U.S. patent application number 10/401494 was filed with the patent office on 2004-12-23 for synthesis of epothilones, intermediates thereto and analogues thereof.
Invention is credited to Balog, Aaron, Bertinato, Peter, Chou, Ting-Chao, Danishefsky, Samuel J., Kamenecka, Ted, Meng, DongFang, Savin, Kenneth A., Sorensen, Erik J., Su, Dai-Shi.
Application Number | 20040260098 10/401494 |
Document ID | / |
Family ID | 27578090 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040260098 |
Kind Code |
A1 |
Danishefsky, Samuel J. ; et
al. |
December 23, 2004 |
Synthesis of epothilones, intermediates thereto and analogues
thereof
Abstract
The present invention provides convergent processes for
preparing epothilone A and B, desoxyepothilones A and B, and
analogues thereof, useful in the treatment of cancer and cancer
which has developed a multidrug-resistant phenotype. Also provided
are intermediates useful for preparing said epothilones.
Inventors: |
Danishefsky, Samuel J.;
(Englewood, NJ) ; Bertinato, Peter; (Old Lyme,
CT) ; Su, Dai-Shi; (New York, NY) ; Meng,
DongFang; (New York, NY) ; Chou, Ting-Chao;
(Paramus, NJ) ; Kamenecka, Ted; (New York, NY)
; Sorensen, Erik J.; (San Diego, CA) ; Balog,
Aaron; (New York, NY) ; Savin, Kenneth A.;
(New York, NY) |
Correspondence
Address: |
Choate, Hall & Stewart
Exchange Place
53 State Street
Boston
MA
02109
US
|
Family ID: |
27578090 |
Appl. No.: |
10/401494 |
Filed: |
March 28, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10401494 |
Mar 28, 2003 |
|
|
|
10062376 |
Feb 1, 2002 |
|
|
|
6603023 |
|
|
|
|
10062376 |
Feb 1, 2002 |
|
|
|
09680493 |
Oct 5, 2000 |
|
|
|
09680493 |
Oct 5, 2000 |
|
|
|
09257072 |
Feb 24, 1999 |
|
|
|
6204388 |
|
|
|
|
09680493 |
Oct 5, 2000 |
|
|
|
08986025 |
Dec 3, 1997 |
|
|
|
6242469 |
|
|
|
|
60075947 |
Feb 25, 1998 |
|
|
|
60092319 |
Jul 9, 1998 |
|
|
|
60097733 |
Aug 24, 1998 |
|
|
|
60032282 |
Dec 3, 1996 |
|
|
|
60033767 |
Jan 14, 1997 |
|
|
|
60047566 |
May 22, 1997 |
|
|
|
60047941 |
May 29, 1997 |
|
|
|
60055533 |
Aug 13, 1997 |
|
|
|
Current U.S.
Class: |
546/281.7 ;
548/181; 548/215; 548/311.1; 548/465 |
Current CPC
Class: |
A61K 31/704 20130101;
A61K 31/704 20130101; C07D 493/04 20130101; C07D 313/00 20130101;
A61K 45/06 20130101; A61K 31/337 20130101; C07C 69/007 20130101;
C07D 277/24 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; C07F 7/1892 20130101; A61K 2300/00 20130101; A61K 31/337
20130101; A61K 31/425 20130101; C07C 69/738 20130101; C07F 7/1804
20130101; A61K 31/70 20130101; A61K 31/335 20130101; A61K 31/335
20130101; A61K 31/475 20130101; A61K 31/70 20130101; C07D 413/06
20130101; A61K 31/425 20130101; C07C 59/76 20130101; C07D 417/14
20130101; A61K 31/475 20130101; C07D 417/06 20130101 |
Class at
Publication: |
546/281.7 ;
548/181; 548/215; 548/311.1; 548/465 |
International
Class: |
C07D 417/02; C07D
413/02; C07D 45/02 |
Goverment Interests
[0002] This invention was made with government support under grants
CA-28824, CA-39821, CA-GM 72231, GM-18248, CA-62948, and AI0-9355
from the National Institutes of Health, and grant CHE-9504805 from
the National Science Foundation.
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 1997 |
WO |
PCT/US97/22381 |
Claims
What is claimed is:
1. A method of preparing a desoxyepothilone having the structure:
85wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CX--, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein Z is O,
N(OR.sub.3) or N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched alkyl; and
wherein n is 0, 1, 2, or 3; which comprises treating an epothilone
having a structure: 86wherein R, R.sub.0, R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R', R", X, Y, Z and n are defined as for
the desoxyepothilone, under suitable conditions so as to
deoxygenate the epothilone, and thereby to provide the
desoxyepothilone.
2. The method of claim 1 wherein desoxyepothilone has the
structure: 87wherein R is H, methyl, ethyl, n-propyl, n-butyl,
n-hexyl, 88or (CH.sub.2).sub.3--OH.
3. The method of claim 1 wherein the epothilone is deoxygenated
using a zinc/copper couple.
4. The method of claim 1 wherein the epothilone is deoxygenated in
the presence of a polar solvent comprising isopropanol and
water.
5. A method of preparing a desoxyepothilone having the structure:
89wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein Z is O,
N(OR.sub.3) or N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched chain alkyl;
and wherein n is 0, 1, 2, or 3; which comprises treating an
epothilone having a structure: 90wherein R, R.sub.0, R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R', R", X, Y, Z and n are
defined as for the desoxyepothilone, under suitable conditions so
as to deoxygenate the epothilone, and thereby to provide the
desoxyepothilone.
6. The method of claim 5 wherein the desoxyepothilone has the
structure: 91wherein R is H, methyl, ethyl, n-propyl, n-butyl,
n-hexyl or hydroxypropyl.
7. The method of claim 5 wherein the epothilone is deoxygenated
using a zinc/copper couple.
8. The method of claim 5 wherein the epothilone is deoxygenated in
the presence of a polar solvent comprising isopropanol and
water.
9. A method of preparing a desoxyepothilone having the structure:
92wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; and wherein Z is
O, N(OR.sub.3) or N--NR.sub.4R.sub.5 where R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched alkyl; and
wherein n is 0, 1, 2, or 3; which comprises treating a protected
desoxyepothilone having the structure: 93wherein R.sub.A is a
linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted
aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl,
triarylsilyl, linear or branched acyl, substituted or unsubstituted
aroyl or benzoyl; and wherein R.sub.B is hydrogen,
t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)
alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, triarysilyl, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl; under suitable
conditions to form the desoxyepothilone.
10. The method of claim 9 wherein n is 3 and R" is
2-methyl-1,3-thiazoliny- l.
11. The method of claim 9 wherein R.sub.A is TES and R.sub.B is
Troc.
12. The method of claim 9 wherein the treating step comprises
contacting the protected desoxyepothilone (i) with SmX.sub.2, where
X is Cl, Br or I, in the presence of a polar nonaqueous solvent
selected from the group consisting of tetrahydrofuran, p-dioxane,
diethyl ether, acetonitrile and N,N-dimethylformamide, and
optionally in the presence of N,N-dimethyl-N'-propylurea or
hexamethylphosphoramide and (ii) with a source of fluoride ion
selected from the group consisting of tetra-n-methylammonium
fluoride, tetra-n-butylammonium fluoride and HF.pyridine.
13. A method of preparing a protected desoxyepothilone having the
structure: 94wherein R, R.sub.0, and R' are independently H, linear
or branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.A is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyloxy- carbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; which
comprises cyclocondensing a hydroxy acid desoxyepothilone precursor
having the structure: 95wherein R, R.sub.0, R.sub.A, R.sub.B, R',
R" and n are defined as above; under suitable conditions to form
the protected desoxyepothilone.
14. The method of claim 13 wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl.
15. The method of claim 13 wherein R.sub.A is TES and R.sub.B is
Troc.
16. The method of claim 13 wherein the hydroxy acid
desoxyepothilone precursor is cyclocondensed using a
cyclocondensing reagent selected from the group consisting of
acetic anhydride, pentafluorophenol, 2,4-dichlorobenzoyl chloride
and 2,4,6-trichlorobenzoyl chloride.
17. The method of claim 13 wherein the hydroxyacid is
cyclocondensed using 2,4,6-trichlorobenzoyl chloride in the
presence of a tertiary amine selected from the group consisting of
triethyl amine, tri-n-propylamine, diisopropylethylamine and
diethylisopropylamine, and optionally in the presence of pyridine
or N,N-dimethylaminopyridine.
18. A method of preparing a hydroxy acid desoxyepothilone precursor
having the structure: 96wherein R, R.sub.0, and R' are
independently H, linear or branched chain alkyl, optionally
substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or
branched alkyl or cyclic acetal, fluorine, NR.sub.1R.sub.2,
N-hydroximino, or N-alkoxyimino, wherein R.sub.1 and R.sub.2 are
independently H, phenyl, benzyl, linear or branched chain alkyl;
wherein R" is --CY.dbd.CHX, or H, linear or branched chain alkyl,
phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.A is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl) alkyloxycarbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; which
comprises selectively etherifying and hydrolyzing a hydroxy ester
desoxyepothilone precursor having the structure: 97wherein R,
R.sub.0, R.sub.B, R.sub.C, R', R" and n are defined as above; and
wherein R.sub.C is tertiary-alkyl; under suitable conditions to
form the hydroxy acid desoxyepothilone precursor.
19. The method of claim 18 wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl.
20. The method of claim 18 wherein R.sub.A is TES and R.sub.B is
Troc.
21. The method of claim 18 wherein the selective etherifying step
comprises contacting the hydroxy ester desoxyepothilone precursor
with a silylating reagent to form an ether intermediate, and the
hydrolyzing step comprises contacting the ether intermediate with a
protic acid or tetra-n-butylammonium fluoride.
22. The method of claim 21 wherein the silylating reagent is TESOTf
in the presence of 2,6-lutidine.
23. The method of claim 21 wherein the protic acid is HCl in the
presence of methyl alcohol.
24. A method of preparing a hydroxy ester desoxyepothilone
precursor having the structure: 98wherein R, R.sub.0, and R' are
independently H, linear or branched chain alkyl, optionally
substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or
branched alkyl or cyclic acetal, fluorine, NR.sub.1R.sub.2,
N-hydroximino, or N-alkoxyimino, wherein R.sub.1 and R.sub.2 are
independently H, phenyl, benzyl, linear or branched chain alkyl;
wherein R" is --CY.dbd.CHX, or H, linear or branched chain alkyl,
phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.B is hydrogen, t-butyloxycarbonyl,
amyloxycarbonyl, (trialkylsilyl)alkyloxycar- bonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; and wherein
R.sub.C is tertiary-alkyl; which comprises reducing a hydroxy
ketoester desoxyepothilone precursor having the structure:
99wherein P, R, R.sub.0, R.sub.B, R.sub.C, R', R" and n are defined
as above; under suitable conditions to form the hydroxy ester
desoxyepothilone precursor.
25. The method of claim 24 wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl.
26. The method of claim 24 wherein R.sub.A is TES and R.sub.B is
Troc.
27. The method of claim 24 wherein the reducing step comprises
contacting the hydroxy ketoester desoxyepothilone precursor with a
stereospecific reducing reagent.
28. The method of claim 24 wherein the stereospecific reducing
reagent comprises hydrogen gas at from about 900 pounds per square
inch to about 2200 pounds per square inch in the presence of
(R)-(BINAP)RuCl.sub.2 and optionally in the presence of HCl and an
alcohol selected from the group consisting of MeOH, EtOH, and
i-PrOH.
29. A method of preparing a hydroxy ketoester desoxyepothilone
precursor having the structure: 100wherein P is H; wherein R,
R.sub.0, and R' are independently H, linear or branched chain
alkyl, optionally substituted by hydroxy, alkoxy, carboxy,
carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.B is hydrogen, t-butyloxycarbonyl,
amyloxycarbonyl, (trialkylsilyl)alkyloxy- carbonyl,
(dialkylarylsilyl) alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.C is tertiary-alkyl; which comprises deprotecting a
protected ketoester desoxyepothilone precursor having the
structure: 101wherein R, R.sub.0, R.sub.A, R.sub.B, R.sub.C, R', R"
and n are defined as above; and wherein P is a linear or branched
alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl,
trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or triarylsilyl;
under suitable conditions to form the hydroxy ketoester
desoxyepothilone precursor.
30. The method of claim 29 wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl.
31. The method of claim 29 wherein R.sub.A is TES and R.sub.B is
Troc.
32. The method of claim 29 wherein P is TBS.
33. The method of claim 29 wherein the deprotecting step comprises
contacting the protected ketoester desoxyepothilone precursor with
a protic acid.
34. The method of claim 33 wherein the protic acid is HCl in methyl
alcohol or ethyl alcohol.
35. A method of preparing a protected ketoester desoxyepothilone
precursor having the structure: 102wherein P is a linear or
branched alkyl, alkoxyalkyl, substituted or unsubstituted
aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or
triarylsilyl; wherein R, R.sub.0, and R' are independently H,
linear or branched chain alkyl, optionally substituted by hydroxy,
alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic
acetal, fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino,
wherein R.sub.1 and R.sub.2 are independently H, phenyl, benzyl,
linear or branched chain alkyl; wherein R" is --CY.dbd.CHX, or H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein X is H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or
linear or branched chain alkyl; wherein n is 2 or 3; wherein
R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyl-oxycarbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.C is tertiary-alkyl; which comprises coupling a
terminal vinyl enol ether ester having the structure: 103wherein R,
R.sub.0, R.sub.B, R.sub.C, and R' are defined as above; wherein m
is 0, 1 or 2; and wherein R.sub.D is linear or branched alkyl,
benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, linear
or branched acyl, substituted or unsubstituted aroyl or benzoyl;
with a protected halovinyl or metalvinyl compound having the
structure: 104wherein R, P and R" are defined as above; and wherein
Q is a halide or a metal; under suitable conditions to form the
protected ketoester desoxyepothilone precursor.
36. The method of claim 35 wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl.
37. The method of claim 35 wherein R.sub.A is TES and R.sub.B is
Troc.
38. The method of claim 35 wherein P is TBS or TES.
39. The method of claim 35 wherein Q is iodine or bromine.
40. The method of claim 35 wherein R.sub.D is methyl or TES.
41. The method of claim 35 wherein the coupling step comprises
contacting the terminal vinyl enol ether ester and the protected
halovinyl compound with noble metal complex capable of effecting a
Suzuki coupling.
42. The method of claim 35 wherein the noble metal complex is
Pd(dppf).sub.2Cl.sub.2 in the presence of Ph.sub.3As and
Cs.sub.2CO.sub.3.
43. A method of preparing a terminal vinyl enol ether ester having
the structure: 105wherein R.sub.0 and R' are independently H,
linear or branched chain alkyl, optionally substituted by hydroxy,
alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic
acetal, fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino,
wherein R.sub.1 and R.sub.2 are independently H, phenyl, benzyl,
linear or branched chain alkyl; wherein m is 0, 1 or 2; wherein
R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyl-oxycarbonyl,
(dialkylarylsilyl)alky-loxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; wherein
R.sub.C is tertiary-alkyl; and wherein R.sub.D is linear or
branched alkyl, benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted
or unsubstituted aroyl or benzoyl; which comprises: (a) treating a
keto enol ester having the structure: 106under suitable conditions
to form an enolate enol ester having the structure: 107wherein M is
Li, Na or K; and (b) coupling the enolate enol ester with a vinyl
aldehyde having the structure: 108wherein m, and R.sub.0 and R' are
as defined above; under suitable conditions to form the terminal
vinyl enol ether ester.
44. The method of claim 43 wherein the treating step comprises
contacting the keto enol ester with a strong nonnucleophilic base
selected from the group consisting of lithium diethylamide, lithium
diethylamide, lithium diisopropylamide, lithium hydride, sodium
hydride, potassium hydride and potassium t-butoxide.
45. The method of claim 44 wherein the treating step is effected in
a polar nonaqueous solvent selected from the group consisting of
tetrahydrofuran, diethyl ether, di-n-propyl ether and
dimethylformamide at a temperature from about -100.degree. C. to
about +10.degree. C.
46. The method of claim 44 wherein the temperature is from about
-20.degree. C. to -40.degree. C.
47. The method of claim 43 wherein the coupling step comprises
contacting the enolate enol ester with the vinyl aldehyde at a
temperature from about -130.degree. C. to about -78.degree. C.
Description
[0001] This application is based on U.S. Provisional Applications
Ser. Nos. 60/075,947, 60/092,319, and 60/097,733, filed Feb. 25,
1998, Jul. 9, 1998, and Aug. 24, 1998, respectively, the contents
of which are hereby incorporated by reference into this
application, and is a continuation-in-part of U.S. Ser.
No.08/986,025, filed Dec. 3, 1997, which was based on U.S.
Provisional Applications Ser. Nos. 60/032,282, 60/033,767,
60/047,566, 60/047,941, and 60/055,533, filed Dec. 3, 1996, Jan.
14, 1997, May 22, 1997, May 29, 1997, and Aug. 13, 1997,
respectively, the contents of which are hereby incorporated by
reference into this application.
FIELD OF THE INVENTION
[0003] The present invention is in the field of epothilone
macrolides. In particular, the present invention relates to
processes for the preparation of epothilones A and B,
desoxyepothilones A and B, and analogues thereof which are useful
as highly specific, non-toxic anticancer therapeutics. In addition,
the invention provides methods of inhibiting multidrug resistant
cells. The present invention also provides novel compositions of
matter which serve as intermediates for preparing the
epothilones.
[0004] Throughout this application, various publications are
referred to, each of which is hereby incorporated by reference in
its entirety into this application to more fully describe the state
of the art to which the invention pertains.
BACKGROUND OF THE INVENTION
[0005] Epothilones A and B are highly active anticancer compounds
isolated from the Myxobacteria of the genus Sorangium. The full
structures of these compounds, arising from an x-ray
crystallographic analysis were determined by Hofle. G. Hofle et
al., Angew. Chem. Int. Ed. Engl., 1996, 35, 1567. The total
synthesis of the epothilones is an important goal for several
reasons. Taxol.RTM. is already a useful resource in chemotherapy
against ovarian and breast cancer and its range of clinical
applicability is expanding. G. I. Georg et al., Taxane Anticancer
Agents; American Cancer Society: San Diego, 1995. The mechanism of
the cytotoxic action of Taxol.RTM., at least at the in vitro level,
involves stabilization of microtubule assemblies. P. B. Schiff et
al., Nature (London), 1979, 277, 665. A series of complementary in
vitro investigations with the epothilones indicated that they share
the mechanistic theme of the taxoids, possibly down to the binding
sites to their protein target. D. M. Bollag et al., Cancer Res.,
1995, 55, 2325. Moreover, the epothilones surpass Taxol.RTM. in
terms of cytotoxicity and far surpass it in terms of in vitro
efficacy against drug resistant cells. Since multiple drug
resistance (MDR) is one of the serious limitations of Taxol.RTM.
(L. M. Landino and T. L. MacDonald in The Chemistry and
Pharmacology of Taxol.RTM. and its Derivatives, V. Farin, Ed.,
Elsevier: New York, 1995, ch. 7, p. 301), any agent which promises
relief from this problem merits serious attention. Furthermore,
formulating the epothilones for clinical use is more
straightforward than Taxol.RTM..
[0006] Accordingly, the present inventors undertook the total
synthesis of the epothilones, and as a result, have developed
efficient processes for synthesizing epothilones A and B, the
corresponding desoxyepothilones, as well as analogues thereof. The
present invention also provides novel intermediates useful in the
synthesis of epothilones A and B and analogues thereof,
compositions derived from such epothilones and analogues, purified
compounds of epothilones A and B, and desoxyepothilones A and B, in
addition to methods of use of the epothilone analogues in the
treatment of cancer. Unexpectedly, certain epothilones have been
found to be effective not only in reversing multi-drug resistance
in cancer cells, both in vitro- and in vivo, but have been
determined to be active as collateral sensitive agents, which are
more cytotoxic towards MDR cells than normal cells, and as
synergistic agents, which are more active in combination with other
cytotoxic agents, such as vinblastin, than the individual drugs
would be alone at the same concentrations. Remarkably, the
desoxyepothilones of the invention have exceptionally high
specificity as tumor cytotoxic agents in vivo, more effective and
less toxic to normal cells than the principal chemotherapeutics
currently in use, including Taxol.RTM., vinblastin, adriamycin and
camptothecin.
SUMMARY OF THE INVENTION
[0007] One object of the present invention is to provide processes
for the preparation of epothilones A and B, and desoxyepothilones A
and B, and related compounds useful as anticancer therapeutics.
Another object of the present invention is to provide various
compounds useful as intermediates in the preparation of epothilones
A and B as well as analogues thereof.
[0008] A further object of the present invention is to provide
synthetic methods for preparing such intermediates. An additional
object of the invention is to provide compositions useful in the
treatment of subjects suffering from cancer comprising any of the
analogues of the epothilones available through the preparative
methods of the invention optionally in combination with
pharmaceutical carriers.
[0009] A further object of the invention is to provide methods of
treating subjects suffering from cancer using any of the analogues
of the epothilones available through the preparative methods of the
invention optionally in combination with pharmaceutical
carriers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1(A) shows a retrosynthetic analysis for epothilone A
and B.
[0011] FIG. 1(B) provides synthesis of compound 11. (a)
t-BuMe.sub.2OTf, 2,6-lutidine, CH.sub.2Cl.sub.2, 98%; (b) (1) DDQ,
CH.sub.2Cl.sub.2/H.sub.- 2O, 89%; (2) (COCl).sub.2, DMSO,
CH.sub.2Cl.sub.2, -78.degree. C.; then Et.sub.3N, -78.degree.
C..fwdarw.rt, 90%; (c) MeOCH.sub.2PPh.sub.3Cl, t-BuOK, THF,
0.degree. C..fwdarw.rt, 86%; (d) (1) p-TsOH, dioxane/H.sub.2O,
50.degree. C., 99%; (2) CH.sub.3PPh.sub.3Br, NaHMDS, PhCH.sub.3,
0.degree. C..fwdarw.rt, 76%; (e) Phl(OCOCF.sub.3).sub.2, MeOH/THF,
rt, 0.25 h, 92%.
[0012] FIG. 2 provides key intermediates in the preparation of
12,13-E- and -Z-deoxyepothilones.
[0013] FIG. 3(A) provides syntheses of key iodinated intermediates
used to prepare hydroxymethylene- and hydroxypropylene-substituted
epothilone derivatives.
[0014] FIG. 3(B) provides methods of preparing hydroxymethylene-
and hydroxypropylene-substituted epothilone derivatives, said
methods being useful generally to prepare 12,13-E epothilones
wherein R is methyl, ethyl, n-propyl, and n-hexyl from the
corresponding E-vinyl iodides.
[0015] FIG. 3(C) shows reactions leading to benzoylated
hydroxymethyl-substituted desoxyepothilone and
hydroxymethylene-substitut- ed epothilone (epoxide).
[0016] FIG. 4(A) provides synthesis of compound 19. (a) DHP, PPTS,
CH.sub.2Cl.sub.2, rt: (b) (1) Me.sub.3SiCCLi, BF.sub.3.OEt.sub.2,
THF, -78.degree. C.; (2) MOMCl, I--Pr.sub.2NEt,
Cl(CH.sub.2).sub.2Cl, 55.degree. C.; (3) PPTS, MeOH, rt; (c) (1)
(COCl).sub.2, DMSO, CH.sub.2Cl.sub.2, -78.degree. C.; then
Et.sub.3N, -78.degree. C..fwdarw.rt; (2) MeMgBr, Et.sub.2O,
0.degree. C..fwdarw.rt, (3) TPAP, NMO, 4 .ANG. mol. sieves,
CH.sub.2Cl.sub.2, 0.degree. C..fwdarw.rt; (d) 16, n-BuLi, THF,
-78.degree. C.; then 15, THF, -78.degree. C..fwdarw.rt; (e) (1)
N-iodosuccinimide, AgNO.sub.3, (CH.sub.3).sub.2CO; (2) Cy.sub.2BH,
Et.sub.2O, AcOH; (f) (1) PhSH, BF.sub.3.OEt.sub.2,
CH.sub.2Cl.sub.2, rt; (2) Ac.sub.2O, pyridine, 4-DMAP,
CH.sub.2Cl.sub.2, rt.
[0017] FIG. 4(B) presents synthesis of compound 1. (a) 11, 9-BBN,
THF, rt; then PdCl.sub.2(dppf).sub.2, Cs.sub.2CO.sub.3, Ph.sub.3As,
H.sub.2O, DMF, 19, rt, 71%; (b) p-TsOH, dioxane/H.sub.2O,
50.degree. C.; (c) KHMDS, THF, -78.degree. C., 51%; (d) (1)
HF-pyridine, pyridine, THF, rt, 97%; (2) t-BuMe.sub.2 SiOTf,
2,6-lutidine, CH.sub.2Cl.sub.2, -25.degree. C., 93%; (3)
Dess-Martin periodinane, CH.sub.2Cl.sub.2, 87%; (4) HF-pyridine,
THF, rt, 99%; (e) dimethyldioxirane, CH.sub.2Cl.sub.2, 0.5 h,
-50.degree. C., 45% (.gtoreq.20:1).
[0018] FIG. 5 shows a scheme of the synthesis of the "left wing" of
epothilone A.
[0019] FIG. 6 provides a scheme of an olefin metathesis route to
epothilone A and other analogues.
[0020] FIG. 7 illustrates a convergent strategy for a total
synthesis of epothilone A (1) and the glycal cyclopropane
solvolysis strategy for the introduction of geminal methyl
groups.
[0021] FIG. 8 provides an enantioselective synthesis of compound
15B.
[0022] FIG. 9 shows the construction of epothilone model systems
20B, 21B, and 22B by ring-closing olefin metathesis.
[0023] FIG. 10 illustrates a sedimentation test for natural,
synthetic and desoxyepothilone A.
[0024] FIG. 11 illustrates a sedimentation test for natural,
synthetic and desoxyepothilone A after cold treatment at 4.degree.
C.
[0025] FIG. 12 illustrates (A) structures of epothilones A (1) and
B (2) and (B) of Taxol.RTM. (1A).
[0026] FIG. 13 shows a method of elaborating acyclic stereochemical
relationships based on dihydropyrone matrices.
[0027] FIG. 14 shows the preparation of intermediate 4A.
[0028] FIG. 15 shows an alternative enantioselective synthesis of
compound 17A.
[0029] FIG. 16 provides a synthetic pathway to intermediate 13C.
(a) 1. tributyl allyltin, (S)-(-)-BINOL, Ti(Oi-Pr).sub.4,
CH.sub.2Cl.sub.2, -20.degree. C., 60%, >95% e.e.; 2. Ac.sub.2O,
Et.sub.3N, DMAP, CH.sub.2Cl.sub.2, 95%; (b) 1. OsO.sub.4, NMO,
acetone/H.sub.2O, 0.degree. C.; 2. NaIO.sub.4, THF/H.sub.2O; (c)
12, THF, -20.degree. C., Z isomer only, 25% from 10; (d)
Pd(dppf).sub.2, Cs.sub.2CO.sub.3, Ph.sub.3As H.sub.2O, DMF, rt.
77%.
[0030] FIG. 17 provides a synthetic pathway to intermediate
epothilone B (2). (a) p-TsOH, dioxane/H.sub.2O, 55.degree. C., 71%;
(b) KHMDS, THF, -78.degree. C., 67%, .alpha./.beta.: 1.5:1; (c)
Dess-Martin periodinane, CH.sub.2Cl.sub.2; (d) NaBH.sub.4, MeOH,
67% for two steps; (e) 1. HF.pyridine, pyridine, THF, rt, 93%; 2.
TBSOTf, 2,6-lutidine, CH.sub.2Cl.sub.2, -30.degree. C., 89%; 3.
Dess-Martin periodinane, CH.sub.2Cl.sub.2, 67%; (f) HF.pyridine,
THF, rt, 80%; (g) dimethyldioxirane, CH.sub.2Cl.sub.2, -50.degree.
C., 70%.
[0031] FIG. 18 provides a synthetic pathway to a protected
intermediate for 8-desmethyl deoxyepothilone A.
[0032] FIG. 19 provides a synthetic pathway to 8-desmethyl
deoxyepothilone A, and structures of
trans-8-desmethyl-desoxyepothiolone A and a trans-iodoolefin
intermediate thereto.
[0033] FIG. 20 shows (top) structures of epothilones A and B and
8-desmethylepothilone and (bottom) a synthetic pathway to
intermediate TBS ester 10 used in the preparation of
desmethylepothilone A. (a) (Z)-Crotyl-B[(-)-lpc].sub.2, -78.degree.
C., Et.sub.2O, then 3N NaOH, 30% H.sub.2O.sub.2; (b) TBSOTf,
2,6-lutidine, CH.sub.2Cl.sub.2 (74% for two steps, 87% ee); (c)
O.sub.3, CH.sub.2Cl.sub.2/MeOH, -78.degree. C., then DMS, (82%);
(d) t-butyl isobutyrylacetate, NaH, BuLi, 0.degree. C., then 6
(60%, 10:1); (e) Me.sub.4NBH(OAc).sub.3, -10.degree. C. (50%, 10:1
.alpha./.beta.) or NaBH.sub.4, MeOH, THF, 0.degree. C., (88%, 1:1
.alpha./.beta.); (f) TBSOTf, 2,6-lutidine, -40.degree. C., (88%);
(g) Dess-Martin periodinane, (90%); (h) Pd(OH).sub.2, H.sub.2, EtOH
(96%); (t) DMSO, oxalyl chloride, CH.sub.2Cl.sub.2, -78.degree. C.
(78%); (j) Methyl triphenylphosphonium bromide, NaHMDS, THF,
0.degree. C. (85%); (k) TBSOTf, 2,6-lutidine, CH.sub.2Cl.sub.2, rt
(87%).
[0034] FIG. 21 shows a synthetic pathway to 8-desmethylepothilone
A. (a) Pd(dppf).sub.2Cl.sub.2, Ph.sub.3As, Cs.sub.2CO.sub.3,
H.sub.2O, DMF, rt (62%); (b) K.sub.2CO.sub.3, MeOH, H.sub.2O (78%);
(c) DCC, 4-DMAP, 4-DMAP.HCl, CHCl.sub.3 (78%); (d) HF.pyr, THF, rt
(82%), (e) 3,3-dimethyl dioxirane, CH.sub.2Cl.sub.2, -35.degree. C.
(72%, 1.5:1).
[0035] FIG. 22 shows a synthetic pathway to prepare epothilone
analogue 27D.
[0036] FIG. 23 shows a synthetic pathway to prepare epothilone
analogue 24D.
[0037] FIG. 24 shows a synthetic pathway to prepare epothilone
analogue 19D.
[0038] FIG. 25 shows a synthetic pathway to prepare epothilone
analogue 20D.
[0039] FIG. 26 shows a synthetic pathway to prepare epothilone
analogue 22D.
[0040] FIG. 27 shows a synthetic pathway to prepare epothilone
analogue 12-hydroxy ethylepothilone.
[0041] FIG. 28 shows the activity of epothilone analogues in a
sedimentation test in comparison with DMSO, epothilone A and/or B.
Structures 17-20, 22, and 24-27 are shown in FIGS. 29-37,
respectively. Compounds were added to tubulin (1 mg/ml) to a
concentration of 10 .mu.M. The quantity of microtubules formed with
epothilone A was defined as 100%.
[0042] FIG. 29 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #17.
[0043] FIG. 30 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #18.
[0044] FIG. 31 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #19.
[0045] FIG. 32 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #20.
[0046] FIG. 33 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #22.
[0047] FIG. 34 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #24.
[0048] FIG. 35 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #25.
[0049] FIG. 36 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #26.
[0050] FIG. 37 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #27.
[0051] FIG. 38 provides a graphical representation of the effect of
fractional combinations of cytotoxic agents.
[0052] FIG. 39 shows epothilone A and epothilone analogues #1-7.
Potencies against human leukemia CCRF-CEM (sensitive) and
CCRF-CEM/VBL MDR (resistant) sublines are shown in round and square
brackets, respectively.
[0053] FIG. 40 shows epothilone B and epothilone analogues #8-16.
Potencies against human leukemia CCRF-CEM (sensitive) and
CCRF-CEM/VBL MDR (resistant) sublines are shown in round and square
brackets, respectively.
[0054] FIG. 41 shows epothilone analogues #17-25. Potencies against
human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR
(resistant) sublines are shown in round and square brackets,
respectively.
[0055] FIG. 42(A) shows epothilone analogues #26-34. Potencies
against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR
(resistant) sublines are shown in round and square brackets,
respectively. (B) shows epothilone analogues #35-46.Potencies
against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR
(resistant) sublines are shown in round and square brackets,
respectively. (C) shows epothilone analogues #4749.
[0056] FIG. 43(A) shows antitumor activity of desoxyepothilone B
against MDR MCF-7/Adr xenograft in comparison with Taxol.RTM..
Control (.diamond-solid.); desoxyepothilone B (.box-solid.; 35
mg/kg); Taxol.RTM. (.tangle-solidup.; 6 mg/kg); adriamycin (x; 1.8
mg/kg); i.p. Q2Dx5; start on day 8. (B) shows antitumor activity of
epothilone B against MDR MCF-7/Adr xenograft in comparison with
Taxol.RTM.. Control (.diamond-solid.); epothilone B (.box-solid.;
25 mg/kg; non-toxic dose); Taxol.RTM. (.tangle-solidup.; 6 mg/kg;
half LD.sub.50); adriamycin (x; 1.8 mg/kg); i.p. Q2Dx5; start on
day 8.
[0057] FIG. 44(A) shows toxicity of desoxyepothilone B in
B6D2F.sub.1 mice bearing B16 melanoma. Body weight was determined
at 0, 2, 4, 6, 8, 10 and 12 days. Control (.tangle-solidup.);
desoxyepothilone B (.smallcircle.; 10 mg/kg QDx8; 0 of 8 died);
desoxyepothilone B (.circle-solid.; 20 mg/kg QDx6; 0 of 8 died).
Injections were started on day 1. (B) shows toxicity of epothilone
B in B6D2F.sub.1 mice bearing B16 melanoma. Body weight was
determined at 0, 2, 4, 6, 8, 10 and 12 days. Control
(.tangle-solidup.); epothilone B (.smallcircle.; 0.4 mg/kg QDx6; 1
of 8 died of toxicity); epothilone B (.circle-solid.; 0.8mg/kg
QDx5; 5 of 8 died). Injections were started on day 1.
[0058] FIG. 45(A) shows comparative therapeutic effect of
desoxyepothilone B and Taxol.RTM. on nude mice bearing MX-1
xenoplant. Tumor, s.c.; drug administered i.p., Q2Dx5, start on day
7. control (.diamond-solid.); Taxol.RTM. (.quadrature.; 5 mg/kg,
one half of LD.sub.50); desoxyepothilone B (.DELTA.; 25 mg/kg;
nontoxic dose). (B) shows comparative therapeutic effect of
desoxyepothilone B and Taxol.RTM. on nude mice bearing MX-1
xenoplant. Tumor, s.c.; drug administered i.p., Q2Dx5, start on day
7. control (.diamond-solid.); Taxol.RTM. (.quadrature.; 5 mg/kg,
one half of LD.sub.50, given on days 7, 9, 11, 13, 15; then 6
mg/kg, given on days 17, 19, 23, 24, 25); desoxyepothilone B (n=3;
.DELTA., x, *; 25 mg/kg, nontoxic dose, given to three mice on days
7, 9, 11, 13, 15; then 35 mg/kg, given on days 17, 19, 23, 24,
25).
[0059] FIG. 46 shows the effect of treatment with desoxyepothilone
B (35 mg/kg), Taxol.RTM. (5 mg/kg) and adriamycin (2 mg/kg) of nude
mice bearing human MX-1 xenograft on tumor size between 8 and 18
days after implantation. Desoxyepothilone B (.quadrature.),
Taxol.RTM. (.DELTA.), adriamycin (X), control (.diamond-solid.);
i.p. treatments were given on day 8, 10, 12, 14 and 16.
[0060] FIG. 47 shows the relative toxicity of epothilone B
(.quadrature.; 0.6 mg/kg QDx4; i.p.) and desoxyepothilone B
(.DELTA.; 25 mg/kg QDx4; i.p.) versus control (.diamond-solid.) in
normal nude mice. Body weight of mice was determined daily after
injection. For epothilone B, 8 of 8 mice died of toxicity on days
5, 6, 6, 7, 7, 7, 7, and 7; for desoxyepothilone B, all six mice
survived.
[0061] FIG. 48 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #43.
[0062] FIG. 49 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #45.
[0063] FIG. 50 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #46.
[0064] FIG. 51 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #47.
[0065] FIG. 52 shows a high resolution .sup.1H NMR spectrum of
epothilone analogue #48.
[0066] FIG. 53(A) shows an approach to the preparation of
desoxyepothilones. (B) represents a key step involving dianion
addition to an aldehyde reactant.
[0067] FIG. 54(A) illustrates the acylation of t-butyl
4-methylpentan-3-on-1-ate to provide t-butyl
4,4-dimethylheptan-3,5-dion-- 1-ate. (B) exemplifies a dianion
addition to 2-methylpent-4-enal, diacylation and selective
saponification to form a key intermediate 11-carbon
diketoester.
[0068] FIG. 55 shows the preparation of a key intermediate
hydroxyacid in the preparation of desoxyepothilones. The final
synthetic steps leading to various desoxyepothilones from the
hydroxyacid are found in, e.g., FIGS. 21-26. R is selected from the
group consisting of H, Me, Et, Pr, Hx, CH.sub.2OH and
(CH.sub.2).sub.3OH. Ar is selected from the group consisting of
phenyl, tolyl, xylyl, thiazolyl, 2-methylthiazolyl, pyrryl and
pyridyl, and is either unsubstituted or substituted with an
C.sub.1-6 alkyl, phenyl or benzyl group. Conditions for the Noyori
reduction are disclosed in Taber et al., Tetrahedron Lett., 1991,
32, 4227, and Noyori et al., J.Amer.Chem.Soc., 1987, 109, 5856, the
contents of which are incorporated herein by reference. Conditions
for the DDQ deprotection are disclosed in Horita et al.,
Tetrahedron, 1986, 42, 3021, the contents of which is incorporated
herein by reference.
[0069] FIG. 56 illustrates an application of the Noyori reduction
of a substrate with C-15 hydroxyl useful in the preparation of
epothilone analogues.
[0070] FIG. 57 exemplifies a dianion addition to a coupled aldehyde
intermediate useful in the preparation of epothilone analogues.
[0071] FIG. 58 provides an application of the Noyori reduction of a
coupled substrate useful in the preparation of epothilone
analogues.
[0072] FIG. 59 shows the preparation of desoxyepothilone analogues
by deoxygenation of the epoxide using Zn/Cu couple, exemplified by
the conversion of desoxyepothilone B from epothilone B. In a sample
procedure, Zn/Cu couple is added to a solution of epothilone B (6
mg, 0.012 mmol) in i-PrOH (0.3 mL) and water (3 drops). The
suspension was heated to 90.degree. C. for 13 hours, cooled to room
temperature, filtered through a pad of Celite.TM. and concentrated.
Flash chromatography afforded 1.5 mg of epothilone B (75%
conversion) and 3.2 mg of desoxyepothilone B (73% yield, as a
mixture of cis and trans isomers in a 0.7:1 ratio).
[0073] FIG. 60 illustrates the therapeutic effect of dEpoB,
Taxol.RTM. and adriamycin in nude mice bearing the human mammary
carcinoma MX-1 xenograft. MX-1 tissue preparation 100 .mu.l/mouse
was implanted s.c. on day 0. Every other day i.p. treatments were
given on day 8, 10, 12, 14 and 16 with dEpoB 35 mg/kg
(.box-solid.), Taxol.RTM. 5 mg/kg (.tangle-solidup.), adriamycin (2
mg/kg (x), and vehicle (DMSO, 30 .mu.l) treated control
(.diamond-solid.). For Taxol.RTM., 2/10 mice died of toxicity on
day 18. For adriamycin, 1/10 mice died of toxicity on day 22. For
dEpoB, 10/10 mice survived and were subjected to the second cycle
of treatment at 40 mg/kg on day 18, 20, 22, 24 and 26. This led to
3/10 mice tumor-free up to day 80, whereas 7/10 mice were with
markedly suppressed tumors and were sacrificed on day 50.
[0074] FIG. 61(A) shows a procedure for preparing intermediate 49.
.sup.aNaH, THF, 25.degree. C., then 0.degree. C., then propionyl
chloride, -50.degree. C., 71%; .sup.bNaH, 0.degree. C. then TESOTf,
-50.degree. C., 78%; LDA, THF, -33.degree. C., 5 min.
[0075] FIG. 61(B) shows a procedure for preparing desoxyepothilone
B. .sup.aTrocCl, pyridine, CH.sub.2Cl.sub.2, 0.degree.
C..fwdarw.25.degree. C.; then 0.5N HCl in MeOH, 0.degree. C., 87%;
.sup.b9-BBN, THF, 50 then 51, (Pd(dppf).sub.2)Cl.sub.2, Ph.sub.3As,
Cs.sub.2CO.sub.3, H.sub.2O, DMF; .sup.c0.4N HCl in MeOH (50% for
two steps); .sup.d(R)-(BINAP)RuCl.su- b.2, H.sub.2 (1200 psi),
MeOH, HCl, 25.degree. C., 7 h, 88%, >95:5; .sup.eTESOTf,
2,6-lutidine, CH.sub.2Cl.sub.2, -78-25.degree. C., then HCl/MeOH,
77%; .sup.f2,4,6-trichlorobenzoyl chloride, TEA, 4-DMAP, toluene,
78%; .sup.8Sml.sub.2, cat. NiI.sub.2, THF, -78.degree. C., 95%;
.sup.hHF.pyridine, THF, 98%; .sup.i2,2-dimethyldioxirane,
CH.sub.2Cl.sub.2, -50.degree. C., 98%, >20:1.
[0076] FIG. 62 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing MX-1 with Q2Dx5 i.v. 6 h infusion, using dEpoB
30 mg/kg (x), Taxol.RTM. 15 mg/kg (.box-solid.), Taxol.RTM. 24
mg/kg (.DELTA.) and control (.circle-solid.).
[0077] FIG. 63 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing MCF-7/Adr with Q2Dx5 i.v. 6 h infusion, using
dEpoB 30 mg/kg (x), Taxol.RTM. 15 mg/kg (.box-solid.), Taxol.RTM.
24 mg/kg (.DELTA.) and control (.circle-solid.).
[0078] FIG. 64 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing CCRF/Taxol.RTM. with Q2Dx5 i.v. 6 h infusion,
using dEpoB 30 mg/kg (x), Taxol.RTM. 20 mg/kg (.box-solid.),
Taxol.RTM. 24 mg/kg (.DELTA.) and control (.circle-solid.).
Treatment on days 6, 8, 10, 12 and 14.
[0079] FIG. 65 shows a procedure for preparing desoxyepothilone B
(14E).
[0080] FIG. 66 shows a procedure for preparing intermediate 8E.
[0081] FIG. 67 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing CCRF/CEM tumor with Q2Dx4 i.v. 6 h infusion,
using dEpoB 30 mg/kg (.quadrature.)), Taxol.RTM. 20 mg/kg
(.DELTA.), and control (.diamond.). Treatment on days 21, 23, 25,
and 27.
[0082] FIG. 68 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing CCRF/Taxol.RTM. with Q2Dx5 i.v. 6 h infusion,
using using dEpoB 30 mg/kg (.quadrature.), Taxol.RTM. 20 mg/kg
(.DELTA.) and control (.diamond.). Treatment on days 19, 21, 23,
25, 27, 39, 41, 43, 45 and 47.
[0083] FIG. 69 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing SK-OV-3 Tumor with Q2Dx6 i.v. 6 h infusion,
using dEpoB 30 mg/kg (.DELTA.), Taxol.RTM. 15 mg/kg (.quadrature.),
and control (.diamond.). Treatment on days 10, 12, 14, 16, 18 and
20.
[0084] FIG. 70 shows changes in body weight following treatment
with desoxyepothilone B and Taxol.RTM. in nude mice bearing SK-OV-3
Tumor by i.v. infusion, using dEpoB 30 mg/kg (.quadrature.),
Taxol.RTM. 15 mg/kg (.DELTA.) and control (.diamond.).
[0085] FIG. 71 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing PC-3 Human Prostate Carcinoma with Q2Dx3 i.v.
