U.S. patent application number 09/825856 was filed with the patent office on 2002-12-19 for epothilone derivatives and methods for making and using the same.
Invention is credited to Ashley, Gary, Metcalf, Brian.
Application Number | 20020193361 09/825856 |
Document ID | / |
Family ID | 25245070 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020193361 |
Kind Code |
A1 |
Ashley, Gary ; et
al. |
December 19, 2002 |
Epothilone derivatives and methods for making and using the
same
Abstract
The present invention relates to 16-membered macrocyclic
compounds. In one aspect of the present invention, compounds of the
formula 1 are provided wherein: R.sup.1, R.sup.2, R.sup.3, and
R.sup.5 are each independently hydrogen, C.sub.1-C.sub.10 alkyl,
C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or
alkylaryl; R.sup.4 is hydrogen, halogen, C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.10 hydroxyalkyl, C.sub.1-C.sub.10 haloalkyl, aryl,
--C(.dbd.O)R.sup.6, --C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where
R.sup.6 and R.sup.7 are each independently hydrogen,
C.sub.1-C.sub.10 aliphatic, aryl or alkylaryl; W is O, NR.sup.8
where R.sup.8 is hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or alkylaryl; X is O,
CH.sub.2 or a carbon-carbon double bond; Y is absent or a
C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, or
C.sub.2-C.sub.10 alkynyl; and Ar is aryl; provided that
10,11-dehydroepothilone C is excluded.
Inventors: |
Ashley, Gary; (Alameda,
CA) ; Metcalf, Brian; (Moraga, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
25245070 |
Appl. No.: |
09/825856 |
Filed: |
April 3, 2001 |
Current U.S.
Class: |
514/183 ;
514/450; 540/461; 549/269 |
Current CPC
Class: |
C07D 417/06 20130101;
C07D 313/00 20130101; C07D 413/06 20130101; C12P 17/181 20130101;
C07D 491/04 20130101; C12N 15/74 20130101; C12N 1/205 20210501;
C12R 2001/01 20210501; C12N 15/52 20130101; C12N 1/20 20130101;
C12N 9/00 20130101; C07D 405/06 20130101; C12P 17/16 20130101 |
Class at
Publication: |
514/183 ;
514/450; 540/461; 549/269 |
International
Class: |
A61K 031/365; C07D
313/20 |
Claims
What is claimed is:
1. A purified compound of the formula 109wherein: R.sup.1, R.sup.2,
R.sup.3, and R.sup.5 are each independently hydrogen,
C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10
alkynyl, aryl or alkylaryl; R.sup.4 is hydrogen, halogen
C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 hydroxyalkyl,
C.sub.1-C.sub.10 haloalkyl, aryl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen, C.sub.1-C.sub.10 aliphatic, aryl
or alkylaryl; W is O, NR.sup.8 where R.sup.8 is hydrogen,
C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10
alkynyl, aryl or alkylaryl; X is O, CH.sub.2 or a carbon-carbon
double bond; Y is absent or a C.sub.1-C.sub.10 alkyl,
C.sub.2-C.sub.10 alkenyl, or C.sub.2-C.sub.10 alkynyl; and Ar is
aryl; provided that 10,11-dehydroepothilone C is excluded.
2. The compound as in claim 1 wherein R.sup.1, R.sup.2, R.sup.3,
and R.sup.5 are each independently hydrogen or C.sub.1-C.sub.5
alkyl; R.sup.4 is hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5
hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen or C.sub.1-C.sub.5 alkyl; W is O or
NR.sup.8 where R.sup.8 is hydrogen or C.sub.1-C.sub.5 alkyl; X is
O, CH.sub.2 or a carbon-carbon double bond; Y is absent or
C.sub.2-C.sub.5 alkenyl; and Ar is a heteroaryl.
3. The compound as in claim 1 wherein R.sup.1, R.sup.2, R.sup.3,
and R.sup.5 are each independently hydrogen, methyl or ethyl;
R.sup.4 is hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl,
fluoromethyl or fluoroethyl; W is O or NH; X is O, CH.sub.2 or a
carbon-carbon double bond; Y is absent or C.sub.2-C.sub.5 alkenyl;
and Ar is an aryl selected from the group consisting of phenyl,
pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thienyl,
pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, quinolyl,
isoquinolyl, quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl,
benzoimidazolyl, and benzopyrazolyl.
4. The compound as in claim 1 wherein R.sup.1, R.sup.2, R.sup.3,
and R.sup.5 are each independently hydrogen or methyl; R.sup.4 is
hydrogen, methyl, ethyl, or fluoromethyl; W is O or NH; X is O,
CH.sub.2 or a carbon-carbon double bond; Y is absent or
C.sub.2-C.sub.3 alkenyl; and Ar is an aryl selected from the group
consisting of thiazolyl, oxazolyl, quinolyl, isoquinolyl,
quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl, benzoimidazolyl,
and benzopyrazolyl.
5. A purified compound of the formula 110wherein: W is O or
NR.sup.8 where R.sup.8 is hydrogen or C.sub.1-C.sub.5 alkyl; X is
O, CH.sub.2 or a carbon-carbon double bond; R.sup.4 is hydrogen,
C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 hydroxyalkyl,
C.sub.1-C.sub.5 haloalkyl, --C(.dbd.O)R.sup.6, --C(.dbd.O)OR.sup.6,
--NR.sup.6R.sup.7 where R.sup.6 and R.sup.7 are each independently
hydrogen or C.sub.1-C.sub.5 alkyl; and, R.sup.9 is aryl or
R.sup.11CH.dbd.C(R.sup.10)-- wherein R.sup.11 is aryl and R.sup.10
is hydrogen, halide, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5
hydroxylalkyl, or C.sub.1-C.sub.5 haloalkyl, provided that 10,
11-dehydroepothilone C is excluded.
6. The compound as in claim 5 wherein: W is O or NH; and, X is
CH.sub.2 or a carbon-carbon double bond; R.sup.4 is hydrogen,
methyl, ethyl, hydroxymethyl, hydroxyethyl, fluoromethyl or
fluoroethyl; and, R.sup.9 is a bicyclic heteroaryl or
R.sup.11CH.dbd.C(R.sup.10)-- wherein R.sup.11 is
2-methyl-1,3-thiazolinyl, 2-methyl-13, oxazolinyl,
2-hydroxymethyl-1,3-thiazolinyl, or 2-hydroxymethyl-1,3-oxazolinyl
and R.sup.10 is hydrogen or methyl.
7. The compound as in claim 5 wherein W is O or NH; and, X is
CH.sub.2 or a carbon-carbon double bond; R.sup.4 is hydrogen,
methyl, ethyl, hydroxymethyl, hydroxyethyl, fluoromethyl or
fluoroethyl; and, R.sup.9 is selected from the group consisting of
111
8. A purified compound of the formulas 112wherein: R.sup.1,
R.sup.2, R.sup.3, and R.sup.5 are each independently hydrogen or
C.sub.1-C.sub.5 alkyl; and, R.sup.4 is hydrogen, C.sub.1-C.sub.5
alkyl, C.sub.1-C.sub.5 hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl,
--C(.dbd.O)R.sup.6, --C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where
R.sup.6 and R.sup.7 are each independently hydrogen or
C.sub.1-C.sub.5 alkyl; R.sup.12 is hydrogen, halide,
C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 hydroxyalkyl,
C.sub.1-C.sub.5 haloalkyl, --(CH.sub.2).sub.mC(.dbd.O)R.sup.6,
--(CH.sub.2).sub.mC(.dbd.O)OR.sup.6,
--(CH.sub.2).sub.mNR.sup.6R.sup.7 where m is 0, 1 or 2, and R.sup.6
and R.sup.7 are each independently hydrogen or C.sub.1-C.sub.5
alkyl; provided that 10,11-dehydroepothilone C is excluded.
9. The compound as in claim 8 wherein R.sup.1, R.sup.2, R.sup.3,
and R.sup.5 are each independently hydrogen, methyl or ethyl;
R.sup.4 is methyl, ethyl, hydroxymethyl, fluoromethyl,
hydroxyethyl, or fluoroethyl; and, R.sup.12 is hydrogen,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 hydroxyalkyl,
C.sub.1-C.sub.3 haloalkyl, --(CH.sub.2).sub.mC(.dbd.O)R.sup- .6,
--(CH.sub.2).sub.mC(.dbd.O)OR.sup.6,
--(CH.sub.2).sub.mNR.sup.6R.sup.7 where m is 0 or 1, and R.sup.6
and R.sup.7 are each independently hydrogen or methyl.
10. The compound as in claim 8 wherein R.sup.1, R.sup.3, and
R.sup.5 are each hydrogen; R.sup.2 is methyl; R.sup.4 is methyl,
ethyl, or fluoromethyl; and, R.sup.12 is methyl, hydroxymethyl,
fluoromethyl, and aminomethyl.
11. A purified compound selected from the group consisting of
113
12. A method of treating a condition characterized by cellular
hyperproliferation comprising: administering to a subject in need
thereof a therapeutically effective amount of a compound of the
formula 114wherein: R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or alkylaryl; R.sup.4 is
hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 hydroxyalkyl,
C.sub.1-C.sub.10 haloalkyl, aryl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen, C.sub.1-C.sub.10 aliphatic, aryl
or alkylaryl; W is O, NR.sup.8 where R.sup.8 is hydrogen,
C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10
alkynyl, aryl or alkylaryl; X is O, CH.sub.2 or a carbon-carbon
double bond; Y is absent or a C.sub.1-C.sub.10 alkyl,
C.sub.2-C.sub.10 alkenyl, or C.sub.2-C.sub.10 alkynyl; and Ar is
aryl; provided that 10,11-dehydroepothilone C is excluded.
13. The method as in claim 12 wherein R.sup.1, R.sup.2, R.sup.3,
and R.sup.5 are each independently hydrogen or methyl; R.sup.4 is
hydrogen, methyl, ethyl, or fluoromethyl; W is O or NH; X is O,
CH.sub.2 or a carbon-carbon double bond; Y is absent or
C.sub.2-C.sub.3 alkenyl; and Ar is an aryl selected from the group
consisting of thiazolyl, oxazolyl, quinolyl, isoquinolyl,
quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl, benzoimidazolyl,
and benzopyrazolyl.
14. The method as in claim 12 wherein the compound is selected from
the group consisting of 115
15. The method as in claim 12 wherein the condition is cancer.
16. The method as in claim 15 wherein the condition is a cancer of
the head or neck.
17. The method as in claim 15 wherein the condition is a cancer of
the liver or biliary tree.
18. The method as in claim 15 wherein the condition is an
intestinal cancer.
19. The method as in claim 15 wherein the condition is ovarian
cancer.
20. The method as in claim 15 wherein the condition is lung
cancer.
21. The method as in claim 15 wherein the condition is a
sarcoma.
22. The method as in claim 15 wherein the condition is a neoplasm
of the central nervous system.
23. The method as in claim 15 wherein the condition is a
lymphoma.
24. The method as in claim 12 wherein the condition is
psoriasis.
25. The method as in claim 12 wherein the condition is rheumatoid
arthritis.
26. The method as in claim 12 wherein the condition is multiple
sclerosis.
27. The method as in claim 12 where the condition is
atherosclerosis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to U.S. Ser. No. ______
filed on the same day as the present application by inventors
Robert Arslanian, John Carney, and Brian Metcalf entitled
EPOTHILONE COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME
(Morrison & Foerster Docket No. 30062-20066.00), which is
incorporated herein by reference.
BACKGROUND
[0002] Epothilone A (R.dbd.H) and Epothilone B (R.dbd.CH.sub.3) are
produced by Sorangium cellulosum strain So ce 90, the structures of
which are shown below, and were the first of several epothilones to
be isolated and characterized. Hofle et al., 1996, Angew. Chem.
Int. Ed. Engl. 35(13/14): 1567-1569. 2
[0003] Epothilone A and epothilone B possess many of the
advantageous properties of taxol. As a result, there is significant
interest in these and structurally related compounds as potential
chemotherapeutic agents. The desoxy counterparts of epothilones A
and B are known as epothilone C (R.dbd.H) and epothilone D
(R.dbd.CH.sub.3), and also exhibit similar anti-tumor activity but
with less cytotoxicity. The structures of epothilones C and D are
shown below. 3
[0004] Although other naturally occurring epothilones have been
described in the literature, these compounds are produced in
exceedingly small amounts. For example, PCT publication WO 99/65913
describes 39 naturally occurring epothilones obtained from
Sorangium cellulosum So ce 90 of which epothilones A, B, C, and D
together account for approximately 98.9% of the total epothilones
produced. The 35 other naturally occurring epothilone compounds
together account for the remaining 1.1% and include epothilone
C.sub.6 (which may also be referred to as 10, 11-dehydroepothilone
C) and whose structure is shown below 4
[0005] Due to the increasing interest in epothilones as anti-cancer
agents, novel derivatives of these compounds are needed and desired
to more fully develop their therapeutic potential.
SUMMARY
[0006] The present invention relates to 16-membered macrocyclic
compounds. In one aspect of the present invention, 16-membered
macrocyclic compounds having a conjugated diene are provided. In
another aspect of the present invention, compounds of the following
formula 5
[0007] are provided wherein:
[0008] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or alkylaryl;
[0009] R.sup.4 is hydrogen, C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.10 hydroxyalkyl, C.sub.1-C.sub.10 haloalkyl, aryl,
--C(.dbd.O)R.sup.6, --C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where
R.sup.6 and R.sup.7 are each independently hydrogen,
C.sub.1-C.sub.10 aliphatic, aryl or alkylaryl;
[0010] W is O, NR.sup.8 where R.sup.8 is hydrogen, C.sub.1-C.sub.10
alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or
alkylaryl;
[0011] X is O, CH.sub.2 or a carbon-carbon double bond;
[0012] Y is absent or a C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, or C.sub.2-C.sub.10 alkynyl; and
[0013] Ar is aryl; provided that 10,11-dehydroepothilone C is
excluded. In another aspect of the present invention, methods for
using the inventive compounds are provided.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The present invention relates to novel compounds that are
useful for the treatment of cancer and other conditions
characterized by abnormal cellular proliferation in a subject in
need thereof.
[0015] Definitions
[0016] Statements regarding the scope of the present invention and
definitions of terms used herein are listed below. The definitions
apply to the terms as they are used throughout this specification,
unless otherwise limited in specific instances, either individually
or as part of a larger group.
[0017] All stereoisomers of the inventive compounds are included
within the scope of the invention, as pure compounds as well as
mixtures thereof. Individual enantiomers, diastereomers, geometric
isomers, and combinations and mixtures thereof are all encompassed
by the present invention. Furthermore, some of the crystalline
forms for the compounds may exist as polymorphs and as such are
included in the present invention. In addition, some of the
compounds may form solvates with water (i.e., hydrates) or common
organic solvents, and such solvates are also encompassed within the
scope of this invention.
[0018] Protected forms of the inventive compounds are included
within the scope of the present invention. A variety of protecting
groups are disclosed, for example, in T. H. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, Third Edition, John
Wiley & Sons, New York (1999), which is incorporated herein by
reference in its entirety. For example, a hydroxy protected form of
the inventive compounds are those where at least one of the
hydroxyl groups is protected by a hydroxy protecting group.
Illustrative hydroxyl protecting groups include but not limited to
tetrahydropyranyl; benzyl; methylthiomethyl; ethylthiomethyl;
pivaloyl; phenylsulfonyl; triphenylmethyl; trisubstituted silyl
such as trimethyl silyl, triethylsilyl, tributylsilyl,
tri-isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl,
methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl and
the like; acyl and aroyl such as acetyl, pivaloylbenzoyl,
4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl and the
like. Keto groups in the inventive compounds may similarly be
protected.
[0019] The present invention includes within its scope prodrugs of
the compounds of this invention. In general, such prodrugs are
functional derivatives of the compounds that are readily
convertible in vivo into the required compound. Thus, in the
methods of treatment of the present invention, the term
"administering" shall encompass the treatment of the various
disorders described with the compound specifically disclosed or
with a compound which may not be specifically disclosed, but which
converts to the specified compound in vivo after administration to
a subject in need thereof. Conventional procedures for the
selection and preparation of suitable prodrug derivatives are
described, for example, in "Design of Prodrugs", H. Bundgaard ed.,
Elsevier, 1985.
[0020] As used herein, the term "aliphatic" refers to saturated and
unsaturated straight chained, branched chain, cyclic, or polycyclic
hydrocarbons that may be optionally substituted at one or more
positions. Illustrative examples of aliphatic groups include alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl
moieties. The term "alkyl" refers to straight or branched chain
saturated hydrocarbon substituent. "Alkenyl" refers to a straight
or branched chain hydrocarbon substituent with at least one
carbon-carbon double bond. "Alkynyl" refers to a straight or
branched chain hydrocarbon substituent with at least one
carbon-carbon triple bound.
[0021] The term "aryl" refers to monocyclic or polycyclic groups
having at least one aromatic ring structure that optionally include
one ore more heteroatoms and preferably include three to fourteen
carbon atoms. Aryl substituents may optionally be substituted at
one or more positions. Illustrative examples of aryl groups include
but are not limited to: furanyl, imidazolyl, indanyl, indenyl,
indolyl, isooxazolyl, isoquinolinyl, naphthyl, oxazolyl,
oxadiazolyl, phenyl, pyrazinyl, pyridyl, pyrimidinyl, pyrrolyl,
pyrazolyl, quinolyl, quinoxalyl, tetrahydronaphththyl, tetrazoyl,
thiazoyl, thienyl, thiophenyl, and the like.
[0022] The aliphatic (i.e., alkyl, alkenyl, etc.) and aryl moieties
may be optionally substituted with one or more substituents,
preferably from one to five substituents, more preferably from one
to three substituents, and most preferably from one to two
substituents. The definition of any substituent or variable at a
particular location in a molecule is independent of its definitions
elsewhere in that molecule. It is understood that substituents and
substitution patterns on the compounds of this invention can be
selected by one of ordinary skill in the art to provide compounds
that are chemically stable and that can be readily synthesized by
techniques known in the art as well as those methods set forth
herein. Examples of suitable substituents include but are not
limited to: alkyl, alkenyl, alkynyl, aryl, halo; trifluoromethyl;
trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy; heterocyclooxy;
oxo; alkanoyl (--C(.dbd.O)-alkyl which is also referred to as
"acyl")); aryloxy; alkanoyloxy; amino; alkylamino; arylamino;
aralkylamino; cycloalkylamino; heterocycloamino; disubstituted
amines in which the two amino substituents are selected from alkyl,
aryl, or aralkyl; alkanoylamino; aroylamino; aralkanoylamino;
substituted alkanoylamino; substituted arylamino; substituted
aralkanoylamino; thiol; alkylthio; arylthio; aralkylthio;
cycloalkylthio; heterocyclothio; alkylthiono; arylthiono;
aralkylthiono; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl;
sulfonamido (e.g., SO.sub.2NH.sub.2); substituted sulfonamido;
nitro; cyano; carboxy; carbamyl (e.g., CONH.sub.2); substituted
carbamyl (e.g., --C(.dbd.O)NRR' where R and R' are each
independently hydrogen, alkyl, aryl, aralkyl and the like);
alkoxycarbonyl, aryl, substituted aryl, guanidino, and heterocyclo
such as indoyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl,
pyridyl, pyrimidyl and the like. Where applicable, the substituent
may be further substituted such as with, alkyl, alkoxy, aryl,
aralkyl, halogen, hydroxy and the like.
