U.S. patent application number 13/841078 was filed with the patent office on 2014-09-04 for processes for preparing tubulysins.
The applicant listed for this patent is Endocyte, Inc.. Invention is credited to Christopher P. LEAMON, Iontcho R. VLAHOV.
Application Number | 20140249315 13/841078 |
Document ID | / |
Family ID | 51421261 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140249315 |
Kind Code |
A1 |
VLAHOV; Iontcho R. ; et
al. |
September 4, 2014 |
PROCESSES FOR PREPARING TUBULYSINS
Abstract
Processes for preparing tubulysins and derivatives thereof are
described. In addition, processes for preparing unnatural
tubulysins are described.
Inventors: |
VLAHOV; Iontcho R.; (West
Lafayette, IN) ; LEAMON; Christopher P.; (West
Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Endocyte, Inc.; |
|
|
US |
|
|
Family ID: |
51421261 |
Appl. No.: |
13/841078 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61771429 |
Mar 1, 2013 |
|
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61793082 |
Mar 15, 2013 |
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Current U.S.
Class: |
546/194 ;
546/209 |
Current CPC
Class: |
C07D 417/12 20130101;
C07D 417/14 20130101; C07K 5/0821 20130101; C07K 5/0606
20130101 |
Class at
Publication: |
546/194 ;
546/209 |
International
Class: |
C07D 417/14 20060101
C07D417/14; C07D 417/12 20060101 C07D417/12 |
Claims
1. A process for preparing a compound of the formula ##STR00114##
or a pharmaceutically acceptable salt thereof; wherein Ar.sub.1 is
optionally substituted aryl or optionally substituted heteroaryl;
R.sub.1 is hydrogen, alkyl, arylalkyl or a pro-drug forming group;
R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl; R.sub.12
is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which
is optionally substituted; 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; 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 ##STR00115## 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 ##STR00116## or
the steps of a) preparing a compound of formula (E1), where X.sub.1
is a leaving group, from a compound of formula E ##STR00117## 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 ##STR00118## or 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 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;
##STR00119## or the step of treating the compound AF with a metal
hydroxide or a metal carbonate; ##STR00120## or 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
##STR00121## or the steps of c) forming an active ester
intermediate from a compound of formula AH ##STR00122## and d)
reacting the active ester intermediate with a compound of the
formula I; ##STR00123## or one or more combinations thereof.
2. The process of claim 1 wherein R.sub.4 is optionally substituted
alkyl.
3. The process of claim 1 er-2 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
##STR00124##
4. 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 ##STR00125##
5. The process of claim 1 comprising the steps of a) preparing a
compound of formula (E1), where X.sub.1 is a leaving group, from a
compound of formula E ##STR00126## 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
##STR00127##
6. The process of claim 1 comprising the step of treating 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 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;
##STR00128##
7. The process of claim 1 comprising the step of treating the
compound AF with a metal hydroxide or a metal carbonate;
##STR00129##
8. The process of claim 1 comprising 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
##STR00130##
9. The process of claim 1 comprising the steps of c) forming an
active ester intermediate from a compound of formula AH
##STR00131## and d) reacting the active ester intermediate with a
compound of the formula I ##STR00132##
10. The process of claim 1 wherein R.sub.1 is hydrogen, benzyl, or
C1-C4 alkyl.
11. The process of claim 1 wherein R.sub.2 is C1-C8 alkyl or C3-C8
cycloalkyl.
12. 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.
13. The process of claim 1 wherein R.sub.3 is C1-C4 alkyl.
14. The process of claim 1 wherein Ar.sub.1 is phenyl or
hydroxyphenyl.
15. The process of claim 1 wherein R.sub.4 is C1-C8 alkyl or C3-C8
cycloalkyl.
16. The process of claim 1 wherein R.sub.5 is branched C3-C6 or
C3-C8 cycloalkyl.
17. The process of claim 1 wherein R.sub.6 is branched C3-C6 or
C3-C8 cycloalkyl.
18. The process of claim 1 wherein R.sub.7 is C1-C6 alkyl.
19. The process of claim 1 wherein R.sub.12 is
CH.sub.2CH.dbd.CH.sub.2, or CH.sub.2(CH.sub.2)nCH.sub.3, where n=1,
2, 3, 4, 5, or 6.