6 or 18 h infusion, using dEpoB 30 mg/kg, 18 h (x), dEpoB 40 mg/kg,
6 h (*), dEpoB 50 mg/kg, 6 h (.smallcircle.) Taxol.RTM. 15 mg/kg, 6
h (.quadrature.), Taxol.RTM. 24 mg/kg, 6 h (.DELTA.) and control
(). Treatment on days 5, 7 and 9.
[0086] FIG. 72 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing CCRF-CEM/VBL with Q2Dx5 i.v. 6 h infusion,
using using dEpoB 30 mg/kg (.quadrature.), Taxol.RTM. 20 mg/kg
(.DELTA.) and control (.diamond.). Treatment on days 19, 21, 23,
25, 27, 39, 41, 43, 45, 47 and 53.
[0087] FIG. 73 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing HT-29 Colon Adenocarcinoma with Q2Dx6 i.v. 6 h
infusion, using dEpoB 30 mg/kg (.DELTA.), Taxol.RTM. 15 mg/kg
(.quadrature.) and control (.diamond.).
[0088] FIG. 74 shows the therapeutic effect of dEpoB and Taxol.RTM.
in nude mice bearing A549 Human Lung Carcinoma with Q2Dx3 i.v. 6 or
18 h infusion, using dEpoB 30 mg/kg 18 h (x), dEpoB 40 mg/kg, 6 h
(*), dEpoB 50 mg/kg, 6 h (.smallcircle.), Taxol.RTM. 15 mg/kg, 6 h
(.quadrature.), Taxol.RTM. 24 mg/kg, 6 h (.DELTA.) and control ().
Treatment on days 7, 9 and 11.
[0089] FIG. 75 shows the Epi stability in plasma of dEpoB human
(.DELTA.) and mice (.circle-solid.).
DETAILED DESCRIPTION OF THE INVENTION
[0090] As used herein, the term "linear or branched chain alkyl"
encompasses, but is not limited to, methyl, ethyl, propyl,
isopropyl, t-butyl, sec-butyl, cyclopentyl or cyclohexyl. The alkyl
group may contain one carbon atom or as many as fourteen carbon
atoms, but preferably contains one carbon atom or as many as nine
carbon atoms, and may be substituted by various groups, which
include, but are not limited to, acyl, aryl, alkoxy, aryloxy,
carboxy, hydroxy, carboxamido and/or N-acylamino moieties.
[0091] As used herein, the terms "alkoxycarbonyl", "acyl" and
"alkoxy" encompass, but are not limited to, methoxycarbonyl,
ethoxycarbonyl, propoxycarbonyl, n-butoxycarbonyl,
benzyloxycarbonyl, hydroxypropylcarbonyl, aminoethoxycarbonyl,
sec-butoxycarbonyl and cyclopentyloxycarbonyl. Examples of acyl
groups include, but are not limited to, formyl, acetyl, propionyl,
butyryl and penanoyl. Examples of alkoxy groups include, but are
not limited to, methoxy, ethoxy, propoxy, n-butoxy, sec-butoxy and
cyclopentyloxy.
[0092] As used herein, an "aryl" encompasses, but is not limited
to, a phenyl, pyridyl, pyrryl, indolyl, naphthyl, thiophenyl or
furyl group, each of which may be substituted by various groups,
which include, but are not limited, acyl, aryl alkoxy, aryloxy,
carboxy, hydroxy, carboxamido or N-acylamino moieties. Examples of
aryloxy groups include, but are not limited to, a phenoxy,
2-methylphenoxy, 3-methylphenoxy and 2-naphthoxy. Examples of
acyloxy groups include, but are not limited to, acetoxy,
propanoyloxy, butyryloxy, pentanoyloxy and hexanoyloxy.
[0093] The subject invention provides chemotherapeutic analogues of
epothilone A and B, including a compound having the structure:
1
[0094] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CHY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and wherein X is
H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or
linear or branched chain alkyl; wherein Z is O, N(OR.sub.3) or
N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and R.sub.5 are
independently H or a linear or branched alkyl; and wherein n is 0,
1, 2, or 3. In one embodiment, the invention provides the compound
having the structure: 2
[0095] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,
CH.sub.2OH, or (CH.sub.2).sub.3OH.
[0096] The invention also provides a compound having the structure:
3
[0097] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CHY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and wherein X is
H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or
linear or branched chain alkyl; wherein Z is O, N(OR.sub.3) or
N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and R.sub.5 are
independently H or a linear or branched chain alkyl; and wherein n
is 0, 1, 2, or 3. In a certain embodiment, the invention provides a
compound having the structure: 4
[0098] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl or
CH.sub.2OH.
[0099] In addition, the invention provides a compound having the
structure: 5
[0100] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CHY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and wherein X is
H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or
linear or branched chain alkyl; wherein Z is O, N(OR.sub.3) or
N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and R.sub.5 are
independently H or a linear or branched chain alkyl; and wherein n
is 0, 1, 2, or 3. In particular, the invention provides a compound
having the structure: 6
[0101] wherein R is H, methyl, ethyl, n-propyl, n-butyl, CH.sub.2OH
or (CH.sub.2).sub.3OH.
[0102] The invention further provides a compound having the
structure: 7
[0103] wherein R, R.sub.0 and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
fluorine, NR.sub.1R.sub.2, N-hydroximino or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CHY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and wherein X is
H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or
linear or branched chain alkyl; wherein Z is O, N(OR.sub.3) or
N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and R.sub.5 are
independently H or a linear or branched chain alkyl; and wherein n
is 0, 1, 2 or 3.
[0104] The invention also provides a compound having the structure:
8
[0105] The subject invention also provides various intermediates
useful for the preparation of the chemotherapeutic compounds
epithilone A and B, as well as analogues thereof. Accordingly, the
invention provides a key intermediate to epothilone A and its
analogues having the structure: 9
[0106] wherein R is hydrogen, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl; wherein R' is
hydrogen, methyl, ethyl, n-propyl, n-hexyl, 10
[0107] CH.sub.2OTBS or (CH.sub.2).sub.3-OTBDPS; and X is a halide.
In one embodiment, the subject invention provides a compound of the
above structure wherein R is acetyl and X is iodo.
[0108] The subject invention also provides an intermediate having
the structure: 11
[0109] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein X is oxygen, (OR).sub.2, (SR).sub.2,
--(O--(CH.sub.2).sub.n--O)--, --(O--(CH.sub.2).sub.n--S)-- or
--(S--(CH.sub.2).sub.n--S)--; and wherein n is 2, 3 or 4. 12
[0110] wherein R is H or methyl.
[0111] Another analogue provided by the invention has the
structure: 13
[0112] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,
CH.sub.2OH, or (CH.sub.2).sub.3OH.
[0113] Additionally, the subject invention provides an analogue
having the structure: 14
[0114] wherein R is H or methyl. The scope of the present invention
includes compounds wherein the C.sub.3 carbon therein possesses
either an R or S absolute configuration, as well as mixtures
thereof.
[0115] The subject invention further provides an analogue of
epothilone A having the structure: 15
[0116] The subject invention also provides synthetic routes to
prepare the intermediates for preparing epothilones. Accordingly,
the invention provides a method of preparing a Z-iodoalkene ester
having the structure: 16
[0117] wherein R is hydrogen, a linear or branched alkyl,
alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, which
comprises (a) coupling a compound having the structure: 17
[0118] with a methyl ketone having the structure: 18
[0119] wherein R' and R" are independently a linear or branched
alkyl, alkoxyalkyl, substituted or unsubstituted aryl or benzyl,
under suitable conditions to form a compound having the structure:
19
[0120] (b) treating the compound formed in step (a) under suitable
conditions to form a Z-iodoalkene having the structure: 20
[0121] and (c) deprotecting and acylating the Z-iodoalkene formed
in step (b) under suitable conditions to form the Z-iodoalkene
ester. The coupling in step (a) may be effected using a strong base
such as n-BuLi in an inert polar solvent such as tetrahydrofuran
(THF) at low temperatures, typically below -50.degree. C., and
preferably at -78.degree. C. The treatment in step (b) may comprise
sequential reaction with N-iodosuccinimide in the presence of
Ag(I), such as silver nitrate, in a polar organic solvent such as
acetone, followed by reduction conditions, typically using a
hydroborating reagent, preferably using Cy.sub.2BH. Deprotecting
step (c) involves contact with a thiol such as thiophenol in the
presence of a Lewis acid catalyst, such as boron
trifluoride-etherate in an inert organic solvent such as
dichloromethane, followed by acylation with an acyl halide, such as
acetyl chloride, or an acyl anhydride, such as acetic anhydride in
the presence of a mild base such as pyridine and/or
4-dimethyaminopyridine (DMAP) in an inert organic solvent such as
dichloromethane.
[0122] The subject invention also provides a method of preparing a
Z-haloalkene ester having the structure: 21
[0123] wherein R is hydrogen, a linear or branched alkyl,
alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein X is a halogen, which comprises (a) oxidatively cleaving a
compound having the structure: 22
[0124] under suitable conditions to form an aldehyde intermediate;
and (b) condensing the aldehyde intermediate with a halomethylene
transfer agent under suitable conditions to form the Z-haloalkene
ester. In one embodiment of the method, X is iodine. In another
embodiment, the method is practiced wherein the halomethylene
transfer agent is Ph.sub.3P.dbd.CHl or
(Ph.sub.3P.sup.+CH.sub.2l)I.sup.-. Disubstituted olefins may be
prepared using the haloalkylidene transfer agent
Ph.sub.3P.dbd.CR'I, wherein R' is hydrogen, methyl, ethyl,
n-prop-yl, n-hexyl, 23
[0125] CO.sub.2Et or (CH.sub.2).sub.3OTBDPS. The oxidative step (a)
can beperformed using a mild oxidant such as osmium tetraoxide at
temperatures of about 0.degree. C., followed by treatment with
sodium periodate, or with lead tetraacetate/sodium carbonate, to
complete the cleavage of the terminal olefin, and provide a
terminal aldehyde. Condensing step (b) occurs effectively with a
variety of halomethylenating reagents, such as Wittig reagents.
[0126] The subject invention further provides a method of preparing
an optically pure compound having the structure: 24
[0127] wherein R is hydrogen, a linear or branched alkyl,
alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, which
comprises: (a) condensing an allylic organometallic reagent with an
unsaturated aldehyde having the structure: 25
[0128] under suitable conditions to form an alcohol, and,
optionally concurrently therewith, optically resolving the alcohol
to form an optically pure alcohol having the structure: 26
[0129] (b) alkylating or acylating the optically pure alcohol
formed in step (a) under suitable conditions to form the optically
pure compound. In one embodiment of the method, the allylic
organometallic reagent is an allyl(trialkyl)stannane. In another
embodiment, the condensing step is effected using a reagent
comprising a titanium tetraalkoxide and an optically active
catalyst. In step (a) the 1,2-addition to the unsaturated aldehyde
may be performed using a variety of allylic organometallic
reagents, typically with an allyltrialkylstannane, and preferably
with allyltri-n-butylstannane, in the presence of chiral catalyst
and molecular sieves in an inert organic solvent such as
dichloromethane. Preferably, the method may be practiced using
titanium tetraalkoxides, such as titanium tetra-n-propoxide, and
S-(-)BINOL as the optically active catalyst. Alkylating or
acylating step (b) is effected using any typical alkylating agent,
such as alkylhalide or alkyl tosylate, alkyl triflate or alkyl
mesylate, any typical acylating agent, such as acetyl chloride,
acetic anhydride, benzoyl chloride or benzoyl anhydride, in the
presence of a mild base catalyst in an inert organic solvent, such
as dichloromethane.
[0130] The subject invention also provides a method of preparing an
open-chain aldehyde having the structure: 27
[0131] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, which
comprises: (a) cross-coupling a haloolefin having the structure:
28
[0132] wherein R is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, and X
is a halogen, with a terminal olefin having the structure: 29
[0133] wherein (OR'").sub.2 is (OR.sub.0).sub.2, (SR.sub.0).sub.2,
--(O--(CH.sub.2).sub.n--O)--, --(O--(CH.sub.2).sub.n--S)-- or
--(S--(CH.sub.2).sub.n--S)-- where R.sub.0 is a linear or branched
alkyl, substituted or unsubstituted aryl or benzyl; and wherein n
is 2, 3 or 4, under suitable conditions to form a cross-coupled
compound having the structure: 30
[0134] wherein Y is CH(OR*).sub.2 where R* is a linear or branched
alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl; and
(b) deprotecting the cross-coupled compound formed in step (a)
under suitable conditions to form the open-chain compound.
Cross-coupling step (a) is effected using reagents known in the art
which are suited to the purpose. For example, the process may be
carried out by hydroborating the pre-acyl component with 9-BBN. The
resulting mixed borane may then be cross-coupled with an
organometallic catalyst such as PdCl.sub.2(dppf).sub.2, or any
known equivalent thereof, in the presence of such ancillary
reagents as cesium carbonate and triphenylarsine. Deprotecting step
(b) can be carried out with a mild acid catalyst such as p-tosic
acid, and typically in a mixed aqueous organic solvent system, such
as dioxane-water. The open-chain compound can be cyclized using any
of a variety of non-nucleophilic bases, such as potassium
hexamethyldisilazide or lithium diethyamide.
[0135] The subject invention also provides a method of preparing an
epothilone having the structure: 31
[0136] which comprises: (a) deprotecting a cyclized compound having
the structure: 32
[0137] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, under
suitable conditions to form a deprotected cyclized compound and
oxidizing the deprotected cyclized compound under suitable
conditions to form a desoxyepothilone having the structure: 33
[0138] and (b) epoxidizing the desoxyepothilone formed in step (a)
under suitable conditions to form the epothilone. Deprotecting step
(a) is effected using a sequence of treatments comprising a
catalyst such as HF-pyridine, followed by t-butyldimethylsilyl
triflate in the presence of a base such as lutidine. Dess-Martin
oxidation and further deprotection with a catalyst such as
HF-pyridine provides the desoxyepothilone. The latter compound can
then be epoxidized in step (b) using any of a variety of
epoxidizing agents, such acetic peracid, hydrogen peroxide,
perbenzoic acid, m-chloroperbenzoic acid, but preferably with
dimethyldioxirane, in an inert organic solvent such as
dichloromethane.
[0139] The subject invention further provides a method of preparing
an epothilone precursor having the structure: 34
[0140] wherein R.sub.1 is hydrogen or methyl; wherein X is O, or a
hydrogen and OR", each singly bonded to carbon; and wherein
R.sub.0, R' and R" are independently hydrogen, a linear or branched
alkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl, which comprises (a)
coupling a compound having the structure: 35
[0141] wherein R is an acetyl, with an aldehyde having the
structure: 36
[0142] wherein Y is oxygen, under suitable conditions to form an
aldol intermediate and optionally protecting the aldol intermediate
under suitable conditions to form an acyclic epthilone precursor
having the structure: 37
[0143] (b) subjecting the acylic epothilone precursor to conditions
leading to intramolecular olefin metathesis to form the epothilone
precursor. In one embodiment of the method, the conditions leading
to intramolecular olefin metathesis require the presence of an
organometallic catalyst. In a certain specific embodiment of the
method, the catalyst contains Ru or Mo. The coupling step (a) may
be effected using a nonnucleophilic base such as lithium
diethylamide or lithium diisopropylamide at subambient
temperatures, but preferably at about -78.degree. C. The olefin
metathesis in step (b) may be carried out using any catalyst known
in the art suited for the purpose, though preferably using one of
Grubbs's catalysts.
[0144] In addition, the present invention provides a compound
useful as an intermediate for preparing epothilones having the
structure: 38
[0145] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsityl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein X is oxygen, (OR*).sub.2, (SR*).sub.2,
--(O--(CH.sub.2).sub.n--O)--, --(O--(CH.sub.2).sub.n--S)-- or
--(S--(CH.sub.2).sub.n--S)--; wherein R* is a linear or branched
alkyl, substituted or unsubstituted aryl or benzyl; wherein
R.sub.2B is a linear, branched or cyclic boranyl moiety; and
wherein n is 2, 3 or 4. In certain embodiments, the invention
provides the compound wherein R' is TBS, R" is TPS and X is
(OMe).sub.2. A preferred example of R.sub.2B is derived from
9-BBN.
[0146] The invention also provides the compound having the
structure: 39
[0147] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein X is oxygen, (OR).sub.2, (SR).sub.2,
--(O--(CH.sub.2).sub.n--O)--, --(O--(CH.sub.2).sub.n--S)-- or
--(S--(CH.sub.2).sub.n--S)--; and wherein n is 2, 3 or 4. In
certain embodiments, the invention provides the compound wherein R'
is TBS, R" is TPS and X is (OMe).sub.2.
[0148] The invention further provides a desmethylepothilone
analogoue having the structure: 40
[0149] wherein R is H or methyl.
[0150] The invention provides a compound having the structure:
41
[0151] wherein R is H or methyl.
[0152] The invention also provides a trans-desmethyldeoxyepothilone
analogue having the structure: 42
[0153] wherein R is H or methyl.
[0154] The invention also provides a trans-epothilone having the
structure: 43
[0155] wherein R is H or methyl.
[0156] The invention also provides a compound having the structure:
44
[0157] wherein R is hydrogen, a linear or branched alkyl,
alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein R' is hydrogen, methyl, ethyl, n-propyl, n-hexyl, 45
[0158] CO.sub.2Et or (CH.sub.2).sub.3OTBDPS. and X is a halogen. In
certain embodiments, the invention provides the compound wherein R
is acetyl and X is iodine.
[0159] The invention additionally provides a method of preparing an
open-chain aldehyde having the structure: 46
[0160] wherein R is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R' and R" are independently hydrogen, a linear or branched
alkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl, which comprises:
[0161] (a) cross-coupling a haloolefin having the structure: 47
[0162] wherein X is a halogen, with a terminal borane having the
structure: 48
[0163] wherein R*.sub.2B is a linear, branched or cyclic alkyl or
substituted or unsubstituted aryl or benzyl boranyl moiety; and
wherein Y is (OR.sub.0).sub.2, (SR.sub.0).sub.2,
--(O--(CH.sub.2).sub.n--O)--, --(O--(CH.sub.2).sub.n--S)-- or
--(S--(CH.sub.2).sub.n--S)-- where R.sub.0 is a linear or branched
alkyl, substituted or unsubstituted aryl or benzyl; and wherein n
is 2, 3 or 4, under suitable conditions to form a cross-coupled
compound having the structure: 49
[0164] and
[0165] (b) deprotecting the cross-coupled compound formed in step
(a) under suitable conditions to form the open-chain aldehyde. In
certain embodiments, the invention provides the method wherein R is
acetyl; R' is TBS; R" is TPS; R*.sub.2B is derived from 9-BBN; and
Y is (OMe).sub.2. Cross-coupling step (a) is effected using
reagents known in the art which are suited to the purpose. For
example, the mixed borane may be cross-coupled with an
organometallic catalyst such as PdCl.sub.2(dppf).sub.2, or any
known equivalent thereof, in the presence of such reagents as
cesium carbonate and triphenylarsine. Deprotecting step (b) can be
carried out using a mild acid catalyst such as p-tosic acid,
typically in a mixed aqueous organic solvent system, such as
dioxane-water.
[0166] The invention also provides a method of preparing a
protected epothilone having the structure: 50
[0167] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkyl-arylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, which
comprises:
[0168] (a) monoprotecting a cyclic diol having the structure:
51
[0169] under suitable conditions to form a cyclic alcohol having
the structure: 52
[0170] and
[0171] (b) oxidizing the cyclic alcohol formed in step (a) under
suitable conditions to form the protected epothilone. In certain
embodiments, the invention provides the method wherein R' and R"
are TBS. The monoprotecting step (a) may be effected using any of a
variety of suitable reagents, including TBSOTf in the presence of a
base in an inert organic solvent. The base may be a
non-nucleophilic base such as 2,6-lutidine, and the solvent may be
dichloromethane. The reaction is conducted at subambient
temperatures, preferably in the range of -30.degree. C. The
oxidizing step (b) utilizes a selective oxidant such as Dess-Martin
periodinane in an inert organic solvent such as dichloromethane.
The oxidation is carried out at ambient temperatures, preferably at
20-25.degree. C.
[0172] The invention further provides a method of preparing an
epothilone having the structure: 53
[0173] which comprises:
[0174] (a) deprotecting a protected cyclic ketone having the
structure: 54
[0175] wherein R' and R" are independently hydrogen, a linear or
branched alkyl, substituted or unsubstituted aryl or benzyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl, under
suitable conditions to form a desoxyepothilone having the
structure: 55
[0176] and (b) epoxidizing the desoxyepothilone formed in step (a)
under suitable conditions to form the epothilone. In certain
embodiments, the invention provides the method wherein R' and R"
are TBS. Deprotecting step (a) is carried out by means of a
treatment comprising a reagent such as HF.pyridine. The deprotected
compound can be epoxidized in step (b) using an epoxidizing agent
such acetic peracid, hydrogen peroxide, perbenzoic acid,
m-chloroperbenzoic acid, but preferably with dimethyldioxirane, in
an inert organic solvent such as dichloromethane.
[0177] The invention also provides a method of preparing a cyclic
diol having the structure: 56
[0178] wherein R' is a hydrogen, a linear or branched alkyl,
substituted or unsubstituted aryl or benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl, which comprises:
[0179] (a) cyclizing an open-chain aldehyde having the structure:
57
[0180] wherein R is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R" is a hydrogen, a linear or branched alkyl, substituted
or unsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, a linear or branched acyl, substituted or
unsubstituted aroyl or benzoyl under suitable conditions to form an
enantiomeric mixture of a protected cyclic alcohol having the
structure: 58
[0181] said mixture comprising an .alpha.- and a .beta.-alcohol
component;
[0182] (b) optionally isolating and oxidizing the .alpha.-alcohol
formed in step (a) under suitable conditions to form a ketone and
thereafter reducing the ketone under suitable conditions to form an
enantiomeric mixture of the protected cyclic alcohol comprising
substantially the .beta.-alcohol; and
[0183] (c) treating the protected cyclic alcohol formed in step (a)
or (b) with a deprotecting agent under suitable conditions to form
the cyclic diol. In certain embodiments, the invention provides the
method wherein R' is TBS and R" is TPS. Cyclizing step (a) is
performed using any of a variety of mild nonnucleophilic bases such
as KHMDS in an inert solvent such as THF. The reaction is carried
out at subambient temperatures, preferably between -90.degree. C.
and -50.degree. C., more preferably at -78.degree. C. Isolation of
the unnatural alpha-OH diastereomer is effected by any usual
purification method, including any suitable type of chromatography
or by crystallization. Chromatographic techniques useful for the
purpose include high pressure liquid chromatography, countercurrent
chromatography or flash chromatography. Various column media are
suited, including, inter alia, silica or reverse phase support. The
beta-OH derivative is then oxidized using a selective oxidant, such
as Dess-Martin periodinane. The resulting ketone is the reduced
using a selective reductant. Various hydridoborane and aluminum
hydride reagents are effective. A preferred reducing agent is
sodium borohydride. Treating step (c ) may be effected using a
variety of deprotecting agents, including HF-pyridine.
[0184] The present invention provides a method of preparing a
desoxyepothilone having the structure: 59
[0185] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CX--, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein Z is O,
N(OR.sub.3) or N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched alkyl; and
wherein n is 0, 1, 2, or 3; which comprises treating an epothilone
having a structure: 60
[0186] wherein R, R.sub.0, R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R', R", X, Y, Z and n are defined as for the
desoxyepothilone, under suitable conditions so as to deoxygenate
the epothilone, and thereby to provide the desoxyepothilone. In one
embodiment, the method is effected wherein the desoxyepothilone has
the structure: 61
[0187] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,
62
[0188] or (CH.sub.2).sub.3--OH. In another embodiment, the method
is effected wherein the epothilone is deoxygenated using a
zinc/copper couple. Preferably, the method is carried out wherein
the epothilone is deoxygenated in the presence of a polar solvent
comprising isopropanol and water.
[0189] The present invention further provides a method of preparing
a desoxyepothilone- having the structure: 63
[0190] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein Z is O,
N(OR.sub.3) or N--NR.sub.4R.sub.5, wherein R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched chain alkyl;
and wherein n is 0, 1, 2, or 3; which comprises treating an
epothilone having a structure: 64
[0191] wherein R, R.sub.0, R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R', R", X, Y, Z and n are defined as for the
desoxyepothilone, under suitable conditions so as to deoxygenate
the epothilone, and thereby to provide the desoxyepothilone. In one
embodiment, the method is performed wherein the desoxyepothilone
has the structure: 65
[0192] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl or
hydroxypropyl. Preferably, the method is effected wherein the
epothilone is deoxygenated using a zinc/copper couple. Favorably,
the epothilone is deoxygenated in the presence of a polar solvent
comprising isopropanol and water.
[0193] The present invention also provides a method of preparing a
desoxyepothilone having the structure: 66
[0194] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; and wherein Z is
O, N(OR.sub.3) or N--NR.sub.4R.sub.5 where R.sub.3, R.sub.4 and
R.sub.5 are independently H or a linear or branched alkyl; and
wherein n is 0, 1, 2, or 3; which comprises treating a protected
desoxyepothilone having the structure: 67
[0195] wherein R.sub.A is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyloxy- carbonyl, (dialkylarylsilyl)
alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, triarysilyl, a linear or branched acyl,
substituted or unsubstituted aroyl or benzoyl; under suitable
conditions to form the desoxyepothilone. In a certain embodiment,
the invention provides a method wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl. Preferably, the method is effected
wherein R.sub.A is TES and R.sub.B is Troc. In a certain other
embodiment, the invention provides a method wherein the treating
step comprises contacting the protected desoxyepothilone (i) with
SmX.sub.2, where X is Cl, Br or I, in the presence of a polar
nonaqueous solvent selected from the group consisting of
tetrahydrofuran, p-dioxane, diethyl ether, acetonitrile and
N,N-dimethylformamide, and optionally in the presence of
N,N-dimethyl-N'-propylurea or hexamethylphosphoramide and (ii) with
a source of fluoride ion selected from the group consisting of
tetra-n-methylammonium fluoride, tetra-n-butylammonium fluoride and
HF.pyridine.
[0196] The present invention also provides a method of preparing a
protected desoxyepothilone having the structure: 68
[0197] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.A is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyloxy- carbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; which
comprises cyclocondensing a hydroxy acid desoxyepothilone precursor
having the structure: 69
[0198] wherein R, R.sub.0, R.sub.A, R.sub.B, R', R" and n are
defined as above; under suitable conditions to form the protected
desoxyepothilone. In particular, the method is carried out wherein
n is 3 and R" is 2-methyl-1,3-thiazolinyl. Preferably, R.sub.A is a
trialkylsilyl, and is more preferably, TES. R.sub.B is favorably
trichloroethyloxycarbonyl (Troc). According to the method, the
hydroxy acid desoxyepothilone precursor is cyclocondensed using a
cyclocondensing reagent selected from the group consisting of
acetic anhydride, pentafluorophenol, 2,4-dichlorobenzoyl chloride
and, preferably, 2,4,6-trichlorobenzoyl chloride. In addition, the
hydroxyacid is favorably cyclocondensed using
2,4,6-trichlorobenzoyl chloride in the presence of a tertiary amine
selected from the group consisting of triethyl amine,
tri-n-propylamine, diisopropylethylamine and
diethyliso-propylamine, and optionally in the presence of pyridine
or N,N-dimethylaminopyridine.
[0199] The present invention further provides a method of preparing
a hydroxy acid desoxyepothilone precursor having the structure:
70
[0200] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.A is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl;
wherein R.sub.B is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl) alkyloxycarbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; which
comprises selectively etherifying and hydrolyzing a hydroxy ester
desoxyepothilone precursor having the structure: 71
[0201] wherein R, R.sub.0, R.sub.B, R.sub.C, R', R" and n are
defined as above; and wherein R.sub.C is tertiary-alkyl; under
suitable conditions to form the hydroxy acid desoxyepothilone
precursor. In one embodiment, the invention is practiced wherein n
is 3 and R" is 2-methyl-1,3-thiazolinyl. Preferably, R.sub.A is TES
and R.sub.B is Troc. In accord with invention, the method is
performed wherein the selective etherifying step comprises
contacting the hydroxy ester desoxyepothilone precursor with a
silylating reagent to form an ether intermediate, and the
hydrolyzing step comprises contacting the ether intermediate with a
protic acid or tetra-n-butylammonium fluoride. Favorably, the
silylating reagent is TESOTf in the presence of 2,6-lutidine. The
protic acid is typically HCl in the presence of an alkyl alcohol,
preferably, methyl alcohol or ethyl alcohol.
[0202] The present invention also provides a method of preparing a
hydroxy ester desoxyepothilone precursor having the structure:
72
[0203] wherein R, R.sub.0, and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.B is hydrogen, t-butyloxycarbonyl,
amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl,
(dialkylarylsilyl)alkyl- oxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; and wherein
R.sub.C is tertiary-alkyl; which comprises reducing a hydroxy
ketoester desoxyepothilone precursor having the structure: 73
[0204] wherein P, R, R.sub.0, R.sub.B, R.sub.C, R', R" and n are
defined as above; under suitable conditions to form the hydroxy
ester desoxyepothilone precursor. In one embodiment, the invention
provides the method wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl. In particular, R.sub.A is a trialkylsily
group, and is, preferably, TES. R.sub.B is Troc. In accord with the
invention, the reducing step comprises contacting the hydroxy
ketoester desoxyepothilone precursor with a stereospecific reducing
reagent. The stereospecific reducing reagent favorably comprises
hydrogen gas at from about 900 pounds per square inch to about 2200
pounds per square inch in the presence of (R)-(BINAP)RuCl.sub.2 and
optionally in the presence of HCl and an alcohol selected from the
group consisting of MeOH, EtOH, and I-PrOH. More preferably, the
hydrogen gas pressure is 1200 psi.
[0205] The present invention further provides a method of preparing
a hydroxy ketoester desoxyepothilone precursor having the
structure: 74
[0206] wherein P is H; wherein R, R.sub.0, and R' are independently
H, linear or branched chain alkyl, optionally substituted by
hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl
or cyclic acetal, fluorine, NR.sub.1R.sub.2, N-hydroximino, or
N-alkoxyimino, wherein R.sub.1 and R.sub.2 are independently H,
phenyl, benzyl, linear or branched chain alkyl; wherein R" is
--CY.dbd.CHX, or H, linear or branched chain alkyl, phenyl,
2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.B is hydrogen, t-butyloxycarbonyl,
amyloxycarbonyl, (trialkylsilyl)alkyloxycar- bonyl,
(dialkylarylsilyl) alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.C is tertiary-alkyl; which comprises deprotecting a
protected ketoester desoxyepothilone precursor having the
structure: 75
[0207] wherein R, R.sub.0, R.sub.A, R.sub.B, R.sub.C, R', R" and n
are defined as above; and wherein P is a linear or branched alkyl,
alkoxyalkyl, substituted or unsubstituted aryloxyalkyl,
trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or triarylsilyl;
under suitable conditions to form the hydroxy ketoester
desoxyepothilone precursor. In one embodiment, n is 3 and R" is
2-methyl-1,3-thiazolinyl. R.sub.A is typically trialkylsilyl, and,
preferably, is TES. R.sub.B is favorably Troc. The method is
effectively practiced wherein P is TBS. In accord with the
invention, the deprotecting step comprises contacting the protected
ketoester desoxyepothilone precursor with a protic acid.
Preferably, the protic acid is HCl in methyl alcohol or ethyl
alcohol.
[0208] The present invention also provides a method of preparing a
protected ketoester desoxyepothilone precursor having the
structure: 76
[0209] wherein P is a linear or branched alkyl, alkoxyalkyl,
substituted or unsubstituted aryloxyalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl or triarylsilyl; wherein R,
R.sub.0, and R' are independently H, linear or branched chain
alkyl, optionally substituted by hydroxy, alkoxy, carboxy,
carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein R" is --CY.dbd.CHX, or H, linear or
branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,
3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,
2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H,
linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,
2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl;
wherein Y is H or linear or branched chain alkyl; wherein n is 2 or
3; wherein R.sub.B is hydrogen, t-butyloxycarbonyl,
amyloxycarbonyl, (trialkylsilyl)alkyl-ox- ycarbonyl,
(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or
branched acyl, substituted or unsubstituted aroyl or benzoyl; and
wherein R.sub.C is tertiary-alkyl; which comprises coupling a
terminal vinyl enol ether ester having the structure: 77
[0210] wherein R, R.sub.0, R.sub.B, R.sub.C, and R' are defined as
above; wherein m is 0, 1 or 2; and wherein R.sub.D is linear or
branched alkyl, benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, linear or branched acyl, substituted or
unsubstituted aroyl or benzoyl; with a protected halovinyl or
metalvinyl compound having the structure: 78
[0211] wherein R, P and R" are defined as above; and wherein Q is a
halide or a metal; under suitable conditions to form the protected
ketoester desoxyepothilone precursor. In one embodiment, the
invention provides the method wherein n is 3 and R" is
2-methyl-1,3-thiazolinyl. In another embodiment, the method is
effectively performed wherein R.sub.A is a trialkylsilyl group, and
is, preferably, TES , and R.sub.B is Troc. P is favorably TBS or
TES, and Q is iodine or bromine. R.sub.D is typically methyl or
TES. In accord with the invention, the coupling step comprises
contacting the terminal vinyl enol ether ester and the protected
halovinyl compound with noble metal complex capable of effecting a
Suzuki coupling. For this step, the noble metal complex is
effectively chosen as Pd(dppf).sub.2Cl.sub.2 in the presence of
Ph.sub.3As and Cs.sub.2CO.sub.3.
[0212] The present invention also provides a method of preparing a
terminal vinyl enol ether ester having the structure: 79
[0213] wherein R.sub.0 and R' are independently H, linear or
branched chain alkyl, optionally substituted by hydroxy, alkoxy,
carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,
fluorine, NR.sub.1R.sub.2, N-hydroximino, or N-alkoxyimino, wherein
R.sub.1 and R.sub.2 are independently H, phenyl, benzyl, linear or
branched chain alkyl; wherein m is 0, 1 or 2; wherein R.sub.B is
hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,
(trialkylsilyl)alkyl-oxycarbonyl,
(dialkylarylsilyl)alky-loxycarbonyl, benzyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched
acyl, substituted or unsubstituted aroyl or benzoyl; wherein
R.sub.C is tertiary-alkyl; and wherein R.sub.D is linear or
branched alkyl, benzyl, trialkylsilyl, dialkylarylsilyl,
alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted
or unsubstituted aroyl or benzoyl; which comprises:
[0214] (a) treating a keto enol ester having the structure: 80
[0215] under suitable conditions to form an enolate enol ester
having the structure: 81
[0216] wherein M is Li, Na or K; and
[0217] (b) coupling the enolate enol ester with a vinyl aldehyde
having the structure: 82
[0218] wherein m, and R.sub.0 and R' are as defined above; under
suitable conditions to form the terminal vinyl enol ether ester. In
accord with the invention, the treating step comprises contacting
the keto enol ester with a strong nonnucleophilic base selected
from the group consisting of lithium diethylamide, lithium
diethylamide, lithium diisopropylamide, lithium hydride, sodium
hydride, potassium hydride and potassium t-butoxide. Preferably,
the treating step is effected in a polar nonaqueous solvent
selected from the group consisting of tetrahydrofuran, diethyl
ether, di-n-propyl ether and dimethylformamide at a temperature
from about -100.degree. C. to about +10.degree. C. More preferably,
the temperature is from about -20.degree. C. to -40.degree. C. The
coupling step as practiced in the invention comprises contacting
the enolate enol ester with the vinyl aldehyde at a temperature
from about -130.degree. C. to about -78.degree. C.
[0219] In addition, the invention provides a method of treating
cancer in a subject suffering therefrom comprising administering to
the subject a therapeutically effective amount of any of the
analogues related to epothilone B disclosed herein optionally in
combination with an additional chemotherapeutic agent and/or with a
pharmaceutically suitable carrier. The method may be applied where
the cancer is a solid tumor or leukemia. In particular, the method
is applicable where the cancer is breast cancer or melanoma.
[0220] The subject invention also provides a pharmaceutical
composition for treating cancer comprising any of the analogues of
epothilone disclosed hereinabove, as an active ingredient,
optionally though typically in combination with an additional
chemotherapeutic agent and/or a pharmaceutically suitable carrier.
The pharmaceutical compositions of the present invention may
further comprise other therapeutically active ingredients.
[0221] The subject invention further provides a method of treating
cancer in a subject suffering therefrom comprising administering to
the subject a therapeutically effective amount of any of the
analogues of epothilone disclosed hereinabove and a
pharmaceutically suitable carrier. The method is especially useful
where the cancer is a solid tumor or leukemia.
[0222] The compounds taught above which are related to epothilones
A and B are useful in the treatment of cancer, and particularly, in
cases where multidrug resistance is present, both in vivo and in
vitro. The ability of these compounds as non-substrates of MDR in
cells, as demonstrated in the Tables below, shows that the
compounds are useful to treat, prevent or ameliorate cancer in
subjects suffering therefrom.
[0223] The magnitude of the therapeutic dose of the compounds of
the invention will vary with the nature and severity of the
condition to be treated and with the particular compound and its
route of administration. In general, the daily dose range for
anticancer activity lies in the range of 0.001 to 25 mg/kg of body
weight in a mammal, preferably 0.001 to 10 mg/kg, and most
preferably 0.001 to 1.0 mg/kg, in single or multiple doses. In
unusual cases, it may be necessary to administer doses above 25
mg/kg.
[0224] Any suitable route of administration may be employed for
providing a mammal, especially a human, with an effective dosage of
a compound disclosed herein. For example, oral, rectal, topical,
parenteral, ocular, pulmonary, nasal, etc., routes may be employed.
Dosage forms include tablets, troches, dispersions, suspensions,
solutions, capsules, creams, ointments, aerosols, etc.
[0225] The compositions include compositions suitable for oral,
rectal, topical (including transdermal devices, aerosols, creams,
ointments, lotions and dusting powders), parenteral (including
subcutaneous, intramuscular and intravenous), ocular (ophthalmic),
pulmonary (nasal or buccal inhalation) or nasal administration.
Although the most suitable route in any given case will depend
largely on the nature and severity of the condition being treated
and on the nature of the active ingredient. They may be
conveniently presented in unit dosage form and prepared by any of
the methods well known in the art of pharmacy.
[0226] In preparing oral dosage forms, any of the unusual
pharmaceutical media may be used, such as water, glycols, oils,
alcohols, flavoring agents, preservatives, coloring agents, and the
like in the case of oral liquid preparations (e.g., suspensions,
elixers and solutions); or carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents, etc., in the case of
oral solid preparations are preferred over liquid oral preparations
such as powders, capsules and tablets. If desired, capsules may be
coated by standard aqueous or non-aqueous techniques. In addition
to the dosage forms described above, the compounds of the invention
may be administered by controlled release means and devices.
[0227] Pharmaceutical compositions of the present invention
suitable for oral administration may be prepared as discrete units
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient in powder or granular
form or as a solution or suspension in an aqueous or nonaqueous
liquid or in an oil-in-water or water-in-oil emulsion. Such
compositions may be prepared by any of the methods known in the art
of pharmacy. In general compositions are prepared by uniformly and
intimately admixing the active ingredient with liquid carriers,
finely divided solid carriers, or both and then, if necessary,
shaping the product into the desired form. For example, a tablet
may be prepared by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient in a
free-flowing form such as powder or granule optionally mixed with a
binder, lubricant, inert diluent or surface active or dispersing
agent. Molded tablets may be made by molding in a suitable machine,
a mixture of the powdered compound moistened with an inert liquid
diluent.
[0228] Methods of preparation of intermediates are disclosed in
U.S. patent application Ser. Nos. 60/032,282, 60/033,767,
60/047,566, 60/047,941, and 60/055,533, filed Dec. 3, 1996, Jan.
14, 1997, May 22, 1997, May 29, 1997, and Aug. 13, 1997,
respectively, the contents of which are hereby incorporated by
reference into this application.
[0229] The present invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described in
the claims which follow thereafter. It will be understood that the
processes of the present invention for preparing epothilones A and
B, analogues thereof and intermediates thereto encompass the use of
various alternate protecting groups known in the art. Those
protecting groups used in the disclosure including the Examples
below are merely illustrative.
EXAMPLE 1
[0230] THP Glycidol; 13:
[0231] A solution of (R)-(+)-glycidol 12 (20 g; 270 mmol) and
freshly distilled 3,4-dihydro-2H-pyran (68.1 g; 810 mmol) in
CH.sub.2Cl.sub.2 (900 ml) was treated with pyridinium
p-toluenesulfonate (2.1 g; 8.36 mmol) at rt and the resulting
solution was stirred for 16 h. Approximately 50% of the solvent was
then removed in vacuo and the remaining solution was diluted with
ether (1 L). The organic layer was then washed with two portions of
saturated aqueous sodium bicarbonate (500 ml), dried
(Na.sub.2SO.sub.4), filtered, and concentrated. Purification of the
residue by flash chromatography (silica, 25.fwdarw.50%
ether:hexanes) afforded THP glycidol 13 (31.2 g; 73%) as a
colorless liquid: IR(film): 2941, 1122, 1034 cm.sup.-1; .sup.1H
NMR(CDCl.sub.3, 500 MHz).delta.4.66(t, J=3.5 Hz, 1H), 4.64 (t,
J=3.5 Hz, 1H), 3.93 (dd, J=11.7, 3.1 Hz, 1H), 3.86 (m, 2H), 3.73
(dd, J=11.8, 5.03 Hz, 1H), 3.67 (dd, J=11.8, 3.4 Hz, 1H), 3.51 (m,
2H), 3.40 (dd, J=11.7, 6.4, 1H), 3.18 (m, 2H), 2.80 (m, 2H), 2.67
(dd, J=5.2, 2.7 Hz, 1H), 2.58 (dd, J=5.0, 2.7 Hz, 1H), 1.82 (m,
2H), 1.73 (m, 2H), 1.52 (m, 4H); .sup.13C NMR (CDCl.sub.3, 125
MHz).delta.98.9, 98.8, 68.5, 67.3, 62.4, 62.2, 50.9, 50.6, 44.6,
44.5, 30.5, 30.4, 25.4, 19.3, 19.2; [.alpha.].sub.D=+4.98 (c=2.15,
CHCl.sub.3).
EXAMPLE 2
[0232] Alcohol 13a:
[0233] Trimethylsilylacetylene (32.3 g; 329 mmol) was added via
syringe to THF (290 ml), and the resulting solution was cooled to
-78.degree. C. and treated with n-butyllithium (154 ml of a 1.6 M
solution in hexanes; 246.4 mmol). After 15 min, boron trifluoride
diethyl etherate (34.9 g; 246 mmol) was added, and the resulting
mixture was stirred for 10 min. A solution of epoxide 13 (26 g;
164.3 mmol) in THF (130 ml) was then added via a cannula and the
resulting solution was stirred for 5.5 h at -78.degree. C. The
reaction was quenched by the addition of saturated aqueous sodium
bicarbonate solution (250 ml) and the solution was allowed to warm
to rt. The mixture was then diluted with ether (600 ml) and washed
successively with saturated aqueous sodium bicarbonate solution
(250 ml), water (250 ml), and brine (250 ml). The organic layer was
then dried (Na.sub.2SO.sub.4), filtered, and concentrated in vacuo.
Purification of the residue by flash chromatography (silica, 20%
ether:hexanes) provided alcohol 13a (34 g; 76%).
EXAMPLE 3
[0234] MOM Ether 13b:
[0235] A solution of alcohol 13a (24 g; 88.9 mmol) and
N,N-diisopropylethylamine (108 ml; 622 mmol) in anhydrous
1,2-dichloroethane (600 ml) was treated with chloromethyl methyl
ether (17 ml; 196 mmol), and the resulting mixture was heated to
55.degree. C. for 28 h. The dark mixture was then cooled to rt and
treated with saturated aqueous sodium bicarbonate solution (300
ml). The layers were separated, and the organic layer was washed
successively with saturated aqueous sodium bicarbonate solution
(200 ml) and brine (200 ml). The organic layer was then dried
(MgSO.sub.4) and filtered through a pad of silica gel (ether
rinse). Purification of the residue by flash chromatography
(silica, 20.fwdarw.30% ether:hexanes) afforded MOM ether 13b (23.7
g; 85%) as a pale yellow oil.
