituted
aryl or optionally substituted heteroaryl; L is selected from the
group consisting of
[0053] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00023##
where
[0054] p is an integer from about 1 to about 3, m is an integer
from about 1 to about 4, and * indicates the points of
attachment;
[0055] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0056] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0057] R.sub.1 is hydrogen, optionally substituted alkyl,
optionally substituted arylalkyl or a pro-drug forming group;
R.sub.5 and R.sub.6 are as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.3 is optionally
substituted alkyl; R.sub.2 and R.sub.4 are as described in the
various embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.7
is optionally substituted alkyl;
[0058] wherein the process comprises the step of forming an active
ester intermediate from a compound of formula AH; and reacting the
active ester intermediate with a compound of the formula I to give
a compound of the formula AT.
##STR00024##
It is to be understood that Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.12,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 may each include
conventional protection groups on the optional substituents.
DETAILED DESCRIPTION
[0059] In one embodiment, a process is described for preparing a
compound of formula B, wherein R.sub.5 and R.sub.6 are as described
in the various embodiments herein, such as being selected from
optionally substituted alkyl or optionally substituted cycloalkyl;
and R.sub.8 is C1-C6 n-alkyl; wherein the process comprises the
step of treating a compound of formula A with triethylsilyl
chloride and imidazole in an aprotic solvent.
##STR00025##
[0060] In the previously reported preparations of the intermediate
silyl ether of formula 2, use of a large excess of triethylsilyl
trifluoromethylsulfonate (TESOTf) and lutidine is described (see,
for example, Peltier, et al., 2006). It was found that the reported
process makes it necessary to submit the product of the reaction to
a chromatographic purification step. Contrary to that reported, it
has been surprisingly discovered herein that the less reactive
reagent TESCl may be used. It has also been surprisingly discovered
herein that although TESCl is a less reactive reagent, it may
nonetheless be used in nearly stoichiometric amounts in the
processes described herein. It is appreciated herein that the use
of the less reactive TESCl may also be advantageous when the
process is performed on larger scales, where higher reactivity
reagents may represent a safety issue. It has also been discovered
that the use of TESCl in nearly stoichiometric amounts renders the
chromatographic purification step unnecessary. In an alternative of
the embodiment, the process is performed without subsequent
purification. In another alternative of the foregoing embodiments,
and each additional embodiment described herein, R.sub.5 is
isopropyl. In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.6 is sec-butyl.
In another alternative of the foregoing embodiments, and each
additional embodiment described herein, R.sub.8 is methyl. In
another alternative of the foregoing embodiments, and each
additional embodiment described herein, the silyl ether is TES.
[0061] In an illustrative example of the processes described
herein, a process for preparing the silyl ether 4 in high yield is
described wherein compound 1 is treated with 1.05 equivalent of
TESCl and 1.1 equivalent of imidazole.
##STR00026##
In one alternative of the foregoing example, the compound 4 is not
purified by chromatography.
[0062] In another embodiment, a process is described for preparing
a compound of formula C, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.8 is
C1-C6 n-alkyl; and R.sub.2 is selected from the group consisting of
optionally substituted alkyl and optionally substituted cycloalkyl;
wherein the process comprises the step of treating a compound of
formula B with from about 1 equivalent to about 1.5 equivalent of
base and from about 1 equivalent to about 1.5 equivalent of a
compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.
##STR00027##
[0063] In another embodiment, the process of the preceding
embodiment is described wherein the compounds of formulae B and C
have the stereochemistry shown in the following scheme for B' and
C'.
##STR00028##
[0064] In another illustrative embodiment, the ether analog of C'
can be used to prepare the tubulysins, the tubulysin conjugates,
and the tubulysin linker compounds described herein.
[0065] In another illustrative embodiment, the process of any one
of the preceding embodiments is described wherein about 1
equivalent to about 1.3 equivalent of a compound of the formula
ClCH.sub.2OC(O)R.sub.2 is used. In another illustrative example,
the process of any one of the preceding embodiments is described,
wherein about 1.2 equivalent of a compound of the formula
ClCH.sub.2OC(O)R.sub.2 is used. In another illustrative example,
the process of any one of the preceding embodiments is described
wherein R.sub.2 is n-propyl. In another alternative of the
foregoing embodiments, and each additional embodiment described
herein, R.sub.2 is CH.sub.2CH(CH.sub.3).sub.2,
CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.3,
CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
[0066] In an illustrative example of the processes described
herein, a process for preparing the N,O-acetal 5 is described. In
another illustrative example, compound 2 is treated with 1.1
equivalent of potassium hexamethyldisilazane (KHMDS) and 1.2
equivalent of chloromethyl butanoate in a nonprotic solvent at
about -45.degree. C. In another illustrative example, the product
formed by any of the preceding examples may be used without
chromatographic purification.
##STR00029##
[0067] The ether analogs of compound 5 can also be used to prepare
the tubulysins, tubulysin linker compounds
##STR00030##
[0068] In another embodiment, a process is described for preparing
a compound of formula D, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and cycloalkyl; R.sub.8 is C1-C6 n-alkyl; R.sub.2
is selected from the group consisting of optionally substituted
alkyl and cycloalkyl; and R.sub.7 is optionally substituted alkyl;
wherein the process comprises the steps of
[0069] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E; and
[0070] b) treating a compound of formula C under reducing
conditions with the compound of formula E1.
##STR00031##
[0071] In one illustrative example, a mixture of compound 5 and the
pentafluorophenyl ester of D-N-methyl-pipecolic acid is reduced
using H.sub.2 and a palladium-on-charcoal catalyst (Pd/C) to yield
compound 6. It has been discovered herein that epimerization of the
active ester of pipecolic acid can occur during reaction or during
its preparation or during the reduction under the previously
reported reaction conditions. For example, contrary to prior
reports indicating that epimerization does not occur (see, for
example, Peltier, 2006), upon repeating those reported processes on
a larger scale it was found here that substantial amounts of
epimerized compounds were formed. In addition, it was discovered
herein that substantial amounts of rearrangement products formed by
the rearrangement of the butyryl group to compound 8a were formed
using the reported processes. Finally, it was discovered herein
that the typical yields of the desired products using the
previously reported processes were only about half of that
reported. It has been discovered herein that using
diisopropylcarbodiimide (DIC) and short reaction times lessens that
amount of both the unwanted by-product resulting from the
epimerization reaction and the by-product resulting from the
rearrangement reaction. In another alternative of the foregoing
embodiments, and each additional embodiment described herein, n is
3. In another alternative of the foregoing embodiments, and each
additional embodiment described herein, R.sub.7 is methyl.
[0072] In one illustrative example, it was found that limiting the
reaction time for the preparation of pentafluorophenyl
D-N-methyl-pipecolate to about 1 hour lessened the formation of the
diastereomeric tripeptide 9a. It has also been discovered that
using dry 10% Pd/C as catalyst, rather than a more typically used
wet or moist catalyst, lessens the amount of epimer 9 formed during
the reduction. It has also been discovered that using dry 10% P/C
and/or shorter reaction times also lessens the formation of
rearranged amide 8.
##STR00032##
[0073] In another embodiment the ether analogs of 5 and 6 can be
used to prepare the tubulysins, tubulysin conjugates, and tubulysin
linker compounds described herein. It has been previously reported
that removal of the protecting group from the secondary hydroxyl
group leads to an inseparable mixture of the desired product 5a and
a cyclic O,N-acetal side-product, 5b, as shown in the following
scheme.
##STR00033##
Further, upon repeating the reported process, it has been
discovered herein that removal of the methyl ester using basic
conditions, followed by acetylation of the hydroxyl group leads to
an additional previously unreported side-product, iso-7a. That
additional side-product is difficult to detect and difficult to
separate from the desired compound 7a. Without being bound by
theory, it is believed herein that iso-7a results from
rearrangement of the butyrate group from the N-hydroxymethyl group
to the secondary hydroxyl group, as shown below.
##STR00034##
[0074] It has been discovered that reordering the two deprotection
steps and using different conditions for each deprotection reaction
results in improved yields of compounds of formula H, such as
compound 7a, after introduction of the R.sub.4CO group on the
secondary hydroxyl group, as further described below.
[0075] In another embodiment, a process is described for preparing
a compound of formula AF, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
contacting compound D with an alcohol, R.sub.12OH, where R.sub.12
is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and a transesterification catalyst. In
one embodiment the transesterification catalyst is trifluoroacetic
acid (TFA). In another embodiment, the transesterification catalyst
is selected from the group consisting of
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the catalyst is (R.sub.13).sub.2SnO.
Illustrative examples of R.sub.13 are methyl, n-butyl, n-octyl,
phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.
##STR00035##
It is to be understood that R.sub.5, R.sub.6, R.sub.12, and R.sub.7
may each include conventional protection groups on the optional
substituents.
[0076] In another embodiment, a process is described for preparing
a compound of formula F, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.2 is
selected from the group consisting of optionally substituted alkyl
and optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating compound D with a hydrolase enzyme.
##STR00036##
In another embodiment, the preceding process wherein the treating
step comprises adding a solution of compound D in a water miscible
solvent to a buffered solution containing the hydrolase enzyme at a
rate which minimizes precipitation of the ester. In another
embodiment the ester is added over a period of from about 24 hours
to about 100 hours. In another embodiment the ester is added over a
period of from about 48 hours to about 100 hours. In another
alternative of the foregoing embodiments, and each additional
embodiment described herein, R.sub.8 is methyl. In another
embodiment, the embodiment of any one of the preceding embodiments
wherein the hydrolase enzyme is an esterase is described. In
another embodiment, the embodiment of any of the preceding
embodiments wherein the esterase is a pig liver esterase is
described.
[0077] In another embodiment, a process is described for preparing
a compound of formula F, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.2 is
selected from the group consisting of optionally substituted alkyl
and optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating compound D with a trialkyltin hydroxide. In one
illustrative embodiment, the trialkyltin hydroxide is trimethyltin
hydroxide.
[0078] In an illustrative example, a solution of compound 4 in
dimethyl sulfoxide (DMSO) is added over a period of 90 hours, to a
buffered solution of pig liver esterase. In another illustrative
example, the buffer is a phosphate buffer. In another illustrative
example, the solution of the enzyme has a pH of 6.5 to 8.5. In
another illustrative, example the solution of the enzyme has a pH
of 7.4 to 7.8. It is appreciated that the buffering material used
can be any buffer compatible with the hydrolase enzyme used to
remove the ester.
##STR00037##
[0079] In another illustrative example, a solution of methyl ester
6 and trimethyltin hydroxide in 1,2-dichloroethane was heated to
yield acid 7.
[0080] Tripeptide methyl ester 6 (reported in an earlier patent
application [1]) was treated with trimethyltin hydroxide to yield
corresponding acid 7. The triethylsilyl group was removed by
treatment with hydrogen flouride-triethyl amine complex, and
acetylated, resulting in acetyl-tripeptide acid 8.
##STR00038##
[0081] In another embodiment, a process is described for preparing
a compound of formula G, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.2 is
selected from the group consisting of optionally substituted alkyl
and optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
treating the silyl ether of compound F with a non basic fluoride
reagent. It has been discovered herein that use of basic conditions
can lead to the production of a by-product arising from the
rearrangement of the ester group to give compound G'.
##STR00039##
[0082] In an illustrative example, compound 7 is treated with
Et.sub.3N.3HF to cleave the TES-ether in the preparation of the
corresponding alcohol 6'. It is to be understood that other
non-basic fluoride reagents to cleave the silyl ether of compounds
F may be used in the methods and processes described herein,
including but not limited to pyridine.HF, and the like to cleave
the TES-ether.
[0083] In another embodiment, a process is described for preparing
a compound of formula AG, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; and R.sub.7 is optionally
substituted alkyl; wherein the process comprises the step of
contacting compound F with an alcohol, R.sub.12OH, where R.sub.12
is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and a transesterification catalyst. In
one embodiment the transesterification catalyst is trifluoroacetic
acid (TFA). In another embodiment, the transesterification catalyst
is selected from the group consisting of
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the catalyst is (R.sub.13).sub.2SnO.
Illustrative examples of R.sub.13 are methyl, n-butyl, n-octyl,
phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.
##STR00040##
It is to be understood that R.sub.2, R.sub.5, R.sub.6, R.sub.2, and
R.sub.12 may each include conventional protection groups on the
optional substituents.
[0084] In another embodiment, a process is described for preparing
a compound of formula BG, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 is as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.12 is as described in the
various embodiments herein, such as being selected from alkyl,
alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
arylalkyl or heteroarylalkyl, each of which is optionally
substituted; and R.sub.2 is optionally substituted alkyl; wherein
the process comprises the step of contacting compound AF with a
metal hydroxide or carbonate. Illustrative examples of a metal
hydroxide or carbonate include LiOH, Li.sub.2CO.sub.3, NaOH,
Na.sub.2CO.sub.3, KOH, K.sub.2CO.sub.3, Ca(OH).sub.2, CaCO.sub.3,
Mg(OH).sub.2, MgCO.sub.3, and the like.
##STR00041##
It is to be understood that R.sub.5, R.sub.6, R.sub.7, and R.sub.12
may each include conventional protection groups on the optional
substituents.
[0085] In another embodiment, a process is described for preparing
a compound of formula H, wherein R.sub.5 and R.sub.6 are each
independently selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.2
and R.sub.4 are independently selected from the group consisting of
optionally substituted alkyl and optionally substituted cycloalkyl;
and R.sub.7 is optionally substituted alkyl; wherein the process
comprises the step of treating a compound of formula G with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group. It is appreciated that the resulting product may
contain varying amounts of the mixed anhydride of compound H and
R.sub.4CO.sub.2H. In another embodiment, the process described in
the preceding embodiment further comprises the step of treating the
reaction product with water to prepare H, free of or substantially
free of anhydride. In another embodiment, the process of the
preceding embodiments wherein X.sub.2 is R.sub.4CO.sub.2, is
described. In another embodiment, the process of any one of the
preceding embodiments wherein R.sub.4 is C1-C4 alkyl is described.
In another alternative of the foregoing embodiments, and each
additional embodiment described herein, R.sub.4 is methyl. In
another embodiment, the process of any one of the preceding
embodiments wherein R.sub.6 is sec-butyl is described. In another
embodiment, the process of any one of the preceding embodiments
wherein R.sub.7 is methyl is described. In another embodiment, the
process of any one of the preceding embodiments wherein R.sub.5 is
iso-propyl is described.
##STR00042##
[0086] In an illustrative example, compound 6' is treated with
acetic anhydride in pyridine. It has been discovered herein that
shortening the time for this step of the process improves the yield
of compound H by limiting the amount of the previously undescribed
alternative acylation side products, such as formula 7a that are
formed. It is appreciated that the resulting product may contain
varying amounts of the mixed anhydride of 8 and acetic acid. In
another embodiment, treatment of the reaction product resulting
from the preceding step with water in dioxane yields compound 8,
free of or substantially free of anhydride. It is to be understood
that other solvents can be substituted for dioxane in the
hydrolysis of the intermediate mixed anhydride. Alternatively, the
step may be performed without solvent.
##STR00043##
[0087] In another embodiment, a process is described for preparing
a compound of formula AH, wherein R.sub.5 and R.sub.6 are as
described in the various embodiments herein, such as being selected
from optionally substituted alkyl or optionally substituted
cycloalkyl; R.sub.2 and R.sub.4 are as described in the various
embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; R.sub.12 is
as described in the various embodiments herein, such as being
selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; and R.sub.2 is optionally substituted
alkyl; wherein the process comprises the step of treating a
compound of formula BG with an acylating agent of formula
R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving group.
##STR00044##
It is to be understood that R.sub.4, R.sub.5, R.sub.6, and R.sub.2
may each include conventional protection groups on the optional
substituents.
[0088] In another embodiment, a process for preparing compound I is
described.
##STR00045##
where PG is a protecting group, LG is a leaving group, and
Ar.sub.1, Ar.sub.2, L, and R.sub.3 are as described in any of the
embodiments described herein.
[0089] In one illustrative example, mixed carbonate 11 was prepared
from 4-nitropyridyldisulfide ethanol 12 and 4-nitrophenyl
chloroformate as shown. Boc-Tut-hydrazide 13 was prepared from
corresponding acid 10 and coupled with mixed carbonate 11 to yield
activated Boc-Tut fragment 14.
##STR00046##
[0090] In another embodiment, a process is described for preparing
a tubulysin linker derivative T, wherein An is optionally
substituted aryl; Ar.sub.2 is optionally substituted aryl or
optionally substituted heteroaryl; L is selected from the group
consisting of
[0091] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00047##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0092] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0093] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0094] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl; R.sub.3 is optionally substituted alkyl;
R.sub.2 and R.sub.4 are independently selected from the group
consisting of optionally substituted alkyl and optionally
substituted cycloalkyl; and R.sub.7 is optionally substituted
alkyl;
[0095] wherein the process comprises the steps of
[0096] c) forming an active ester intermediate from a compound of
formula H; and
[0097] d) reacting the active ester intermediate with a compound of
the formula I.
##STR00048##
[0098] In one embodiment, compound H is treated with an excess
amount of active ester forming agent and pentafluorophenol to form
the pentafluorophenol ester of compound H, followed by removal of
the excess active ester forming agent prior to the addition of
compound I. In another alternative of the foregoing embodiments,
and each additional embodiment described herein, Ar.sub.1 is
phenyl. In another alternative of the foregoing embodiments, and
each additional embodiment described herein, Ar.sub.1 is
substituted phenyl. In another alternative of the foregoing
embodiments, and each additional embodiment described herein,
Ar.sub.1 is 4-substituted phenyl. In another alternative of the
foregoing embodiments, and each additional embodiment described
herein, Ar.sub.1 is R.sub.A-substituted phenyl. In another
alternative of the foregoing embodiments, and each additional
embodiment described herein, Ar.sub.1 is 4-hydroxyphenyl, or a
hydroxyl protected form thereof. In another alternative of the
foregoing embodiments, and each additional embodiment described
herein, Ar.sub.2 is substituted pyridyl. In another alternative of
the foregoing embodiments, and each additional embodiment described
herein, Ar.sub.2 is substituted 2-pyridyl. In another alternative
of the foregoing embodiments, and each additional embodiment
described herein, Ar.sub.2 is 3-nitro-2-pyridyl. In another
alternative of the foregoing embodiments, and each additional
embodiment described herein, R.sub.3 is methyl.
[0099] In an illustrative example, compound 8 is treated with an
excess amount of a polymeric version of a carbodiimide and
pentafluorophenol to form the pentafluorophenyl ester of 8, the
polymeric carbodiimide is removed by filtration; and compound 9 is
added to the solution to yield tubulysin B linker derivative 2. In
another embodiment, the process of any one of the preceding
embodiments wherein the polymeric carbodiimide is
polystyrene-CH.sub.2--N.dbd.C.dbd.N-cyclohexane (PS-DCC) is
described.
##STR00049##
[0100] In another embodiment, the ether analog of compound 8 can be
converted to the ether analog of compound 2, via the ether analog
of compound 15, where R is allyl, or
CH.sub.2(CH.sub.2).sub.nCH.sub.3, and n is 1, 2, 3, 4, 5, or 6.