[0023] The terms "alkylaryl" or "arylalkyl" refer to an aryl group
with an aliphatic substituent that is bonded to the compound
through the aliphatic group. An illustrative example of an
alkylaryl or arylalkyl group is benzyl, a phenyl with a methyl
group that is bonded to the compound through the methyl group
(--CH.sub.2Ph where Ph is phenyl).
[0024] The term "acyl" refers to --C(.dbd.O)R where R is an
aliphatic group, preferably a C.sub.1-C.sub.6 moiety.
[0025] The term "alkoxy" refers to --OR wherein O is oxygen and R
is an aliphatic group.
[0026] The term "aminoalkyl" refers to --RNH.sub.2 where R is an
aliphatic moiety.
[0027] The terms "halogen," "halo", or "halide" refer to fluorine,
chlorine, bromine and iodine.
[0028] The term "haloalkyl" refers to --RX where R is an aliphatic
moiety and X is one or more halogens.
[0029] The term "hydroxyalkyl" refers to --ROH where R is an
aliphatic moiety.
[0030] The term "oxo" refers to a carbonyl oxygen (.dbd.O).
[0031] In addition to the explicit substitutions at the
above-described groups, the inventive compounds may include other
substitutions where applicable. For example, the lactone or lactam
backbone or backbone substituents may be additionally substituted
(e.g., by replacing one of the hydrogens or by derivatizing a
non-hydrogen group) with one or more substituents such as
C.sub.1-C.sub.5 aliphatic, C.sub.1-C.sub.5 alkoxy, aryl, or a
functional group. Illustrative examples of suitable functional
groups include but are not limited to: acetal, alcohol, aldehyde,
amide, amine, boronate, carbamate, carboalkoxy, carbonate,
carbodiimide, carboxylic acid, cyanohydrin, disulfide, enamine,
ester, ether, halogen, hydrazide, hydrazone, imide, imido, imine,
isocyanate, ketal, ketone, nitro, oxime, phosphine, phosphonate,
phosphonic acid, quaternary ammonium, sulfenyl, sulfide, sulfone,
sulfonic acid, thiol, and the like.
[0032] The term "purified" as used herein to refer to a compound of
the present invention, means that the compound is in a preparation
in which the compound forms a major component of the composition,
such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%, about 95% or more by weight of the components in the
composition.
[0033] The term "subject" as used herein, refers to an animal,
preferably a mammal, who has been the object of treatment,
observation or experiment and most preferably a human who has been
the object of treatment and/or observation.
[0034] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue system,
animal or human that is being sought by a researcher, veterinarian,
medical doctor or other clinician, which includes alleviation of
the symptoms of the disease or disorder being treated.
[0035] The term "composition" is intended to encompass a product
comprising the specified ingredients in the specified amounts, as
well as any product that results, directly or indirectly, from
combinations of the specified ingredients in the specified
amounts.
[0036] The term "pharmaceutically acceptable salt" is a salt of one
or more of the inventive compounds. Suitable pharmaceutically
acceptable salts of the compounds include acid addition salts which
may, for example, be formed by mixing a solution of the compound
with a solution of a pharmaceutically acceptable acid such as
hydrochloric acid, sulfuric acid, fumaric acid, maleic acid,
succinic acid, acetic acid, benzoic acid, citric acid, tartaric
acid, carbonic acid or phosphoric acid. Furthermore, where the
compounds of the invention carry an acidic moiety, suitable
pharmaceutically acceptable salts thereof may include alkali metal
salts (e.g., sodium or potassium salts); alkaline earth metal salts
(e.g., calcium or magnesium salts); and salts formed with suitable
organic ligands (e.g., ammonium, quaternary ammonium and amine
cations formed using counteranions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl
sulfonate). Illustrative examples of pharmaceutically acceptable
salts include but are not limited to: acetate, adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate,
bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate,
camphorate, camphorsulfonate, camsylate, carbonate, chloride,
citrate, clavulanate, cyclopentanepropionate, digluconate,
dihydrochloride, dodecylsulfate, edetate, edisylate, estolate,
esylate, ethanesulfonate, formate, fumarate, gluceptate,
glucoheptonate, gluconate, glutamate, glycerophosphate,
glycolylarsanilate, hemisulfate, heptanoate, hexanoate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,
isothionate, lactate, lactobionate, laurate, lauryl sulfate,
malate, maleate, malonate, mandelate, mesylate, methanesulfonate,
methylsulfate, mucate, 2-naphthalenesulfonate, napsylate,
nicotinate, nitrate, N-methylglucamine ammonium salt, oleate,
oxalate, pamoate (embonate), palmitate, pantothenate, pectinate,
persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate,
pivalate, polygalacturonate, propionate, salicylate, stearate,
sulfate, subacetate, succinate, tannate, tartrate, teoclate,
tosylate, triethiodide, undecanoate, valerate, and the like.
[0037] The term "pharmaceutically acceptable carrier" is a medium
that is used to prepare a desired dosage form of the inventive
compound. A pharmaceutically acceptable carrier includes solvents,
diluents, or other liquid vehicle; dispersion or suspension aids;
surface active agents; isotonic agents; thickening or emulsifying
agents, preservatives; solid binders; lubricants and the like.
Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of
Pharmaceutical Excipients, Third Edition, A. H. Kibbe, ed. (Amer.
Pharmaceutical Assoc. 2000), both of which are incorporated herein
by reference in their entireties, disclose various carriers used in
formulating pharmaceutical compositions and known techniques for
the preparation thereof.
[0038] The term "pharmaceutically acceptable ester" is an ester
that hydrolzyes in vivo into a compound of the present invention or
a salt thereof. Illustrative examples of suitable ester groups
include, for example, those derived from pharmaceutically
acceptable aliphatic carboxylic acids such as formates, acetates,
propionates, butyrates, acrylates, and ethylsuccinates.
[0039] Compounds of the Present Invention
[0040] In one aspect of the present invention, the compounds are a
16 membered macrocycle having a conjugated diene. The macrocycle
may be a cyclic ester or lactone, a cyclic amide or lactam, or a
cyclic thioester.
[0041] In another aspect of the present invention, compounds of the
following formula 6
[0042] are provided wherein:
[0043] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or alkylaryl;
[0044] R.sup.4 is hydrogen, halogen, C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.10 hydroxyalkyl, C.sub.1-C.sub.10 haloalkyl, aryl,
--C(.dbd.O)R.sup.6, --C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where
R.sup.6 and R.sup.7 are each independently hydrogen,
C.sub.1-C.sub.10 aliphatic, aryl or alkylaryl;
[0045] W is O, NR.sup.8 where R.sup.8 is hydrogen, C.sub.1-C.sub.10
alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, aryl or
alkylaryl;
[0046] X is O, CH.sub.2 or a carbon-carbon double bond;
[0047] Y is absent or a C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10
alkenyl, or C.sub.2-C.sub.10 alkynyl; and
[0048] Ar is aryl; provided that 10,11-dehydroepothilone C is
excluded.
[0049] In one embodiment, compounds of formula I are provided
wherein:
[0050] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen or C.sub.1-C.sub.5 alkyl;
[0051] R.sup.4 is hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5
hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen or C.sub.1-C.sub.5 alkyl;
[0052] W is O or NR.sup.8 where R.sup.8 is hydrogen or
C.sub.1-C.sub.5 alkyl;
[0053] X is O, CH.sub.2 or a carbon-carbon double bond;
[0054] Y is absent or C.sub.2-C.sub.5 alkenyl; and
[0055] Ar is a heteroaryl.
[0056] In another embodiment, compound of formula I are provided
wherein:
[0057] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen, methyl or ethyl;
[0058] R.sup.4 is hydrogen, methyl, ethyl, hydroxymethyl,
hydroxyethyl, fluoromethyl or fluoroethyl;
[0059] W is O or NH;
[0060] X is O, CH.sub.2 or a carbon-carbon double bond;
[0061] Y is absent or C.sub.2-C.sub.5 alkenyl; and
[0062] Ar is an aryl selected from the group consisting of phenyl,
pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thienyl,
pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, quinolyl,
isoquinolyl, quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl,
benzoimidazolyl, and benzopyrazolyl.
[0063] In another embodiment, compound of formula I are provided
wherein:
[0064] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen or methyl;
[0065] R.sup.4 is hydrogen, methyl, ethyl, or fluoromethyl;
[0066] W is O or NH;
[0067] X is O, CH.sub.2 or a carbon-carbon double bond;
[0068] Y is absent or C.sub.2-C.sub.3 alkenyl; and
[0069] Ar is an aryl selected from the group consisting of
thiazolyl, oxazolyl, quinolyl, isoquinolyl, quinoxalyl, indolyl,
benzothiazolyl, benzoxazolyl, benzoimidazolyl, and
benzopyrazolyl.
[0070] In another aspect of the present invention, compounds of the
following formula 7
[0071] are provided wherein:
[0072] W is O or NR.sup.8 where R.sup.8 is hydrogen or
C.sub.1-C.sub.5 alkyl;
[0073] X is O, CH.sub.2 or a carbon-carbon double bond;
[0074] R.sup.4 is hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5
hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen or C.sub.1-C.sub.5 alkyl; and,
[0075] R.sup.9 is aryl or R.sup.11CH.dbd.C(R.sup.10)-- wherein
R.sup.11 is aryl and R.sup.10 is hydrogen, halide, C.sub.1-C.sub.5
alkyl, C.sub.1-C.sub.5 hydroxylalkyl, or C.sub.1-C.sub.5
haloalkyl.
[0076] In another embodiment, compounds of formula II are provided
wherein:
[0077] W is O or NH; and,
[0078] X is CH.sub.2 or a carbon-carbon double bond;
[0079] R.sup.4 is hydrogen, methyl, ethyl, hydroxymethyl,
hydroxyethyl, fluoromethyl or fluoroethyl; and,
[0080] R.sup.9 is a bicyclic heteroaryl or
R.sup.11CH.dbd.C(R.sup.10)-- wherein R.sup.11 is
2-methyl-1,3-thiazolinyl, 2-methyl-13, oxazolinyl,
2-hydroxymethyl-1,3-thiazolinyl, or 2-hydroxymethyl-1,3-oxazolinyl
and R.sup.10 is hydrogen or methyl.
[0081] In another embodiment, compounds of formula II are provided
wherein:
[0082] W is O or NH; and,
[0083] X is CH.sub.2 or a carbon-carbon double bond;
[0084] R.sup.4 is hydrogen, methyl, ethyl, hydroxymethyl,
hydroxyethyl, fluoromethyl or fluoroethyl; and,
[0085] R.sup.9 is selected from the group consisting of 8
[0086] In another aspect of the present invention, compounds of the
following formulas 9
[0087] are provided wherein:
[0088] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen or C.sub.1-C.sub.5 alkyl; and,
[0089] R.sup.4 is hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5
hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl, --C(.dbd.O)R.sup.6,
--C(.dbd.O)OR.sup.6, --NR.sup.6R.sup.7 where R.sup.6 and R.sup.7
are each independently hydrogen or C.sub.1-C.sub.5 alkyl;
[0090] R.sup.12 is hydrogen, halide, C.sub.1-C.sub.5 alkyl,
C.sub.1-C.sub.5 hydroxyalkyl, C.sub.1-C.sub.5 haloalkyl,
--(CH.sub.2).sub.mC(.dbd.O)R.sup.6,
--(CH.sub.2).sub.mC(.dbd.O)OR.sup.6,
--(CH.sub.2).sub.mNR.sup.6R.sup.7 where m is 0, 1 or 2, and R.sup.6
an R.sup.7 are each independently hydrogen or C.sub.1-C.sub.5
alkyl; provided that 10,11-dehydroepothilone C is excluded.
[0091] In one embodiment, compounds are of formulas III or IV
wherein
[0092] R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are each
independently hydrogen, methyl or ethyl;
[0093] R.sup.4 is methyl, ethyl, hydroxymethyl, fluoromethyl,
hydroxyethyl, or fluoroethyl; and,
[0094] R.sup.12 is hydrogen, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
hydroxyalkyl, C.sub.1-C.sub.3 haloalkyl,
--(CH.sub.2).sub.mC(.dbd.O)R.sup- .6,
--(CH.sub.2).sub.mC(.dbd.O)OR.sup.6,
--(CH.sub.2).sub.mNR.sup.6R.sup.7 where m is 0 or 1, and R.sup.6
and R.sup.7 are each independently hydrogen or methyl.
[0095] In another embodiment, compounds are of formulas III or IV
wherein
[0096] R.sup.1, R.sup.3, and R.sup.5 are each hydrogen;
[0097] R.sup.2 is methyl;
[0098] R.sup.4 is methyl, ethyl, or fluoromethyl; and,
[0099] R.sup.12 is methyl, hydroxymethyl, fluoromethyl, and
aminomethyl.
[0100] In another aspect of the present invention, the following
compounds are provided: 10
[0101] The compounds of the present invention are cytotoxic agents
and may be used in any suitable manner including but not limited to
as anti-cancer agents. An illustrative assay for assessing the
degree of cytotoxicy and tubulin polymerization is described in
Example 1.
[0102] Synthetic Methods
[0103] General principles of organic chemistry including functional
moieties and reactivity and common protocols are described by for
example in Advanced Organic Chemistry 3rd Ed. by Jerry March (1985)
which is incorporated herein by reference in its entirety. In
addition, it will be appreciated by one of ordinary skill in the
art that the synthetic methods described herein may use a variety
of protecting groups whether or not they are explicitly described.
A "protecting group" as used herein means a moiety used to block
functional moiety such as oxygen, sulfur, or nitrogen so that a
reaction can be carried out selectively at another reactive site in
a multifunctional compound. General principles including specific
functional groups and their uses are described for example in T. H.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,
3rd edition, John Wiley & Sons, New York (1999).
[0104] In one aspect of the present invention, two fragments are
coupled together and subsequently cyclized to form 16 membered
macrocycles of the present invention. In one embodiment, fragments
A and B are joined together under Heck coupling conditions (a
palladium catalyst such as
(diphenylphosphineferrocenyl)dichloropalladium; a base such as
cesium carbonate or 9-borabicyclo[3.3.1]nonane; and
triphenylarsine). In another embodiment, fragments A and B' (an
alkyne form of fragment B) are joined together under Suzuki
coupling reaction conditions. Fragment B' is treated with a borane
such as catechol borane or 9-borabicyclo[3.3.1]nona- ne, and then
fragments B' and A, a palladium catalyst such as
(diphenylphosphineferrocenyl)dichloropalladium, a base such as
cesium carbonate, and triphenylarsine are reacted together. Both
methods are illustrated schematically by Scheme 1 where Ar, W, Y,
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are as described
previously and Z is a protected hydroxy group or a protected amino
group that is capable of becoming W upon deprotection and
cyclization. 11
[0105] Fragment A is made using a number of methods. Scheme 2A
illustrates one embodiment where R.sup.5 is hydrogen and Z is a
protected hydroxy group and Ar--Y, and R.sup.4 are as described
previously. 12
[0106] Briefly, (2R)-N-acetyl-2,10-camphorsultam is treated with a
dialkylboron triflate such as diethylborontriflate and a base such
as diisopropyl ethylamine and then reacted with Ar--Y--CHO in an
Oppolzer aldol condensation. The resulting alcohol is protected by
reacting the compound with triethylsilyl triflate and lutidine and
reduced to form the aldehyde. Fragment A is formed by extending the
aldehyde in a Wittig reaction by treating the aldehyde with a
phosphonium salt such as iodoethyltriphenylphosphonium iodide
prepared in situ from ethyltriphenylphosphonium iodide.
[0107] Scheme 2B illustrates another embodiment where Ar--Y,
R.sup.4 and R.sup.5 are as described previously. This method is
preferred where R.sup.5 is a non-hydrogen moiety. 13
[0108] Aldehyde Ar--Y--CHO is treated with
diisopinocampheyl-allylborane in a Brown asymmetric allylation. The
resulting alcohol is protected with triethylsilyl triflate and
lutidine and the alkene is oxidized to an aldehyde. The aldehyde is
extended as in Scheme 2A in a Wittig reaction with a phosphonium
salt such as iodoethyltriphenylphosphonium iodide prepared in situ
from ethyltriphenylphosphonium iodide.
[0109] Scheme 3 illustrates another embodiment where Z is a
protected amino group and Ar--Y, R.sup.4 and R.sup.5 are as
described previously. 14
[0110] Briefly, the hydroxy protected form of fragment A of the
opposite configuration as that described in Schemes 2A and 2B is
deprotected and treated with diphenylphosphoryl azide and
diazabicycloundecene to form the azido-version of fragment A.
[0111] Fragment B where R.sup.1 is hydrogen is prepared as
described by Scheme 4A. 15
[0112] A 1,1-diisopropoxy-2,2-dimethyl-3-alkanone is extended in an
aldol condensation reaction. The resulting hydroxyl group is
protected with trichloroethoxycarbonyl chloride, and the acetal
protecting group is removed to yield a C.sub.3-C.sub.11
Intermediate of fragment B. This intermediate aldehyde is extended
in another aldol reaction and protected to yield fragment B.
[0113] Fragment B' (where R.sup.3 is hydrogen) and where R.sup.1 is
hydrogen is prepared as described by Scheme 4B. 16
[0114] The method is similar to that described by Scheme 4A except
that the first aldol reaction is performed using the alkynal 17
[0115] instead of the alkenal 18
[0116] Fragments B and B' where R.sup.1 is a non-hydrogen is
prepared as described by Scheme 5. 19
[0117] The alkene and alkyne versions of the C.sub.3-C.sub.11
Intermediate are prepared as described by Schemes 4A and 4B
respectively. The C.sub.3-C.sub.11 Intermediate is treated with
N-propionyl-benzyloxazolidi- none in an Evans aldol reaction. The
resulting alcohol is protected with the silyl group and then the
benzyloxazolidinone is hydrolyzed. Esterification with with
tert-butanol yields fragment B.
[0118] Fragments A and B can be joined together in a Heck coupling
reaction to form a diene as shown by Scheme 6A. 20
[0119] Alternatively, Fragments A and B' can be joined together in
a Suzuki coupling reaction to form the same diene as shown by
Scheme 6B. 21
[0120] Compounds of the present invention that are cyclic lactones
are made from the coupling of fragment A (where Z is a protected
hydroxyl group) and fragment B (or B') as illustrated by Scheme 7A.
22
[0121] The diene product of either the Heck coupling of fragments A
and B or the Suzuki coupling of fragments A and B' is subjected to
an ester exchange and then partially deprotected. The resulting
product is lactonized and deprotected to yield compounds
corresponding to formula I where W is O.