EXAMPLE 4
[0236] Alcohol 14:
[0237] A solution of THP ether 13b (20 g; 63.7 mmol) in methanol
(90 ml) was treated with pyridinium p-toluenesulfonate (4.0 g; 15.9
mmol) and the resulting mixture was stirred at rt for 16 h. The
reaction was then quenched by the addition of saturated aqueous
sodium bicarbonate solution (100 ml), and the excess methanol was
removed in vacuo. The residue was diluted with ether (300 ml), and
the organic layer was washed successively with saturated aqueous
sodium bicarbonate solution (200 ml) and brine (200 ml). The
organic layer was dried (MgSO.sub.4), filtered, and concentrated.
Purification of the residue by flash chromatography (silica,
40.fwdarw.50% ether:hexanes) provided alcohol 14 (13.1 g; 95%) as a
colorless oil.
EXAMPLE 5
[0238] Alcohol 14a:
[0239] To a cooled (-78.degree. C.) solution of oxalyl chloride
(24.04 ml of a 2.0 M solution in CH.sub.2Cl.sub.2; 48.08 mmol) in
CH.sub.2Cl.sub.2 (165 ml) was added anhydrous DMSO (4.6 ml; 64.1
mmol) in dropwise fashion. After 30 min, a solution of alcohol 14
(6.93 g; 32.05 mmol) in CH.sub.2Cl.sub.2 (65 ml+10 ml rinse) was
added and the resulting solution was stirred at -78.degree. C. for
40 min. Freshly distilled triethylamine (13.4 ml; 96.15 mmol) was
then added, the cooling bath was removed, and the mixture was
allowed to warm to 0.degree. C. The reaction mixture was then
diluted with ether (500 ml), and washed successively with two
portions of water (250 ml) and one portion of brine (250 ml). The
organic layer was then dried (MgSO.sub.4), filtered, and
concentrated.
[0240] The crude aldehyde (6.9 g) prepared in the above reaction
was dissolved in ether (160 ml) and cooled to 0.degree. C.
Methylmagnesium bromide (32.1 ml of a 3.0 M solution in butyl
ether; 96.15 mmol) was then added, and the solution was allowed to
warm slowly to rt. After 10 h, the reaction mixture was cooled to
0.degree. C. and the reaction was quenched by the addition of
saturated aqueous ammonium chloride solution. The mixture was
diluted with ether (200 ml) and washed successively with water (150
ml) and brine (150 ml). The organic layer was dried (MgSO.sub.4),
filtered, and concentrated. Purification of the residue by flash
chromatography (silica, 40.fwdarw.50% ether hexanes) provided
alcohol 14a (6.3 g; 85% from 14).
EXAMPLE 6
[0241] Ketone 15:
[0242] A solution of alcohol 14 (1.0 g; 4.35 mmol), 4 A mol.
sieves, and N-methylmorpholine-N-oxide (1.0 g; 8.7 mmol) in
CH.sub.2Cl.sub.2 (20 ml) at rt was treated with a catalytic amount
of tetra-n-propylammonium perruthenate, and the resulting black
suspension was stirred for 3 h. The reaction mixture was then
filtered through a pad of silica gel (ether rinse), and the
filtrate was concentrated in vacuo. Purification of the residue by
flash chromatography (silica, 10% ether:hexanes) afforded ketone 15
(924 mg; 93%) as a light yellow oil.
EXAMPLE 7
[0243] Alkene 17:
[0244] A cooled (-78.degree. C.) solution of phosphine oxide 16
(1.53 g; 4.88 mmol) in THF (15.2 ml) was treated with
n-butyllithium (1.79 ml of a 2.45 M solution in hexanes). After 15
min, the orange solution was treated with a solution of ketone 15
(557 mg; 2.44 mmol) in THF (4.6 ml). After 10 min, the cooling bath
was removed, and the solution was allowed to warm to rt. The
formation of a precipitate was observed as the solution warmed. The
reaction was quenched by the addition of saturated aqueous ammonium
chloride solution (20 ml). The mixture was then poured into ether
(150 ml) and washed successively with water (50 ml) and brine (50
ml). The organic layer was dried (Na.sub.2SO.sub.4), filtered, and
concentrated. Purification of the residue by flash chromatography
(silica, 10% ether:hexanes) afforded alkene 17 (767 mg; 97%) as a
colorless oil: IR(film): 2956, 2177, 1506, 1249, 1149, 1032, 842,
cm.sup.-1; .sup.1H NMR(CDCl.sub.3, 500 MHz).delta.6.95(s, 1H),
6.53(s, 1H), 4.67(d, J=6.7 Hz, 1H), 4.57 (d, J=6.8 Hz, 1H), 4.29
(dd, J=8.1, 5.4 Hz, 1H), 3.43 (s, 3H), 2.70 (s, 3H), 2.62 (dd,
J=16.9, 8.2 Hz, 1H), 2.51 (dd, J=17.0, 5.4 Hz, 1H), 2.02 (s, 3H);
.sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.164.4, 152.5, 137.1,
121.8, 116.2, 103.7, 93.6, 86.1, 79.6, 55.4, 25.9, 19.1, 13.5;
[.alpha.].sub.D =-27.3 (c=2.2, CHCl.sub.3).
EXAMPLE 8
[0245] Alkynyl Iodide Formation:
[0246] To a solution of the alkyne 17 (3.00 g, 9.29 mmol) in
acetone (100 mL) at 0.degree. C. was added NIS (2.51 g; 11.2 mmol)
and AgNO.sub.3 (0.160 g; 0.929 mmol). The mixture was then slowly
warmed to rt. After 1.5 h, the reaction was poured into Et.sub.2O
(250 mL) and washed once with sat bisulfite (40 mL), once with sat
NaHCO.sub.3 (40 mL), once with brine (40 mL) and dried over
anhydrous MgSO.sub.4. Purification by flash chromatography on
silica gel using gradient elution with hexanes/ethyl acetate
(10:1-7:1) gave 2.22 g (64%) of the iodide 17a as an amber oil.
EXAMPLE 9
[0247] Reduction of the Alkynyl Iodide:
[0248] BH.sub.3.DMS (0.846 mL, 8.92 mmol) was added to a solution
of cyclohexene (1.47 mL, 17.9 mmol) in Et.sub.2O (60 mL) at
0.degree. C. The reaction was then warmed to rt. After 1 h, the
iodide x (2.22 g, 5.95 mmol) was added to Et.sub.2O. After 3 h,
AcOH (1.0 mL) was added. After 30 additional min, the solution was
poured into sat NaHCO.sub.3 and extracted with Et.sub.2O
(3.times.100 mL). The combined organics were then washed once with
brine (50 mL) and dried over anhydrous MgSO.sub.4. Purification by
flash chromatography on silica gel eluting with hexanes/ethyl
acetate (6:1) gave 1.45 g (65%) of the vinyl iodide 18 as a yellow
oil.
EXAMPLE 10
[0249] MOM Removal:
[0250] To a solution of iodide 18 (1.45 g, 3.86 mmol) in
CH.sub.2Cl.sub.2 (40 mL) at rt was added thiophenol (1.98 mL, 19.3
mmol) and BF.sub.3.degree.Et.sub.2O (1.90 mL, 15.43 mmol). After 22
h, the reaction was poured into EtOAc (150 mL) and washed with 1N
NaOH (2.times.50 mL) and dried over anhydrous MgSO.sub.4.
Purification by flash chromatography on silica gel using gradient
elution with hexanes/ethyl acetate (4:1-2:1-1:1) gave 1.075 g (86%)
of the alcohol 18a as a pale yellow oil.
EXAMPLE 11
[0251] Acetate Formation:
[0252] To a solution of alcohol 18a (1.04 g, 3.15 mmol) in
CH.sub.2Cl.sub.2 (30 mL) was added pyridine (2.52 mL, 25.4 mmol),
acetic anhydride (1.19 mL, 12.61 mmol) and DMAP (0.005 g). After 1
h, the volatiles were removed in vacuo. Purification of the
resulting residue by flash chromatography on silica gel eluting
with hexanes/ethyl acetate (7:1) gave 1.16 g (99%) of the acetate
19 as a pale yellow oil. IR(film):1737, 1368, 1232, 1018 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 500 MHz).delta.6.97 (s, 1H), 6.53 (s, 1H),
6.34 (dd, J=17.5, 1.0 Hz, 1 H), 6.18 (dd, J=13.7, 6.9 Hz, 1h), 5.40
(t, J=6.4 Hz, 1H), 2.70 (s, 3h), 2.61 (m, 2H), 2.08 (2s, 6H).
.sup.13C NMR (CDCl.sub.3, 125 MHz).delta.169.8, 164.4, 152.2,
136.4, 136.1, 120.6, 116.4, 85.1, 38.3, 21.0, 19.1, 14.7;
[.alpha.].sub.D=-28.8 (c=1.47, CHCl.sub.3).
EXAMPLE 12
[0253] To a solution of alcohol 4 (2.34 g, 3.62 mmol) and
2,6-lutidine (1.26 mL, 10.86 mmol) in CH.sub.2Cl.sub.2 (23 mL) at
0.degree. C. was treated with TBSOTf (1.0 mL, 4.34 mmol). After
stirrring for 1.5 h at 0.degree. C. the reaction mixture was
quenched with MeOH (200 uL) and the mixture stirred an additional 5
min. The reaction mixture was diluted with Et.sub.2O (100 mL) and
washed successively with 1 N HCl (25 mL), water (25 mL), and brine
(25 mL). The solution was dried over MgSO.sub.4, filtered, and
concentrated. The residue was purified by flash chromatography on
silica gel eluting with 5% Et.sub.2O in hexanes to provide compund
7 (2.70 g, 98%) as a colorless foam.
EXAMPLE 13
[0254] A solution of compound 7 (2.93 g, 3.85 mmol) in
CH.sub.2Cl.sub.2/H.sub.2O (20:1, 80 mL) was treated with DDQ (5.23
g, 23.07 mmol) and the resulting suspension was stirred at room
temperature for 24 h. The reaction mixture was diluted with
Et.sub.2O (200 mL) and washed with aqueous NaHCO.sub.3 (2.times.40
mL). The aqueous layer was extracted with Et.sub.2O (3.times.40 mL)
and the combined organic fractions were washed with brine (50 mL),
dried over MgSO.sub.4, filtered, and concentrated. Purification of
the crude oil by flash chromatography on silica gel eluting with
30% ether in hexanes afforded alcohol 7A (2.30 g, 89%) as a
colorless oil: IR (film) 3488, 1471, 1428, 1115, 1054 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 500 MHz).delta.7.70 (6H, dd, J=8.0, 1.5
Hz), 7.44 (9H, s), 4.57 (1H, d, J=3.5 Hz), 4.19 (1H, s), 3.67 (1H,
d, J=8.5 Hz), 3.06 (1H, dd, J=11.5, 5.0 Hz), 2.89 (1H, dd, J=11.5,
5.0 Hz), 2.68 (1H, d, J=13.5 Hz), 2.59 (1H, d, J=13.5 Hz), 2.34
(1H, dt, J=12.0, 2.5 Hz), 2.11 (1H, m), 1.84 (1H, dt, J=12.0, 2.5
Hz), 1.76 (2H, m), 1.59 (2H, m), 1.34 (3H, s), 1.13 (3H, d, J=7.5
Hz), 1.10 (3H, s), 0.87 (9H, s), 0.84 (3H, d, J=12.0 Hz), 0.02 (3H,
s), 0.01 (3H, s); .sup.13C NMR (CDCl.sub.3, 125 MHz).delta.136.18,
134.66, 130.16, 127.84, 78.41, 75.91, 63.65, 59.69, 45.43, 45.09,
37.72, 30.84, 30.50, 26.23, 25.89, 22.42, 21.05, 18.40, 15.60,
14.41, -3.23, -3.51; [.alpha.].sub.D=-0.95 (c=0.173,
CHCl.sub.3).
EXAMPLE 14
[0255] To a solution of oxalyl chloride (414 .mu.L, 4.74 mmol) in
CH.sub.2Cl.sub.2 (40 mL) at -78.degree. C. was added dropwise DMSO
(448 uL, 6.32 mmol) and the resulting solution was stirred at
-78.degree. C. for 30 min. Alcohol 7a (2.12 g, 3.16 mmol) in
CH.sub.2Cl.sub.2 (20 mL) was added and the resulting white
suspension was stirred at -78.degree. C. for 45 min. The reaction
mixture was quenched with Et.sub.3N (2.2 mL, 15.8 mmol) and the
solution was allowed to warm to 0.degree. C. and stirred at this
temperature for 30 min. The reaction mixture was diluted with
Et.sub.2O (100 mL) and washed successively with aqueous NH.sub.4Cl
(20 mL), water (20 mL), and brine (20 mL). The crude aldehyde was
purified by flash chromatography on silica gel eluting with 5%
Et.sub.2O in hexanes to provide aldehyde 8 (1.90 g, 90%) as a
colorless oil.
EXAMPLE 15
[0256] A solution of (methoxymethyl)triphenylphosphonium chloride
(2.97 g, 8.55 mmol) in THF (25 mL) at 0.degree. C. was treated with
KO'Bu (8.21 mL, 1M in THF, 8.1 mmol). The mixture was stirred at
0.degree. C. for 30 min. Aldehyde 8 (3.1 g, 4.07 mmol) in THF (10
mL) was added and the resulting solution was allowed to warm to
room temperature and stirred at this temperature for 2 h. The
reaction mixture was quenched with aqueous NH.sub.4Cl (40 mL) and
the resulting solution extracted with Et.sub.2O (3.times.30 mL).
The combined Et.sub.2O fractions were washed with brine (20 ml),
dried over MgSO.sub.4, filtered, and concentrated. The residue was
purified by flash chromatography on silica gel eluting with 5%
Et.sub.2O in hexanes to yield compound 9 (2.83 g, 86%) as a
colorless foam.
EXAMPLE 16
[0257] To a solution of compound 9 (2.83 g, 3.50 mmol) in
dioxane/H.sub.2O (9:1, 28 mL) was added pTSA.H.sub.2O (1.0 g, 5.30
mmol) and the resulting mixture was heated to 50.degree. C. for 2
h. After cooling to room temperature the mixture was diluted with
Et.sub.2O (50 mL) and washed with aqueous NaHCO.sub.3 (15 mL),
brine (20 ml), dried over MgSO.sub.4, filtered, and concentrated to
provide aldehyde 9a (2.75 g, 99%) as a colorless foam.
EXAMPLE 17
[0258] Methyltriphenylphosphonium bromide (1.98 g, 5.54 mmol) in
THF (50 mL) at 0.degree. C. was treated with lithium
bis(trimethylsilyl)amide (5.04 mL, 1M in THF, 5.04 mmol) and the
resulting solution was stirred at 0.degree. C. for 30 min. Aldehyde
9a (2.0 g, 2.52 mmol) in THF (5.0 mL) was added and the mixture was
allowed to warm to room temperature and stirred at this temperature
for 1 h. The reaction mixture was quenched with aqueous NH.sub.4Cl
(15 mL) and extracted with Et.sub.2O (3.times.20 mL). The combined
Et.sub.2O fractions were washed with brine (15 mL), dried over
MgSO.sub.4, filtered, and concentrated. The residue was purified by
flash chromatography on silica gel eluting with 5% Et.sub.2O in
hexanes to afford compound 10 (1.42 g, 76%) as a colorless
foam.
EXAMPLE 18
[0259] A solution of compound 10 (1.0 g, 1.34 mmol) in MeOH/THF
(2:1, 13 mL) was treated with [bis(trifluoroacetoxy)iodobenzene]
(865 mg, 2.01 mmol) at room temperature. After 15 min the reaction
mixture was quenched with aqueous NaHCO.sub.3 (25 mL). The mixture
was extracted with Et.sub.2O (3.times.25 mL) and the combined
Et.sub.2O fractions were washed with brine, dried over MgSO.sub.4,
filtered, and concentrated. Purification of the residue by flash
chromatography on silica gel eluting with 5% Et.sub.2O in hexanes
provided compound 11 (865 mg, 92%) as a colorless foam: IR (film)
1428, 1252, 1114, 1075, 1046 cm.sup.-1; .sup.1H NMR (CDCl.sub.3,
500 MHz).delta.7.61 (6H, dd, J=7.9, 1.4 Hz), 7.38 (9H, s), 5.47
(1H, m), 4.87 (1H, d, J=10.0 Hz), 4.76 (1H, d, J=15.9 Hz), 4.30
(1H, d, J=3.7 Hz), 3.95 (1H, s), 3.56 (1H, dd, J=7.5, 1.4 Hz), 3.39
(3H, s), 2.84 (3H, s), 2.02 (1H, m), 1.64 (2H, m), 1.34 (1H, m),
1.11 (3H, s), 1.02 (3H, d, J=7.4 Hz), 0.90 (3H, s), 0.85 (9H, s),
0.62 (3H, d, J=6.8 Hz), -0.04 (3H, s), -0.05 (3H, s); .sup.13C NMR
(CDCl.sub.3, 125 MHz).delta.138.29, 135.79, 135.04, 129.86, 127.78,
114.98, 110.49, 60.11, 55.57, 46.47, 43.91, 36.82, 34.21, 26.26,
19.60, 18.60, 17.08, 16.16, 13.92, -2.96, -3.84;
[.alpha.].sub.D=+1.74 (c=0.77, CHCl.sub.3).
EXAMPLE 19
[0260] Suzuki Coupling:
[0261] To a solution of olefin 11 (0.680 g, 1.07 mmol) in THF (8.0
mL) was added 9-BBN (0.5 M soln in THF, 2.99 mL, 1.50 mmol). In a
separate flask, the iodide 19 (0.478 g, 1.284 mmol) was dissolved
in DMF (10.0 mL). CSCO.sub.3 (0.696 g, 2.14 mmol) was then added
with vigorous stirring followed by sequential addition of
Ph.sub.3As (0.034 g, 0.111 mmol), PdCl.sub.2(dppf).sub.2 (0.091 g,
0.111 mmol) and H.sub.2O (0.693 mL, 38.5 mmol). After 4 h, then
borane solution was added to the iodide mixture in DMF. The
reaction quickly turned dark brown in color and slowly became pale
yellow after 2 h. The reaction was then poured into H.sub.2O (100
mL) and extracted with Et.sub.2O (3.times.50 mL). The combined
organics were washed with H.sub.2O (2.times.50 mL), once with brine
(50 mL) and dried over anhydrous MgSO.sub.4. Purification by flash
chromatography on silica gel eluting with hexanes/ethyl acetate
(7:1) gave 0.630 g (75%) of the coupled product 20 as a pale yellow
oil.
EXAMPLE 20
[0262] Hydrolysis of Dimethyl Acetal 21:
[0263] The acetate 20 (0.610 g, 0.770 mmol) was dissolved in
dioxane/H.sub.2O (9:1, 15 mL) and p-TSA.H.sub.2O (0.442 g, 2.32
mmol) was added. The mixture was then heated to 55.degree. C. After
3 h, the mixture was cooled to rt and poured into Et.sub.2O. This
solution was washed once with sat NaHCO.sub.3 (30 mL), once with
brine (30 mL) and dried over anhydrous MgSO.sub.4. Purification by
flash chromatography on silica gel eluting with hexanes/ethyl
acetate (7:1) gave 0.486 g (85%) of the aldehyde 21 as a pale
yellow oil. IR (film) 1737, 1429, 1237, 1115, 1053cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 500 MHz).delta.9.74 (1H, s), 7.61 (6H, dd,
J=7.8, 1.2 Hz), 7.38 (9H, m), 6.94 (1H, s), 6.53 (1H, s), 5.39 (1H,
m), 5.31 (1H, m), 5.29 (1H, t, J=6.9 Hz), 4.61 (1H, d, J=4.3 Hz),
3.50 (1H, dd, J=5.2, 2.6 Hz), 2.70 (3H, s), 2.48 (2H, m), 2.14 (1H,
m), 2.09 (3H, s), 2.07 (3H, s), 1.83 (2H, m), 1.41 (1H, m), 1.18
(1H, m), 1.01 (3H, s), 0.99 (3H, s), 0.91 (3H, d, J=7.4 Hz), 0.85
(9H, s), 0.69 (1H, m), 0.58 (3H, d, J=6.8 Hz), -0.05 (3H, s), -0.06
(3H, s); .sup.13C NMR (CDCl.sub.3, 125 MHz).delta.205.46, 170.01,
164.49, 152.46, 137.10, 135.60, 134.22, 132.55, 130.65, 127.84,
123.82, 120.66, 116.19, 81.09, 78.47, 76.73, 51.66, 43.14, 38.98,
30.99, 30.42, 27.63, 26.10, 21.15, 20.92, 20.05, 19.15, 18.49,
15.12, 14.70, 12.75, -3.25, -4.08; [.alpha.].sub.D=-18.7 (c=0.53,
CHCl.sub.3).
EXAMPLE 21
[0264] Aldol:
[0265] To a solution of the acetate-aldehyde 21 (84 mg,0.099 mmol)
in THF at -78.degree. C. was added KHMDS (0.5M in toluene, 1.0 ml,
0.5 mmol)) dropwise. The resulting solution was stirred at
-78.degree. C. for 30 min. Then the reaction mixure was cannulated
to a short pad of silica gel and washed with ether. The residue was
purified by flash chromatography (silica, 12% EtOAc in hexane) to
give the lactone 22 (37 mg of 3-S and 6 mg of 3-R, 51%) as white
foam.
EXAMPLE 22
[0266] Monodeprotection:
[0267] Lactone 22 (32 mg, 0.0376 mmol) was treated with 1 ml of
pyridine buffered HF.pyridine-THF solution at room temperture for 2
h. The reaction mixure was poured into saturated aqueous
NaHCO.sub.3 and extracted with ether. The organic layer was washed
in sequence with saturated CuSO.sub.4 (10 ml.times.3) and saturated
NaHCO.sub.3 (10 ml), then dried over Na.sub.2SO.sub.4 and
concentrated under vacuum. The residue was purified by flash
chromatography (silica, 25% EtOAc in hexane) and to give diol 22a
(22 mg, 99%) as white foam.
EXAMPLE 23
[0268] TBS-Protection:
[0269] To a cooled (-30.degree. C.) solution of diol 22a (29 mg,
0.0489 mmol) and 2,6-lutidine (0.017 ml, 0.147 mmol) in anhydrous
CH.sub.2Cl.sub.2 (1 ml) was added TBSOTf (0.015 ml, 0.0646 mmol).
The resulting solution was then stirred at -30.degree. C. for 30
min. The reaction was quenched with 0.5M HCl (10 ml) and extracted
with ether (15 ml). Ether layer was washed with saturated
NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and concentrated in vacuo.
Purifiction of the residue by flash chromatogrphy (silica, 8% EtOAc
in hexane) afforded TBS ether 22B (32 mg, 93%) as white foam.
EXAMPLE 24
[0270] Ketone Formation:
[0271] To a solution of alcohol 22B (30 mg, 0.0424 mmol) in
CH.sub.2Cl.sub.2 (2.0 mL) at 25.degree. C. was added Dess-Martin
periodinane (36 mg, 0.0848 mmol) in one portion. The resulting
solution was then allowed to stir at 25.degree. C. for 1.5 h. The
reaction was quenched by the addition of 1:1 saturated aqueous
sodium bicarbonate: sodium thiosulfate (10 ml) and stirred for 5
min. The mixture was then extracted with ether (3.times.15 ml). The
organic layer was dried (Na.sub.2SO.sub.4), filtered, and
concentrated in vacuo. Purification of the residue by flash
chromatography (silica, 8% EtOAc in hexane) provided ketone 22C (25
mg, 84%) as white foam. IR(film): 2928, 1745, 1692, 1254, 1175, 836
cm.sup.-1; .sup.1H NMR(CDCl.sub.3, 500 MHz).delta.6.97 (s, 1H),
6.57 (s, 1H), 5.53 (dt, J=3.4, 11.1 Hz, 1H), 5.37 (dd, J=16.4, 9.9
Hz, 1H), 5.00 (d, J=10.3 Hz, 1H), 4.02 (d, J=9.7 Hz, 1H), 3.89 (d,
J=8.7 Hz, 1H), 3.00 (m, 1H), 2.82 (d, J=6.5 Hz, 1H), 2.71 (m, 5H),
2.36 (q, J=10.7 Hz, 1H), 2.12 (, 3H), 2.07 (dd, J-8.2, 1H), 1.87
(bs, 1H), 1.49 (m, 3H), 1.19 (m, 5H), 1.14 (s, 3H), 1.08 (d, J=6.8
Hz, 3H), 0.94 (m, 12H), 0.84 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H),
0.07 (s, 3H), -0.098 (s, 3H); .sup.13C NMR (CDCl.sub.3, 125
MHz).delta.218.7, 170.1, 164.5, 152.6, 137.9, 133.9, 124.8, 119.6,
115.9, 72.7, 53.2, 43.9, 41.0, 40.3, 32.9, 32.3, 28.4, 27.1, 26.3,
26.1, 26.0, 19.2, 19.1, 18.3, 18.2, 17.1, 16.0, 15.2, 14.3, -4.2,
-4.4, -4.6, -4.8; [.alpha.].sub.D=-21.93 (c=1.4, CHCl.sub.3).
EXAMPLE 25
[0272] Desoxy Compound:
[0273] To a solution of TBS ether 22C (27 mg, 0.038 mmol) in THF(1
ml) at 25.degree. C. in a plastic vial was added dropwise
HF.pyridine (0.5 ml). The resulting solution was allowed to stir at
25.degree. C. for 2 h. The reaction mixture was diluted with
chloroform (2 ml) and very slowly added to satured sodium
bicarbonate (20 ml) . The mixture was extracted with CHCl.sub.3 (20
ml.times.3). The organic layer was dried (Na.sub.2SO.sub.4),
filtered, and concentrated in vacuo. Purification of the residue by
flash chromatography (silica, 30% EtOAc in hexane) provided diol 23
(18 mg, 99%) as white foam: IR(film): 3493, 2925, 1728, 1689, 1249
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 500 MHz).delta.6.96 (s, 1H),
6.59 (s, 1H), 5.44 (dt, J=4.3, 10.4 Hz, 1H), 5.36 (dt, J=5.1, 10.2
Hz, 1H), 5.28 (dd, J=1.7, 9.8 Hz, 1H), 4.11 (d, J=7.2 Hz, 1H), 3.74
(s, 1H), 3.20 (d, J=4.5 Hz, 1H), 3.14 (dd, J=2.2, 6.8 Hz, 1H), 3.00
(s, 1H), 2.69 (m, 4H), 2.49 (dd, J=11.3, 15.1 Hz, 1H), 2.35 (dd,
J-2.5, 15.1 Hz, 1H), 2.27 (m, 1H), 2.05 (m, 1H), 2.04 (s, 3H), 2.01
(m, 1H) 1.75 (m, 1H), 1.67 (m, 1H), 1.33 (m, 4H), 1.21 (s, 1H),
1.19 (m, 2H), 1.08 (d, J=7.0 Hz, 3H), 1.00 (s, 3H), 0.93 (d, J=7.1
Hz, 3H); .sup.13C NMR (CDCl.sub.3, 125 MHz).delta.226.5, 176.5,
171.1, 158.2, 144.7, 139.6, 131.1, 125.7, 122.0, 84.6, 80.2, 78.6,
59.4, 47.9, 45.4, 44.6, 38.5, 37.9, 33.7, 33.6, 28.7, 25.1, 25.0,
21.9, 21.7, 19.6; [.alpha.]=-84.7 (c=0.85, CHCl.sub.3).
EXAMPLE 26
[0274] Epothilone:
[0275] To a cooled (-50.degree. C.) solution of desoxyepothilone (9
mg, 0.0189 mmol) in dry CH.sub.2Cl.sub.2 (1 ml) was added freshly
prepared dimethyldioxirane (0.95 ml, 0.1 M in acetone). The
resulting solution was allowed to warm up to -30.degree. C. for 2
h. A stream of nitrogen was then bubbled through the solution to
remove excess DMDO. The residue was purified by flash
chromatography (silica, 40% EtOAc in hexane) and afforded
epothilone A (4.6 mg, 49%) as colorless solid and 0.1 mg of
cis-epoxide diastereomer. This material was identical with the
natural epothilone A in all respects.
EXAMPLE 27
[0276] Procedure for Ring-Closing Olefin Metathesis:
[0277] To a stirred solution of diene 24 (5 mg, 0.0068 mmol) in dry
benzene (1.5 mL) was added Grubbs's catalyst (2.8 mg, 0.0034 mmol).
After 12 h, an additional portion of catalyst was added (2.8 mg).
After an additional 5 h, the reaction was concentrated.
Purification by silica gel chromatography eluting with
hexanes/ethyl acetate (11:1) gave the lactone 23 (3.5 mg, 94%, 2:1
E/Z).
EXAMPLE 28
[0278] Preparation of Compound 19:
[0279] Alcohol 2A:
[0280] A mixture of (S)-(-)-1,1.sup.1-bi-2-naphthol (259 mg. 0.91
mmoL), Ti(O-i-Pr).sub.4 (261 .mu.L; 0.90 mmol), and 4 .ANG. sieves
(3.23 g) in CH.sub.2Cl.sub.2 (16 mL) was heated at reflux for 1 h.
The mixture was cooled to rt and aldehyde 1 was added. After 10
min. the suspension was cooled to -78.degree. C., and allyl
tributyltin (3.6 mL; 11.60 mmol) was added. The reaction mixture
was stirred for 10 min at -78.degree. C. and then placed in a
-20.degree. C. freezer for 70 h. Saturated NaHCO.sub.3 (2 mL) was
added, and the mixture was stirred for 1 h, poured over
Na.sub.2SO.sub.4, and then filtered through a pad of MgSO.sub.4 and
celite. The crude material was purified by flash chromatography
(hexanes/ethyl acetate, 1:1) to give alcohol 2A as a yellow oil
(1.11 g; 60%).
EXAMPLE 29
[0281] Acetate 3A:
[0282] To a solution of alcohol 2A (264 mg; 1.26 mmol) in
CH.sub.2Cl.sub.2 (12 mL) was added DMAP (15 mg: 0.098 mmol),
Et.sub.3N (0.45 mL; 3.22 mmol), and Ac.sub.2O (0.18 mL; 1.90 mmol).
After 2 h, the reaction mixture was quenched by 20 mL of H.sub.2O,
and extracted with EtOAC (4.times.20 mL). The combined organic
layer was dried with MgSO.sub.4, filtered, and concentrated. Flash
chromatrography (EtOAC/hexanes, 1:3) afforded acetate 3A as a
yellow oil (302 mg; 96%).
EXAMPLE 30
[0283] Vinyl Iodide 19:
[0284] To a solution of acetate 3A (99 mg; 0.39 mmol) in acetone at
0.degree. C. was added H.sub.2O (4 drops), OsO.sub.4 (2.5% wt. in
butyl alcohol; 175 .mu.L; 0.018 mmol), and
N-methyl-morpholine-N-oxide (69 mg; 0.59 mmol). The mixture was
stirred at 0.degree. C. for 2 h and 45 min and then quenched with
Na.sub.2SO.sub.3. The solution was poured to 10 mL of H.sub.2O and
extracted with EtOAc (5.times.10 mL) The combined organic layer was
dried over MgSO.sub.4, filtered, and concentrated.
[0285] To a solution of this crude product in THF/H.sub.2O (4 mL,
3:1) was added NaIO.sub.4(260 mg; 1.22 mmol). After 1.25 h, the
reaction mixture was then quenched with 10 mL of H.sub.2O and
concentrated. The residue was extracted with EtOAc (5.times.10 mL).
The organic layer was dried over MgSO.sub.4, filtered, and
concentrated. Flash chromatography (EtOAc/hexanes, 1:1) gave a
yellow oil (80 mg) which contained unidentified by-product(s). This
mixture was used without further purification.
[0286] To a solution of (Ph.sub.3P.sup.+CH.sub.2I)I.sup.- (100 mg;
0.19 mmol) in 0.25 mL of THF at rt was added 0.15 mL (0.15 mmol) of
NaHMDS (1M in THF). To the resulting solution at -78.degree. C. was
added HMPA (22 .mu.L; 0.13 mmol) and the product from previous step
(16 mg) in THF (0.25 mL). The reaction mixture was then stirred at
rt for 30 min. After the addition of hexanes (10 mL), the solution
was extracted with EtOAc (4.times.10 mL). The combined EtOAC layer
was dried (MgSO.sub.4), filtered, and concentrated. Preparative TLC
(EtOAc/hexanes, 2.3) afforded vinyl iodide 19 as a yellow oil (14
mg; 50% for three steps).
EXAMPLE 31
[0287] Iodoolefin Acetate 8C:
[0288] To a suspension of ethyltriphenylphosphonium iodide (1.125
g, 2.69 mmol) in THF (10 mL) was added nBuLi (2.5 M soln in
hexanes, 1.05 mL, 2.62 mmol) at rt. After disappearance of the
solid material, the solution was added to a mixture of iodine
(0.613 g, 2.41 mmol) in THF (20 mL) at -78.degree. C. The resulting
suspension was vigorously stirred for 5 min at -78.degree. C., then
warmed up -20.degree. C., and treated with sodium
hexamethyldisilazane (1 M soln in THF, 2.4 mL, 2.4 mmol). The
resulting red solution was stirred for 5 min followed by the slow
addition of aldehyde 9C (0.339 g, 1.34 mmol). The mixture was
stirred at -20.degree. C. for 40 min, diluted with pentane (50 mL),
filtered through a pad of celite, and concentrated. Purification of
the residue by flash column chromatography (hexanes/ethyl acetate,
85:15) gave 0.202 g (25% overall from vinyl acetate 10C) of the
vinyl iodide 8C as a yellow oil. IR (film): 2920, 1738, 1234
cm.sup.-1; .sup.1H NMR (CDCl.sub.3): .delta. 6.98 (s, 1H), 6.56 (s,
1H), 5.42 (dd, J=5.43, 6.57 Hz, 1H), 5.35 (t, J=6.6 Hz, 1H), 2.71
(s, 3H), 2.54 (q, J=6.33, 2H), 2.50 (s, 3H), 2.09 (s, 6H); .sup.13C
NMR (CDCl.sub.3): .delta. 170.1, 164.6, 152.4, 136.9, 130.2, 120.6,
116.4, 103.6, 40.3, 33.7, 21.2, 19.2, 14.9; [.alpha.].sub.D
=-20.7.degree. (c=2.45, CHCl.sub.3).
EXAMPLE 32
[0289] Acetal 13C:
[0290] To a solution of olefin "7C" (0.082 g, 0.13 mmol) in
THF-(0.5 mL) was added 9-BBN (0.5 M soln in THF, 0.4 mL, 0.2 mmol).
After stirring at rt. for 3.5 h, an additional portion of 9-BBN
(0.5 M soln in THF, 0.26 mL, 0.13 mmol) was added. In a separate
flask, iodide 8C (0.063 g, 0.16 mmol) was dissolved in DMF (0.5
mL). Cs.sub.2CO.sub.3 (0.097 g, 0.30 mmol) was then added with
vigorous stirring followed by sequential addition of
PdCl.sub.2(dppf).sub.2 (0.018 g, 0.022 mmol), Ph.sub.3As (0.0059 g,
0.019 mmol), and H.sub.20 (0.035 mL, 1.94 mmol). After 6 h, then
borane solution was added to the iodide mixture in DMF. The
reaction quickly turned dark brown in color and slowly became pale
yellow after 3 h. The reaction was then poured into H.sub.2O (10
mL) and extracted with Et.sub.2O (3.times.15 mL). The combined
organic layers were washed with H.sub.2O (3.times.15 mL), brine
(1.times.20 mL), dried over MgSO.sub.4, filtered, and concentrated.
Flash column chromatography (hexanes/ethyl acetate, 9:1) gave 0.089
g (77%) of the coupled product 13C as a yellow oil.
EXAMPLE 33
[0291] Aldehyde 14C:
[0292] Acetal 13C (0.069 g, 0.077 mmol) was dissolved in
dioxane/H.sub.2O (9:1, 1 mL) and pTSA.H.sub.2O (0.045 g, 0.237
mmol) was added. The mixture was then heated to 55.degree. C. After
3 h, the mixture was cooled to rt, poured into Et.sub.2O, and
extracted with Et.sub.2O (4.times.15 mL). The combined ether
solutions were washed with sat NaHCO.sub.3 (1.times.30 mL), brine
(1.times.30 mL), dried over MgSO.sub.4, filtered, and concentrated.
Flash column chromatography (hexanes/ethyl acetate, 3:1) gave 0.046
g (71%) of the aldehyde 14C as a pale yellow oil.
EXAMPLE 34
[0293] Macrocycle 15C-(SR):
[0294] To a solution of aldehyde 14C (0.021 g, 0.024 mmol) in THF
(5 mL) at -78.degree. C. was added KHMDS (0.5 M soln in toluene,
0.145 mL, 0.073 mmol). The solution was stirred at -78.degree. C.
for 1 h, then quenched with sat'd NH.sub.4Cl, and extracted with
ether (3.times.15 mL). The combined organic layers were dried with
MgSO.sub.4, filtered, and concentrated. Flash column chromatography
(hexanes/ethyl acetate, 7:1) gave 0.008 g of the desired
.alpha.-alcohol 15C-(S) and 0.006 g of -alcohol 15C-(R) (67% total
) as pale yellow oils.
EXAMPLE 35
[0295] Macrocycle 15C-(S):
[0296] To a solution of .beta.-alcohol 15C-(R) (0.006 g, 0.0070
mmol) in 0.5 mL of CH.sub.2Cl.sub.2 at rt. was added Dess-Martin
periodinane (0.028g, 0.066 mmol). After 0.5 h, an additional
portion of Dess-Martin periodinane (0.025 mg, 0.059 mmol) was
added. The resulting solution was stirred at rt for additional 1 h,
then treated with ether (2 mL) and sat'd
Na.sub.2S.sub.2O.sub.3/sat'd NaHCO.sub.3 (3 mL, 1:1), poured into
H.sub.2O (20 mL), and extracted with ether (4.times.10 mL). The
combined ether solutions were washed with H.sub.2O (1.times.30 mL),
brine (1.times.30 mL), dried with MgSO.sub.4, filtered, and
concentrated. To a solution of crude ketone 15C' in MeOH/THF (2 mL,
1:1) at -78.degree. C. was added NaBH.sub.4 (0.015 g, 0.395 mmol).
The resulting solution was stirred at rt for 1 h, quenched with sat
NH.sub.4Cl, and extracted with ether (3.times.15 mL). The organic
layers were dried with MgSO.sub.4, filtered, and concentrated.
Flash column chromatography (hexanes/ethyl acetate, 9:1) gave
0.0040 g (67%) of the a-alcohol 15C-(S) as a pale yellow oil and
0.0006 g of .beta.-alcohol 15C-(R).
EXAMPLE 36
[0297] Diol 15C":
[0298] The silyl ether 15C-(S) (0.010 g, 0.012 mmol) was dissolved
in HF.pyridine/pyridine/THF (1 mL). The solution was stirred at rt.
for 2 h, then diluted with Et.sub.2O (1 mL), poured into a mixture
of Et.sub.2O/sat. NaHCO.sub.3 (20 mL, 1:1), and extracted with
Et.sub.2O (4.times.10 mL). The Et.sub.2O solutions were washed with
sat CuSO.sub.4 (3.times.30 mL), sat NaHCO.sub.3 (1.times.30 mL),
brine (1.times.30 mL), dried with MgSO.sub.4, filtered, and
concentrated. Flash column chromatography (hexanes/ethyl acetate,
9:1) gave 0.0066 g (93%) of the diol 15C" as a pale yellow oil.
EXAMPLE 37
[0299] Alcohol 15C'":
[0300] To a solution of diol 15C" (0.0066 g, 0.011 mmol) in 0.5 mL
of CH.sub.2Cl.sub.2 at -78.degree. C. was added 2,6-lutidine (7
.mu.L, 0.060 mmol) and TBSOTf (5 .mu.L, 0.022 mmol). The resulting
solution was stirred at -30.degree. C. for 0.5 h, then quenched
with H.sub.2O (5 mL), and extracted with Et.sub.2O (4.times.10 mL).
The ether solutions were washed with 0.5 M HCl (1.times.10 mL),
sat'd NaHCO.sub.3 (1.times.10 mL), dried over MgSO.sub.4, filtered,
and concentrated. Flash column chromatography (hexanes/ethyl
acetate, 93:7) gave 0.0070 g (89%) of the alcohol 15C'" as a pale
yellow oil.
EXAMPLE 38
[0301] Ketone 16C:
[0302] To a solution of alcohol 15C'" (0.006 g, 0.0083 mmol) in 0.5
mL of CH.sub.2Cl.sub.2 at rt. was added Dess-Martin periodinane
(0.030 g, 0.071 mmol). After 1.25 h, another portion of Dess-Martin
periodinane (0.025 mg, 0.059 mmol) was added. The resulting
solution was stirred at rt for additional 0.75 h, treated with
ether (1 mL) and sat'd Na.sub.2S.sub.2O.sub.3/sat'd NaHCO.sub.3 (2
mL, 1:1), poured into H.sub.2O (20 mL), and extracted with ether
(4.times.10 mL). The ether solution was washed with sat NaHCO.sub.3
(1.times.20 mL), dried with MgSO.sub.4, filtered, and concentrated.
Flash column chromatography (hexanes/ethyl acetate, 9:1) gave
0.0040 g (67%) of the ketone 16C as a pale yellow oil.
EXAMPLE 39
[0303] Desoxyepothiolone B 2C:
[0304] To a solution of ketone 16C (0.004 g, 0.0056 mmol) in THF
(0.35 mL) was added HF.pyridine (0.25 mL) dropwise over 20 min. The
solution was stirred at rt for 1.5 h, diluted with CHCl.sub.3 (2
mL), poured into sat'd NaHCO.sub.3/CHCl.sub.3 (20 mL, 1:1) slowly,
and extracted with CHCl.sub.3 (4.times.10 mL). The combined
CHCl.sub.3 layers were dried with MgSO.sub.4, filtered, and
concentrated. Flash column chromatography (hexanes/ethyl acetate,
3:1) gave 0.0022 g (80%) of the desoxyepothilone B 2C as a pale
yellow oil.
EXAMPLE 40
[0305] Epothilone B 2:
[0306] To a solution of desoxyepothilone B (0.0022 g, 0.0041 mmol)
in CH.sub.2Cl.sub.2 (0.25 mL) at -50.degree. C. was added
dimethyldioxirane (0.1 mL, 0.0095 mmol) dropwise. The resulting
solution was stirred at -50.degree. C. for 1 h. The
dimethyldioxirane and solvent were removed by a stream of N.sub.2.
The residue was purified by flash column chromatography
(hexanes/ethyl acetate, 1:1) gave 0.0015 g (70%) of epothiolone B
(2) as a pale yellow oil which was identical with an authentic
sample in .sup.1H NMR, IR, mass spectrum, and [.alpha.].sub.D.
EXAMPLE 41
[0307] 8-Desmethylepothilone A
[0308] Crotylation Product:
[0309] To a stirred mixture of potassium tert-butoxide (1.0 M soln
in THF, 50.4 mL, 50.4 mmol), THF (14 mL), and cis-2-butene (9.0 mL,
101 mmol) at -78.degree. C. was added n-BuLi (1.6 M, in hexanes,
31.5 mL, 50.4 mmol). After complete addition of n-BuLi, the mixture
was stirred at -45.degree. C. for 10 min and then cooled to
-78.degree. C. (+)-B-Methoxydiisopinocam- pheylborane (19.21 g,
60.74 mmol) was then added dropwise in Et.sub.2O (10 mL). After 30
min, BF.sub.3 Et.sub.2O (7.47 mL, 60.74 mmol) was added followed by
aldehyde 4D (9.84 g, 60.74 mmol) in THF (15 mL) generating a
viscous solution which could not be stirred. The mixture was shaken
vigorously every 10 min to ensure homogeneity. After 3 h at
-78.degree. C., the reaction was treated with 3N NaOH (36.6 mL, 110
mmol) and 30% H.sub.2O.sub.2 (15 mL) and the solution brought to
reflux for 1 h. The reaction was poured into Et.sub.2O (300 mL) and
washed with H.sub.2O (100 mL), brine (30 mL) and dried over
anhydrous MgSO.sub.4. The crude material was placed in a
bulb-to-bulb distillation apparatus to remove the ligand from the
desired product. Heating at 80.degree. C. at 2 mm Hg removed 90% of
the lower boiling ligand. Further purification of the alcohol 4D
was achieved by flash chromatography on silica gel eluting with
Et.sub.2O in CH.sub.2Cl.sub.2 (2%-4%) to give pure alcohol 4D as a
clear oil. The erythro selectivty was >50:1 as judged by .sup.1H
NMR spectroscopy. The product was determined to be 87% ee by
formation of the Mosher ester: IR (film): 3435, 2861, 1454, 1363,
1099 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.34 (5H,
m), 5.80 (1H, m), 5.09 (1H, dd, J=1.6, 8.3 Hz), 5.04 (1H, d, J=1.6
Hz), 4.52 (2H, s), 3.51 (2H, t, J=5.8 Hz), 3.47 (1H, m), 2.27 (2H,
m), 1.73 (3H, m), 1.42 (1H, m), 1.04 (3H, d, J=6.9 Hz); .sup.13C
NMR (CDCl.sub.3, 100 MHz) .delta. 141.1, 138.2, 128.3, 127.6,
127.5, 115.0, 74.5, 72.9, 70.4, 43.7, 31.3, 26.5, 14.6.
