The present invention provides convergent processes for preparing epothilone A and B, desoxyepothilones A and B, and analogues thereof. Also provided are analogues related to epothilone A and B and intermediates useful for preparing same. The present invention further provides novel compositions based on analogues of the epothilones and methods for the treatment of cancer and cancer which has developed a multidrug-resistant phenotype.
|
7. A purified compound having the structure:
##STR00068##
wherein R1 is H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independently H, phenyl, benzyl, linear or branched chain alkyl,
wherein R1 is other than H, methyl, ethyl, n-propyl, n-hexyl, CH2OH, (CH2)3OH, or
##STR00069##
59. A method for treating cancer in a subject comprising:
administering to a subject a therapeutically effective amount of a compound having the structure:
##STR00094##
wherein R1 is H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independently H, phenyl, benzyl, linear or branched chain alkyl,
said method optionally further comprising administering a cytotoxic agent.
30. A pharmaceutical composition comprising:
a compound having the structure:
##STR00081##
wherein R1 is H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independently H, phenyl, benzyl, linear or branched chain alkyl,
wherein R1 is other than H, methyl, ethyl, n-propyl, n-hexyl, CH2OH, (CH2)3OH, or
##STR00082##
and
a pharmaceutically acceptable carrier,
said composition optionally further comprising a cytotoxic agent.
or a compound having the structure:
##STR00065##
wherein R1, R2, and R3 are each independently H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independendy H, phenyl, benzyl, linear or branched chain alkyl;
R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, wherein X is H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, and Y is H or linear or branched chain alkyl;
Z is O, N(OR6) or N—NR7R8, wherein R6, R7 and R8 are independently H or a linear or branched chain alkyl or alkoxy; and
n is 0, 1, 2, or 3; wherein when the compound is of the formula:
##STR00066##
R1 is other than H, methyl, ethyl, n-propyl, n-hexyl, CH2OH, (CH2)3OH, or
##STR00067##
53. A method of treating cancer in a subject comprising:
administering to the subject a therapeutically effective amount of a compound having the structure:
##STR00092##
or a compound having the structure:
##STR00093##
wherein R1, R2, and R3 are each independently H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independently H, phenyl, benzyl, linear or branched chain alkyl;
R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, wherein X is H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, and Y is H or linear or branched chain alkyl;
Z is O, N(OR6) or N—NR7R8, wherein R6, R7 and R8 are independently H or a linear or branched chain alkyl or alkoxy; and
n is 0, 1, 2, or 3,
said method optionally further comprising administering a cytotoxic agent.
24. A pharmaceutical composition comprising:
a compound having a structure:
##STR00077##
or a compound having a structure:
##STR00078##
wherein R1, R2, and R3 are each independantly H, or linear or branched chain alkyl, which alkyl may be singly or multiply substituted by hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted carboxy, carboxaldehyde, substituted or unsubstituted, linear or branched alkyl, substituted or unsubstituted cyclic acetal, fluorine, NR4R5, N-hydroximino, or N-alkoxyimino, wherein R4 and R5 are independently H, phenyl, benzyl, linear or branched chain alkyl;
R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, wherein X is H, linear or branched chain alkyl, phenyl, or 2-methyl-1,3-thiazol-4-yl, and Y is H or linear or branched chain alkyl;
Z is O, N(OR6) or N—NR7R8, wherein R6, R7 and R8 are independently H or a linear or branched chain alkyl or alkoxy; and
n is 0, 1, 2, or 3; and
wherein when the compound is of the formula:
##STR00079##
R1 is other than H, methyl, ethyl, n-propyl, n-hexyl, CH2OH, (CH2)3OH, or
##STR00080##
and
a pharmaceutically acceptable carrier,
said composition optionally further comprising a cytotoxic agent.
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|||||||||||||||||||||||||||
This application is a continuation application filed under 37 C.F.R. §1.53(b) of application Ser. No. 08/986,025, filed Dec. 3, 1997 now U.S. Pat. No. 6,242,469, the entire contents of which are hereby incorporated by reference, which is based on U.S. Provisional Application Serial 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.