##STR00050##
[0101] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (T2), wherein Ar.sub.1 is
optionally substituted aryl; Ar.sub.2 is optionally substituted
aryl or optionally substituted heteroaryl; L is selected from the
group consisting of
[0102] --(CR.sup.2).sub.pCR.sup.aR.sup.b--,
##STR00051##
where
[0103] p is an integer from about 1 to about 3, m is an integer
from about 1 to about 4, and * indicates the points of
attachment;
[0104] Ra, Rb, and R are each independently selected in each
instance from the group consisting of hydrogen and alkyl; or any
two of R.sup.a, R.sup.b, and R are taken together with the attached
carbon atom(s) to form a carbocyclic ring;
[0105] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0106] R.sub.1 is hydrogen, optionally substituted alkyl,
optionally substituted arylalkyl or a pro-drug forming group;
R.sub.5 and R.sub.6 are as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.3 is optionally
substituted alkyl; R.sub.2 and R.sub.4 are as described in the
various embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.7
is optionally substituted alkyl;
[0107] wherein the process comprises the step of contacting
compound T, with an alcohol, R.sub.12OH, where R.sub.12 is alkyl,
alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
arylalkyl or heteroarylalkyl, each of which is optionally
substituted; and a transesterification catalyst.
[0108] In one embodiment the transesterification catalyst is
trifluoroacetic acid (TFA). In another embodiment, the
transesterification catalyst is selected from the group consisting
of (R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted. In
another embodiment, the catalyst is (R.sub.13).sub.2SnO.
Illustrative examples of R.sub.13 are methyl, n-butyl, n-octyl,
phenyl, o-MeO-phenyl, p-MeO-phenyl, phenethyl, and benzyl. It is to
be understood that Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.2, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, and R.sub.12 may each include
conventional protection groups on the optional substituents.
##STR00052##
[0109] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (T2), wherein Ar.sub.1 is
optionally substituted aryl; Ar.sub.2 is optionally substituted
heteroaryl; L is selected from the group consisting of
[0110] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00053##
where
[0111] p is an integer from about 1 to about 3, m is an integer
from about 1 to about 4, and * indicates the points of
attachment;
[0112] Ra, Rb, and R are each independently selected in each
instance from the group consisting of hydrogen and alkyl; or any
two of R.sup.a, R.sup.b, and R are taken together with the attached
carbon atom(s) to form a carbocyclic ring;
[0113] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0114] R.sub.1 is hydrogen, optionally substituted alkyl,
optionally substituted arylalkyl or a pro-drug forming group;
R.sub.5 and R.sub.6 are as described in the various embodiments
herein, such as being selected from optionally substituted alkyl or
optionally substituted cycloalkyl; R.sub.3 is optionally
substituted alkyl; R.sub.2 and R.sub.4 are as described in the
various embodiments herein, such as being selected from optionally
substituted alkyl or optionally substituted cycloalkyl; and R.sub.7
is optionally substituted alkyl;
[0115] wherein the process comprises the step of forming an active
ester intermediate from a compound of formula AH; and reacting the
active ester intermediate with a compound of the formula Ito give a
compound of the formula AT.
##STR00054##
It is to be understood that Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.12,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 may each include
conventional protection groups on the optional substituents.
[0116] In another embodiment, a compound having formula D, wherein
the compound is free of or substantially free of a compound having
formula C-1 is described, where in R.sub.2, R.sub.5, R.sub.6,
R.sub.7, and R.sub.8 are as described in any of the embodiments
described herein. Without being bound by theory, it is believed
herein that compounds C-1 are formed from the corresponding
compounds C via an acyl transfer.
##STR00055##
[0117] In another embodiment, compound 4, free of or substantially
free of compound 8a and/or compound iso-6 is described. In another
embodiment, an optically pure form of compound 6 is formed.
##STR00056##
[0118] In another embodiment, a compound H, wherein the compound H
is free of or substantially free, of a compound having the formula
Oxazine-2 is described.
##STR00057##
[0119] In another embodiment, a compound F is described wherein
R.sub.2, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are as described in
the any of the embodiments described herein.
##STR00058##
[0120] In another embodiment, the compound having formula 7 is
described.
##STR00059##
[0121] In another embodiment a compound G, where the compound is
free of or substantially free of a compound G' is described,
wherein R.sub.2, R.sub.5, R.sub.6, and R.sub.7 are as described in
any of the embodiments described herein.
##STR00060##
[0122] In another embodiment, compound 6' is described, wherein
compound 6' is free of or substantially free of the isomer of G'
shown below
##STR00061##
[0123] In another embodiment, compound 8 is described, wherein
compound 8 is free of or substantially free of compound 8b is
described
##STR00062##
[0124] In another embodiment, a compound I is described wherein
Ar.sub.1, Ar.sub.2, R.sub.3 and L are as described in any of the
embodiments described herein.
##STR00063##
[0125] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, L is
--(CR.sub.2).sub.pCR.sup.aR.sup.b--;
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, ** indicates the attachment point to the
carbonyl group, and * indicates the point of attachment to
SAr.sub.2; R.sup.a, R.sup.b, and R are each independently selected
from the group consisting of hydrogen and alkyl; or any two of
R.sup.a, R.sup.b, and R are taken together with the attached carbon
atom(s) to form a carbocyclic ring. In another embodiment of each
of the embodiments described herein, R.sup.a and R.sup.b are
hydrogen. In another embodiment of each of the embodiments
described p is 1. In another embodiment of the foregoing
embodiments and each additional embodiment described herein R.sup.a
and R.sup.b are hydrogen and p is 1.
[0126] In another embodiment, a compound H is described wherein
R.sup.4 is Me and R.sup.2, R.sub.5, R.sub.6, and R.sub.2 are as
described in any of the embodiments described herein; and the
compound H is free of or substantially free of the compound H
wherein R.sub.4 and R.sub.2 are both Me.
##STR00064##
[0127] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.5 is
isopropyl.
[0128] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.6 is
sec-butyl.
[0129] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.8 is methyl.
[0130] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.2 is
CH.sub.2CH(CH.sub.3).sub.2, CH.sub.2CH.sub.2CH.sub.3,
CH.sub.2CH.sub.3, CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
[0131] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, n is 3.
[0132] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.7 is methyl.
[0133] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.8 is methyl.
[0134] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.4 is methyl.
[0135] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, Ar.sub.1 is phenyl. In
another alternative of the foregoing embodiments, and each
additional embodiment described herein, Ar.sub.1 is substituted
phenyl. In another alternative of the foregoing embodiments, and
each additional embodiment described herein, Ar.sub.1 is
4-substituted phenyl. In another alternative of the foregoing
embodiments, and each additional embodiment described herein,
Ar.sub.1 is R.sub.A-substituted phenyl. In another alternative of
the foregoing embodiments, and each additional embodiment described
herein, Ar.sub.1 is 4-hydroxyphenyl, or a hydroxyl protected form
thereof.
[0136] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.3 is methyl.
[0137] Illustrative embodiments of the invention are further
described by the following enumerated clauses:
[0138] 1. A process for preparing a compound of the formula
##STR00065##
or a salt or solvate thereof; wherein
[0139] Ar.sub.1 is optionally substituted aryl or optionally
substituted heteroaryl;
[0140] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0141] L is selected from the group consisting of
[0142] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00066##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0143] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0144] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0145] Y is acyloxy or R.sub.12O;
[0146] R.sub.3 is optionally substituted alkyl;
[0147] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0148] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0149] R.sub.7 is optionally substituted alkyl;
[0150] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted; and
[0151] n is 1, 2, 3, or 4;
[0152] wherein the process comprises
[0153] the step of treating a compound of formula A with
triethylsilyl chloride and imidazole in an aprotic solvent, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00067##
or
[0154] the step of treating a compound of formula B with a base and
a compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.; wherein the molar ratio of the compound of the
formula ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from
about 1 to about 1.5, where R.sub.8 is C1-C6 unbranched alkyl
##STR00068##
or
[0155] the steps of
[0156] a) preparing a compound of formula (E1) from a compound of
formula (E), where X.sub.1 is a leaving group
##STR00069##
and
[0157] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00070##
or
[0158] the step of treating compound of formula D with a hydrolase
enzyme or with a trialkyltin hydroxide, where R.sub.8 is C1-C6
unbranched alkyl
##STR00071##
or
[0159] the step of treating a compound of formula F1 with a
non-basic fluoride reagent
##STR00072##
or
[0160] the step of treating a compound of formula G1 with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00073##
or
[0161] the steps of
[0162] c) forming an active ester intermediate from a compound of
formula H1
##STR00074##
and
[0163] d) reacting the active ester intermediate with a compound of
the formula I
##STR00075##
or combinations thereof
[0164] 1A. A process for preparing a compound of the formula
##STR00076##
or a salt thereof, wherein
[0165] Ar.sub.1 is optionally substituted aryl or optionally
substituted heteroaryl;
[0166] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0167] L is selected from the group consisting of
[0168] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00077##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0169] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0170] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0171] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0172] R.sub.3 is optionally substituted alkyl;
[0173] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0174] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0175] R.sub.2 is optionally substituted alkyl; and
[0176] n is 1, 2, 3, or 4;
[0177] wherein the process comprises
[0178] the step of treating a compound of formula A with
triethylsilyl chloride and imidazole in an aprotic solvent, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00078##
or
[0179] the step of treating a compound of formula B with a base and
a compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.; wherein the molar ratio of the compound of the
formula ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from
about 1 to about 1.5, where R.sub.8 is C1-C6 unbranched alkyl
##STR00079##
or
[0180] the steps of
[0181] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E
##STR00080##
and
[0182] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00081##
or
[0183] the step of treating compound D with a hydrolase enzyme or
with a trialkyltin hydroxide, where R.sub.8 is C1-C6 unbranched
alkyl
##STR00082##
or
[0184] the step of treating the silyl ether of compound F with a
non-basic fluoride reagent
##STR00083##
or
[0185] the step of treating a compound of formula G with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00084##
or
[0186] the steps of
[0187] c) forming an active ester intermediate from a compound of
formula H
##STR00085##
and
[0188] d) reacting the active ester intermediate with a compound of
the formula I
##STR00086##
or combinations thereof
[0189] 1B. A process for preparing a compound of the formula
##STR00087##
or a salt thereof, wherein
[0190] Ar.sub.1 is optionally substituted aryl;
[0191] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0192] L is selected from the group consisting of
[0193] --(CR.sub.2).sub.pCR.sup.aR.sup.b--,
##STR00088##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0194] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0195] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0196] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0197] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted;
[0198] R.sub.3 is optionally substituted alkyl;
[0199] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0200] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0201] R.sub.7 is optionally substituted alkyl; and
[0202] n is 1, 2, 3, or 4;
[0203] wherein the process comprises
[0204] the step of treating a compound of formula A with
triethylsilyl chloride and imidazole in an aprotic solvent, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00089##
or
[0205] the step of treating a compound of formula B with a base and
a compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.; wherein the molar ratio of the compound of the
formula ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from
about 1 to about 1.5, where R.sub.8 is C1-C6 unbranched alkyl
##STR00090##
or
[0206] the steps of
[0207] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E
##STR00091##
and
[0208] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1., where
R.sub.8 is C1-C6 unbranched alkyl
##STR00092##
or
[0209] the step of treating compound D with a hydrolase enzyme or
with a trialkyltin hydroxide, where R.sub.8 is C1-C6 unbranched
alkyl
##STR00093##
or
[0210] the step of contacting compound D with an alcohol
R.sub.12OH; and a transesterification reagent selected from TFA or
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, or (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted or
[0211] the step of contacting compound AF with water and an
alkaline salt;
##STR00094##
or
[0212] the step of treating a compound of formula BG with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00095##
or
[0213] the steps of
[0214] c1) forming an active ester intermediate from a compound of
formula AH
##STR00096##
and
[0215] d1) reacting the active ester intermediate with a compound
of the formula I
##STR00097##
or combinations thereof
[0216] 1C. A compound of the formula
##STR00098##
[0217] or a salt or solvate thereof
wherein
[0218] Ar.sub.1 is optionally substituted aryl or optionally
substituted heteroaryl;
[0219] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0220] L is selected from the group consisting of
[0221] --(CR.sup.2).sub.pCR.sup.aR.sup.b--,
##STR00099##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0222] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0223] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0224] R.sub.3 is optionally substituted alkyl; and
[0225] R.sub.9 is hydrogen or an amine protecting group.
[0226] 1D. The process or compound of any one of the previous
clauses wherein Y is acyloxy.
[0227] 1E. The process or compound of any one of the previous
clauses wherein Y is R.sub.2C(O)O, where R.sub.2 is selected from
the group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl.
[0228] 2. The process of any one of the previous clauses comprising
the step of treating a compound of formula A with triethylsilyl
chloride and imidazole in an aprotic solvent, where R.sub.8 is
C1-C6 unbranched alkyl
##STR00100##
[0229] 3. The process of any one of the previous clauses comprising
the step of treating a compound of formula B with a base and a
compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.; wherein the molar ratio of the compound of the
formula ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from
about 1 to about 1.5, where R.sub.8 is C1-C6 unbranched alkyl
##STR00101##
[0230] 4. The process of any one of the previous clauses comprising
the steps of
[0231] a) preparing a compound of formula (E1) from a compound of
formula E, where X.sub.1 is a leaving group
##STR00102##
and
[0232] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00103##
[0233] 5. The process of any one of the previous clauses comprising
the step of treating compound D with a hydrolase enzyme or a
trialkyltin hydroxide, where R.sub.8 is C1-C6 unbranched alkyl
##STR00104##
[0234] 5A The process of any one of the previous clauses comprising
the step of contacting compound D with an alcohol R.sub.12OH; and a
transesterification reagent selected from
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, (R.sub.13).sub.3SnOSn(R.sub.13).sub.3, or a
combination thereof, where R.sub.13 is independently selected from
alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally
substituted.
[0235] 5B. The process of any one of the previous clauses
comprising the step of contacting compound AF with water and an
alkaline salt;
##STR00105##
[0236] 6. The process of any one of the previous clauses comprising
the step of treating a compound of formula G1 with an acylating
agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a leaving
group
##STR00106##
[0237] 6A. The process of any one of the previous clauses
comprising the step of treating a compound of formula G with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00107##
[0238] 6B. The process of any one of the previous clauses
comprising the step treating a compound of formula BG with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00108##
[0239] 7. The process of any one of the previous clauses comprising
the steps of
[0240] c) forming an active ester intermediate from a compound of
formula H1
##STR00109##
and
[0241] d) reacting the active ester intermediate with a compound of
the formula I
##STR00110##
[0242] 7A. The process of any one of the previous clauses
comprising the steps of
[0243] c) forming an active ester intermediate from a compound of
formula H
##STR00111##
and
[0244] d) reacting the active ester intermediate with a compound of
the formula I
##STR00112##
[0245] 7B. The process of any one of the previous clauses
comprising the steps of
[0246] c) forming an active ester intermediate from a compound of
formula AH
##STR00113##
and
[0247] d) reacting the active ester intermediate with a compound of
the formula I
##STR00114##
[0248] 8. The process or compound of any one of the preceding
clauses wherein R.sub.2 is C1-C8 alkyl or C3-C8 cycloalkyl. 9. The
process or compound of any one of the preceding clauses wherein
R.sub.2 is n-butyl.
[0249] 10. The process or compound of any one of the preceding
clauses wherein R.sub.3 is C1-C4 alkyl.
[0250] 11. The process or compound of any one of the preceding
clauses wherein R.sub.3 is methyl.
[0251] 12. The process or compound of any one of the preceding
claims wherein Ar.sub.1 is optionally substituted aryl.
[0252] 12A. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is phenyl or hydroxyphenyl.
[0253] 13. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is 4-hydroxyphenyl.
[0254] 14. The process or compound of any one of the preceding
clauses wherein R.sub.4 is C1-C8 alkyl or C3-C8 cycloalkyl.
[0255] 15. The process or compound of any one of the preceding
clauses wherein R.sub.4 is methyl.
[0256] 16. The process or compound of any one of the preceding
clauses wherein R.sub.5 is branched C3-C6 or C3-C8 cycloalkyl.
[0257] 17. The process or compound of any one of the preceding
clauses wherein R.sub.5 is iso-propyl.
[0258] 18. The process or compound of any one of the preceding
clauses wherein R.sub.6 is branched C3-C6 or C3-C8 cycloalkyl.
[0259] 19. The process or compound of any one of the preceding
clauses wherein R.sub.5 is sec-butyl.
[0260] 20. The process or compound of any one of the preceding
clauses wherein R.sub.7 is C1-C6 alkyl.
[0261] 21. The process or compound of any one of the preceding
clauses wherein R.sub.7 is methyl.
[0262] 22. The process or compound of any one of the preceding
claims wherein Ar.sub.2 is optionally substituted heteroaryl.
[0263] 22A. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is substituted pyridyl.
[0264] 23. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is substituted 2-pyridyl.
[0265] 24. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is 3-nitro-2-pyridyl.
[0266] 25. The process or compound of any one of the preceding
clauses wherein R.sub.2 is CH.sub.2CH(CH.sub.3).sub.2,
CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.3,
CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
[0267] 26. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is substituted phenyl.
[0268] 27. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is 4-substituted phenyl.
[0269] 28. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is R.sub.A-substituted phenyl.
[0270] 29. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is 4-hydroxyphenyl, or a hydroxyl
protected form thereof
[0271] 30. The process or compound of any one of the preceding
clauses wherein L is --(CR.sup.2).sub.pCR.sup.aR.sup.b--.
[0272] 30. The process or compound of any one of the preceding
clauses wherein L is --(CR.sub.2).sub.pCR.sup.aR.sup.b--, p is 1,
and each of R.sup.a and R.sup.b is methyl.
[0273] 31. The process or compound of any one of the preceding
clauses wherein L is
##STR00115##
[0274] 32. The process or compound of any one of the preceding
clauses wherein L is
##STR00116##
[0275] 32A. The process or compound of any one of claims 1 to 7
wherein O-L-S is O--(CR.sub.2).sub.pCR.sup.aR.sup.b--S.
[0276] 32B. The process or compound of any one of claims 1 to 7
wherein O-L-S is
##STR00117##
[0277] 32C. The process or compound of any one of claims 1 to 7
wherein O-L-S is
##STR00118##
[0278] 33. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are each hydrogen.
[0279] 34. The process or compound of any one of the preceding
clauses wherein p is 1.
[0280] 35. The process or compound of any one of the preceding
clauses wherein m is 1.
[0281] 35A. The process or compound of any one of the preceding
clauses wherein the transesterification catalyst is selected from
(R.sub.13).sub.8Sn.sub.4O.sub.2(NCS).sub.4,
(R.sub.13).sub.2Sn(OAc).sub.2, (R.sub.13).sub.2SnO,
(R.sub.13).sub.2SnCl.sub.2, (R.sub.13).sub.2SnS,
(R.sub.13).sub.3SnOH, and (R.sub.13).sub.3SnOSn(R.sub.13).sub.3,
where R.sub.13 is independently selected from alkyl, arylalkyl,
aryl, or cycloalkyl, each of which is optionally substituted.
[0282] 35B. The process or compound of any one of the preceding
clauses wherein the transesterification catalyst is
(R.sub.13).sub.2SnO.
[0283] 35C. The process or compound of any one of the preceding
clauses wherein the transesterification catalyst is
(n-Bu).sub.2SnO.
[0284] 35D. The process or compound of any one of the preceding
clauses wherein the transesterification catalyst is TFA.
[0285] 35E. The process or compound of any one of the preceding
clauses wherein the alkaline salt is a metal hydroxide or a metal
carbonate.