[0122] Compounds of the present invention that are cyclic lactams
are made from the coupling of fragment A (where Z is a protected
amino group such as N.sub.3) and fragment B (or B') as illustrated
by Schemes 7B and 7C. 23
[0123] The diene product of either the Heck coupling of fragments A
and B or the Suzuki coupling of fragments A and B' is subjected to
a Staudinger reduction and the resulting amine is protected. The
resulting product is treated with trifluoroacetic acid and then
cyclized to form the cyclic lactam. Deprotection of the Troc
protecting group yields compounds corresponding to formula I where
W is NH.
[0124] Methods for making N-alkyl lactam derivatives are described
by Scheme 7C. 24
[0125] The amino-carboxyacid is made as described by Scheme 7B and
then subjected to reductive amination. The resulting product is
then cyclized and deprotected to yield compounds corresponding to
formula I where W is NR.sup.8.
[0126] In another aspect of the present invention, the inventive
compounds are made from modified versions of fragments A and B
(designated as A" and B") using Stille coupling. 25
[0127] As shown by Scheme 8, the Stille coupling reaction of
fragments A" and B" makes the same intermediate as that produced
from the previously described Heck coupling or Suzuki coupling
reactions. This intermediate can be cyclized under conditions
similar to that described by Schemes 7A, 7B, and 7C to make the
compounds of the present invention.
[0128] Fragment A" can be made as described by Scheme 9 by
stannylation of fragment A. 26
[0129] Fragment B" where R.sup.3 is hydrogen is made by can be made
starting with fragment B'. 27
[0130] As shown by Scheme 10A, fragment B' is treated with catechol
borane in a hydroboration reaction and then iodinated to yield
fragment B" where R.sup.3 is hydrogen.
[0131] Fragment B" where R.sup.3 is a non-hydrogen is also made
starting from fragment B' 28
[0132] As shown by Scheme 10B, fragment B' is treated with
zirconocene dichloride and trialkylaluminum in a Schwartz reaction
to yield fragment B" where R.sup.3 is alkyl.
[0133] Biological Methods
[0134] In another aspect of the present invention, a subset of the
inventive compounds is made using biological methods. In one
embodiment, 10, 11-dehydroepothilone D whose structure is shown
below 29
[0135] is isolated from a strain of Myxococcus xanthus, K111-40-1.
This strain expresses the epothilone polyketide synthase but not an
active epoK gene product so produces primarily epothilone D and
lesser amounts of epothilone C and 10, 11-dehydroepothilone D.
Strain K111-40-1 (PTA-2712) was deposited with the American Type
Culture Collection ("ATCC"), 10801 University Blvd., Manassas, Va.,
20110-2209 USA on Nov. 21, 2000. In another embodiment, 10,
11-dehydroepothilone D may be isolated from M. xanthus strain
K111-72-4.4 that expresses the epothilone polyketide synthase and
contains an epoK gene with an inactivating in frame deletion.
Strain K111-72-4.4 (PTA-2713) also was deposited with the ATCC on
Nov. 21, 2000. Methods for fermentation of these strains,
purification of 10, 11-dehydroepothilone D produced by these
strains, and recombinant strains that make 10, 11-dehydroepothilone
D are described in related application U.S. Ser. No. ______ filed
on the same day as the present application by inventors Robert
Arslanian, John Carney and Brian Metcalf entitled EPOTHILONE
COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME (Morrison &
Foerster Docket No. 30062-30031.xx).
[0136] Recombinant techniques can be used to make subset of the
compounds of the present invention. These compounds include those
with substitutents at the C-2 and/or C-6 and/or C-8 and/or C-10
and/or C-14 positions that differ from the naturally occurring
epothilones A-D. Procedures for making these kinds of changes in
heterologous hosts such as Myxococcus xanthus, Steptomyces
lividians, and Pseudomonas fluorescens are described in U.S. Ser.
No. 09/443,501 filed Nov. 19, 1999 entitled RECOMBINANT METHODS AND
MATERIALS FOR PRODUCING EPOTHILONE AND EPOTHILONE DERIVATIVES which
is incorporated herein by reference. Among other things, the
application provides the nucleotide sequence of the epothilone PKS
and modification enzyme genes cloned from Sorangium cellulosum
SMP44; cosmids containing overlapping fragments of the epothilone
PKS and modification enzyme genes; plasmid pairs having the full
complement of epoA, epoB, epoC, epoD, epoE, epoF, epoK, and epoL
genes; and heterologous host cells for making epothilones and
epothilone derivatives. Cosmids, pKOS35-70.1A2 (ATCC 203782),
pKOS35-70.4 (ATCC 203781), pKOS35-70.8A3 (ATCC 203783), and
pKOS35-79.85 (ATCC 203780); plasmid pair, pKOS039-124R (PTA-926)
and pKOS039-126R (PTA-927); and strain K111-32.25 (PTA-1700)
derived from Myxococcus xanthus containing all the epothilone genes
and their promoters, have been deposited with the ATCC on Apr. 14,
2000. Additional procedures for making epothilones in Myxococcus
xanthus are described in: U.S. Ser. No. 09/560,367 filed Apr. 28,
2000 entitled PRODUCTION OF POLYKETIDES; U.S. Ser. No. ______ filed
Sep. 14, 2000 entitled PRODUCTION OF POLYKETIDES (Morrison &
Foerster Docket No. 30062-30031.21); U.S. Ser. No. ______ filed
Dec. 12, 2000 entitled PRODUCTION OF POLYKETIDES (Morrison &
Foerster Docket No. 30062-30031.22); and U.S. Ser. No. ______ filed
Feb. 13, 2001 entitled PRODUCTION OF POLYKETIDES (Morrison &
Foerster Docket No. 30062-30031.23), all of which are also
incorporated herein by reference. Illustrative examples of
compounds that may be made using recombinant techniques include:
2-methyl-10,11-dehydroepothilone C or D;
6-desmethyl-10,11-dehydroepothilone C or D;
8-desmethyl-10,11-dehydroepot- hilone C or D;
10-methyl-10,11-dehydroepothilone C or D; and
14-methyl-10,11-dehydroepothilone C or D.
[0137] In another aspect of the present invention, biologically
derived strategies are used to modify certain compounds of the
present invention regardless of whether the compounds are made
biologically or by de novo chemical synthesis. In one embodiment, a
microbially-derived hydroxylase is used to hydroxylate a terminal
alkane, particularly an alkyl substituent of the thiazole moiety of
the inventive compounds. Protocols for effectuating such a
transformation are described for example by PCT Publication No. WO
00/39276 which is incorporated herein in its entirety by reference.
Example 26 describes in greater detail the hydroxylation of the
C-20 methyl of 10,11-dehydroepothilone D to
21-hydroxy-10,11-dehydroe- pothilone D. This general method can be
readily adapted for making corresponding 21-hydroxy derivatives
from other compounds of the invention.
[0138] In another embodiment, Epo K, a P450 epoxidase that performs
the epoxidation reaction in host cells that naturally produce
epothilones or another epoxylase may be used to make 12, 13-epoxy
versions of the compounds of the present invention. A general
method for using EpoK for epoxidation is described by Example 5 of
PCT publication WO 00/31247 which is incorporated herein by
reference. Example 27 describes in greater detail the epoxidation
of 10,11-dehydroepothilone D to 10,11-dehydroepothilone B, the
general method which can be readily adapted for making
corresponding 12,13-epoxide derivatives from other compounds of the
invention.
[0139] Alternatively, the epoxidation reaction can occur by
contacting an epothilone compound containing a double bond at a
position that corresponds to the bond between carbon-12 and carbon
13 to a culture of cells that expresses a functional Epo K. Such
cells include the myxobacterium Sorangium cellulosum. In
particularly preferred embodiments, the Sorangium cellulosum
expresses Epo K but does not contain a functional epothilone
polyketide synthase ("PKS") gene. Such strains may be made by
mutagenesis where one or more mutations in the epothilone PKS gene
render it inoperative. Such mutants can occur naturally (which may
be found by screening) or can be directed using either mutagens
such as chemicals or irradation or by genetic manipulation. A
particularly effective strategy for making strains with an
inoperative epothilone PKS is homologous recombination as described
by PCT publication WO 00/31247.
[0140] Formulation
[0141] A composition of the present invention generally comprises
an inventive compound and a pharmaceutically acceptable carrier.
The inventive compound may be free form or where appropriate as
pharmaceutically acceptable derivatives such as prodrugs, and salts
and esters of the inventive compound.
[0142] The composition may be in any suitable form such as solid,
semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug
Delivery Systems, 5.sup.th edition, Lippicott Williams &
Wilkins (1991) which is incorporated herein by reference. In
general, the pharmaceutical preparation will contain one or more of
the compounds of the invention as an active ingredient in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral, or parenteral application. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, pessaries, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers that
can be used include water, glucose, lactose, gum acacia, gelatin,
mannitol, starch paste, magnesium trisilicate, talc, corn starch,
keratin, colloidal silica, potato starch, urea, and other carriers
suitable for use in manufacturing preparations, in solid,
semi-solid, or liquified form. In addition, auxiliary stabilizing,
thickening, and coloring agents and perfumes may be used.
[0143] In one embodiment, the compositions containing an inventive
compound are Cremophor.RTM.-free. Cremophor.RTM. (BASF
Aktiengesellschaft) is a polyethoxylated castor oil which is
typically used as a surfactant in formulating low soluble drugs.
However, because Cremophor.RTM. can case allergic reactions in a
subject, compositions that minimize or eliminate Cremophor.RTM. are
preferred. Formulations of epothilone A or B that eliminate
Cremophor.RTM. are described for example by PCT Publication WO
99/39694 which is incorporated herein by reference and may be
adapted for use with the inventive compounds.
[0144] Where applicable, the inventive compounds may be formulated
as microcapsules and nanoparticles. General protocols are described
for example, by Microcapsules and Nanoparticles in Medicine and
Pharmacy by Max Donbrow, ed., CRC Press (1992) and by U.S. Pat.
Nos. 5,510,118; 5,534,270; and 5,662,883 which are all incorporated
herein by reference. By increasing the ratio of surface area to
volume, these formulations allow for the oral delivery of compounds
that would not otherwise be amenable to oral delivery.
[0145] The inventive compounds may also be formulated using other
methods that have been previously used for low solubility drugs.
For example, the compounds may form emulsions with vitamin E or a
PEGylated derivative thereof as described by WO 98/30205 and
00/71163which are incorporated herein by reference. Typically, the
inventive compound is dissolved in an aqueous solution containing
ethanol (preferably less than 1% w/v). Vitamin E or a
PEGylated-vitamin E is added. The ethanol is then removed to form a
pre-emulsion that can be formulated for intravenous or oral routes
of administration. Another strategy involves encapsulating the
inventive compounds in liposomes. Methods for forming liposomes as
drug delivery vehicles are well known in the art. Suitable
protocols include those described by U.S. Pat. Nos. 5,683,715;
5,415,869, and 5,424,073 which are incorporated herein by reference
relating to another relatively low solubility cancer drug taxol and
by PCT Publication WO 01/10412 which is incorporated herein by
reference relating to epothilone B. Of the various lipids that may
be used, particularly preferred lipids for making
epothilone-encapsulated liposomes include phosphatidylcholine and
polyethyleneglycol-derivitized distearyl phosphatidylethanolamine.
Example 28 provides an illustrative protocol for making liposomes
containing 10, 11-dehydroepothilone D, the general method which can
be readily adapted to make liposomes containing other compounds of
the present invention.
[0146] Yet another method involves formulating the inventive
compounds using polymers such as polymers such as biopolymers or
biocompatible (synthetic or naturally occurring) polymers.
Biocompatible polymers can be categorized as biodegradable and
non-biodegradable. Biodegradable polymers degrade in vivo as a
function of chemical composition, method of manufacture, and
implant structure. Illustrative examples of synthetic polymers
include polyanhydrides, polyhydroxyacids such as polylactic acid,
polyglycolic acids and copolymers thereof, polyesters polyamides
polyorthoesters and some polyphosphazenes. Illustrative examples of
naturally occurring polymers include proteins and polysaccharides
such as collagen, hyaluronic acid, albumin, and gelatin.
[0147] Another method involves conjugating the compounds of the
present invention to a polymer that enhances aqueous solubility.
Examples of suitable polymers include polyethylene glycol,
poly-(d-glutamic acid), poly-(1-glutamic acid), poly-(1-glutamic
acid), poly-(d-aspartic acid), poly-(1-aspartic acid),
poly-(1-aspartic acid) and copolymers thereof. Polyglutamic acids
having molecular weights between about 5,000 to about 100,000 are
preferred, with molecular weights between about 20,000 and 80,000
being more preferred and with molecular weights between about
30,000 and 60,000 being most preferred. The polymer is conjugated
via an ester linkage to one or more hydroxyls of an inventive
epothilone using a protocol as essentially described by U.S. Pat.
No. 5,977,163 which is incorporated herein by reference, and by
Example 29. Preferred conjugation sites include the hydroxyl off
carbon-21 in the case of 21-hydroxy-derivatives of the present
invention. Other conjugation sites include the hydroxyl off carbon
3 and the hydroxyl off carbon 7.
[0148] In another method, the inventive compounds are conjugated to
a monoclonal antibody. This strategy allows the targeting of the
inventive compounds to specific targets. General protocols for the
design and use of conjugated antibodies are described in Monoclonal
Antibody-Based Therapy of Cancer by Michael L. Grossbard, ed.
(1998) which is incorporated herein by reference.
[0149] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the subject treated and the particular mode of
administration. For example, a formulation for intravenous use
comprises an amount of the inventive compound ranging from about 1
mg/mL to about 25 mg/mL, preferably from about 5 mg/mL to 15 mg/mL,
and more preferably about 10 mg/mL. Intravenous formulations are
typically diluted between about 2 fold and about 30 fold with
normal saline or 5% dextrose solution prior to use.
[0150] Methods to Treat Cancer
[0151] In one aspect of the present invention, the inventive
compounds are used to treat cancer. In one embodiment, the
compounds of the present invention are used to treat cancers of the
head and neck which include tumors of the head, neck, nasal cavity,
paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx,
hypopharynx, salivary glands, and paragangliomas. In another
embodiment, the compounds of the present invention are used to
treat cancers of the liver and biliary tree, particularly
hepatocellular carcinoma. In another embodiment, the compounds of
the present invention are used to treat intestinal cancers,
particularly colorectal cancer. In another embodiment, the
compounds of the present invention are used to treat ovarian
cancer. In another embodiment, the compounds of the present
invention are used to treat small cell and non-small cell lung
cancer. In another embodiment, the compounds of the present
invention are used to treat breast cancer. In another embodiment,
the compounds of the present invention are used to treat sarcomas
which includes fibrosarcoma, malignant fibrous histiocytoma,
embryonal rhabdomysocarcoma, leiomysosarcoma, neurofibrosarcoma,
osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part
sarcoma. In another embodiment, the compounds of the present
invention are used to treat neoplasms of the central nervous
systems, particularly brain cancer. In another embodiment, the
compounds of the present invention are used to treat lymphomas
which include Hodgkin's lymphoma, lymphoplasmacytoid lymphoma,
follicular lymphoma, mucosa-associated lymphoid tissue lymphoma,
mantle cell lymphoma, B-lineage large cell lymphoma, Burkitt's
lymphoma, and T-cell anaplastic large cell lymphoma.
[0152] The method comprises administering a therapeutically
effective amount of an inventive compound to a subject suffering
from cancer. The method may be repeated as necessary either to
contain (i.e. prevent further growth) or to eliminate the cancer.
Clinically, practice of the method will result in a reduction in
the size or number of the cancerous growth and/or a reduction in
associated symptoms (where applicable). Pathologically, practice of
the method will produce at least one of the following: inhibition
of cancer cell proliferation, reduction in the size of the cancer
or tumor, prevention of further metastasis, and inhibition of tumor
angiogenesis.
[0153] The compounds and compositions of the present invention can
be used in combination therapies. In other words, the inventive
compounds and compositions can be administered concurrently with,
prior to, or subsequent to one or more other desired therapeutic or
medical procedures. The particular combination of therapies and
procedures in the combination regimen will take into account
compatibility of the therapies and/or procedures and the desired
therapeutic effect to be achieved.
[0154] In one embodiment, the compounds and compositions of the
present invention are used in combination with another anti-cancer
agent or procedure. Illustrative examples of other anti-cancer
agents include but are not limited to: (i) alkylating drugs such as
mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan,
Ifosfamide; (ii) antimetabolites such as methotrexate; (iii)
microtubule stabilizing agents such as vinblastin, paclitaxel,
docetaxel, and discodermolide; (iv) angiogenesis inhibitors; (v)
and cytotoxic antibiotics such as doxorubicon (adriamycin),
bleomycin, and mitomycin. Illustrative examples of other
anti-cancer procedures include: (i) surgery; (ii) radiotherapy; and
(iii) photodynamic therapy.
[0155] In another embodiment, the compounds and compositions of the
present invention are used in combination with an agent or
procedure to mitigate potential side effects from the inventive
compound or composition such as diarrhea, nausea and vomiting.
Diarrhea may be treated with antidiarrheal agents such as opioids
(e.g. codeine, diphenoxylate, difenoxin, and loeramide), bismuth
subsalicylate, and octreotide. Nausea and vomiting may be treated
with antiemetic agents such as dexamethasone, metoclopramide,
diphenyhydramine, lorazepam, ondansetron, prochlorperazine,
thiethylperazine, and dronabinol. For those compositions that
includes polyethoxylated castor oil such as Cremopho.RTM.,
pretreatment with corticosteroids such as dexamethasone and
methylprednisolone and/or H.sub.1 antagonists such as
diphenylhydramine HCl and/or H.sub.2 antagonists may be used to
mitigate anaphylaxis. Illustrative formulations for intravenous use
and pretreatment regiments are described by Examples 30 and 31
respectively.
[0156] Methods of Treating of Non-cancer, Cellular
Hyperproliferative Disorders
[0157] In another aspect of the present invention, the inventive
compounds are used to treat non-cancer disorders that are
characterized by cellular hyperproliferation. In one embodiment,
the compounds of the present invention are used to treat psoriasis,
a condition characterized by the cellular hyperproliferation of
keratinocytes which builds up on the skin to form elevated, scaly
lesions. The method comprises administering a therapeutically
effective amount of an inventive compound to a subject suffering
from psoriasis. The method may be repeated as necessary either to
decrease the number or severity of lesions or to eliminate the
lesions. Clinically, practice of the method will result in a
reduction in the size or number of skin lesions, diminution of
cutaneous symptoms (pain, burning and bleeding of the affected
skin) and/or a reduction in associated symptoms (e.g., joint
redness, heat, swelling, diarrhea, abdominal pain). Pathologically,
practice of the method will result in at least one of the
following: inhibition of keratinocyte proliferation, reduction of
skin inflammation (for example, by impacting on: attraction and
growth factors, antigen presentation, production of reactive oxygen
species and matrix metalloproteinases), and inhibition of dermal
angiogenesis.