EXAMPLE 42
[0310] TBS Ether 5D:
[0311] Alcohol 4D (5.00 g, 21.4 mmol) was dissolved in
CH.sub.2Cl.sub.2 (150 mL) and 2,6-lutidine (9.97 mL, 85.6 mmol) was
added. The mixture was cooled to 0.degree. C. and TBSOTf (9.83 mL,
42.8 mmol) was slowly added. The reaction was then warmed to rt.
After 1 h, the reaction was poured into Et.sub.2O (300 mL) and
washed once with 1 N HCl (50 mL), once with sat NaHCO.sub.3 (50
mL), once with brine (30 mL) and dried over anhydrous MgSO.sub.4.
Purification by flash chromatography on silica gel eluting with
hexanes/diethyl ether (97:3) gave 6.33 g (85%) of pure olefin 5D as
a clear oil: IR (film): 1472, 1361, 1255, 1097, 1068 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.30 (5H, m), 5.81 (1H,
m), 4.97 (1H, dd, J=1.4, 4.8 Hz), 4.94 (1H, d, J=1.1 Hz), 3.51 (1H,
q, J=5.1 Hz), 3.41 (2H, dt, j=2.1, 6.6 Hz), 2.27 (1H, q, J=5.5 Hz),
1.68 (1h, m), 1.55 (1H, m), 1.41 (2H, m), 0.93 (3H, d, J=6.9 Hz),
0.85 (9H, s), -0.01 (6H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz)
.delta. 141.2, 138.6, 128.3, 127.6, 127.4, 113.9, 75.6, 72.7, 70.6,
42.7, 30.1, 25.9, 25.4, 18.1, 15.1, -4.3, -4.4.
EXAMPLE 43
[0312] Aldehyde 6D:
[0313] The olefin 5 (4.00 g, 11.49 mmol) was dissolved in 1:1
MeOH/CH.sub.2Cl.sub.2 (100 mL). Pyridine (4.0 mL) was then added
and the mixture cooled to -78.degree. C. Ozone was then bubbled
through the reaction for 10 minutes before the color turned light
blue in color. Oxygen was then bubbled through the reaction for 10
min. Dimethyl sulfide (4.0 mL) was then added and the reaction
slowly warmed to rt. The reaction was stirred overnight and then
the volatiles were removed in vacuo. Purification by flash
chromatography on silica gel eluting with hexanes/ethyl acetate
(9:1) gave 3.31 g (82%) of the aldehyde 6D as a clear oil: IR
(film): 2856, 1727, 1475, 1361, 1253, 1102 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 9.76 (1H, s), 7.33 (5H, m), 4.50 (2H,
s), 4.11 (1H, m), 3.47 (2H, m), 2.46 (1H, m), 1.50-1.70 (4H, band),
1.05 (3H, d, J=7.0 Hz), 0.86 (9H, s), 0.06 (3H, s), 0.03 (3H, s);
.sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 204.8, 138.3, 128.2,
127.4, 127.3, 72.7, 71.7, 69.9, 51.1, 31.1, 25.9, 25.6, 17.8, 7.5,
-4.4, -4.8.
EXAMPLE 44
[0314] Dianion Addition Product 7D:
[0315] The tert-butyl isobutyrylacetate (0.653 g, 3.51 mmol) was
added to a suspension of NaH (60% in mineral oil, 0.188 g, 4.69
mmol) in THF (50 mL) at rt. After 10 min, the mixture was cooled to
0.degree. C. After an additional 10 min, n-BuLi (1.6 M in hexanes,
2.20 mL, 3.52 mmol) was slowly added. After 30 min, the aldehyde 6D
(1.03 g, 2.93 mmol) was added neat. After 10 min, the reaction was
quenched with H.sub.2O (10 mL) and extracted with Et.sub.2O
(2.times.75 mL). The combined organics were washed once with brine
(30 mL) and dried over anhydrous MgSO.sub.4. The crude reaction
mixture contained a 15:1 ratio of diastereomers at C5. Purification
by flash chromatography on silica gel eluting with hexanes/ethyl
acetate (9:1.fwdarw.7:1) gave 0.723 g (47%) of the desired alcohol
7D as a clear oil: IR (film): 3531, 2953, 1739, 1702, 1367, 1255,
1153 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.33 (5H,
m), 4.49 (2H, s), 3.75 (1H, d, J=2.6 Hz), 3.71 (1H, m), 3.62 (1H,
d, J=16.0 Hz), 3.53 (1H, d, J=16.0 Hz), 3.44 (2H, t, J=5.1 Hz),
2.70 (1H, d, J=2.6 Hz), 1.83 (1H, m), 1.55 (4H, m), 1.46 (9H, s),
1.17 (3H, s), 1.11 (3H, s), 0.89 (9H, s), 0.82 (3H, d, J=7.0 Hz),
0.09 (6H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 208.9,
167.3, 138.4, 128.3, 127.6, 127.5, 81.3, 79.5, 78.7, 72.8, 70.1,
52.4, 47.6, 35.8, 30.6, 28.2, 25.9, 25.8, 22.6, 20.5, 17.9, 7.05,
-4.0, -4.5.
EXAMPLE 45
[0316] Directed Reduction:
[0317] To a solution of tetramethylammonium triacetoxyborohydride
(1.54 g, 5.88 mmol) in acetonitrile (4.0 mL) was added anhydrous
AcOH (4.0 mL). The mixture was stirred at rt for 30 min before
cooling to -10.degree. C. A solution of the ester 7D (0.200 g, 0.39
mmol) in acetonitrile (1.0 mL) was added to the reaction and it was
stirred at -10.degree. C. for 20 h. The reaction was quenched with
1 N sodium-potassium tartrate (10 mL) and stirred at rt for 10 min.
The solution was then poured into sat NaHCO.sub.3 (25 mL) and
neutralized by the addition of solid Na.sub.2CO.sub.3. The mixture
was then extracted with EtOAc (3.times.30 mL) and the organics were
washed with brine (20 mL) and dried over anydrous MgSO.sub.4.
Purification by flash chromatography on silica gel eluting with
hexanes/ethyl acetate (4:1) gave 0.100 g (50%) of the diol as 10:1
ratio of diastereomeric alcohols.
EXAMPLE 46
[0318] Monoprotection of the Diol:
[0319] The diol (1.76 g, 3.31 mmol) was dissolved in
CH.sub.2Cl.sub.2 (100 mL) and cooled to 0.degree. C. 2,6-lutidine
(12.2 mL, 9.92 mmol) was added followed by TBSOTf (1.14 mL, 4.96
mmol) and the reaction slowly warmed to rt. After 1 h, the reaction
was poured into Et.sub.2O (300 mL) and washed once with 1N HCl (50
mL), once with sat NaHCO.sub.3 (50 mL), once with brine (30 mL) and
dried over anhydrous MgSO.sub.4. Purification by flash
chromatography on silica gel eluting with hexanes/ethyl acetate
(20:1.fwdarw.15:1) gave 2.03 g (95%) of the alcohol 8D as a clear
oil, which was used as a mixture of diastereomers.
EXAMPLE 47
[0320] C5 Ketone Formation:
[0321] The alcohol 8D (2.03 g, 3.14 mmol) was dissolved in
CH.sub.2Cl.sub.2 (50 mL) and Dess-Martin periodinane (2.66 g, 6.28
mmol) was added. After 2 h, a 1:1 mixture of sat'd NaHCO.sub.3/sat
Na.sub.2S.sub.2O.sub.3 (20 mL) was added. After 10 min, the mixture
was poured into Et.sub.2O (300 mL) and the organic layer was washed
with brine (30 mL) and dried over anhydrous MgSO.sub.4.
Purification by flash chromatography on silica gel eluting with
hexanes/ethyl acetate (15:1) gave 1.85 g (91%) of the ketone
(benzyl ether) as a clear oil, which was used as a mixture of
diastereomers.
EXAMPLE 48
[0322] Debenzylation:
[0323] The ketone (benzyl ether) (1.85 g, 2.87 mmol) was dissolved
in EtOH (50 mL), and Pd(OH).sub.2 (0.5 g) was added. The mixture
was then stirred under an atmosphere of H.sub.2. After 3 h, the
reaction was purged with N.sub.2 and then filtered through a pad of
celite rinsing with CHCl.sub.3 (100 mL). Purification by flash
chromatography on silica gel eluting with ethyl acetate in hexanes
(12%.fwdarw.15%) gave 1.43 g (90%) of the diastereomeric alcohols
as a clear oil. The C3 diastereomers were separated by flash
chromatography on TLC-grade SiO.sub.2 eluting with ethyl acetate in
hexanes (15%):
[0324] Alpha isomer: IR (film): 3447, 1732, 1695, 1254, 1156
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 4.24 (1H, dd,
J=3.6, 5.8 Hz), 3.83 (1H, m), 3.53 (1H, m), 3.06 (1H, t, J=7.1 Hz),
2.36 (1H, dd, J=3.6, 17.2 Hz), 2.12 (1H, dd, J=3.9, 17.2 Hz), 1.68
(1H, t, J=5.4 Hz), 1.54 (2H, m), 1.41 (1H, m), 1.37 (9H, s), 1.31
(1H, m), 1.16 (3H, s), 1.02 (3H, s), 0.99 (3H, d, J=6.8 Hz), 0.84
(9H, s), 0.81 (9H, s), 0.05 (3H, s), 0.01 (6H, s), -0.01 (3H, s);
.sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 217.7, 171.3, 80.57,
73.5, 73.1, 63.0, 53.4, 26.8, 41.2, 32.1, 28.1, 28.0, 26.0, 25.9,
23.1, 19.8, 18.1 (overlapping), 15.3, -4.0, -4.3 (overlapping),
-4.8.
[0325] Beta isomer: IR (film): 3442, 2857, 1732, 1700, 1472, 1368,
1255 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 4.45 (1H,
t, J=5.3 Hz), 3.86 (1H, m), 3.52 (2H, q, J=5.9 Hz), 3.01 (1H, m),
2.28 (1H, dd, J=4.3, 17.1 Hz), 2.16 (1H, dd, J=5.5, 17.1 Hz), 1.67
(1H, t, J=5.6 Hz), 1.56 (2H, m), 1.44 (1H, m), 1.37 (9H, s),1.34
(1H, m), 1.13 (3H, s), 0.97 (3H, d, J=7.4 Hz), 0.96 (3H, s), 0.83
(9H, s), 0.79 (9H, s), 0.01 (3H, s), 0.00 (6H, s), -0.07 (3H, s);
.sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 217.1, 171.2, 80.6,
73.5, 72.1, 62.9, 63.9, 46.4, 41.2, 32.0, 28.1, 28.0, 26.0, 25.9,
21.5, 19.5, 18.2, 18.1, 15.8, -4.0, -4.3, -4.4, -4.7.
EXAMPLE 49
[0326] Aldehyde Formation:
[0327] DMSO (0.177 mL, 2.50 mmol) was added to a mixture of oxalyl
chloride (0.11 mL, 1.25 mmol) in CH.sub.2Cl.sub.2 (15 mL) at
-78.degree. C. After 10 min, the alcohol (0.531 g, 0.96 mmol) was
added in CH.sub.2Cl.sub.2 (4 mL). After 20 min, TEA (0.697 mL, 5.00
mmol) was added to the reaction followed by warming to rt. The
reaction was then poured into H.sub.2O (50 mL) and extracted with
Et.sub.2O (3.times.50 mL). The organics were washed once with
H.sub.2O (30 mL), once with brine (30 mL) and dried over anhydrous
MgSO.sub.4. The aldehyde was used in crude form.
EXAMPLE 50
[0328] Wittig Olefination to Give 9D:
[0329] NaHMDS (1.0 M soln in THF, 1.54 mL, 1.54 mmol) was added to
a suspension of methyl triphenylphosphonium bromide (0.690 g, 1.92
mmol) in THF (20 mL) at 0.degree. C. After 1 h, the crude aldhyde
(0.96 mmol) was added in THF (5 mL). After 15 min at 0.degree. C.,
H.sub.2O (0.1 mL) was added and the reaction poured into hexanes
(50 mL). This was filtered through a plug of silica gel eluting
with hexanes/Et.sub.2O (9:1, 150 mL). The crude olefin 9D was
further purified by flash chromatography on silica gel eluting with
ethyl acetate in hexanes (5%) to give 0.437 g (83% for two steps)
of the olefin 9D as a clear oil: IR (film): 2857, 1732, 1695, 1472,
1368, 1255, 1156 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 5.72 (1H, m), 4.91 (2H, m), 4.25 (1H, dd, J=3.9, 5.4 Hz),
3.81 (1H, m), 3.05 (1H, m), 2.38 (1H, dd, J=7.9, 17.2 Hz), 2.12
(1H, dd, J=6.6, 17.2 Hz), 2.04 (2H, q, J=7.5 Hz), 1.47 (1H, m),
1.39 (9H, s), 1.34 (1H, m), 1.20 (3H, s), 1.00 (3H, s), 3.00 (3H,
d, J=6.7 Hz), 0.85 (9H, s), 0.83 (9H, s), 0.07 (3H, s), 0.00 (6H,
s), -0.05 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta.
217.5, 172.1, 137.9, 114.0, 80.4, 74.0, 73.0, 53.0, 46.9, 41.3,
35.1, 29.0, 28.1, 26.0, 25.9, 22.8, 20.2, 18.2 (overlapping), 14.9,
-4.1, -4.2, -4.3, -4.8.
EXAMPLE 51
[0330] TBS Ester 10D:
[0331] The olefin 9D (0.420 g, 0.76 mmol) was dissolved in
CH.sub.2Cl.sub.2 (15 mL) and treated successively with 2,6-lutidine
(1.33 mL, 11.4 mmol) and TBSOTf (1.32 mL, 5.73 mmol). After 7 h,
the reaction was poured into Et.sub.2O (100 mL) and washed
successively with 0.2N HCl (25 mL), brine (20 mL) and dried over
anhydrous MgSO.sub.4. The residue was purified by flash
chromatography on a short pad of silica gel with fast elution with
hexanes/ethyl acetate (20:1) to give the TBS ester 10D as a clear
oil. The purification must be done quickly to avoid hydrolysis of
the silyl ester: IR (film): 2930, 1721, 1695, 1472, 1254, 1091
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 5.73 (1H, m),
4.91 (2H, m), 4.25 (1H, dd, J=3.8, 5.4 Hz) 3.80 (1H, q, J=6.8 Hz),
3.06 (1H, m), 2.50 (1H, dd, J=3.7, 17.3 Hz), 2.19 (1H, dd, J=5.7,
17.3 Hz), 2.04 (2H, dd, J=7.6, 15.3 Hz), 1.49 (1H, m), 1.36 (1H,
m), 1.21 (3H, s), 1.00 (3H, d, J=6.8 Hz), 0.88 (9H, s), 0.85 (9H,
s), 0.83 (9H, s), 0.22 (3H, s), 0.22 (3H, s), 0.21 (3H, s), 0.06
(3H, s), 0.01 (6H, s), -0.05 (3H, s); .sup.13C NMR (CDCl.sub.3, 100
MHz) .delta. 217.3, 172.3, 138.5, 114.4, 74.5, 73.0, 53.2, 46.9,
41.8, 35.1, 29.0, 26.0, 25.7, 25.5, 22.8, 20.4, 18.2, 18.1, 17.5,
14.9, -2.9, -4.0, -4.2, -4.3, -4.8, -4.9.
EXAMPLE 52
[0332] Suzuki Coupling:
[0333] The acetate acid 13D was purified by flash chromatography on
silica gel eluting with hexanes/ethyl acetate (7:1.fwdarw.4:1).
This was further purified by preparative-TLC eluting with
hexanes/ethyl acetate (2:1) to remove unreacted vinyl iodide 12D
from the acetate acid 13D. Isolated yield of the acid was 0.297 g
(62% based on 90% purity with borane residues).
EXAMPLE 53
[0334] Hydrolysis of Acetate Acid 13D:
[0335] The acetate 13D (0.220 g, 0.297 mmol) was dissolved in
MeOH/H.sub.2O (2:1, 15 mL) and K.sub.2CO.sub.3 (0.300 g) was added.
After 3 h, the reaction was diluted with sat NH.sub.4Cl (20 mL) and
extracted with CHCl.sub.3 (5.times.20 mL). The hydroxy-acid 14D was
purified by flash chromatography on silica gel eluting with
hexanes/ethyl acetate (4:1.fwdarw.2:1) to give 0.146 g (70%) of the
pure hydroxy acid 14D. IR (film): 3510-2400, 1712, 1694, 1471,
1254, 1093 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.
6.96 (1H, s), 6.66 (1H, s), 5.55 (1H, m), 5.38 (1H, m), 4.38 (1H,
dd, J=3.4, 6.1 Hz), 4.19 (1H, t, J=7.5 Hz), 3.84 (1H, m), 3.05 (1H,
t, J=7.0 Hz), 2.72 (3H, s), 2.49 (1H, dd, J=3.2, 16.4 Hz), 2.42
(2H, m), 2.33 (1H, dd, J=6.2, 16.4 Hz), 2.07 (2H, m), 2.02 (3H, s),
1.33 (4H, m), 1.19 (3H, s), 1.14 (3H, s), 1.06 (3H, d, J=6.7 Hz),
0.89 (9H, s), 0.88 (9H, s), 0.11 (3H, s), 0.07 (3H, s), 0.04 (6H,
s); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 217.8, 176.6, 164.9,
152.5, 141.7, 132.9, 125.0, 119.0, 15.3, 73.5, 73.3, 53.4, 47.0,
40.1, 35.8, 33.2, 29.8, 27.4, 26.0, 25.9, 24.5, 19.0, 18.1, 15.2,
14.3, -4.0, -4.2, -4.2, -4.7.
EXAMPLE 54
[0336] Macrolactonization:
[0337] DCC (0.150 g, 0.725 mmol), 4-DMAP (0.078 g, 0.64 mmol) and
4-DMAP.HCl (0.110 g, 0.696 mmol) were dissolved in CHCl.sub.3 (80
mL) at 80.degree. C. To this refluxing solution was added by
syringe pump the hydroxy acid 14D (0.020 g, 0.029 mmol) and DMAP
(0.010 g) in CHCl.sub.3 (10 mL) over 20 h. The syringe needle was
placed at the base of the condensor to ensure proper addition.
After 20 h, the reaction was cooled to 50.degree. C. and AcOH
(0.046 mL, 0.812 mmol) was added. After 2 h, the reaction was
cooled to rt and washed with sat NaHCO.sub.3 (30 mL), brine (30 mL)
and dried over anhydrous Na.sub.2SO.sub.4. The lactone 15D was
purified by flash chromatography on silica gel eluting with
hexanes/ethyl acetate (20:1.fwdarw.15:1) to give 0.014 g (75%): IR
(film): 2929, 1741, 1696, 1254, 1097 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 6.95 (1H, s), 6.55 (1H, s), 5.48 (1H,
m), 5.37 (1H, m), 5.16 (1H, d, J=9.8 Hz), 4.17 (1H, d, J=8.3 Hz),
4.07 (1H, t, J=7.2 Hz), 3.02 (1H, t, J=7.2 Hz), 2.77 (1H, m), 2.70
(3H, s), 2.64 (2H, m), 2.29 (1H, m), 2.15 (1H, m), 2.12 (3H, s),
1.92 (1H, m), 1.71 (1H, m), 1.44 (2H, m), 1.26 (1H, m), 1.17 (3H,
s), 1.12 (3H, s), 1.11 (3H, d, J=7.0 Hz), 0.91 (9H, s), 0.85 (9H,
s), 0.09 (3H, s), 0.06 (6H, s), -0.04 (3H, s); .sup.13C NMR
(CDCl.sub.3, 100 MHz) .delta. 215.2, 171.9, 164.5, 152.5, 138.0,
133.5, 123.8, 120.0, 116.7, 79.4, 76.2, 72.5, 53.5, 47.4, 39.9,
34.5, 31.9, 31.5, 30.2, 27.7, 26.1, 25.9, 24.1, 23.8, 23.1, 22.6,
19.2, 18.5, 18.2, 16.3, 14.9, 14.1, -3.7, -4.2, -4.7, -5.2.
EXAMPLE 55
[0338] Desmethyldesoxyepothilone A (16D):
[0339] To the lactone 15D (0.038 g, 0.056 mmol) in THF (2.0 mL) was
added HF.pyridine (1.0 mL). After 2 h, the reaction was poured into
sat NaHCO.sub.3 (30 mL) and extracted with CHCl.sub.3 (5.times.20
mL). The organics were dried over Na.sub.2SO.sub.4. The crude diol
16D was purified by flash chromatography on silica gel eluting with
hexanes/ethyl acetate (3:1.fwdarw.2:1) to give 0.023 g (89%): IR
(film): 3501, 2933, 1734, 1684, 1290, 1248, 1045 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 6.95 (1H, s), 6.59 (1H, s), 5.40
(2H, m), 5.23 (1H, dd, J=1.4, 9.5 Hz), 4.38 (1H, bd, J=11.1 Hz),
3.78 (1H, t, J=6.9 Hz), 3.59 (1H, bs), 3.47 (1H, s), 2.99 (1H, q,
j=7.0 Hz), 2.68 (3H, s), 2.66 (1H, m), 2.46 (1H, dd, J=11.4, 14.4
Hz), 2.26 (1H, dd, J=2.2, 14.4 Hz), 2.22 (1H, m), 2.06 (3H, s),
1.96 (1H, m), 1.49 (3H, m), 1.35 (3H, m), 1.30 (3H, s), 1.15 (3H,
d, J=6.9 Hz), 1.06 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz)
.delta. 221.5, 170.3, 165.1, 151.8, 139.1, 132.8, 125.2, 119.1,
115.5, 78.4, 72.5, 70.8, 53.8, 42.7, 39.6, 32.3, 31.8, 28.3, 26.8,
24.8, 23.1, 19.0, 17.2, 16.0, 11.1.
EXAMPLE 56
[0340] Epoxide Formation:
[0341] Diol 16D (0.008 g, 0.017 mmol) was dissolved in
CH.sub.2Cl.sub.2 (1.0 mL) and cooled to -60.degree. C.
Dimethyldioxirane (0.06 M, 0.570 mL, 0.0034 mmol) was then slowly
added. The reaction temperature was slowly warmed to -25.degree. C.
After 2 h at -25.degree. C., the volatiles were removed from the
reaction at -25.degree. C. under vacuum. The resulting residue was
purified by flash chromatography on silica gel eluting with MeOH in
CH.sub.2Cl.sub.2 (1%.fwdarw.2%) to give a 1.6:1 mixture of
cis-epoxides 3D and the diastereomeric cis-epoxide (0.0058 g, 74%).
The diastereomeric epoxides were separated by preparative-TLC
eluting with hexanes/ethyl acetate (1:1) after 3 elutions to give
pure diastereomers:
[0342] Betaepoxide3D: IR (film): 3458, 2928, 1737, 1685, 1456,
1261, 1150, 1043, 1014 cm.sup.-1; .sup.1H NMR (CD.sub.2Cl.sub.2,
500 MHz) .delta. 7.01 (1H, s), 6.56 (1H, s), 5.35 (1H, dd, J=2.3,
9.6 Hz), 4.30 (1H, dd, J=3.0, 5.7 Hz), 3.85 (1H, m), 3.81 (1H, d,
J=5.7 Hz), 3.42 (1H, d, J=2.0 Hz), 3.03 (1H, q, J=6.8 Hz), 2.97
(1H, m), 2.88 (1H, m), 2.67 (3H, s), 2.46 (1H, dd, J=9.0, 14.5 Hz),
2.33 (1H, dd, J=2.6, 14.5 Hz), 2.13 (1H, dt, J=3.0, 15.0 Hz), 2.08
(3H, s), 1.82 (1H, m), 1.52 (6H, m), 1.41 (1H, m), 1.33 (3H, s),
1.21 (4H, m), 1.12 (3H, d, J=7.0 Hz), 1.06 (3H, s); .sup.13C NMR
(CD.sub.2Cl.sub.2, 125 MHz) .delta. 221.9, 170.6, 165.6, 152.2,
138.3, 120.2, 116.6, 77.3, 73.4, 69.9, 57.7, 55.3, 43.7, 39.7,
32.6, 32.0, 29.8, 27.2, 25.7, 24.7, 22.5, 19.2, 19.0, 15.6, 15.6,
11.5;
[0343] Alpha epoxide: IR (film): 3439, 2918, 1735, 1684, 1455,
1262, 1048, 1014 cm.sup.-1; .sup.1H NMR (CD.sub.2Cl.sub.2, 500 MHz)
.delta. 7.02 (1H, s), 6.56 (1H, s), 5.62 (1H, d, J=8.1 Hz), 4.33
(1H, dd, J=2.7, 11.0 Hz), 3.85 (1H, t, J=5.9 Hz), 3.27 (1H, d,
J=5.3 Hz), 3.11 (1H, m), 3.07 (1H, d, J=7.0 Hz), 3.04 (1H, s), 2.87
(1H, m), 2.68 (3H, s), 2.46 (1H, dd, J=11.1, 14.1 Hz), 2.35 (1H,
dd, J=2.3, 14.1 Hz), 2.11 (3H, s), 2.06 (1H, ddd, J=1.9, 4.5, 15.1
Hz), 1.87 (1H, m), 1.52 (6H, m), 1.38 (2H, m), 1.29 (3H, s), 1.08
(3H, d, J=6.9 Hz), 1.03 (3H, s); .sup.13C NMR (CD.sub.2Cl.sub.2,
125 MHz) .delta. 222.1, 170.2, 165.3, 152.5, 137.6, 119.7, 116.7,
76.7, 72.9, 70.6, 57.1, 55.1, 44.7, 40.0, 32.1, 31.4, 30.0, 26.6,
25.5, 24.7, 21.3, 19.3, 18.7, 15.7, 11.5.
EXAMPLE 57
[0344] Experimental Data for C-12 Hydroxy Epothilone Analogs
[0345] Propyl Hydroxy Compound 43:
[0346] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 6.96 (1H, s), 6.59
(1H, s), 5.16-5.23 (2H, band), 4.28 (1H, m), 3.72 (1H, m), 3.63
(2H, t, J=6.3 Hz), 3.17 (1H, dq, J=2.2, 0.5 Hz), 3.02 (1H, s), 2.70
(3H, s), 2.65 (2H, m), 2.46 (1H, dd, J=10.9, 14.6 Hz), 2.29 (2H,
m), 1.98-2.09 (6H, band), 1.60-1.91 (6H, band), 1.35 (3H, s), 1.33
(3H, s), 1.18 (3H, d, J=6.8 Hz), 1.07 (3H, s), 1.01 (3H, d, J=7.1
Hz); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 220.69, 170.29,
165.00, 151.81, 141.63, 138.93, 120.64, 118.81, 115.52, 78.53,
77.23, 73.93, 71.85, 62.26, 53.63, 41.57, 39.54, 37.98, 32.33,
32.14, 31.54, 30.75, 29.67, 25.27, 22.89, 18.92, 17.67, 15.98,
15.74, 13.28; MS e/m 536.2, calc 535.29.
[0347] Hydroxy Methyl Compound 46:
[0348] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 6.97 (1H, s), 6.63
(1H, s), 5.43 (1H, dd, J=5.7, 9.1 Hz), 5.24 (1H, d, J=7.4 Hz), 4.31
(1H, d, J=9.7 Hz), 4.05 (2H, dd, J=7.3, 31.0 Hz), 3.87 (1H, bs),
3.69 (1H, bs), 3.17 (1H, dd, J=2.0, 6.9 Hz), 3.03 (1H, s), 2.69
(3H, s), 2.63 (1H, m), 2.45 (1H, dd, J=11.2, 14.6 Hz), 2.37 (1H,
m), 2.25 (2H, m), 2.11 (1H, m), 2.05 (3H, s), 1.78 (1H, m), 1.70
(1H, m), 1.35 (3H, s), 1.34 (2H, m), 1.29 (1H, m), 1.18 (3H, d,
J=6.8 Hz), 1.06 (3H, s), 1.00 (3H, d, J=7.0 Hz); .sup.13C NMR
(CDCl.sub.3, 100 MHz) .delta. 220.70, 170.16, 165.02, 151.63,
141.56, 138.41, 121.33, 118.65, 115.33, 77.74, 77.25, 74.11, 71.37,
65.75, 53.86, 41.52, 39.52, 37.98, 31.46, 27.70, 25.10, 22.86,
18.74, 17.20, 16.17, 15.63, 13.41.
EXAMPLE 58
[0349] 4,4-Dimethyl-3,5-dioxoheptanoate, tert-butyl ester 47.
[0350] t-Butyl 4-methyl-3-oxo-4-methyl pentanoate (22.5 g, 121
mmol) was added dropwise in 20 mL of dry THF to a slurry of NaH
(6.29 g, 60% dispersion in mineral oil, 157.2 mmol) in 500 mL of
dry THF. The reaction mixture was stirred at 0.degree. C. for 30
min and then the cold bath was cooled to -50.degree. C. Freshly
distilled propionyl chloride (12.3 g, 133.0 mmol) was added rapidly
(neat) via syringe to the cold solution. The reaction was monitored
by TLC and the cold bath was maintained below -30.degree. C. until
the reaction was complete. After 1 hr, the reaction was quenched by
pouring into a solution of saturated aqueous NH.sub.4Cl and
subjected to an aqueous workup. Flash column chromatography with 2%
EtOAc/hexanes afforded the desired tricarbonyl 42 (16.4 g, 67.8
mmol) in 56% yield; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
12.43 (s, 0.20H), 5.07 (s, 0.20H), 3.36 (s, 1.6H), 2.47 (q, J=7.13
Hz, 2H), 1.44 (s, 9H), 1.35 (s, 6H), 1.03 (t, J=7.18 Hz, 3H);
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 209.7, 202.9, 166.1,
81.91, 62.53, 43.34, 31.67, 27.82 (3), 21.03 (2), 7.82; IR (neat)
3411.8, 1742.6, 1718.5, 1702.0, 1644.2, 1460.6, 1156.1
cm.sup.-1.
[0351] 4,4-Dimethyl-5-oxo-3-triethylsilyloxy-2-heptenoate,
tert-butyl ester 48. The ester 47 (5.79 g, 23.9 mmol) in THF was
added to a suspension of NaH (60% in mineral oil, 1.24 g, 31.1
mmol) in THF (200 mL) at 0.degree. C. After 20 min, the reaction
was cooled to -50.degree. C. and TESOTf (5.95 mL, 26.32 mmol) was
added. After an additional 20 min, the reaction was poured into
saturated aqueous NaHCO.sub.3 (300 mL). This mixture was extracted
with Et.sub.2O (2.times.200 mL) and the combined organic layers
were dried over anhydrous MgSO.sub.4O. The resulting oil was
purified by flash column chromatography on SiO2 eluting with
Et.sub.2O/hexanes (1:20 to 1:15) to give 6.65 g (78%) of the
desired enol ether 48 as a clear oil; .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 5.16 (s, 1H), 2.48 (q, J=7.2 Hz, 2H), 1.45 (s,
9H), 1.24 (s, 6H), 1.02 (t, J=7.2 Hz, 3H), 0.95 (t, J=8.1 Hz, 9H),
0.74 (q, J=8.1 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
211.2, 169.4, 165.1, 97.77, 78.93, 55.54, 30.33, 28.21, 22.94,
8.15, 6.78, 6.02; IR (neat) 1712, 1619, 1384, 1243, 1150
cm.sup.-1.
EXAMPLE 59
[0352]
(6R,7R,8S)-7-Hydroxy-5-oxo-4,4,6,8-tetramethyl-3-triethylsilyloxy-2-
,10-undecadienoate, tert-butyl ester 49.
[0353] The keto enol ether 48 (7.80 g, 22.0 mmol) in 175 mL of dry
THF was cooled to -30.degree. C. in a cold bath
(CO.sub.2(s)/CH.sub.3CN) and then, a cooled solution of LDA (27.2
mmol, 0.90 M in THF) was added dropwise via syringe over 5 min.
Immediately after the addition of the keto enol ether, the reaction
vessel was placed in a -120.degree. C. cold bath (N2(liq)/pentane)
and the reaction mixture was stirred for 10 min. Then, the aldehyde
(2.0 g, 20.0 mmol) was added via syringe in 1 mL of dry THF. The
reaction was complete after 30 min and was quenched by pouring into
a solution of saturated aqueous NaHCO.sub.3. The desired aldol
product 49 (6.0 g, 13.2 mmol) was isolated in 60% yield (yield of
the major product of a 5.5:1 mixture of diastereomers, epimeric at
C-8) after flash column chromatography with 6-8% Et.sub.2O/hexanes;
(major diastereomer, higher Rf); .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 5.78 (m, 1H), 5.22 (m, 1H), 5.05 (m, 2H), 3.37 (m, 2H),
3.18 (q, J=7.08 Hz, 1H), 2.52 (m, 1H), 1.85 (dt, J=14.0, 8.37 Hz,
1H), 1.62 (m, 1H), 1.55 (s, 3H), 1.46 (s, 9H), 1.22 (s, 3H), 1.25
(s, 3H), 1.04 (d, J=6.90 Hz, 3H), 0.95 (t, J=7.94 Hz, 6H), 0.76 (q,
J=8.26 Hz, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 217.8,
168.1, 164.6, 137.1, 116.2, 99.03, 79.21, 74.82, 56.74, 40.65,
37.39, 35.06, 28.24, 22.53, 22.29, 14.77, 10.54, 6.95, 6.04; (minor
diastereomer, lower Rf) .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
5.73 (m, 1H), 5.23 (s, 1H), 5.02 (m, 2H), 3.43 (d, J=8.74 Hz, 1H),
3.21 (m, 2H), 2.06 (m, 1H), 1.84 (m, 1H), 1.62 (m, 1H), 1.55 (s,
3H), 1.46 (s, 9H), 1.27 (s, 3H), 1.24 (s, 3H), 1.06 (d, J=6.91 Hz,
3H), 0.96 (t, J=8.06 Hz, 9H), 0.77 (q, J=7.53 Hz, 6H); .sup.13C NMR
(100 MHz, CDCl.sub.3): .delta. 217.8, 168.5, 165.0, 136.8, 116.8,
99.46, 79.62, 75.47, 57.17, 41.43, 37.86, 35.58, 30.70, 28.66,
28.31, 22.90, 22.74, 16.53, 11.77, 7.37, 6.44.
EXAMPLE 60
[0354] (6R,
7R,8S)-7-Trichloroethoxyethylcarbonate-3,5-dioxo-4,4,6,8-tetra-
methyl-10-und ecenoate, tert-butyl ester 50.
[0355] The alcohol 49 (1.61 g, 3.55 mmol) was dissolved in 20 mL of
dry CH.sub.2Cl.sub.2 and cooled to 0.degree. C. in an ice bath.
Then, pyridine (1.12 g, 14.2 mmol) and
trichloroethoxyethylcarbonoyl chloride (TrocCl) (1.50 g, 7.10 mmol)
were added via syringe in that order. The reaction was stirred at
0.degree. C. for 5 min and then the ice bath was removed and the
reaction was allowed to come to rt and stir for 30 minutes. After
this period of time, TLC analysis showed the complete consumption
of the starting material. The reaction mixture was cooled to
0.degree. C. and the TES enol ether was hydrolyzed by the addition
of 20 mL of 0.5 M methanolic HCl. The reaction mixture was stirred
for 5 min at 0.degree. C. and then quenched by pouring into a
solution of saturated aqueous NaHCO.sub.3. The desired tricarbonyl
50 (1.54 g, 3.01 mmol) was isolated after an aqueous workup and
flash column chromatography with 7-9% Et.sub.2O/hexanes; .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 12.63 (s, 0.25H), 5.70 (m, 1H),
5.15 (s, 0.25H), 5.08-4.88 (m, 2H), 4.91 (dd, J=6.60, 5.01 Hz,
0.30H), 4.78 (m, 1H), 4.77 (dd, J=7.86, 3.58 Hz, 0.70H), 4.72 (dd,
J=11.8, 9.66 Hz, 1H), 3.48 (d, J=16.2 Hz, 0.75H), 3.42 (d, J=16.2
Hz, 0.75H), 3.36 (m, 0.30H), 3.30 (m, 0.70H), 1.88 (m, 2H), 1.50
(s, 3H), 1.46 (s, 9H), 1.39 (s, 3H), 1.12 (d, J=6.88 Hz, 0.70H),
1.10 (d, J=6.88 Hz, 1.3H), 0.93 (d, J=6.63 Hz, 1.3H), 0.88
(d,)=6.86 Hz, 0.70H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
210.5, 209.5, 203.16, 178.3, 172.6, 166.2, 154.1, 135.9, 135.6,
117.2, 116.9, 94.69, 94.56, 90.69, 82.68, 81.98, 81.65, 81.53,
63.58, 54.34, 46.56, 41.99, 41.62, 36.41, 35.84, 34.49, 34.44,
31.56, 28.23 (3), 27.94 (3), 22.62, 22.08, 21.56, 20.80, 15.95,
15.58, 14.09, 13.02, 12.98, 11.35; IR (neat) 1757.9, 1718.9,
1700.2, 1642.2, 1620.7, 1250.6, 1156.3 cm.sup.-1.
EXAMPLE 61
[0356] TBS Vinyl Iodide 51.
[0357] n-BuLi (1.6 M in hexanes, 7.69 mL, 12.3 mmol) was added to a
suspension of ethyl triphenylphosphonium iodide (5.15 g, 12.3 mmol)
in THF (50 mL) at 25.degree. C. After 20 min, the clear red
solution was transferred dropwise via syringe to a vigorously
stirred solution of 12 (3.12 g, 12.3 mmol) in THF (100 mL) at
-78.degree. C. The resulting pale yellow suspension was stirred
rapidly and warmed to 20.degree. C. Then, NaHMDS (1.0 M soln in
THF, 12.3 mL, 12.3 mmol) was added dropwise via syringe. During the
addition of the NaHMDS, the reaction mixture changed from a
yellow-orange slurry and to bright red solution. The TBS aldehyde
(D.-S. Su et al., Angew.Chem.Int.Ed.Engl., 1997, 36, 757; 2.00 g,
6.15 mmol) was then added in THF. After 30 min, the reaction
mixture was poured into hexanes (100 mL) and H.sub.2O (0.5 mL) was
added. The solution was then passed through a plug of SiO2 eluting
with 2:1 hexanes/Et.sub.2O. The iodide was purified by flash
chromatography on SiO2 eluting with hexanes/ethyl acetate (20:1 to
15:1) to give the vinyl iodide 51 (1.46 g, 55%) as a yellow oil:
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.95 (s, 1H), 6.50 (s,
1H), 5.45 (dt, J=1.5, 6.8 Hz, 1H), 4.22 (t, J=6.4 Hz, 1H), 2.73 (s,
3H), 2.48 (s, 3H), 2.39 (m, 2H), 2.02 (s, 3H), 0.90 (s, 9H), 0.06
(3, s), 0.02 (3, s); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
164.86, 153.46, 142.17, 132.54, 119.23, 115.68, 102.79, 77.70,
44.40, 39.09, 26.35, 19.65, 18.63, 14.54, -4.59, 4.84; IR (neat)
2928, 1470, 1252, 1068 cm.sup.-1.
[0358] Post-Suzuki, C-15 Hydroxy Tricarbonyl 52.
[0359] 9-BBN (0.5 M soln in THF, 6.68 mL, 3.34 mmol) was added over
a 45 min period to a solution of the olefin 50 (1.43 g, 2.78 mmol)
in THF (15 mL) at 25.degree. C. After 2 h, TLC analysis revealed
the complete consumption of the starting olefin. In a separate
flask, containing the vinyl iodide 51 (1.20 g, 2.80 mmol) and DMF
(20 mL), were added successively and with vigorous stirring:
Cs.sub.2CO.sub.3 (1.82 g, 5.60 mmol), Pd(dppf).sub.2Cl.sub.2 (0.454
g, 0.56 mmol), AsPh.sub.3 (0.171 g, 0.56 mmol) and H.sub.2O (1.82
mL, 0.1 mmol). Then the borane solution, prepared above, was added
rapidly to the vigorously stirred solution containing the vinyl
iodide. After 2 h, the reaction was complete and the reaction
mixture was poured into Et.sub.2O (300 mL) and washed with H.sub.2O
(3.times.200 mL), brine (1.times.50 mL) and dried over anhydrous
MgSO.sub.4. This crude product was purified by flash column
chromatography on SiO.sub.2 eluting with hexanes/ethyl acetate
(18:1 to 13:1 to 10:1) to afford the TBS protected coupled product
as an impure mixture which was taken on to the next step.
[0360] The crude TBS protected coupled product (.about.2.78 mmol)
was dissolved in 0.36 N HCl in MeOH (30 mL) at 25.degree. C. After
3.5 h, the mixture was poured into a solution of saturated aqueous
NaHCO.sub.3 and extracted with CHCl.sub.3 (4.times.60 mL). The
combined organic layers were washed once with brine (50 mL) and
dried over anhydrous Na.sub.2SO.sub.4. The diol was purified by
flash column chromatography on SiO.sub.2 eluting with hexanes/ethyl
acetate (4:1 to 3:1 to 2:1) to give the pure product 52 as a clear
oil (0.910 g, 46% for two steps): .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 6.96 (s, 1H), 6.56 (s, 1H), 5.16 (t, J=6.9 Hz,
1H), 4.83 (d, J=11.9 Hz, 1H), 4.75 (dd, J=3.4, 8.0 Hz, 1H), 4.70
(d, J=11.9 Hz, 1H), 4.14 (t, J=6.4 Hz, 1H), 3.45 (q, J=13.2 Hz,
2H), 3.32 (m, 1H), 2.72 (s, 3H), 2.32 (t, J=6.5 Hz, 2H), 2.04 (s,
3H), 2.01 (m, 2H), 1.74 (m, 2H), 1.69 (s, 3H), 1.45 (s, 9H), 1.38
(s, 6H), 1.09 (d, J=6.9 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H); .sup.13C
NMR(100 MHz, CDCl.sub.3): .delta. 209.51, 203.04, 166.15, 164.39,
154.14, 152.72, 141.71, 138.24, 120.70, 118.76, 115.28, 94.54,
81.85, 77.31, 76.57, 63.41, 54.16, 46.47, 41.48, 34.56, 33.95,
31.98, 31.53, 27.85, 24.85, 23.45, 21.47, 20.75, 19.04, 15.60,
14.33, 11.35; IR (neat) 3546, 3395, 1756, 1717, 1699, 1644, 1621,
1506, 1456, 1251 cm.sup.-1.
EXAMPLE 62
[0361] Noyori C-3/C-15 Diol Product 53.
[0362] The diketone 52 (0.900 g, 1.27 mmol) was dissolved in 0.12 N
HCl in MeOH (10 mL) at 25.degree. C. The RuBINAP catalyst (0.018 M
in THF, 1.0 mL, 0.018 mmol) was then added and the mixture
transferred to a Parr apparatus. The vessel was purged with H.sub.2
for 5 min and then pressurized to 1200 psi. After 12 h at
25.degree. C., the reaction was returned to atmospheric pressure
and poured into a saturated solution of NaHCO.sub.3. This mixture
was extracted with CHCl.sub.3 (4.times.50 mL) and the combined
organic layers were dried over anhydrous Na.sub.2; SO.sub.4. The
product was purified by flash column chromatography on silica gel
eluting with hexanes/ethyl acetate (4:1 to 2:1) to give 0.75 g
(81%) of the hydroxy ester 53 as a white foam; .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 6.94 (s, 1H), 6.55 (s, 1H), 5.15 (t,
J=6.9 Hz, 1H), 4.85 (t, J=5.3 Hz, 1H), 4.81 (d, J=12.0 Hz, 1H),
4.71 (d, J=12.0 Hz, 1H), 4.12 (m, 2H), 3.43 (m, 2H), 2.70 (s, 3H),
2.37 (dd, J=2.2, 6.2 Hz, 1H), 2.30 (t, J=6.7 Hz, 2H), 2.24 (dd,
J=10.6, 16.2 Hz, 1H), 2.03 (s, 3H), 1.99 (m, 2H), 1.68 (s, 3H),
1.44 (s, 9H), 1.18 (s, 3H), 1.16 (s, 3H), 1.09 (d, J=6.8 Hz, 3H),
0.94 (d, J=6.8 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
215.95, 172.39, 164.39, 154.21, 152.74, 141.70, 138.33, 120.59,
118.77, 115.27, 94.64, 82.98, 81.26, 76.51, 72.78, 51.82, 41.40,
37.36, 34.66, 33.96, 32.08, 31.10, 30.20, 27.96, 25.06, 23.45,
21.73, 21.07, 19.17, 19.01, 16.12, 15.16, 14.33, 12.17; IR (neat)
3434.0, 1757.5, 1704.5, 1249.9, 1152.8 cm.sup.-1.