20. The process of claim 1 wherein the metal hydroxide is LiOH.
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. 61/771,429, filed
Mar. 1, 2013, and U.S. Provisional Application 61/793,082, 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 tubulysins and derivatives thereof. In particular, the
processes pertain to the preparation of unnatural tubulysins.
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, exceeding the cell growth inhibition of any clinically
relevant traditional chemotherapeutic e.g. 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. Accordingly, processes for preparing tubulysins,
including non-naturally occurring tubulysins are needed.
[0004] Tubulysins are described herein. Structurally, tubulysins
often include linear tetrapeptoid backbones, including illustrative
compounds having formula T or AT
##STR00001##
and pharmaceutically acceptable salts thereof; wherein
[0005] Ar.sub.1 is optionally substituted aryl;
[0006] R.sub.1 is hydrogen, alkyl, arylalkyl or a pro-drug forming
group;
[0007] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0008] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted;
[0009] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0010] R.sub.3 is optionally substituted alkyl;
[0011] R.sub.5 and R.sub.6 are each independently selected from the
group consisting of optionally substituted alkyl and optionally
substituted cycloalkyl;
[0012] R.sub.7 is optionally substituted alkyl; and
[0013] n is 1, 2, 3, or 4.
[0014] 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),
##STR00002##
and analogs and derivatives of each of the foregoing. A common
feature in the molecular architecture of the more potent natural
occurring tubulysins is the acid and/or base sensitive
N-acyloxymethyl substituent (or a N,O-acetal of formaldehyde)
represented by R2-C(O) in the formula (T).
[0015] Another illustrative group of tubulysins described herein
are those having formula 1.
##STR00003##
[0016] Structures of Several Natural Tubulysins
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
[0017] 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. Described herein are improved processes for
making tubulysins, or analogs or derivatives thereof, including
compounds of formula (AT).
[0018] In one illustrative embodiment of the invention, processes
for preparing tubulysins, or analogs or derivatives thereof,
including compounds of formula (AT). 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.
##STR00004##
It is to be understood that R.sub.5 and R.sub.6 may each include
conventional protection groups on the optional substituents.
[0019] 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 from about 1 to about 1.5.
##STR00005##
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.
[0020] 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
[0021] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E; and
[0022] b) treating a compound of formula C under reducing
conditions in the presence of the compound of formula E1.
##STR00006##
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.
[0023] 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
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.
##STR00007##
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.
[0024] 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
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.
##STR00008##
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.
[0025] 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.7 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.
##STR00009##
[0026] 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.
[0027] 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.
##STR00010##
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.
[0028] In another embodiment, a process is described for preparing
a tubulysin of formula (AT), wherein Ar.sub.1 is aryl or heteroaryl
each of which is optionally substituted; 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;
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
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.
##STR00011##
It is to be understood that Ar.sub.1, 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.
[0029] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (TL-2), 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
##STR00012##
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 at
least two of R.sup.a, R.sup.b, or R are taken together with the
attached carbon atoms to form a carbocyclic ring;
[0030] 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.7
is optionally substituted alkyl; wherein the process comprises the
step of contacting compound TL, 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 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. 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.
##STR00013##
[0031] In another embodiment, a process is described for preparing
a tubulysin linker derivative of formula (TL-2), wherein Ar.sub.1
is optionally substituted aryl or optionally substituted
heteroaryl; Ar.sub.e is optionally substituted aryl or optionally
substituted heteroaryl; L is selected from the group consisting
of
##STR00014##
wherein
[0032] 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;
[0033] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
at least two of R.sup.a, R.sup.b, or R are taken together with the
attached carbon atoms to form a carbocyclic ring;
[0034] 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;
[0035] 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;
[0036] 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 IL to give
a compound of the formula TL-2.
##STR00015##
[0037] In another embodiment, the process described in any of the
embodiments described herein wherein Ar.sub.1 is optionally
substituted aryl is described.
[0038] In another embodiment, the process described in any of the
embodiments described herein wherein Ar.sub.1 is optionally
substituted heteroaryl is described.
[0039] 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
in any of the embodiments described herein.
DETAILED DESCRIPTION
[0040] 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.
##STR00016##
[0041] 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.