[0158] In another embodiment, the compounds of the present
invention are used to treat multiple sclerosis, a condition
characterized by progressive demyelination in the brain. Although
the exact mechanisms involved in the loss of myelin are not
understood, there is an increase in astrocyte proliferation and
accumulation in the areas of myelin destruction. At these sites,
there is macrophage-like activity and increased protease activity
which is at least partially responsible for degradation of the
myelin sheath. The method comprises administering a therapeutically
effective amount of an inventive compound to a subject suffering
from multiple sclerosis. The method may be repeated as necessary to
inhibit astrocyte proliferation and/or lessen the severity of the
loss of motor function and/or prevent or attenuate chronic
progression of the disease. Clinically, practice of the method will
result in in improvement in visual symptoms (visual loss,
diplopia), gait disorders (weakness, axial instability, sensory
loss, spasticity, hyperreflexia, loss of dexterity), upper
extremity dysfunction (weakness, spasticity, sensory loss), bladder
dysfunction (urgency, incontinence, hesitancy, incomplete
emptying), depression, emotional lability, and cognitive
impairment. Pathologically, practice of the method will result in
the reduction of one or more of the following, such as myelin loss,
breakdown of the blood-brain barrier, perivascular infiltration of
mononuclear cells, immunologic abnormalities, gliotic scar
formation and astrocyte proliferation, metalloproteinase
production, and impaired conduction velocity.
[0159] In another embodiment, the compounds of the present
invention are used to treat rheumatoid arthritis, a multisystem
chronic, relapsing, inflammatory disease that sometimes leads to
destruction and ankyiosis of affected joints. Rheumatoid arthritis
is characterized by a marked thickening of the synovial membrane
which forms villous projections that extend into the joint space,
multilayering of the synoviocyte lining (synoviocyte
proliferation), infiltration of the synovial membrane with white
blood cells (macrophages, lymphocytes, plasma cells, and lymphoid
follicles; called an "inflammatory synovitis"), and deposition of
fibrin with cellular necrosis within the synovium. The tissue
formed as a result of this process is called pannus and, eventually
the pannus grows to fill the joint space. The pannus develops an
extensive network of new blood vessels through the process of
angiogenesis that is essential to the evolution of the synovitis.
Release of digestive enzymes (matrix metalloproteinases (e.g.,
collagenase, stromelysin)) and other mediators of the inflammatory
process (e.g., hydrogen peroxide, superoxides, lysosomal enzymes,
and products of arachadonic acid metabolism) from the cells of the
pannus tissue leads to the progressive destruction of the cartilage
tissue. The pannus invades the articular cartilage leading to
erosions and fragmentation of the cartilage tissue. Eventually
there is erosion of the subchondral bone with fibrous ankylosis and
ultimately bony ankylosis, of the involved joint.
[0160] The method comprises administering a therapeutically
effective amount of an inventive compound to a subject suffering
from rheumatoid arthritis. The method may be repeated as necessary
to accomplish to inhibit synoviocyte proliferation and/or lessen
the severity of the loss of movement of the affected joints and/or
prevent or attenuate chronic progression of the disease.
Clinically, practice of the present invention will result in one or
more of the following: (i) decrease in the severity of symptoms
(pain, swelling and tenderness of affected joints; morning
stiffness, weakness, fatigue, anorexia, weight loss); (ii) decrease
in the severity of clinical signs of the disease (thickening of the
joint capsule, synovial hypertrophy, joint effusion, soft tissue
contractures, decreased range of motion, ankylosis and fixed joint
deformity); (iii) decrease in the extra-articular manifestations of
the disease (rheumatic nodules, vasculitis, pulmonary nodules,
interstitial fibrosis, pericarditis, episcleritis, iritis, Felty's
syndrome, osteoporosis); (iv) increase in the frequency and
duration of disease remission/ symptom-free periods; (v) prevention
of fixed impairment and disability; and/or (vi)
prevention/attenuation of chronic progression of the disease.
Pathologically, practice of the present invention will produce at
least one of the following: (i) decrease in the inflammatory
response; (ii) disruption of the activity of inflammatory cytokines
(such as IL-I, TNFa, FGF, VEGF); (iii) inhibition of synoviocyte
proliferation; (iv) inhibition of matrix metalloproteinase
activity, and/or (v) inhibition of angiogenesis.
[0161] In another embodiment, the compounds of the present
invention are used to threat atherosclerosis and/or restenosis,
particularly in patients whose blockages may be treated with an
endovascular stent. Atheroschlerosis is a chronic vascular injury
in which some of the normal vascular smooth muscle cells ("VSMC")
in the artery wall, which ordinarily control vascular tone
regulating blood flow, change their nature and develop
"cancer-like" behavior. These VSMC become abnormally proliferative,
secreting substances (growth factors, tissue-degradation enzymes
and other proteins) which enable them to invade and spread into the
inner vessel lining, blocking blood flow and making that vessel
abnormally susceptible to being completely blocked by local blood
clotting. Restenosis, the recurrence of stenosis or artery
stricture after corrective procedures, is an accelerated form of
atherosclerosis.
[0162] The method comprises coating a therapeutically effective
amount of an inventive compound on a stent and delivering the stent
to the diseased artery in a subject suffering from atherosclerosis.
Methods for coating a stent with a compound are described for
example by U.S. Pat. Nos. 6,156,373 and 6,120,847. Clinically,
practice of the present invention will result in one or more of the
following: (i) increased arterial blood flow; (ii) decrease in the
severity of clinical signs of the disease; (iii) decrease in the
rate of restenosis; or (iv) prevention/attenuation of the chronic
progression of atherosclerosis. Pathologically, practice of the
present invention will produce at least one of the following at the
site of stent implantation: (i) decrease in the inflammatory
response, (ii) inhibition of VSMC secretion of matrix
metalloproteinases; (iii) inhibition of smooth muscle cell
accumulation; and (iv) inhibition of VSMC phenotypic
differentiation.
[0163] Dosage Levels
[0164] In one embodiment, dosage levels that are administered to a
subject suffering from cancer or a non-cancer disorder
characterized by cellular proliferation are of the order from about
1 mg/m.sup.2 to about 200 mg/m.sup.2 which may be administered as a
bolus (in any suitable route of administration) or a continuous
infusion (e.g. 1 hour, 3 hours, 6 hours, 24 hours, 48 hours or 72
hours) every week, every two weeks, or every three weeks as needed.
It will be understood, however, that the specific dose level for
any particular patient depends on a variety of factors. These
factors include the activity of the specific compound employed; the
age, body weight, general health, sex, and diet of the subject; the
time and route of administration and the rate of excretion of the
drug; whether a drug combination is employed in the treatment; and
the severity of the condition being treated.
[0165] In another embodiment, the dosage levels are from about 10
mg/m.sup.2 to about 150 mg/m.sup.2, preferably from about 10 to
about 75 mg/m.sup.2 and more preferably from about 15 mg/m.sup.2 to
about 50 mg/m.sup.2 once every three weeks as needed and as
tolerated. In another embodiment, the dosage levels are from about
1 mg to about 150 mg/m.sup.2, preferably from about 10 mg/m.sup.2
to about 75 mg/m.sup.2 and more preferably from about 25 mg/m.sup.2
to about 50 mg/m.sup.2 once every two weeks as needed and as
tolerated. In another embodiment, the dosage levels are from about
1 mg/m.sup.2 to about 100 mg/m.sup.2, preferably from about 5
mg/m.sup.2 to about 50 Mg/m.sup.2 and more preferably from about 10
Mg/m.sup.2 to about 25 mg/m.sup.2 once every week as needed and as
tolerated. In another embodiment, the dosage levels are from about
0.1 to about 25 mg/m.sup.2, preferably from about 0.5 to about 15
mg/m.sup.2 and more preferably from about 1 mg/m.sup.2 to about 10
mg/m.sup.2 once daily as needed and tolerated.
[0166] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the present invention and shall not be construed as
being a limitation on the scope of the invention or claims.
EXAMPLE 1
[0167] Biological Activity
[0168] 10,11-Dehydroepothilone D was screened for anticancer
activity in four different human tumor cell lines using
sulforhodamine B (SRB) assay. 10,11-dehydroepothilone D shows
growth inhibitory effect on all four cell lines with IC.sub.50s
ranging from 28 nM to 40 nM. The mechanism of action was determined
by a cell-based tubulin polymerization assay which revealed that
the compound promotes tubulin polymerization. Human cancer cell
lines MCF-7 (breast), NCI/ADR-Res (breast, MDR), SF-268 (glioma),
NCI-H460 (lung) were obtained from National Cancer Institute. The
cells were maintained in a 5% CO2-humidified atmosphere at 37
degree in RPMI 1640 medium (Life Technology) supplemented with 10%
fetal bovine serum (Hyclone) and 2 mM L-glutamine.
[0169] Cytotoxicity of 10, 11-dehydroepothilone D was determined by
SRB assay (Skehan et al., J. Natl. Cancer Inst. 82: 1107-1112
(1990) which is incorporated herein by reference). Cultured cells
were trypsinized, counted and diluted to the following
concentrations per 100 .mu.l with growth medium: MCF-7, 5000;
NCI/ADR-Res, 7500; NCI-H460, 5000; and, SF-268, 7500. The cells
were seeded at 100 .mu.l/well in 96-well microtiter plates. Twenty
hours later, 100 .mu.l of 10, 11-dehydroepothilone D (ranging from
1000 nM to 0.001 nM diluted in growth medium) were added to each
well. After incubation with the compound for 3 days, the cells were
fixed with 100 .mu.l of 10% trichloric acid ("TCA") at 4 degree for
1 hour, and stained with 0.2% SRB/1% acetic acid at room
temperature for 20 minutes. The unbound dye was rinsed away with 1%
acetic acid, and the bound SRB was then extracted by 200 .mu.l of
10 mM Tris base. The amount of bound dye was determined by OD 515
nm, which correlates with the total cellular protein contents. The
data were then analyzed using Kaleida Graph program and the
IC.sub.50's calculated. Epothione D that was chemically synthesized
was tested in parallel for comparison.
[0170] For tubulin polymerization assay, MCF-7 cells were grown to
confluency in 35 mm-culture dishes and treated with 1 .mu.M of
either 10, 11-dehydroepothilone D or epothilone D for 0, 1 or 2
hours at 37 degree (Giannakakou et al., J. Biol. Chem.
271:17118-17125 (1997); Int. J. Cancer 75: 57-63 (1998) which are
incorporated herein by reference). After washing the cells twice
with 2 ml of PBS without calcium or magnesium, the cells were lysed
at room temperature for 5-10 minutes with 300 .mu.l of lysis buffer
(20 mM Tris, PH 6.8, 1 mM MgCl.sub.2, 2 mM EGTA, 1% Triton X-100,
plus protease inhibitors). The cells were scraped and the lysates
transferred to 1.5-ml Eppendof tubes. The lysates were then
centrifuged at 18000 g for 12 minutes at room temperature. The
supernatant containing soluble or unpolymerized (cytosolic) tubulin
were separated from pellets containing insoluble or polymerized
(cytoskeletal) tubulin and transferred to new tubes. The pellets
were then resuspended in 300 .mu.l of lysis buffer. Changes in
tubulin polymerization in the cell were determined by analyzing
same volume of aliquots of each sample with SDS-PAGE, followed by
immunoblotting using an anti-tubulin antibody (Sigma).
[0171] The results of several experiments showed that 10,
11-dehydroepothilone D (designated as "Epo490") has an IC.sub.50 in
the range of 28 nM to 40 nM against four different human tumor
cells lines.
1 TABLE 1 Cell lines EpoD (nM) Epo490 (nM) N = 3 N = 2 MCF-7 21
.+-. 10 28 .+-. 8 NCI/ADR 40 .+-. 12 35 .+-. 9 SF-268 34 .+-. 8 40
.+-. 5 NCI-H460 30 .+-. 2 34 .+-. 1
[0172] Tubulin polymerization assays reveal that 10,
11-dehydroepothilone D has the same mechanism of action as
epothilone D. In MCF-7 cells, 10, 11-dehydroepothilone D strongly
promoted tubulin polymerization at the conditions tested, with
similar kinetics and effect as epothilone D. Other compounds of the
invention may be tested in a similar manner by replacing the
compound of interest for 10, 11-dehydroepothilone D.
EXAMPLE 2
(5S)-2-iodo-5-(6-quinolyl)-5-(triethylsilyloxy)-2-pentene
[0173] 30
(a)
(2R)-N-[(3S)-3-hydroxy-3-(6-quinolyl)propionyl]-2,10-camphorsultam
[0174] 31
[0175] A 1.0 M solution of diethylboron triflate in
CH.sub.2Cl.sub.2 (110 mL) is added slowly to a 0.degree. C.
solution of 25.7 g of (2R)-N-acetyl-2,10-camphorsultam (Oppolzer et
al. 1992, Tetrahedron Letters 33, 2439) in 250 mL of
CH.sub.2Cl.sub.2. A solution of diisopropylethylamine (15 mL) in 75
mL of CH.sub.2Cl.sub.2 is then added dropwise over 20 minutes at
0.degree. C., and the mixture is then cooled to -78.degree. C. A
solution of quinoline-6-carboxaldehyde (15.7 g) in 100 mL of
CH.sub.2Cl.sub.2 is then added at such a rate as to keep the
reaction temperature below -70.degree. C. After an additional 2
hours, the mixture is warmed to ambient temperature and quenched by
addition of 100 mL of 3:1 tetrahydrofuran/water and 750 mL of sat.
aq. NH.sub.4Cl. The mixture is concentrated to a slurry, then
diluted with water, adjusted to pH 8, and extracted with ethyl
acetate. The extract is washed sequentially with water and brine,
then dried over MgSO.sub.4, filtered, and evaporated to yield the
crude adduct. Purification by silica gel chromatography yields pure
(2R)-N-[3-hydroxy-3-(6-quinolyl)propionyl]born-
ane-2,10-camphorsultam.
(b)
(2R)-N-[(3S)-3-(triethylsilyloxy)-3-(6-quinolyl)propionyl]-2,10-campho-
rsultam
[0176] 32
[0177] A solution of
(2R)-N-[3-hydroxy-3-(6-quinolyl)propionyl]-2,10-camph- orsultam
(41.4 gm) in 250 mL of CH.sub.2Cl.sub.2 is cooled on ice and
treated with 2,6-lutidine (13 gm) and triethylsilyl
trifluoromethanesulfonate (29 gm). After stirring for 12 hours, the
mixture is washed with water and dried over MgSO.sub.4, filtered,
and evaporated to yield the crude product. Purification by silica
gel chromatography yields pure
(2R)-N-[3-(triethylsilyloxy)-3-(6-quinolyl)-pr-
opionyl]-2,10-camphorsultam.
(c) (3S)-3-(triethylsilyloxy)-3-(6-quinolyl)propanal
[0178] 33
[0179] A solution of
(2R)-N-[3-(triethylsilyloxy)-3-(6-quinolyl)-propionyl-
]bornane-10,2-sultam (52.8 gm) in 1000 mL of CH.sub.2Cl.sub.2 is
cooled to -78.degree. C., and a 1.0 M solution of
diisobutylaluminum hydride in hexane is added dropwise so as to
keep the reaction temperature below -65.degree. C. The mixture is
stirred for an additional 6 hours at -78.degree. C., then is
quenched by addition of sat. aq. NH.sub.4Cl and allowed to warm to
ambient temperature. The mixture is washed with brine, and the
organic phase is dried over MgSO.sub.4, filtered, and evaporated.
The product is purified by silica gel chromatography.
(d) (5S)-2-iodo-5-(6-quinolyl)-5-(triethylsilyloxy)-2-pentene
[0180] A suspension of ethyltriphenylphosphonium iodide (7.9 gm) in
150 mL of tetrahydrofuran is treated with a 2.5 M solution of
n-butyllithium (7.17 mL) at ambient temperature. The resulting red
solution is transferred via cannula into a vigorously stirred
solution of iodine (4.54 gm) in 150 mL of tetrahydrofuran cooled to
-78.degree. C. The resulting suspension is stirred for 5 minutes,
then gradually warmed to -30.degree. C. A 1.0 M solution of sodium
hexamethyldisilazide (17.3 mL) is then added dropwise to form a red
solution. A solution of
(3S)-3-(triethylsilyloxy)-3-(6-quinolyl)propanal (1.9 gm) in 10 mL
of tetrahydrofuran is then added dropwise, and stirring is
continued at -30.degree. C. for 30 minutes. The mixture is diluted
with ether, filtered through a pad of Celite, and concentrated. The
product is purified by flash chromatography on silica gel.
EXAMPLE 3
(5S)-2-iodo-5-(2-methylbenzothiazol-5-yl)-5-(triethylsilyloxy)-2-pentene
[0181] 34
[0182] Is prepared according to the method of Example 2, replacing
quinoline-6-carboxaldehyde with
2-methylbenzothiazole-5-carboxaldehyde.
EXAMPLE 4
(5S)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-hep-
tadiene (Fragment A: ArY/R.sup.9=(2-methylthiazol-4-yl)propen-2-yl,
R.sup.5=H R.sup.4=Me)
[0183] 35
[0184] Is prepared according to the method of Example 2, replacing
quinoline-6-carboxaldehyde with
2-methyl-3-(2-methylthiazol-4-yl)propenal- .
EXAMPLE 5
(6S)-3-iodo-6-(6-guinolyl)-6-(triethylsilyloxy)-3-hexene
[0185] 36
[0186] A suspension of propyltriphenylphosphonium iodide (8.2 gm)
in 150 mL of tetrahydrofuran is treated with a 2.5 M solution of
n-butyllithium (7.17 mL) at ambient temperature. The resulting red
solution is transferred via cannula into a vigorously stirred
solution of iodine (4.54 gm) in 150 mL of tetrahydrofuran cooled to
-78.degree. C. The resulting suspension is stirred for 5 minutes,
then gradually warmed to -30.degree. C. A 1.0 M solution of sodium
hexamethyldisilazide (17.3 mL) is then added dropwise to form a red
solution. A solution of
(3S)-3-(triethylsilyloxy)-3-(6-quinolyl)propanal (1.9 gm) in 10 mL
of tetrahydrofuran is then added dropwise, and stirring is
continued at -30.degree. C. for 30 minutes. The mixture is diluted
with ether, filtered through a pad of Celite, and concentrated. The
product is purified by flash chromatography on silica gel.
EXAMPLE 6
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyloxy)methylthiazol--
4-yl)-5-(triethylsilyloxy)-2,6-heptadiene
[0187] 37
(a) ethyl
2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxylate
[0188] 38
[0189] To a solution of ethyl
2-hydroxymethyl)-thiazole-4-carboxylate 33 (38.4 g) and pyridine
(41 mL) in 100 mL of CH.sub.2Cl.sub.2 is slowly added
2,2,2-trichloroethyl chloroformate (32 mL, 0.23 mol) at 0.degree.