[0286] 35F. The process or compound of any one of the preceding
clauses wherein the alkaline salt is selected from LiOH,
Li.sub.2CO.sub.3, NaOH, Na.sub.2CO.sub.3, KOH, K.sub.2CO.sub.3,
Ca(OH).sub.2, CaCO.sub.3, Mg(OH).sub.2, or MgCO.sub.3.
[0287] 36. The process or compound of any one of the preceding
clauses wherein the alkaline salt is LiOH.
[0288] 37. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is substituted pyridyl.
[0289] 38. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is substituted 2-pyridyl.
[0290] 39. The process or compound of any one of the preceding
clauses wherein Ar.sub.2 is 3-nitro-2-pyridyl.
[0291] 40. The process or compound of any one of the preceding
clauses wherein R.sub.2 is CH.sub.2CH(CH.sub.3).sub.2,
CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.3,
CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
[0292] 41. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is substituted phenyl.
[0293] 42. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is 4-substituted phenyl.
[0294] 43. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is R.sub.A-substituted phenyl.
[0295] 44. The process or compound of any one of the preceding
clauses wherein Ar.sub.1 is 4-hydroxyphenyl, or a hydroxyl
protected form thereof
[0296] 45. The process or compound of any one of the preceding
clauses wherein L is --(CR.sup.2).sub.pCR.sup.aR.sup.b--.
[0297] 46. The process or compound of any one of the preceding
clauses wherein L is
##STR00119##
[0298] 47. The process or compound of any one of the preceding
clauses wherein L is
##STR00120##
[0299] 47A. The process or compound of any one of the preceding
clauses wherein O-L-S is O--(CR.sup.2).sub.pCR.sup.aR.sup.b--S.
[0300] 47B. The process or compound of any one of the preceding
clauses wherein O-L-S is
##STR00121##
[0301] 47C. The process or compound of any one of the preceding
clauses wherein O-L-S is
##STR00122##
[0302] 48. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are each hydrogen.
[0303] 49. The process or compound of any one of the preceding
clauses wherein p is 1.
[0304] 50. The process or compound of any one of the preceding
clauses wherein m is 1.
[0305] 50A. The process or compound of any one of the preceding
clauses wherein each R is hydrogen.
[0306] 51. The process or compound of any one of the preceding
clauses wherein R.sub.2 is CH.sub.2CH(CH.sub.3).sub.2,
CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.3,
CH.dbd.C(CH.sub.3).sub.2, or CH.sub.3.
[0307] 52. The process or compound of any one of the preceding
clauses wherein R.sup.a is hydrogen and R.sup.b is methyl.
[0308] 52A. The process or compound of any one of the preceding
clauses wherein R is hydrogen.
[0309] 53. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are each methyl.
[0310] 54. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are taken together with the
attached carbon to form cyclopropyl.
[0311] 55. The process or compound of any one of the preceding
clauses wherein R.sup.a is hydrogen and R.sup.b is methyl.
[0312] 55A. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are each methyl.
[0313] 55B. The process or compound of any one of the preceding
clauses wherein R.sup.a and R.sup.b are taken together with the
attached carbon to form cyclopropyl.
[0314] 56. A process for preparing a compound of the formula
##STR00123##
wherein
[0315] Ar.sub.1 is optionally substituted aryl;
[0316] Ar.sub.2 is optionally substituted aryl or optionally
substituted heteroaryl;
[0317] L is selected from the group consisting of
[0318] --(CR.sup.2).sub.pCR.sup.aR.sup.b--,
##STR00124##
where p is an integer from about 1 to about 3, m is an integer from
about 1 to about 4, and * indicates the points of attachment;
[0319] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
any two of R.sup.a, R.sup.b, and R are taken together with the
attached carbon atom(s) to form a carbocyclic ring;
[0320] R.sub.Ar represents 0 to 4 substituents selected from the
group consisting of amino, or derivatives thereof, hydroxy or
derivatives thereof, halo, thio or derivatives thereof, alkyl,
haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,
heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic
acids and derivatives thereof, carboxylic acids and derivatives
thereof;
[0321] where X is hydrogen or alkyl, including C.sub.1-6 alkyl and
C.sub.1-4 alkyl, alkenyl, including C.sub.2-6 alkenyl and C.sub.2-4
alkenyl, cycloalkyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, each of which is optionally substituted; or
CH.sub.2QR.sup.9; where Q is NH, O, or S; or; and R.sup.9 is alkyl,
including C.sub.1-6 alkyl and C.sub.1-4 alkyl, alkenyl, including
C.sub.2-6 alkenyl and C.sub.2-4 alkenyl, cycloalkyl, aryl,
heteroaryl, arylalkyl, or heteroarylalkyl, each of which is
optionally substituted; or R.sup.9 is hydrogen or C(O)R.sup.10,
where R.sup.10 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, aryl,
heteroaryl, arylalkyl, or heteroarylalkyl, each of which is
optionally substituted; or R.sup.9 is C(O)R.sup.20,
S(O).sub.2R.sup.20, or P(O)(OR.sup.20).sub.2; where R.sup.20 is
independently selected in each instance from the group consisting
of H, alkyl, including C.sub.1-6 alkyl and C.sub.1-4 alkyl,
alkenyl, including C.sub.2-6 alkenyl and C.sub.2-4 alkenyl,
cycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, each
of which is optionally substituted; or R.sup.20 is a metal
cation;
[0322] R.sup.3 is optionally substituted alkyl;
[0323] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0324] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0325] R.sub.7 is optionally substituted alkyl; and
[0326] n is 1, 2, 3, or 4;
[0327] wherein the process comprises
[0328] the step of treating a compound of formula A with
triethylsilyl chloride and imidazole in an aprotic solvent, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00125##
or
[0329] the step of treating a compound of formula B with a base and
a compound of the formula ClCH.sub.2OC(O)R.sub.2 in an aprotic
solvent at a temperature from about -78.degree. C. to about
0.degree. C.; wherein the molar ratio of the compound of the
formula ClCH.sub.2OC(O)R.sub.2 to the compound of formula B from
about 1 to about 1.5, where R.sub.8 is C1-C6 unbranched alkyl
##STR00126##
or
[0330] the steps of
[0331] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E
##STR00127##
and
[0332] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1, where
R.sub.8 is C1-C6 unbranched alkyl
##STR00128##
or
[0333] the step of treating compound D with a hydrolase enzyme or
with a trialkyltin hydroxide, where R.sub.8 is C1-C6 unbranched
alkyl
##STR00129##
or
[0334] the step of treating the silyl ether of compound F with a
non-basic fluoride reagent
##STR00130##
or
[0335] the step of treating a compound of formula G with an
acylating agent of formula R.sub.4C(O)X.sub.2, where X.sub.2 is a
leaving group
##STR00131##
or
[0336] the steps of
[0337] c) forming an active ester intermediate from a compound of
formula H
##STR00132##
and
[0338] d) reacting the active ester intermediate with a compound of
the formula I
##STR00133##
or combinations thereof
[0339] 57. The process of clause 56 wherein X is CH.sub.2QR.sup.9A;
where Q is NH, O, or S; and R.sup.9A is alkyl, including C.sub.1-6
alkyl and C.sub.1-4 alkyl, alkenyl, including C.sub.2-6 alkenyl and
C.sub.2-4 alkenyl, cycloalkyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, each of which is optionally substituted; or
R.sup.9A is H or C(O)R.sup.10, where R.sup.10 is C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
each of which is optionally substituted.
[0340] 58. The process of clause 56 wherein X is CH.sub.2QR.sup.9A;
where Q is O; and R.sup.9A is alkyl, including C.sub.1-6 alkyl and
C.sub.1-4 alkyl, alkenyl, including C.sub.2-6 alkenyl and C.sub.2-4
alkenyl, cycloalkyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, each of which is optionally substituted; or
R.sup.9A is H or C(O)R.sup.10, where R.sup.10 is C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
each of which is optionally substituted.
[0341] 59. The process of clause 56 wherein X is CH.sub.2QR.sup.9B;
where Q is NH, O, or S; and R.sup.9B is C(O)R.sup.20,
S(O).sup.2R.sup.20, or P(O)(OR.sup.20).sub.2; where R.sup.20 is
independently selected in each instance from the group consisting
of H, alkyl, including C.sub.1-6 alkyl and C.sub.1-4 alkyl,
alkenyl, including C.sub.2-6 alkenyl and C.sub.2-4 alkenyl,
cycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, each
of which is optionally substituted; or R.sup.20 is a metal
cation.
[0342] 60. The process of clause 56 wherein X is CH.sub.2QR.sup.9B;
where Q is O; and R.sup.9B is C(O)R.sup.20, S(O).sub.2R.sup.20, or
P(O)(OR.sup.20).sub.2; where R.sup.20 is independently selected in
each instance from the group consisting of H, alkyl, including
C.sub.1-6 alkyl and C.sub.1-4 alkyl, alkenyl, including C2-6
alkenyl and C.sub.2-4 alkenyl, cycloalkyl, aryl, heteroaryl,
arylalkyl, or heteroarylalkyl, each of which is optionally
substituted; or R.sup.20 is a metal cation.
[0343] 61. The process of clause 56 wherein X is CH.sub.2QR.sup.9B;
where Q is NH; and R.sup.9B is C(O)R.sup.20, where R.sup.20 is
alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl, each of which is
optionally substituted.
[0344] 62. The process of clause 56 wherein X is
CH.sub.2CH.dbd.CHR.sup.22 or CH.sub.2C(R.sup.22).dbd.CH.sub.2,
where R.sup.22 is C(O)R.sup.20, where R.sup.20 is wherein R.sup.22
is alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl, each of which is
optionally substituted.
[0345] 63. The process of clause 56 wherein X is
CH.sub.2CH(R.sup.a)C(O)R.sup.23, where R.sup.23 is H, or alkyl,
alkenyl, cycloalkyl, aryl, or arylalkyl, each of which is
optionally substituted; R.sup.a is C(O)R.sup.9, C(O)OR.sup.9 or
CN;
[0346] 64. The process of clause 56 wherein X is CH.sub.2OR.sup.25;
where R.sup.25 is H, and alkyl, alkenyl, cycloalkyl, aryl, and
arylalkyl, each of which is optionally substituted; or R.sup.25 is
alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl, each of which is
optionally substituted; or R.sup.25 is alkyl.
[0347] 65. The process of clause 56 wherein X is CH.sub.2OH.
[0348] 66. The process of clause 56 wherein X is CH.sub.2X.sup.3;
where X.sup.3 is halogen, including bromo or iodo,
OS(O).sub.2R.sup.24, OP(O)(OR.sup.24)R.sup.24, or
OP(O)(OR.sup.24).sub.2; where R.sup.24 is independently selected in
each instance from the group consisting of H, and alkyl, alkenyl,
cycloalkyl, aryl, and arylalkyl, each of which is optionally
substituted, and metal cations.
[0349] In one embodiment, one or more of the following
intermediates can be used to prepare a tubulysin, tubulysin
conjugate, and/or a tubulysin linker compound.
##STR00134## ##STR00135##
where R' is Me or R, and R is allyl, or
CH.sub.2(CH.sub.2).sub.2CH.sub.3, where n=1, 2, 3, 4, 5, or 6 and
Ar.sub.2 is as described in various embodiments described
herein.
[0350] In another embodiment, processes for preparing tubulysin
conjugates are described herein. In one aspect, the processes
include the step of reacting compounds (2) and the corresponding
tubulysins described herein that include a 3-nitropyridin-2-ylthio
activating group (Ar.sub.2), such as compounds of the formulae
##STR00136##
or salt and/or solvates thereof, as described herein, with a
binding ligand-linker intermediate, such as are described in WO
2008/112873, the disclosure of which is incorporated herein by
reference. In another aspect, the processes include acetonitrile as
a reaction solvent. In another aspect, the processes include
aqueous phosphate as a reaction medium buffer. Without being bound
by theory, it is believed herein that the use of acetonitrile as a
solvent leads to a more rapid homogenous reaction solution. In
addition, though without being bound by theory, it is believed
herein that a more rapidly formed homogenous reaction medium may
improve the yield and rate at which tubulysin conjugates described
herein are formed. In addition, though without being bound by
theory, it is believed herein that the use of
3-nitropyridin-2-ylthio activating groups may lead to faster
reaction times. In addition, though without being bound by theory,
it is believed herein that the use of 3-nitropyridin-2-ylthio
activating groups may provide better chromatographic separation
between the product tubulysin conjugates and the by-product
3-nitropyridin-2-ylthiol compared to conventional pyridin-2-ylthio
activating groups. In addition, such better chromatographic
separation may allow the use of alternative purification methods.
For example, in addition to preparative HPLC, the tubulysin
conjugates described herein may be purified by rapid pass-through
chromatography methods, such as flash C.sub.18 chromatography,
Biotage.TM. C.sub.18 chromatography systems, and the like.
[0351] Additional methods and processes useful for performing the
above processes are found in U.S. patent application Ser. No.
12/739,579, published as U.S. Application Publication No.
2010/0240701, the disclosure of which is incorporated herein by
reference in its entirety.
[0352] It is to be understood that as used herein, the term
tubulysin refers both collectively and individually to the
naturally occurring tubulysins, and the analogs and derivatives of
tubulysins. Illustrative examples of a tubulysin are shown in Table
1.
[0353] As used herein, the term tubulysin linker derivatives
generally refers to the compounds described herein and analogs and
derivatives thereof. It is also to be understood that in each of
the foregoing, any corresponding pharmaceutically acceptable salt
is also included in the illustrative embodiments described
herein.
[0354] It is to be understood that such derivatives may include
prodrugs of the compounds described herein, compounds described
herein that include one or more protection or protecting groups,
including compounds that are used in the preparation of other
compounds described herein.
[0355] In addition, as used herein the term tubulysin linker
derivatives also refers to prodrug derivatives of the compounds
described herein, and including prodrugs of the various analogs and
derivatives thereof. In addition, as used herein, the term
tubulysin linker derivatives refers to both the amorphous as well
as any and all morphological forms of each of the compounds
described herein. In addition, as used herein, the term tubulysin
linker derivatives refers to any and all hydrates, or other
solvates, of the compounds described herein.
[0356] It is to be understood that each of the foregoing
embodiments may be combined in chemically relevant ways to generate
subsets of the embodiments described herein. Accordingly, it is to
be further understood that all such subsets are also illustrative
embodiments of the invention described herein.
[0357] The compounds described herein may contain one or more
chiral centers, or may otherwise be capable of existing as multiple
stereoisomers. It is to be understood that in one embodiment, the
invention described herein is not limited to any particular
stereochemical requirement, and that the compounds, and
compositions, methods, uses, and medicaments that include them may
be optically pure, or may be any of a variety of stereoisomeric
mixtures, including racemic and other mixtures of enantiomers,
other mixtures of diastereomers, and the like. It is also to be
understood that such mixtures of stereoisomers may include a single
stereochemical configuration at one or more chiral centers, while
including mixtures of stereochemical configuration at one or more
other chiral centers.
[0358] Similarly, the compounds described herein may include
geometric centers, such as cis, trans, (E)-, and (Z)-double bonds.
It is to be understood that in another embodiment, the invention
described herein is not limited to any particular geometric isomer
requirement, and that the compounds, and compositions, methods,
uses, and medicaments that include them may be pure, or may be any
of a variety of geometric isomer mixtures. It is also to be
understood that such mixtures of geometric isomers may include a
single configuration at one or more double bonds, while including
mixtures of geometry at one or more other double bonds.
[0359] As used herein, the term aprotic solvent refers to a solvent
which does not yield a proton to the solute(s) under reaction
conditions. Illustrative examples of nonprotic solvents are
tetrahydrofuran (THF), 2,5-dimethyl-tetrahydrofuran,
2-methyl-tetrahydrofuran, tetrahydropyran, diethyl ether, t-butyl
methyl ether, dimethyl formamide, N-methylpyrrolidinone (NMP), and
the like. It is appreciated that mixtures of these solvents may
also be used in the processes described herein.
[0360] As used herein, an equivalent amount of a reagent refers to
the theoretical amount of the reagent necessary to transform a
starting material into a desired product, i.e. if 1 mole of reagent
is theoretically required to transform 1 mole of the starting
material into 1 mole of product, then 1 equivalent of the reagent
represents 1 mole of the reagent; if X moles of reagent are
theoretically required to convert 1 mole of the starting material
into 1 mole of product, then 1 equivalent of reagent represents X
moles of reagent.
[0361] As used herein, the term active ester forming agent
generally refers to any reagent or combinations of reagents that
may be used to convert a carboxylic acid into an active ester.
[0362] As used herein, the term active ester generally refers to a
carboxylic acid ester compound wherein the divalent oxygen portion
of the ester is a leaving group resulting in an ester that is
activated for reacting with compounds containing functional groups,
such as amines, alcohols or sulfhydryl groups. Illustrative
examples of active ester-forming compounds are
N-hydroxysuccinimide, N-hydroxyphthalimide, phenols substituted
with electron withdrawing groups, such as but not limited to
4-nitrophenol, pentafluorophenol, N,N'-disubstituted isoureas,
substituted hydroxyheteroaryls, such as but not limited to
2-pyridinols, 1-hydroxybenzotriazoles,
1-hydroxy-7-aza-benzotriazoles, cyanomethanol, and the like.
Illustratively, the reaction conditions for displacing the active
ester with a compound having an amino, hydroxy or thiol group are
mild. Illustratively, the reaction conditions for displacing the
active ester with a compound having an amino, hydroxy or thiol
group are performed at ambient or below ambient temperatures.
Illustratively, the reaction conditions for displacing the active
ester with a compound having an amino, hydroxy or thiol group are
performed without the addition of a strong base. Illustratively,
the reaction conditions for displacing the active ester with a
compound having an amino, hydroxy or thiol group are performed with
the addition of a tertiary amine base, such as a tertiary amine
base having a conjugate acid pKa of about 11 or less, about 10.5 or
less, and the like.
[0363] As used herein, the term "alkyl" includes a chain of carbon
atoms, which is optionally branched. As used herein, the term
"alkenyl" and "alkynyl" includes a chain of carbon atoms, which is
optionally branched, and includes at least one double bond or
triple bond, respectively. It is to be understood that alkynyl may
also include one or more double bonds. It is to be further
understood that in certain embodiments, alkyl is advantageously of
limited length, including C.sub.1-C.sub.24, C.sub.1-C.sub.12,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4. It is to be
further understood that in certain embodiments alkenyl and/or
alkynyl may each be advantageously of limited length, including
C.sub.2-C.sub.24, C.sub.2-C.sub.12, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4. It is appreciated herein that
shorter alkyl, alkenyl, and/or alkynyl groups may add less
lipophilicity to the compound and accordingly will have different
pharmacokinetic behavior. Illustrative alkyl groups are, but not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl,
hexyl, heptyl, octyl and the like.
[0364] As used herein, the term "cycloalkyl" includes a chain of
carbon atoms, which is optionally branched, where at least a
portion of the chain in cyclic. It is to be understood that
cycloalkylalkyl is a subset of cycloalkyl. It is to be understood
that cycloalkyl may be polycyclic. Illustrative cycloalkyl include,
but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl,
2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like.