This invention was made with government support under grants CA-28824, CA-39821, CA-GM 72231, CA-62948, and AI0-9355 from the National Institutes of Health, and grant CHE-9504805 from the National Science Foundation. Additionally, the present invention was supported in part by a fellowship from the United States Army to Dongfang Meng (DAMD 17-97-1-7146), and thus the government has certain rights in the invention.
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.
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.
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 Höfle. G. Höfle et al., Angew. Chem. Int. Ed. Engl., 1996, 35, 1567. The total synthesis of the epothilones is an important goal for several reasons. Taxol 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, 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 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 (L. M. Landino and T. L. MacDonald in The Chemistry and Pharmacology of Taxol 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.
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, vinblastin, adriamycin and camptothecin.
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.
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.
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.
FIG. 1(A) shows a retrosynthetic analysis for epothilone A and B.
FIG. 1(B) provides synthesis of compound 11. (a) t-BuMe2OTf, 2,6-lutidine, CH2Cl2, 98%; (b) (1) DDQ, CH2Cl2/H2O, 89%; (2) (COCl)2, DMSO, CH2Cl2, −78° C.; then Et3N, −78° C.→rt, 90%; (c) MeOCH2PPh3Cl, t-BuOK, THF, 0° C.→rt, 86%; (d) (1) p-TsOH, dioxane/H2O, 50° C., 99%; (2) CH3PPh3Br, NaHMDS, PhCH3, 0° C.→rt, 76%; (e) Phl(OCOCF3)2, MeOH/THF, rt, 0.25 h, 92%.
FIGS. 3(A) and 3(B) provide syntheses of key iodinated intermediates used to prepare hydroxymethylene- and hydroxypropylene-substituted epothilone derivatives.
FIGS. 3(C) and 3(D) provide 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.
FIGS. 3(E) and 3(F) show reactions leading to benzoylated hydroxymethyl-substituted desoxyepothilone and hydroxymethylene-substituted epothilone (epoxide).
FIG. 4(A) provides synthesis of compound 19. (a) DHP, PPTS, CHCH2Cl2, rt: (b) (1) Me3SiCCLi, BF3.OEt2, THF, −78° C.; (2) MOMCl, I-Pr2NEt, Cl(CH2)2Cl, 55° C.; (3) PPTS, MeOH, rt; (c) (1) (COCl)2, DMSO, CH2Cl2, −78° C.; then Et3N, −78° C.→rt; (2) MeMgBr, Et2O, 0° C.→rt, (3) TRAP, NMO, 4 Å mol. sieves, CH2Cl2, 0° C.→rt,; (d) 16, n-BuLi, THF, −78° C.; then 15, THF, −78° C.→rt; (e) (1) N-iodosuccinimide, AgNO3, (CH3)2CO; (2) Cy2BH, Et2O, AcOH; (f) (1) PhSH, BF3.OEt2, CH2Cl2, rt; (2) Ac2O, pyridine, 4-DMAP, CH2Cl2, rt.
FIG. 4(B) presents synthesis of compound 1. (a) 11, 9-BBN, THF, rt; then PdCl2(dppf)2, Cs2CO3, Ph3As, H2O, DMF, 19, rt, 71%; (b) p-TsOH, dioxane/H2O, 50° C.; (c) KHMDS, THF, −78° C., 51%; (d) (1) HF-pyridine, pyridine, THF, rt, 97%; (2) t-BuMe2 SiOTf, 2,6-lutidine, CH2Cl2, −25° C., 93%; (3) Dess-Martin periodinane, CH2Cl2, 87%; (4) HF-pyridine, THF, rt, 99%; (e) dimethyidioxirane, CH2Cl2, 0.5 h, −50° C., 45% (≧20: 1).
FIGS. 6(A) and 6(B) provide a scheme of an olefin metathesis route to epothilone A and other analogues.
FIGS. 14(A) and 14(B) show the preparation of intermediate 4A.
FIGS. 18(A) and 18(B) provide a synthetic pathway to a protected intermediate for 8-desmethyl deoxyepothilone A.