EXAMPLE 63
[0363] C-3/C-15 Bis(TES) Carboxylic Acid 54.
[0364] 2,6-Lutidine (0.48 g, 4.5 mmol) and TESOTf (0.59 g, 2.25
mmol) were added successively to a cooled solution of the diol 53
(164 mg, 0.225 mmol) in CH.sub.2Cl.sub.2 (2.5 mL) at -78.degree. C.
The reaction mixture was stirred at -78.degree. C. for 5 min and
then warmed to rt. The reaction was stirred at rt for 6 hr and then
quenched with saturated aqueous NH.sub.4Cl and subjected to an
aqueous workup. The crude product was concentrated in vacuo and
subjected directly to the next set of reaction conditions; .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 6.96 (s, 1H), 6.66 (s, 1H), 5.04
(t, J=6.93 Hz, 1H), 4.90 (d, J=12.0 Hz, 1H), 4.77 (dd, J=7.99, 3.21
Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.46 (m, 1H), 4.10 (dq, J=12.3,
7.11 Hz, 2H), 3.42 (m, 1H), 2.70 (s, 3H), 2.60 (dd, J=16.7, 2.34
Hz, 1H), 2.34 (dd, J=16.7, 7.94 Hz, 1H), 2.27 (dd, J=14.0, 6.97 Hz,
1H), 2.18 (m, 1H), 2.09 (m, 1H), 2.04 (s, 1H), 1.95 (s, 3H), 1.82
(m, 2H), 1.61 (s, 3H), 1.44 (m, 2H), 1.27-1.22 (m, 4H), 1.14 (d,
J=8.45 Hz, 3H), 1.11 (d, J=6.81 Hz, 2H), 1.04 (d, J=6.88 Hz, 2H),
1.15-1.01 (m, 2H), 0.94 (t, J=7.92 Hz, 18H), 0.65-0.57 (m, 12H);
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 215.11, 175.34, 165.00,
154.14, 152.80, 142.60, 136.84, 121.31, 118.79, 114.60, 94.77,
81.60, 79.06, 76.64, 73.87, 54.19, 41.18, 39.56, 35.09, 34.52,
32.29, 31.95, 24.76, 23.62, 22.55, 18.95, 18.64, 15.87, 13.69,
11.33, 6.94, 6.83, 5.07, 4.76; IR (neat) 3100-2390, 1756.8, 1708.8,
1459.3,1250.6, 816.1 cm.sup.-1.
EXAMPLE 64
[0365] C-15 Hydroxy Acid for Macrolactonization 55.
[0366] The crude bis(triethylsilyl)ether 54, prepared above, was
dissolved in 5 mL of dry THF and then cooled to 0.degree. C. Then,
1 mL of 0.12 M HCl/MeOH was added. The reaction mixture was stirred
at 0.degree. C. for 20 min and then checked for completion. TLC
analysis at this time revealed the complete consumption of starting
material. The reaction was quenched by pouring into a solution of
saturated aqueous NaHCO.sub.3 and subjected to an aqueous workup.
Flash column chromatography with 25 to 30:1 CHCl.sub.3/MeOH
afforded the desired carboxylic acid 55 in 77% yield; .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 6.96 (s, 1H), 6.69 (1, s), 5.11 (t,
J=6.9 Hz, 1H), 4.91 (d, J=12.0 Hz, 1H), 4.71 (dd, J=3.1, 8.2 Hz,
1H), 4.64 (d, J=12.0 Hz, 1H), 4.42 (d, J=5.9 Hz, 1H), 4.10 (m, 1H),
3.43 (m, 1H), 2.71 (s, 3H), 2.57 (dd, J=2.1, 10.5 Hz, 1H), 2.25 (m,
3H), 2.11 (m, 1H), 1.98 (s, 3H), 1.95 (m, 2H), 1.72 (m 1), 1.67 (s,
3H), 1.45 (m, 2H), 1.16 (s, 3H), 1.13 (s, 3H), 1.09 (d, J=6.7 Hz,
3H), 0.99 (d, J=6.7 Hz, 3H), 0.95 (t, J=7.9 Hz, 9H), 0.64 (dq,
J=2.3, 7.9 Hz, 6H); .sup.3C NMR (100 MHz, CDCl.sub.3): .delta.
215.11, 176.00 (165.10, 154.18, 152.35, 142.24, 138.55, 120.74,
118.21, 115.02, 94.76, 81.91, 76.86, 76.63, 73.95, 54.08, 41.28,
39.64, 34.73, 34.16, 32.02, 31.67, 24.71, 23.41, 22.49, 19.17,
18.62, 15.71, 14.86, 11.20, 6.93, 5.05); IR (neat) 3400-2390,
1755.9, 1703.8, 1250.4, 735.4 cm.sup.-1.
EXAMPLE 65
[0367] C-3 Triethylsilyl/C-7 Trichloroethoxyethylcarbonate
Macrolactonization Product 56.
[0368] Triethylamine (155 mg, 1.53 mmol) and 2,4,6-trichlorobenzoyl
chloride (312 mg, 1.28 mmol) were added to a solution of the
hydroxy acid 55 (198 mg, 0.256 mmol) in 3.6 mL of dry THF. The
reaction mixture was stirred for 0.25 h at rt and then diluted with
45 mL of dry toluene. The resultant solution was added slowly
dropwise, via syringe pump, over 3 hr to a stirred solution of DMAP
(328 mg, 2.68 mmol) in 145 mL of dry toluene. After the addition of
the substrate was complete, the reaction was stirred for an
additional 0.5 h and then taken up in an equal volume of Et.sub.2O
and washed with 1N HCl (1.times.), saturated aqueous NaHCO.sub.3
(1.times.), and brine (1.times.). The organic layer was dried over
MgSO.sub.4 and concentrated in vacuo. Flash column chromatography
of the crude product with 10% EtOAc/hexanes afforded the desired
macrolactone (153 mg, 0.20 mmol) in 78% yield; .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 6.96 (s, 1H), 6.53 (s, 1H), 5.20 (m, 2H),
5.04 (d, J=10.2 Hz, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.78 (d, J=12.0
Hz, 1H), 4.07 (m, 1H), 3.32 (m, 1H), 2.86-2.63 (m, 3H), 2.70 (5,
3H), 2.48 (m, 1H), 2.11 (s, 3H), 2.04 (dd, J=6.17, 14.7 Hz, 1H),
1.73 (m, 4H), 1.66 (s, 3H), 1.25 (m, 2H), 1.19 (s, 3H), 1.15 (s,
3H), 1.12 (d, J=6.68 Hz, 3H), 1.01 (d, J=6.83 Hz, 3H), 0.89 (t,
J=8.00 Hz, 9H), 0.58 (q, J=7.83 Hz, 6H); .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta. 212.75, 170.66, 164.62, 154.60, 152.52,
140.29, 138.44, 119.81, 119.38, 116.28, 94.84, 86.44, 80.14, 76.59,
76.10, 53.55, 45.89, 39.23, 35.47, 32.39, 31.69, 31.57, 31.16,
29.68, 27.41, 25.00, 23.44, 22.94, 19.23, 18.66, 16.28, 14.83,
6.89, 5.22; IR (neat) 1760.5, 1742.6, 1698.0, 1378.8, 1246.2,
1106.0, 729.8 cm.sup.-1.
EXAMPLE 66
[0369] Sml.sub.2 Mediated Deprotection of Troc Group 57.
[0370] Samarium metal (0.334 g, 2.22 mmol) and iodine (0.51 g, 2.0
mmol) in 25 mL of dry, deoxygenated THF were stirred together
vigorously for 2.5 hr at ambient temperature. During this period of
time, the reaction mixture progressed from a dark orange to an
olive green to deep blue color. The resultant deep blue solution of
Sml2 was used directly in the following reaction. Sml2 (25 mL of a
0.08 M stock solution, 2.0 mmol) was added rapidly via syringe to a
stirred solution of the macrolactone 57 (200 mg, 0.26 mmol) and a
catalytic amount of Nil.sub.2 (10 mg) in 10 mL of dry THF at
-78.degree. C. The resultant deep blue solution was maintained at
-78.degree. C. with continued vigorous stirring for 2.5 hr. TLC
analysis at this time revealed the complete consumption of the
starting material and formation of a single, lower Rf product. The
reaction mixture was quenched with saturated aqueous NaHCO.sub.3
and subjected to an aqueous workup. Flash column chromatography
with 25% EtOAc/hexanes afforded the desired alcohol 57 (143 mg,
0.24 mmol) in 91% yield; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
6.95 (s, 1H), 6.54 (s, 1H), 5.15 (m, 1H), 5.05 (d, J=10.15 Hz, 1H),
4.08 (dd, J=10.1, 2.66 Hz, 1H), 3.87 (m,1H), 3.01 (s,1H), 3.06 (m,
1H), 2.83-2.65 (m, 3H), 2.70 (s, 3H), 2.44 (m, 1H), 2.10 (s, 3H),
2.07 (m, 1H), 1.83 (m, 1H), 1.77 (m, 1H), 1.71 (m, 1H), 1.64 (s,
3H), 1.60 (s, 1H), 1.37 (m, 1H), 1.31 (m, 1H), 1.20 (m, 1H), 1.15
(s, 3H), 1.14 (m, 5H), 1.02 (d, J=7.02 Hz, 3H), 0.89 (t, J=7.97 Hz,
9H), 0.64-0.52 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
218.34, 170.73, 164.59, 152.46, 139.07, 138.49, 120.48, 119.54,
116.00, 79.31, 75.81, 73.48, 53.62, 42.98, 39.48, 39.01, 32.85,
32.41, 31.20, 26.12, 24.26, 22.01, 22.46, 19.18, 16.44, 15.30,
13.99, 6.98 (3), 5.27 (3); IR (neat) 3524.0, 1740.3; 1693.4,
1457.2, 1378.4, 733.2 cm.sup.-1.
EXAMPLE 67
[0371] Desoxyepothilone B 2C.
[0372] The TES protected alcohol 57 (143 mg, 0.24 mmol) was
dissolved in 2 mL of dry THF in a plastic reaction vessel and
cooled to 0.degree. C. in an ice bath. The resultant solution was
treated with 1 mL of HF-pyridine. The reaction mixture was stirred
for 80 min at 0.degree. C. and then quenched by pouring into a
saturated aqueous solution of NaHCO.sub.3. An aqueous workup
followed by flash column chromatography with 10% EtOAc/hexanes
afforded desoxyepothilone B (112 mg, 0.23 mmol) in 95% yield. The
resultant product exhibited a .sup.1H NMR spectrum identical to
that of authentic desoxyepothilone B.
[0373] Total Synthesis of Desoxyepothilone B
EXAMPLE 68
[0374] tert-Butyl 4-methyl-3-oxopentanoate (1E).
[0375] Meldrum's acid (80 g, 555 mmol) was dissolved in 600 mL of
CH.sub.2Cl.sub.2 and cooled to 0.degree. C. Freshly distilled
pyridine (87.8 g, 1.11 mol) was added to the CH.sub.2Cl.sub.2
solution and then iso-butyryl chloride (65.0 g, 610.5 mmol) was
added to the mixture via a pressure equalizing addition funnel. The
reaction was stirred at 0.degree. C. for 1 hr and then warmed to rt
and stirred for 2 hr. Then, the reaction was quenched with water
(200 mL) and washed with 0.5 M HCl (.times.2), water (.times.1),
and brine (.times.1). The organize layer was dried over MgSO.sub.4
and concentrated in vacuo. The crude product was azeotropically
dried with benzene (250 mL), and then dissolved in 200 mL of
benzene and 200 mL of tert-butanol was added. The resultant
reaction was heated at reflux for 4 hr. After this period, the
volatiles were removed in vacuo and the product then distilled on
the high vacuum pump (bp 62-63.degree. C., 0.1 mm Hg). The desired
.beta.-keto ester 1E was obtained (58.6 g, 315.5 mmol) in 57% yield
as a clear, colorless, light oil.
EXAMPLE 69
[0376] tert-Butyl 4,4-dimethy-3,5-dioxoheptanoate (2E).
[0377] .beta.-Keto ester 1E (55.0 g, 295.3 mmol) was added dropwise
in 50 mL of dry THF to a slurry of NaH (9.7 g, 60% dispersion in
mineral oil, 383.9 mmol) in 1.15 L of dry THF. The reaction mixture
was stirred at 0.degree. C. for 30 min and then the cold bath was
cooled to -50.degree. C. Propionyl chloride (27.3 g, 295.3 mmol)
was added rapidly (neat) by syringe to the cold solution. The
reaction was monitored by TLC and the cold bath was maintained
below -30.degree. C. until the reaction was complete. After 1 hr,
the reaction was quenched by pouring into a solution of saturated
aqueous NH.sub.4Cl and subjected to an aqueous workup. The aqueous
layer was extracted with Et.sub.2O (.times.2, 200 mL). Flash column
chromatography with 2% EtOAc/hexanes afforded the desired
tricarbonyl 2E (50.7 g, 209.6 mmol) in 71% yield; .sup.1H NMR (400
MHz, CDCl.sub.3); .delta. 12.43 (s, 0.20H), 5.07 (s, 0.20H), 3.36
(s, 1.6H), 2.47 (q, J=7.13 Hz, 2H), 1.44 (s, 9H), 1.35 (s, 6H),
1.03 (t, J=7.18 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta. 209.7, 202.9, 166.1, 81.91, 62.53, 43.34, 31.67, 27.82 (3),
21.03 (2), 7.82; IR (neat) 3411.8, 1742.6, 1718.5, 1702.0, 1644.2,
1460.6, 1156.1 cm.sup.-1.
EXAMPLE 70
[0378] tert-Butyl-4,4-dimethyl-3-methoxy-5-oxo-2-heptenoate
(3E).
[0379] Trimethylsilyldiazomethane (TMSCHN.sub.2, 46.2 mL of a 2.0 M
solution in THF, 92.4 mmol) was added by syringe to a stirred
solution of the tricarbonyl (16.0 g, 66.0 mmol) and
diisopropylethylamine (Hunig's base, 16.1 mL, 92.4 mmol) in 330 mL
of a 9:1 solution of acetonitrile:methanol at rt. The resultant
reaction mixture was stirred at rt for 18-20 hr. The reaction
mixture was then quenched with saturated aqueous NaHCO.sub.3 and
the enol ether extracted with Et.sub.2O (.times.3, 50 mL). The
combined organic layers were washed with brine and then dried of
MgSO.sub.4, filtered and concentrated in vacuo. Flash column
chromatography of the crude product (2% EtOAc/hexanes) afforded the
desired enol ether 3E (12.5 g, 48.4 mmol) in 74% yield; .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 5.18 (s, 1H), 3.88 (s, 3H), 2.45 (q,
J=7.33 Hz, 2H), 1.48 (s, 9H), 1.25 (s, 6H), 1.02 (t, J=7.21 Hz,
3H).
EXAMPLE 71
[0380]
(67R,7R,8S)-7-Hydroxy-5-oxo-4,4,6,8-tetramethy-3-triethysilyloxy-2,-
10-undecadienoate, tert-butyl ester (6E).
[0381] The keto enol ether 3E (8.0 g, 31.3 mmol) in 750 mL of dry
THF was cooled to -30.degree. C. in a cold bath
(CO.sub.2(s)/CH.sub.3CN) and then a solution of LDA (37.5 mmol,
0.90 M in THF) was added dropwise via syringe over 10 min. The
reaction mixture was stirred at -30 to -33.degree. C. for 20 min.
Then the reaction vessel was placed in a -120.degree. C. cold bath
(N.sub.2(liq)/pentane) and the reaction mixture was stirred for 10
min. Finally the aldehyde 5 (3.6 g, 36.7 mmol; aldehyde 5E was
readily prepared according to the procedure outlined in: Lin,
N.-H., et al., J. Am. Chem. Soc. 1996, 118, 9062.) was added via
syringe in 5 mL of CH.sub.2Cl.sub.2. The reaction was complete
after 10 min and was quenched by pouring into a solution of
saturated aqueous NH.sub.4Cl. The desired aldol product 6E (5.2 g,
14.7 mmol) was isolated in 47% yield (yield of the major product of
a 5.5:1 mixture of diastereomers, epimeric at C-8) after flash
column chromatography with 6-5% EtOAC/hexanes; (major diasteromer,
high R.sub.f); .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 5.78 (m,
1H), 5.18 (s, 1H), 4.98 (m, 2H), 3.90 (s, 3H), 3.37 (m, 1H), 3.35
(s, 1H), 3.35 (s, 1H), 3.12 (q, J=7.74 Hz, 1H), 2.53 (m, 1H), 1.87
(dt, J=13.8, 8.47 Hz, 1H), 1.61 (m, 1H), 1.55 (s, 1H), 1.48 (s,
9H), 1.28 (s, 3H), 1.27 (s, 3H), 1.05 (d, J=6.91 Hz, 3H), 0.079 (d,
J=6.76 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 217.7,
171.2, 164.8, 137.0, 116.3, 97.29, 80.22, 74.67, 62.17, 56.05,
41.05, 37.31, 34.99, 28.13, 22.69, 22.67, 15.00, 10.44; (minor
diastereomer, low R.sub.f): .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 5.76 (m, 1H), 5.19 (s, 1H), 5.06 (m, 2H), 1.48 (s, 3H),
3.41 (m, 1H), 3.17 (m, 1H), 3.12 (m, 1H), 2.11 (m, 1H), 1.86 (m,
1H), 1.63 (m, 1H), 1.48 (s, 9H), 1.28 (s, 3H), 1.27 (s, 3H), 1.07
(s, J=6.91 Hz, 3H), 0.99 (d, J=6.64 Hz, 3H).
EXAMPLE 72
[0382]
(6R,7R,8S)-7-(2,2,2-Trichloroethoxycarbonate)-5-oxo-4,4,6,8-tetrame-
thyl-3-triethylsilyloxy-2,10-undecadienoate, tert-butyl ester.
[0383] Alcohol 6E (5.2 g, 14.7 mmol) was dissolved in 70 mL of dry
CH.sub.2Cl.sub.2 and cooled to 0.degree. C. in an ice bath. Then,
pyridine (4.65 g, 58.8 mmol) and trichloroethoxyethylcarbonoyl
chloride (TrocCl) (6.23 g, 29.4 mmol) were added by syringe in that
order. The reaction was stirred at 0.degree. C. for 5 min and then
the ice bath was removed and the reaction was allowed to come to rt
and stir for 30 minutes. After this period of time, TLC analysis
showed the complete consumption of the starting material. The
reaction mixture was quenched by pouring it into a solution of
saturated aqueous NaHCO.sub.3. Flash column chromatography with 3%
EtOAc/hexanes through a short plug of silica gel afforded the
desired enol ether which was subjected immediately to hydrolysis;
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 5.71 (m, 1H), 5.21 (s,
1H), 5.02 (m, 2H), 4.86 (d, J=11.9 Hz, 1H), 4.85 (m, 1H), 4.72 (d,
J=11.9 Hz, 1H), 3.91 (s, 3H), 3.25 (m, 1H), 2.26 (m, 1H), 1.87 (m,
1H), 1.81 (m, 1H), 1.48 (s, 9H), 1.31 (s, 3H), 1.26 (2, 3H), 1.11
(d, J=6.85 Hz, 3H), 0.91 (d, J=6.64 Hz, 3H).
EXAMPLE 73
[0384]
(6R,7R,8S)-7-Trichloroethoxyethylcarbonate-3,5-dioxo-4,4,6,8-tetram-
ethyl-10-undecenoate, tert-butyl ester (7E).
[0385] The Troc-protected enol ether (as above) was dissolved in
acetone and treated with 300 mg (catalytic) of p-TsOH at rt for 5-6
hrs. The reaction was monitored by TLC and after complete
consumption of the starting enol ether was apparent, the reaction
mixture was quenched with saturated aqueous NaHCO.sub.3. The
desired tricarbonyl 7E (6.8 g, 12.8 mmol), 87% (2 steps) was
isolated after an aqueous workup and flash column chromatography
with 7-9% EtOAc/hexanes; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
12.63 (s, 0.25H), 5.70 (m, 1H), 5.15 (s, 0.25H), 5.08-4.88 (m, 2H),
4.91 (dd, J=6.60, 5.01 Hz, 0.30H), 4.78 (m, 1H), 4.77 (dd, J=7.86,
3.58 Hz, 0.70H), 4.72 (dd, J=11.8, 9.66 Hz, 1H), 3.48 (d, J=16.2
Hz, 0.75H), 3.42 (d, J=16.2 Hz, 0.75H), 3.36 (m, 0.30H), 3.30 (m,
0.70H), 1.88 (m, 2H), 1.50 (s, 3H), 1.46 (s, 9H), 1.39 (s, 3H),
1.12 (d, J=6.88 Hz, 0.70H), 1.10 (d, J=6.88 Hz, 1.3H), 0.93 (d,
J=6.63 Hz, 1.3H), 0.88 (d, J=6.86 Hz, 0.70H); .sup.13C NMR (100
MHz, CDCl.sub.3): .delta. 210.5, 209.5, 203.16, 178.3, 172.6,
166.2, 154.1, 135.9, 135.6, 117.2, 116.9, 94.69, 94.56, 90.69,
82.68, 81.98, 81.65, 81.53, 63.58, 54.34, 46.56, 41.99, 41.62,
36.41, 35.84, 34.49, 34.44, 31.56, 28.23 (3), 27.94 (3), 22.62,
22.08, 21.56, 20.80, 15.95, 15.58, 14.09, 13.02, 12.98, 11.35; IR
(neat) 1757.98, 1718.9, 1700.2, 1642.2, 1620.7, 1250.6, 1156.3
cm.sup.-1.
EXAMPLE 74
[0386] Allylic Alcohol (16E).
[0387] A mixture of (S)-(-)-1,1'-bi-2-naphthol (1.37 g, 4.8 mmol),
Ti(O-i-Pr).sub.4 (1.36 g, 4.8 mmol), and 4 .ANG. sieves (11 g) in
CH.sub.2Cl.sub.2 (300 mL) was heated at reflux for 1 h. The mixture
was cooled to rt and aldehyde 15E (8.0 g, 47.9 mmol; prepared
according to the procedure outlined in an earlier Danishefsky
synthesis of the epothilones: Meng, D.; Sorensen., E. J.;
Bertinato, P.; Danishefsky, S. J. J. Org. Chem. 1996, 61, 7998) was
added. After 10 min, the suspension was cooled to -78.degree. C.,
and allyl tri-n-butyltin (20.9 g, 67.1 mmol) was added. The
reaction mixture was stirred for 10 min at -78.degree. C. and then
placed in a 20.degree. C. freezer for 70 h. Saturated aqueous
NaHCO.sub.C solution (2 mL) was added, and the mixture was stirred
for 1 h, poured over Na.sub.2SO.sub.4, and then filtered through a
pad of MgSO.sub.4 and celite. The crude material was purified by
flash chromatography with EtOAc/hexanes
(5%.fwdarw.10%.fwdarw.15%.fwda- rw.20%.fwdarw.25%.fwdarw.30%, two
column volumes each) to give alcohol 16E as a yellow oil (6.0 g,
88.0 mmol) in 60% yield; [.alpha.].sub.D=-15.9 (c. 4.9,
CHCl.sub.3); IR (film) 3360, 1641, 1509, 1434, 1188, 1017, 914
cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3, 25.degree. C.) .delta.
6.92 (s,1H), 6.55 (s, 1H), 5.82 (m, 1H), 5.13 (dd, J=17.1, 1.3 Hz,
1H), 5.09 (d, J=10.2 Hz, 1H), 4.21 (t, J=6.0 Hz, 1H), 2.76 (br s,
1H), 2.69 (s, 3H), 2.40 (m, 2H), 2.02 (s, 3H); .sup.13C NMR (125
MHz, CDCl.sub.3, 25.degree. C.) .delta. 164.5, 152.6, 141.5, 134.6,
119.2, 117.6, 115.3, 76.4, 39.9, 19.0, 14.2; HRMS calcd. for
C.sub.11H.sub.15NOS: 209.0874 found: 209.0872 (M+H).
EXAMPLE 75
[0388] TBS Allylic Ether (17E).
[0389] Alcohol 16E (5.70 g, 27.3 mmol) was dissolved in 50 mL of
dry CH.sub.2Cl.sub.2 and cooled to -78.degree. C. Then,
2,6-lutidine (7.6 g, 70.9 mmol) and TBSOTf (9.36 g, 35.4 mmol) were
added via syringe successively and in that order. The reaction
mixture was stirred at this temperature for 30 minutes and then
quenched by pouring the reaction mixture into saturated aqueous
NaHCO.sub.3. An aqueous workup followed by flash column
chromatography with 2% EtOAc/hexanes afforded the desired TBS ether
(7.37 g, 22.8 mmol) in 84% yield. The allylic alcohol, 17E, could
also be prepared according to the general procedure outlined in the
following references: (a) Racherla, U. S.; Brown, H. C. J. Org.
Chem. 1991, 56, 401. (b) Yang, Z.; He, Y.; Vourloumis, D.;
Vallberg, H.; Nicolaou, K. C. Angew. Chem., Int. Ed. Engl., 1997,
36, 166.
EXAMPLE 76
[0390] Aldehyde (18E).
[0391] To a solution of TBS ether 17E (7.37 g, 22.8 mmol) in
acetone (150 mL) at 0.degree. C. was added 6.7 g of a 60% solution
of N-methyl-morpholine-N-oxide (NMO) in water (34.2 mmol),
OsO.sub.4 (0.039 M in THF, 6 mL, 0.23 mmol). The resultant mixture
was stirred at 0.degree. C. for 2 h and then quenched with
saturated aqueous Na.sub.2SO.sub.3 solution (100 mL). The solution
was poured into H.sub.2O (100 mL) and extracted with EtOAc
(8.times.50 mL). The combined organic layer was dried over
MgSO.sub.4, filtered, concentrated, and flashed through a short
plug of silica gel to afford (7.8 g, 21.8 mmol) the crude diol in
96% yield.
[0392] To a solution of the crude diol (7.8 g, 21.8 mmol; the
oxidation procedure described here may also be acomplished with
NaIO.sub.4 as outlined in a previous Danishefsky synthesis of the
epothilones: Meng, D., et al., J. Ann. Chem. Soc. 1997, 119,
10073.) in 400 mL benzene at 0.degree. C. was added Pb(OAc).sub.4
(19.4 g, 43.7 mmol) and Na.sub.2CO.sub.3 (9.24 g, 87.2 mmol). The
reaction mixture was stirred at 0.degree. C. for 10 min and then rt
for 1.5 hr. After this period of time, the reaction mixture was
quenched by pouring into brine. The reaction was filtered through
Celite.TM. and then the resultant aqueous layer was extracted with
EtOAc (5.times.50 mL) dried over MgSO.sub.4. Flash column
chromatography on silica gel with 20% EtOAc/hexanes on a short pad
of silica gave the aldehyde 18E as a yellow oil (5.02 g, 15.5 mmol)
in 71% yield.
EXAMPLE 77
[0393] TBS Vinyl Iodide (19E).
[0394] n-BuLi (2.5 M in hexanes, 22.6 mL, 55.4 mmol) was added to a
suspension of ethyl triphenylphosonium iodide (23.2 g, 55.4 mmol)
in THF (100 mL) at 25.degree. C. After 30 min, the clear red
solution was transferred dropwise by syringe to a vigorously
stirred solution of 12 (14.1 g, 55.4 mmol) in THF (1100 mL) at
-78.degree. C. After addition of the Wittig reagent was completed,
the resulting pale yellow suspension was stirred rapidly and warmed
to 20.degree. C. Then, NaHMDS (1.0 M soln in THF, 55.4 mL, 55.4
mmol) was added dropwise by syringe. During the addition of the
NaHMDS, the reaction mixture changed from a yellow-orange slurry
and to bright red solution. Aldehyde 18E (6.0 g, 18.5 mmol) was
then added in THF. After 30 min, the reaction mixture was poured
into hexanes (400 mL) and then 0.5 mL brine was added. The solution
was then passed through a plug of SiO.sub.2 eluting with 2:1
hexanes/Et.sub.2O. The iodide was purified by flash chromatography
on SiO.sub.2 eluting with hexanes/ethyl acetate (20:1 to 15:1) to
give the vinyl iodide 19E (5.0 g, 10.2 mmol, 50%) as a yellow oil:
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.95 (s, 1H), 6.50 (s,
1H), 5.45 (dt, J=1.5, 6.8 Hz, 1H), 4.22 (t, J=6.4 Hz, 1H), 2.73 (s,
3H), 2.48 (s, 3H), 2.39 (m, 2H), 2.02 (s, 3H), 0.90 (s, 9H), 0.06
(3, s), 0.02 (3, s); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
164.86, 153.46, 142.17, 132.54, 119.23; 115.68, 102.79, 77.70,
44.40, 39.09, 26.35, 19.65, 18.63, 14.54, -4.59, -4.84; IR (neat)
2928, 1470, 1252, 1068 cm.sup.-1.
EXAMPLE 78
[0395] Post-Suzuki, C-15 Hydroxy Tricarbonyl (10E).
[0396] 9-BBN (0.5 M soln in THF, 14.1 mL, 7.03 mmol) was added over
a 45 min period to a solution of the olefin 7E (2.78 g, 5.41 mmol)
in THF (25 mL) at 25.degree. C. After 2 h, TLC analysis revealed
the complete consumption of the starting olefin.
[0397] In a separate flask, containing the vinyl iodide 18E (2.65
g, 5.41 mmol) and DMF (45 mL), were added successively and with
vigorous stirring: Cs.sub.2CO.sub.3 (3.52 g, 10.82 mmol);
Pd(dppf).sub.2Cl.sub.2 (1.10 g, 1.35 mmol), AsPh.sub.3 (0.41 g,
1.35 mmol) and H.sub.2O (3.5 mL, 0.19 mol). Then the borane
solution, prepared above, was added rapidly by syringe to the
vigorously stirred solution containing the vinyl ioidide. After 2
h, the reaction TLC analysis revealed that the reaction was
complete. The reaction mixture was poured into Et.sub.2O
(3.times.200 mL), brine (1.times.50 mL) dried over anhydrous
MgSO.sub.4. This crude product was purified by flash column
chromatography on SiO.sub.2 eluting with hexanes/ethyl acetate
(18:1 to 13:1 to 10:1) to afford the TBS protected coupled product
9E as an impure mixture which was taken on to the next step without
further purification.
[0398] The crude TBS protected coupled product 9E was dissolved in
0.5 M HCl in MeOH (30 mL) at 25.degree. C. The reaction was
monitored by TLC for corruption and after 3.5 h (disappearance of
starting TBS ether), the mixture was poured into a solution of
saturated aqueous NaHCO.sub.3 and extracted with CHCl.sub.3
(4.times.60 mL). The combined organic layers were washed once with
brine (50 mL) and dried over with anhydrous MgSO.sub.4. The diol
was purified by flash column chromatography on SiO.sub.2 eluting
with hexanes/ethyl acetate (4:1 to 3:1 to 2:1) to give the pure
product 10E as a clear oil (2.44 g, 3.35 mmol, 62% for two steps):
.sup.1H NMR (400 MHz, CDCl.sub.3: .delta. 6.96 (s, 1H), 6.56 (s,
1H), 5.16 (t, J=6.9 Hz, 1H), 4.83 (d, J=11.9 Hz, 1H), 4.75 (dd,
J=3.4, 8.0 Hz, 1H), 4.70 (d, J=11.9 Hz, 1H), (t, J=6.4 Hz, 1H),
3.45 (q, J=13.2 Hz, 2H), 3.32 (m, 1H), 2.72 (s, 3H), 2.32 (t, J=6.5
Hz, 2H), 2.04 (s, 3H), 2.01 (m, 2H), 1.74 (m, 2H), 1.69 (s, 3H),
1.45 (s, 9H), 1.38 (s, 6H), 1.09 (d, J=6.9 Hz, 3H), 0.93 (d, J=6.9
Hz, 3H); .sup.13C NMR (100 MHz CDCl.sub.3: .delta. 209.51, 203.04,
166.15, 164.39, 154.14, 152.72, 141.71, 138.24, 120.70, 118.76,
115.28, 94.54, 81.85, 77.31, 76.57, 63.41, 54.16, 46.47, 41.48,
34.56, 33.95, 31.98, 31.53, 27.85, 24.85, 23.45, 21.47, 20.75,
19.04, 15.60, 14.33, 11.35; IR (neat) 3546, 3395, 1756, 1717, 1699,
1644, 1621, 1506, 1456, 1251, cm.sup.-1.
EXAMPLE 79
[0399] Noyori C-3/C-15 Diol Product (11E).
[0400] The diketone 10E (1.77 g, 2.43 mmol) was dissolved in 0.12 N
HCl in MeOH (21 mL, 1.3 eq) at 25.degree. C. The (R)-RuBINAP
catalyst (0.045) M in THF, 8.0 mL, 0.36 mmol) was then added and
the mixture transferred to a Parr apparatus. The vessel was purged
with H.sub.2 for 5 min and then pressurized to 1200 psi. After
12-14 h at 25.degree. C., the reaction was returned to atmospheric
pressure and poured into a saturated solution of NaHCO.sub.3. This
mixture was extracted with CHCl.sub.3 (4.times.50 mL) and the
combined organic layers were dried over anhydrous MgSO.sub.4. The
product was purified by flash column chromatography on silica gel
eluting with hexanes/ethyl acetate (4:1 to 2:1) to give 1.42 g
(81%) of the hydroxy ester 11E as a green foam; .sup.1H NMR (400
MHz, CDCl.sub.3: .delta. 6.96 (s, 1H), 6.55 (s, 1H), 5.15 (t, J=6.9
Hz, 1H), 4.85 (t, J=5.3 Hz, 1H), 4.81 (d, J=12.0 Hz, 1H), 4.71 (d,
J=12.0 Hz, 1H), 4.12 (m, 2H), 3.43 (m, 2H), 2.70 (s, 3H), 2.37 (dd,
J=2.2, 6.2 Hz, 1H), 2.30 (t, J=6.7 Hz, 2H), 2.24 (dd, J=10.6, 16.2
Hz, 1H), 2.03 (s, 3H), 1.99 (m, 2H), 1.68 (S, 3h), 1.44 (s, 9h),
1.18 (s, 3h), 1.16 (s, 3h), 1.09 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8
Hz, 3H); .sup.13C NMR (100 MHz CDCl.sub.3): .delta. 215.95, 172.39,
164.39, 154.21, 152.74, 141.70, 138.33, 120.59, 118.77, 115.27,
94.64, 82.98, 81.26, 76.51, 72.78, 51.82, 41.40, 37.36, 34.66,
33.96, 32.08, 31.10, 30.20, 27.96, 25.06, 23.45, 21.73, 21.07,
19.17, 19.01, 16.12, 15.16, 14.33, 12.17; IR (neat) 3434.0, 1757.5,
1704.5, 1249.9, 1152.8 cm.sup.-1.
[0401] C-3/C-15 Bis(TES) Carboxylic Acid.
[0402] 2,6-Lutidine (2.1 g, 19.6 mmol) and TESOTf (2.6 g, 9.8 mmol)
were added successively to a cooled solution of the diol 11E (2.38,
3.26 mmol) in CH.sub.2Cl.sub.2 (30 mL) at -78.degree. C. The
reaction mixture was stirred at -78.degree. C. for 5 min and then
warmed to rt and stirred for 1 hr. Then 2,6-lutidine (4.9 g, 45.6
mmol) and TESOTF (6.0 g, 22.8 mmol) were added successively to a
-78.degree. C. cooled solution. The reaction was stirred at rt for
6 hr and then quenched with saturated aqueous NH.sub.4Cl and
subjected to an aqueous workup. The crude product was concentrated
in vacuo and the 2,6-lutidine removed on high vacuum pump and then
subjected directly to the next set of reaction conditions; .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 6.96 (s, 1H), 6.66 (s, 1H), 5.04
(t, J=6.93 Hz, 1H), 4.90 (d, J=12.0 Hz, 1H), 4.77 (dd, J=7.99, 3.21
Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.46 (m, 1H), 4.10 (dq, J=12.3,
7.11 Hz, 2H), 3.42 (m, 1H), 2.70 (s, 3H), 2.60 (dd, J=16.7, 2.34
Hz, 1H), 2.34 (dd, J=16.7, 7.94 Hz, 1H), 2.27 (dd, J=14.0, 6.97 Hz,
1H), 2.18 (m, 1H), 2.09 (m, 1H), 2.04 (s, 1H), 1.95 (s, 3H), 1.82
(m, 2H), 1.61 (s, 3H), 1.44 (m, 2H), 1.27-1.22 (m, 4H), 1.14 (d,
J=8.45 Hz, 3H), 1.11 (d, J=6.81 Hz, 2H), 1.04 (d, J=6.88 Hz, 2H),
1.15-1.01 (m, 2H), 0.94 (t, J=7.92 Hz, 18H), 0.65-0.57 (m, 12H);
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 215.11, 175.34, 165.00,
154.14, 152.80, 142.60, 136.84, 121.31, 118.79, 114.60, 94.77,
81.60, 79.06, 76.64, 73.87, 54.19, 41.18, 39.56, 35.09, 34.52,
32.29, 31.95, 24.76, 23.62, 22.55, 18.95, 18.64, 15.87, 13.69,
11.33, 6.94, 6.83, 5.07, 4.76; IR (neat) 3100-2390, 1756.8, 1708.8,
1459.3, 1250.6, 816.1 cm.sup.-1.
EXAMPLE 80
[0403] C-15 Hydroxy Acid for Macrolactonization (12E).
[0404] The crude bis(triethylsilyl)ether, prepared above, was
dissolved in 20 mL of dry THF and then cooled to 0.degree. C. Then,
6 ml of 0.12 M HCl/MeOH was added. The reaction mixture was stirred
at 0.degree. C. for 3 min and maintained at 0.degree. C. for the
duration. The reaction was monitored closely by TLC analysis.
Methanolic HCl (0.12 M) was added in small portions, and roughly
1.3 equivalents of 0.12 M HCl was required for the hydrolysis of
the C-15 TBS ether (approximately 30-40 mL). The reaction was
complete in appoximately 30 min. The reaction was quenched by
pouring into a solution of saturated aqueous NaHCO.sub.3 and
subjected to an aqueous workup. Flash column chromatography with
40% EtOAc/hexanes afforded the desired carboxylic acid 12E (1.71 g,
2.20 mmol) in 67% yield; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
6.96 (s, 1H), 6.69 (1, s), 5.11 (t, J=6.9 Hz, 1H), 4.91 (d, J=12.0
Hz, 1H), 4.71 (dd, J=3.1, 8.2 Hz, 1H), 4.64 (d, J=12.0 Hz, 1H),4.42
(d, J=5.9 Hz, 1H), 4.10 (m, 1H), 3.43 (m, 1H), 2.71 (s, 3H), 2.57
(dd, J=2.1, 10.5 Hz, 1H), 2.25 (m, 3H), 2.11 (m, 1H), 1.98 (s, 3H),
1.95 (m, 2H), 1.72 (m, 1), 1.67 (s, 3H), 1.45 (m, 2H), 1.16 (s,
3H), 1.13 (s, 3H), 1.09 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H),
0.95 (t, J=7.9 Hz, 9H), 0.64 (dq, J=2.3, 7.9 Hz, 6H); .sup.13C NMR
(100 MHz, CDCl.sub.3): .delta. 215.11, 176.00 (165.10, 154.18,
152.35, 142.24, 138.55, 120.74, 118.21, 115.02, 94.76, 81.91,
76.86, 76.63, 73.95, 54.08, 41.28, 39.64, 34.73, 34.16, 32.02,
31.67, 24.71, 23.41, 22.49, 19.17, 18.62, 15.71, 14.86, 11.20,
6.93, 5.05); IR (neat) 3400-2390, 1755.9, 1703.8, 1250.4, 735.4
cm.sup.-1.
EXAMPLE 81
[0405] C-3 Triethylsilyl/C-7 Trichloroethoxyethylcarbonate
Macrolactonization Product (13E).
[0406] Triethylamine (155 mg, 1.53 mmol) and 2,4,6-trichlorobenzoyl
chloride (312 mg, 1.28 mmol) were added to a solution of the
hydroxy acid 12E (198 mg, 0.256 mmol) in 3.6 mL of dry THF. The
reaction mixture was stirred for 15 min (and NO LONGER) at rt and
then diluted with 20 mL of dry toluene. The resultant solution was
added slowly dropwise, via syringe pump, over 3 hr to a previously
prepared, stirred solution of DMAP (328 mg, 2.68 mmol) in 300 mL of
dry toluene. After the addition of the substrate was complete, the
reaction was stirred for an additional 0.5 h and then concentrated
in vacuo. Flash column chromatography of the crude product with 10%
EtOAc/hexanes afforded the macrolactone 13E (153 mg, 0.20 mmol) in
78% yield; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.96 (s, 1H),
6.53 (s, 1H), 5.20 (m, 2H), 5.04 (d, J=10.2 Hz, 1H), 4.84 (d,
J=12.0 Hz, 1H), 4.78 (d, J=12.0 Hz, 1H), 4.07 (m, 1H), 3.32 (m,
1H), 2.86-2.63 (m, 3H), 2.70 (s, 3H), 2.48 (m, 1H), 2.11 (s, 3H),
2.04 (dd, J=6.17, 14.7 Hz, 1H), 1.73 (m, 4H), 1.66 (s, 3H), 1.25
(m, 2H), 1.19 (s, 3H), 1.15 (s, 3H), 1.12 (d, J=6.68 Hz, 3H), 1.01
(d, J=6.83 Hz, 3H), 0.89 (t, J=8.00 Hz, 9H), 0.58 (q, J=7.83 Hz,
6H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 212.75, 170.66,
164.62, 154.60, 152.52, 140.29, 138.44, 119.81, 119.38, 116.28,
94.84, 86.44, 80.14, 76.59, 76.10, 53.55, 45.89, 39.23, 35.47,
32.39, 31.69, 31.57, 31.16, 29.68, 27.41, 25.00, 23.44, 22.94,
19.23, 18.66, 16.28, 14.83, 6.89, 5.22; IR (neat) 1760.5, 1742.6,
1698.0, 1378.8, 1246.2, 1106.0, 729.8 cm.sup.-1.
EXAMPLE 82
[0407] Sml.sub.2 Mediated Deprotection of Troc Group.