As used herein, the term "cycloalkenyl" includes a chain of carbon
atoms, which is optionally branched, and includes at least one
double bond, where at least a portion of the chain in cyclic. It is
to be understood that the one or more double bonds may be in the
cyclic portion of cycloalkenyl and/or the non-cyclic portion of
cycloalkenyl. It is to be understood that cycloalkenylalkyl and
cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be
understood that cycloalkyl may be polycyclic. Illustrative
cycloalkenyl include, but are not limited to, cyclopentenyl,
cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to
be further understood that chain forming cycloalkyl and/or
cycloalkenyl is advantageously of limited length, including
C.sub.3-C.sub.24, C.sub.3-C.sub.12, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, and C.sub.5-C.sub.6. It is appreciated herein that
shorter alkyl and/or alkenyl chains forming cycloalkyl and/or
cycloalkenyl, respectively, may add less lipophilicity to the
compound and accordingly will have different pharmacokinetic
behavior.
[0365] As used herein, the term "heteroalkyl" includes a chain of
atoms that includes both carbon and at least one heteroatom, and is
optionally branched. Illustrative heteroatoms include nitrogen,
oxygen, and sulfur. In certain variations, illustrative heteroatoms
also include phosphorus, and selenium. As used herein, the term
"cycloheteroalkyl" including heterocyclyl and heterocycle, includes
a chain of atoms that includes both carbon and at least one
heteroatom, such as heteroalkyl, and is optionally branched, where
at least a portion of the chain is cyclic. Illustrative heteroatoms
include nitrogen, oxygen, and sulfur. In certain variations,
illustrative heteroatoms also include phosphorus, and selenium.
Illustrative cycloheteroalkyl include, but are not limited to,
tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,
morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the
like.
[0366] As used herein, the term "aryl" includes monocyclic and
polycyclic aromatic groups, including aromatic carbocyclic and
aromatic heterocyclic groups, each of which may be optionally
substituted. As used herein, the term "carbaryl" includes aromatic
carbocyclic groups, each of which may be optionally substituted.
Illustrative aromatic carbocyclic groups described herein include,
but are not limited to, phenyl, naphthyl, and the like. As used
herein, the term "heteroaryl" includes aromatic heterocyclic
groups, each of which may be optionally substituted. Illustrative
aromatic heterocyclic groups include, but are not limited to,
pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl,
quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl,
imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl,
oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl,
benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.
[0367] As used herein, the term "amino" includes the group
NH.sub.2, alkylamino, and dialkylamino, where the two alkyl groups
in dialkylamino may be the same or different, i.e. alkylalkylamino.
Illustratively, amino includes methylamino, ethylamino,
dimethylamino, methylethylamino, and the like. In addition, it is
to be understood that when amino modifies or is modified by another
term, such as aminoalkyl, or acylamino, the above variations of the
term amino are included therein. Illustratively, aminoalkyl
includes H.sub.2N-alkyl, methylaminoalkyl, ethylaminoalkyl,
dimethylaminoalkyl, methylethylaminoalkyl, and the like.
Illustratively, acylamino includes acylmethylamino, acylethylamino,
and the like.
[0368] As used herein, the term "amino and derivatives thereof"
includes amino as described herein, and alkylamino, alkenylamino,
alkynylamino, heteroalkylamino, heteroalkenylamino,
heteroalkynylamino, cycloalkylamino, cycloalkenylamino,
cycloheteroalkylamino, cycloheteroalkenylamino, arylamino,
arylalkylamino, arylalkenylamino, arylalkynylamino, acylamino, and
the like, each of which is optionally substituted. The term "amino
derivative" also includes urea, carbamate, and the like.
[0369] As used herein, the term "hydroxy and derivatives thereof"
includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy,
heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy,
cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy,
arylalkenyloxy, arylalkynyloxy, acyloxy, and the like, each of
which is optionally substituted. The term "hydroxy derivative" also
includes carbamate, and the like.
[0370] As used herein, the term "thio and derivatives thereof"
includes SH, and alkylthio, alkenylthio, alkynylthio,
heteroalkylthio, heteroalkenylthio, heteroalkynylthio,
cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio,
cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio,
arylalkynylthio, acylthio, and the like, each of which is
optionally substituted. The term "thio derivative" also includes
thiocarbamate, and the like.
[0371] As used herein, the term "acyl" includes formyl, and
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,
cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,
arylalkenylcarbonyl, arylalkynylcarbonyl, acylcarbonyl, and the
like, each of which is optionally substituted.
[0372] As used herein, the term "carboxylate and derivatives
thereof" includes the group CO.sub.2H and salts thereof, and esters
and amides thereof, and CN.
[0373] The term "optionally substituted" as used herein includes
the replacement of hydrogen atoms with other functional groups on
the radical that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid
is optionally substituted.
[0374] As used herein, the term "optionally substituted aryl"
includes the replacement of hydrogen atoms with other functional
groups on the aryl that is optionally substituted. Such other
functional groups illustratively include, but are not limited to,
amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid
is optionally substituted.
[0375] Illustrative substituents include, but are not limited to, a
radical --(CH.sub.2).sub.xZ.sup.X, where x is an integer from 0-6
and Z.sup.X is selected from halogen, hydroxy, alkanoyloxy,
including C.sub.1-C.sub.6 alkanoyloxy, optionally substituted
aroyloxy, alkyl, including C.sub.1-C.sub.6 alkyl, alkoxy, including
C.sub.1-C.sub.6 alkoxy, cycloalkyl, including C.sub.3-C.sub.8
cycloalkyl, cycloalkoxy, including C.sub.3-C.sub.8 cycloalkoxy,
alkenyl, including C.sub.2-C.sub.6 alkenyl, alkynyl, including
C.sub.2-C.sub.6 alkynyl, haloalkyl, including C.sub.1-C.sub.6
haloalkyl, haloalkoxy, including C.sub.1-C.sub.6 haloalkoxy,
halocycloalkyl, including C.sub.3-C.sub.8 halocycloalkyl,
halocycloalkoxy, including C.sub.3-C.sub.8 halocycloalkoxy, amino,
C.sub.1-C.sub.6 alkylamino, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)amino, alkylcarbonylamino, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylamino, aminoalkyl, C.sub.1-C.sub.6
alkylaminoalkyl, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z.sup.X is
selected from --CO.sub.2R.sup.4 and --CONR.sup.5R.sup.6, where
R.sup.4, R.sup.5, and R.sup.6 are each independently selected in
each occurrence from hydrogen, C.sub.1-C.sub.6 alkyl, and
aryl-C.sub.1-C.sub.6 alkyl.
[0376] The term protecting group generally refers to chemical
functional groups that can be selectively appended to and removed
from functionality, such as amine groups, present in a chemical
compound to render such functionality inert to chemical reaction
conditions to which the compound is exposed. See. e.g., Greene and
Wuts, Protective Groups in Organic Synthesis, 2d edition, John
Wiley & Sons, New York, 1991. Numerous amine protecting groups
are known in the art. Illustrative examples include the
benzyloxycarbonyl (CBz), chlorobenzyloxycarbonyl,
t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc),
isonicotinyloxycarbonyl (i-Noc) groups.
[0377] The term "prodrug" as used herein generally refers to any
compound that when administered to a biological system generates a
biologically active compound as a result of one or more spontaneous
chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or
metabolic chemical reaction(s), or a combination thereof. In vivo,
the prodrug is typically acted upon by an enzyme (such as
esterases, amidases, phosphatases, and the like), simple biological
chemistry, or other process in vivo to liberate or regenerate the
more pharmacologically active drug. This activation may occur
through the action of an endogenous host enzyme or a non-endogenous
enzyme that is administered to the host preceding, following, or
during administration of the prodrug. Additional details of prodrug
use are described in U.S. Pat. No. 5,627,165; and Pathalk et al.,
Enzymic protecting group techniques in organic synthesis,
Stereosel. Biocatal. 775-797 (2000). It is appreciated that the
prodrug is advantageously converted to the original drug as soon as
the goal, such as targeted delivery, safety, stability, and the
like is achieved, followed by the subsequent rapid elimination of
the released remains of the group forming the prodrug.
[0378] Prodrugs may be prepared from the compounds described herein
by attaching groups that ultimately cleave in vivo to one or more
functional groups present on the compound, such as --OH--, --SH,
--CO.sub.2H, --NR.sub.2. Illustrative prodrugs include but are not
limited to carboxylate esters where the group is alkyl, aryl,
aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of
hydroxyl, thiol and amines where the group attached is an acyl
group, an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate.
Illustrative esters, also referred to as active esters, include but
are not limited to 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups
such as acetoxymethyl, pivaloyloxymethyl, .beta.-acetoxyethyl,
.beta.-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl,
(1-aminoethyl)carbonyloxymethyl, and the like;
alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl,
.alpha.-ethoxycarbonyloxyethyl, .beta.-ethoxycarbonyloxyethyl, and
the like; dialkylaminoalkyl groups, including di-lower alkylamino
alkyl groups, such as dimethylaminomethyl, dimethylaminoethyl,
diethylaminomethyl, diethylaminoethyl, and the like;
2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)
pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and
lactone groups such as phthalidyl, dimethoxyphthalidyl, and the
like.
[0379] Further illustrative prodrugs contain a chemical moiety,
such as an amide or phosphorus group functioning to increase
solubility and/or stability of the compounds described herein.
Further illustrative prodrugs for amino groups include, but are not
limited to, (C.sub.3-C.sub.20)alkanoyl;
halo-(C.sub.3-C.sub.20)alkanoyl; (C.sub.3-C.sub.20)alkenoyl;
(C.sub.4-C.sub.7)cycloalkanoyl;
(C.sub.3-C.sub.6)-cycloalkyl(C.sub.2-C.sub.16)alkanoyl; optionally
substituted aroyl, such as unsubstituted aroyl or aroyl substituted
by 1 to 3 substituents selected from the group consisting of
halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with one or more of 1 to 3
halogen atoms; optionally substituted
aryl(C.sub.2-C.sub.16)alkanoyl, such as the aryl radical being
unsubstituted or substituted by 1 to 3 substituents selected from
the group consisting of halogen, (C.sub.1-C.sub.3)alkyl and
(C.sub.1-C.sub.3)alkoxy, each of which is optionally further
substituted with 1 to 3 halogen atoms; and optionally substituted
heteroarylalkanoyl having one to three heteroatoms selected from O,
S and N in the heteroaryl moiety and 2 to 10 carbon atoms in the
alkanoyl moiety, such as the heteroaryl radical being unsubstituted
or substituted by 1 to 3 substituents selected from the group
consisting of halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl, and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with 1 to 3 halogen atoms. The
groups illustrated are exemplary, not exhaustive, and may be
prepared by conventional processes.
[0380] It is understood that the prodrugs themselves may not
possess significant biological activity, but instead undergo one or
more spontaneous chemical reaction(s), enzyme-catalyzed chemical
reaction(s), and/or metabolic chemical reaction(s), or a
combination thereof after administration in vivo to produce the
compound described herein that is biologically active or is a
precursor of the biologically active compound. However, it is
appreciated that in some cases, the prodrug is biologically active.
It is also appreciated that prodrugs may often serves to improve
drug efficacy or safety through improved oral bioavailability,
pharmacodynamic half-life, and the like. Prodrugs also refer to
derivatives of the compounds described herein that include groups
that simply mask undesirable drug properties or improve drug
delivery. For example, one or more compounds described herein may
exhibit an undesirable property that is advantageously blocked or
minimized may become pharmacological, pharmaceutical, or
pharmacokinetic barriers in clinical drug application, such as low
oral drug absorption, lack of site specificity, chemical
instability, toxicity, and poor patient acceptance (bad taste,
odor, pain at injection site, and the like), and others. It is
appreciated herein that a prodrug, or other strategy using
reversible derivatives, can be useful in the optimization of the
clinical application of a drug.
[0381] As used herein, the term "treating", "contacting" or
"reacting" when referring to a chemical reaction means to add or
mix two or more reagents under appropriate conditions to produce
the indicated and/or the desired product. It should be appreciated
that the reaction which produces the indicated and/or the desired
product may not necessarily result directly from the combination of
two reagents which were initially added, i.e., there may be one or
more intermediates which are produced in the mixture which
ultimately leads to the formation of the indicated and/or the
desired product.
[0382] As used herein, the term "composition" generally refers to
any product comprising the specified ingredients in the specified
amounts, as well as any product which results, directly or
indirectly, from combinations of the specified ingredients in the
specified amounts. It is to be understood that the compositions
described herein may be prepared from isolated compounds described
herein or from salts, solutions, hydrates, solvates, and other
forms of the compounds described herein. It is also to be
understood that the compositions may be prepared from various
amorphous, non-amorphous, partially crystalline, crystalline,
and/or other morphological forms of the compounds described herein.
It is also to be understood that the compositions may be prepared
from various hydrates and/or solvates of the compounds described
herein. Accordingly, such pharmaceutical compositions that recite
compounds described herein are to be understood to include each of,
or any combination of, the various morphological forms and/or
solvate or hydrate forms of the compounds described herein.
Illustratively, compositions may include one or more carriers,
diluents, and/or excipients. The compounds described herein, or
compositions containing them, may be formulated in a
therapeutically effective amount in any conventional dosage forms
appropriate for the methods described herein. The compounds
described herein, or compositions containing them, including such
formulations, may be administered by a wide variety of conventional
routes for the methods described herein, and in a wide variety of
dosage formats, utilizing known procedures (see generally,
Remington: The Science and Practice of Pharmacy, (21.sup.st ed.,
2005)).
EXAMPLES
##STR00137##
[0383] Example
[0384] Synthesis of Dipeptide 5. 4.9 g of dipeptide 3 (11.6 mmol)
was dissolved in 60 mL dichloromethane, imidazole (0.87 g, 12.7
mmol) was added to the resulting solution at 0.degree. C. The
reaction mixture was warmed slightly to dissolve all solids and
re-cooled to 0.degree. C. TESCl (2.02 mL, 12.1 mmol) was added
drop-wise at 0.degree. C., the reaction mixture was stirred under
argon and warmed to room temperature over 2 h. TLC (3:1
hexanes/EtOAc) showed complete conversion. The reaction was
filtered to remove the imidazole HCl salt, extracted with
de-ionized water, and the aqueous phase was back washed with
dichloromethane, the combined organic phase was washed with brine,
dried over Na.sub.2SO.sub.4, filtered to remove the
Na.sub.2SO.sub.4, concentrated under reduced pressure,
co-evaporated with toluene and dried under high-vacuum overnight to
give 6.4 g of crude product 4 (vs 5.9 g of theoretical yield).
[0385] The crude product 4 was co-evaporated with toluene again and
used without further purification. TES protected dipeptide was
dissolved in 38 mL THF (anhydrous, inhibitor-free) and cooled to
-45.degree. C. and stirred for 15 minutes before adding KHMDS (0.5
M in toluene, 25.5 mL, 12.8 mmol, 1.1 equiv) drop-wise. After the
addition of KHMDS was complete, the reaction mixture was stirred at
-45.degree. C. for 15 minutes, and chloromethyl butyrate (1.8 mL,
1.2 equiv, 14 mmol) was added. The reaction mixture changed from
light yellow to a bluish color. TLC (20% EtOAc/petroleum ether)
showed the majority of starting material was converted. LC-MS
showed about 7% starting material left. The reaction was quenched
by adding 3 mL MeOH, the mixture was warmed to room temperature and
concentrated under reduced pressure to an oily residue. The residue
was dissolved in petroleum ether and passed through short silica
plug to remove the potassium salt. The plug was washed with 13%
EtOAc/petroleum ether, and the collected eluates were combined and
concentrated under reduced pressure. The crude alkylated product
was passed through an additional silica plug (product/silica=1:50)
and eluted with 13% EtOAc/petroleum ether to remove residual
starting material to give 5.7 g of product 5 (two steps, yield
76%)
##STR00138##
Example
[0386] Synthesis of Tripeptide 6. Alkylated dipeptide 5 (4.3 g, 7.0
mmol), N-methyl pipecolinate (MEP) (4.0 g, 28.0 mmol, 4 equiv) and
pentafluorophenol (5.7 g, 30.8 mmol 4.4 equiv) were added to a
flask. N-methylpyrrolidone (NMP, 86 mL) was added to the mixture.
To the mixture was added diisopropylcarbodiimide (DIC, 4.77 mL,
30.8 mmol, 4.4 equiv) was added to the mixture. The mixture was
stirred at room temperature for 1 h. Pd/C (10%, dry, 1.7 g) was
added. The flask was shaken under hydrogen (30-35 psi) for 5 hours.
The reaction mixture was analyzed by HPLC. The starting material
was found to be less than 3%. The mixture was filtered through
diatomaceous earth. The diatomaceous earth was extracted with 200
mL ethyl acetate. The filtrate and the ethyl acetate extract were
combined and transferred to a separatory funnel and washed with 1%
NaHCO.sub.3/10% NaCl solution (200 mL.times.4). The organic layer
was isolated and evaporated on a rotary evaporator under reduced
pressure. The crude product was dissolved in 40 mL of MeOH/H.sub.2O
(3:1). The crude product solution was loaded onto a Biotage C18
column (Flash 65i, 350 g, 450 mL, 65.times.200 mm) and eluted with
buffer A [10 mM NH.sub.4OAc/ACN (1:0] and B (ACN, acetonitrile).
The fractions were collected and organic solvent was removed by
evaporating on a rotary evaporator. 100 mL of 10% NaCl solution and
100 mL of methyl tert-butyl ether (MTBE) were added to the flask
and the mixture was transferred to a separatory funnel. The organic
layer was isolated and dried over anhydrous Na.sub.2SO.sub.4,
filtered and evaporated on a rotary evaporator to dryness. 2.5 g of
tripeptide intermediate 6 was obtained (yield 50%).
##STR00139##
Example
[0387] Synthesis of Tripeptide Acid 7. To 2 L of 0.05 M phosphate
(pH=7.4) at 30.degree. C. was added 3.6 g of porcine liver esterase
(17 units/mg). 3.0 g of methyl ester 6 was dissolved in 100 mL of
DMSO. The first 50 mL of this solution was added at a rate of 1.1
mL/h, and the second half was added at a rate of 1.2 mL/h via
syringe pump. After the addition was complete, the reaction mixture
was allowed to stir at 30.degree. C. for several hours. HPLC of an
EtOAc extract of the reaction mixture showed the reaction was
complete. The reaction mixture was drained from the reactor in 1 L
portions and extracted with EtOAc (3.times.1 L). The combined
extracts were washed with brine, dried over Mg2SO.sub.4 and
concentrated under reduced pressure. 2.8 g of product 7 was
recovered (95%). The product appeared to be clean by UPLC analysis,
except for pentafluorophenol carried over from the previous
reaction.
[0388] Intermediate 7 spectral data: LCMS (ESI) [M+H].sup.+ 697.3;
.sup.1H NMR (CD3OD) 8.02 (s, 1H), 5.94 (d, J=12.3 Hz, 1H), 5.48 (d,
J=12.3 Hz, 1H), 4.93 (d, J=8.2 Hz, 1H), 4.65 (d, J=8.5 Hz, 1H),
3.63 (s, br, 1H), 2.91 (br, 1H), 2.67 (s, 3H), 2.53-2.14 (m, 3H),
2.14-1.94 (m, 4H), 1.94-1.74 (m, 4H), 1.74-1.50 (m, 4H), 1.28-1.17
(m, 1H), 1.02-0.83 (m, 24H), 0.71-0.55 (m, 6H).