FIGS. 19(A), 19(B), and 19(C) provide a synthetic pathway to 8-desmethyl deoxyepothilone A and a transiodoolefin intermediate thereto.
FIG. 20(A) shows structures of epothilones A and B and 8-desmethylepothilone and FIG. 20(B) shows a synthetic pathway to intermediate TBS ester 10 used in the preparation of desmethylepothilone A. (a) (Z)-Crotyl-B[(−)-1pc]2, −78° C., Et2O, then 3 N NaOH, 30% H2O2; (b) TBSOTf, 2,6-lutidine, CH2Cl2 (74% for two steps, 87% ee); (c) O3, CH2Cl2/MeOH, −78° C., then DMS, (82%); (d) t-butyl isobutyrylacetate, NaH, BuLi, 0° C., then 6 (60%, 10:1); (e) Me4NBH(OAc)3, −10° C. (50%, 10:1 α/β) or NaBH4, MeOH, THF, 0° C., (88%, 1:1 α/β); (f) TBSOTf, 2,6-lutidine, −40° C., (88%); (g) Dess-Martin periodinane, (90%); (h) Pd(OH)2, H2, EtOH (96%); (1) DMSO, oxalyl chloride; CH2Cl2, −78° C. (78%); (0) Methyl triphenylphosphonium bromide, NaHMDS, THF, 0° C. (85%); (k) TBSOTf, 2,6-lutidine, CH2Cl2, rt (87%).
FIGS. 22(A), 22(B) and 22(C) show a synthetic pathway to prepare epothilone analogue 27D.
FIGS. 23(A), 23(B) and 23(C) show a synthetic pathway to prepare epothilone analogue 24D.
FIGS. 24(A) and 24(B) show a synthetic pathway to prepare epothilone analogue 19D.
FIGS. 25(A), 25(B), 25(C) and 25(D) show a synthetic pathway to prepare epothilone analogue 20D.
FIGS. 26(A), 26(B), 26(C) and 26(D) show a synthetic pathway to prepare epothilone analogue 22D.
FIGS. 27(A), 27(B) and 27(C) show a synthetic pathway to prepare epothilone analogue 12-hydroxy ethyl epothilone.
FIGS. 28(A) and 28(B) show 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. 39(A) and 39(B) show 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.
FIGS. 40(A) and 40(B) show 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.
FIGS. 41(A) and 41(B) show 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.
FIGS. 42(C) and 42(D) show epothilone analogues #35-46. Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEMNVBL MDR (resistant) sublines are shown in round and square brackets, respectively.
FIGS. 42(E) shows epothilone analogues #47-49.
FIG. 43(A) shows antitumor activity of desoxyepothilone B against MDR MCF-7/Adr xenograft in comparison with taxol. Control (♦); desoxyepothilone B (▪; 35 mg/kg); taxol (▴; 6 mg/kg); adriamycin (X;1.8 mg/kg); i.p. Q2Dx5; start on day 8.
FIG. 43(B) shows antitumor activity of epothilone B against MDR MCF-7/Adr xenograft in comparison with taxol. Control (♦); epothilone B (▪; 25 mg/kg; non-toxic dose); taxol (▴; 6 mg/kg; half LD50); adriamycin (X;1.8 mg/kg); i.p. Q2Dx5; start on day 8.
FIG. 44(A) shows toxicity of desoxyepothilone B in B6D2F, mice bearing B16 melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10 and 12 days. Control (▴); desoxyepothilone B (o; 10 mg/kg QDx8; 0 of 8 died); desoxyepothilone B (●; 20 mg/kg QDx6; 0 of 8 died). Injections were started on day 1.
FIG. 44(B) shows toxicity of epothilone B in B6D2F, mice bearing B16 melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10 and 12 days. Control (▴); epothilone B (o; 0.4 mg/kg QDx6; 1 of 8 died of toxicity); epothilone B (●; 0.8 mg/kg QDx5; 5 of 8 died). Injections were started on day 1.