[0042] In an illustrative example of the processes described
herein, a process for preparing the silyl ether 2 in high yield is
described wherein compound 1 is treated with 1.05 equivalent of
TESCl and 1.1 equivalent of imidazole.
##STR00017##
In one alternative of the foregoing example, the compound 2 is not
purified by chromatography.
[0043] 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.
##STR00018##
[0044] 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'.
##STR00019##
[0045] 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.
[0046] In an illustrative example of the processes described
herein, a process for preparing the N,O-acetal 3 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.
##STR00020##
[0047] 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
[0048] a) preparing a compound of formula (E1) where X.sub.1 is a
leaving group from a compound of formula E; and
[0049] b) treating a compound of formula C under reducing
conditions with the compound of formula E1.
##STR00021##
[0050] In one illustrative example, a mixture of compound 3 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 4. 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 8 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.
[0051] 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 9. 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.
##STR00022##
[0052] 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 5 and a cyclic
O,N-acetal side-product as shown in the following scheme.
##STR00023##
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-7. That
additional side-product is difficult to detect and difficult to
separate from the desired compound 7. Without being bound by
theory, it is believed herein that iso-7 results from rearrangement
of the butyrate group from the N-hydroxymethyl group to the
secondary hydroxyl group, as shown below.
##STR00024##
[0053] 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
another 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.
##STR00025##
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.
[0054] In an illustrative example, compound 4 is heated with an
alcohol and di-n-butyltin oxide at about 100.degree. C. to yield
ether 10. It is appreciated that a co-solvent may be present. In
one embodiment, the molar ratio (tin oxide)/(compound 10) is about
0.01 to about 0.30, or about 0.02 to about 0.20, or about 0.05 to
about 0.15, or about 0.05 to about 0.10
##STR00026##
[0055] 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.7 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.
##STR00027##
[0056] 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.
[0057] In an illustrative example, compound 10 is treated with
LiOH.H.sub.2O in a mixture of THF and water at about room
temperature to yield compound 11. It is appreciated that the THF
may be replaced with other solvents.
##STR00028##
[0058] In another embodiment, a process is described for preparing
a compound of formula AH, 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 BG 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 AH 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 AH, 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.
##STR00029##
[0059] In an illustrative example, compound 11 is treated with
acetic anhydride in pyridine. It is appreciated that the resulting
product may contain varying amounts of the mixed anhydride of 12
and acetic acid. In another embodiment, treatment of the reaction
product resulting from the preceding step with water in dioxane
yields compound 12, 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.
##STR00030##
[0060] In another embodiment, a process is described for preparing
a tubulysin of formula (AT), wherein Ar.sub.1 is optionally
substituted aryl; 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;
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
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.
##STR00031##
It is to be understood that Ar.sub.1, 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.
[0061] In one embodiment, compound AH is treated with an excess
amount of active ester forming agent and pentafluorophenol to form
the pentafluorophenol ester of compound AH, 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, R.sub.3 is methyl. In another alternative of the foregoing
embodiments, and each additional embodiment described herein,
R.sub.1 is hydrogen.
[0062] In an illustrative example, compound 12 is treated with an
excess amount of a polymeric version of a carbodiimide and
pentafluorophenol to form the pentafluorophenyl ester of 12, the
polymeric carbodiimide is removed by filtration; and amino acid
(S)-tubutyrosine is added to the solution to yield the tubulysin,
compound 13. 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.
##STR00032##
[0063] In another embodiment, a compound AF is described wherein
R.sub.12, R.sub.5, R.sub.6, and R.sub.7 are as described in the any
of the embodiments described herein.
##STR00033##
[0064] In another embodiment, the following compound is described
wherein R.sub.12, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are as
described in the any of the embodiments described herein.
##STR00034##
[0065] In another embodiment, the compound having formula 10 is
described.
##STR00035##
[0066] In another embodiment a compound BG, is described, wherein
R.sub.12, R.sub.5, R.sub.6, and R.sub.7 are as described in any of
the embodiments described herein.
##STR00036##
[0067] In another embodiment, compound 11 is described.
##STR00037##
[0068] In another embodiment, compound 7 is described.
##STR00038##
[0069] In another embodiment, a compound AH is described wherein
R.sub.4 is Me and R.sub.12, R.sub.5, R.sub.6, and R.sub.7 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.