##STR00140##
Example
[0389] Synthesis of Tubulysin B. 1.4 g (2.01 mmol) of tripeptide 7
was dissolved in 8.4 mL THF and 327.4 .mu.L (2.01 mmol) of
3HF.NEt.sub.3 was added and the reaction mixture stirred for 30
minutes. LC-MS analysis (10% to 100% acetonitrile, pH 7 buffer)
confirmed complete deprotection of the TES group. THF was removed
under reduced pressure. The residue was dried under high vacuum for
5 minutes. The crude product was dissolved in 8.4 mL dry pyridine.
2.85 mL (30.15 mmol, 15 equiv) of Ac.sub.2O was added at 0.degree.
C. The resulting clear solution was stirred at room temperature for
3.5 hours. LC-MS analysis (10% to 100% acetonitrile, pH 7.0)
indicated>98% conversion. 56 mL of dioxane/H.sub.2O was added
and the resulting mixture stirred at room temperature for 1 hour.
The mixture was concentrated under reduced pressure. The residue
was co-evaporated with toluene (3.times.) and dried under high
vacuum overnight. Crude product 8 was used directly for the next
reaction.
[0390] Intermediate 8 spectral data: LCMS (ESI) [M+H].sup.+ 625.2;
.sup.1H NMR (CD.sub.3OD) 8.00 (s, 1H), 6.00 (s, br, 1H), 5.84 (d,
J=12.1 Hz, 1H), 5.40 (d, J=12.1 Hz, 1H), 4.63 (d, J=9.1 Hz, 1H),
3.09 (br, 1H), 2.60-2.20 (m, 7H), 2.12 (s, 3H), 2.09-1.86 (m, 3H),
1.80-1.63 (m, 3H), 1.59 (m, 5H), 1.19 (m, 1H), 1.03-0.81 (m, 15H);
.sup.13C NMR (CD3OD) 176.2, 174.2, 172.1, 169.1, 155.5, 125.2,
71.4, 69.6, 56.6, 55.5, 44.3, 37.7, 37.1, 36.4, 32.0, 31.2, 25.6,
23.7, 21.0, 20.9, 20.7, 19.3, 16.5, 14.2, 11.0
[0391] Method A. The crude tripeptide acid 8 was dissolved in 28 mL
EtOAc (anhydrous) and 740 mg (4.02 mmol, 2.0 equiv) of
pentafluorophenol was added, followed by 1.04 g (5.03 mmol, 2.5
equiv) of DCC. The resulting reaction mixture was stirred at room
temperature for 1 hour. LC-MS (5% to 80% acetonitrile, pH=2.0,
formic acid) analysis indicated>95% conversion. The urea
by-product was filtered off, the EtOAc was removed under reduced
pressure, and the residue was dried under high vacuum for 5
minutes. The residue was dissolved in 8.4 mL DMF, and tubutyrosine
hydrochloride salt (Tut-HCl, 678.7 mg, 2.61 mmol, 1.3 equiv) was
added, followed by DIPEA (2.28 mL, 13.07 mmol, 6.5 equiv). The
resulting clear solution was stirred at room temperature for 10
minutes. The reaction mixture was diluted with DMSO and purified on
prep-HPLC(X-bridge column, 10 mM NH.sub.4OAc, pH=6.3, 25% to 100%
acetonitrile). Pure fractions were combined, acetonitrile was
removed under reduced pressure, extracted with EtOAc (3.times.),
and dried over Na.sub.2SO.sub.4. The EtOAc was removed under
reduced pressure and the residue was dried under high vacuum for 1
hour to yield 513 mg of the desired product (31% combined yield
from 6).
[0392] Method B. Tripeptide 8 (229 mg, 0.367 mmol) was dissolved in
EtOAc (anhydrous), 134.9 mg (0.733 mmol, 2.0 equiv) of
pentafluorophenol was added, followed by 970 mg (1.84 mmol, 5.0
equiv) of DCC on the resin. The resulting reaction mixture was
stirred at room temperature for 16 hours. LC-MS analysis
indicated>96% conversion. The reaction mixture was filtered and
concentrated to dryness, dried under high vacuum for 5 minutes. The
residue was dissolved in 3.5 mL DMF, Tut-HCl (123.9 mg, 0.477 mmol,
1.3 equiv) was added, followed by DIPEA (0.42 mL, 2.386 mmole, 6.5
equiv). The resulting clear solution was stirred at room
temperature for 10 minutes. The reaction mixture was diluted with
DMSO, purified on prep-HPLC (X bridgecolumn, 10 mM NH.sub.4OAc, 25%
to 100%, two runs). The pure fractions were combined, the
acetonitrile was removed under reduced pressure, the residue was
extracted with EtOAc (2.times.) and the combined EtOAc extracts
dried over Na.sub.2SO.sub.4. The EtOAc was removed under reduced
pressure. The residue was dried under high vacuum for 1 hour to
yield 175 mg of desired product (58% combined yield from 6).
##STR00141##
Example
[0393] Large Scale Synthesis of Dipeptide 5. 10.2 g of dipeptide 3
(25.6 mmol) was dissolved in 130 mL dichloromethane, imidazole (1.9
g, 28.1 mmol) was added to the resulting solution at 0.degree. C.
The reaction mixture was warmed slightly to dissolve all solids and
re-cooled to 0.degree. C. TESCl (4.5 mL, 26.8 mmol) was added drop
wise at 0.degree. C., the reaction mixture was stirred under argon
and warmed to room temperature over 2 h. TLC (3:1 hexanes/EtOAc)
showed complete conversion. The reaction was filtered to remove the
imidazole HCl salt, extracted with de-ionized water, and the
aqueous phase was back-washed with dichloromethane, the combined
organic phase was washed with brine, dried over Na.sub.2SO.sub.4,
filtered to remove the Na.sub.2SO.sub.4, concentrated under reduced
pressure, co-evaporated with toluene and dried under high-vacuum
overnight to give 12.2 g of product 4.
[0394] The crude product 4 was co-evaporated with toluene again and
used without further purification. TES protected dipeptide was
dissolved in 80 mL THF (anhydrous, inhibitor-free) and cooled to
-45.degree. C. and stirred for 15 minutes before adding KHMDS (0.5
M in toluene, 50 mL, 25.0 mmol, 1.05 equiv) drop-wise. After the
addition of KHMDS was complete, the reaction mixture was stirred at
-45.degree. C. for 15 minutes, and chloromethyl butyrate (3.6 mL,
1.2 equiv, 28.3 mmol) was added. The reaction mixture changed from
light yellow to a bluish color. TLC (20% EtOAc/petroleum ether)
showed the reaction was complete. The reaction was quenched by
adding 20 mL MeOH, the mixture was warmed to room temperature and
concentrated under reduced pressure to an oily residue. The residue
was dissolved in petroleum ether and passed through short silica
plug to remove the potassium salt. The plug was washed with 13%
EtOAc/petroleum ether, and the collected eluents were combined and
concentrated under reduced pressure to give 12.1 g of product 5
(two steps, yield 76%)
##STR00142##
Example
[0395] Large Scale Synthesis of Tripeptide 6. Alkylated dipeptide 5
(7.6 g, 12.4 mmol), N-methyl pipecolinate (MEP) (7.0 g, 48.9 mmol,
4 equiv) and pentafluorophenol (10.0 g, 54.3 mmol 4.4 equiv) were
added to a flask. N-methylpyrrolidone (NMP, 152 mL) was added to
the mixture. To the mixture was added diisopropylcarbodiimide (DIC,
8.43 mL, 54.4 mmol, 4.4 equiv) was added to the mixture. The
mixture was stirred at room temperature for 1 h. Pd/C (10%, dry,
3.0 g) was added. The flask was shaken under hydrogen (30-35 psi)
for 5 hours. The reaction mixture was analyzed by HPLC. The
reaction was complete. The mixture was filtered through celite. The
celite was washed with 500 mL ethyl acetate. The solutions were
combined and transferred to a separatory funnel and washed with 1%
NaHCO.sub.3/10% NaCl solution (250 mL.times.4). The organic layer
was isolated and evaporated on a rotary evaporator under reduced
pressure. The crude product was dissolved in dichloromethane and
the urea was filtered. The crude product solution was loaded onto a
Teledyne Redisep Silica Column (330 g) and purified with
EtOAc/petroleum ether on CombiFlash flash chromatography system.
The fractions were collected and organic solvent was removed by
evaporating to give 5.0 g of the tripeptide (61%). NMRand mass
spectral data were consistent with those measured for the
Example
##STR00143##
Example
[0396] Large Scale Synthesis of Tripeptide Acid 7. To 2 L of 0.05 M
phosphate (pH=7.4) at 30.degree. C. was added 3.6 g of porcine
liver esterase (17 units/mg). 3.0 g of methyl ester 6 was dissolved
in 100 mL of DMSO. The first 50 mL of this solution was added at a
rate of 1.1 mL/h, and the second half was added at a rate of 1.2
mL/h via syringe pump. After the addition was complete, the
reaction mixture was allowed to stir at 30.degree. C. for several
hours. HPLC of an EtOAc extract of the reaction mixture showed the
reaction was complete. The reaction mixture was drained from the
reactor in 1 L portions and extracted with 94% EtOAc-6% MeOH
(vol./vol.) solution (3.times.1 L). The combined extracts were
washed with brine, dried over Na.sub.2SO.sub.4 and concentrated
under reduced pressure. 2.8 g of product 6 was recovered (95%). The
product appeared to be clean by UPLC analysis, except for
pentafluorophenol carried over from the previous reaction.
##STR00144##
Example
[0397] Large Scale Synthesis of Tubulysin B. 3.0 g (4.30 mmol) of
tripeptide 7 was dissolved in 18 mL THF and 0.70 mL (4.30 mmol) of
3HF.NEt.sub.3 was added and the reaction mixture stirred for 30
minutes. LC-MS analysis (10% to 100% acetonitrile, pH 7 buffer)
confirmed complete deprotection of the TES group. THF was removed
under reduced pressure. The residue was dried under high vacuum for
5 minutes. The crude product was dissolved in 18 mL dry pyridine.
6.11 mL (64.50 mmol, 15 equiv) of Ac.sub.2O was added at 0.degree.
C. The resulting clear solution was stirred at room temperature for
5 hours. LC-MS analysis (10% to 100% acetonitrile, pH 7.0)
indicated>98% conversion. 117 mL of dioxane/H.sub.2O was added
and the resulting mixture stirred at room temperature for 1 hour.
The mixture was concentrated under reduced pressure. The residue
was co-evaporated with toluene (3.times.) and dried under high
vacuum overnight. Crude product 8 was used directly for the next
reaction. LCMS (ESI) [M+H].sup.+ 625.2; the NMR spectral data was
consistent with structure 8.
[0398] Method B. The crude tripeptide acid 8 (2.67 g, 4.30 mmol)
was dissolved in 43 mL of DCM (anhydrous), 1.59 g (8.6 mmol, 2.0
equiv) of pentafluorophenol was added, followed by 9.33 g (21.5
mmol, 5.0 equiv) of DCC on the resin. The resulting reaction
mixture was stirred at room temperature for 16 hours. LC-MS
analysis indicated>96% conversion. The reaction mixture was
filtered and concentrated to dryness, dried under high vacuum for 5
minutes. The residue was dissolved in 16.5 mL DMF, Tut-HCl (1.45 g,
5.59 mmol, 1.3 equiv) was added, followed by DIPEA (4.88 mL, 27.95
mmol, 6.5 equiv). The resulting clear solution was stirred at room
temperature for 10 minutes. The reaction mixture was purified on
prep-HPLC (X-bridge column, 50 mM NH.sub.4HCO.sub.3, 25% to 100%,
six runs). The pure fractions were combined, the acetonitrile was
removed under reduced pressure, the residue was extracted with
EtOAc (2.times.) and the combined EtOAc extracts dried over
Na.sub.2SO.sub.4. The EtOAc was removed under reduced pressure. The
residue was dried under high vacuum for 1 hour to yield 1.35 g of
desired product (38% combined yield from 6). NMR spectral data was
consistent with the tubulysin B.
##STR00145##
Example
[0399] Synthesis of 3-nitro-2-disulfenylethanol 12. A three-necked
500 mL flask was dried and argon purged, then fitted with an
addition funnel 3-Nitro-2-sulfenyl chloride pyridine 12a (5.44 g,
27.11 mmol, 1.4 equiv) was added to the flask and dissolved in 200
mL of CH.sub.2Cl.sub.2. The solution was cooled to 0.degree. C.
Mercaptoethanol (1.33 mL, 18.98 mmol) was diluted with 50 mL of
CH.sub.2Cl.sub.2 and placed in the addition funnel. The
2-mercaptoethanol solution was then added drop wise slowly over the
course of 15 minutes. The reaction progress was monitored by TLC
(Rf 0.4 in 5% CH.sub.3OH/CH.sub.2Cl.sub.2). Solvent was removed
under reduced pressure and dried. The crude product was purified
over silica gel (5% CH.sub.3OH/CH.sub.2Cl.sub.2). The fractions
were collected and solvent was removed by evaporating on a rotary
evaporator and dried. 3.4 g of 3-nitro-2-disulfenylethanol 12 was
obtained (77% yield).
##STR00146##
Example
[0400] Synthesis of
4-nitrophenyl-(3'-nitropyridin-2'-yl)disulfenylethyl carbonate 11.
A 250 mL Round-Bottomed Flask was dried and argon purged.
3-Nitro-2-disulfenylethanol 12 (3.413 g, 14.69 mmol) was added and
dissolved in 45 mL of CH.sub.2Cl.sub.2. 4-Nitrophenylchloroformate
(3.663 g, 17.63 mmol, 1.2 equiv) was added, along with
triethylamine (2.9 mL, 20.57 mmol, 1.4 equiv), and the mixture
stirred under argon overnight. The mixture was concentrated under
reduced pressure and dried. The residue was purified by silica (30%
EtOAc/petroleum ether) and the fractions were collected, solvent
was removed under reduced pressure, and dried. 2.7 g of
4-nitrophenyl-(3'-nitropyridin-2'-yl)disulfenylethyl carbonate 11
was obtained (47% yield).
##STR00147##
Example
[0401] Synthesis of 2-(Boc-tubutyrosine (Tut))hydrazinecarboxylic
acid (3' nitropyridyl-2'-yl)disulfanylethyl ester 14. 10.67 g (33
mmol) of Boc-Tut-acid 10 was dissolved in 100 mL anhydrous THF,
17.24 g (33 mmol) of PyBop, and 17.50 mL (99 mmol, 3.0 equiv) of
DIPEA were added. The reaction mixture stirred for few minutes, 1.0
mL (31.68 mmol, 0.96 equiv) of hydrazine was added and stirred for
15 minutes. LC-MS analysis (X-Bridge shield RP18, 3.5 .mu.m column;
gradient 10% to 100% acetonitrile in 6 min, pH 7.4 buffer)
confirmed the hydrazide 13 formation. 14.47 g (36.3 mmol, 1.1
equiv) of 4-nitrophenyl-(3'-nitropyridin-2'-yl)disulfenylethyl
carbonate 11 was added. The resulting clear solution was stirred at
room temperature for 24 hours. LC-MS analysis (X-Bridge shield
RP18, 3.5 .mu.m column; gradient 30% to 100% acetonitrile in 9 min,
pH 7.4 buffer) indicated>98% conversion. The reaction mixture
was diluted with EtOAc (.about.1.0 L), washed with sat. NH.sub.4Cl
(400 mL), sat. NaHCO.sub.3 solution (3.times.300 mL), and brine
(300 mL). The organic layer was dried over Na.sub.2SO.sub.4 (100
g), and concentrated under reduced pressure. The crude product was
loaded onto a Teledyne Redisep Gold Silica Column and eluted with
MeOH/CH.sub.2Cl.sub.2 (330 g column; 0 to 10% gradient) using a
CombiFlash chromatography system. The fractions were collected and
solvent was removed under reduced pressure and dried. 16.10 g of
2-(Boc-Tut)hydrazinecarboxylic acid (3'
nitropyridyl-2'-yl)disulfanylethyl ester 14 was obtained (82%
yield).
##STR00148##
Example
[0402] Synthesis of azido methylbutyrate dipeptide 5. 10.83 g of
dipeptide 3 (27.25 mmol) was dissolved in 100 mL dichloromethane
and imidazole (2.05 g, 1.1 eq.) was added. The reaction mixture was
stirred at room temperature to dissolve all solids and cooled in
the ice bath for 10 min. TESCl (4.8 mL, 1.05 eqiv.) was added
drop-wise at 0.degree. C., stirred under argon, and warmed to room
temperature over 1.5 h. TLC (3:1 hexanes/EtOAc) showed complete
conversion. The reaction was filtered to remove the imidazole HCl
salt. 125 mL dichloromethane was added to the filtrate, and the
resulting solution was extracted with 250 mL brine. The brine layer
was extracted with 125 mL dichloromethane. The combined organic
phase was washed with 250 mL brine, separated, dried over 45.2 g of
Na.sub.2SO.sub.4, and filtered. The resulting solution was
concentrated under reduced pressure, co-evaporated with toluene
(2.times.5 mL) and dried over high-vacuum overnight to give 14.96 g
of crude product 4.
[0403] The crude product 4 was used without further purification.
TES protected dipeptide was dissolved in 100 mL THF (anhydrous,
inhibitor-free), cooled to -45.degree. C., and stirred at
-45.degree. C. for 15 minutes before adding KHMDS (0.5 M in
toluene, 61 mL, 1.05 equiv.), drop-wise. After the addition of
KHMDS was finished, the reaction was stirred at -45.degree. C. for
20 minutes, and chloromethyl butyrate (4.4 mL, 1.1 equiv.) was
added. The reaction mixture was stirred at -45.degree. C. for
another 20 minutes. The reaction was quenched with 25 mL MeOH and
warmed to room temperature. 250 mL EtOAc and 250 mL brine were
added to the reaction mixture, and the organic phase was separated.
The solvent was evaporated to reduce the volume of solution. The
solution was passed through 76.5 g silica in a 350 mL sintered
glass funnel The silica plug was washed with 500 mL EtOAc/petroleum
ether (1:4). The filtrate and the wash were concentrated to oily
residue and dried under high vacuum to give 16.5 g product 5 as a
light yellow wax.
##STR00149##
Example
[0404] Synthesis of tripeptide methyl ester 6. Based on 16.5 g of
alkylated dipeptide 5 (26.97 mmol), N-methyl pipecolinate (MEP)
(5.51 g, 1.4 equiv.) and pentafluorophenol (7.63 g, 1.5 equiv.)
were added to a 300 mL hydrogenation flask. NMP (115 mL) was then
added, followed by EDC (7.78 g, 1.5 equiv.). The mixture was
stirred at room temperature for overnight. 16.5 g of alkylated
dipeptide 5 was dissolved in 16.5 mL NMP, transferred the solution
into the hydrogenation flask, washed the residual 5 with 8 mL NMP,
and transferred into the hydrogenation flask. Dry 10% Pd/C (1.45,
0.05 eq.) was added. The reaction mixture was vacuumed/back filled
with hydrogen 3 times, and the flask was shaken under hydrogen
(.about.35 psi) for 3.5 hours. The reaction mixture was analyzed by
HPLC. The reaction mixture was filtered through 40 g of celite in a
350 mL sintered glass funnel and washed with 250 mL of EtOAc. The
filtrate and the wash were transferred to a separatory funnel and
washed with a 1% NaHCO.sub.3/10% NaCl solution (200 mL.times.3).