C. After the resulting mixture is stirred for 30 minutes, the
reaction is quenched by the addition of 10% aq NaHCO3. The organic
layer is separated, and the aqueous layer is extracted with
CH.sub.2Cl.sub.2. The combined organic extracts are washed with 2 N
HCl, 10% aq NaHCO.sub.3, and brine, dried (Na.sub.2SO.sub.4), and
concentrated. The residue is then recrystallized in ethanol (50 mL)
to yield a light yellow solid. The mother liquor is concentrated
and chromatographed to afford an additional amount of product.
(b)
2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-carboxaldehyde
[0190] 39
[0191] To a solution of ethyl
2-(2,2,2-trichloroethoxycarbonyloxymethyl)th- iazole-4-carboxylate
(23 g) in CH.sub.2Cl.sub.2 (200 mL) is added a solution of
diisobutylaluminum hydride (1.0 M in CH.sub.2Cl.sub.2, 120 mL) at
-78.degree. C. over 30 minutes. The resulting mixture is kept at
-78.degree. C. for 10 hours. The excess hydride is quenched with
acetic acid (5 mL) and the reaction is warmed to ambient
temperature, and the mixture is stirred with sat. aq. Rochelle's
salt (150 mL) until the suspension clears. The organic layer is
washed with 10% aq NaHCO.sub.3 and brine, dried (Na.sub.2SO.sub.4),
filtered, and concentrated. Purification by column chromatography
on SiO.sub.2 (toluene/ethyl acetate, 6:1) affords the product as a
light yellow syrup.
(c)
2-methyl-3-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)pro-
penal
[0192] 40
[0193] A mixture of
2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazole-4-c-
arboxaldehyde (21.0 g) and
2-(triphenylphosphoranylidene)propionaldehyde (20.6 g) in 300 mL of
benzene is heated at reflux for 3 hours, then cooled to ambient
temperature and concentrated. Purification by flash column
chromatography on SiO2 (hexanes/ethyl acetate, 4:1) yields the
product as a clear oil.
(d)
(4S)-4-hydroxy-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)--
thiazol-4-yl)-1,5-hexadiene
[0194] 41
[0195] A solution of
2-methyl-3-(2-((2,2,2-trichloroethoxycarbonyloxymethy-
l)thiazol-4-yl)propenal (9.20 g) in 50 mL of anhydrous ether is
cooled to -100.degree. C. A pentane solution of
(+)-diisopinocampheylallylborane (1.5 equiv) is added dropwise to
the vigorously stirred aldehyde solution. After the addition is
complete, the reaction mixture is stirred for 1.5 hours and warmed
to -50.degree. C. A solution of 30% aq H2O2 (20 mL) and 10% aq
NaHCO3 (50 mL) is added, and the resulting turbid mixture is
stirred at 25.degree. C. for 8 hours. The organic layer is
separated, and the aqueous layer is extracted with ether. The
combined organic layers are washed with satd aq
Na.sub.2S.sub.2O.sub.3 and brine, dried (MgSO.sub.4), filtered, and
concentrated. Purification by flash column chromatography on
SiO.sub.2 (hexanes/ethyl acetate, 10:1) affords the alcohol as a
clear oil.
(e)
(4S)-4-(triethylsilyloxy)-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyl-
oxymethyl)-thiazol-4-yl)-1,5-hexadiene
[0196] 42
[0197] Triethylsilyl trifluromethanesulfonate (15 mL) is added
dropwise to a -78.degree. C. solution of
(4S)-4-hydroxy-5-methyl-6-(2-(2,2,2-trichlor-
oethoxycarbonyloxymethyl)-thiazol -4-yl)-1,5-hexadiene (7.65 g) and
2,6-lutidine (10 mL) in 50 mL of CH.sub.2Cl.sub.2. After the
addition, the reaction mixture is allowed to warm to ambient
temperature and is stirred for 5 hours. The reaction mixture is
poured into 2 N HCl and extracted with ether. The combined organic
layers were washed with 10% aq NaHCO.sub.3 and brine, dried
(Na.sub.2SO.sub.4), filtered, and concentrated. Flash
chromatography on SiO.sub.2 (hexanes/ethyl acetate, 20:1) provides
the product as a colorless oil.
(f)
(3S)-3-(triethylsilyloxy)-4-methyl-5-(2-(2,2,2-trichloroethoxycarbonyl-
oxy-methyl)-thiazol-4-yl)-pent-4-enal
[0198] 43
[0199] Osmium tetraoxide (1 wt % in THF, 20.3 mL) is added to a
mixture of
(4S)-4-(triethylsilyloxy)-5-methyl-6-(2-(2,2,2-trichloroethoxycarbonyloxy-
methyl)-thiazol-4-yl) -1,5-hexadiene (20.6 g), H.sub.2O (21 mL),
and N-methylmorpholine N-oxide (50% in THF, 10 mL, 0.048 mol) in
tert-butanol (155 mL) at 0.degree. C. After the resulting mixture
is stirred for 12 hours, Na.sub.2SO.sub.3 (10 g) and water (5 mL)
are added. The resulting solution is stirred at 25.degree. C. for
30 minutes and then extracted with ether. The combined extracts are
washed with brine, dried (Na.sub.2SO.sub.4), filtered, and
concentrated. Purification by flash chromatography on SiO.sub.2
provides a 1:1 diastereomeric mixture of the diol as a colorless,
viscous oil. Lead tetraacetate (19.1 g) is added portionwise over 5
minutes to a suspension of the diol (18.0 g) and Na.sub.2CO.sub.3
(8.67 g) in 500 mL of benzene at 0.degree. C. After stirring for 15
minutes, the mixture is filtered through a SiO.sub.2 pad to afford
the aldehyde product. The product is directly subjected to the next
reaction.
(g)
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyloxy)methylthia-
zol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene
[0200] To a suspension of ethyltriphenylphosphonium iodide (7.90 g)
in THF (150 mL) is added n-butyllithium (7.17 mL, 2.5 M in hexane)
at ambient temperature. After disappearance of the solid material,
the red solution is cannulated into a vigorously stirred solution
of iodine (4.54 g) in THF (150 mL) at -78.degree. C. The resulting
dark brown suspension is stirred for 5 minutes and allowed to warm
gradually to -30.degree. C. A solution of sodium
hexamethyldisilazide (17.3 mL, 1.0 M in THF) is added dropwise to
afford a dark red solution. A solution of
(3S)-3-(triethylsilyloxy)-4-methyl-5-(2-(2,2,2-trichloroethoxy-carbonylox-
y-methyl)-thiazol-4-yl))-pent-4-enal (3.10 g) in THF (10 mL) is
slowly added, and stirring is continued at -30.degree. C. for 30
minutes. The reaction mixture is diluted with pentane (100 mL),
filtered through a pad of Celite, and concentrated. Purification by
flash column chromatography on SiO.sub.2 (hexane/ethyl acetate,
15:1) affords the vinyl iodide as a yellow syrup.
EXAMPLE 7
(4S,5S)-2-iodo-4,6-dimethyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)--
2,6-heptadiene
[0201] 44
(a)
(3S,4S)-4-hydroxy-3,5-dimethyl-6-(2-methylthiazol-4-yl))-1,5-hexadiene
[0202] 45
[0203] A solution of 2-methyl-3-(2-methylthiazol-4-yl))propenal
(4.3 g) in 50 mL of anhydrous ether is cooled to -100.degree. C. A
pentane solution of (+)-diisopinocampheyl-trans-crotylborane (1.5
equiv) is added dropwise to the vigorously stirred aldehyde
solution. After the addition is complete, the reaction mixture is
stirred for 1.5 hours and warmed to -50.degree. C. A solution of
30% aq H2O2 (20 mL) and 10% aq NaHCO3 (50 mL) is added, and the
resulting turbid mixture is stirred at 25.degree. C. for 8 hours.
The organic layer is separated, and the aqueous layer is extracted
with ether. The combined organic layers are washed with satd aq
Na.sub.2S.sub.2O.sub.3 and brine, dried (MgSO.sub.4), filtered, and
concentrated. Purification by flash column chromatography on
SiO.sub.2 (hexanes/ethyl acetate, 10:1) affords the alcohol as a
clear oil.
(b)
(3S,4S)-4-(triethylsilyloxy)-3,5-dimethyl-6-(2-methylthiazol-4-yl))-1,-
5-hexadiene
[0204] 46
[0205] Triethylsilyl trifluromethanesulfonate (15 mL) is added
dropwise to a -78.degree. C. solution of
(3S,4S)-4-hydroxy-3,5-dimethyl-6-(2-methylth-
iazol-4-yl)-1,5-hexadiene (4.3 g) and 2,6-lutidine (10 mL) in 50 mL
of CH.sub.2Cl.sub.2. After the addition, the reaction mixture is
allowed to warm to ambient temperature and is stirred for 5 hours.
The reaction mixture is poured into 2 N HC1 and extracted with
ether. The combined organic layers were washed with 10% aq
NaHCO.sub.3 and brine, dried (Na.sub.2SO.sub.4), filtered, and
concentrated. Flash chromatography on SiO.sub.2 (hexanes/ethyl
acetate, 20:1) provids the product as a colorless oil.
(c)
(2R,3S)-3-(triethylsilyloxy)-2,4-dimethyl-5-(2-methylthiazol-4-yl))-pe-
nt-4-enal
[0206] 47
[0207] Osmium tetraoxide (1 wt % in THF, 20.3 mL) is added to a
mixture of
(3S,4S)-4-(triethylsilyloxy)-3,5-dimethyl-6-(2-methylthiazol-4-yl)-1,5-he-
xadiene (13.5 g), H.sub.2O (21 mL), and N-methylmorpholine N-oxide
(50% in THF, 10 mL, 0.048 mol) in tert-butanol (155 mL) at
0.degree. C. After the resulting mixture is stirred for 12 hours,
Na.sub.2SO.sub.3 (10 g) and water (5 mL) are added. The resulting
solution is stirred at 25.degree. C. for 30 minutes and then
extracted with ether. The combined extracts are washed with brine,
dried (Na.sub.2SO.sub.4), filtered, and concentrated. Purification
by flash chromatography on SiO.sub.2 provides a 1:1 diastereomeric
mixture of the diol as a colorless, viscous oil. Lead tetraacetate
(19.1 g) is added portionwise over 5 minutes to a suspension of the
diol (18.0 g) and Na.sub.2CO.sub.3 (8.67 g) in 500 mL of benzene at
0.degree. C. After stirring for 15 minutes, the mixture is filtered
through a SiO.sub.2 pad to afford the aldehyde product. The product
is directly subjected to the next reaction.
(d)
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyloxy)methylthia-
zol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene
[0208] To a suspension of ethyltriphenylphosphonium iodide (7.90 g)
in THF (150 mL) is added n-butyllithium (7.17 mL, 2.5 M in hexane)
at ambient temperature. After disappearance of the solid material,
the red solution is cannulated into a vigorously stirred solution
of iodine (4.54 g) in THF (150 mL) at -78.degree. C. The resulting
dark brown suspension is stirred for 5 minutes and allowed to warm
gradually to -30.degree. C. A solution of sodium
hexamethyldisilazide (17.3 mL, 1.0 M in THF) is added dropwise to
afford a dark red solution. A solution of
(2R,3S)-3-(triethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)-pent-4-ena-
l (2.0 g) in THF (10 mL) is slowly added, and stirring is continued
at -30.degree. C. for 30 minutes. The reaction mixture is diluted
with pentane (100 mL), filtered through a pad of Celite, and
concentrated. Purification by flash column chromatography on
SiO.sub.2 (hexane/ethyl acetate, 15:1) affords the vinyl iodide as
a yellow syrup.
EXAMPLE 8
(5S)-5-azido-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-heptadiene
[0209] 48
(a)
(4R)-4-hydroxy-3,5-dimethyl-6-(2-methylthiazol-4-yl))-1,5-hexadiene
[0210] 49
[0211] A solution of 2-methyl-3-(2-methylthiazol-4-yl))propenal
(4.3 g) in 50 mL of anhydrous ether is cooled to -100.degree. C. A
pentane solution of (-)-diisopinocampheylallylborane (1.5 equiv) is
added dropwise to the vigorously stirred aldehyde solution. After
the addition is complete, the reaction mixture is stirred for 1.5
hours and warmed to -50.degree. C. A solution of 30% aq
H.sub.2O.sub.2 (20 mL) and 10% aq NaHCO.sub.3 (50 mL) is added, and
the resulting turbid mixture is stirred at 25.degree. C. for 8
hours. The organic layer is separated, and the aqueous layer is
extracted with ether. The combined organic layers are washed with
satd aq Na.sub.2S.sub.2O.sub.3 and brine, dried (MgSO.sub.4),
filtered, and concentrated. Purification by flash column
chromatography on SiO.sub.2 (hexanes/ethyl acetate, 10:1) affords
the alcohol as a clear oil.
(b)
(4R)-4-(triethylsilyloxy)-5-methyl-6-(2-methylthiazol-4-yl))-1,5-hexad-
iene
[0212] 50
[0213] Triethylsilyl trifluromethanesulfonate (15 mL) is added
dropwise to a -78.degree. C. solution of
(4R)-4-hydroxy-5-methyl-6-(2-methylthiazol-4- -yl))-1,5-hexadiene
(4.3 g) and 2,6-lutidine (10 mL) in 50 mL of CH.sub.2Cl.sub.2.
After the addition, the reaction mixture is allowed to warm to
ambient temperature and is stir-red for 5 hours. The reaction
mixture is poured into 2 N HCl and extracted with ether. The
combined organic layers were washed with 10% aq NaHCO.sub.3 and
brine, dried (Na.sub.2SO.sub.4), filtered, and concentrated. Flash
chromatography on SiO.sub.2 (hexanes/ethyl acetate, 20:1) provids
the product as a colorless oil.
(c)
(3R)-3-(triethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)-pent-4-ena-
l
[0214] 51
[0215] Osmium tetraoxide (1 wt % in THF, 20.3 mL) is added to a
mixture of
(4R)-4-(triethylsilyloxy)-5-methyl-6-(2-methylthiazol-4-yl)-1,5-hexadiene
(13.5 g), H.sub.2O (21 mL), and N-methylmorpholine N-oxide (50% in
THF, 10 mL, 0.048 mol) in tert-butanol (155 mL) at 0.degree. C.
After the resulting mixture is stirred for 12 hours,
Na.sub.2SO.sub.3 (10 g) and water (5 mL) are added. The resulting
solution is stirred at 25.degree. C. for 30 minutes and then
extracted with ether. The combined extracts are washed with brine,
dried (Na.sub.2SO.sub.4), filtered, and concentrated. Purification
by flash chromatography on SiO.sub.2 provides a 1:1 diastereomeric
mixture of the diol as a colorless, viscous oil. Lead tetraacetate
(19.1 g) is added portionwise over 5 minutes to a suspension of the
diol (18.0 g) and Na.sub.2CO.sub.3 (8.67 g) in 500 mL of benzene at
0.degree. C. After stirring for 15 minutes, the mixture is filtered
through a SiO.sub.2 pad to afford the aldehyde product. The product
is directly subjected to the next reaction.
(d)
(5R)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-
-heptadiene
[0216] 52
[0217] To a suspension of ethyltriphenylphosphonium iodide (7.90 g)
in THF (150 mL) is added n-butyllithium (7.17 mL, 2.5 M in hexane)
at ambient temperature. After disappearance of the solid material,
the red solution is cannulated into a vigorously stirred solution
of iodine (4.54 g) in THF (150 mL) at -78.degree. C. The resulting
dark brown suspension is stirred for 5 minutes and allowed to warm
gradually to -30.degree. C. A solution of sodium
hexamethyldisilazide (17.3 mL, 1.0 M in THF) is added dropwise to
afford a dark red solution. A solution of
(3R)-3-(triethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)-pent-4-enal
(2.0 g) in THF (10 mL) is slowly added, and stirring is continued
at -30.degree. C. for 30 minutes. The reaction mixture is diluted
with pentane (100 mL), filtered through a pad of Celite, and
concentrated. Purification by flash column chromatography on
SiO.sub.2 (hexane/ethyl acetate, 15:1) affords the vinyl
iodide.
(e)
(5R)-5-hydroxy-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-heptadiene
[0218] 53
[0219] A solution of
(5R)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(trie-
thylsilyloxy)-2,6-heptadiene (2.28 g) in 50 mL of 3:1:1 acetic
acid/THF/water is stirred at ambient temperature for 8 hoursours,
then concentrated. The residue is dissolved in ethyl acetate,
washed sequentially with sat. NaHCO.sub.3 and brine, then dried
over MgSO.sub.4, filtered, and evaporated. The product is purified
by SiO.sub.2 chromatography (30% ethyl acetate/hexanes).
(f)
(5S)-5-azido-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-heptadiene
[0220] A solution of
(5R)-5-hydroxy-2-iodo-6-methyl-7-(2-methylthiazol-4-y-
l)-2,6-heptadiene (1.74 g) in 30 mL of toluene is cooled to
0.degree. C. and treated with diphenylphosphoryl azide (1.65 g) and
1,8-diazabicyclo[5.4.0]undec-7-ene (0.91 g) for 2 hours. The mix is
warmed to ambient temperature and diluted with ethyl acetate. The
solution is washed sequentially with water, sat. NaHCO.sub.3, and
brine, then dried over MgSO.sub.4, filtered, and evaporated. The
product is purified by SiO.sub.2 chromatography (7.5% ethyl
acetate/hexanes).
EXAMPLE 9
(5S)-5-azido-2-iodo-5-(6-quinolyl)-2-pentene
[0221] 54
(a)
(2S)-N-[(3R)-3-hydroxy-3-(6-quinolyl)propionyl]-2,10-camphorsultam
[0222] 55
[0223] A 1.0 M solution of diethylboron triflate in
CH.sub.2Cl.sub.2 (110 mL) is added slowly to a 0.degree. C.
solution of 25.7 gm of (2S)-N-acetyl-2,10-camphorsultam in 250 mL
of CH.sub.2Cl.sub.2. A solution of diisopropylethylamine (15 mL) in
75 mL of CH.sub.2Cl.sub.2 is then added dropwise over 20 minutes at
0.degree. C., and the mixture is then cooled to -78.degree. C. A
solution of quinoline-6-carboxaldehyde (15.7 g) in 100 mL of
CH.sub.2Cl.sub.2 is then added at such a rate as to keep the
reaction temperature below -70.degree. C. After an additional 2
hours, the mixture is warmed to ambient temperature and quenched by
addition of 100 mL of 3:1 tetrahydrofuran/water and 750 mL of sat.
aq. NH.sub.4Cl. The mixture is concentrated to a slurry, then
diluted with water, adjusted to pH 8, and extracted with ethyl
acetate. The extract is washed sequentially with water and brine,
then dried over MgSO.sub.4, filtered, and evaporated to yield the
crude adduct. Purification by silica gel chromatography yields pure
product.