##STR00039##
[0070] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.5 is
isopropyl.
[0071] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.6 is
sec-butyl.
[0072] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.8 is methyl.
[0073] 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.
[0074] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.12 is
CH.sub.2CH.dbd.CH.sub.2, or CH.sub.2(CH.sub.2)nCH.sub.3, where n is
1, 2, 3, 4, 5, or 6.
[0075] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.12 is
CH.sub.2CH.dbd.CH.sub.2, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
[0076] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, n is 3.
[0077] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.7 is methyl.
[0078] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.4 is methyl.
[0079] 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.
[0080] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.3 is methyl.
[0081] In another alternative of the foregoing embodiments, and
each additional embodiment described herein, R.sub.1 is
hydrogen.
[0082] Illustrative embodiments of the invention are further
described by the following enumerated clauses:
[0083] 1. A process for preparing a compound of the formula
##STR00040##
[0084] or a pharmaceutically acceptable salt thereof; wherein
Ar.sub.1 is optionally substituted aryl or optionally substituted
heteroaryl;
[0085] R.sub.1 is hydrogen, alkyl, arylalkyl or a pro-drug forming
group;
[0086] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0087] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted;
[0088] R.sub.3 is optionally substituted alkyl;
[0089] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0090] 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;
and n is 1, 2, 3, or 4;
[0091] 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
##STR00041##
or
[0092] 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
##STR00042##
or
[0093] the steps of a) preparing a compound of formula (E1), where
X.sub.1 is a leaving group, from a compound of formula E
##STR00043##
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
##STR00044##
or
[0094] 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 selected from TFA or 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;
##STR00045##
or
[0095] the step of treating the compound AF with a metal hydroxide
or a metal carbonate;
##STR00046##
or
[0096] 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
##STR00047##
or
[0097] the steps of c) forming an active ester intermediate from a
compound of formula AH
##STR00048##
and d) reacting the active ester intermediate with a compound of
the formula I
##STR00049##
or
[0098] one or more combinations thereof.
[0099] 2. The process of clause 1 wherein Ar.sub.1 is optionally
substituted aryl.
[0100] 3. The process of clause 1 wherein Ar.sub.1 is optionally
substituted heteroaryl.
[0101] 4. A process for preparing a compound having formula
(TL-2)
##STR00050##
wherein
[0102] L is selected from the group consisting of
##STR00051##
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;
[0103] R.sup.a, R.sup.b, and R are each independently selected in
each instance from the group consisting of hydrogen and alkyl; or
at least two of R.sup.a, R.sup.b, or R are taken together with the
attached carbon atoms to form a carbocyclic ring;
[0104] 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;
[0105] wherein the process comprises the step of contacting a
compound having formula (TL)
##STR00052##
with 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 selected from TFA or 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.
[0106] 5. The process of any one of the preceding clauses wherein
R.sub.4 is optionally substituted alkyl.
[0107] 6. The process of any one of the preceding 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
##STR00053##
[0108] 7. The process of any one of the preceding 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
##STR00054##
[0109] 8. The process of any one of the preceding clauses
comprising the steps of
a) preparing a compound of formula (E1), where X.sub.1 is a leaving
group, from a compound of formula E
##STR00055##
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
##STR00056##
[0110] 9. The process of any one of the preceding clauses
comprising the step of treating 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 selected from TFA or 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;
##STR00057##
[0111] 10. The process of any one of the preceding clauses
comprising the step of treating the compound AF with a metal
hydroxide or a metal carbonate;
##STR00058##
[0112] 11. The process of any one of the preceding clauses
comprising 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
##STR00059##
[0113] 12. The process of any one of the preceding clauses
comprising the steps of
c) forming an active ester intermediate from a compound of formula
AH
##STR00060##
and d) reacting the active ester intermediate with a compound of
the formula I
##STR00061##
[0114] 13. A process for preparing a compound of the following
formula
##STR00062##
the process comprising the step of contacting a compound of the
formula
##STR00063##
with an acid and R.sub.12OH, wherein R.sub.2, R.sub.5, R.sub.6,
R.sub.8, and R.sub.12 are as described in any of the embodiments
described herein.