FIG. 45(A) shows comparative therapeutic effect of desoxyepothilone B and taxol on nude mice bearing MX-1 xenoplant. Tumor, s.c; drug administered i.p., Q2Dx5, start on day 7. control (♦); Taxol (□; 5 mglkg, one half of LD50); desoxyepothilone B (Δ; 25 mg/kg; nontoxic dose).
FIG. 45(B) shows comparative therapeutic effect of desoxyepothilone B and taxol on nude mice bearing MX-1 xenoplant. Tumor, s.c.; drug administered i.p., Q2Dx5, start on day 7. control (♦); Taxol (□; 5 mg/kg, one half of LD50, given on days 7, 9, 11, 13, 15; then 6 mg/kg, given on days 17, 19, 23, 24, 25); desoxyepothilone B (n-3; Δ, 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).
As used herein, the term “linear or branched chain alkyld” 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.
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.
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.
The subject invention provides chemotherapeutic analogues of epothilone A and B, including a compound having the structure:
##STR00001##
wherein R, R0, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR1R2, N-hydroximino, or N-alkoxyimino, wherein R1 and R2 are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═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(OR3) or N—NR4R5, wherein R3, R4 and R5 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:
##STR00002##
wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH2OH, or (CH2)3OH.
The invention also provides a compound having the structure:
##STR00003##
wherein R, R0, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR1R2, N-hydroximino, or N-alkoxyimino, wherein R1 and R2 are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —OTY═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 6indolyl; wherein Y is H or linear or branched chain alkyl; wherein Z is O, N(OR3) or N—NR4R5 wherein R3, R4 and R5 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:
##STR00004##
wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl or CH2OH.
In addition, the invention provides a compound having the structure:
##STR00005##
wherein R, R0, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR1R2, N-hydroximino, or N-alkoxyimino, wherein R1 and R2 are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is -CHY═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(OR3) or N—NR4R5, wherein R3, R4 and R5 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:
##STR00006##
wherein R is H, methyl, ethyl, n-propyl, n-butyl, CH2OH or (CH2)3OH.
The invention further provides a compound having the structure:
##STR00007##
wherein R, R5 and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR1R2, N-hydroximino or N-alkoxyimino, wherein R1 and R2 are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═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(OR3) or N—NR4R5, wherein R3, R4 and R5 are independently H or a linear or branched chain alkyl; and wherein n is 0, 1, 2 or 3.
The invention also provides a compound having the structure: ##STR00008##
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:
##STR00009##
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,
##STR00010##
CH2OTBS or (CH2)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.
The subject invention also provides an intermediate having the structure:
##STR00011##
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)2, (SR)2, —(O—(CH2)n—O)—, —(O—(CH2)n—S)— or —(S—(CH2)n—S)—; and wherein n is 2, 3 or 4.
##STR00012##
wherein R is H or methyl.
Another analogue provided by the invention has the structure:
##STR00013##
wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH2OH,
For CCRF-CEM/VBL, the relative potency ordering is:
Desoxy Epothilone A≧Epothilone A>>Taxol>Triol Analog>Model System I
For CCRF-CEM/VM, the relative potency ordering is:
Taxol=Epothilone A>Desoxy Epothilone A>>Model System I>Triol Analog
It is concluded that CCRF-CEM/VM cells are collaterally sensitive to certain epothilone compounds.
TABLE 3
Relative Efficacy of Epothilone Compounds Against
The DC-3F Hamster Lung Cell Growth and Against
DC-3F MDR Sublines Resistant Actinomylin D
IC50 in μM
COMPOUNDS
DC-3F
DC-3F/ADH
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
0.01061
0.0198
0.042
EPOTHILONE [1]
TAXOL
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
(0.005816 nm)
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).
The relative potency of the compounds are as follows:
DC-3F: Actinomycin D>Vinblastine≧Epothilone A (0.0036 μM)>Desoxy epothilone>VP-16>Taxol (0.09 μM)>Model system I and triol analog
DC-3F/ADX: Desoxyepothilone≧Epothilone A (0.06 μM)>Actinomycin D>Model system I>Vinblastine>triol analog>viablastine>taxol (32.0 μM)
DC-3F/ADX cells (8379-fold resistant to actinomycin D) are >338 fold (ca. 8379 fold) resistant to Taxol, VP-16, Vinblastine and Actinomycin D but <20 fold resistant to epothilone compounds.