The organic layer was isolated and dried over 45.2 g of
Na.sub.2SO.sub.4. The solution was filtered and rotovaped under
reduced pressure. A sticky amber residue was obtained and dried
under high vacuum overnight to give 19.3 g of crude product. The
crude product was dissolved in 10 mL of dichloromethane, split into
two portions, and purified with a 330 g Teledyne Redisep Silica
Gold column. The combined fractions of two purifications were
evaporated and dried under high vacuum to give 7.64 g of 6 as a
pale yellow solid (overall yield: 39% over 3 steps from compound
3).
##STR00150##
Example
[0405] Alternative Synthesis of tripeptide acid 7. Methyl ester 6
(6.9 g, 9.7 mmol) was dissolved in 1,2-dichloroethane (193 mL) and
added to a round bottomed flask, equipped with a stir bar and
condenser. To this solution was added trimethyltin hydroxide (24.6
g, 14 eq.). The mixture was heated at 70.degree. C. for 5 hours.
LC-MS analysis indicated that the desired product had been formed
and <15% of starting methyl ester 6 remained. The reaction was
cooled in an ice bath for 30 minutes. The resulting precipitate was
then removed by filtration. The filtrate was stored overnight at
-20.degree. C. The filtrate was then divided into two portions and
each was subjected the chromatography procedure which follows.
[0406] Each portion was concentrated under reduced pressure and
then placed under high vacuum for 30 min. The concentrate was then
immediately dissolved in acetonitrile (95 mL). To this solution was
then added an ammonium bicarbonate solution (95 mL; 50 mM, pH=7).
This solution was loaded onto a Biotage SNAP C18 reverse phase
cartridge (400 g, KP-C18-HS) and eluted with 50 mM ammonium
bicarbonate and acetonitrile (1:1 to 100% ACN) using a Biotage
chromatography system. Fractions were analyzed by LC-MS. Pure
fractions were combined and ACN was removed under reduced pressure.
The resulting aqueous suspension was extracted with EtOAc
(3.times.). The combined organic layers were washed with brine,
dried over anhydrous Na.sub.2SO.sub.4, and concentrated under
reduced pressure. Purification of the two portions resulted in the
recovery of 7 (4.6 g, 65%).
##STR00151##
Example
[0407] Synthesis of acetyl tripeptide acid 8. In a round bottomed
flask, tripeptide acid 7 (3.9 g, 5.6 mmol) was dissolved in
anhydrous THF (23 mL). To this solution was added 3 HF.TEA complex
(1.8 mL, 2 eq.). The reaction was stirred at room temperature for 1
hour. LC-MS analysis indicated complete conversion to the desired
des-TES product 7a. The solvent was removed under reduced pressure
and the residue was placed on the high vacuum for 40 minutes. The
resulting residue was then dissolved in pyridine (26 mL), and
acetic anhydride (7.9 mL, 15 eq.) and DMAP (25 mg) were added. The
reaction was stirred at room temperature for 1 hour. LC-MS analysis
indicated complete conversion to the desired acetyl tripeptide acid
8. To the reaction mixture was then added a 1:1 solution of
1,4-dioxane/water (150 mL). The reaction was stirred for 1 hour at
which point the solvents were removed under high vacuum rotovap. To
the residue was added toluene and the solvent was removed under
vacuum (80 mL, 3.times.). The resulting crude 8 was dried under
high vacuum overnight. The crude material was then dissolved in ACN
(72 mL). Sodium phosphate buffer (50 mM, pH=7.8, 288 mL) was then
added, and the pH of the resulting suspension was adjusted to
neutral using saturated sodium bicarbonate solution. This solution
was loaded onto a Biotage SNAP C18 reverse phase cartridge (400 g,
KP-C18-HS) and eluted with water and acetonitrile (20% ACN to 65%
ACN) using a Biotage chromatography system. Fractions were analyzed
by LC-MS. Clean fractions were combined, the ACN was removed, and
the aqueous solution was placed on the freeze dryer, resulting in
purified acetyl tripeptide 8 (2.5 g, 71%).
##STR00152##
Example
[0408] Synthesis of 2-(tubulysin B)hydrazinecarboxylic acid (3'
nitropyridyl-2'-yl)disulfanylethyl ester 2. The activated
Boc-Tut-fragment 14 (2.63 g, 4.42 mmol, 1.1 equiv) was treated with
TFA/CH.sub.2Cl.sub.2 (42 mL; 1:1) and stirred for 30 minutes. LC-MS
analysis (X-Bridge shield RP18, 3.5 .mu.m column; gradient 10% to
100% acetonitrile in 6 min, pH 7.4 buffer) confirmed the product
formation. TFA was removed under reduced pressure, co-evaporated
with CH.sub.2Cl.sub.2 (3.times.30 mL) and activated Tut-derivative
9 was dried under high vacuum for 18 h. In another flask, the
tripeptide acid 8 (2.51 g, 4.02 mmol) was dissolved in 70 mL
CH.sub.2Cl.sub.2 (anhydrous) and 1.48 g (8.04 mmol, 2.0 equiv) of
pentafluorophenol in 5 mL of CH.sub.2Cl.sub.2 was added, followed
by 8.74 g (20.1 mmol, 5.0 equiv) of DCC-resin. The resulting
reaction mixture was stirred at room temperature for 20 hours.
LC-MS analysis (X-Bridge shield RP18, 3.5 .mu.m column; gradient
10% to 100% acetonitrile in 6 min, pH 7.4 buffer) indicated>99%
conversion. The DCC-resin was filtered off, the CH.sub.2Cl.sub.2
was removed under reduced pressure, and the pentafluorophenol
activated product 15 was dried under high vacuum for 10 minutes.
The residue was dissolved in 16.7 mL DMF, and DIPEA (12.6 mL, 72.36
mmol, 18.0 equiv) was added. Tut-fragment trifluoroacetic acid salt
9 in DMF (8.5 mL) was added slowly over 5 min. The resulting clear
solution was stirred at room temperature for 1 h. LC-MS analysis
(X-Bridge shield RP18, 3.5 .mu.m column; gradient 10% to 100%
acetonitrile in 6 min, pH 7.4 buffer) confirmed the product
formation. The reaction mixture was diluted with EtOAc (700 mL),
washed with brine (300 mL, 2.times.100 mL), dried over
Na.sub.2SO.sub.4 (75 g), concentrated, and dried for 15 hours. The
crude product was dissolved in CH.sub.2Cl.sub.2 (25 mL) and loaded
onto a Teledyne Redisep Gold Silica Column and eluted with
MeOH/CH.sub.2Cl.sub.2 (330 g column; 0 to 5% gradient) using
Combiflash chromatographic system. The fractions were collected and
solvent was removed by evaporating on a rotary evaporator and
dried. 3.91 g of 2-(tubulysin B)hydrazinecarboxylic acid (3'
nitropyridyl-2'-yl)disulfanylethyl ester 2 was obtained (89%
yield).
Example
[0409] General Synthesis of Disulfide Containing Tubulysin
Conjugates. A binding ligand-linker intermediate containing a thiol
group is taken in deionized water (ca. 20 mg/mL, bubbled with argon
for 10 minutes prior to use) and the pH of the suspension was
adjusted with aqueous phosphate (bubbled with argon for 10 minutes
prior to use) to a pH of about 7.0 (the suspension may become a
solution when the pH increased). Additional deionized water is
added (ca. 20-25%) to the solution as needed, and to the aqueous
solution is added immediately a solution of compound (2) in
acetonitrile (ca. 20 mg/mL). The reaction mixture becomes
homogenous quickly. After stirring under argon, e.g. for 45
minutes, the reaction mixture is diluted with 2.0 mM sodium
phosphate buffer (pH 7.0, ca 150 volume percent) and the
acetonitrile is removed under vacuum. The resulting suspension is
filtered and the filtrate may be purified by preparative HPLC.
Fractions are lyophilized to isolate the conjugates. The foregoing
method is equally applicable for preparing other tubulysin
conjugates by the appropriate selection of the tubulysin starting
compound, including tubulysin starting compounds having a
3-nitropyridin-2-ylthio activating group.
[0410] Illustrative binding ligand-linker intermediates are
described in WO 2008/112873, the disclosure of which is
incorporated herein by reference.
##STR00153##
[0411] EC0488. This binding ligand-linker intermediate was prepared
by SPPS according to the general peptide synthesis procedure
described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00002 Reagents mmol equivalent MW amount
H-Cys(4-methoxytrityl)- 0.10 0.17 g 2-chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH
0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g
Fmoc-Glu-OtBu 0.19 1.9 425.47 0.080 g N.sup.10TFA-Pteroic Acid 0.16
1.6 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq of AA
PyBOP 1.0 eq of AA
[0412] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0413] Cleavage step. Reagent: 92.5% TFA, 2.5% H2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3.times. (10 min, 5 min, 5 min) with argon
bubbling, drain, wash the resin once with cleavage reagent, and
combine the solution. Rotavap until 5 ml remains and precipitate in
diethyl ether (35 mL). Centrifuge, wash with diethyl ether, and
dry. About half of the crude solid (.about.100 mg) was purified by
HPLC.
[0414] HPLC Purification step. Column. Waters Xterra Prep MS C18 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 25 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give 43 mg EC0488 (51% yield). .sup.1H NMR and LC/MS (exact mass
1678.62) were consistent with the product.
Example
[0415] Synthesis of EC0531.
##STR00154##
[0416] EC0531 is prepared according to the processes described
herein from compound (2) and EC0488 in 73% yield.
##STR00155## ##STR00156##
[0417] The ether analog of compound 103 can also be prepared.
Reductive condensation of that ether analog with MEP yields 105
directly.
##STR00157##
Example
[0418] Compound 102. 4.9 g of dipeptide 101 (11.6 mmol) was
dissolved in dichloromethane (60 mL) and imidazole (0.87 g, 12.7
mmol) was added to the resulting solution at 0.degree. C. The
reaction mixture was warmed slightly to dissolve all the solids and
cooled back to 0.degree. C. Triethylsilyl chloride (TESCl) (2.02
mL, 12.1 mmol) was added drop-wise at 0.degree. C., stirred under
argon, and warmed to room temperature over 2 h. TLC (3:1
hexanes/EtOAc) showed complete conversion. The reaction was
filtered to remove the imidazole HCl salt, extracted with
de-ionized water, and the aqueous phase was back-washed with
dichloromethane. The combined organic phase was washed with brine,
dried over Na.sub.2SO.sub.4, filtered, and concentrated under
reduced pressure. The resulting residue was co-evaporated with
toluene and dried over high-vacuum overnight to give 6.4 g of crude
product 102 (5.9 g theoretical yield).
##STR00158##
Example
[0419] Compound 103. The crude product 102 was co-evaporated with
toluene again and then dissolved in THF (38 mL, anhydrous,
inhibitor-free), cooled to -45.degree. C., and stirred for 15
minutes before adding potassium hexamethyldisilazide (KHMDS) (0.5 M
in toluene, 25.5 mL, 12.8 mmol, 1.1 equiv), drop-wise. After the
addition of KHMDS was finished, the reaction was stirred at
-45.degree. C. for 15 minutes, and chloromethyl butyrate (1.8 mL,
1.2 equiv, 14 mmol) was added. The reaction mixture changed from
light yellow to a blueish color. TLC (20% EtOAc/petroleum ether)
showed the majority of starting material converted. LC-MS showed
about 7% starting material left. The reaction was quenched by
adding MeOH (3 mL), warmed to room temperature, and concentrated to
an oily residue. The residue was dissolved in petroleum ether and
passed through short silica plug. The plug was washed with 13%
EtOAc/petroleum ether, and the eluents was concentrated. The
alkylated product was passed through silica plug
(product/silica=1:50) and washed by 13% EtOAc/petroleum ether to
remove residual starting material to give 5.7 g of product 103 (two
steps with yield 76%).
##STR00159##
Example
[0420] Compound 104. Alkylated dipeptide 103 (4.3 g, 7.0 mmol),
N-methyl pipecolinate (MEP) (4.0 g, 28.0 mmol, 4 equiv), and
pentafluorophenol (PFP) (5.7 g, 30.8 mmol 4.4 equiv) were added to
a flask. N-Methyl-2-pyrrolidone (NMP) (86 mL) was then added,
followed by N,N'-diisopropylcarbodiimide (DIC) (4.77 mL, 30.8 mmol,
4.4 equiv). The mixture was stirred at room temperature for 1 h.
Pd/C (10%, dry, 1.7 g) was added. The flask was shaken under
hydrogen (30-35 psi) for 5 hours. The reaction mixture was analyzed
by HPLC. The starting material was found to be less than 3%. The
mixture was filtered through celite. The celite was washed with
ethyl acetate (200 mL). The combined filtrate was transferred to
separatory funnel and washed with 1% NaHCO.sub.3/10% NaCl solution
(200 mL.times.4). The organic layer was isolated and rotavaped
under reduced pressure. The crude product was dissolved in 40 mL of
MeOH/H.sub.2O (3:1). The crude product solution was loaded onto a
Biotage C18 column (Flash 65i, 350 g, 450 mL, 65.times.200 mm) and
eluted with buffer A [10 mM NH.sub.4OAc/ACN (1:1)] and B (ACN). The
fractions were collected and organic solvent was removed under
reduced pressure. A 10% NaCl solution (100 mL) and methyl t-butyl
ether (MTBE) (100 mL) were added to the flask and the mixture was
transferred to a separatory funnel. The organic layer was isolated
and dried over anhydrous Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure. 2.5 g of tripeptide
intermediate 104 was obtained (yield 50%).
##STR00160##
Example
[0421] Compound 105a. Compound 104 (50 mg, 0.07 mmol) in allyl
alcohol (5 mL) was treated with di-n-butyltin oxide (1.75 mg, 0.007
mmol, 10% mol). The reaction mixture was heated to reflux for 22
hrs till the reaction was complete. The reaction was concentrated
and purified with HPLC in 10-100% ACN/NH.sub.3HCO.sub.3 buffer
(pH7.0) to give the title compound (32.4 mg, yield 65%). LCMS:
[M+H].sup.+ m/z=707.73. .sup.1H NMR (CD.sub.3OD, .delta. in ppm):
8.35 (s, 1H), 6.01 (m, 2H); 5.2-5.5 (m, 3H), 5.14 (d, J=10.26 Hz,
1H), 5.04 (d, J=5.87 Hz, 1H), 4.88 (s, 3H), 4.82 (d, J=5.5 Hz, 2H),
4.70 (d, J=8.79 Hz, 1H), 4.50 (d, J=10.26 Hz, 1H), 4.42 (b, 1H),
4.06 (s, 2H), 2.92 (d, J=11.36 Hz, 1H), 2.55 (d, J=9.17 Hz, 1H),
1.95-2.20 (m, 7H), 1.45-1.82 (m, 7H), 1.22 (m, 2H), 0.82-1.00 (m,
17H), 0.77 (d, J=6.23 Hz, 3H), 0.59-0.70 (m, 6H); .sup.13C NMR
(CD.sub.3OD, .delta. in ppm): 176.97, 175.08, 174.09, 160.95,
146.02, 134.13, 132.05, 127.94, 117.38, 116.37, 73.85, 70.32,
69.14, 68.40, 65.34, 56.89, 55.20, 53.55, 43.35, 40.37, 36.38,
31.59, 30.15, 24.80, 24.27, 22.93, 19.09, 18.71, 15.31, 9.52, 5.77,
4.41.
##STR00161##
Example
[0422] Compound 106a. Compound 105a (15.3 mg, 0.02 mmol) was
subjected to hydrolysis with LiOH.H.sub.2O (0.99 mg, 0.024 mmol) in
4:1 THF/H.sub.2O (2.5 mL) for 19 hrs at room temperature (rt). The
reaction was purified with HPLC in 10-100% ACN/NH.sub.3HCO.sub.3
buffer (pH7.0) to provide compound 106a (9.2 mg, yield 83%). LCMS:
[M+H].sup.+ m/z=553.55. .sup.1H NMR (CD.sub.3OD, .delta. in ppm):
7.94 (s, 1H), 6.00 (m, 1H), 5.1-5.4 (m, 3H), 4.68 (d, J=9.09 Hz,
2H), 4.10 (d, J=3.81 Hz, 2H), 2.80 (b, 1H), 2.56 (s, 2H), 1.4-2.2
(m, 1H), 1.20 (m, 1H), 0.80-0.99 (m, 13H); .sup.13C NMR
(CD.sub.3OD, .delta. in ppm): 17.90, 167.53, 153.18, 134.05,
123.09, 116.53, 68.63, 67.25, 54.85, 54.44, 42.10, 37.75, 36.53,
30.60, 29.13, 24.26, 23.25, 21.37, 20.32, 19.53, 14.72, 9.51.
##STR00162##
Example
[0423] Compound 107a. To compound 106a (9.2 mg, 0.017 mmol) in
pyridine (1 mL) was added acetic anhydride (15.7 .mu.L, 0.165 mmol)
and a catalytic amount of 4-dimethylamino pyridine (0.053 M in
pyridine, 5 .mu.L) at rt under argon. The reaction was stirred for
24 hrs. To the reaction mixture was added 0.4 mL of dioxane/water
(1:1) and stirred for 10 min, and then the solvent was removed in
vacuo. The residue was purified with HPLC in 10-100%
ACN/NH.sub.3HCO.sub.3 buffer (pH7.0) to provide the product 7a 10.4
mg (quantitative yield).
[0424] LCMS: [M+H].sup.+ m/z=595.59. .sup.1H NMR (CD.sub.3OD,
.delta. in ppm): 7.96 (s, 1H), 5.8-6.0 (m, 2H), 5.33 (d, J=17.59
Hz, 1H), 5.19 (d, J=10.56 Hz, 1H), 4.71 (d, J=9.23 Hz, 2H), 4.05
(d, J=5.71 Hz, 2H), 3.30 (m, 6H), 2.50 (b, 4H), 2.10 (s, 3H),
1.40-2.00 (m, 7H), 1.20 (m, 1H), 0.80-1.02 (m, 11H); .sup.13C NMR
(CD.sub.3OD, .delta. in ppm): 175.11, 170.44, 167.29, 153.45,
133.92, 123.40, 116.79, 116.55, 68.62, 67.82, 67.11, 54.75, 54.16,
42.39, 36.31, 36.12, 34.91, 30.55, 29.26, 24.09, 23.26, 21.25,
20.24, 19.48, 19.20, 14.78, 9.56.