[0115] 14. A process for preparing a compound of the following
formula
##STR00064##
the process comprising the step of contacting a compound of the
formula
##STR00065##
with a transesterification catalyst 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 and R.sub.12OH, wherein R.sub.2, R.sub.5, R.sub.6,
R.sub.8, R.sub.12, and R.sub.13 are as described in any of the
embodiments described herein.
[0116] 15. A process for preparing a compound of the following
formula
##STR00066##
the process comprising the step of contacting a compound of the
formula
##STR00067##
with a base and R.sub.12OCH.sub.2X, where X is Cl or Br; and
wherein R.sub.2, R.sub.5, R.sub.6, R.sub.8, R.sub.12, and R.sub.13
are as described in any of the embodiments described herein. In
another embodiment, R.sub.12OCH.sub.2X is
n-C.sub.5H.sub.11OCH.sub.2Br.
[0117] 16. A process for preparing a compound of the following
formula
##STR00068##
the process comprising the step of contacting a compound of the
formula
##STR00069##
with an acid and R.sub.12OH, wherein R.sub.14 is Et.sub.3Si or
R.sub.4C(O), and R.sub.2, R.sub.4, R.sub.5, R.sub.6, R.sub.8, and
R.sub.12 are as described in any of the embodiments described
herein.
[0118] 17. A process for preparing a compound of the following
formula
##STR00070##
the process comprising the step of contacting a compound of the
formula
##STR00071##
with a transesterification catalyst 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; and R.sub.12OH, wherein R.sub.2, R.sub.5, R.sub.6,
R.sub.8, R.sub.12, and R.sub.13 are as described in any of the
embodiments described herein.
[0119] 18. A process for preparing a compound of the following
formula
##STR00072##
the process comprising the step of contacting a compound of the
formula
##STR00073##
with an acid and R.sub.12OH, wherein n, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, Ar.sub.1, Ar.sub.2, L and
R.sub.12 are as described in any of the embodiments described
herein.
[0120] 19. The process of any one of the preceding clauses wherein
R.sub.1 is hydrogen, benzyl, or C1-C4 alkyl.
[0121] 19A. The process of any one of the preceding clauses wherein
R.sub.1 is hydrogen.
[0122] 20. The process of any one of the preceding clauses wherein
R.sub.2 is C1-C8 alkyl or C3-C8 cycloalkyl.
[0123] 20A. The process of any one of the preceding clauses wherein
R.sub.2 is n-butyl.
[0124] 20B. The process 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.
[0125] 21. The process of any one of the preceding clauses wherein
R.sub.3 is C1-C4 alkyl.
[0126] 21A. The process of any one of the preceding clauses wherein
R.sub.3 is methyl.
[0127] 22. The process of any one of the preceding clauses wherein
Ar.sub.1 is phenyl or hydroxyphenyl.
[0128] 22A. The process of any one of the preceding clauses wherein
Ar.sub.1 is 4-hydroxyphenyl.
[0129] 23. The process of any one of the preceding clauses wherein
R.sub.4 is C1-C8 alkyl or C3-C8 cycloalkyl.
[0130] 23A. The process of any one of the preceding clauses wherein
R.sub.4 is methyl.
[0131] 24. The process of any one of the preceding clauses wherein
R.sub.5 is branched C3-C6 or C3-C8 cycloalkyl.
[0132] 24A. The process of any one of the preceding clauses wherein
R.sub.5 is iso-propyl.
[0133] 25B. The process of any one of the preceding clauses wherein
R.sub.5 is sec-butyl.
[0134] 26. The process of any one of the preceding clauses wherein
R.sub.6 is branched C3-C6 or C3-C8 cycloalkyl.
[0135] 27. The process of any one of the preceding clauses wherein
R.sub.7 is C1-C6 alkyl.
[0136] 27A. The process of any one of the preceding clauses wherein
R.sub.7 is methyl.
[0137] 28. The process of any one of the preceding clauses wherein
R.sub.12 is CH.sub.2CH.dbd.CH.sub.2, or
CH.sub.2(CH.sub.2)nCH.sub.3, where n=1, 2, 3, 4, 5, or 6.