(b)
(2S)-N-[(3R)-3-(triethylsilyloxy)-3-(6-quinolyl)propionyl]-2,10-campho-
rsultam
[0224] 56
[0225] A solution of
(2S)-N-[(3R)-3-hydroxy-3-(6-quinolyl)propionyl]-2,10--
camphorsultam (41.4 gm) in 250 mL of CH.sub.2Cl.sub.2 is cooled on
ice and treated with 2,6-lutidine (13 gm) and triethylsilyl
trifluoromethanesulfonate (29 gm). After stirring for 12 hours, the
mixture is washed with water and dried over MgSO.sub.4, filtered,
and evaporated to yield the crude product. Purification by silica
gel chromatography yields pure product.
(c) (3R)-3-(triethylsilyloxy)-3-(6-quinolyl)propanal
[0226] 57
[0227] A solution of
(2S)-N-[(3R)-3-(triethylsilyloxy)-3-(6-quinolyl)-prop-
ionyl]-2,10-camphorsultam (52.8 gm) in 1000 mL of CH.sub.2Cl.sub.2
is cooled to -78.degree. C., and a 1.0 M solution of
diisobutylaluminum hydride in hexane is added dropwise so as to
keep the reaction temperature below -65.degree. C. The mixture is
stirred for an additional 6 hours at -78.degree. C., then is
quenched by addition of sat. aq. NH.sub.4Cl and allowed to warm to
ambient temperature. The mixture is washed with brine, and the
organic phase is dried over MgSO.sub.4, filtered, and evaporated.
The product is purified by silica gel chromatography.
(d) (5R)-2-iodo-5-(6-quinolyl)-5-(triethylsilyloxy)-2-pentene
[0228] 58
[0229] A suspension of ethyltriphenylphosphonium iodide (7.9 gm) in
150 mL of tetrahydrofuran is treated with a 2.5 M solution of
n-butyllithium (7.17 mL) at ambient temperature. The resulting red
solution is transferred via cannula into a vigorously stirred
solution of iodine (4.54 gm) in 150 mL of tetrahydrofuran cooled to
-78.degree. C. The resulting suspension is stirred for 5 minutes,
then gradually warmed to -30.degree. C. A 1.0 M solution of sodium
hexamethyldisilazide (17.3 mL) is then added dropwise to form a red
solution. A solution of
(3R)-3-(triethylsilyloxy)-3-(6-quinolyl)propanal (1.9 gm) in 10 mL
of tetrahydrofuran is then added dropwise, and stirring is
continued at -30.degree. C. for 30 minutes. The mixture is diluted
with ether, filtered through a pad of Celite, and concentrated. The
product is purified by flash chromatography on silica gel.
(e) (5R)-2-iodo-5-(6-quinolyl)-5-hydroxy-2-pentene
[0230] 59
[0231] A solution of
(5R)-2-iodo-5-(6-quinolyl)-5-(triethylsilyloxy)-2-pen- tene (2.26
g) in 50 mL of 3:1:1 acetic acid/THF/water is stirred at ambient
temperature for 8 hours, then concentrated. The residue is
dissolved in ethyl acetate, washed sequentially with sat.
NaHCO.sub.3 and brine, then dried over MgSO.sub.4, filtered, and
evaporated. The product is purified by SiO.sub.2
chromatography.
(e) (5S)-5-azido-2-iodo-5-(6-quinolyl)-2-pentene
[0232] A solution of (5R)-2-iodo-5-(6-quinolyl)-5-hydroxy-2-pentene
(1.74 g) in 30 mL of toluene is cooled to 0.degree. C. and treated
with diphenylphosphoryl azide (1.65 g) and
1,8-diazabicyclo[5.4.0]undec-7-ene (0.91 g) for 2 hours. The mix is
warmed to ambient temperature and diluted with ethyl acetate. The
solution is washed sequentially with water, sat. NaHCO.sub.3, and
brine, then dried over MgSO.sub.4, filtered, and evaporated. The
product is purified by SiO.sub.2 chromatography.
EXAMPLE 10
Tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7--
(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
[0233] 60
(a)
(4R,5S,6S)-1,1-diisopropoxy-5-hydroxy-2,2,4,6-tetramethyl-8-nonen-3-on-
e
[0234] 61
[0235] A solution of 1,1-diisopropoxy-2,2,-dimethyl-3-pentanone
(3.29 g) in 15 mL of THF is added slowly to a solution of lithium
diisopropylamide (15.7 mmol) in 20 mL of THF cooled to -78.degree.
C., the mixture is stirred for 30 minutes, warmed to 40.degree. C.
and stirred for 30 minutes, then recooled to -78.degree. C. A
solution of (2S)-2-methyl-4-pentenal (16.36 mmol) in 2 mL of
CH.sub.2Cl.sub.2 is added and the mixture is stirred for 1 hour at
-78.degree. C. Saturated aq. NH.sub.4Cl is added and the mixture is
warmed to ambient temperature and extracted with ethyl acetate. The
extract is dried over Na.sub.2SO.sub.4, filtered, and evaporated.
The residue is purified by silica gel chromatography (2% ethyl
acetate/hexanes) to separate the two diastereomeric products.
(b)
(4R,5S,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4-
,6-tetramethyl-8-nonen-3-one
[0236] 62
[0237] Trichloroethyl chloroformate (2.5 mL) and pyridine (2.95 mL)
are added to a solution of
(4R,5S,6S)-1,1-diisopropoxy-5-hydroxy-2,2,4,6-tetr-
amethyl-8-nonen-3-one (3.0 g) in 40 mL of CH.sub.2Cl.sub.2 at
0.degree. C., and the mixture is stirred for 5 hours before pouring
into sat. aq. NaCl and extracting with CH.sub.2Cl.sub.2. The
extract is dried over Na.sub.2SO.sub.4, filtered, and evaporated.
The product is purified by chromatography on SiO.sub.2 (2% ethyl
acetate/hexanes).
(c)
(4R,5S,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4,6-tetramet-
hyl-8-nonenal
[0238] 63
[0239] A mixture of
(4R,5S,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxyca-
rbonyloxy)-2,2,4,6-tetramethyl-8-nonen-3-one (4.58 g) and
p-toluenesulfonic acid monohydrate (0.45 g) in 100 mL of 3:1
THF/water is heated at reflux for 7 hours. The mixture is cooled
and poured into sat. aq. NaHCO.sub.3, then extracted with ethyl
acetate. The extract is dried over Na.sub.2SO.sub.4, filtered, and
evaporated. The product is purified by chromatography on SiO.sub.2
(3% ethyl acetate/hexanes).
(d) tert-butyl
(3S,6R,7S,8S)-5-oxo-3-hydroxy-4,4,6,8-tetramethyl-7-(2,2,2--
trichloroethoxycarbonyloxy)-10-undecenoate
[0240] 64
[0241] Tert-butyl acetate (0.865 mL) is added to a solution of
lithium diisopropylamide (7.52 mmol) in 30 mL of ether at
-78.degree. C., and the mixture is stirred for 1 hour. A solution
of bis(1,2:5,6-di-O-isopropylid-
ene-.alpha.-L-glucofuranos-3-O-yl)cyclopentadienyltitanium chloride
(8.34 mmol) in 90 mL of ether is added dropwise over 40 minutes,
and the reaction is stirred for an additional 30 minutes at
-78.degree. C., warmed to -30.degree. C. and kept for 45 minutes,
then recooled to -78.degree. C. A solution of
(4R,5S,6S)-3-oxo-5-(2,2,2-trichloroethoxycar-
bonyloxy)-2,2,4,6-tetramethyl-8-nonenal (2.57 g) in 15 mL of ether
is added over 10 minutes and the reaction is continued for 2 hours
before addition of 14 mL of 5 M water in THF. The mix is stirred
for 1 hour, then filtered through Celite. The filtrate is washed
with sat. aq. NaCl, and the brine layer is back extracted with
ether. The organic phases are combined, dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by chromatography on SiO.sub.2 (7% ethyl acetate/hexanes).
(e) Tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethy-
l-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
[0242] A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-hydroxy-4,4,6,8-tetr-
amethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate (1.8
g), imidazole (0.48 g), and triethylsilyl chloride (0.68 g) in 5 mL
of dimethylformamide is stirred for 2 hours at ambient temperature,
then poured into water and extracted with ether. The extract is
washed with sat. aq. NaCl, dried over MgSO.sub.4, filtered, and
evaporated. The product is purified by chromatography on SiO.sub.2
(20:1 toluene/ethyl acetate).
EXAMPLE 11
Tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,6,8,10-pentamethyl-
-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
[0243] 65
[0244] Prepared according to the method of Example 10, substituting
(2S)-2,4-dimethyl -4-pentenal in place of
(2S)-2-methyl-4-pentenal.
EXAMPLE 12
Tert-butyl
(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4.6,8-pentameth-
yl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
[0245] 66
(a)
(4S)-3-[(2S,3S,6R,7S,8S)-5-oxo-3-hydroxy-2,4,4,6,8-pentamethyl-7-(2,2,-
2-trichloroethoxycarbonyloxy)-10-undecenoyl]-4-benzyloxazolidinone
[0246] 67
[0247] A solution of (4S)-3-propionyl-4-benzyloxazolidinone (91
mmol) in 200 mL of CH.sub.2Cl.sub.2 at 0.degree. C. is treated
sequentially with dibutylboron triflate (27 mL) and triethylamine
(16.7 mL) at such a rate so as to keep the reaction temperature
below 3.degree. C. After stirring for 30 minutes, the mixture is
cooled to -78.degree. C. and treated with a solution of
(4R,5S,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,4-
,6-tetramethyl-8-nonenal (90 mmol) in 10 mL of CH.sub.2Cl.sub.2.
After 1 hour, the mixture is allowed to warm slowly to 0.degree. C.
and stirred an additional 1 hour. The reaction is quenched by
addition of 100 mL of pH 7 phosphate buffer and 300 mL of methanol.
A mixture of 2:1 methanol/30% aq. H.sub.2O.sub.2 (300 mL) is then
added while maintaining the temperature below 10.degree. C. After
stirring for 1 hour, the mixture is concentrated under vacuum to a
slurry, which is extracted with ether. The extract is washed
sequentially with 5% NaHCO.sub.3 and brine, dried over MgSO.sub.4,
filtered, and evaporated. The product is isolated by silica gel
chromatography.
(b)
(4S)-3-[(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4,6,8-pentamet-
hyl-7-(2,2,2)-trichloroethoxycarbonyloxy)-10-undecenoyl]-4-benzyloxazolidi-
none
[0248] 68
[0249] A solution of
(4S)-3-[(2S,3S,6R,7S,8S)-5-oxo-3-hydroxy-2,4,4,6,8-pe-
ntamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoyl]-4-benzyloxaz-
olidinone (2.6 g), imidazole (0.48 g), and triethylsilyl chloride
(0.68 g) in 5 mL of dimethylformamide is stirred for 2 hours at
ambient temperature, then poured into water and extracted with
ether. The extract is washed with sat. aq. NaCl, dried over
MgSO.sub.4, filtered, and evaporated. The product is purified by
chromatography on SiO.sub.2.
(c)
(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4,6,8-pentamethyl-7-(2-
,2,2-trichloroethoxycarbonyloxy)-10-undecenoic acid
[0250] 69
[0251] A solution of
(4S)-3-[(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2-
,4,4,6,8-pentamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoyl]-4-
-benzyloxazolidinone (21.5 g) in 125 mL of 4:1 THF/water is cooled
on ice and treated with 10.2 mL of 30% H.sub.2O.sub.2 followed by a
solution of lithium hydroxide (0.96 g) in 50 mL of water. After 1
hour, a solution of sodium sulfite (12.6 g) in 75 mL of water is
added, and the mixture is adjusted to pH 3 and concentrated under
vacuum. The resulting slurry is extracted with ethyl acetate. The
extract is washed with brine, dried over MgSO.sub.4, filtered, and
evaporated. The product is purified by chromatography on
SiO.sub.2.
(d) tert-butyl
(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4,6,8-penta-
methyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
[0252] A solution of
(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4,6,8-
-pentamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoic
acid (11.8 g), tert-butanol (4.5 g), and 4-dimethylaminopyridine
(0.2 g) in 20 mL of CH.sub.2Cl.sub.2 is cooled on ice and treated
with dicyclohexylcarbodiimide (4.6 g) over a 5-minute period. The
mixture is allowed to warm to ambient temperature and is stirred
for 3 hours. The slurry is diluted with CH.sub.2Cl.sub.2 and
filtered, and the filtrate is washed sequentially with sat. aq.
citric acid, 5% NaHCO.sub.3, and brine. The solution is dried over
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by chromatography on SiO.sub.2.
EXAMPLE 13
Tert-butyl
(3S,7R,8S)-5-oxo-3-(triethylsilyloxy)-4,4,8-trimethyl-7-(2,2,2--
trichloroethoxycarbonyloxy)-10-undecenoate
[0253] 70
(a)
(5R,6S)-1,1-diisopropoxy-5-hydroxy-2,2,6-trimethyl-8-nonen-3-one
[0254] 71
[0255] A solution of 1,1-diisopropoxy-2,2,-dimethyl-3-butanone
(3.08 g) in 15 mL of ether is added slowly to a solution of lithium
diisopropylamide (15.7 mmol) in 20 mL of ether cooled to
-78.degree. C., the mixture is stirred for 30 minutes, warmed to
-40.degree. C. and stirred for 30 minutes, then recooled to
-78.degree. C. A solution of
bis(1,2:5,6-di-O-isopropylidene-.alpha.-D-glucofuranos-3-O-yl)cyclopentad-
ienyltitanium chloride (17.4 mmol) in 180 mL of ether is added
dropwise over 40 minutes, and the reaction is stirred for an
additional 30 minutes at -78.degree. C., warmed to -30.degree. C.
and kept for 45 minutes, then recooled to -78.degree. C. A solution
of (2S)-2-methyl-4-pentenal (16.36 mmol) in 30 mL of ether is added
over 10 minutes and the mixture is stirred for 2 hours at
-78.degree. C. A 5 M solution of water in THF (28 mL) is added, the
mixture is stirred for 1 hour and then filtered through Celite. The
filtrate is washed with sat. aq. NaCl, and the brine layer is back
extracted with ether. The organic phases are combined, dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by chromatography on SiO.sub.2.
(b)
(5R,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,6-tr-
imethyl-8-nonen-3-one
[0256] 72
[0257] Trichloroethyl chloroformate (2.5 mL) and pyridine (2.95 mL)
are added to a solution of
(5R,6S)-1,1-diisopropoxy-5-hydroxy-2,2,6-trimethyl- -8-nonen-3-one
(2.9 g) in 40 mL of CH.sub.2Cl.sub.2 at 0.degree. C., and the
mixture is stirred for 5 hours before pouring into sat. aq. NaCl
and extracting with CH.sub.2Cl.sub.2. The extract is dried over
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by chromatography on SiO.sub.2.
(c)
(5R,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbonyloxy)-2,2,6-trimethyl-8-n-
onenal
[0258] 73
[0259] A mixture of
(5R,6S)-1,1-diisopropoxy-5-(2,2,2-trichloroethoxycarbo-
nyloxy)-2,2,6-trimethyl-8-nonen-3-one (4.3 g) and p-toluenesulfonic
acid monohydrate (0.45 g) in 100 mL of 3:1 THF/water is heated at
reflux for 7 hours. The mixture is cooled and poured into sat. aq.
NaHCO.sub.3, then extracted with ethyl acetate. The extract is
dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product
is purified by chromatography on SiO.sub.2.
(d) tert-butyl
(3S,7R,8S)-5-oxo-3-hydroxy-4,4,8-trimethyl-7-(2,2,2-trichlo-
roethoxy-carbonyloxy)-10-undecenoate
[0260] 74
[0261] Tert-butyl acetate (0.865 mL) is added to a solution of
lithium disopropylamide (7.52 mmol) in 30 mL of ether at
-78.degree. C., and the mixture is stirred for 1 hour. A solution
of bis(1,2:5,6-di-O-isopropylid-
ene-.alpha.-L-glucofuranos-3-O-yl)cyclopentadienyltitanium chloride
(8.34 mmol) in 90 mL of ether is added dropwise over 40 minutes,
and the reaction is stirred for an additional 30 minutes at
-78.degree. C., warmed to -30.degree. C. and kept for 45 minutes,
then recooled to -78.degree. C. A solution of
(5R,6S)-3-oxo-5-(2,2,2-trichloroethoxycarbon-
yl-oxy)-2,2,6-trimethyl-8-nonenal (2.4 g) in 15 mL of ether is
added over 10 minutes and the reaction is continued for 2 hours
before addition of 14 mL of 5 M water in THF. The mix is stirred
for 1 hour, then filtered through Celite. The filtrate is washed
with sat. aq. NaCl, and the brine layer is back extracted with
ether. The organic phases are combined, dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by chromatography on SiO.sub.2.
(e) tert-butyl
(3S,7R,8S)-5-oxo-3-(triethylsilyloxy)-4,4,8-trimethyl-7-(2,-
2,2-trichloroethoxy-carbonyloxy)-10-undecenoate
[0262] A solution of tert-butyl
(3S,7R,8S)-5-oxo-3-hydroxy-4,4,8-trimethyl-
-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate (1.7 g),
imidazole (0.48 g), and triethylsilyl chloride (0.68 g) in 5 mL of
dimethylformamide is stirred for 2 hours at ambient temperature,
then poured into water and extracted with ether. The extract is
washed with sat. aq. NaCl, dried over MgSO.sub.4, filtered, and
evaporated. The product is purified by chromatography on
SiO.sub.2.
EXAMPLE 14
Tert-butyl
(3S,6R,7S,8S-5-oxo-3-(triethylsilyloxy)-4,4,6,8-tetramethyl-7-(-
2,2,2-trichloroethoxycarbonyloxy)-10-undecynoate
[0263] 75
[0264] Prepared according to the method of Example 10, substituting
(2S)-2-methyl-4-propynal for (2S)-2-methyl-4-propenal.
EXAMPLE 15
Tert-butyl
(2S,3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-2,4,4,6,8-pentameth-
yl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecynoate
[0265] 76
[0266] Prepared according to the method of Example 12, substituting
(2S)-2-methyl-4-propynal for (2S)-2-methyl-4-propenal.
EXAMPLE 16
Tert-butyl
(3S,7S,8S)-5-oxo-3-(triethylsilyloxy)-4,4,8-trimethyl-7-(2,2,2--
trichloroethoxycarbonyloxy)-10-undecynoate
[0267] 77
[0268] Prepared according to the method of Example 13, substituting
(2S)-2-methyl-4-propynal for (2S)-2-methyl-4-propenal.
EXAMPLE 17
21-hydroxy-epothilone D (12,13-Desoxyepothilone F)
[0269] 78
(a) tert-butyl
(3S,6R,7S,8S,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilyloxy)--
17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16--
hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-12,16-dienoate
[0270] 79
[0271] A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethylsilyloxy)-4-
,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undecenoate
(2.12 g) in 4 mL of THF is added to a solution of
9-borabicyclononane dimer (490 mg) in 2 mL of THF, the mixture is
stirred for 1 hour at ambient temperature, and then water (0.25 ML)
is added to destroy excess 9-BBN. The resulting solution is added
to a vigorously stirred mixture of
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxymethyl)thiazo-
l-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene (2.0 g), cesium
carbonate (1.49 g), triphenylarsine (0.188 g), and
(dppf)PdCl.sub.2.CH.sub.2Cl.sub.- 2 (0.25 g) in 2 mL of degassed
dimethylformamide cooled to 0.degree. C. The reaction is stirred
for 15 hours, then poured into 10% NaHSO.sub.4 and extracted with
ethyl acetate. The organic phase is separated, washed sequentially
with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4, filtered,
and evaporated. The product is purified by flash chromatography on
SiO.sub.2 (10:1 hexanes/ethyl acetate).