##STR00163##
Example
[0425] Compound 107a (10.4 mg, 0.017 mmol) was dissolved in
anhydrous methylene chloride (4 mL) and to this solution was added
DCC-resin (2.3 mmol/g, 0.038 g, 0.087 mmol) and followed by
pentafluorophenol (PFP, 6.26 mg, 0.034 mmol) at rt under argon. The
reaction was stirred for 19 hrs at rt. The reaction mixture was
filtered and the solution was concentrated. The residue was
redissolved in dry DMF (4 mL). Then,
(2S,4R)-4-amino-5-(4-hydroxyphenyl)-2-methylpentanoic acid (Tut
acid) was added into the solution, followed by DIPEA (8.9 .mu.L,
0.051 mmol). When completed, the reaction was concentrated in vacuo
and the residue was purified with HPLC. Product 108a was obtained
(13.1 mg, 96% yield). LCMS: [M+H].sup.+ m/z=800.88. .sup.1H NMR
(CD.sub.3OD, 6 in ppm): 8.08 (s, 1H), 7.02 (d, J=8.43 Hz, 2H), 6.68
(d, J=8.06 Hz, 2H), 5.99 (d, J=10.99 Hz, 1H), 5.80 (m, 1H), 5.38
(d, J=9.53 Hz, 1H), 5.31 (d, J=17.23 Hz, 1H), 5.13 (d, J=10.63 Hz,
1H), 4.66 (d, J=8.79 Hz, 1H), 4.55 (d, J=10.28 Hz, 1H), 4.30 (b,
2H), 4.00 (b, 2H), 3.16 (b, 2H), 2.80 (d, J=5.86 Hz, 2H), 2.40 (b,
4H), 2.10-2.30 (b, 2H), 1.40-1.90 (b, 6H), 1.23 (s, 3H), 1.17 (d,
J=6.96 Hz, 3H), 1.05 (d, J=6.23 Hz, 2H), 0.94 (d, J=6.97 Hz, 2H),
0.90 (d, J=7.70 Hz, 2H), 0.79 (d, J=6.6 Hz, 3H); .sup.13C NMR
(CD.sub.3OD, .delta. in ppm): 179.24, 174.88, 170.97, 170.43,
170.20, 161.29, 155.62, 149.30, 133.70, 130.23, 128.44, 123.54,
116.41, 114.72, 69.92, 68.15, 67.87, 54.96, 53.92, 49.27, 42.40,
39.62, 37.72, 36.91, 36.08, 35.29, 31.01, 29.51, 29.33, 24.08,
23.72, 21.93, 19.40, 19.34, 18.89, 17.24, 15.00, 9.34.
##STR00164##
Example
[0426] Compound 114a. Compound 107a (26.4 mg, 0.044 mmol) was
dissolved in anhydrous methylene chloride (5 mL) and to this
solution was added DCC-resin (2.3 mmol/g, 0.096 g, 0.22 mmol),
followed by pentafluorophenol (PFP, 16.4 mg, 0.089 mmol) at rt
under argon. The reaction was stirred for 19 hrs at rt. The
reaction was filtered and concentrated and the residue was
redissolved in dry DMF (5 mL).
2-((3-nitropyridin-2-yl)disulfanyl)ethyl
2-((2S,4R)-4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2-methylpen-
tanoyl)hydrazinecarboxylate (40.0 mg, 0.067 mmol) was deprotected
with TFA/DCM (1:1, 5 mL, 1 drop of TIPS as scavenger) at rt for 1
hr. The solvent was removed under reduced pressure, 5 mL more of
DCM was added, and then the solvent was co-evaporated to dryness.
The residue was dissolved in dry DMF (2 mL) and was added to the
solution of PFP ester intermediate in DMF made above after the
addition of DIPEA (23.2 .mu.L, 0.13 mmol) at rt under argon. The
reaction was stirred for 19 hrs and diluted with EtOAc (20 mL). The
organic phase was washed with water (5 mL.times.3) and brine. The
organic layer was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated after filtration to give the crude product 114a (52.8
mg), which could be used for conjugation with folate. LCMS:
[M+H].sup.+ m/z=1072.92.
##STR00165##
Example
[0427] Compound 105b. Compound 4 (75.9 mg, 0.11 mmol) in n-butanol
(4 mL) was treated with n-Bu.sub.2SnO (2.12 mg, 0.0085 mmol, 8.0
mol %) at rt and the reaction was heated to 100.degree. C. for 2
days. The solvent was reduced to a minimum and the product was
purified with CombiFlash (Teledyne Redisep Silica column, eluted
with 0 to 15% of MeOH/DCM) to give 44.0 mg (56%) of intermediate
105b. LCMS: [M+H].sup.+ m/z=739.61. .sup.1H NMR (CDCl.sub.3,
.delta. in ppm): 8.07 (s, 1H), 7.02 (d, J=9.68 Hz, 1H), 5.27 (d,
J=9.67 Hz, 1H), 5.02 (dd, J=8.36, 2.64 Hz, 1H), 4.69 (t, J=9.23 Hz,
4.20-4.40 (m, 4H), 3.47 (td, J=6.6, 1.76 Hz, 2H), 2.88 (d, J=11.44
Hz, 1H), 2.46 (dd, J=10.55, 3.08 Hz, 2H), 1.90-2.24 (m, 8H),
1.10-1.79 (m, 18H), 0.80-1.00 (m, 19H), 0.58-0.78 (m, 6H).
##STR00166##
Example
[0428] Compound 106b. The same procedure as for compound 106a was
followed. 106b (11.7 mg, 35%) was obtained from intermediate 105b
(44.0 mg). LCMS: [M+H].sup.+ m/z=569.51. .sup.1H NMR (CDCl.sub.3
drops of CD.sub.3OD, .delta. in ppm) 8.00 (s, 1H), 5.23 (b, 1H),
4.80 (b, 1H), 4.58 (d, J=8.80 Hz, 1H), 4.42 (b, 1H), 3.45 (t,
J=6.38 Hz, 1H), 3.33 (b, 3H), 2.15-2.40 (m, 3H), 1.80-2.10 (m, 2H),
1.40-1.79 (m, 4H), 1.04-1.38 (m, 3H), 0.60-1.02 (m, 9H).
##STR00167##
Example
[0429] Compound 107b. In a 10 mL round bottom flask, 106b (11.7 mg,
0.021 mmol) and acetic anhydride (20 .mu.L, 0.212 mmol) were
dissolved in pyridine (imp. To this solution was added a catalytic
amount of dimethylaminopyridine (1 mg, 0.008 mmol). This solution
was stirred at room temperature for 16 h under Argon. LCMS (10-100%
ACN, 50 mM NH.sub.4HCO.sub.3 pH7) indicated all of the starting
material had been consumed and product had been formed. To the
flask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL) and
the solution was stirred for 10 min to hydrolyze any potential
diacetate side product. The reaction mixture was concentrated under
reduced pressure, then purified by preparative HPLC (10-100% ACN,
50 mM NH.sub.4HCO.sub.3 pH7) to yield 107b (9.6 mg, 76%). LCMS:
[M+H].sup.+=611.53. .sup.1H H NMR (CDCl.sub.3 w/2 drops
CD.sub.3OD):
[0430] 7.97 (s, 1H) 5.83 (d, J=9.9 Hz, 1H) 5.28 (s, 1H) 4.58 (d,
J=9.0 Hz, 1H) 4.24 (d, J=9.3 Hz, 2H) 3.42 (m, 3H) 2.60-2.95 (br,
7H) 2.20-2.58 (br, 6H) 1.76-2.20 (br, 11H) 1.40-1.56 (br, 12H)
1.02-1.20 (br, 12H) 0.40-1.10 (br, 27H) 0.04 (s, 8H). .sup.13C NMR:
175.04, 170.53, 67.78, 53.74, 44.33, 36.79, 35.64, 31.69, 29.89,
24.86, 20.96, 20.49, 19.52, 15.95, 13.99, 10.65, 1.21
##STR00168##
Example
[0431] Compound 108b. In a 25 mL round bottom flask, 107b (9.6 mg,
0.016 mmol) and pentafluorophenol (28.2 mg, 0.153 mmol) were
dissolved in dry dichloromethane (5 mL). N-cyclohexylcarbodiimide,
N'-methyl polystyrene (33.4 mg, 2.3 mmol/g, 0.077 mmol) was added
and the reaction mixture was stirred at room temperature for 16 h
under Argon. LCMS (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7)
indicated all of the starting material had been consumed and
activated intermediate had been formed. The reaction mixture was
filtered and concentrated under reduced pressure, and the residue
was dissolved in a solution of N,N-dimethylformamide (2 mL) and
N,N-diisopropylethylamine (8mL, 0.046 mmol). PFP ester intermediate
(6.0 mg, 0.023 mmol) was added and the reaction mixture was stirred
at room temperature for 2 h under argon. LCMS (10-100% ACN, 50 mM
NH.sub.4HCO.sub.3 pH7) indicated all of the activated intermediate
had been consumed and product had been formed. The reaction mixture
was purified by preparative HPLC (10-100% ACN, 50 mM
NH.sub.4HCO.sub.3 pH7) to yield 108b (4.7 mg, 37%). LCMS:
[M+H].sup.+ m/z=816.71. .sup.1H NMR (CDCl.sub.3, .delta. in ppm):
8.04 (s, 1H) 7.05 (d, J=8.4 Hz, 2H) 6.80 (d, J=8.4 Hz, 2H) 5.90 (m,
1H) 5.38 (d, J=10.2 Hz, 1H) 4.63 (t, J=9.3 Hz, 1H) 4.38 (br, 1H)
4.27 (d, J=9.9 Hz, 1H) 3.48 (m, 1H) 3.34 (m, 2H) 2.86 (m, 6H) 2.56
(m, 3H) 2.23 (s, 3H) 2.16 (s, 3H) 1.22-2.10 (br, 16H) 1.12 (d,
J=6.9 Hz, 3H) 1.03 (d, J=6.6 Hz, 3H) 0.88 (m, 14H). .sup.13C NMR:
174.90, 170.44, 161.73, 155.52, 149.37, 130.77, 128.56, 124.33,
115.91, 70.40, 69.69, 67.62, 55.45, 53.70, 49.25, 44.61, 40.40,
36.94, 36.69, 35.93, 31.77, 31.16, 30.06, 24.94, 23.14, 21.08,
20.74, 20.20, 19.55, 17.78, 16.08, 14.05, 10.70
##STR00169##
Example
[0432] Compound 105c. Compound 4 (73.9 mg, 0.10 mmol) in n-pentanol
(4 mL) was treated with n-Bu.sub.2SnO (2.10 mg, 0.0083 mmol, 8.0
mol %) at rt and the reaction was heated to 100.degree. C. for 2
days. The solvent was reduced to a minimum and the product was
purified with CombiFlash (Teledyne Redisep Silica column, eluted
with 0 to 15% of MeOH/DCM) to give 51.2 mg (64%) of intermediate
105c. LCMS: [M+H].sup.+ m/z=767.64. .sup.1H NMR (CDCl.sub.3,
.delta. in ppm): 8.07 (m, 1H), 7.06 (t, J=9.23 Hz, 1H), 5.95 (d,
J=12.3 Hz, 1H), 5.43 (d, J=12.32 Hz, 1H), 5.26 (d, J=9.68 Hz, 1H),
5.03 (dd, J=8.36, 2.64 Hz, 1H), 4.93 (dd, J=8.36, 6.24 Hz, 1H),
4.71 (dd, J=15.83, 8.80 Hz, 1H), 4.20-4.33 (m, 3H), 3.46 (m, 1H),
2.88 (d, J=11.43 Hz, 1H), 2.30-2.60 (m, 2H), 2.20 (s, 2H),
1.95-2.18 (m, 3H), 1.50-1.80 (m, 6H), 1.10-1.44 (m, 6H), 0.80-1.04
(m, 13H), 0.50-0.77 (m, 6H).
##STR00170##
Example
[0433] Compound 106c. The same procedure as for compound 106a was
followed, intermediate 106c (14.9 mg, 38%) was obtained from 105c
(51.2 mg). LCMS: [M+H].sup.+ m/z=583.56. .sup.1H NMR (CD.sub.3OD,
.delta. in ppm): 7.97 (s, 1H), 5.27 (d, J=9.67 Hz, 1H), 4.67 (d,
J=9.23 Hz, 1H), 4.58 (d, J=9.68 Hz, 1H), 3.53 (m, 3H), 2.80 (b,
1H), 2.58 (b, 4H), 1.48-2.18 (m, 13H), 1.10-1.42 (m, 6H), 0.70-1.08
(m, 18H).
##STR00171##
Example
[0434] Compound 107c. In a 10 mL round bottom flask, 106c (14.9 mg,
0.026 mmol) and acetic anhydride (20 .mu.L, 0.212 mmol) were
dissolved in pyridine (1 mL). This this solution was added a
catalytic amount of dimethylaminopyridine (1 mg, 0.008 mmol). This
solution was stirred at room temperature for 16 h under argon. LCMS
(10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) indicated all of the
starting material had been consumed and product had been formed. To
the flask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL)
and the solution was stirred for 10 min to hydrolyze any potential
diacetate side product. The reaction mixture was concentrated under
reduced pressure, then purified by preparative HPLC (10-100% ACN,
50 mM NH.sub.4HCO.sub.3 pH7) to yield 107c (4.8 mg, 30%). LCMS:
[M+H].sup.+ m/z=625.58. .sup.1H NMR (CDCl.sub.3 w/2 drops
CD.sub.3OD) 7.98 (s, 1H) 5.82 (d, J=10.8 Hz, 1H) 5.26 (s, 1H) 4.57
(d, J=8.4 Hz, 1H) 4.23 (d, J=8.4 Hz, 2H) 3.42 (m, 3H) 2.60-2.92
(br, 8H) 2.15-2.40 (br, 4H) 1.90-2.12 (m, 7H) 1.38-1.90 (br, 14H)
1.00-1.38 (br, 13H) 0.50-1.00 (br, 22H), 0.03 (s, 13H). .sup.13C
NMR: 175.15, 150.56, 125.47, 69.55, 68.09, 55.33, 53.71, 44.59,
36.77, 35.74, 31.34, 30.19, 29.86, 29.32, 28.51, 24.84, 22.85,
22.55, 20.86, 20.40, 19.91, 15.94, 14.10, 10.63, 1.17
##STR00172##
Example
[0435] Compound 108c. In a 25 mL round bottom flask, 107c (4.8 mg,
0.008 mmol) and pentafluorophenol (14.1 mg, 0.077 mmol) were
dissolved in dry dichloromethane (5 mL). N-cyclohexylcarbodiimide,
N-methyl polystyrene (16.7 mg, 2.3 mmol/g, 0.038 mmol) was added
and the reaction mixture was stirred at room temperature for 16 h
under Argon. LC-MS (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7)
indicated all of the starting material had been consumed and
activated intermediate had been formed. The reaction mixture was
filtered and concentrated under reduced pressure, and the residue
was dissolved in a solution of N,N-dimethylformamide (2 mL) and
N,N-diisopropylethylamine (4 .mu.L, 0.023 mmol). PFP ester
intermediate (3.0 mg, 0.012 mmol) was added and the reaction
mixture was stirred at room temperature for 2 h under Argon. LC-MS
(10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) indicated all of the
activated intermediate had been consumed and product had been
formed. The reaction mixture was purified by preparative HPLC
(10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) to yield 108c (1.1 mg,
17%). LCMS: [M+H].sup.+ m/z=830.76. .sup.1H NMR (CDCl.sub.3 w/2
drops CD.sub.3OD): 8.00 (s, 1H) 7.01 (d, J=8.7 Hz, 2H) 6.74 (d,
J=8.4 Hz, 2H) 5.89 (d, J=12.6 Hz, 1H) 5.25 (d, J=9.0 Hz, 1H) 4.55
(d, J=8.7 Hz, 1H) 4.30 (m, 3H) 3.39 (m, 3H) 3.21 (m, 2H) 2.81 (m,
3H) 2.04-2.60 (br, 45H) 1.76-2.04 (m, 5H) 1.34-1.76 (br, 9H) 1.20
(m, 6H) 1.12 (d, J=7.2 Hz, 4H) 1.01 (d, J=6.3 Hz, 3H) 0.89 (t,
J=7.1 Hz, 6H) 0.78 (m, 6H)
##STR00173##
Example
[0436] Compound 114b. In a 5 mL round bottom flask, 113 (10.0 mg,
0.009 mmol) was dissolved in a solution of trifluoroacetic acid
(125mL, 1.632 mmol) and dichloromethane (0.5 mL) and stirred at
room temperature for 1 hr under argon, then 1-butanol (200mL, 2.186
mmol) added and reaction mixture stirred at room temperature for 30
min under argon. LCMS (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7)
indicated all of the starting material had been consumed and
product had been formed. The reaction mixture was purified by
preparative HPLC (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) to
yield 114b (3.2 mg, 32%). LCMS: [M+H].sup.+ m/z=1088.79. .sup.1H
NMR (CDCl.sub.3w/2 drops CD.sub.3OD): 8.86 (s, 1H) 8.47 (d, J=8.0
Hz, 1H) 7.99 (s, 1H) 7.31 (d, J=9.5 Hz, 2H) 7.01 (d, J=7.5 Hz, 2H)
6.73 (d, J=8.5 Hz, 2H) 5.94 (d, J=10.5 Hz, 1H) 5.34 (d, J=10.0 Hz,
1H) 4.58 (m, 3H) 4.38 (t, J=6.0 Hz, 4H), 4.27 (d, J=10.0 Hz, 2H)
3.37 (m, 2H) 3.18 (m, 2H) 3.09 (t, J=6.3 Hz, 3H) 2.70-2.90 (br, 6H)
2.43 (dd, J=11.0 Hz, 3.0 Hz, 2H) 2.26-2.36 (br, 4H) 2.12-2.22 (br,
10H) 2.02-2.12 (br, 2H) 1.86-2.02 (br, 11H) 1.69-1.80 (br, 6H)
1.54-1.69 (br, 10H) 1.34-1.52 (br, 12H) 1.09-1.34 (br, 16H) 1.047
(dd, J=15.0 Hz, 6.5 Hz, 19H) 0.88 (m, 19H) 0.75 (m, 17H). .sup.13C
NMR: 174.95, 174.59, 170.64, 170.23, 161.92, 156.91, 156.06,
153.88, 149.00, 133.77, 130.79, 123.92, 120.98, 115.53, 69.95,
69.61, 67.03, 63.82, 55.32, 53.21, 44.78, 41.42, 40.40, 36.84,
36.38, 35.62, 35.22, 31.54, 31.40, 30.37, 24.99, 24.66, 23.20,
20.68, 20.24, 19.56, 19.27, 17.69, 15.71, 13.72, 10.35
##STR00174##
Example
[0437] Compound 114c. In a 5 mL round bottom flask, 113 (10.0 mg,
0.009 mmol) was dissolved in a solution of trifluoroacetic acid
(125 .mu.L, 1.632 mmol) and dichloromethane (0.5 mL) and stirred at
room temperature for 1 hr under argon, then 1-pentanol (200 .mu.L,
1.840 mmol) added and reaction mixture stirred at room temperature
for 30 min under argon. LC-MS (10-100% ACN, 50 mM NH.sub.4HCO.sub.3
pH7) indicated all of the starting material had been consumed and
product had been formed. The reaction mixture was purified by
preparative HPLC (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) to
yield 114c (3.6 mg, 36%). LCMS: [M+H].sup.+ m/z=1102.77.