[0138] 28A. The process of any one of the preceding clauses wherein
R.sub.12 is CH.sub.2CH.dbd.CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2CH.sub.3, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
[0139] 29. The process of any one of the preceding clauses wherein
Ar.sub.1 is substituted phenyl.
[0140] 29A. The process of any one of the preceding clauses wherein
Ar.sub.1 is 4-substituted phenyl.
[0141] 29B. The process of any one of the preceding clauses wherein
Ar.sub.1 is R.sub.A-substituted phenyl.
[0142] 29C. The process of any one of the preceding clauses wherein
Ar.sub.1 is 4-hydroxyphenyl, or a hydroxyl protected form
thereof.
[0143] 30. The process of any one of the preceding clauses wherein
R.sub.13 is CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
[0144] 31. The process of any one of the preceding clauses wherein
the metal hydroxide is LiOH.
[0145] 32. A compound of the formula
##STR00074##
[0146] or a pharmaceutically acceptable salt thereof; wherein
Ar.sub.1 is optionally substituted aryl;
[0147] R.sub.1 is hydrogen, alkyl, arylalkyl or a pro-drug forming
group;
[0148] R.sub.2 is selected from the group consisting of optionally
substituted alkyl and optionally substituted cycloalkyl;
[0149] R.sub.12 is alkyl, alkenyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl,
each of which is optionally substituted;
[0150] R.sub.3 is optionally substituted alkyl;
[0151] R.sub.4 is optionally substituted alkyl or optionally
substituted cycloalkyl;
[0152] 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;
and n is 1, 2, 3, or 4.
[0153] 33. A compound of formula
##STR00075##
[0154] 35. A compound of formula
##STR00076##
[0155] 35. A compound of formula
##STR00077##
[0156] 36. A compound of formula
##STR00078##
[0157] 37. The compound of any one of the preceding clauses wherein
R.sub.1 is hydrogen, benzyl, or C1-C4 alkyl.
[0158] 37A. The compound of any one of the preceding clauses
wherein R.sub.1 is hydrogen.
[0159] 38. The compound of any one of the preceding clauses wherein
R.sub.2 is C1-C8 alkyl or C3-C8 cycloalkyl.
[0160] 38A. The compound of any one of the preceding clauses
wherein R.sub.2 is n-butyl.
[0161] 38B. The 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.
[0162] 39. The compound of any one of the preceding clauses wherein
R.sub.3 is C1-C4 alkyl.
[0163] 39A. The compound of any one of the preceding clauses
wherein R.sub.3 is methyl.
[0164] 40. The compound of any one of the preceding clauses wherein
Ar.sub.1 is phenyl or hydroxyphenyl.
[0165] 40A. The compound of any one of the preceding clauses
wherein Ar.sub.1 is 4-hydroxyphenyl.
[0166] 40B. The compound of any one of the preceding clauses
wherein Ar.sub.1 is substituted phenyl.
[0167] 40C. The compound of any one of the preceding clauses
wherein Ar.sub.1 is 4-substituted phenyl.
[0168] 40D. The compound of any one of the preceding clauses
wherein Ar.sub.1 is R.sub.A-substituted phenyl.
[0169] 40E. The compound of any one of the preceding clauses
wherein Ar.sub.1 is 4-hydroxyphenyl, or a hydroxyl protected form
thereof.
[0170] 41. The compound of any one of the preceding clauses wherein
R.sub.4 is C1-C8 alkyl or C3-C8 cycloalkyl.
[0171] 41A. The compound of any one of the preceding clauses
wherein R.sub.4 is methyl.
[0172] 42. The compound of any one of the preceding clauses wherein
R.sub.5 is branched C3-C6 or C3-C8 cycloalkyl.
[0173] 42A. The compound of any one of the preceding clauses
wherein R.sub.5 is iso-propyl.
[0174] 42B. The compound of any one of the preceding clauses
wherein R.sub.5 is sec-butyl.
[0175] 43. The compound of any one of the preceding clauses wherein
R.sub.6 is branched C3-C6 or C3-C8 cycloalkyl.
[0176] 44. The compound of any one of the preceding clauses wherein
R.sub.7 is C1-C6 alkyl.
[0177] 44A. The compound of any one of the preceding clauses
wherein R.sub.7 is methyl.