(b)
(3S,6R,7S,8S,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-(2,-
2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hexamethy-
l-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-12,16-dienoic
acid
[0272] 80
[0273] A solution of tert-butyl
(3S,6R,7S,8S,12Z,15S,16E)-5-oxo-3,15-bis(t-
riethylsilyloxy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-y-
l)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-
-12,16-dienoate (2.86 g) in 12 mL of CH.sub.2Cl.sub.2 is treated
with 2,6-lutidine (0.86 mL) and triethylsilyl
trifluoromethanesulfonate (0.98 g) at 0.degree. C. for 30 minutes,
then at ambient temperature for 10 hours. The mixture is diluted
with 50 mL of ethyl acetate and poured into 20 mL of 1 N HCl. The
organic phase is separated, washed with pH 7 phosphate buffer,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
residue is dissolved in 5 mL of THF and treated with 0.5 mL of 0.1
N HCl in methanol. The reaction is monitored by thin-layer
chromatography, with additional aliquots of methanolic HCl being
added to achieve complete reaction. When complete, the reaction is
poured into 15 mL of pH 7 phosphate buffer and extracted with ethyl
acetate. The extract is washed with brine, dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by flash chromatography on SiO.sub.2 (1:1 hexanes/ethyl
acetate).
(c)
7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-epothil-
one D
[0274] 81
[0275] A solution of
(3S,6R,7S,8S,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)
-15-hydroxy-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4-
,4,6,8,12,16-hexamethyl
-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-12- ,16-dienoic
acid (0.426 g) in 9 mL of THF is treated with triethylamine (0.36
mL) and 2,4,6-trichlorobenzoyl chloride (0.528 g). After 15
minutes, 40 mL of toluene is added, and the resulting solution is
added via syringe pump over 3 hours to a solution of
4-dimethylaminopyridine (0.525 g) in 400 mL of toluene. After an
additional 1 hour, the mixture is filtered through Celite and
concentrated. The product is purified by flash chromatography on
SiO.sub.2 (2:1 hexanes/ethyl acetate).
(d) 21-hydroxy-3-O-(triethylsilyl)-epothilone D
[0276] 82
[0277] A solution
of7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(trieth-
ylsilyl)-epothilone D (0.196 g) in 1 mL of THF is added to a
stirred suspension of activated zinc dust (0.261 g) in 2 mL of
acetic acid. After stirring for 1.5 hours, the mixture is diluted
with ethyl acetate and filtered. The filtrate is washed
sequentially with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4,
filtered, and evaporated. The product is purified by flash
chromatography on SiO.sub.2 (2:1 hexanes/ethyl acetate).
(e) 21-hydroxy-epothilone D
[0278] A solution of21-hydroxy-3-O-(triethylsilyl)-epothilone D (82
mg) in 2 mL of THF in a polyethylene vessel and treated with 1.5 mL
of HF.pyridine for 1 hour at 0.degree. C. and 30 minutes at ambient
temperature, then diluted with 30 mL of ethyl acetate and poured
into 20 mL of sat. aq. NaHCO.sub.3. The organic phase is separated
and washed sequentially with 1 N HCl, 10% NaHCO.sub.3, and brine,
then dried over MgSO.sub.4, filtered, and evaporated. The product
is purified by flash chromatography on SiO.sub.2 (1:2 hexanes/ethyl
acetate).
EXAMPLE 18
10,11-dehydroepothilone D
[0279] 83
(a) tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilylo-
xy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroe-
thoxycarbonyloxy)-heptadeca-10,12,16-trienoate (Method A)
[0280] 84
[0281] Method A: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls-
ilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undec-
enoate (2.12 g) in 4 mL of THF is added to a vigorously stirred
mixture of
(5S)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-he-
ptadiene (1.4 g), cesium carbonate (1.49 g), triphenylarsine (0.188
g), and (dppf)PdCl.sub.2.CH.sub.2Cl.sub.2 (0.25 g) in 2 mL of
degassed dimethylformamide cooled to 0.degree. C. The reaction is
stirred for 15 hours, then poured into 10% NaHSO.sub.4 and
extracted with ethyl acetate. The organic phase is separated,
washed sequentially with 10% NaHCO.sub.3 and brine, dried over
MgSO.sub.4, filtered, and evaporated. The product is purified by
flash chromatography on SiO.sub.2 (10:1 hexanes/ethyl acetate).
[0282] Method B: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls- ilyloxy)
-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-unde-
cynoate (2.1 g) in 4 mL of THF is added to a 1.0 M solution of
catecholborane in THF (3.3 mL), the mixture is stirred for 2 hour
at 60.degree. C. The resulting solution is added to a vigorously
stirred mixture of
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxyme-
thyl)thiazol -4-yl)-5-(triethylsilyloxy)-2,6-heptadiene (2.0 g),
cesium carbonate (1.49 g), triphenylarsine (0.188 g), and
(dppf)PdCl.sub.2.CH.sub.2Cl.sub.2 (0.25 g) in 2 mL of degassed
dimethylformamide cooled to 0.degree. C. The reaction is stirred
for 15 hours, then poured into 10% NaHSO.sub.4 and extracted with
ethyl acetate. The organic phase is separated, washed sequentially
with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4, filtered,
and evaporated. The product is purified by flash chromatography on
SiO.sub.2 (10:1 hexanes/ethyl acetate).
(b)
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17-
-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxyc-
arbonyloxy)-heptadeca-10,12,16-trienoic acid
[0283] 85
[0284] A solution of tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-b-
is(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7--
(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate
(2.38 g) in 12 mL of CH.sub.2Cl.sub.2 is treated with 2,6-lutidine
(0.86 mL) and triethylsilyl trifluoromethanesulfonate (0.98 g) at
0.degree. C. for 30 minutes, then at ambient temperature for 10
hours. The mixture is diluted with 50 mL of ethyl acetate and
poured into 20 mL of 1 N HCl. The organic phase is separated,
washed with pH 7 phosphate buffer, dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The residue is dissolved in 5 mL of THF
and treated with 0.5 mL of 0.1 N HCl in methanol. The reaction is
monitored by thin-layer chromatography, with additional aliquots of
methanolic HCl being added to achieve complete reaction. When
complete, the reaction is poured into 15 mL of pH 7 phosphate
buffer and extracted with ethyl acetate. The extract is washed with
brine, dried with Na.sub.2SO.sub.4, filtered, and evaporated. The
product is purified by flash chromatography on SiO.sub.2 (1:1
hexanes/ethyl acetate).
(c)
7-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-dehydroe-
pothilone D
[0285] 86
[0286] A solution of
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilylo-
xy)-15-hydroxy-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-
-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoic acid (0.4
g) in 9 mL of THF is treated with triethylamine (0.36 mL) and
2,4,6-trichlorobenzoyl chloride (0.528 g). After 15 minutes, 40 mL
of toluene is added, and the resulting solution is added via
syringe pump over 3 hours to a solution of 4-dimethylaminopyridine
(0.525 g) in 400 mL of toluene. After an additional 1 hour, the
mixture is filtered through Celite and concentrated. The product is
purified by flash chromatography on SiO.sub.2 (2:1 hexanes/ethyl
acetate).
(d) 3-O-(triethylsilyl)-10,11-dehydroepothilone D
[0287] 87
[0288] A solution
of7-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl-
)-10,11-dehydroepothilone D (0.18 g) in 1 mL of THF is added to a
stirred suspension of activated zinc dust (0.261 g) in 2 mL of
acetic acid. After stirring for 1.5 hours, the mixture is diluted
with ethyl acetate and filtered. The filtrate is washed
sequentially with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4,
filtered, and evaporated. The product is purified by flash
chromatography on SiO.sub.2 (2:1 hexanes/ethyl acetate).
(e) 10,11-dehydroepothilone D
[0289] A solution of 3-O-(triethylsilyl)-10,11-dehydroepothilone D
(80 mg) in 2 mL of THF in a polyethylene vessel and treated with
1.5 mL of HF.pyridine for 1 hour at 0.degree. C. and 30 minutes at
ambient temperature, then diluted with 30 mL of ethyl acetate and
poured into 20 mL of sat. aq. NaHCO.sub.3. The organic phase is
separated and washed sequentially with 1 N HCl, 10% NaHCO.sub.3,
and brine, then dried over MgSO.sub.4, filtered, and evaporated.
The product is purified by flash chromatography on SiO.sub.2 (1:2
hexanes/ethyl acetate).
EXAMPLE 19
21-hydroxy-10,11-dehydro-epothilone D
[0290] 88
(a) tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-bis(triethylsilylo-
xy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12-
,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-tri-
enoate
[0291] 89
[0292] Method A: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls-
ilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undec-
enoate (2.12 g) in 4 mL of THF is added to a vigorously stirred
mixture of
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxymethyl)thiazo-
l-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene (2.0 g), cesium
carbonate (1.49 g), triphenylarsine (0.188 g), and
(dppf)PdCl.sub.2.CH.sub.2Cl.sub.- 2 (0.25 g) in 2 mL of degassed
dimethylformamide cooled to 0.degree. C. The reaction is stirred
for 15 hours, then poured into 10% NaHSO.sub.4 and extracted with
ethyl acetate. The organic phase is separated, washed sequentially
with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4, filtered,
and evaporated. The product is purified by flash chromatography on
SiO.sub.2 (10:1 hexanes/ethyl acetate).
[0293] Method B: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls-
ilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undec-
ynoate (2.1 g) in 4 mL of THF is added to a 1.0 M solution of
catecholborane in THF (3.3 mL), the mixture is stirred for 2 hour
at 60.degree. C. The resulting solution is added to a vigorously
stirred mixture of
(5S)-2-iodo-6-methyl-7-(2-(2,2,2-trichloroethoxycarbonyl-oxyme-
thyl)thiazol-4-yl)-5-(triethylsilyloxy)-2,6-heptadiene (2.0 g),
triphenylarsine (0.188 g), and (dppf)PdCl.sub.2.CH.sub.2Cl.sub.2
(0.25 g) in 2 mL of degassed dimethylformamide cooled to 0.degree.
C. The reaction is stirred for 15 hours, then poured into 10%
NaHSO.sub.4 and extracted with ethyl acetate. The organic phase is
separated, washed sequentially with 10% NaHCO.sub.3 and brine,
dried over MgSO.sub.4, filtered, and evaporated. The product is
purified by flash chromatography on SiO.sub.2 (10:1 hexanes/ethyl
acetate).
(b)
(3S,6R,7S,8S,10E,12Z,5S,16E)-5-oxo-3-(triethylsilyloxy)-15-hydroxy-17--
(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-4,4,6,8,12,16-hex-
amethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoic
acid
[0294] 90
[0295] A solution of tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3,15-b-
is(triethylsilyloxy)-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-
-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-hepta-
deca-10,12,16-trienoate (2.8 g) in 12 mL of CH.sub.2Cl.sub.2 is
treated with 2,6-lutidine (0.86 mL) and triethylsilyl
trifluoromethanesulfonate (0.98 g) at 0.degree. C. for 30 minutes,
then at ambient temperature for 10 hours. The mixture is diluted
with 50 mL of ethyl acetate and poured into 20 mL of 1 N HCl. The
organic phase is separated, washed with pH 7 phosphate buffer,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
residue is dissolved in 5 mL of THF and treated with 0.5 mL of 0.1
N HCl in methanol. The reaction is monitored by thin-layer
chromatography, with additional aliquots of methanolic HCl being
added to achieve complete reaction. When complete, the reaction is
poured into 15 mL of pH 7 phosphate buffer and extracted with ethyl
acetate. The extract is washed with brine, dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by flash chromatography on SiO.sub.2 (1:1 hexanes/ethyl
acetate).
(c)
7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triethylsilyl)-10,11-d-
ehydro-epothilone D
[0296] 91
[0297] A solution of
(3S,6R,7S,8S,10E,12Z,15S,16E)-5-oxo-3-(triethylsilylo-
xy)-15-hydroxy-17-(2-(2,2,2-trichloroethoxycarbonyloxymethyl)thiazol-4-yl)-
-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-1-
0,12,16-trienoic acid (0.42 g) in 9 mL of THF is treated with
triethylamine (0.36 mL) and 2,4,6-trichlorobenzoyl chloride (0.528
g). After 15 minutes, 40 mL of toluene is added, and the resulting
solution is added via syringe pump over 3 hours to a solution of
4-dimethylaminopyridine (0.525 g) in 400 mL of toluene. After an
additional 1 hour, the mixture is filtered through Celite and
concentrated. The product is purified by flash chromatography on
SiO.sub.2 (2:1 hexanes/ethyl acetate).
(d) 21-hydroxy-3-O-(triethylsilyl)-10,11-dehydro-epothilone D
[0298] 92
[0299] A solution of
7,21-bis-O-(2,2,2-trichloroethoxycarbonyl)-3-O-(triet-
hylsilyl)-10,11-dehydro-epothilone D (0.19 g) in 1 mL of THF is
added to a stirred suspension of activated zinc dust (0.261 g) in 2
mL of acetic acid. After stirring for 1.5 hours, the mixture is
diluted with ethyl acetate and filtered. The filtrate is washed
sequentially with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4,
filtered, and evaporated. The product is purified by flash
chromatography on SiO.sub.2 (2:1 hexanes/ethyl acetate).
(e) 21-hydroxy-10,11-dehydro-epothilone D
[0300] A solution of
21-hydroxy-3-O-(triethylsilyl)-10,11-dehydro-epothilo- ne D (80 mg)
in 2 mL of THF in a polyethylene vessel and treated with 1.5 mL of
HF-pyridine for 1 hour at 0.degree. C. and 30 minutes at ambient
temperature, then diluted with 30 mL of ethyl acetate and poured
into 20 mL of sat. aq. NaHCO.sub.3. The organic phase is separated
and washed sequentially with 1 N HCl, 10% NaHCO.sub.3, and brine,
then dried over MgSO.sub.4, filtered, and evaporated. The product
is purified by flash chromatography on SiO.sub.2 (1:2 hexanes/ethyl
acetate).
EXAMPLE 20
21-fluoro-10,11-dehydroepothilone D
[0301] 93
(a) 21-(p-toluenesulfonyloxy)-10,11-dehydroepothilone D
[0302] 94
[0303] A solution of 21-hydroxy-10,11-dehydro-epothilone D (40 mg)
in 2 mL of CH.sub.2Cl.sub.2 is treated with pyridine (1 mL),
4-dimethylaminopyridine (1 mg), and p-toluenesulfonyl chloride (24
mg) at 0.degree. C. for 30 minutes. The reaction is diluted with
ethyl acetate and washed sequentially with 1 N HCl, sat.
NaHCO.sub.3, and brine, then dried over MgSO.sub.4, filtered, and
evaporated. The product is purified by SiO.sub.2 chromatography
(40% ethyl acetate/hexanes).
(b) 21-fluoro-10,11-dehydroepothilone D
[0304] A solution of
21-(p-toluenesulfonyloxy)-10,11-dehydroepothilone D (66 mg) in 5 mL
of acetonitrile is treated with tetrabutylammonium
(triphenylsilyl)difluorosilicate (216 mg) at reflux for 10 hours.
The mixture is cooled and evaporated, and the residue is
chromatographed on SiO.sub.2 (40% ethyl acetate/hexanes) to provide
the product.
EXAMPLE 21
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-5,5,7,9,13-pentamethyl-1-
5-(6-quinolyl)-1-oxacyclodeca-11,13-diene
[0305] 95
[0306] Is prepared according to the method of Example 18 replacing
(6S)-2-iodo-5-(6-quinolyl)-5-(triethylsilyloxy)-2-pentene for
(5S)-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-5-(triethylsilyloxy)-2,6-he-
ptadiene.
EXAMPLE 22
12,13-methylene-10,11-dehydroepothilone D
[0307] 96
(a) 3,7-bis-O-(tert-butyldimethylsilyl)-10,11-anhydroepothilone
D
[0308] 97
[0309] A solution of 10,11-dehydroepothilone D (530 mg) and
2,6-lutidine (0.61 mL) in 30 mL of CH.sub.2Cl.sub.2 at 0.degree. C.
is treated with tert-butyldimethylsilyl triflate (0.9 mL) for 1
hour, then queched by pouring into sat. NaHCO.sub.3 and extracted
with CH.sub.2Cl.sub.2. The extract is dried over Na.sub.2SO.sub.4,
filtered, and concentrated. The product is purified by SiO.sub.2
chromatography (10% ethyl acetate/hexanes).
(b)
12,13-(dibromomethylene)-3,7-bis-O-(tert-butyldimethylsilyl)-10,11-deh-
ydroepothilone D
[0310] 98
[0311] Benzyltriethylammonium chloride (7 mg), ethanol (20 uL), and
50% aq. NaOH are added sequentially to a solution of
3,7-bis-O-(tert-butyldim- ethylsilyl)-10,11-dehydroepothilone D
(230 mg) in 2.7 mL of CHBr.sub.3, and the mixture is stirred
vigorously at 45.degree. C. for 18 hours. The reaction is cooled to
ambient temperature, poured into sat. NH.sub.4Cl, and extracted
with CH.sub.2Cl.sub.2. The extract is dried over Na.sub.2SO.sub.4,
filtered, and concentrated. The product is purified by SiO.sub.2
chromatography (5% ethyl acetate/hexanes).
(c)
12,13-methylene-3,7-bis-O-(tert-butyldimethylsilyl)-10,11-dehydroepoth-
ilone D
[0312] 99
[0313] A solution of
12,13-(dibromomethylene)-3,7-bis-O-(tert-butyldimethy-
lsilyl)-10,11-dehydroepothilone D (30 mg) in 0.5 mL of hexane is
treated with tributyltin hydride (80 uL) and azobisisobutyronitrile
(1 mg) at reflux for 9 hours. The mixture is cooled, concentrated,
and chromatographed on SiO.sub.2 to yield the product.
(d) 12,13-methylene-10,11-dehydroepothilone D
[0314] A solution of
12,13-methylene-3,7-bis-O-(tert-butyldimethylsilyl)-1-
0,11-dehydroepothilone D (73 mg) in 1 mL of THF is treated with 0.5
mL of a 1 M solution of tetrabutylammonium fluoride in THF for 1
hour. The mixture is diluted with ethyl acetate, washed
sequentially with water and brine, then dried over
Na.sub.2SO.sub.4, filtered, and evaporated. The product is purified
by SiO.sub.2 chromatography.