##STR00175##
Example
[0438] Compound 117b. In a 25 mL round bottom flask, 114b (3.2 mg,
0.003 mmol) was dissolved in dimethylsulfoxide (2 mL). A solution
of 116 (4.9 mg, 0.003 mmol) in 20 mM, pH7, sodium phosphate buffer
(2 mL) was added dropwise, stirring at room temperature with argon
bubbling for 30 min. LCMS (10-100% ACN, 50 mM NH.sub.4HCO.sub.3
pH7) indicated all of the starting material had been consumed and
product had been formed. The reaction mixture was purified by
preparative HPLC (10-100% ACN, 50 mM NH.sub.4HCO.sub.3 pH7) to
yield 117b (4.3 mg, 56%). LCMS: [M+H].sup.+ m/z=1306.82. .sup.1H
NMR (9:1 DMSO-d.sub.6:D.sub.2O): 8.60 (s, 1H) 8.14 (s, 1H) 7.59 (d,
J=8.5 Hz, 2H) 6.94 (d, J=7.5 Hz, 2H) 6.60 (dd, J=13.3 Hz, 8.8 Hz,
3H) 5.77 (d, J=11.5 Hz, 1H) 5.20 (d, J=9.5 Hz, 1H) 4.46 (m, 3H)
4.00-4.40 (br, 12H) 3.48-3.62 (br, 11H) 3.28-3.48 (br, 12H)
3.10-3.28 (br, 4H) 2.80-3.08 (br, 7H) 2.60-3.80 (br, 3H) 2.48 (s,
1H) 2.26-2.40 (br, 2H) 2.00-2.26 (br, 19H) 1.58-2.00 (br, 20H)
1.28-1.58 (br, 8H) 1.18 (q, J=7.5 Hz, 3H) 0.84-1.10 (br, 8H) 0.75
(m, 9H) 0.60 (d, J=6.5 Hz, 3H). .sup.13C NMR: 175.25, 174.93,
174.36, 173.59, 173.22, 172.76, 172.70, 172.02, 171.85, 171.67,
170.85, 170.34, 169.68, 166.48, 161.94, 160.67, 156.42, 155.80,
154.22, 150.98, 149.56, 149.21, 149.08, 130.64, 129.17, 128.69,
128.08, 124.89, 122.00, 115.31, 111.84, 72.31, 72.23, 71.82, 71.69,
69.84, 69.74, 68.21, 66.59, 63.52, 63.09, 55.04, 53.74, 53.56,
53.23, 52.96, 52.48, 46.11, 43.63, 42.39, 37.43, 35.69, 35.41,
35.19, 32.17,
##STR00176##
Example
[0439] Compound 109. 1.1 g of dipeptide 1 (2.77 mmole), was mixed
with 53 mg (0.21 mmole, 0.08 eq) of n-Bu.sub.2SnO in 15 mL of
benzyl alcohol and heated to 130.degree. C. for 21/2 hours, then
100.degree. C. overnight. LC/MS showed no starting material left.
The reaction mixture was loaded onto a 330 g of Combiflash column,
purified with petroleum ether/EtOAc to give some clean fractions.
Mixed fractions were repurified to give a combined yield of 0.67 g
(51%) of pure benzyl ester 109. LCMS: [M+H].sup.+ m/z=474.46.
.sup.1H NMR (CDCl.sub.3, .delta. in ppm): 8.12 (s, 1H), 7.46-7.43
(m, 2H), 7.40-7.32 (m, 3H), 6.68 (d, J=9.6 Hz, 1H), 5.41 (d, J=12.3
Hz, 1H), 5.36 (d, J=12.3 Hz, 1H), 5.24 (d, J=4.5 Hz, 1H), 4.87 (m,
1H), 4.02-3.90 (m, 2H), 2.24-2.13 (m, 2H), 1.88-1.78 (m, 2H),
1.42-1.30 (m, 2H), 1.07 (d, J=6.9 Hz, 3H), 0.97-0.90 (m, 9H).
.sup.13C NMR (CDCl.sub.3, .delta. in ppm): 176.1, 170.2, 161.3,
146.5, 135.7, 128.6, 128.5, 128.4, 127.8, 69.6, 68.8, 66.9, 51.6,
41.1, 38.6, 31.8, 24.1, 19.7, 18.3, 16.0, 11.7.
##STR00177##
Example
[0440] Compound 110. 0.67 g (1.42 mmole) of dipeptide benzyl ester
109 was dissolved in 5 mL dichloromethane. To this solution was
added 263 .mu.L of TESCl (236 mg, 1.56 mmole, 1.1 eq), and 117 mg
(1.72 mmole, 1.2 eq) of imidazole. The reaction was stirred at
0.degree. C. and solid formed. After 2 hours, the solid was
filtered away and the filtrate was concentrated. The residue was on
the Combiflash (24 g of silica column) with petroleum ether/EtOAC.
After concentration, 763 mg (92%) of the desired product 110 was
recovered. .sup.1H NMR (CDCl.sub.3, .delta. in ppm): 8.12 (s, 1H),
7.46-7.43 (m, 2H), 7.40-7.32 (m, 3H), 6.68 (d, J=8.4 Hz, 1H), 5.41
(d, J=12.3 Hz, 1H), 5.36 (d, J=12.3 Hz, 1H), 5.13 (t, J=5.7 Hz,
1H), 4.03-3.95 (m, 1H), 3.83 (d, 1H), 2.20-2.05 (m, 1H), 1.95-1.86
(m, 2H), 1.48-1.38 (m, 1H), 1.30-1.20 (m, 2H), 1.03 (d, 3H),
0.96-0.82 (m, 18H), 0.65 (t, 6H). .sup.13C NMR (CDCl.sub.3, .delta.
in ppm): 178.2, 168.4, 161.1, 146.5, 135.7, 128.6, 128.5, 128.4,
127.7, 70.7, 70.1, 66.9, 51.3, 39.9, 38.3, 31.6, 24.2, 18.3, 17.6,
16.0, 11.5, 6.8, 4.6.
##STR00178##
Example
[0441] Compound 111. 746 mg (1.27 mmole) of TES protected dipeptide
benzyl ester 110 was dissolved in 8 mL of THF (anhydrous,
inhibitor-free) and cooled to -45.degree. C. After 15 minutes of
cooling, 2.8 mL of 0.5 M KHMDS (1.1 eq., 1.4 mmole) in toluene
solution was added dropwise. After an additional 15 mins, 175 .mu.L
of chloromethyl butyrate (1.1 eq., 1.4 mmole) was added dropwise.
After 30 mins, TLC showed only a trace amount of starting material
left. After 2 hours, the reaction mixture was quenched 1 mL MeOH,
and allowed to warm to room temperature. The reaction was extracted
with EtOAc/brine. The organic layer was washed with brine and then
concentrated to give 759 mg (87%) of crude product 111. LCMS:
[M+Na].sup.+ m/z=710.57. .sup.1H NMR (CDCl.sub.3, .delta. in ppm)
8.10 (s, 1H), 7.44-7.40 (m, 2H), 7.39-7.30 (m, 3H), 5.43 (d, J=12.3
Hz, 1H), 5.37 (d, J=12.3 Hz, 1H), 5.35 (s, 2H), 4.98 (t, J=5.1 Hz,
1H), 4.40-4.20 (br, 1H), 3.52 (d, J=16.0 Hz, 1H), 2.42-2.38 (t,
J=6.7 Hz, 2H), 2.25-2.05 (m, 2H), 1.78-1.72 (m, 2H), 1.68-1.55 (m,
3H), 1.30-1.20 (m, 1H), 1.00-0.85 (m, 24H), 0.65 (t, 6H). .sup.13C
NMR (CDCl.sub.3, .delta. in ppm) 177.6, 173.0, 171.0, 161.1, 146.6,
135.7, 128.6, 128.42, 128.36, 127.6, 77.2, 70.8, 66.8, 63.5, 40.9,
35.9, 34.9, 31.1, 25.0, 20.1, 19.5, 18.1, 15.7, 13.6, 10.5, 6.8,
4.7.
##STR00179##
Example
[0442] Compound 112. 239 mg of MEP (1.67 mmole, 1.5 eq), 316 mg of
EDC (1.65 mmole, 1.5 eq), and 300 mg of pentafluorophenol (1.63
mmole, 1.5 eq) were dissolved in 8 mL of N-methyl-2-pyrrolidone.
The reaction was stirred overnight. 759 mg (1.1 mmole) of the
alkylated dipeptide 111 in 1 mL NMP was then added. An additional
0.8 mL of NMP was used to rinse the flask/syringe to transfer
residue to the hydrogenation flask. 60 mg (0.05 eq) of 10% Pd/C was
then added and the reaction mixture was hydrogenated at 35 PSI,
overnight. LC/MS showed the major product is the benzyl ester,
along with 10% free acid. The reaction was filtered through celite,
and the filter cake was washed with EtOAc. The filtrate was
extracted with brine, washed with brine, and concentrated.
Combiflash purification with petroleum ether/EtOAc resulted in the
recovery of 215 mg (25%) of pure benzyl ester 112. LCMS:
[M+H].sup.+ m/z=787.66. .sup.1H NMR (CDCl.sub.3, .delta. in ppm):
8.09 (s, 1H), 7.44-7.40 (m, 2H), 7.39-7.30 (m, 3H), 7.07 (d, J=15.5
Hz, 1H), 5.93 (d, J=12.3 Hz, 1H), 5.42 (d, J=12.3 Hz, 1H), 5.34 (s,
2H), 4.93 (dd, J=8.4, 2.7 Hz, 1H), 4.70-4.60 (m, 1H), 4.50-4.30
(br, 1H), 2.88 (m, 1H), 2.60-2.28 (m, 4H), 2.21 (s, 3H), 2.08-1.89
(m, 4H), 1.80-1.40 (m, 8H), 1.36-1.1.07 (m, 3H), 1.00-0.80 (m,
21H), 0.77 (d, 3H), 0.65 (t, 6H). .sup.13C NMR (CDCl.sub.3, .delta.
in ppm): 177.5, 175.1, 174.1, 173.0, 161.1, 146.5, 135.8, 128.6,
128.4, 128.3, 127.6, 77.2, 70.7, 69.5, 69.2, 66.7, 57.3, 55.4,
53.5, 53.4, 44.8, 41.3, 36.8, 35.9, 31.4, 30.3, 25.0, 24.7, 23.2,
20.2, 19.4, 18.1, 16.2, 13.6, 10.6, 6.8, 5.1, 4.7.
##STR00180## ##STR00181##
Example
[0443] Synthesis of EC1759. Paraformaldehyde (1.5 g, 1.25 eq) was
added to 16 mL of TMSBr. The resulted suspension was cooled to
0.degree. C., and 1-pentanol (4.36 mL, 40 mmole, 1 equiv.) was
added dropwise. The reaction was stirred at 0.degree. C. and warmed
up to room temperature. After overnight, TMSBr was evaporated under
reduced pressure. Vacuum distillation of the residue was carried
out at 7 mm Hg pressure, the fraction came out at 56.degree. C. was
the desired product EC1759 (4.3 g, 59%).
Example
[0444] Synthesis of EC1760. 1.58 g (3.09 mmole) TES-dipeptide
EC0997 was dissolved in 12 mL THF (anhydrous, inhibitor-free). The
resulted solution was cooled to -45.degree. C. To the solution, 6.5
mL of 0.5 M KHMDS in toulene (3.25 mmole, 1.05 equiv.) was added
dropwise. After finishing the addition, the reaction mixture was
stirred at -45.degree. C. for 15 minutes. 600 .mu.L of bromomethyl
pentyl ether EC1759 (4.1 mmole, 1.33 equiv.) was added dropwise.
The reaction mixture was warmed from -45.degree. C. to -10.degree.
C. in 90 minutes, then quenched with 10% NaCl/1% NaHCO.sub.3
aqueous solution, extracted with EtOAc. The organic phase was
washed with 10% NaCl/1% NaHCO.sub.3 aqueous solution three times,
then brine. The separated organic phase was dried over
Na.sub.2SO.sub.4 Na.sub.2SO.sub.4 was filtered off and the solvent
was evaporated under vacuum to give 2.4 g of crude product. The
crude product was purified with EtOAc/petroleum ether to give 1.47
g of product EC1760 (78%)
Example
[0445] Synthesis of EC1761. 0.38 g of MEP (2.65 mmole, 1.4 equiv.)
was suspended in 1.2 mL NMP, 0.53 g of PFP (2.88 mmole, 1.5 equiv.)
and 0.55 g of EDC (2.87 mmole, 1.5 equiv.) were added. The reaction
mixture was stirred overnight in a hydrogenation vessel.
[0446] 1.17 g (1.91 mmole) of alkylated dipeptide EC1760 was
dissolved in 0.3 mL NMP and transferred to the above hydrogenation
vessel, and the residue of the dipeptide was rinsed with 0.3 mL NMP
and transferred to the hydrogenation vessel. 154 mg of 10% Pd/C
(dry, 0.05 equiv.) was added to the solution. The hydrogenation was
carried out at 35 PSI. After 5 hrs, LC/MS showed there was no
starting material. The reaction mixture was filtered through celite
pad and the reaction vessel was washed with EtOAc and filtered
through celite pad. The combined solution was washed with 10%
NaCl/1% Na.sub.2CO.sub.3 solution to remove PFP, then with brine.
The organic phase was dried over Na.sub.2SO.sub.4. Na.sub.2SO.sub.4
was filtered off and the solvent was evaporated under vacuum to
give 1.20 g (88%) of crude product EC1761.
Example
[0447] Synthesis of EC1602. 1.17 g (1.65 mmole) of tripeptide ester
EC1761 was dissolved in 15 mL MeOH, the solution was cooled to
0.degree. C. 300 mg of LiOH hydrate (7.15 mmole, 4.3 equiv.)
dissolved in 5 mL H.sub.2O was added to the ester solution, the
resulted reaction mixture was stirred and warmed up to room
temperature in 2 hours. LC/MS showed no starting material left.
MeOH was removed using rotary evaporator, and the residual was
worked up by extraction between EtOAc/brine. The organic phase was
dried over Na.sub.2SO.sub.4. Na.sub.2SO.sub.4 was filtered off and
the solvent was evaporated under vacuum to give 0.80 g (83%) of
crude product EC1602.
Example
[0448] Synthesis of EC1633. 0.80 g (1.37 mmole) of tripeptide acid
EC1602 was dissolved in 6.4 mL of pyridine, the solution was cooled
to 0.degree. C. 6.0 mg (0.049 mmole, 0.035 equiv) DMAP was added
and then 2 mL of acetic anhydride (21.2 mmole, 15.5 equiv) was
added, the reaction mixture was warmed up to room temperature in 5
hours and stored in -20 0.degree. C. for 2 days. 20 mL dioxane/20
mL H.sub.2O was added to the reaction mixture at 0.degree. C. and
stirred for 1 hour. The solvent was evaporated under reduced
pressure. 20 mL of phosphate buffer (20 mM) and 5 mL acetonitrile
were added to the residue, the pH of the resulted solution was
adjusted to 5.4 using saturated NaHCO.sub.3 solution. The solution
was loaded on Biotage 120 g C18 column. The flask containing the
crude product was rinsed with 1 mL acetonitrile/5 mL phosphate
buffer and loaded on the column. The purification was done using a
gradient from 20% ACN/80% water to 70% ACN/30%. The fractions
containing the desired product were combined and ACN was evaporated
under reduced pressure. There were white precipitate coming out
from solution, brine was added to the suspension and EtOAc was used
to extract the desired product. The organic phase was dried over
Na.sub.2SO.sub.4. Na.sub.2SO.sub.4 was filtered off and the solvent
was evaporated under vacuum to give 0.49 g (57%) of product
EC1633
##STR00182##
##STR00183## ##STR00184##
Example
[0449] General Procedures:
[0450] Synthesis of EC1623 (Scheme 2). EC1008 (I: R.sub.1=n-propyl.
103 mg) was dissolved in anhydrous dichloromethane (DCM, 2.0 mL)
and to this solution was added trifluoroacetic acid (TFA, 0.50 mL).
The resulting solution was stirred at ambient temperature under
argon for 20 minutes, and to which was added 1-pentanol (0.72 mL).
The reaction mixture was stirred at ambient temperature for 3
minutes, concentrated on a Buchi Rotavapor at 30.degree. C. for 10
minutes, residue stirred at ambient temperature under high vacuum
for 75 minutes, and to which was added saturated NaHCO.sub.3
solution (10 mL) with vigorous stirring, followed by addition of
acetonitrile (ACN, 3.0 mL). The resulting white suspension was
stirred at ambient temperature for 3 minutes and let stand to
settle. The top clear solution was loaded onto a Biotage SNAP 12 g
KP-C18-HS column on a Biotage system. The white solid left in the
reaction flask was dissolved in water (5.0 mL) and the solution was
also loaded onto the Biotage column. The remaining solid stuck on
the glass wall of the reaction flask was dissolved in ACN (2.0 mL).
To this solution was added water (6.0 mL) and the resulting cloudy
solution was loaded onto the same Biotage column. The reaction
mixture was eluted following these parameters: Flow rate: 15
mL/min. A: water; B: CAN. Method: 25% B 2 CV (column volume),
25-50% B 3 CV, and 50% B 5 CV (1 CV=15 mL). Fractions containing
the desired product was collected and freeze-dried to afford EC1623
(II: R=n-pentyl. 95.9 mg) as a white powder.
[0451] Synthesis of EC1662 (Scheme 3).
[0452] Step 1: Anhydrous DCM (5.0 mL) was added to a mixture of
EC1623 (II: R=n-pentyl. 114 mg), pentafluorophenol (PFP, 67.3 mg),
and DCC-resin (2.3 mmol/g, 396 mg) and the suspension was stirred
at ambient temperature under argon for 23 hours. The resin was
filtered off and washed with anhydrous DCM (3.0 mL) and the
combined filtrates were concentrated under reduced pressure to give
a residue, which was vacuumed at ambient temperature for 1 hour
prior to use in Step 3.
[0453] Step 2: EC1426 (114 mg) was dissolved in anhydrous DCM (1.5
mL) and to which was added TFA (0.50 mL). The resulting solution
was stirred at ambient temperature under argon for 70 minutes and
concentrated under reduced pressure to give a residue, which was
co-evaporated with anhydrous DCM (2.0 mL.times.3) and vacuumed at
ambient temperature for 9 hours prior to use in Step 3.
[0454] Step 3: The residue from Step 1 was dissolved in anhydrous
DCM (1.5 mL) and to this solution was added DIPEA (0.50 mL)
followed by a solution of the residue from Step 2 dissolved in
anhydrous dimethylformamide (DMF, 1.5 mL). The resulting solution
was stirred at ambient temperature under argon for 1 hour, diluted
with ethyl acetate (EtOAc, 60 mL), and washed with brine (20
mL.times.3). The organic layer was separated, dried
(Na.sub.2SO.sub.4), and concentrated under reduced pressure to give
a residue, which was vacuumed at ambient temperature for 2 hours,
dissolved in DCM (3.5 mL), and loaded onto a 24 g silica gel column
on a CombiFlash system for purification. The materials were eluted
with 0-5% MeOH in DCM to afford EC1662 (III: R=n-pentyl. 171 mg) as
a white solid.
Example
[0455] Synthesis of EC1664 (Scheme 3). A solution of EC1454
(SPACER-SH; See FIG. 1 for structure. 44.1 mg.) in 20 mM phosphate
buffer (pH 7.0, 4.0 mL) was added to a solution of EC1662 (24.1 mg)
in MeOH (4.8 mL), followed by addition of saturated
Na.sub.2SO.sub.4 (0.30 mL). The reaction mixture was stirred at
ambient temperature under argon for 30 minutes and the solution was
injected onto a preparative HPLC (A: 50 M NH.sub.4HCO.sub.3 buffer,
pH 7.0; B: CAN. Method: 10-80% B in 20 minutes.) for purification.
Fractions containing the desired product were collected and
freeze-dried to afford EC1664 (IV: R=n-pentyl. 42.8 mg) as a fluffy
yellow solid.
##STR00185##
##STR00186##
[0456] The ether analogs of compounds III and 112 can be used to
prepare the tubulysin intermediates described herein.
* * * * *