In general, these results are similar to those for CCRF-CEM cells.
TABLE 4
Three Drug Combination Analysis
(Based on the Mutually Exclusive Assumption -
Classical Isobologram Method)
Drug A: EPOTHILONE B
(#8) (μM)
Drug B: TAXOL (μM)
Drug C: VINBLASTINE
(μM)
Conditions:
CCRF-CEM, 3 DRUG COMBINATION,
RATIO (A:B:C: 1:5:1); EPOTHILONE +
TAXOL + VINBLASTINE; EXPOSURE
TIME 72 HRS; XTT ASSAY.
Combination Index* Values at:
Dm
(IC50)
Parameters
Drug
ED50
ED75
ED90
ED95
(μM)
m
r
A
−00061
1.71561
.98327
B
−00109
2.14723
.98845
C
−00061
1.76186
.9919
A +
1.51545
1.38631
1.27199
1.20162
−00146
2.41547
.97168
B
B +
1.43243
1.33032
1.23834
1.18091
.00138
2.35755
.95695
C
A +
.74395
.68314
.62734
.59204
.00045
2.0098
.96232
C
A +
1.37365
1.32001
1.27285
1.24412
.00122
2.11202
.93639
B +
C
VBL → microtubule depolymerization
Taxol → microtubule
polymerization
Epo-B → microtubule
polymerization
Epothilone B and Taxol have a similar mechanism of action
(polymerization) but Epothilone B synergizes VBL whereas Taxol
antagonizes VBL.
Taxol + VBL → Antagonism
EpoB + Taxol → Antagonism
EpoB + VBL → Synergism
EpoB + Taxol + VBL → Antagonism
*Combination index values <1, =1, and >1 indicate synergism, additive effect, and antagonism, respectively.
TABLE 5
Relative cytotoxicity of epothilone compounds in vitro.
IC50 in μM
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
(52.7X)§
0.00036
0.00004)
(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)
(35.3X)
25.89
4.73)
(117.4X)
TAXOL
***
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.
§Number in parentheses indicates relative resistance (fold) when compared with parent cell line.
TABLE 6
Relative potency of epothilone compounds in vitro.
IC50 in μM
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
β-Epo
8-demethyl
16
7.47
16.48
0.967
α-Epo
EPOTHILONE A
***
0.0024
(0.0027 ±
0.0211
(0.020 ±
0.006
(0.00613 ±
(Natural)
0.0031
0.0003)
0.0189
0.001)
(7.4X)
0.00625
0.0001) (2.3X)
EPOTHILONE B
****
0.00017
0.0017
(7.0X)
0.00077
(Natural)
EPOTHILONE B
(Synthetic)
0.00055
0.0031
(0.00213 ±
0.0018
(0.00126 ±
EPOTHILONE B
(0.00035 ±
0.00055)
0.0003)
(Synthetic, larger
0.0003)
quantity synthesis)
0.0021
(6.1X)
0.0012
(3.6X)
(25.9 mg)
0.00033
TABLE 7
Relative cytotoxicity of epothilone compounds in vitro.
IC50
CEM
CEM/VBL
epothilone A
0.0029
μM
0.0203
μM
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
TABLE 8
Chemotherapeutic Effect of Epothilone B, Taxol & Vinblastine in
CB-17 Scid Mice Bearing Human CCRF-CEM and CCRF-CEM/VBL Xenograft1
Average weight change
Average tumor volume
Tumor
Drug2
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
1.03
1.00
1.00
1.00
Epo B
0.74
24.7
−0.1
−0.7
−1.4
+0.3
1.0
0.53
0.48
0.46
1.05
25.0
+0.1
−1.5
−2.4
+0.1
1.0
0.46
0.35
0.43
Taxol
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
EpoB
0.7
25.8
+0.1
−0.7
−1.0
−0.2
1.0
0.32
0.40
0.33
1.08
26.0
−0.2
−1.3
−2.1
−0.5
1.0
0.41
0.27
0.31
Taxol
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
1CCRF-CEM and CCRF-CEM/VBL tumor tissue 50 ul/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.