[0408] Samarium metal (0.52 g, 3.43 mmol) and iodine (0.78 g, 3.09
mmol) in 40 mL of dry, deoxygenated THF were stirred together
vigorously at reflux for 2.5 hr. During this period of time, the
reaction mixture progressed from a dark orange to an olive green to
deep blue color. The resultant deep blue solution of Sml.sub.2 was
used directly in the following reaction. A catalytic amount of
Nil.sub.2 (10 mg) was added in one portion to the vigorously
stirted solution of Sml.sub.2. The reaction mixture was stirred 5
min at rt and then cooled to -78.degree. C. in a a dry ice/acetone
bath. Then, the macrolactone 13E (297 mg, 0.386 mmol), in 10 mL of
dry THF, was added over 1 min to the rapidly stirred, cold solution
of Sml.sub.2/Nil.sub.2. The resultant deep blue solution was
maintained at -78.degree. C. with continued vigorous stirring for 1
hr. TLC analysis at this time revealed the complete consumption of
the starting material and formation of a single, lower R.sub.f
product. The reaction mixture was quenched with saturated aqueous
NaHCO.sub.3 and subjected to an aqueous workup. Flash column
chromatography with 25% EtOAc/hexanes afforded the C-7 alcohol (204
mg, 0.343 mmol) in 89% yield; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 6.95 (s,1H), 6.54 (s,1H), 5.15 (m, 1H), 5.05 (d, J=10.15
Hz, 1H), 4.08 (dd, J=10.1, 2.66 Hz, 1H), 3.87 (m, 1H), 3.01 (s,
1H), 3.06 (m, 1H), 2.83-2.65 (m, 3H), 2.70 (s, 3H), 2.44 (m, 1H),
2.10 (s, 3H), 2.07 (m, 1H), 1.83 (m, 1H), 1.77 (m, 1H), 1.71 (m,
1H), 1.64 (s, 3H), 1.60 (s, 1H), 1.37 (m, 1H), 1.31 (m, 1H), 1.20
(m, 1H), 1.15 (s, 3H), 1.14 (m, 5H), 1.02 (d, J=7.02 Hz, 3H), 0.89
(t, J=7.97 Hz, 9H), 0.64-0.52 (m, 6H); .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta. 218.34, 170.73, 164.59, 152.46, 139.07,
138.49, 120.48, 119.54, 116.00, 79.31, 75.81, 73.48, 53.62, 42.98,
39.48, 39.01, 32.85, 32.41, 31.20, 26.12, 24.26, 22.01, 22.46,
19.18, 16.44, 15.30, 13.99, -6.98 (3), 5.27 (3); IR (neat) 3524.0,
1740.3, 1693.4, 1457.2, 1378.4, 733.2 cm.sup.-1.
EXAMPLE 83
[0409] Desoxyepothilone B (12E).
[0410] The C-3 TES protected alcohol (204 mg, 0.343 mmol) was
dissolved in 6 mL of dry THF in a plastic reaction vessel and
cooled to 0.degree. C. in an ice bath. The resultant solution was
treated with 3 mL of HF-pyridine. The reaction mixture was stirred
for 80 min at 0.degree. C. and then quenched by pouring into a
saturated aqueous solution of NaHCO.sub.3. An aqueous workup
followed by flash column chromatography with 10% EtOAc/hexanes
afforded desoxyepothilone B 12E (160 mg, 0.32 mmol) in 95% yield.
The resultant product exhibited a .sup.1H NMR spectrum identical to
desoxyepothilone B prepared as described hereinabove.
[0411] Discussion
[0412] Total Synthesis of (-)-Epothilone A.
[0413] The first known method for preparing epothilone A (1) is
provided by this invention. Carbons 9 through 11 insulate domains
of chirality embracing carbons 3 through 8 on the acyl side of the
macrolactone, and carbons 12 through 15 on the alkyl side.
Transmitting stereochemical information from one of the segments to
the other is unlikely. Thus, the approach taken deals with the
stereochemistry of each segment individually. In the acyl segment,
this strategy required knowledge of both the relative and absolute
configurations of the "polypropionate-like" network. In the alkyl
segment, two possibilities emerge. In one instance, the C12-C13
epoxide would be included in the construct undergoing merger with
the acyl related substructure. In that case it would be necessary
to secure the relative stereochemical relationship of carbons 15,
13 and 12. It was necessary to consider the the possibility that
the epoxide would be deleted from the alkyl-side moiety undergoing
coupling. This approach would only be feasible if the epoxide could
be introduced with acceptable stereocontrol after closure of the
macrocycle. The synthesis of compound 4, which contains most of the
requisite stereochemical information required for the acyl
fragment, is described above. This intermediate is prepared by a
novel oxidatively induced solvolytic cleavage of the
cyclopropanopyran 3. Also described above is a construct containing
the alkyl side coupling partner embodying the absolute and relative
stereochemistry at carbons 15, 13 and 12, which differs from the
alternative approach set forth below.
[0414] In considering the union of the alkyl and acyl domains,
several potential connection sites were available. At some point,
an acylation would be required to establish an ester (or lactone)
bond (see bold arrow 2). Furthermore, an aldol construction was
required to fashion a C2-C3 connection. Determining the exact
timing of this aldol step required study. It could be considered in
the context of elongating the C3-C9 construct to prepare it for
acylation of the C-15 hydroxyl. Unexpectedly, it was discovered
that the macrolide could be closed by an unprecedented
macroaldolization. (For a previous instance of a keto aldehyde
macroaldolization, see: C. M. Hayward, et al., J. Am. Chem. Soc.,
1993, 175, 9345.) This option is implied by bold arrow 3 in FIG.
1(A).
[0415] The first stage merger of the acyl and alkyl fragments (see
bold arrow 1) posed a difficult synthetic hurdle. It is recognized
in the art (P. Bertinato, et al., J. Org. Chem., 1996, 61, 8000;
vide infra) that significant resistance is encountered in
attempting to accomplish bond formation between carbons 9 and 10 or
between carbons 10 and 11, wherein the epoxide would be included in
the alkyl coupling partner. These complications arose from
unanticipated difficulties in fashioning acyl and alkyl reactants
with the appropriate complementarity for merger across either of
these bonds. An initial merger between carbons 11 and 12 was
examined. This approach dictated deletion of the oxirane linkage
from the O-alkyl coupling partner. After testing several
permutations, generalized systems 5 and 6 were examined to enter
the first stage coupling reaction. The former series was to be
derived from intermediate 4. A de novo synthesis of a usable
substrate corresponding to generalized system 5 would be necessary
(FIG. 1 (B)).
[0416] The steps leading from 4 to 11 are shown in Scheme 2.
Protection of the future C-7 alcohol (see compound 7) was followed
by cleavage of the benzyl ether and oxidation to aldehyde 8.
Elongation of the aldehyde to the terminal allyl containing
fragment 10 proceeded through end ether 9 (mixture of E and Z
geometrical isomers). Finally, the dithiane linkage was oxidatively
cleaved under solvolytic trapping conditions, giving rise to
specific coupling component 11. G. Stork; K. Zhao, Tetrahedron
Lett. 1989, 30, 287.
[0417] The synthesis of the alkyl fragment started with
commercially available (R)-glycidol 12 which was converted, via its
THP derivative 13, to alcohol 14. After cleavage of the
tetrahydropyran blocking group, the resultant alcohol was smoothly
converted to the methyl ketone 15, as shown. The latter underwent
an Emmons-type homologation with phosphine oxide 16. D.Menget al.,
J. Org. Chem., 1996, 61, 7998. This Emmons coupling provided a ca.
8:1 mixture of olefin stereoismoers in favor of trans-17. The
resultant alkyne 17 was then converted, via compound 18 to
Z-iodoalkene 19 (see FIG. 4(A)). E. J. Corey et al., J. Am. Chem.
Soc., 1985, 107, 713.
[0418] The critical first stage coupling of the two fragments was
achieved by a B-alkyl Suzuki carbon-carbon bond construction. N.
Miyaura et al., J. Am. Chem. Soc., 1989, 111, 314; N. Miyaura and
A. Suzuki, Chem. Rev., 1995, 95, 2457. Thus, hydroboration of the
pre-acyl fragment 11 was accomplished by its reaction with 9-BBN.
The resultant mixed borane cross-coupled to iodoolefin 19, under
the conditions indicated, to give 20 in 71% yield. (FIG. 4(B)) Upon
cleavage of the acetal, aldehyde 21 was in hand.
[0419] The availability of 21 permitted exploration of the strategy
in which the methyl group of the C-1 bound acetoxy function would
serve as the nucleophilic component in a macroaldolization. Cf. C.
M. Hayward et al., supra. Deprotonation was thereby accomplished
with potassium hexamethyldisilazide in THF at -78.degree. C.
Unexpectedly, these conditions give rise to a highly
stereoselective macroaldolization, resulting in the formation of
the C-3 (S)-alcohol 22, as shown. The heavy preponderance of 22 was
favored when its precursor potassium aldolate is quenched at ca.
0.degree. C. When the aldolate was protonated at lower temperature,
higher amounts of the C-3 (R) compound were detected. In fact,
under some treatments, the C-3 (R) epimer predominates. It is
therefore possible to generate highly favorable C-3(R):C-3(S)
ratios in analytical scale quenches. In preparative scale
experiments, the ratio of 22 to its C-3 epimer is 6:1.
[0420] With compound 22 in ready supply, the subgoal of obtaining
desoxyepothilone (23) was feasible. This objective was accomplished
by selective removal of the triphenylsilyl (TPS) group in 22,
followed, sequentially, by selective silylation of the C-3 alcohol,
oxidation of the C-5 alcohol, and, finally, fluoride-induced
cleavage of the two silyl ethers.
[0421] Examination of a model made possible by the published
crystal structure of epothilone (Hofle et al., supra), suggested
that the oxirane is disposed on the convex periphery of the
macrolide. Oxidation of 23 was carried out with dimethyl dioxirane
under the conditions shown. The major product of this reaction was
(-)epothilone A (1), the identity of which was established by nmr,
infrared, mass spectral, optical rotation and chromotaraphic
comparisons with authentic material. Hofle et al., supra. In
addition to epothilone A (1), small amounts of a diepoxide mixture,
as well as traces of the diastereomeric cis C12-C13 monoepoxide
(.gtoreq.20:1) were detected.
[0422] The method of synthesis disclosed herein provides workable,
practical amounts of epothilone A. More importantly, it provides
routes to congeners, analogues and derivatives not available from
the natural product itself.
[0423] Studies Toward a Synthesis of Epothilone A: Use of
Hydropyran Templates For the Management of Acyclic Stereochemical
Relationships.
[0424] The synthesis of an enantiomerically pure equivalent of the
alkoxy segment (carbons 9-15) was carried out in model studies. The
key principle involves transference of stereochemical bias from an
(5)-lactaldehyde derivative to an emerging dihydropyrone. The
latter, on addition of the thiazole moiety and disassembly,
provides the desired acyclic fragment in enantiomerically pure
form.
[0425] Various novel structural features of the epothilones make
their synthesis challenging. The presence of a thiazole moiety, as
well as a cis epoxide, and a geminal dimethyl grouping are key
problems to be overcome. An intriguing feature is the array of
three contiguous methylene groups which serves to insulate the two
functional domains of the molecules. The need to encompass such an
achiral "spacer element" actually complicates prospects for
continuous chirality transfer and seems to call for a strategy of
merging two stereochemically committed substructures. The present
invention provides a synthesis of compound 4A (FIG. 14), expecting
that, in principle, such a structure could be converted to the
epothilones themselves, and to related screening candidates.
[0426] The identification of compound 4A as a synthetic
intermediate served as an opportunity to illustrate the power of
hydropyran matrices in addressing problems associated with the
control of stereochemistry in acyclic intermediates. The synthesis
of dihydropyrones was previously disclosed through what amounts to
overall cyclocondensation of suitably active dienes and aldehydic
heterodienophiles. Danishefsky, S. J. Aldrichimica Acta, 1986, 19,
59. High margins of steroselectivity can be realized in assembling
(cf. 5A+6A.fwdarw.7A) such matrices (FIG. 13). Moreover, the
hydropyran platforms service various stereospecific reactions (see
formalism 7A.fwdarw.8A). Furthermore, the products of these
reactions are amenable to ring opening schemes, resulting in the
expression of acyciic fragments with defined stereochemical
relationships (cf. 8A.fwdarw.9A). Danishefsky, S. J. Chemtracts,
1989, 2, 273.
[0427] The present invention provides the application of two such
routes for the synthesis of compound 4A. Route 1, which does not
per se involve control of the issue of absolute configuration,
commences with the known aldehyde 10A. Shafiee, A., et al., J.
Heterocyclic Chem., 1979, 16, 1563; Schafiee, A.; Shahocini, S. J.
Heterocyclic Chem., 1989, 26, 1627. Homologation, as shown,
provided enal 12A. Cyclocondensation of 12A with the known diene
(Danishefsky, S. J.; Kitahara, T. J. Am. Chem. Soc., 1974, 96,
7807), under BF.sub.3 catalysis, led to racemic dihydropyrone 13A.
Reduction of 13A under Luche conditions provided compound 14A.
Luche, J.-L. J. Am. Chem. Soc., 1978, 100, 2226. At this point it
was feasible to take advantage of a previously introduced lipase
methodology for resolution of glycal derivatives through
enzymatically mediated kinetic resolution. Berkowitz, D. B. and
Danishefsky, S. J. Tetrahedron Lett., 1991, 32, 5497; Berkowitz, D.
B.; Danishefsky, S.).; Schulte, G. K. J. Am. Chem. Soc., 1992, 114,
4518. Thus, carbinol 14A was subjected to lipase 30, in the
presence of isopropenyl acetate, following the prescriptions of
Wong (Hsu, S.-H., et al., Tetrahedron Lett., 1990, 31, 6403) to
provide acetate 15A in addition to the enantiomerically related
free glycal 16A. Compound 15A was further advanced to the PMB
protected system 17A. At this juncture, it was possible to use
another reaction type previously demonstrated by the present
inventors. Thus, reaction of 17A with dimethyidioxirane
(Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem. Int. Ed. Engl.,
1996, 35, 1381) generated an intermediate (presumably the
corresponding glycal epoxide) which, upon treatment with sodium
metaperiodate gave rise to aldehyde formate 18A. Allylation of 18A
resulted in the formation of carbinol 19A in which the formate
ester had nicely survived. (For a review of allylations, see:
Yamamoto, Y.; Asao, N. Chem. Rev. 1993, 93, 2207.) However, 19A was
accompanied by its anti stereoisomer (not shown here) [4:1].
Mesylation of the secondary alcohol, followed by deprotection (see
19A.fwdarw.20A) and cyclization, as indicated, gave compound
4A.
[0428] In this synthesis, only about half of the dihydropyrone was
secured through the process of kinetic resolution. While, in
theory, several of the synthetic stratagems considered contemplate
use of each enantiomer of 15A to reach epothilone itself, another
route was sought to allow for full enantiomeric convergence. The
logic of this route is that the chirality of a "dummy" asymmetric
center is communicated to the emerging pyran following previously
established principles of tunable diastereoselection in the
cyclocondensation reaction. (Danishefsky, supra) Cyclo-condensation
of lactaldehyde derivative 21A (Heathcock, C. H., et al., J. Org.
Chem., 1980, 45, 3846) with the indicated diene, under ostensible
chelation control, afforded 22A. The side chain ether could then be
converted to the methyl ketone 25A as shown (see
22A.fwdarw.23A.fwdarw.24A.fwdarw.25A). Finally, an Emmons
condensations (for example, see: Lythgoe, B., et al., Tetrahedron
Lett., 1975, 3863; Toh, H. T.; Okamura, W. H. J. Org. Chem., 1983,
48, 1414; Baggiolini, E. G., et al., J. Org. Chem., 1986, 51, 3098)
of 25A with the phoshphine oxide 26A was transformed to phosphine
oxide 26A according to the procedure described in Toh, supra) as
shown in FIG. 15 gave rise to 27A. (The known
2-methyl-4-chloromethylthiazole (see Marzoni, G. J. Heterocyclic
Chem., 1986, 23, 577.) A straightforward protecting group
adjustment then afforded the previously encountered 17A. This route
illustrates the concept of stereochemical imprinting through a
carbon center which eventually emerges in planar form after
conferring enantioselection to subsequently derived stereocenters.
The use of the dihydropyrone based logic for securing the
stereochemical elements of the epothilones, as well as the
identification of a possible strategy for macrocyclization will be
described in the following section.
[0429] Studies Toward a Synthesis of Epothilone A: Sterocontrolled
Assembly of the Acyl Region and Models for Macrocyclization.
[0430] Ring-forming olefin metathesis has been employed to
construct 16-membered ring congeners related to epothilone A. A
stereospecific synthesis of the C3-C9 sector of the acyl fragment
was achieved by exploiting a novel oxidative opening of a
cyclopropanated glycal.
[0431] Disclosed in the previous section is a synthesis of the
"alkoxy" segment of epothilone (1) (see compound 2B, FIG. 7)
encompassing carbons 10 to 21. In this section the synthesis of
another fragment encoding the stereochemical information of acyl
section carbons 3 to 9. It was envisioned that the aldehydo center
(C.sub.3) of the formal target 3B would serve as an attachment site
to a nucleophilic construct derived from compound 2B (requiring
placement of a 2 carbon insert, as suggested in FIG. 7), through
either inter- or intramolecular means. In such a context, it would
be necessary to deal independently with the stereochemistry of the
secondary alcohol center eventually required at C.sub.3. One of the
interesting features of system 3B is the presence of geminal methyl
groups at carbon 4 (epothilone numbering).
[0432] Again, use is made of a dihydropyran strategy to assemble a
cyclic matrix corresponding, after appropriate disassembly, to a
viable equivalent of system 3B. The expectation was to enlarge upon
the dihydropyran paradigm to include the synthesis of gem-dimethyl
containing cyclic and acyclic fragments. The particular reaction
type for this purpose is generalized under the heading of
transformation of 4B.fwdarw.5B (see FIG. 7). Commitment as to the
nature of the electrophile E is avoided. Accordingly, the question
whether a reduction would or would not be necessary in going from
structure type 5B to reach the intended generalized target 3B is
not addressed.
[0433] The opening step consisted of a stereochemically tuneable
version of the dienealdehyde cyclocondensation reaction (FIG. 8;
Danishefsky, S. J., Aldrichimica Acta, 1986, 19, 59), in this
instance drawing upon chelation control in the merger of the
readily available enantiomerically homogenous aldehyde 6B with the
previously known diene 7B. Danishefsky, S. J., et al., J. Am. Chem.
Soc. 1979, 101, 7001. Indeed, as precedent would have it, under the
influence of titanium tetrachloride there was produced
substantially a single isomer shown as compound 8B. In the usual
and stereochemically reliable way (Danishefsky, S. J., Chemtracts
Org. Chem. 1989, 2, 273), the dihydropyrone was reduced to the
corresponding glycal, 9B. At this point, it was feasible to utilize
a directed Simmons-Smith reaction for the conversion of glycal 9B
to cyclopropane 10B. Winstein, S.; Sonnenberg, J. J. Am. Chem.
Soc., 1961, 83, 3235; Dauben, W. G.; Berezin, G. H. J. Am. Chem.
Soc., 1963, 85, 468; Furukawa, J., et al., Tetrahedron, 1968, 24,
53; For selected examples, see Soeckman, R. K. Jr.: Charette, A.
B.; Asberom, T.; Johnston, B. H. J. Am. Chem. Soc., 1991, 113,
5337; Timmers, C. M.; Leeuwenurgh, M. A.; Verheijen, J. C.; Van der
Marel, G. A.; Van Boom, J. H. Tetrahedron: Asymmetry, 1996, 7, 49.
This compound is indeed an interesting structure in that it
corresponds in one sense to a cyclopropano version of a
C-glycoside. At the same time, the cyclopropane is part of a
cyclopropylcarbinyl alcohol system with attendant possibilities for
rearrangement. Wenkert, E., et al., J. Amer. Chem. Soc., 1970, 92,
7428. It was intended to cleave the C-glycosidic bond of the
cyclopropane in a fashion which would elaborate the geminal methyl
groups, resulting in a solvent-derived glycoside with the desired
aldehyde oxidation state at C-3 (see hypothesized transformation
4B.fwdarw.5B, FIG. 7). In early efforts, the non-oxidative version
of the projected reaction (i.e. E.sup.+=H.sup.+) could not be
reduced to practice. Instead, products clearly attributable to the
ring expanded system 11 were identified. For example, exposure of
10B to acidic methanol gave rise to an epimeric mixture of
seven-membered mixed-acetals, presumably through the addition of
methanol to oxocarbenium ion 11B.
[0434] However, the desired sense of cyclopropane opening, under
the influence of the ring oxygen, was achieved by subjecting
compound 10B to oxidative opening with N-iodosuccinimide. (For
interesting Hg(II)-induced solvolyses of cyclopropanes that are
conceptually similar to the conversion of 10B to 12B, see: Collum,
D. B.; Still, W. C.; Mohamadi, F. J. Amer. Chem. Soc., 1986, 108,
2094; Collum, D. B.; Mohamadi, F.; Hallock, J. S.; J. Amer. Chem.
Soc., 1983, 105, 6882. Following this precedent, a Hg(II)-induced
solvolysis of cyclopropane 10B was achieved, although this
transformation proved to be less efficient than the reaction shown
in FIG. 8.) The intermediate iodomethyl compound, obtained as a
methyl glycoside 12B, when exposed to the action of
tri-n-butyltinhydride gave rise to pyran 13B containing the geminal
methyl groups. Protection of this alcohol (see 13B.fwdarw.14B),
followed by cleavage of the glycosidic bond, revealed the acyclic
dithiane derivative 15B which can serve as a functional version of
the hypothetical aldehyde 3B.
[0435] Possible ways of combining fragments relating to 2B and 3B
in a fashion to reach epothilone and congeners thereof were
examined. In view of the studies of Schrock (Schrock, R. R., et
al., J. Am. Chem. Soc., 1990, 112, 3875) and Grubbs (Schwab, P. et
al., Angew. Chem. Int. Ed. Engl., 1995; 34, 2039; Grubbs, R. H.;
Miller, S. J. Fu, G. C. Acc. Chem. Res., 1995, 28, 446; Schmalz,
H.-C., Angew. Chem. Int. Ed. Engl., 1995, 34, 1833) and the
disclosure of Hoveyda (Houri, A. F., et al., J. Am. Chem. Soc.,
1995, 117, 2943), wherein a complex lactam was constructed in a key
intramolecular olefin macrocyclization step through a molybdenum
mediated intramolecuar olefin in metathesis reaction (Schrock,
supra; Schwab, supra), the possibility of realizing such an
approach was considered. (For other examples of ring-closing
metathesis, see: Martin, S. F.; Chen, H.-J.; Courtney, A. K.; Lia,
Y.; Ptzel, M.; Ramser, M N.; Wagman, A. S. Tetrahedron, 1996, 52,
7251; Furstner, A.; Langemann, K. J. Org. Chem., 1996, 61,
3942.)
[0436] The matter was first examined with two model
.omega.-unsaturated acids 16B and 17B which were used to acylate
alcohol 2B to provide esters 18B and 19B, respectively (see FIG.
9). These compounds did indeed undergo olefin metathesis
macrocyclization in the desired manner under the conditions shown.
In the case of substrate 18B, compound set 20B was obtained as a
mixture of E- and Z-stereoisomers [ca. 1:1]. Diimide reduction of
20B was then conducted to provide homogeneous 22B. The olefin
methathesis reaction was also extended to compound 19B bearing
geminal methyl groups corresponding to their placement at C4 of
epothilone A. Olefin metathesis occurred, this time curiously
producing olefin 21B as a single entity in 70% yield
(stereochemisty tentatively assigned as Z.) Substantially identical
results were obtained through the use of Schrock's molybdenum
alkylidene metathesis catalyst.
[0437] As described above, olefin metathesis is therefore amenable
to the challenge of constructing the sixteen membered ring
containing both the required epoxy and thiazolyl functions of the
target system. It is pointed out that no successful olefin
metathesis reaction has yet been realized from seco-systems bearing
a full compliment of functionality required to reach epothilone.
These negative outcomes may merely reflect a failure to identify a
suitable functional group constraint pattern appropriate for
macrocylization.
[0438] The Total Synthesis of Epothilone B: Extension of the Suzuki
Coupling Method
[0439] The present invention provides the first total synthesis of
epothilone A (1). D. Meng, et al., J. Org. Chem, 1996, 67, 7998 P.
Bertinato, et al., J. Org. Chem, 1996, 61, 8000. A. Balog, et al.,
Angew. Chem. Int. Ed. Engl., 1996, 35, 2801. D. Meng, et al., J.
Amer. Chem. Soc., 1997, 119, 10073. (For a subsequent total
synthesis of epothilone A, see: Z. Yang, et al., Angew. Chem. Int.
Ed. Engl., 1997, 36, 166.) This synthesis proceeds through the
Z-desoxy compound (23) which underwent highly stereoselective
epoxidation with 2,2-dimethyidioxirane under carefully defined
conditions to yield the desired .beta.-epoxide. The same
myxobacterium of the genus Sorangium which produces 23 also
produces epothilone B (2). The latter is a more potent agent than
23, both in antifungal screens and in cytotoxicity/cell nucleus
disintegration assays. G. Hofle, et al., Angew. Chem. Int. Ed.
Engl. 1996, 35, 1567; D. M. Bollag, et al., Cancer Res. 1995, 55,
2325.
[0440] An initial goal structure was desoxyepothilone B (2C) or a
suitable derivative thereof. Access to such a compound would enable
the study of the regio- and stereoselectivity issues associated
with epoxidation of the C12-C13 double bond. A key issue was the
matter of synthesizing Z-tri-substituted olefinic precursors of 2C
with high margins of stereoselection. A synthetic route to the
disubstituted system (A. Balog, et al., Agnew. Chem. Int. Ed.
Engl., 1996, 35, 2801) employed a palladium-mediated B-alkyl Suzuki
coupling (N. Miyaura, et al., J. Am. Chem. Soc. 1989, 111, 314.
(For a review, see: N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95,
2457) of the Z-vinyl iodide 19 (FIG. 4(A)) with borane 7C derived
from hydroboration of compound 11 (FIG. 1(A)) with 9-BBN (FIG.
4(B)).)
[0441] A preliminary approach was to apply the same line of
thinking to reach a Z-tri-substituted olefin (FIG. 17) en route to
2C. Two issues had to be addressed. First, it would be necessary to
devise a method to prepare vinyl iodide 8C, the tri-substituted
analog of 19. If this goal could be accomplished, a question
remained as to the feasibility of conducting the required B-alkyl
Suzuki coupling reaction to reach a Z-tri-substituted olefin. The
realization of such a transformation with a "B-alkyl" (as opposed
to a "B-alkenyl" system) at the intermolecular level, and where the
vinyl iodide is not of the .beta.-iodoenoate (or .beta.-iodoenone)
genre, was not precedented. (For some close analogies which differ
in important details from the work shown here, see: N. Miyaura, et
al., Bull. Chem. Soc. Jpn. 1982, 55, 2221; M. Ohba, et al.,
Tetrahedron Lett., 1995, 36, 6101; C. R. Johnson, M. P. Braun, J.
Am. Chem. Soc. 1993, 715, 11014.)
[0442] The synthesis of compound 8C is presented in FIG. 16. The
route started with olefin 10C which was prepared by catalytic
asymmetric allylation of 9C (G. E. Keck, et al., J. Am. Chem. Soc.,
1993, 115, 8467) followed by acetylation. Site-selective
dihydroxylation of 10C followed by cleavage of the glycol generated
the unstable aldehyde 11C. Surprisingly, the latter reacted with
phosphorane 12C (J. Chen, et al., Tetrahedron Lett., 1994, 35,
2827) to afford the Z-iodide 8C albeit in modest overall yield.
Borane 7C was generated from 11 as described herein. The coupling
of compound 7C and iodide 8C (FIG. 16) could be conducted to
produce the pure Z-olefin 13C.
[0443] With compound 13C in hand, protocols similar to those
employed in connection with the synthesis of 23 could be used. (A.
Balog, et al., Angew. Chem. Int. Ed. Engl., 1996, 35, 2801). Thus,
cleavage of the acetal linkage led to aldehyde 14C which was now
subjected to macroaldolization (FIG. 17). The highest yields were
obtained by carrying out the reaction under conditions which
apparently equilibrate the C3 hydroxyl group. The 3R isomer was
converted to the required 3S epimer via reduction of its derived
C3-ketone (see compound 15C). The kinetically controlled aldol
condensation leading to the natural 3S configuration as discribed
in the epothilone A series was accomplished. However, the overall
yield for reaching the 3S epimer is better using this protocol.
Cleavage of the C-5 triphenyisilyl ether was followed sequentially
by monoprotection (t-butyldimethylsilyl) of the C3 hydroxyl,
oxidation at C5 (see compound 16C), and, finally, cleavage of the
silyl protecting groups to expose the C3 and C7 alcohols (see
compound 2C).
[0444] It was found that Z-desoxyepothilone B (2C) undergoes very
rapid and substantially regio- and stereoselective epoxidation
under the conditions indicated (although precise comparisons are
not available, the epoxidation of 2C appears to be more rapid and
regioselective than is the case with 23) (A. Balog, et al., Angew.
Chem. Int. Ed. Engl., 1996, 35, 2801), to afford epothilone B (2)
identical with an authentic sample (.sup.1H NMR, mass spec, IR,
[.alpha.].sub.D). Accordingly, the present invention dislcoses the
first total synthesis of epothilone B. Important preparative
features of the present method include the enantioselective
synthesis of the trisubstituted vinyl iodide 8C, the
palladium-mediated stereospecific coupling of compounds 7C and 8C
to produce compound 13C (a virtually unprecedented reaction in this
form), and the amenability of Z-desoxyepothilone B (2C) to undergo
regio- and stereoselective epoxidation under appropriate
conditions.
[0445] Desmethylepothilone A
[0446] Total syntheses of epothilones A and B have not been
previously disclosed. Balog, A., et al., Angew. Chem., Int. Ed.
Engl. 1996, 35, 2801; Nicolaou, K. C., et al., Angew. Chem., Int.
Ed. Engl. 1997, 36, 166. Nicolaou, K. C., et al., Angew. Chem.,
Int. Ed. Engl. 1997, 36, 525; Schinzer, D., et al., Angew. Chem.,
Int. Ed. Engl. 1997, 36, 523. Su, D. -S., et al., Angew. Chem. Int.
Ed. Engl. 1997, 36, 757. The mode of antitumor action of the
epothilones closely mimics that of Taxol.RTM. (paclitaxel). Hofle,
G., et al., H. Angew. Chem., Int. Ed. Engl. 1996, 35, 1567.
Although Taxol.RTM. is a clinically proven drug, its formulation
continues to be difficult. In addition, Taxol.RTM. induces the
multidrug resistance (MDR) phenotype. Hence, any novel agent that
has the same mechanism of action as Taxol.RTM. and has the prospect
of having superior therapeutic activity warrants serious study.
Bollag, D. M., et al., Cancer Res. 1995, 55, 2325.
[0447] The present invention provides epothilone analogs that are
more effective and more readily synthesized than epothilone A or B.
The syntheses of the natural products provide ample material for
preliminary biological evaluation, but not for producing adequate
amounts for full development. One particular area where a
structural change could bring significant relief from the
complexities of the synthesis would be in the deletion of the C8
methyl group from the polypropionate domain (see target system 3D).
The need to deal with this C8 chiral center complicates all of the
syntheses of epothilone disclosed thus far. Deletion of the C8
methyl group prompts a major change in synthetic strategy related
to an earlier dienealdehyde cyclocondensation route. Danishefsky,
S. J. Chemtracts 1989, 2, 273; Meng, D., et al., J. Org. Chem.
1996, 61, 7998; Bertinato, P., et al., J. Org. Chem. 1996, 61,
8000.
[0448] As shown in FIG. 20, asymmetric crotylation (87% ee) of 4D
(Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919),
followed by protection led to TBS ether 5D. The double bond was
readily cleaved to give aldehyde 6D. The aldehyde was coupled to
the dianion derived from t-butyl isobutyrylacetate to provide 7D.
The ratio of the C.sub.5S (7D): C.sub.5R compound (not shown) is ca
10:1. That the Weiler-type .beta.-ketoester dianion chemistry
(Weiler, L. J. Am. Chem. Soc. 1970, 92, 6702.; Weiler, L.; Huckin,
S. N. J. Am. Chem. Soc. 1974, 96, 1082) can be conducted in the
context of the isobutyryl group suggested several alternate
approaches for still more concise syntheses. Directed reduction of
the C.sub.3 ketone of 7D following literature precedents (Evans, D.
A., et al., J. Org. Chem. 1991, 56, 741), followed by selective
silylation of the C.sub.3 hydroxyl gave a 50% yield of a 10:1 ratio
of the required C.sub.35 (see compound 8D) to C.sub.3R isomer (not
shown). Reduction with sodium borohydride afforded a ca. 1:1
mixture of C.sub.3 epimers. The carbinol, produced upon
debenzylation, was oxidized to an aldehyde which, following
methylenation through a simple Wittig reaction, afforded olefin 9D.
Treatment of this compound with TBSOTf provided ester 10D which was
used directly in the Suzuki coupling with the vinyl iodide 12D.
[0449] The hydroboration of 10D with 9-BBN produced intermediate
11D which, on coupling with the vinyl iodide 12D and in situ
cleavage of the TBS ester led to 13D (FIG. 21). After
deacetylation, the hydroxy acid 14D was in hand. Macrolactonization
of this compound (Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50,
2394) produced 15D which, after desilylation, afforded
C.sub.8-desmethyldesoxyepothilone (16D). Finally, epoxidation of
this compound with dimethyldioxirane produced the goal structure
3D. The stereoselectivity of epoxidation was surprisingly poor
(1.5:1) given that epoxidation of desoxyepothilone A occurred with
>20:1 stereoselectivity. Deletion of the C.sub.8 methyl group
appears to shift the conformational distribution of 16D to forms in
which the epoxidation by dimethyl dioxirane is less
.beta.-selective. It is undetermined whether the effect of the C,
methyl on the stereoselectivity of epoxidation by dimethydioxirane
and the dramatic reduction of biological activity are related.
[0450] Compounds 3D and 16D were tested for cytotoxicity in cell
cultures and assembly of tubulin in the absence of GTP. Microtubule
protein (MTP) was purified from calf brains by two cycles of
temperature dependent assembly and disassembly. Weisenberg, R. C.
Science 1972, 177, 1104. In control assembly experiments, MTP (1
mg/mL) was diluted in assembly buffer containing 0.1 M MES
(2-(N-morpholino) ethanesulfonic acid), 1 mM EGTA, 0.5 mM
MgCl.sub.2, 1 mM GTP and 3M glycerol, pH 6.6. The concentration of
tubulin in MTP was estimated to be about 85%. Assembly was
monitored spectrophotometrically at 350 nm, 35.degree. C. for 40
min by following changes in turbidity as a measure of polymer mass.
Gaskin, F.; Cantor, C. R.; Shelanksi, M. L. J. Mol. Biol. 1974, 89,
737. Drugs were tested at a concentration of 10 .mu.M, in the
absence of GTP. Microtubule formation was verified by electron
microscopy. To determine the stability of microtubules assembled in
the presence of GTP or drug, turbidity was followed for 40 min
after the reaction temperature was shifted to 4.degree. C.
[0451] Cytotoxicity studies showed drastically reduced activity in
the 8-desmethyl series. Compounds 3D and 16D were approximately 200
times less active than their corresponding epothilone A
counterparts (see Table 1). Recalling earlier SAR findings at both
C.sub.3 and C.sub.5, in conjunction with the findings disclosed
herein, the polypropionate sector of the epothilones emerges as a
particularly sensitive locus of biological function. Su, D.-S., et
al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757; Meng, D., et al.,
J. Am. Chem. Soc. 1997, 119.
1TABLE 1 Relative efficacy of epothilone compounds against
drug-sensitive and resistant human leukemic CCRF-CEM cell
lines..sup.a CCRF-CEM CCRF-CEM/VBL CCRF-CEM/VM.sub.1 Compound
IC.sub.50 (.mu.M).sup.b IC.sub.50 (.mu.M).sup.b IC.sub.50
(.mu.M).sup.b 16D 5.00 5.75 6.29 3D 0.439 2.47 0.764 epothilone A
0.003 0.020 0.003 desoxyepothilone A 0.022 0.012 0.013 epothilone B
0.0004 0.003 0.002 desoxyepothilone B 0.009 0.017 0.014 paclitaxel
0.002 3.390 0.002 .sup.aThe cytotoxicities of test compounds were
determined by the growth of human lymphoblastic leukemic cells
CCRF-CEM, or their sublines resistant to vinbiastine and Taxol
.RTM. (CCRF-CEM/VBL) or resistant to etoposide (CCRF-CFM/VM-1).
XTT-microculture tetrazolium/formazan assays were used. .sup.bThe
IC.sub.50 values were calculated from 5-6 concentrations based on
the median-effect plot using computer software.
[0452] Desoxyepothiline B: an Effective Microtubule-Targeted
Antitumor Agent with a Promising in Vivo Profile Relative to
Epothilone b
[0453] The epothilones have been synthesized as herein disclosed
and evaluated for antitumor potential in vitro and in vivo.
Epothilones and paclitaxel are thought to share similar mechanisms
of action in stabilizing microtubule arrays as indicated by binding
displacement studies, substitution for Taxol.RTM. in
Taxol.RTM.-dependent cell growth, and electron microscopic
examinations. Cell growth inhibitory effects have been determined
in two rodent and three human tumor cell lines and their drug
resistant sublines. While Taxol.RTM. showed as much as 1970-fold
cross-resistance to the sublines resistant to Taxol.RTM.,
adriamycin, vinblastine or actinomycin D, most ephothilones exhibit
little or no cross-resistance. In multidrug resistant
CCRF-CEM/VBL.sub.100 cells, the 50% cell growth inhibitory
concentrations (IC.sub.50 values) for epothilone A, epothilone B,
desoxyepothilone A, desoxy epothilone B and Taxol.RTM. were 0.02,
0.002, 0.012, 0.017 and 4.14 .mu.M, respectively. In vivo studies,
using i.p. administration, indicate that the parent, epothilone B,
is highly toxic to mice with little therapeutic effect when
compared with lead compound desoxyepothilone B (2540 mg/kg, Q2Dx5,
i.p.), which showed far superior therapeutic effect and lower
toxicity than paclitaxel, doxorubicin, camptothecin or vinblastine
(at maximal tolerated doses) in parallel experiments. In nude mice
bearing a human mammary carcinoma xenograft (MX-1), marked tumor
regression and cures have been obtained with desoxyepothilone
B.
[0454] The isolation of the naturally occurring macrolides
epothilone A and epothilone B from the myoxobacteria Sorangium
cellulosum (Hoefle, G., et al. Angew.Chem.Int.Ed.Engl. 1996, 35,
567-1569; Gerth, K., et al. J. Antibiot. 1996, 49, 560-563) and the
subsequent demonstration of their ability to stabilize microtubule
arrays in vitro elicited considerable interest in this class of
compounds (Bollag, D. M., et al., Cancer Res. 1995, 55, 2325-2333;
Su, D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096;
Meng, D., et al., J.Am. Chem. Soc.1997, 119, 2733-2734; Muhlradt,
P. F. & Sasse, F. Cancer Res. 1997, 57, 3344-3346; Service, R.
E. Science 1996, 274, 2009). We have recently conducted the total
synthesis of these natural products as well as over 45 related
analogs (Meng, D., et al., J. Am. Chem. Soc. 1997, 119,
10073-10092; Su, D-S., et al., Angew. Chem. Int. Ed. Engl. 1997,
36, 757-759; Chou, T.-C., Zhang, X.-G., & Danishefsky, S. J.
Proc. Am. Assoc. Cancer Res. 1998, 39, 163-164) in order to
investigate their chemical structure-biological activity
relationships (Su, D.-S, et al., Agnew. Chem. Int. Ed. Engl. 1997,
36, 2093-2096). The studies disclosed herein allowed the
characterization of the epothilone structure in three zones. Thus,
in the C-1-8 acyl sector, the present inventors have determined
that structural changes are not tolerated in terms of in vitro
cytoxocity and microtubule stabilizing ability. This stands in
contrast to the C-9- 15 O-alkyl sector and the C-15 pendant aryl
sectors wherein considerable modification of structures is
tolerated (Su, D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36,
2093-2096; Meng, D., et al., 1997, J.Am.Chem.Soc. 119,
10073-10092). Described herein are the results of in vitro and in
vivo experiments on the Z-12,13 desoxy version of epothilone B
(desoxyepothilone B).
[0455] It has been shown that the natural epothilones A and B have
a similar mechanism of action to paclitaxel (Taxol.RTM.) although
structurally diverse (Su, D.-S., et al., Angew. Chem. Int. Ed.
Engl. 1997, 36, 2093-2096; Meng, D., et al., J.Am.Chem.Soc. 1997,
19, 2733-2734; Schiff, P. B., Fant, J. & Horwitz, S. B. Nature
1979, 277, 665-667; Landino, L. M. & MacDonald, T. L., in: The
Chemistry and Pharmacology of Taxol and Its Derivatives, Favin, V.,
ed., Elsevier, N.Y. 1995, Chapter 7, p. 301). Paclitaxel, isolated
from the Pacific yew tree (Taxus brevifolia), has been widely used
clinically to treat a variety of solid cancers including neoplasms
of ovary, breast, colon and lung (Landino, L. M. & MacDonald,
T. L. id.; Rose, W. C. Anti-Cancer Drugs, 1992, 3, 311-321;
Rowinsky, E. K., et al., Seminars Oncol. 1993, 20, 1-15).
Epothilones A and B as well as Taxol.RTM. stabilize microtubule
assemblies as demonstrated by binding displacement, substitution
for paclitaxel in paclitaxel-dependent cell growth, and electron
microscopic examinations (Bollag, D. M., et al., Cancer Res. 1995,
55, 2325-2333) Despite these similarities, the epothilones are more
water soluble than paclitaxel, thereby offering potentially
distinct advantages for formulation. Epothilones are more potent
than paclitaxel in inhibiting cell growth, especially against cells
expressing P-glycoprotein (Pgp) that are multidrug resistant (MDR),
including cross-resistance to paclitaxel (Bollag, D. M., id.; Su,
D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36,
2093-2096).
[0456] Materials and Methods
[0457] All stock solutions of the above (except VBL in saline) were
prepared using dime thylsulfoxide (DMSO) as a solvent and were
further diluted to desired concentrations for experimental use. The
final concentration of DMSO in tissue culture was 0.25% (v/v) or
less to avoid solvent cytotoxicity. For in vivo studies, paclitaxel
in Cremophor-EtOH was further diluted with DMSO as needed.
Vinblastine sulfate (Velban) (Eli Lilly & Co. Indianapolis,
Ind.), and doxorubicin or adriamycin HCl (DX or Adr) (Pharmacia,
Columbus, Ohio) in saline were diluted with DMSO as needed. DMSO
was used as a vehicle for epothilones. Each mouse received >40
.mu.L DMSO in all experiments.
[0458] Cell Lines
[0459] The CCRF-CEM human T-cell acute lymphoblastic leukemia cell
line and its vinblastine-resistant (CCRF-CEM/VBL.sub.100) and
teniposide-resistant (CCRF-CEM/VM.sub.1) sublines (Cass, C. E., et
al., 1989, Cancer Res. 49, 5798-5804; Danks, M. K., Yalowich, J.
C., & Beck, W. T. 1987, Cancer Res. 47, 1297-1301) were used.
CCRF-CEM/Taxol.RTM. was developed by the present inventors
following continuous exposure of CCRF-CEM cells with increasing
concentrations of paclitaxel (at IC.sub.50-IC.sub.90) for ten
months. The fresh medium with paclitaxel was replenished every
week. The CCRF-CEM/Taxol.RTM. exhibited 57-fold resistance to
paclitaxel (IC.sub.50=0.0021 .mu.M, see Table 1A). The DC-3F
hamster lung fibroblast cell line and its actinomycin D-selected
sublines (DC-3F/ADII and DC-3F/ADX) were obtained from the Memorial
Sloan-Kettering Cancer Center (MSKCC). The murine leukemic P388/0
and its doxorubicin-selected subline (P388/DX) as well as human
neuroblastoma SK-N-As and its doxorubicin-selected subline
(SK-N-FI/Adr) were obtained from MSKCC.
[0460] The drug-resistant cell lines were continuously cultured in
the presence of the selecting agent, AD, DX, VBL or VM to maintain
the drug resistant phenotypes. Each sub-cell line was cultured for
one to two passages in an appropriate concentration (e.g.
IC.sub.50) of the drug, which was then removed from the media and
the cells were rested in fresh media for a minimum of 4 days before
each assay. All cells were cultured in RPMI 1640-10% FBS at
37.degree. C., 5% CO.sub.2 (see below).
[0461] Cytotoxicity Assays
[0462] The cells were cultured at an initial density of
5.times.10.sup.4 cells/mL. They were maintained in a 5%
CO.sub.2-humidified atmosphere at 37.degree. C. in RPMI-1640 medium
(GIBCO-BRL, Gaithersburg, Md.) containing penicillin (100 U/mL),
streptomycin (100 mg/mL) (GIBCO-BRL) and 100% heat inactivate fetal
bovine serum. Culture for cell suspension (such as for CCRF-CEM,
P388 and sublines), were performed by the XTT-microculture
tetrazonium method (Scudiero, D. A. et al., Cancer Res. 1988, 48,
4827-4833) in duplicate in 96-well microtiter plates.