EXAMPLE 23
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-5,5,7,9,13-pentamethyl-1-
5-methylthiazol-4-yl)propen-2-yl)-1-azacyclohexadeca-11,13-diene
[0315] 100
(a) tert-butyl
(3S,6R,7S,8S,10E,12Z,5S,16E)-15-azido-5-oxo-3-(triethylsily-
loxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichlor-
oethoxycarbonyloxy)-heptadeca-10,12,16-trienoate (Method A)
[0316] 101
[0317] Method A: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls-
ilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undec-
enoate (2.12 g) in 4 mL of THF is added to a vigorously stirred
mixture of
(5S)-5-azido-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-heptadiene
(1.1 g), cesium carbonate (1.49 g), triphenylarsine (0.188 g), and
(dppf)PdCl2-CH.sub.2Cl.sub.2 (0.25 g) in 2 mL of degassed
dimethylformamide cooled to 0.degree. C. The reaction is stirred
for 15 hours, then poured into 10% NaHSO.sub.4 and extracted with
ethyl acetate. The organic phase is separated, washed sequentially
with 10% NaHCO.sub.3 and brine, dried over MgSO.sub.4, filtered,
and evaporated. The product is purified by flash chromatography on
SiO.sub.2 (10:1 hexanes/ethyl acetate).
[0318] Method B: A solution of tert-butyl
(3S,6R,7S,8S)-5-oxo-3-(triethyls-
ilyloxy)-4,4,6,8-tetramethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-10-undec-
ynoate (2.1 g) in 4 mL of THF is added to a 1.0 M solution of
catecholborane in THF (3.3 mL), the mixture is stirred for 2 hour
at 60.degree. C. The resulting solution is added to a vigorously
stirred mixture of
(5S)-5-azido-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-hept-
adiene (1.1 g), cesium carbonate (1.49 g), triphenylarsine (0.188
g), and (dppf)PdCl.sub.2.CH.sub.2Cl.sub.2 (0.25 g) in 2 mL of
degassed dimethylformamide cooled to 0.degree. C. The reaction is
stirred for 15 hours, then poured into 10% NaHSO.sub.4 and
extracted with ethyl acetate. The organic phase is separated,
washed sequentially with 10% NaHCO.sub.3 and brine, dried over
MgSO.sub.4, filtered, and evaporated. The product is purified by
flash chromatography on SiO.sub.2.
(b) tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-(tert-butoxycarbonylamino)-
-5-oxo-3-(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexame-
thyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate
[0319] 102
[0320] A solution of tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-azido-5-o-
xo-3-(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-
-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate
(4.1 g) in 100 mL of THF is treated with triphenylphosphine (2.81
g) at 40.degree. C. for 19 hours, then treated with 2 mL of water
and heated at 65.degree. C. for 4 hours. Silica gel (70 g) is added
and the mixture is concentrated under vacuum. Silica gel
chromatography (1.5% methanol in CHCl.sub.3+0.5% Et.sub.3N) yields
the intermediate amine. The amine (3.3 g) is dissolved in 100 mL of
acetonitrile and treated with tert-butyl pyrocarbonate (1.37 g) and
triethylamine (0.91 g) for 16 hours at ambient temperature. The
solution is diluted with ethyl acetate and washed sequentially with
1 N HCl, sat. NaHCO.sub.3, and brine, then dried over MgSO.sub.4,
filtered, and evaporated. SiO.sub.2 chromatography provides the
product.
(c)
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-amino-3-hydroxy-5-oxo-3-yl)-4,4,6,8,1-
2,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-tr-
ienoic acid
[0321] 103
[0322] A solution of tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-(tert-but-
oxycarbonylamino)-5-oxo-3-(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4-
,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12-
,16-trienoate (0.8 g) in 20 mL of CH.sub.2Cl.sub.2 is treated with
trifluoroacetic acid (10 mL) at ambient temperature for 2 hours,
then is concentrated. The crude product is used without
purification.
(d)
(4S,7R,8S,9S,11E,12Z,15S)-4-hydroxy-2,6-dioxo-5,5,7,9,13-pentamethyl-1-
5-methylthiazol-4-yl)propen-2-yl)-8-(2,2,2-trichloroethoxycarbonyloxy)-1-a-
zacyclohexadeca-11,13-diene
[0323] 104
[0324] The crude product from step (c) above is dissolved in 10 mL
of dimethylformamide and then diluted with 500 mL of
CH.sub.2Cl.sub.2. This is treated with 1-hydroxy-7-azabenzotriazole
(0.36 g), diisopropylethylamine (1.02 g), and
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-- tetramethyluronium
hexafluorophosphate (1.03 g) for 16 hours at ambient temperature.
The mixture is washed with water, then dried over MgSO.sub.4,
filtered, and evaporated. The residue is dissolved in 30 mL of
acetic acid/THF/water (3:1:1) for 30 minutes, then evaporated,
neutralized with NaHCO.sub.3, and extracted with ethyl acetate. The
extract is dried over MgSO.sub.4, filtered, and evaporated. The
product is isolated by SiO.sub.2 chromatography.
(e)
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-5,5,7,9,13-pentameth-
yl-15-(1-(2-methylthiazol-4-yl)propen-2-yl)-1-azacyclohexadeca-11,13-diene
[0325] A solution of
(4S,7R,8S,9S,11E,12Z,15s)-4-hydroxy-2,6-dioxo-5,5,7,9-
,13-pentamethyl-15-(1-(2-methylthiazol-4-yl)propen-2-yl)-8-(2,2,2-trichlor-
oethoxycarbonyloxy)-1-azacyclohexadeca-11,13-diene (0.18 g) in 1 mL
of THF is added to a stirred suspension of activated zinc dust
(0.261 g) in 2 mL of acetic acid. After stirring for 1.5 h, the
mixture is diluted with ethyl acetate and filtered. The filtrate is
washed sequentially with 10% NaHCO.sub.3 and brine, dried over
MgSO.sub.4, filtered, and evaporated. The product is purified by
flash chromatography on SiO.sub.2.
EXAMPLE 24
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-5,5,7,9,13-pentamethyl
-15-1-(2-methylthiazol-4-yl)propen-2-yl)-1-azacyclohexadeca-11,13-diene
[0326] 105
[0327] Is prepared according to the method of Example 23,
substituting (5S)-5-azido-2-iodo-5-(6-quinolyl)-2-pentene in place
of
(5S)-5-azido-2-iodo-6-methyl-7-(2-methylthiazol-4-yl)-2,6-heptadiene.
EXAMPLE 25
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-1,5,5,7,9,13-hexamethyl--
15-(1-(2-methylthiazol-4-yl)propen-2-yl)-1-azacyclohexadeca-11,13-diene
[0328] 106
(a)
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-(methylamino)-3-hydroxy-5-oxo-3-17-(2-
-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-7-(2,2,2-trichloroethoxycarb-
onyloxy)-heptadeca-10,12,16-trienoic acid
[0329] 107
[0330] A solution of tert-butyl
(3S,6R,7S,8S,10E,12Z,15S,16E)-15-amino-5-o-
xo-3-(triethylsilyloxy)-17-(2-methylthiazol-4-yl)-4,4,6,8,12,16-hexamethyl-
-7-(2,2,2-trichloroethoxycarbonyloxy)-heptadeca-10,12,16-trienoate
(0.68 g) in 5 mL of methanol is treated with acetaldehyde (50 mg),
acetic acid (60 mg), and sodium cyanoborohydride (200 mg) for 12
hours at ambient temperature. The mixture is evaporated to dryness.
The crude product is used without purification.
(b)
(4S,7R,8S,9S,11E,12Z,15S)-4-hydroxy-2,6-dioxo-1,5,5,7,9,13-hexamethyl--
15-(1-(2-methylthiazol-4-yl)propen-2-yl)-8-(2,2,2-trichloroethoxycarbonylo-
xy)-1-azacyclohexadeca-11,13-diene
[0331] 108
[0332] The crude product from step (a) above is dissolved in 10 mL
of dimethylformamide and then diluted with 500 mL of
CH.sub.2Cl.sub.2. This is treated with 1-hydroxy-7-azabenzotriazole
(0.36 g), diisopropylethylamine (1.02 g), and
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-- tetramethyluronium
hexafluorophosphate (1.03 g) for 16 hours at ambient temperature.
The mixture is washed with water, then dried over MgSO.sub.4,
filtered, and evaporated. The residue is dissolved in 30 mL of
acetic acid/THF/water (3:1:1) for 30 minutes, then evaporated,
neutralized with NaHCO.sub.3, and extracted with ethyl acetate. The
extract is dried over MgSO.sub.4, filtered, and evaporated. The
product is isolated by SiO.sub.2 chromatography.
(c)
(4S,7R,8S,9S,11E,12Z,15S)-4,8-dihydroxy-2,6-dioxo-1,5,5,7,9,13-hexamet-
hyl-(2-methylthiazol-4-yl)propen-2-yl)-1-azacyclohexadeca-11,13-diene
[0333] A solution of
(4S,7R,8S,9S,11E,12Z,15S)-4-hydroxy-2,6-dioxo-1,5,5,7-
,9,13-hexamethyl-15-(1-(2-methylthiazol-4-yl)propen-2-yl)-8-(2,2,2-trichlo-
roethoxycarbonyloxy)-1-azacyclohexadeca-11,13-diene (0.18 g) in 1
mL of THF is added to a stirred suspension of activated zinc dust
(0.261 g) in 2 mL of acetic acid. After stirring for 1.5 hours, the
mixture is diluted with ethyl acetate and filtered. The filtrate is
washed sequentially with 10% NaHCO.sub.3 and brine, dried over
MgSO.sub.4, filtered, and evaporated. The product is purified by
flash chromatography on SiO.sub.2.
EXAMPLE 26
[0334] Microbial Transformation of C-20 Methyl to C-20
Hydroxymethyl
[0335] This example describes the microbial transformation of C-20
methyl to C-20 hydroxymethyl of 10,11-dehydroepothilone D. A frozen
vial (approximately 2 ml) of Amycolata autotrophica ATCC 35203 or
Actinomyces sp. strain PTA-XXX as described by PCT Publication No.
WO 00/39276 is used to inoculate 1 500 ml flask containing 100 mL
of medium. The vegetative medium consists of 10 g of dextrose, 10 g
of malt extract, 10 g of yeast extract, and 1 g of peptone in liter
of deionized water. The vegetative culture is incubated for three
days at 28.degree. C. on a rotary shaker operating at 250 rpm. One
mL of the resulting culture is added to each of sixty-two 500 mL
flasks containing the transformation medium which as the same
composition as the vegetative medium. The cultures are incubated at
28.degree. C. and 250 rpm for 24 hours. 10, 11-dehydroepothilone D
is dissolved in 155 ml of ethanol and the solution is distributed
to the sixty-two flasks. The flasks are then returned to the shaker
and incubated for an additional 43 hours at 28.degree. C. and 250
rpm. The reaction culture is then processed to recover
21-hydroxy-10,11-dehydroepothilone D (which also may be referred to
as 10,11-dehydro-12, 13-desoxy epothilone F).
EXAMPLE 27
[0336] Epoxidation Using EpoK
[0337] This example describes the enzymatic epoxidation of
10,11-dehydroepothilone D using EpoK to convert
10,11-dehydroepothilone D into 10,11-dehydroepothilone B. Briefly,
the epoK gene product was expressed in E. coli as a fusion protein
with a polyhistidine tag (his tag) and purified as described by PCT
publication, WO 00/31247 which is incorporated herein by reference.
The reaction consists of 50 mM Tris (pH7.5), 21 .mu.M spinach
ferredoxin, 0.132 units of spinach ferredoxin: NADP.sup.+
oxidoreductase, 0.8 units of glucose-6-phosphate dehydrogenase, 1.4
mM NADP, and 7.1 mM glucose-6-phosphate, 100 .mu.M or 200 .mu.M
10,11-dehydroepothilone D, and 1.7 pM amino terminal histidine
tagged EpoK or 1.6 .mu.M carboxy terminal histidine tagged EpoK in
a 100 .mu.L volume. The reactions are incubated at 30.degree. C.
for 67 minutes and stopped by heating at 90.degree. C. for 2
minutes. The insoluble material is removed by centrifugation, and
50 .mu.L of the supernatant containing the desired product is
analyzed by LC/MS.
EXAMPLE 28
[0338] Liposomal Composition
[0339] This example describes liposomal compositions containing 10,
11-dehydroepothilone D. A mixture of lipids and
10,11-dehydroepothilone D are dissolved in ethanol and the solution
is dried as a thin film by rotation under reduced pressure. The
resultant lipid film is hydrated by addition of the aqueous phase
and the particle size of the epothilone-derivative containing
liposomes is adjusted to the desired range. Preferably, the mean
particle diameter is less than 10 microns, preferably from about
0.5 to about 4 microns. The particle size may be reduced to the
desired level, for example, by using mills (e.g., air-jet mill,
ball mill, or vibrator mill), microprecipitation, spray-drying,
lyophillization, high-pressure homogenization, recrystrytallization
from supercritical media, or by extruding an aqueous suspension of
the liposomes through a series of membranes (e.g., polycarbonate
membranes) having a selected uniform pore size. In one embodiment,
the liposomal composition comprises: an inventive compound (1.00
mg); phosphatidylcholine (16.25 mg); cholesterol (3.75 mg);
polyethyleneglycol derivatized distearyl phosphatidylethanolamine
(5.00 mg); lactose (80.00 mg); citric acid (4.20 mg); tartaric acid
(6.00 mg); NaOH (5.44 mg); water (up to 1 mL). In another
embodiment, the liposomal composition comprises: an inventive
compound (1.00 mg); phosphatidylcholine (19.80 mg); cholesterol
(3.75 mg); distearyl phosphatidylcholine (1.45 mg); lactose (80.00
mg); citric acid (4.20 mg); tartaric acid (6.00 mg); NaOH (5.44
mg); water (up to 1 mL). In yet another embodiment, the liposomal
composition comprises: an inventive compound (1.00 mg);
1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (17.50 mg);
1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol, Na (7.50 mg);
lactose (80 mg); citric acid (4.20 mg); tartaric acid (6.00 mg);
NaOH (5.44 mg); water (up to 1 mL). Liposomal compositions
containing other compounds of the present invention are prepared
using conditions similar to those described above.
EXAMPLE 29
[0340] This example describes the preparation of a poly-glutamic
acid-21-hydroxy-10,11-dehydroepothilone D conjugate.
Poly(1-glutamic acid) ("PG") sodium salt (MW 34 K, Sigma, 0.35 g)
is dissolved in water. The pH of the queous solution is adjusted to
2 using 0.2 M HCl. The precipitate is collected, dialyzed against
distilled water, and lyophilized to yile 0.29 g of PG. To a
solution of PG (75 mg, repeating unit FW 170, 0.44 mmol) in dry DME
(1.5 mL) is added 20 mg of 21-hydroxy-10, 11-dehydroepothilone D,
15 mg of dicyclohexylcarbodiimide ("DCC") and a trace amount of
dimethylaminopyridine. The reaction is allowed to proceed at room
temperature for four hours or until completed as indicated by thin
layer chromatography. The reaction mixture is poured into
chloroform and the resulting precipitate is collected and dried in
a vacuum to yield approximately 65 mg of
PG-21-hydroxy-10,11-dehydroepothil- one D conjugate. Changing the
weight ratio of inventive compound to PG in the starting materials
results in polymeric conjugates of various concentrations of
21-hydroxyl-10, 11-dehydroepothilone D. Conjugates of other
compounds of the present invention are prepared using conditions
similar to those described above.
EXAMPLE 30
[0341] Intravenous Formulation
[0342] This example describes an intravenous formulation of
10,11-dehydroepothilone D. The formulation contains 10 mg/mL of 10,
11-dehydroepothilone D in a vehicle containing 30% propylene
glycol, 20% Creomophor EL, and 50% ethanol. The vehicle is prepared
by measuring ethanol (591.8 g) to a beaker containing a stir bar;
adding Creomophor EL (315.0 g) to the solution and mixing for ten
minutes; and then adding propylene glycol (466.2 g) to the solution
and mixing for another ten minutes. 10, 11-dehydroepothilone D (1
g) is added to a 1 L volumetric flask containing 400-600 mL of the
vehicle and mixed for five minutes. After 10, 11-dehydroepothilone
D is in solution, the volume is brought to 1 L; allowed to mix for
another ten minutes; and filtered through a 0.22 .mu.m Millipore
Millipak filter. The resulting solution is used to aseptically fill
sterile 5 mL vials using a metered peristaltic pump to a targeted
fill volume of 5.15 mL/vial. The filled vials are immediately
stoppered and crimped.
[0343] The vial containing 10 mg/mL of 10, 11-dehydroepothilone D
is diluted in normal saline or 5% dextrose solution for
administration to patients and administered in non-PVC, non-DEHP
bags and administration sets. The product is infused over a one to
six hour period to deliver the desired dose.
[0344] In one embodiment, the formulation is diluted twenty fold in
sterile saline prior to intravenous infusion. The final infusion
concentration is 0.5 mg/mL of the inventive compound, 1.5%
propylene glycol, 1% Chremophor EL, and 2.5% ethanol which is
infused over a one to six hour period to deliver the desired
dose.
[0345] Intravenous formulations containing other compounds of the
present invention may be prepared and used in a similar manner.
EXAMPLE 31
[0346] Pretreatment for Cremophor.RTM. Toxicity
[0347] This example describes a pretreatement regiment for for
Cremophor.RTM. toxicity. Formulations of 10, 11-dehydroepothilone D
or another compound of the invention that includes Cremophor.RTM.
may cause toxicity in patients. Pretreatment with steroids can be
used to prevent anaphylaxis. Any suitable corticosterioid or
combination of corticosteroid with H.sub.1 antagonists and/or
H.sub.2 antagonists may be used. In one embodiment, a subject is
premedicated with an oral dose of 50 mg of diphenylhydramine and
300 mg of cimetidine one hour prior to treatment with the inventive
compound in a Cremophor.RTM. containing formulation. In another
embodiment, the subject is premedicated with an intravenous
administration of 20 mg of dexamethasone at least one half hour
prior to treatment with the inventive compound in a Cremophor.RTM.
containing formulation. In another embodiment, the subject is
premedicated with an intravenous administration of 50 mg of
diphenylhydramine, 300 mg of cimetidine and 20 mg of dexamethasone
at least one half hour prior to treatment with the inventive
compound in a Cremophor.RTM. containing formulation. In yet another
embodiment, the weight of the subject is taken into account and the
subject is pretreated with an administration of diphenylhydramine
(5 mg/kg, i.v.); cimetidine (5 mg/kg, i.v).; and dexamethasone (1
mg/kg, i.m.) at least one half hour prior to the treatment with the
inventive compound in a Cremophor.RTM. containing formulation.
[0348] All scientific and patent publications referenced herein are
hereby incorporated by reference. The invention having now been
described by way of written description and example, those of skill
in the art will recognize that the invention can be practiced in a
variety of embodiments, that the foregoing description and example
is for purposes of illustration and not limitation of the following
claims.
* * * * *