2Epo B, epothilone B; VBL, vinblastine.
3The tumor volumes for each group on day 7 was about 1 mm3. The average volumes of CCRF-CEM control group on day 12, 17 and 22 were 19, 76 and 171 mm3, and of CCRF-CEM/VBL control group were 35, 107 and 278 mm3, respectively.
4Two mice died of drug toxicity on day 19 & 20.
5Three mice died of drug toxicity on day 18, 19 and 21.
6One mouse died of drug toxicity on day 17.
In summary, epothilones and taxol have similar modes of action by stabilizing polymerization of microtubules. However, epothilones and taxol have distinct novel chemical structures.
MDR cells are 1500-fold more resistant to taxol (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. Desoxyepothilone B and compd #24 are only 3-4-fold less potent than Taxol and compound #27 is >2-fold more potent than Taxol. Finally, Taxol and vinblastine showed antagonism against CCRF-CEM tumor cells, whereas the combination of Epo B+vinblastine showed synergism.
Relative Cytotoxicity of Epothilones against Human Leukemic Cells in Vitro is in the order as follows:
CCRF-CEM Leukemic Cells
Epo B (IC50=0.00035 μM; Rel. Value=1)>VBL (0.00063;1/1.8)>#27(0.0010;1/2.9)>Taxol (0.0021; 1/6)>Epo A (0.0027; 1/7.7)>#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)>#(0.96; 1/2943)
CCRF-CEM/VBL MDR Leukemic Cells
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)x#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 (4.14; 1/11828 [1/1971.4])>VP-16 (10.33; 1/29514 [1/4919]) *Potency in parentheses is relative to Epo B in CCRF-CEM cells. **Potency in square brackets is relative to Epo B in CCRF-CEM/VBL MDR cells.
As shown in Table 9, treatment of MX-1 xenograft-bearing nude mice with desoxyepothilone B (35 mg/kg, 0/10 lethality), taxol (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 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
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 at 5 mg/kg for five more doses resulted in continued tumor growth at a moderate rate, and 2 out of 10 mice died of toxicity.
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;
TABLE 9
Therapeutic Effect of Desoxyepothilone B, Taxol, and Adriamycin in Nude Mice Bearing Human MX-1 Xenografta
Average Body Weight Change
Average Tumor Volume
Tumor
Dose
(g)
(T/C)
Disappear-
Drug
(mg/kg)
Day 8
10
12
14
16
18
Day 10
12
14
16
18
Died
ance
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
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
2b
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
3c
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
aMX-1 tissue 100 μl/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 mm3, respectively.
bOne mouse died of toxicity on day 22.
cFour mice died of toxicity on day 24.
TABLE 10
Extended Experiment of Desoxyepothilone B, Taxol, Cisplatin and Cyclophophamide in Nude Mice Bearing Human MX-1 Xenografta
Average Body Weight Change
Average Tumor
Dose
(g)
Tumor 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
2/10b
2/10
3/10
5/10
5/10
44(5/10)
0/10
Taxol
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
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 on day 18, 20, 22, 24 and 26; control group mice were sacrificed.
bOne of the mice tumor reappeared on day 20.
TABLE 11
Toxicity of Epothilone B and
Desoxyepothilone B in normal nude mice.