[0463]
2',3'-Bis(methoxy-4-nitro-5-sufophenyl)-5-[(phenylamino)carbonyl]-2-
H-tetrazolium hyudroxide (XTT) was prepared at 1 mg/mL in prewarmed
(37.degree. C.) medium without serum. Phenazine methosulfate (PMS)
and fresh XTT were mixed together to obtain 0.025 mM PMS-XTT
solution (25 .mu.L of the stock 5 mM PMS was added per 5 mL of 1
mg/mL XTT). Following a 72 h incubation, 50 .mu.L of the assay
aliquots were added to each well of the cell culture. After
incubation at 37.degree. C. for 4 h, absorbance at 450 nm and 630
nm was measured with a microplate reader (EL340, Bio-Tek
Instruments, Inc., Winooski, Vt.).
[0464] The cytotoxicity of the drug toward the monolayer cell
cultures (such as DC-3F, MCF-7, SK-N-As and sublines) was
determined in 96-well microtiter plates by the SRB method as
described by Skehan and co-workers (Skehan, P., et al., J. Natl.
Cancer Inst. 1990, 82, 1107-1112) for measuring the cellular
protein content. Cultures were fixed with trichloroacetic acid and
then stained for 30 min with 0.4% suforhodamine B dissolved in 1%
acetic acid. Unbound dye was removed by acetic acid washes, and the
protein-bound dye was extracted with an unbuffered Tris base
(tris(hydroxy-methyl)aminomethane) for determination of absorbance
at 570 nm in a 96-well microtiter plate reader. The experiments
were carried out in duplicate. Each run entailed six to seven
concentrations of the tested drugs. Data were analyzed with the
median-effect plot (Chou, T.-C. & Talalay, P. T. (1984) Adv.
Enzyme Regul. 22, 27-55) using a previously described computer
program (Chou, J., & Chou T.-C. 1987, Dose-effect analysis with
microcomputers: Quantitation of ED.sub.50, synergism, antagonism,
low-dose risk, reception-ligand binding and enzyme kinetics, IBM-PC
software and manual, Biosoft, Cambridge, U.K.).
[0465] Stability of Desoxyepothilone B in Plasma
[0466] HPLC Method.
[0467] Sample Preparation. To 300 microliters of spiked plasma are
added 30 microliters of methanol. The mixture is agitated and
allowed to stand for 2 minutes. Then 600 microliters of methanol
are added. The mixture is centrifuged. The supernatant is removed
for analysis by HPLC. Analyses were performed under the following
chrmotographic conditions: Column: Nova-Pak C18, 15 cm. Eluant: 50%
acetonitrile/water with 0.8% triethylamine, 0.2% phosphoric acid.
Detection: UV (250 nm).
[0468] Animals
[0469] Athymic nude mice (nu/nu) were used for MX-1 and MCF-7/Adr
human mammary carcinoma xenografts. Mice were obtained from Taconic
Laboratory Animals and Service (Germantown, N.Y.: outbred, Swiss
background). Male mice 6-8 weeks old, weighing 20-25 g were
used.
[0470] Results
[0471] Structure-Activity Relationships
[0472] To determine structure-activity relationships of epothilones
(Su, D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36,
2093-2096), the susceptibility of CCRF-CEM leukemic cells and the
respective drug-resistant sublines CCRF-CEM/VBL.sub.100 (Pgp-MDR
cells) (Cass, C. E., et al., Cancer Res. 1989, 49, 5798-5804) and
CCRF/CEM/VM.sub.1 (cells with a mutated topo 11 gene) (Danks, M.
K., Yalowich, J. C., & Beck, W. T. Cancer Res. 1987, 47,
1297-1301) to epothilones A and B and desoxyepthilone B (Table 1A)
were determined. Although/VBL.sub.100 is 527-fold resistant to VBL
and 1970-fold resistance to paclitaxel, the epothilones A and B
exhibited only 6.1.about.7.4-fold resistance, while
desoxyepothilones A and B evidenced only 0.6.about.1.8-fold
resistance. Using paclitaxel as the selecting agent,
CCRF-CEM/Taxol.RTM. was grown 57-fold resistant and found to be
10.9-fold resistance to VBL. By contrast, DX, AD and VP-16 showed
only 2.3-4.5 fold resistance, and epothilones A and B showed very
little resistance (i.e., 1.4.about.3.1-fold) and desoxyepothilones
A and B displayed almost no resistance (i.e., 0.7.about.1.7 fold)
(Table 1A). CCRF-CEM/VM.sub.1 cells that were 117-fold resistant to
etoposide were sensitive to all epothilones or desoxyepothilones
listed in Table 1A with only 0.6-3.6 fold resistance.
2TABLE 1A Susceptibility of CCRF-CEM and its drug resistant
sublines to epothilone derivatives. (C) (A) (B) CCRF- (D) CCRF-
CCRF- CEM/ CCRF Compound CEM CEM/VBL.sub.100 Taxol .RTM.
CEM/VM.sub.1 (B)(A) (C)(A) (D)(A) IC.sub.50 (.mu.M)* Epo A 0.0027
0.020 0.0037 0.0061 7.4 1.4 2.3 Epo B 0.00035 0.0021 0.0011 0.0013
6.1 3.1 3.6 dEpo A 0.0220 0.012 0.0150 0.013 0.55 0.7 0.59 dEpo B
0.0095 0.017 0.0162 0.014 1.8 1.7 1.5 Taxol .RTM. 0.0021 4.140
0.120 0.0066 1971 57 3.1 Vinblastine 0.0063 0.332 0.0069 0.00041
527 10.9 0.7 Etoposide 0.290 10.30 1.32 34.4 35 4.5 117 Adriamycin
0.036 1.74 0.082 0.128 48 2.3 3.6 Actinomycin 0.00035 0.038 0.0013
0.00027 109 3.7 0.8 D *Cell growth inhibition was measured by XTT
tetrazonium assay (Scudiero, D.A. et al., Cancer Res. 1988, 48,
4827-4833) following 72 h incubation for cell growth as described
previously. The IC.sub.50 values were determined with 6-7
concentrations of each drug using a computer program (Chou, T.-C.
& Talalay, P.T. (1984) Adv. Enzyme Regul. 22, 27-55); Chou, J.,
& Chou T.-C., Dose-effect analysis with microcomputers:
Quantitation of ED.sub.50, synergism, # antagonism, low-dose risk,
reception-ligand binding and enzyme kinetics, 1987, IBM-PC software
and manual, Biosoft, Cambridge, U.K.)
[0473] Toxicity of dEpoB vs. EpoB
[0474] The toxicity of EpoB and dEpoB was compared in normal
athymic nude mice on the daily i.p. schedule. EpoB at 0.6 mg/kg,
QDX4, i.p. led to lethality in all eight mice. In contrast, in the
group treated with dEpoB 25 mg/kg, QDX5, i.p., zero of six mice
died. It was also observed that the vehicle treated control group
showed a steady increase in body weight and the dEpoB treated mice
maintained approximately the same average body weight, whereas the
EpoB treated group showed steady decreases in body weight until
death. These results indicated a higher toxicity for both EpoB and
dEpoB than in tumor bearing nude mice when the treatment schedule
was Q2Dx5, i.p. (see Tables 1C and 1D). In the preliminary studies,
for the non-tumor bearing nude mice receiving EpoB 0.6 mg/kg or
dEpoB 25 mg/kg, QDx4, i.p., there were no apparent changes in
hematological cell counts or blood chemistry parameters except for
a 43% decrease in lymphocytes. Similar leukopenia was found with
paclitaxel. Some obstructive fecal mass in the large intestine was
noted following Epo treatments in the preliminary study. No gross
pathological abnormalities were observed in other organs.
3TABLE 1B Toxicity of Epothilone B, and Desoxyepothilone B in
normal nude mice. Dose schedule and route of Number Number of Group
administration of mice mice died Control 4 0 Epothilone B 0.6
mg/kg, QD .times. 4, 8 8* i.p. Desoxyepothilone 25 mg/kg, QD
.times. 4, 6 0 B i.p. *Mice died of toxicity on day 5, 6, 6, 7, 7,
7, 7, 7
[0475] Comparison of Different Routes of Administration
[0476] Nude mice bearing human ovarian adenocarcinoma, SK-OV3, and
human mammary adenocarcinoma, MX-1, were treated with dEpoB, both
i.p. (DMSO as solvent) and i.v. (cremophor and EtOH, 1:1), with
Taxol.RTM., i.p. and i.v. (both with clinical samples in cremophor
and EtOH as specified by the manufacturer), and with EpoB, i.v.
(used cremophor and EtOH, 1:1). As shown in Table 6, for Q2Dx5
schedule, dEpoB, i.p. (35 mg/kg) and Taxol.RTM. i.v. (15 mg/kg)
both yield potent therapeutic effects against MX-1 with tumor-size
on day 19, treated/control=0.02 and 0.01, respectively (see Table
1F). For the ovarian tumor a lesser therapeutic effect was seen,
tumor size on day 21, treated/control=0.28 for both drugs (see
Table 1G). For EpoB i.v., at 0.6 mg/kg there was less therapeutic
effect and more toxicity than for dEpoB and Taxol.RTM.. In
contrast, dEpoB, i.v. (15 mg/kg) and Taxol.RTM., i.p. (5 mg/kg)
showed more toxicity and less therapeutic effect against both
tumors. Thus, dEpoB showed the best results when given i.p. and
Taxol.RTM. gave better results when given i.v. in cremophor and
EtOH.
[0477] In Vitro Effect Against Various Tumor Sublines
[0478] Further susceptibility evaluations were conducted for
epothilones A and B and desoxyepothilones A and B in four
additional tumor cell lines and four of their drug resistant
sublines (Table 1C). Hamster lung tumor cells DC-3F/ADX, that were
selected 13,000-fold resistant to AD, were found to be 328-fold
resistant to paclitaxel and 1 24-fold resistant to DX when compared
with the parent cell line (DC-3F). In contrast, epothilones A and B
and desoxyepothilone A showed only 3.9.about.28-fold resistance,
and epothilones A and B and desoxyepothilone B showed no
cross-resistance (0.9-fold resistance).
[0479] Murine leukemic P388/Adr cells that were selected 482-fold
resistant to DX, were found to be 111-fold resistant to paclitaxel.
However, epothilones A and B showed less than 6-fold resistance,
and for desoxyepothilone A and B there was no cross-resistance
(<0.6-fold resistance).
[0480] Human neuroblastoma cells, SK-N-F1, that were selected as
18-fold resistant to DX, were found to be 80-fold resistant to
paclitaxel. By contrast, epothilone B was 25-fold resistant, while
the resistance of epothilone A and desoxyepothilones A and B was
only between 1.9 and 3.1.
[0481] Human mammary carcinoma cells, MCF-7/Adr, that were selected
3.8-fold resistant to DX, were found to be 46-fold resistant to
paclitaxel. In contrast, compounds epothilones A and B and
desoxyepothilone B was 3.1.about.5.4-fold resistant, and dEpoB
showed only 2.4-fold resistant. Overall, dEpoB was the least
cross-resistant among epothilones and desoxyepothilones in various
drug-resistant tumor sublines. By contrast, paclitaxel suffers from
marked cross-resistance in tumor cells that were selected to be
resistant to VBL, DX or AD. In three out of five cell lines
studied, cross-resistance to paclitaxel was even greater than that
of the selecting agents.
[0482] Therapeutic Effects against MX-1 Xenografts
[0483] Therapeutic effects of compounds epothilone A and
desoxyepothilone B, paclitaxel, VBL and CPT were evaluated in
athymic nude mice bearing human mammary adenocarcinoma MX-1
xenografts (Table 1D). Desoxyepothilone B at a 15 mg/kg dose i.p.
on days 7, 9, 11, 13 and 15 produced a 50-60% tumor volume
reduction when compared to the control group. A higher dose of
drug, 25 mg/kg, produced as much as 96% average tumor volume
reduction measured two days after the last treatment (i.e., on day
17). These effects were achieved with no lethality nor body weight
reduction. Furthermore, with a 25 mg/kg dose, one out of six mice
was tumor-free on day 35 after tumor implantation (i.e. on day 35).
In contrast, after treatment with EpoB (0.3 mg/kg and 0.6 mg/kg,
i.p., on days 7, 9, 11, 13 and 15), the average body weight
decreased over 1 g and 2 g, respectively. In the case of 0.6 mg/kg
treatment, three out of seven mice died of toxicity. Despite the
apparent toxicity at these doses, EpoB appeared to have only
marginal therapeutic effect, as only 16% to 26% tumor volume
reduction was observed (Table 1D). The parallel experiments for
paclitaxel led to a lower therapeutic effect. In animals treated
with paclitaxel, 5 mg/kg, there was 55% reduction in tumor volume
and no decrease in average body weight. At a dose of 10 mg/kg,
paclitaxel showed a 89% tumor reduction, however, four out of seven
mice died of toxicity. For DX (2.about.3 mg/kg) and CPT
(1.5.about.3 mg/kg) i.e., near the maximal tolerated doses,
inferior results were obtained when compared with dEpoB. Thus,
dEpoB even at non-toxic dose had the best therapeutic effect among
the five compounds studied under the same experimental
conditions.
[0484] In a separate experiment, MX-1 xenograft-bearing mice were
treated with dEpoB, 35 mg/kg, Q2Dx5, i.p. beginning on day 8 after
tumor implantation (FIG. 60). On day 16, two out of ten mice had no
detectable tumor. These ten mice were further treated with dEpo B,
40 mg/kg, Q2Dx5 beginning on day 18. At the end of treatment on day
26, five out of ten mice had no detectable tumor, and three
remained tumor-free on day 60. There was body weight reduction
during treatments but no lethality occurred. In a parallel
experiment, ten mice were treated with paclitaxel, 5 mg/kg, Q2Dx5,
i.p. from day 8 to day 16, followed by a second cycle of treatment
in the same manner from day 18 to day 26. The tumor sizes were
reduced but continued to grow during treatment and by day 24, the
average tumor size was 2285.+-.597 mm.sup.3 (n=10). In a parallel
experiment, DX was given 2 mg/kg, Q2Dx5, i.p. (FIG. 60), and
reduced therapeutic effect was seen compared to dEpoB or
paclitaxel. No data after day 18 is shown because the tumor burden
in the control group was excessive and the mice in this group were
sacrificed.
[0485] Therapeutic Effects Against MCF-7/Adr Xenografts
[0486] The therapeutic effects of dEpoB, Taxol.RTM., DX and CPT
were also evaluated in nude mice bearing xenografts of human
mammary adenocarcinoma resistant to DX (MCF-7/Adr) (Table 1E). As
indicated earlier in Table 1B for the cytotoxicity results in
vitro, MCF-7/Adr cells selected to be 3.8-fold resistant to DX were
found to be 46-fold resistant to paclitaxel, and only 2.4-fold
resistant to dEpoB. For in vivo studies, each drug was given Q2Dx5,
i.p. beginning on day 8 after tumor implantation. Paclitaxel 12
mg/kg and DX 3 mg/kg were highly toxic to the nude mice with 317
and 3/6 lethality, respectively. CPT 3 mg/kg led to moderate
toxicity without lethality, and dEpoB 35 mg/kg showed negligible
toxicity as shown by minimal body weight changes (Table 1E). CPT at
3 mg/kg reduced 57% of tumor size on day 17 (p<0.05 when
compared with control group). Desoxyepothilone B at 35 mg/kg
significantly suppressed tumor size by 66-73% when compared with
the control group (p<0.005.about.0.05), without complete tumor
regression. In contrast, paclitaxel 5 mg/kg, and DX 2 mg/kg,
produced slight growth suppression of this drug-resistant tumor
which was not significantly different from the control group (see
Table 1D). Thus, dEpoB stands out as the superior drug among the
four tested against this drug-resistant tumor.
4TABLE 1C Comparison of in vitro growth inhibition potency of
epothilone derivatives against various parent and drug resistant
tumor cell lines. DC- P388/ SK-N SK-N MCF-7 Compound DC-3F 3F/ADX
P388/0 Adr -As -Fl MCF-7 /Adr IC.sub.50 (.mu.M)* Epo A 0.0037 0.053
0.0018 0.0010 0.012 0.023 0.0030 0.0094 (14.5 .times.).sup.# (5.3
.times.).sup.# (1.9 .times.).sup.# (3.1 .times.).sup.# Epo B 0.0006
0.017 0.00029 0.0016 0.004 0.010 0.0005 0.0027 (28 .times.) (5.5
.times.) (25 .times.) (5.4 .times.) dEpo A 0.011 0.042 0.0213
0.0125 0.073 0.223 0.032 0.144 (3.9 .times.) (0.59 .times.) (3.1
.times.) (4.5 .times.) dEpo B 0.00097 0.00091 0.0068 0.0042 0.021
0.046 0.0029 0.0071 (0.9 .times.) (0.62 .times.) (2.2 .times.) (2.4
.times.) Taxol .RTM. 0.095 32.0 0.0029 0.326 0.0016 0.130 0.0033
0.150 (338 .times.) (111 .times.) (80 .times.) (46 .times.)
Actinomycin 0.00044 0.572 0.00015 0.0012 0.00085 0.0119 0.00068
0.00167 D (13000 .times.) (8 .times.) (14 .times.) (2.5 .times.)
0.018 2.236 0.0055 2.65 0.077 1.42 0.057 0.216 Adriamycin (124
.times.) (482 .times.) (18.4 .times.) (3.8 .times.) *Cell growth
inhibition was measured by protein staining SRB assay (Skehan, P.,
et al., J. Natl. Cancer Inst. 1990, 82, 1107-1112) following 72 h
incubation as described previously. The IC.sub.50 values were
determined with 6-7 concentrations of each drug using a computer
program (Chou, T.-C. & Talalay, P.T., Adv. Enzyme Regul. 1984,
22, 27-55; Chou, J., & Chou T.-C., Dose-effect analysis with
microcomputers: Quantitation of ED.sub.50, # synergism, antagonism,
low-dose risk, reception-ligand binding and enzyme kinetics, 1987,
IBM-PC software and manual. Biosoft, Cambridge, U.K.).
.sup.#Numbers in parentheses are folds of resistance based on the
IC.sub.50 ratio when compared with the corresponding parent cell
lines except for P388/0 and P388/Adr, XTT assay (Scudiero, D.A., et
al., Cancer Res. 1988, 48, 4827-4833) was used.
[0487]
5TABLE 1D Therapeutic effect of desoxyopothilone B, epothilone B,
Taxol .RTM., vinblastine and camptothecin in nude mice bearing
human MX-1 zenograft. Average Body Weight Change Average Tumor Size
Dose (g) (T/C) Toxicity Drug (mg/kg) Day 7 11 13 15 17 Day 11 13 15
17 Death N Control 27.2 +0.8 +1.1 +1.9 +0.6 1.00 1.00 1.00 1.00 0/8
8 dEpo B 15 27.1 +0.8 +1.1 +1.6 +1.5 0.65 0.46 0.49** 0.41** 0/6 6
25.sup.# 27.0 +0.4 +0.7 +1.0 +0.7 0.38* 0.11** 0.05*** 0.04**** 0/6
6 Epo B 0.3 26.9 +0.5 +0.4 -0.3 -1.2 1.00 0.71 0.71 0.84 0/7 7
(0.6.sup..dagger. 27.4 -0.3 -1.3 -2.1 -2.1 1.08 0.73 0.81 0.74
3/7).sup.## 7 Taxol .RTM. 5 26.9 -0.1 +0.4 +1.1 +1.2 0.54 0.46
0.40* 0.45** 0/7 7 10.sup..dagger-dbl. 27.6 -2.7 -1.1 -0.3 +2.2
0.43 0.37 0.12 0.11 4/7.sup.## 7 Vinblastine 0.2 25.7 +0.6 +1.4
+2.3 +2.9 0.65 0.54 0.56 0.88 0/7 7 (0.4.sup. 26.4 +0.8 +0.5 +1.9
+2.1 0.80 0.56 0.83 0.88 1/7).sup.## 7 Campothecin 1.5 27.4 -0.9
-0.7 -0.4 +1.0 0.61 0.45* 0.32* 0.36** 0/7 7 MX-1 tissue 50
.mu.l/mouse was implanted s.c. onday 0. Every other day i.p.
treatments were given on days 7, 9, 11, 13, 15; Theaverage tumor
volumes of the control group on day 11, 13, 15 and 17 were 386 .+-.
120, 915 .+-. 245, 1390 .+-. 324, and 1903 .+-. 319 mm.sup.3 (mean
.+-. SEM), respectively; *P < 0.05, **P < 0.01, ***P <
0.005, ****P < 0.001; .sup.#One out of six mice with no
detectable tumor on day 35; .dagger.Three mice died of drug
toxicity on day 17; .dagger-dbl.Four mice died of drug toxicity on
day 13, 13, 13, 15; .sup.One mouse died of drug toxicity on day 15;
.sup.##P values were not shown due to toxic lethality.
[0488]
6TABLE 1F Therapeutic effects of desoxyopothilone B, epothilone B,
taxol, adriamycin and camptothecin in nude mice bearing MDR human
MCF-7/Adr tumor. Average Body Weight Change Average Tumor Size Dose
(g) (T/C) Toxicity Drug (mg/kg) Day 8 11 13 15 17 Day 11 13 15 17
Death N Control 0 25.0 +2.0 +2.6 +3.1 +3.7 1.00 1.00 1.00 1.00 0/8
8 dEpo B 35 25.0 0.3 +0.7 +0.6 +0.8 0.31** 0.27*** 0.30*** 0.34*
0/8 8 Taxol .RTM. 6 25.3 +1.7 +1.8 +0.8 +0.9 0.57 0.66 0.85 0.90
0/7 7 (12 24.5 -0.7 -1.3 -2.4 0 0.50 0.51 0.32 0.40 3/7 7).sup.#
Adriamycin 2 25.6 +0.2 -0.4 -0.6 -0.4 0.70 0.68 0.84 0.78 0/8 8 (3
24.6 +0.5 -1.3 -3.2 -1.6 0.66 0.83 0.57 0.53 3/6 6).sup.#
Campothecin 1.5 24.4 +1.1 +0.9 +1.7 +1.4 1.08 0.72 0.61 0.72 0/8 8
(3 24.5 -0.6 -0.4 -0.8 -0.9 0.95 0.76 0.61 0.43* 0/6 6 MCE-7/Adr
cell 3 .times. 10.sup.6 /mouse was implanted s.c. on day 0. Every
other day ip treatments were given on days 8, 10, 12, 14 and 16.
The average tumor size of control group on day 11, 13, 15 and 17
was 392" .+-. 84, 916 .+-. 210, 1499 .+-. 346, and 2373 .+-. 537
mm.sup.3 respectively (mean .+-. SEM). *<P0.05, **P < 0.01,
***P < 0.005; .sup.#P values were not shown due to
lethality.
[0489]
7TABLE 1F Therapeutic effects of desoxyopothilone B, Epo B and
Taxol .RTM. in nude mice bearing MX-1 tumors using different
vehicles and different routes of administration..sup.a Average Body
Weight Change Average Tumor Size Dose (g) (T/C) Tumor Toxicity
Drug/Route (mg/kg) Day 9 13 15 17 19 Day 13 15 17 19 Disapp. Death
Control 0 26.4 -0.2 -0.4 +0.2 +0.8 1.00 1.00 1.00 1.00 0/6 0/6 dEpo
B/i.p. 35 27.8 -1.7 -2.1 -2.1 -2.4 0.35 0.14 0.04 0.02 3/6 0/6 dEpo
B/i.v. 15 27.0 0 -0.6 -1.1 -2.6 0.47 0.30 0.10 0.04 0/6 4/6.sup.b
Epo B/i.p. 0.6 27.0 -0.9 -0.5 -3.3 -3.4 0.67 0.63 0.61 0.51 0/6 0/6
Taxol .RTM./i.p. 5 27.4 -1.1 -2.0 -1.0 -0.2 0.59 0.72 0.59 0.55 0/6
0/6 Taxol .RTM./i.v. 15 27.2 -0.6 -0.8 -0.8 -0.9 0.36 0.13 0.04
0.01 2/6 0/6 .sup.a50 .mu.g tumor tissue was implanted s.c. on day
0. Every other day i.p. or i.v. treatments were given on days 9,
11, 13, 15 and 17. The average tumor size of control group on day
13, 15, 17 and 19 was 274, 378, 677, 1139 mm.sup.3 respectively.
.sup.b4/6 mice died of drug toxicity on day 23, 23, 23, 25.
[0490]
8TABLE 1G Therapeutic effects of desoxyopothilone B, Epo B and
Taxol .RTM. in nude mice bearing SK-OV-3 tumors using different
vehicles and different routes of administration..sup.a Average Body
Weight Change Average Tumor Size Dose (g) (T/C) Tumor Toxicity
Drug/Route (mg/kg) Day 9 13 15 17 19 Day 15 17 19 21 Disapp. Death
Control 0 26.4 -0.2 -0.4 +0.2 +0.8 1.00 1.00 1.00 1.00 0/6 0/6 dEpo
B/i.p. 35 27.8 -1.7 -2.1 -2.1 -2.4 0.57 0.33 0.35 0.28 0/6 0/6 dEpo
B/i.v. 15 27.0 0 -0.6 -1.1 -2.6 0.86 0.56 0.50 0.44 0/6 0/6.sup.b
Epo B/i.p. 0.6 27.0 -0.9 -0.5 -3.3 -3.4 0.75 0.69 0.88 0.77 0/6 0/6
Taxol .RTM./i.p. 5 27.4 -1.1 -2.0 -1.0 -0.2 0.69 0.60 0.49 0.40 0/6
0/6 Taxol .RTM./i.v. 15 27.2 -0.6 -0.8 -0.8 -0.9 0.97 0.67 0.42
0.28 0/6 0/6 .sup.a50 .mu.g tumor tissue was implanted s.c. on day
0. Every other day i.p. or i.v. treatments were given on days 9,
11, 13,15 and 17. The average tumor size of control group on day
13, 15, 17 and 19 was 274, 378, 677, 1139 mm.sup.3 respectively.
.sup.b4/6 mice died of drug toxicity on day 23, 23, 23, 25.
[0491] Discussion
[0492] Two classes of naturally occurring compounds, epothilones
and paclitaxel, which are apparently structurally dissimilar, show
similar modes of action in stabilizing microtubule assemblies.
These similarities include binding tubulin, substitution for
paclitaxel in maintaining paclitaxel-dependent cell growth in a
resistant cell line, and similar morphologic changes as determined
by electron microscopic examination of the drug-microtubule
complex. There are differences, however, between the two classes of
compounds. These differences are most prominently exhibited by the
lack of cross-resistance in cytotoxicity between the epothilones
and paclitaxel even in CCRF-CEM/Taxol.RTM. cells (Table 1A).
Furthermore, in CCRF/CEM/VBL.sub.100, the cells were 527-fold
resistant to vinblastine and 1971-fold resistance to paclitaxel,
but were only 6.1-fold resistant to EpoB and 1.8-fold resistant to
dEpoB (Table 1A). In DC-3F/ADX cells, there was 13,000-fold
resistance to actinomycin D and 338-fold resistance to paclitaxel.
However, these cells were only 28-fold resistance to EpoB and had
no resistance to dEpoB (i.e., 0.9-fold resistance or collateral
sensitivity) (Table 1B). Paclitaxel showed a higher degree of
cross-resistance in these cell lines than other MDR-drugs such as
doxorubicin, actinomycin D, vinblastine or etoposide. In some cases
the degrees of resistance to paclitaxel were even greater than
those of the resistance-selecting agent (e.g., CCRF-CEM/VBL.sub.100
in Table 1A, and SK-N-FI and MCF/7-Adr in Table 1B). In contrast,
among all compounds tested, dEpoB showed the least cross-resistance
in several drug-resistant cell lines (e.g. DC-3F/Adr) and even
showed slight collateral sensitivity (e.g. DC-3F/ADX and P388/Adr
in Table 1B). Parallel cancer chemotherapeutic studies for EpoB,
dEpoB, Taxol.RTM. and other drugs were performed under the same
experimental conditions (i.e., treatment schedule, Q2D; solvent
vehicle, DMSO; and route of administration, i.p.) in animals.
[0493] The i.p. route of other formulations for administration of
dEpo B is far better tolerated than the i.v. method. Even though
EpoB is the most potent, it is by no means the best candidate for
cancer therapy in terms of therapeutic index (i.e. the therapeutic
efficacy at tolerable dosage, or the ratio of toxic dose vs the
therapeutic dose). Desoxyepothilone B, lacking the epoxide
functionality, exhibited far superior therapeutic results in vivo
as compared to the more potent EpoB. Similarly, the present
therapeutic results for dEpoB in MX-1 xenografts were far better
than those for EpoB, paclitaxel, doxorubicin, vinblastine or
camptothecin. In addition, the effects of dEpo B on MCF-7/Adr
xenografts were significantly better than those for paclitaxel,
doxorubicin and camptothecin. In view of the finding that the
epothilones have little or no cross-resistance against MDR tumor
cells in vitro, the special therapeutic advantage of such compounds
might be in their use against MDR-resistant tumors.
[0494] Novel Aldol Condensation with 2-Methyl-4-pentenal:
Application to Preparation of Epothilone B and Desoxyepothilone
B
[0495] Stereoselectivity poses a potential hindrance to enhancing
access to multicomponent libraries. Nicolaou, K. C., et al., J. Am.
Chem. Soc. 1997, 119, 7960; Nicolaou, K. C., et al., J. Am. Chem.
Soc. 1997, 119, 7974. Nicolaou, K. C., et al., Angew. Chem. Int.
Ed. Engl. 1997, 36, 2097. However, stereoselectivity holds the
attraction that it allows for accumulation of substantial
quantities of fully synthetic key epothilones of correct
configuration. Comparable harvesting of needed amounts of material
through the stereo-random olefin metathesis route (Yang, Z., Y., et
al., Angew. Chem. Int. Ed. Engl. 1997, 36, 166; Nicolaou, K. C., et
al., Angew. Chem. Int. Ed. Engl. 1997, 36, 525; Nicolaou, K. C., et
al., Nature 1997, 387, 268; Nicolaou, K. C., et al., J. Am. Chem.
Soc. 1997, 119, 7960; Nicolaou, K. C. et al., J. Am. Chem. Soc.
1997, 119, 7974; Nicolaou, K. C., et al., Angew. Chem. Int. Ed.
Engl. 1996, 35, 2399. Schinzer, D., et al., Angew. Chem. Int. Ed.
Engl. 1997, 36, 523; Meng, D., et al., J. Am. Chem. Soc. 1997, 119,
2733) would be virtually prohibitive. Biological studies in
xenograft mice provided herein identified some significant toxicity
problems with the highly potent epothilone B. Remarkably, in vivo
studies in the intraperitoneal mode of injection demonstrate that
the less potent 12,13-deoxyepothilone B is well tolerated and is
virtually curative against a variety of xenograft tumors. Chou,
T.-C., et al., Proc. Nat'l Acad. Sci. USA 1998, 0000.
Desoxyepothilone B has clinical advantages relative to paclitaxel,
particularly as regards vulnerability to the phenomenon of multiple
drug resistance. The preparative route disclosed herein retains the
advantages of high stereoselectivity throughout, and provides an
improved approach to the previously difficult C1-C11 domain. FIG.
53 provides a global overview of the problem.
[0496] The route disclosed herein is based on four findings. The
first is the ease of formation and the synthetic utility of the
Z-lithium enolate (48A) readily produced from 59 as shown in FIG.
61(A). In this easily obtained construct, the critical enolate of
the ethyl ketone is fashioned in the context of a putative
.beta.,.delta.-diketoester ensemble embracing carbons 1-6 of the
target (cf. structure 47). The advantages of this direct approach
for synthetic economy are apparent.
[0497] The second and most surprising finding undergirding this
synthesis is that the sense of addition of enolate 48A to readily
available S-aldehyde 58 provides the desired C7-C8 anti
relationship with good diastereofacial selectivity in conjunction
with the expected C6-C7 syn relationship (by Ik-addition). C. H.
Heathcock, in Asymmetric Synth. J. D. Morrison, ed., Academic
Press, New York, 1984, Vol. 3, p.111-212; see compound 49 and its
stereoisomer. The stereochemistry of the minor diastereomer was
presumed to be C7-C8 syn, but was not rigorously proven. This was
based on precedent (Mori, I., et al., J. Org. Chem., 1990, 55,
1114), assuming that this isomer arises from facial selectivity in
the aldol reaction, not as a consequence of the E-enolate of 48A.
Those assignments also rely to varying extents on the Heathcock
precedent. The 5.5:1 outcome for the diastereofacial selectivity of
this aldol reaction is counter to expectations arising from the
traditional models first advanced by Cram and Felkin. (For
transition state models in diastereomeric carbonyl addition
reactions see: D. J. Cram, F. A. Elhafez, J. Am. Chem. Soc. 1952,
74, 5828; D. J. Cram, K. R. Kopecky, J. Am. Chem. Soc. 1959, 81,
2748; J. W. Cornforth, R. H., et al., J. Am. Chem. Soc. 1959, 81,
112; G. J. Karabatsos, J. Am. Chem. Soc. 1967, 89, 1367; M.
Cherest, H. Felkin, N. Prudent, Tetrahedron Lett. 1968, 2199; N. T.
Ahn, O. Eisenstein, Nouv. J. Chem. 1977, 1, 61; A. S. Cieplak, J.
Am. Chem. Soc. 1981, 103, 4540; E. P. Lodge, C. H. Heathcock J. Am.
Chem. Soc. 1987, 109, 2819.) These extensively invoked
formulations, which differ widely in their underlying
conformational assumptions and stereochemical treatments, usually
converge in terms of their predicted outcome.
[0498] The high anti:syn diastereofacial ratio arises from a
peculiar characteristic of aldehyde 58 and likely reflects the
relationship of its vinyl and formyl groups. It is not, apparently
the result of a gross property of enolate 48A. Indeed, the same
enolate, with the benchmark aldehyde phenylpropanal 60a, performs
in the expected fashion (C. H. Heathcock in Asymmetric Synth. J. D.
Morrison, ed., Academic Press, New York, 1984, Vol. 3, p.111-212),
yielding an 11:1 ratio of 61a:62a. Furthermore, with aldehyde 60b,
the dihydro version of 58, the C7 to C8 anti:syn (61b:62b) ratio
drops to 1:1.3. Moreover, when the distance between the vinyl and
formyl groups is extended, as in 60c, selectivity is also
compromised. By contrast the phenyl and dimethylallyl analogs of 58
(60d and 60e) bearing the same relationship of unsaturated groups
as in 58, exhibit good anti-antiselectivity (see products 61d and
61e as well as 62d and 62e). Also, aldehyde 60f, a substrate known
for its tendency to favor the anti-diastereofacial product (M. T.
Reetz, Angew. Chem. Int. Ed. Engl. 1984, 23, 556) on the basis of
presumed chelation control, performs normally with enolate 48A
affording a 1:4 ratio of 61f:62f.
[0499] With respect to the impact of the strong anti:syn
diastereofacial selectivity in the aldol reaction of 58 and 48A on
the overall efficiency of the synthesis, the rather favorable
result in establishing the C7-C8 bond opened the possibility that
the C1-C7 fragment could be entered into the synthesis as an
achiral block. Accordingly, it would be necessary to gain control
over the eventual stereochemistry at C3. This subgoal was to be
accomplished by the implementation of any asymmetric, reagent
controlled Noyori reduction (vide infra). Noyori, R., et al., J.
Am. Chem. Soc. 1987, 109, 5856; Taber, D. F., Silverbert, L.,
Tetrahedron Lett. 1991, 32, 4227; Ager, D. J., Laneman, S.,
Tetrahedron Asymmetry 1997, 8, 3327.
[0500] The third critical element was the finding that the key
B-alkyl Suzuki merger which controls the geometry of the
trisubstituted double bond can be conducted successfully even on
the elaborate 51, obtained from 49. The cognate substrate for the
Suzuki reaction was the previously described vinyl iodide, 51 (Su,
D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757). The
remarkable coupling step, afforded the Z-olefin 52A and thence, 52
after removal of the C15 silyl protecting group (FIG. 61(B)). The
.beta.,.delta.-disketo ester array in 52 responded well to
asymmetric catalytic reduction under modified Noyori conditions
(Taber, D., Silverbert, L. J., Tetrahedron Lett. 1991, 32, 4227) to
give the diol 53 (88%, >95:5). Strict regiochemical and
diastereofacial control in the Noyori reduction was very dependent
on the amount of acid present in the reaction. Without acid, the
rate of reduction dropped off as well as the selectivity in the
reduction. Further, the carbonyl at C-5 was never reduced under
these conditions but was absolutely necessary for the reduction of
the C-3 carbonyl. When C-5 was in the alcohol oxidation state, no
reduction was seen. The conversion of 53 to desoxyepothilone B and
thus epothilone B was accomplished by methodologies set forth
herein. Balog, A., et al., Angew. Chem. Int. Ed. Engl. 1996, 35,
2801; Su, D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757;
Meng, D., et al., J. Am. Chem Soc. 1997, 119, 10073.
[0501] Biological Results
[0502] In the tables which follow, model system I is
desoxyepothilone. Model system 2 has the structure: 83
[0503] wherein R' and R" are H.
[0504] Model system 3 has the structure: 84
[0505] As shown in Table 2A, CCRF-CEM is the parent cell line.
CCRF-CEM/VBL (MDR cell line) is 1143-fold resistant to Taxol.RTM..
CCRF-CEM/VM (Topo II mutated cell line) only 1.3-fold resistant to
Taxol.RTM..
[0506] In terms of relative potency, synthetic Epothilone is
roughly the same as natural Epothilone A. For CCRF-CEM cells, the
ordering is:
[0507] Taxol.RTM.=Epothilone A>Desoxy Epothilone A>>Triol
Analog>>Model System I
[0508] For CCRF-CEM/VBL, the relative potency ordering is:
[0509] Desoxy Epothilone A.gtoreq.Epothilone
A>>Taxol.RTM.>Triol Analog>Model System I
[0510] For CCRF-CEM/VM, the relative potency ordering is:
[0511] Taxol.RTM.=Epothilone A>Desoxy Epothilone A>>Model
System I>Triol Analog
[0512] It is concluded that CCRF-CEM/VM cells are collaterally
sensitive to certain epothilone compounds.
9TABLE 2 Relative Efficacy of Epothilone Compound Against
HumanLeukemic CCRF-CEM Cell Growth and Against CCRF-CEM MDR
Sublines Resistant to Taxol .RTM. or Etoposide IC.sub.50 in .mu.M
CCRF- CCRF-CEM/ CCRF-CEM/ COMPOUND CEM VLB VM-1 EPOTHILONE A
NATURAL 0.0035 0.0272 0.0034 EPOTHILONE A SYNTHETIC 0.0029 0.0203
0.0034 MODEL SYSTEM I [3] 271.7 22.38 11.59 TRIOL ANALOG [2] 14.23
6.28 43.93 DESOXY EPOTHILONE [1] 0.002 0.012 0.013 Taxol .RTM.
0.0023 2.63 0.0030 VINBLASTINE 0.00068 0.4652 0.00068 VP-16
(ETOPOSIDE) 0.2209 7.388 34.51
[0513]
10TABLE 2A Relative Potency of Epothilone Compounds Against Human
Leukemic CCRF Sublines CCRF-CEM/VBL CCRF-CEM/VM, (MDR Cell Line)
(Topo II gene mutated cell line) CCRF-CEM (Taxol .RTM.
Resistant)-(1143 fold) (Taxol .RTM. Sensitive) (Parent Cell Line)
(Vinblastine Resistant) (VP-16 resistant) IC.sub.50 [IC.sub.50
IC.sub.50 [IC.sub.50 IC.sub.50 [IC.sub.50 (.mu.M) relative to
(.mu.M) relative to (.mu.M) relative to COMPOUND (A) Epolthilone A]
(B) Epolthilone A(B)(A)] (C) Epolthilone A (C)(A)] Taxol .RTM.
0.0023 [0.72] 2.63 [109.6] (1143).sup.a 0.0030 [0.88] (1.30).sup.a
MODEL 271.7 [84906] 22.38 [932.5] (0082).sup.b 11.59 [3409]
(0.043).sup.b SYSTEM I TRIAL ANALOG 14.23 [4447] 6.28 [261.7]
(0.44).sup.b 43.93 [12920] (3.09).sup.a DESOXYEPO- 0.022 [6.9]
0.012 [0.5] (0.55).sup.b 0.013 [3.82] (0.59).sup.b THILONE A
EPOTHILONE A 0.0032 [1] 0.024 [1] (7.5).sup.a 0.0034 [1]
(1.06).sup.a .sup.a(B)/(A) or (C)/(A) ratio >1 indicates fold of
resistance when compared with the parent cell line. .sup.b(B)/(A)
or (C)/(A) ratio <1 indicates fold of collateral sensitivity
when compared with the parent cell line.
[0514]
11TABLE 3 Relative Efficacy of Epothilone Compounds Against The
DC-3F Hamster Lung Cell Growth and Against DC-3F MDR Sublines
Resistant Actinomylin D IC.sub.50 in .mu.M COMPOUNDS DC-3F
DC-3F/ADII DC-3F/ADX EPOTHILONE A 0.00368 0.01241 0.0533 NATURAL
EPOTHILONE A 0.00354 0.0132 0.070 SYNTHETIC MODEL SYSTEM I [3] 9.52
3.004 0.972 TRIOL ANALOG [2] 10.32 4.60 4.814 DESOXY EPOTHILONE
0.01061 0.0198 0.042 [1] Taxol .RTM. 0.09469 3.205 31.98
VINBLASTINE 0.00265 0.0789 1.074 VP-16 (Etoposide) 0.03386 0.632
12.06 ACTINOMYCIN-D 0.000058 0.0082 0.486 (0.05816 nm)
[0515] Concerning Table 3, experiments were carried out using the
cell lines DC-3F (parent hamster lung cells), DC-3F/ADII (moderate
multidrug-resistant (MDR) cells) and DC-3F/ADX (very strong MDR
cells).
[0516] The relative potency of the compounds are as follows:
12 DC-3F: Actinomycin D > Vinbfastine .gtoreq. Epothilone A
(0.0036 .mu.M) > Desoxy epothilone > VP-16 > Taxol .RTM.
(0.09 .mu.M) > Model system I and triol analog DC-3F/ADX:
Desoxyepothilone .gtoreq. Epothilone A (0.06 .mu.M) >
Actinomycin D > Model system I > Vinblastine > triol
analog > viablastine > Taxol .RTM. (32.0 .mu.M)
[0517] DC-3F/ADX cells (8379-fold resistant to actinomycin D) are
>338 fold (ca. 8379 fold) resistant to Taxol.RTM., VP-16,
Vinblastine and Actinomycin D but <20 fold resistant to
epothilone compounds.
[0518] In general, these results are similar to those for CCRF-CEM
cells.
13TABLE 4 Three Drug Combination Analysis (Based on the Mutually
Exclusive Assumotion - Classical IsoboloRram Method) Drug A:
EPOTHILONE B (#8) (.mu.M) Drug B: Taxol .RTM. (.mu.M) Drug C:
VINBLASTINE (.mu.M) Conditions: CCRF-CEM, 3 DRUG COMBINATION, RATIO
(A:B:C: 1:5:1); EPOTHILONE + Taxol .RTM. + VINBLASTINE; EXPOSURE
TIME 72 HRS; XTT ASSAY. Combination Index* Values at: Parameters
Drug ED50 ED75 ED90 ED95 Dm (IC.sub.50) (.mu.M) m r A -00061
1.71561 .98327 B -00109 2.14723 .98845 C -00061 1.76186 .9919 A + B
1.51545 1.38631 1.27199 1.20162 -00146 2.41547 .97168 B + C 1.43243
1.33032 1.23834 1.18091 .00138 2.35755 .95695 A + C .74395 .68314
.62734 .59204 .00045 2.0098 .96232 A + B + 1.37365 1.32001 1.27285
1.24412 .00122 2.11202 .93639 C VBL .fwdarw. microtubule
depolymerization Taxol .RTM. .fwdarw. microtubule polymerization
Epo-B.fwdarw. microtubule polymerization Epothilone B and Taxol
.RTM. have a similar mechanism of action (polymerization) but
Epothilone B synergizes VBL whereas Taxol .RTM. antagonizes VBL.
Taxol .RTM. + VBL .fwdarw. Antagonism EpoB + Taxol .RTM. .fwdarw.
Antagonism EpoB + VBL .fwdarw. Synergism EpoB + Taxol .RTM. + VBL
.fwdarw. Antagonism *Combination index values <1, = 1, and >1
indicate synergism, additive effect, and antagonism,
respectively.