[0178] 45. The compound of any one of the preceding clauses wherein
R.sub.12 is CH.sub.2CH.dbd.CH.sub.2, or
CH.sub.2(CH.sub.2)nCH.sub.3, where n=1, 2, 3, 4, 5, or 6.
[0179] 45A. The compound of any one of the preceding clauses
wherein R.sub.12 is CH.sub.2CH.dbd.CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2CH.sub.3, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3.
[0180] 46. The compound selected from the group consisting of
##STR00079## ##STR00080## ##STR00081## ##STR00082##
where n=1, 2, 3, 4, 5, or 6.
[0181] In any of the embodiments described herein, the acid
selected for the conversion of the NCH.sub.2OC(O)R.sub.2 moiety to
the NCH.sub.2OR.sub.12 moiety is TFA.
[0182] In any of the embodiments described herein, the catalyst
selected for the conversion of the NCH.sub.2OC(O)R.sub.2 moiety to
the NCH.sub.2OR.sub.12 moiety is (n-Bu).sub.2SnO.
[0183] 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.
[0184] As used herein, the term tubulysin 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.
[0185] 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.
[0186] In addition, as used herein the term tubulysin 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 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 refers to any and all hydrates, or other solvates, of the
compounds described herein.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
Illustratively, such particularly limited length alkyl groups,
including C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may
be referred to as lower alkyl. 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. Illustratively, such particularly limited length
alkenyl and/or alkynyl groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenyl and/or alkynyl. 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. In embodiments of the invention described herein, it is
to be understood, in each case, that the recitation of alkyl refers
to alkyl as defined herein, and optionally lower alkyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkenyl refers to
alkenyl as defined herein, and optionally lower alkenyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkynyl refers to
alkynyl as defined herein, and optionally lower alkynyl.
Illustrative alkyl, alkenyl, and alkynyl 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, and the corresponding groups
containing one or more double and/or triple bonds, or a combination
thereof.
[0195] 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.
[0196] 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.
[0197] As used herein, the term "aryl" includes monocyclic and
polycyclic 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.
[0198] 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.
[0199] 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,
heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino,
heteroarylalkynylamino, acylamino, and the like, each of which is
optionally substituted. The term "amino derivative" also includes
urea, carbamate, and the like.
[0200] 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, heteroaryloxy, heteroarylalkyloxy,
heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like,
each of which is optionally substituted. The term "hydroxy
derivative" also includes carbamate, and the like.
[0201] 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, heteroarylthio, heteroarylalkylthio,
heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the
like, each of which is optionally substituted. The term "thio
derivative" also includes thiocarbamate, and the like.
[0202] As used herein, the term "acyl" includes formyl, and
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,
cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,
arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,
heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,
heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of
which is optionally substituted.
[0203] As used herein, the term "carboxylic acid and derivatives
thereof" includes the group CO.sub.2H and salts thereof, and esters
and amides thereof, and CN.
[0204] 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, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, 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, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0205] As used herein, the terms "optionally substituted aryl" and
"optionally substituted heteroaryl" include the replacement of
hydrogen atoms with other functional groups on the aryl or
heteroaryl that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0206] 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,
aryl-C.sub.1-C.sub.6 alkyl, and heteroaryl-C.sub.1-C.sub.6
alkyl.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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
##STR00083##
[0213] Example
Synthesis of Dipeptide 3
[0214] 4.9 g of dipeptide 1 (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 2 (vs 5.9 g of theoretical yield).
[0215] The crude product 2 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 blueish 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 3 (two steps, yield
76%)
##STR00084##
Example
Synthesis of Tripeptide 4
[0216] Alkylated dipeptide 3 (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 (1:1)] 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 4 was obtained (yield 50%).
##STR00085##
Example
Large Scale Synthesis of Dipeptide 3
[0217] 10.2 g of dipeptide 1 (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 2.
[0218] The crude product 2 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 blueish 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 3
(two steps, yield 76%)
##STR00086##
Example
Large Scale Synthesis of Tripeptide 4
[0219] Alkylated dipeptide 3 (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%). NMR and mass
spectral data were consistent with those measured for the
Example
##STR00087##
Also described herein, is the conversion of 4 to 10 (R remains Me)
by contacting 4 with TFA and an alcohol. In some illustrative
examples of compound 10, R is allyl, or
CH.sub.2(CH.sub.2)nCH.sub.3, where n is 1, 2, 3, 4, 5, or 6.