[0519]
14TABLE 5 Relative cytotoxicity of epothilone compounds in vitro.
IC.sub.50 in .mu.M Compounds CCRF-CEM CCRF-CEM/VLB CCRF-CEM/VM-1
VINBLASTINE ****0.0008 0.44 0.00049 0.0006 (0.00063 0.221 (0.332
0.00039 (0.00041 0.0005 + 0.00008) 0.336 = 0.063 0.00036 = 0.00004)
(52.7X).sup..sctn. (0.7X) VP-16 0.259 6.02 35.05 0.323 (0.293 9.20
(10.33 42.24 (34.39 0.296 .+-. 0.019) 15.76 .+-. 2.87) 25.89 .+-.
4.73) (35.3X) (117.4X) Taxol .RTM. ***0.0021 4.14 0.0066 #17 *0.090
0.254 #18 1157.6 >>1 #19 0.959 >>1 #20 *0.030 0.049 #21
-- -- #22 *0.098 0.146 #23 -- -- #24 ***0.0078 0.053 #25 *0.021
0.077 #26 *0.055 0.197 #27 0.0010 0.0072 Epothilone A (Syn) 0.0021
0.015 Epothilone B (Syn) 0.00042 0.0017 *Number of asterisks
denotes relative potency. .sup..sctn.Number in parentheses
indicates relative resistance (fold) when compared with parent cell
line.
[0520]
15TABLE 6 Relative potency of epothilone compounds in vitro.
IC.sub.50 in .mu.M Compounds CCRF-CEM CCRF-CEM/VBL CCRF-CEM/VM-1
Desoxy Epo. A 1 *0.022 0.012 0.013 2 14.23 6.28 43.93 3 271.7 22.38
11.59 4 2.119 43.01 2.76 5 >20 35.19 98.04 Trans- A 6 0.052
0.035 0.111 7 7.36 9.82 9.65 Syn-Epo.-B 8 ****0.00082 0.0029 0.0044
Natural B 9 ****0.00044 0.0026 0.0018 Desoxy Epo. B 10 ***0.0095
0.017 0.014 Trans. Epo. B 11 *0.090 0.262 0.094 12 0.794 >5
>5 13 11.53 5.63 14.46 8-desmethyl 14 5.42 5.75 6.29 desoxy-Epo
8-desmethyl 15 0.96 5.95 2.55 Mix-cis Epo 8-desmethyl 15 0.439 2.47
0.764 .beta.-Epo 8-demethyl 16 7.47 16.48 0.976 .alpha.-Epo
EPOTHILONE A ***0.0024 (0.0027 0.0211 (0.020 0.006 (0.00613 {close
oversize brace} (Natural) 0.0031 .+-. 0.0003) 0.0189 .+-. 0.001)
0.00625) .+-.0.0001) (7.4X) (2.3X) EPOTHILONE B ****0.00017 0.0017
(7.0X) 0.00077 (Natural) EPOTHILONE B 0.00055 0.0031 (0.00213
0.0018 (0.00126 (Synthetic) EPOTHILONE B (0.00035 .+-.0.00055)
.+-.0.0003) (Synthetic, larger .+-.0.0003) 0.0021 (6.1X) 0.0012
(3.6X) quantity synthesis) 0.00033 (25.9 mg)
[0521]
16TABLE 7 Relative cytotoxicity of epothilone compounds in vitro.
IC.sub.50 CEM CEM/VBL epothilone A 0.0029 .mu.M 0.0203 .mu.M
desoxyepothilone 0.022 0.012 2 14.2 6.28 3 271.7 22.4 4 2.1 43.8 5
>20 35.2 6 0.052 0.035 7 7.4 9.8 synthetic epothilone B 0.00082
0.00293 natural epothilone B 0.00044 0.00263 desoxyepothilone B
0.0095 0.0169 11 0.090 0.262 12 0.794 >5 13 11.53 5.63 14 5.42
5.75 15 0.439 2.47 16 7.47 16.48 17 0.090 0.254 18 1157.6 >>1
19 0.959 >>1 20 0.030 0.049 21 Not Available -- 22 0.098
0.146 23 Not Available -- 24 0.0078 0.053 25 0.0212 0.077 26 0.0545
0.197 27 0.0010 0.0072
[0522]
17TABLE 8 Chemotherapeutic Effect of Epothilone B, Taxol .RTM.
& Vinblastine in CB-17 Scid Mice Bearing Human CCRF-CEM and
CCRF-CEM/VBL Xenograft.sup.1 Average weight change Average tumor
volume Tumor Drug.sup.2 Dose Day 0 Day 7 Day 12 Day 17 Day 22 Day 7
Day 12 Day 17 Day 22 CCRF-CEM 0 24.4 +0.2 +0.4 +0.1 +0.5 1.0.sup.3
1.00 1.00 1.00 Epo B 0.7.sup.4 24.7 -0.1 -0.7 -1.4 +0.3 1.0 0.53
0.48 0.46 1.0.sup.5 25.0 +0.1 -1.5 -2.4 0.1 1.0 0.46 0.35 0.43
Taxol .RTM. 2.0 25.1 -0.1 -1.1 -1.5 -0.3 1.0 0.39 0.29 0.28 4.0
25.1 -0.2 -1.7 -1.9 -0.3 1.0 0.37 0.23 0.19 VBL 0.2 25.9 +0.2 -0.8
-1.5 -0.3 1.0 0.45 0.25 0.29 0.4 25.0 -0.1 -1.4 -1.8 -0.7 1.0 0.31
0.27 0.30 CCRF-CEM 0 26.3 -0.3 +0.1 -0.3 +0.4 1.0 1.00 1.00 1.00
/VBL Epo B 0.7 25.8 +0.1 -0.7 -1.0 -0.2 1.0 0.32 0.40 0.33
1.0.sup.6 26.0 -0.2 -1.3 -2.1 -0.5 1.0 0.41 0.27 0.31 Taxol .RTM.
2.0 26.1 0 -0.9 -1.5 -0.1 1.0 0.60 0.58 0.70 4.0 26.0 0 -1.4 -1.6
-0.9 1.0 0.79 0.55 0.41 VBL 0.2 25.9 -0.3 -0.8 -1.4 -0.3 1.0 0.86
0.66 0.67 0.4 25.9 0 -1.2 -1.8 -0.5 1.0 1.02 0.57 0.62
.sup.1CCRF-CEM and CCRF-CEM/VBL tumor tissue 50 .mu.l/mouse
implanted S.C. on day 0, Treatments i.p., QD on day 7, 8, 9, 10, 14
and 15. There were seven CB-17 scid male mice in each dose group
and control. .sup.2Epo B, epothilone B; VBL, vinblastine. .sup.3The
tumor volumes for each group on day 7 was about 1 mm.sup.3. The
average volumes of CCRF-CEM control group on day 12, 17 and 22 were
19, 76 and 171 mm.sup.3, and of CCRF-CEMNBL control group were 35,
107 and 278 mm.sup.3, respectively. .sup.4Two mice died of drug
toxicity on day 19 & 20. .sup.5Three mice died of drug toxicity
on day 18, 19 and 21. .sup.6One mouse died of drug toxicity on day
17.
[0523] In summary, epothilones and Taxol.RTM. have similar modes of
action by stabilizing polymerization of microtubules. However,
epothilones and Taxol.RTM. have distinct novel chemical
structures.
[0524] MDR cells are 1500-fold more resistant to Taxol.RTM.
(CCRF-CEM/VBL cells), epothilone A showed only 8-fold resistance
and epothilone B showed only 5-fold resistance. For CCRF-CEM cells,
Epo B is 6-fold more potent than Epo A and 10-fold more potent than
Taxol.RTM.. Desoxyepothilone B and compd #24 are only 3-4-fold less
potent than Taxol.RTM. and compound #27 is >2-fold more potent
than Taxol.RTM.. Finally, Taxol.RTM. and vinblastine showed
antagonism against CCRF-CEM tumor cells, whereas the combination of
Epo B+vinblastine showed synergism.
[0525] Relative Cytotoxicity of Epothilones against Human Leukemic
Cells in Vitro is in the order as follows:
[0526] CCRF-CEM Leukemic Cells
[0527] EpoB (IC.sub.50=0.00035 .mu.M; Rel. Value=1)>VBL(0.00063;
1/1.8)>#27(0.0010; 1/2.9)>Taxol.RTM. (0.0021; 1/6) >Epo A
(0.0027; 117.7)>#24(0.0078; 1/22.3)>#10 (0.0095;
1/27.1)>#25 (0.021; 1/60)>#1 (0.022; 1/62.8)>#20 (0.030;
1/85.7)>#6 (0.052; 1/149)>#26 0.055; 1/157)>#17 (0.090;
1/257)>VP-16 (0.29; 1/8.29)>#15 (0.44; 1/1257)>#19 (0.96;
1/2943)
[0528] CCRF-CEM/VBL MDR Leukemic Cells
[0529] Epo B (0.0021; 1/6* [1]**)>#27 (0.0072; 1/20.6)>#1
(0.012; 1/34.3)>#10 (0.017; 1/48.6)>Epo A (0.020; 1/57.1
[1/9.5])>#6 (0.035)>#20 (0.049)>#24 (0.053)>#25
(0.077)>#22 (0.146)>#26 (0.197)>#17 (0.254)>#11
(0.262)>VBL (0.332; 1/948.6 [1/158.1])>Taxol.RTM. (4.14;
1/11828 [1/1971.4])>VP-16 (10.33; 1/29514 [1/4919])
[0530] *Potency in parentheses is relative to Epo B in CCRF-CEM
cells.
[0531] **Potency in square brackets is relative to Epo B in
CCRF-CEM/VBL MDR cells.
[0532] As shown in Table 9, treatment of MX-1 xenograft-bearing
nude mice with desoxyepothilone B (35mg/kg, 0/10 lethality),
Taxol.RTM. (5 mg/kg, 2/10 lethality; 10 mg/kg, 2/6 lethality) and
adriamycin (2 mg/kg, 1/10 lethality; 3 mg/kg, 4/6 lethality) every
other day, i.p. beginning day 8 for 5 doses resulted in a far
better therapeutic effect for desoxyepothilone B at 35 mg/kg than
for Taxol.RTM. at 5 mg/kg and adrimycin at 2 mg/kg with tumor
volume reduction of 98%, 53% and 28%, respectively. For the
desoxyepothilone B-treated group, 3 out of 10 mice were found with
tumor non-detectable on day 18. (See FIG. 46)
[0533] Extended treatment with desoxyepothilone B (40 mg/kg, i.p.)
beginning day 18 every other day for 5 more doses resulted in 5 out
of 10 mice with tumor disappearing on day 28 (or day 31). See Table
10. By contrast, the extended treatment with Taxol.RTM. at 5 mg/kg
for five more doses resulted in continuted tumor growth at a
moderate rate, and 2 out of 10 mice died of toxicity.
[0534] Toxicity studies with daily i.p. doses of desoxyepothilone B
(25 mg/kg, a very effective therapeutic dose as indicated in
earlier experiments) for 4 days to six mice resulted in no
reduction in average body weight. (Table 13; FIG. 47) By contrast,
epothilone B (0.6 mg/kg, i.p.) for 4 days to eight mice resulted in
33% reduction in average body weight; all eight mice died of
toxicity between day 5 and day 7.
[0535] As evident from Table 15, desoxyepothilone B performs
significantly better than Taxol.RTM., vinblastine, adriamycin and
camptothecin against MDR tumor xenografts (human mammary
adeoncarcinoma MCF-7/Adr xenografts). This drug-resistant tumor
grows very aggressively and is refractory to Taxol.RTM. and
adriamycin at half their lethal doses. Taxol.RTM. at 6 mg/kg i.p.
Q2Dx5 reduced tumor size only 10% while adriamycin resulted in only
a 22% reduction on day 17. Whereas, desoxyepothilone B at 35 mg/kg
reduced tumor size by 66% on day 17 and yet showed no reduction in
body weight or apparent toxicity. Even at the LD.sub.50 dosage for
Taxol.RTM. (12 mg/kg) or adriamycin (3 mg/kg), desoxyepothilone B
still performed more effectively. By comparison, camptothecin at
1.5 and 3.0 mg/kg reduced tumor size by 28% and 57%, respectively.
Overall, in comparison with the four important anticancer drugs in
current use, i.e., Taxol.RTM., adriamycin, vinblastine and
camptothecin, desoxyepothilone B showed superior chemotherapeutic
effect against MDR xenografts.
[0536] In vivo therapeutic results in nude mice for dEpoB and
Taxol.RTM. are reported in Tables 19-21. As shown in the Tables, 6
hr i.v. infusion via tail vein provided a good therapeutic profile
with remarkably low toxicity.
[0537] For mammary MX-1 xenograft (non-MDR), desoxyepothilone B was
as effective as Taxol.RTM.. Both drugs were administered by 6 hr
i.v. infusion and both achieved full cure.
[0538] For the MDR-mammary MCF-7/Adr xenograft, the therapeutic
effect of desoxyepothilone B was far better than Taxol.RTM.,
although Q2Dx5 did not achieve a cure.
[0539] For CCRF-CEM/Taxol.RTM. (57-fold resistant to Taxol.RTM. in
vitro, in-house developed cell line), Taxol.RTM. did not show
significant therapeutic effect in this nude mice xenograft whereas
desoxyepothilone B achieved a full cure.
[0540] Prolonged (6 hr.) i.v. infusion allowed higher doses (e.g.,
30 mg/kg, Q2Dx5) to be administered (without lethality) than i.v.
bolus injection of desoxyepothilone B, and yet reduced drug
toxicity.
[0541] Accordingly, the present inventors have found
desoxyepothilone B to have excellent properties for therapeutic
application as an MDR agent and moreover as a general anticancer
agent.
18TABLE 9 Therapeutic Effect of Desoxyepothilone B, Taxol .RTM.,
and Adriamycin in Nude Mice Bearing Human MX-1 Xenografta Average
Body Weight Change Average Tumor Volume Tumor Dose (g) (T/C)
Disappear- #Mice Drug (mg/kg) Day 8 10 12 14 16 18 Day 10 12 14 16
18 ance Died Control 0 24.6 -0.1 +1.0 +1.0 +1.3 +1.8 1.00 1.00 1.00
1.00 1.00 0/10 0/10 Desoxyepothilone B 35 23.0 -0.1 +0.7 -0.3 -1.7
-1.6 0.42 0.28 0.07 0.04 0.02 0/10 3/10 Taxol .RTM. 5 24.0 -1.3
-0.8 -1.4 -1.9 -1.8 0.58 0.36 0.34 0.42 0.47 2/10 0/10 10 24.3 -1.0
-1.0 -2.3 -3.5 -3.8 0.85 0.40 0.21 0.20 0.12 2/6 1/6 Adriamycin
2.sup.b 23.9 +0.3 0 -1.4 -1.9 -2.0 0.94 0.88 1.05 0.69 0.72 1/10
0/10 3.sup.c 22.4 +1.3 -0.2 -1.5 -2.1 -2.3 0.72 0.54 0.56 0.51 0.36
4/6 0/6 .sup.aMX-1 tissue 100 .mu.l/mouse was implanted s.c on day
0. Every other day i.p. treatments were given on day 8, 10, 12, 14
and 16. The average tumor volume of control group on day 10, 12,
14, 16 and 18 were 78, 151, 372, 739 and 1257 mm.sup.3,
respectively. .sup.bOne mouse died of toxicity on day 22.
.sup.cFour mice died of toxicity on day 24.
[0542]
19TABLE 10 Extended Experiment of Desoxyepotbilone B, Taxol .RTM.,
Cisplatin and Cyclophosphamide in Nude Mice Bearing Human MX-1
Xenograft.sup.a Average Body Tumor Average Tumor Dose Weight Change
(g) Disappearance Disappearance Drug (mg/kg) Day 8 20 22 24 26 28
Day 20 22 24 26 28 Duration (Day) #Died Desoxyepo B 40 23.0 -1.7
-2.4 -2.4 -1.4 -1.2 .sup.11 2/10.sup.b 2/10 3/10 5/10 5/10 44(5/10)
0/10 Taxol .RTM. 5 24.0 -1.6 -0.3 +0.1 -0.6 -0.4 0/10 0/10 0/10
0/10 0/10 2/10 10 No extended test 1/6 on day 16 Reappeared on 2/6
day 38 .sup.aExtended experiment was carried out after 5 times
injection (on day 8, 10, 12, 14 and 16). Every other day i.p.
treatments were given continuously: Desoxyepothilone B and Taxol
.RTM. on day 18, 20, 22, 24 and 26; control group mice were
sacrificed. .sup.bOne of the mice tumor reappeared on day 20.
[0543]
20TABLE 11 Toxicity of Epothilone B and Desoxyepothilone B in
normal nude mice. Dose and Group Schedule Number Duration (mg/kg)
of mice Died Disappearance Control 4 0 Epothilone B.sup.a 0.6 QD
.times. 4 8 8 Desoxyepothilone B 25 QD .times. 4 6 0 .sup.aMice
died of toxicity on day 5, 6, 6, 7, 7, 7, 7, 7
[0544]
21TABLE 12 Therapeutic Effect of Epothilone B, Desoxyepothilone B
and Taxol .RTM. in B6D2F.sub.1 Mice Bearing B16 Melanom.sup.a
Average Weight Average Tumor Dose Change (g) Volume (T/C) #Mice
Drug (mg/kg) Day 0 3 5 7 9 11 Day 5 7 9 11 Died Control 0 26.5 -02
0 -0.2 0 +1.0 1.00 1.00 1.00 1.00 0/15 Epothilone B 0.4 QDx6.sup.b
27.1 -0.2 -0.6 -1.1 -3.4 -3.9 1.08 1.07 1.27 1.07 1/8 0.8
QDx5.sup.c 27.0 0 -0.8 -3.1 -4.7 -4.7 0.57 0.89 0.46 0.21 5/8
Desoxyepothilone B 10 QDx8 27.0 -0.7 -0.9 -1.1 -1.5 -0.3 0.23 0.22
0.51 0.28 0/6 20 QD1-4,7-8 26.9 -1.3 -2.2 -1.3 -1.6 -0.8 0.59 0.63
0.58 0.33 0/6 Taxol .RTM. 4 QDx8 26.7 +0.1 +0.2 +0.3 +0.4 +0.8 0.62
0.39 0.56 0.51 0/8 6.5 QDxB 26.7 +0.1 +0.3 +0.3 +0.4 +1.7 0.19 0.43
0.20 0.54 0/8 .sup.aB16 melanoma cells 1.2 .times. 10.sup.6/mouse
was implanted S.C. on day 0. Daily treatments start on day 1 after
inoculation. Number of mice in each group: Control, 15; Epothilone
B, 8; Desoxythilone B, 5 and Taxol .RTM., 8. The average tumor
volume of control group on day 5, 7, 9 and 11 were 16, 138, 436 and
1207 mm.sup.3, respectively. See FIGS. 44(a) and (b). .sup.bOne
mouse died of toxicity on day 10. .sup.cFive mice died of toxicity
on day 8, 10, 10, 11, 12. One moribund mouse was sacrificed for
toxicological examinations on day 11.
[0545]
22TABLE 13 Therapeutic Effect of Desoxyepothilone B, Epothilone B,
Taxol .RTM.and Vinblastine in Nude Mice Bearing Human MX-1
Xenograft.sup.a. Average Body Average Tumor Dose Weight Change (g)
Volume (T/C) Drug (mg/kg) Day 7 11 13 15 17 Day 11 13 15 17 Note
Control 27.9 +0.8 +1.1 +1.9 0.6 1.00 1.00 1.00 1.00 0/8 died
Desoxyepothilone B 15 27.1 +0.8 +1.1 +1.6 +1.5 0.65 0.46 0.49 0.41
0/6 died .sup. 25.sup.b 27.0 +0.4 +0.7 +1.0 +0.7 0.38 0.11 0.05
0.04 0/6 died (1/6 cured on day 35) Epothilone B 0.3 26.9 +0.5 +0.4
-0.3 -1.2 1.00 0.71 0.71 0.84 0/7 died .sup. 0.6.sup.c 27.4 -0.3
-1.3 -2.1 -2.1 1.08 0.73 0.81 0.74 3/7 died Taxol .RTM. 5 26.9 -0.1
+0.4 +1.1 +1.2 0.54 0.46 0.40 0.45 0/7 died .sup. 10.sup.d 27.6
--2.7 -1.1 -0.3 +2.2 0.43 0.37 0.12 0.11 4/7 died Vinblastine 0.2
25.7 +0.6 +1.4 +2.3 +2.9 0.65 0.54 0.56 0.88 0/7 died .sup.
0.4.sup.c 26.4 +0.8 +0.5 +1.9 +2.1 0.80 0.56 0.83 0.88 1/7 died
.sup.aMX-1 tissue 50 .mu.l/mouse was implanted s.c. on day 0. Every
other day i.p. treatments were given on day 7, 9, 11, 13 and 15.
Number of mice in each group: Control, 8; Desoxyepothilone B, 6;
Epothilone B, 7; Taxol .RTM., 7 and Vinblastine, 7. The average
tumor volume of control group on day 11, 13, 15 and 17 were 386,
915, 1390 and 1903 mm.sup.3, respectively. See FIG. 45. .sup.bOne
out of six mice with no detectable tumor on day 35. .sup.cThree
mice died of drug toxicity on day 17. Every other day i.p.
treatments were given except day 15. .sup.dFour mice died of drug
toxicity on day 13, 13, 13, 15. .sup.eOne mouse died of drug
toxicity on day 15.
[0546]
23TABLE 14 Toxicity of Hematology and Chemistry of Desoxyepothilone
B, and Taxol .RTM. in Nude Mice Bearing Human MX-1 Xenograft.sup.a
Hematology.sup.b Chemistry.sup.b WBC Dose Total Neutrophils Lymph
RBC PLT GOT GPT Drug (mg/kg ip) (10.sup.3/mm.sup.3) (%) (%)
(10.sup.3/mm.sup.3) (10.sup.6/mm.sup.3) (U/L) (U/L) Control 12.9 38
61 8.1 800 (n = 4) 203 45 (n = 4) Desoxyepo- 25 and 35.sup.c 11.8
48 48 8.4 700 (n = 6) 296 55 (n = 3) thilone B Taxol .RTM. 5 and
6.sup.d 10.9 51 48 6.1 1083 (n = 5) 438 79 (n = 5) Normal
range.sup.c 6.91.about.12.9 8.25.about.40.8 62.about.90
10.2.about.12.0 190.about.340 260 138.7 .sup.aMinced MX-1 tumor
tissue 50 .mu.l/mouse was implanted s.c. on day 0. .sup.bAll assays
were determined on day 30; averaged values were given.
.sup.cDesoxyepothilone B 25 mg/kg was given i.p on day 7, 9, 11,
13, 15; 35 mg/kg on day 17, 19, 23, 24, 25. .sup.dTaxol .RTM.5mg/kg
was given i.p. on day 7, 9, 11, 13, 15; 6 mg/kg on day 17, 19, 23,
24, 25. .sup.eNormal ranges are for wiid type deer mice and
C.sub.3/HeJ mice (obtained from clinical, biochemical and
hematological Reference values in Normal Experimental Animals,
Brtjm Mitruka, ed., Masson Publishing USA, Inc., N.Y., 1977, and
from Clinical Chemistry of Laboratory Animals, Weter F. Loeb, ed.,
Pergamon Press, 1989)
[0547]
24TABLE 15 Therapeutic Effect of Desoxyepothilone B, Taxol .RTM.,
Adriamycin, and Camptothecin in Nude Mice Bearing MDR Human
MCF-7/Adr Tumor. Average Body Average Tumor Dose Weight Change (g)
Volume (T/C) Drug (mg/kg) Day 8 11 13 15 17 Day 11 13 15 17 Died
Control 0 25.0 +2.0 +2.6 +3.1 +3.7 1.00 1.00 1.00 1.00 0/8
DesoxyEpoB 35 25.0 +0.3 +0.7 +0.6 +0.8 0.31 0.27 0.30 0.34 0/8
Taxol .RTM. 6 25.3 +1.7 +1.8 +0.8 +0.9 0.57 0.66 0.85 0.90 0/8 12
24.5 +0.7 -1.3 -2.4 0 0.50 0.51 0.32 0.40 3/6 Adriamycin 1.8 25.6
+0.2 -0.4 -0.6 -0.4 0.70 0.68 0.84 0.78 0/8 3 24.6 +0.5 -1.5 -3.2
-1.6 0.66 0.83 0.57 0.53 3/6 Camptothecin 1.5 24.4 +1.1 +0.9 +1.7
+1.4 1.08 0.72 0.61 0.72 0/8 3.0 24.5 -0.6 -0.4 -0.8 -0.9 0.95 0.76
0.61 0.43 0/6 .sup.aMCF-7/Adr cell 3 .times. 10.sup.6/mouse was
implanted s.c. on day 0. Every other day i.p. treatments were given
on day 8, 10, 12, 14 and 16. The average tumor volume of control
group on day 11, 13, 15 and 17 were 392, 919, 1499 and 2372
mm.sup.3, respectively.
[0548]
25TABLE 16 Extended Experiment of Desoxyepothilone B, Taxol .RTM.
in Nude Mice Bearing Human MX-1 Xenograft.sup.a Average Body Tumor
Average Tumor Dose Weight Change (g) Disappearance Disappearance
Drug (mg/kg) Day 8 20 22 24 26 28 Day 20 22 24 26 28 Duration (Day)
Died Desoxyepo B 40 23.0 -1.7 -2.4 -2.4 -1.4 -1.2 .sup. 2.10.sup.b
2/10 3/10 5/10 5/10 44(5/10) 0/10 Taxol .RTM. 5 24.0 -1.6 -0.3 +0.1
-0.6 -0.4 0/10 0/10 0/10 0/10 0/10 2/10 10 No Extended Test 1/6 on
day 16, Reappear on day 38 2/6.sub.(0/6) .sup.aExtended experiment
was going on after 5 times injection (on day 8, 10, 12, 14 and 16).
Every other day i.p. treatments were given continuously:
Desoxyepothilone B and Taxol .RTM. on day 18, 20, 22, 24 and 26;
Control group mice were sacrificed. .sup.bIn one of the mice, a
tumor reappeared on day 20.
[0549] As evident from table 16, extended treatment of nude mice
bearing human MX-1 xenografts with desoxyepothilone B results in
complete tumor disappearance, with no mortality in any test
animals. In conclusion, treatment with desoxyepothilone B shows
remarkable specificity with respect to tumor toxicity, but very low
normal cell toxicity.
26TABLE 17 Therapeutic Effects of Desoxyepothilone B, Taxol .RTM.
in Nude Mice Bearing MX-1 Xenograft. Treatment Schedule # Died of
toxicity CONTROL 8 10 12 14 16 18 20 0/10 or Size 19 78 151 372 739
1257 1991 Sacrificed (n = 10) .+-.2 .+-.8 .+-.15 .+-.55 .+-.123
.+-.184 .+-.331 DESOXYEPOTHILONE B edule 35 mg/kg on day 40 mg/kg
on day No Treatment 8 10 12 14 16 18 20 22 24 26 28 30 45 47 50 60
0/10 nor Size use 1 15 15 40 40 15 32 30 30 30 30 0 0 0 24 S* --
use 2 23 23 15 15 15 15 30 48 48 0 30 48 900 1200 S -- use 3 15 60
90 105 105 126 96 150 180 0 48 64 600 600 S -- use 4 21 38 38 0 0
10 8 8 8 8 0 0 0 0 0 0 use 5 12 23 50 12 0 4 0 0 0 0 0 0 0 0 0 0
use 6 15 40 32 8 8 8 8 12 12 12 12 30 120 120 S -- use 7 21 30 15
15 8 8 8 8 8 8 8 8 180 280 S -- use 8 20 48 70 15 15 8 8 0 0 0 0 0
0 8 S -- use 9 25 50 40 15 8 0 0 0 0 0 0 0 0 0 4 4 use 10 20 38 38
38 38 25 25 25 0 0 15 15 100 100 S -- Taxol .RTM. se hedule 5 mg/kg
on day 5 mg/kg on day 8 10 12 14 16 18 20 22 24 26 28 30 45 47 50
60 2/10 mor Size 17 45 54 128 311 596 1114 1930 2285 S (n = 10)
.+-.2 .+-.7 .+-.13 .+-.42 .+-.115 .+-.151 .+-.346 .+-.569 .+-.597
Extended studies .fwdarw. Extended observations .fwdarw. Experiment
ended *S: Sacrificed due to tumor burden
[0550]
27TABLE 18 Toxicity of Epothilone B and Desoxyepothilone B in
normal nude mice Dose and Schedule Group (mg/kg) Number of mice
Died Control 4 0 Epothilone B.sup.a 0.6 QD .times. 4 8 8
Desoxyepothilone B 25 QD .times. 4 6 0 .sup.aMice died of toxicity
on day, 5, 6, 6, 7, 7, 7, 7, 7
[0551]
28TABLE 19 Therapuetic effects of desoxyepothilone B (dEpo B) and
Taxol .RTM. in nude mice bearing MX-1 xenograft.sup.a Route.sup.b
Average Body Average Tumor Dose i.v. Weight (g) Size (T/C) Tumor
(mg/kg) infusion Day 8 14 16 18 20 Day 14 16 18 20 Disappearance
rol 0 30.2 +0.8 +1.7 +2.6 +3.2 1.00 1.00 1.00 1.00 0/5 B 30 Q2Dx5
30.3 -2.7 -4.0 -4.5 -6.8 0.19 0.10 0.03 TD.sup.c 3/3 l .RTM. 15
Q2Dx5 30.8 0 -1.1 -1.6 -1.4 0.06 0.01 TD TD 4/4 24 Q2Dx5 28.5 -4.8
-5.3 -6.0 -6.2 0.03 TD TD TD 4/4 MX-1 human mammary carcinoma
tissue 50 .mu.g was implanted s.c. into mice on day 0. Every other
day i.v. infusion were given on day 8, 10, 12, 14 and 16. The
average tumor volume of control group on day 14, 16, 18 and 20 were
170 .+-. 10, 246 .+-. 29, 345 .+-. 42 and 529 .+-. 65 mm.sup.3
(mean "SEM), respectively. The i.v. infusion was for 6 hrs. The
vehicle used was in 100 Fl (Cremophor + EtOH = 1:1) + 4 ml saline.
TD: Tumor disapperance
[0552]
29TABLE 20 Therapuetic effects of desoxyepoB (dEpo B) and Taxol
.RTM. in nude mice bearing MCF-7 xenograft.sup.a Route.sup.b
Average Body Average Tumor Dose i.v. Weight (g) Size (T/C) Toxicity
(mg/kg) infusion Day 8 14 16 18 Day 14 16 18 death rol 0 30.2 +0.8
+1.7 +2.6 1.00 1.00 1.00 0/5 B 30 Q2Dx5 30.3 -2.7 -4.0 -4.5 0.16***
0.15 0.13*** 0/3 l .RTM. 15 Q2Dx5 30.8 0 -1.1 -1.6 0.81 0.89 0.76
0/4 24 Q2Dx5 28.5 -4.8 -5.3 -6.0 0.73 0.71* 0.73* 0/4 MCF-7/Adr
human mammary carcinoma tissue 50 .mu.g was implanted s.c. into
mice on day 0. Every other day i.v. infusion were given on day 8,
10, 12, 14 and 16. The average tumor volume of control group on day
14, 16, and 18 were 1281 .+-. 145, 1767 .+-. 161, and 2381 .+-. 203
mm.sup.3 (mean .+-. SEM), respectively. The i.v. infusion n was for
6 hrs. The vehicle used was in 100 .mu.l (Cremophor + EtOH = 1:1) +
4 ml saline. TD: Tumor disapperance
[0553] In further tests, desoxyepothilone B showed similar
anticancer efficacy as Taxol.RTM. in regular human tumor xenographs
in nude mice, represented in Table 21. However, in drug-resistant
tumors, desoxyepothilone B is by far better cancer therapeutic
agent when compared with Taxol.RTM. in all tumors tested.
Desoxyepothilone B is not only superior to Taxol.RTM. in many
respects but is also a better therapeutic agent than epothilone B,
camptothecin, vinblastine, adriamycin or VP-16 (etoposide) against
many other tumors. See Table 21.
[0554] Stability of Desoxyepothilone B in Plasma
[0555] As shown in FIG. 75, desoxyepothilone B is surprisingly and
unexpectedly significantly more stable in human serum plasma than
in mouse plasma. Desoxyepothilone B is relatively unstable in mouse
plasma with a short half-life of about 15 or 20 min, but is very
stable in human plasma with a half-life of about 75 hours. It is
therefore likely that desoxyepothilone B can be particularly
favorable in treating patients in view of the long lasting effects
and the absence of a need to use prolonged i.v. infusions.
30TABLE 20A Therapuetic effects of desoxyepoB (dEpo B) and Taxol
.RTM. in nude mice bearing human ovarian UL3-C tumor..sup.a Average
Body Average Tumor Dose Weight (g) Size (T/C) Drug (mg/kg) Day 19
27 29 31 33 Day 27 29 31 33 Control 0 30.9 +0.9 +0.8 +0.4 +0.1 1.00
1.00 1.00 1.00 dEpo B 30 30.0 -1.5 -4.0 -3.6 -2.7 0.19 0.13 0.03
0.03 Taxol .RTM. 20 31.3 -2.9 -3.3 -2.4 -2.7 0.10 0.08 0.03 0.04
.sup.aUL3-C tissue (50 mg) was implanted s.c. into mice on day 0.
I.V. infusions were given on day 19, 21, 23, 25 and 27. The average
tumor volume of the control group on day 27, 29, 31 and 33 were
349, 378, 488 and 66 mm.sup.3, respectively.
[0556]
31TABLE 21 Comparison Between dEpoB and Taxol .RTM. Chemotherapy in
Nude Mice Lowest Average Therapeutic Effects Doses Tumor Size Tumor
dEpoB vs Tumor Drugs Schedule (T/C) Disappearance Taxol .RTM. A549
dEpoB 40 mg/kg Q2D .times. 3 0.01 1/3 .congruent. Lung Taxol .RTM.
15 mg/kg Q2D .times. 3 0.01 2/4 MX-1 dEpoB 30 mg/kg Q2D .times. 6 0
5/5 .congruent. Taxol .RTM. 15 mg/kg Q2D .times. 6 0 5/5 HT-29
dEpoB 30 mg/kg Q2D .times. 6 0.02 0 .ltoreq. Colon Taxol .RTM. 15
mg/kg Q2D .times. 6 0.01 0 UL3-C dEpoB 30 mg/kg Q2D .times. 5 0.03
0 .gtoreq. Ovary Taxol .RTM. 15 mg/kg Q2D .times. 5 0.04 0 PC-3
dEpoB 40 mg/kg Q2D .times. 3 0.12 0 << Prostate Taxol .RTM.
15 mg/kg Q2D .times. 3 0.02 0 SK-OV-3 dEpoB 30 mg/kg Q2D .times. 6
0.17 0 << Ovary Taxol .RTM. 15 mg/kg Q2D .times. 6 0.02 1/4
MCF-7/Adr dEpoB 30 mg/kg Q2D .times. 5 0.11 0 >> Breast Taxol
.RTM. 24 mg/kg Q2D .times. 5 0.71 0 CCRF/Taxol dEpoB 30 mg/kg Q2D
.times. 6 0 3/3 >>> Leukemia Taxol .RTM. 15 mg/kg Q2D
.times. 6 1.09 0 CCRF/VBL dEpoB 30 mg/kg Q2D .times. 5 0 2/2
>>> Leukemia Taxol .RTM. 15 mg/kg Q2D .times. 5 0.76 0
CCRF/CEM dEpoB 30 mg/kg Q2D .times. 5 0 2/2 .congruent. Leukemia
Taxol .RTM. 15 mg/kg Q2D .times. 5 0 2/2
[0557]
32TABLE 22 Comparison of Cancer Chemotherapeutic Effects Route of
Therapeutic effect Tumor Administation Rank Order B16 Melanoma i.p.
dEpoB > Taxol .RTM. >> EpoB MX-1 i.p. dEpoB >
Camptothecin > Taxol .RTM. > VBL, EpoB MX-1 i.p. dEpoB >
Adriamycin > Taxol .RTM. SK-OV-3 i.v./i.p. Taxol
.RTM..apprxeq.dEpoB PC-3 i.v./i.p. Taxol .RTM. > dEpoB >>
Adriamycin MCF-7/Adr i.p. dEpoB >> Camptothecin >
Adriamycin > Taxol .RTM. MX-1 i.v. infusion Taxol
.RTM..apprxeq.dEpoB MCF-7/Adr i.v. infusion dEpoB >> VP-16,
Taxol .RTM. > VBL, Adriamycin CCRF-CEM i.v. infusion
dEpoB.apprxeq.Taxol .RTM. CCRF-CEM/Taxol .RTM. i.v. infusion dEpoB
>>> Taxol .RTM. CCRF-CEM/VBL i.v. infusion dEpoB
>>> Taxol .RTM. SK-OV-3 i.v. infusion Taxol .RTM. >
dEpoB HT-29 i.v. infusion Taxol .RTM. .gtoreq. dEpoB A549 i.v.
infusion Taxol .RTM., dEpoB, VBL > VP-16 PC-3 i.v. infusion
Taxol .RTM., VBL .gtoreq. dEpoB >> VP-16
[0558]
33TABLE 23 Therapeutic Effects of DesoxyepoB and Taxol .RTM. in
Nude Mice Bearing Human Lymphoblastic Leukemia CCRF-CEM.sup.a.
Average Body Weight Changes (g) Drug Dose Day 21 23 25 27 29 31 33
35 37 39 41 Control 0 29.0 +0.1 -1.1 -0.2 +0.8 +0.1 End End End End
End DesoxyepoB 40 26.6 -1.4 -3.6 -3.9 -5.2 -4.2 -3.1 -2.3 -1.2 -0.2
+0.9 Taxol .RTM. 20 29.0 -0.1 -1.9 -2.9 -3.0 -3.6 -2.6 -0.5 +1.2
+1.3 +2.1 Average Tumor Size (T/C) Proportion of Tumor
Disappearance (n/total) Drug Dose Day 21 23 25 27 29 31 33 35 37 39
41 Control 0 1.00 1.00 1.00 1.00 1.00 End End End End End End 0/5
0/5 0/5 0/5 0/5 DesoxyepoB 40 1.10 0.73 0.31 0.09 0 0 0 0 ND.sup.b
ND ND 0/3 0/3 0/3 0/3 3/3 3/3 3/3 3/3 1/3 1/3 0/3 Taxol .RTM. 20
1.17 0.78 0.44 0.11 0 0 0 0 ND ND ND 0/4 0/4 0/4 1/4 4/4 4/4 4/4
1/4 0/4 0/4 0.4 .sup.aCCRF-CEM tissue (50 .mu.g) was implanted s.c.
into mice on day 0. I.V. infusion 6 hr were given on day 21, 23, 25
and 27. The average tumor volumes of the control group on day 23,
25, 27, 29 and 31 were 376 .+-. 100 427 .+-. 130 685 .+-. 144, 850
.+-. 155, and 1062 .+-. 165 mm.sup.3 (mean .+-. SEM), respectively.
.sup.bTumor reappeared. "End": Test ended because of tumor
burden.
[0559]
34TABLE 24 Therapeutic Effects of dEpoB, Taxol .RTM., VBL and VP-16
in Nude Mice Bearing Human Lung Carcinoma A549 Dose i.v. Average
Body Average Tumor (mg/kg) infusion Weight (g) Size (mm.sup.3)
Tumor Drugs Q2D .times. 3 (hrs) Day 7 11 13 15 Day 11 13 15 17
Disappearance Control 0 6 26.4 +1.3 +0.6 +0.2 96 128 306 553 0/4
dEpoB 30 18 29.3 -2.0 -5.8 -5.6 17 21 26 21 1/3 40 6 27.8 -1.6 -3.2
-2.4 31 19 4 4 1/3 50 6 27.1 -2.0 -3.2 -2.5 33 19 6 6 1/2 Taxol
.RTM. 15 6 27.6 -1.2 -0.8 +0.5 49 32 7 6 2/4 24 6 28.5 -2.1 -2.7
-0.9 14 8 0 0 4/4 VBL 2 i.v. inj 25.5 +13.4 +2.3 +3.2 11 11 11 7
2/3 VP-16 25 i.v. inj 27.2 +0.1 -0.2 -2.2 59 150 275 536 0/3
[0560]
35TABLE 25 Therapeutic Effects of dEpoB, Taxol .RTM., VBL and VP-16
in Nude Mice Bearing Human Prostate Adenocarcinoma PC-3 i.v.
Average Body Weight (g) Average Tumor Size (TC) Dose infusion Day
Day Tumor Drugs (mg/kg) (hrs) 5 9 11 13 9 11 13 Disappearance
Control 0 6 26.5 +1.4 +0.5 +0.1 1.00 1.00 1.00 0/5 dEpoB 30 18 29.4
-2.2 -5.9 -5.8 0.17 0.06 0.06 0/3 40 6 28.3 -1.7 -3.4 -2.7 0.40
0.19 0.12 0/3 50 6 28.1 -2.1 -3.0 -2.6 0.37 0.13 0.03 0/3 Taxol
.RTM. 16 6 26.6 -1.1 -0.9 +0.4 0.34 0.09 0.02 0/4 24 6 28.8 -2.2
-2.8 -0.8 0.18 0.05 0.06 2/4 VBL 2 i.v. inj 23.5 +1.4 +2.1 +3.0
0.37 0.13 0.03 1/3 VP-16 25 i.v. inj 24.9 +0.3 -0.1 -2.4 1.00 1.12
0.88 0/3 PC-3 tissue (50 .mu.g) was implanted s.c into mice on day
0. I.V. infusions were given on day 5, 7 and 9. The average tumor
volumes of control group subjects on 9, 11 day were 766 .+-. 50,
1457 .+-. 180 and 2441 .+-. 221 mm.sup.3 (mean .+-. SEM),
respectively.
[0561]
36TABLE 26 Therapeutic Effects of DesoxyepoB and Taxol .RTM. in
Nude Mice Bearing Human Lymphoblastic Leukemia CCRF-CEM. Average
Body Weight Change (g) Average Tumor Size (T/C) Drug Dose Day Day
Tumor Q2D .times. 4 21 23 25 27 29 31 23 25 27 29 31 Disappearance
Control 0 29.0 +0.1 -1.1 -0.2 +0.8 +0.1 1.00 1.00 1.00 1.00 1.00
0/5 DesoxyepoB 40 26.6 -1.4 -3.6 -3.9 -5.2 -4.2 0.68 0.30 0.09 0 0
3/3 Taxol 20 29.0 -0.1 -1.9 -2.9 -3.0 -3.6 0.78 0.44 0.11 0 0 4/4
CCRF-CEM tissue (50 .mu.g) was implanted s.c into mice on day 0.
I.V. infusion 6 hr were given on day 21, 23, 25 and 27. The average
tumor volume of the control group day 23, 25, 27, 29 and 31 were
376 .+-. 100 427 .+-. 130, 685 .+-. 144, 850 .+-. 155, and 1062
.+-. 165 mm.sup.3 (mean .+-. SEM), respectively.
[0562]
37TABLE 27 Comparison of Therapeutic Effects and Toxicity of dEpoB
and Paclitaxel with Different Solvents, Routes and Schedules of
Administration Using a Non-MDR MX-1 Xenograft.sup.a Therapeutic
Effect against MX-1 Xenograft.sup.d Toxicity toward Nude Mice.sup.e
Route Solvent.sup.b Dose (mg/kg).sup.c dEpoB Paclitaxel DepoB
Paclitaxel i.p. DMSO 35 Q2D .times. 5 + + + + + + + + + + + + + +
Cremophor 15 Q2D .times. 5 + + + + + + + + + + + + --EtOH i.v. DMSO
15 Q2D .times. 5 + + + + + + + + + + + injection 24 Q2D .times. 5 +
+ + + + + + + + + + i.v. Cremophor infusion --EtOH 6 hr 30 Q2D
.times. 5 + + + + + + + + + + + + + + 24 hr 60 Q4D .times. 2 + + +
+ + ND.sup.f + + + ND.sup.f .sup.aApproximate relative scale: +,
marginal; + +, little; + + +, moderate; + + + +, substantial; + + +
+ +, marked. .sup.bDMDO: dimethylsulfoxide; Cremophor:EtOH (1:1).
.sup.cThe dose of dEpoB. .sup.dBased on tumor volume reduction when
compared with the untreated control. .sup.eBased on body weight
decrease or lethality. .sup.fNot done.
* * * * *