##STR00088##
Example
[0220] Compound 4 (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.
##STR00089##
Example
[0221] Compound 10a (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 11a (9.2 mkg, 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, 11H), 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.
##STR00090##
Example
[0222] To compound 11a (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 12a 10.4 mg (quantitative
yield). 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.
##STR00091##
Example
[0223] Compound 12a (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 13a was obtained
(13.1 mg, 96% yield). LCMS: [M+H].sup.+ m/z=800.88. .sup.1H NMR
(CD.sub.3OD, .delta. 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.
##STR00092##
Example
[0224] Compound 12a (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 15a (52.8
mg), which could be used for conjugation with folate. LCMS:
[M+H].sup.+ m/z=1072.92.
##STR00093##
Example
[0225] 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 10b. 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).
##STR00094##
Example
[0226] The same procedure as compound 11a was followed. 11b (11.7
mg, 35%) was obtained from intermediate 10b (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).
##STR00095##
Example
[0227] In a 10 mL round bottom flask, 11b (11.7 mg, 0.021 mmol) and
acetic anhydride (20 .mu.L, 0.212 mmol) were dissolved in pyridine
(1 mL). 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 12b (9.6 mg, 76%). LCMS:
[M+H].sup.+=611.53. .sup.1H NMR (CDCl.sub.3 w/2 drops CD.sub.3OD):
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, .sup.1H) 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
##STR00096##
Example
[0228] In a 25 mL round bottom flask, 12b (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 (8 .mu.L, 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 13b (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
##STR00097##
Example
[0229] 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 10b. 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).
##STR00098##
Example
[0230] The same procedure as for compound 11a was followed,
intermediate 11c (14.9 mg, 38%) was obtained from 10c (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).
##STR00099##
Example
[0231] In a 10 mL round bottom flask, 11c (14.9 mg, 0.026 mmol) and
acetic anhydride (20 .mu.L, 0.212 mmol) were dissolved in pyridine
(1 mL). 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 12c (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
##STR00100##
Example
[0232] In a 25 mL round bottom flask, 12c (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 13c (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)
##STR00101##
Example
[0233] In a 5 mL round bottom flask, 14 (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-butanol (200 .mu.L, 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 15b (3.2 mg,
32%). LCMS: [M+H].sup.+ m/z=1088.79. .sup.1H NMR (CDCl.sub.3 w/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
##STR00102##
Example
[0234] In a 5 mL round bottom flask, 14 (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 15c (3.6 mg,
36%). LCMS: [M+H].sup.+ m/z=1102.77.
##STR00103##
Example
[0235] In a 25 mL round bottom flask, 15b (3.2 mg, 0.003 mmol) was
dissolved in dimethylsulfoxide (2 mL). A solution of 16 (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 17b (4.3 mg,
56%). LCMS: [M+H].sup.+ m/z=1306.82. .sup.1H NMR (9:1
DMSO-d6: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,
##STR00104##
Example
[0236] 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 18. LCMS: [M+H].sup.+ m/z=474.46. .sup.1H NMR
(CDCl.sub.3, 6 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, 6
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.
##STR00105##
Example
[0237] 0.67 g (1.42 mmole) of dipeptide benzyl ester 18 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 19 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.
##STR00106##
Example
[0238] 746 mg (1.27 mmole) of TES protected dipeptide benzyl ester
19 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 20. 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.
##STR00107##
Example
[0239] 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 20 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
benzyl ester 21. 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, 6 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.
##STR00108## ##STR00109##
Example
Synthesis of EC1759
[0240] 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
Synthesis of EC1760
[0241] 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
Synthesis of EC1761
[0242] 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. 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
Synthesis of EC1602
[0243] 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
Synthesis of EC1633
[0244] 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 H2O 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.
##STR00110## ##STR00111## ##STR00112##
Example
General Procedures
Synthesis of EC1623 (Scheme 2)
[0245] 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.
Example
Synthesis of EC1662 (Scheme 3)
[0246] 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.
[0247] 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.
[0248] 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.
Synthesis of EC1664 (Scheme 3)
[0249] 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.
##STR00113##
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