Dose and
Number
Dis-
Schedule
of
appear-
Group
(mg/kg)
mice
Died
ance
Duration
Control
4
0
Epothilone Ba
0.6 QD × 4
8
8
Desoxyepothilone B
25 QD × 4
6
0
aMice died of toxicity on day 5, 6, 6, 7, 7, 7, 7, 7
TABLE 12
Therapeutic Effect of Epothilone B, Desoxyepothilone B and Taxol in B6D2F, Mice Bearing B16 Melanomaa
Average Weight Change
Average Tumor Volume
Dose
(g)
(T/C)
# Mice
Drug
(mg/kg)
Day 0
3
5
7
9
11
Day 5
7
9
11
Died
Control
0
26.5
−0.2
0
−0.2
0
+1.0
1.00
1.00
1.00
1.00
0/15
Epothilone B
0.4 QD × 6b
27.1
−0.2
−0.6
−1.1
−3.4
−3.9
1.08
1.07
1.27
1.07
1/8
0.8 QD × 5c
27.0
0
−0.8
−3.1
−4.7
−4.7
0.57
0.89
0.46
0.21
5/8
Desoxyepothilone B
10 QD × 8
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
4 QD × 8
26.7
+0.1
+0.2
+0.3
+0.4
+0.8
0.62
0.39
0.56
0.51
0/8
6.5 QD × 8
26.7
+0.1
+0.3
+0.3
+0.4
+1.7
0.19
0.43
0.20
0.54
0/8
aB16 melanoma cell 1.2 × 106/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, 8. The average tumor volume of control group on day 5, 7, 9 and 11 were 16, 138, 436 and 1207 mm3, respectively. See FIGS. 44(a) and (b).
bOne mouse died of toxicity on day 10.
cFive mice died of toxicity on day 8, 10, 10, 11, 12. One moribund mouse was sacrificed for toxicological examinations on day 11.
TABLE 13
Therapeutic Effect of Desoxyepothilone B,
Epothilone B, Taxol, and Vinblastine in Nude Mice Bearing Human MX-1 Xenografta
Average Body Weight Change
Average Tumor Volume
Dose
(g)
(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
25b
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
0.6c
27.4
−0.3
−1.3
−2.1
−2.1
1.08
0.73
0.81
0.74
3/7 died
Taxol
5
26.9
−0.1
+0.4
+1.1
+1.2
0.54
0.46
0.40
0.45
0/7 died
10d
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
0.4c
26.4
+0.8
+0.5
+1.9
+2.1
0.80
0.56
0.83
0.88
1/7 died
aMX-1 tissue 50 μl/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, 7 and Vinblastine, 7. The average tumor volume of control group on day 11, 13, 15 and 17 were 386, 915, 1390 and 1903 mm3, respectively See
bOne out of six mice with no detectable tumor on day 35.
cThree mice died of drug toxicity on day 17. Every other day i.p. treatments were given except day 15.
dFour mice died of drug toxicity on day 13, 13, 13, 15.
eOne mouse died of toxicity on day 15.
TABLE 14
Toxicity of Hematology and
Chemistry of Desoxyepothilone B, and Taxol in Nude Mice Bearing Human MX-1 Xenografta
Hematologyb
WBC
Chemistryb
Dose
Total
Neutrophils
Lymph
RBC
PLT
GOT
GPT
Drug
(mg/kg ip)
(103/mm3)
(%)
(%)
(103/mm3)
(103/mm3)
(U/L)
(U/L)
Control
12.9
38
61
8.1
800 (n = 4)
203
45 (n = 4)
Desoxyepo-
25 and 35c
11.8
48
48
8.4
700 (n = 6)
296
55 (n = 3)
thilone B
Taxol
5 and 6d
10.9
51
48
6.1
1083 (n = 5)
438
79 (n = 5)
Normal rangee
6.91˜12.9
8.25˜40.8
62˜90
10.2˜12.0
190˜340
260
138.7
aMinced MX-1 tumor tissue 50 μl/mouse was implanted s.c. on day 0.
bAll assays were determined on day 30; averaged values were given.
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.
dTaxol 5 mg/kg was given i.p. on day 7, 9, 11, 13, 15; 6 mg/kg on day 17, 19, 23, 24, 25.
eNormal ranges are for wild type deer mice and C3/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)
TABLE 15
Therapeutic Effect of Desoxyepothilone B, Taxol, Adriamycin, and Camptothecin in Nude Mice
Bearing MDR Human MCF-7/Adr Tumor.
Average Body Weight Change
Average Tumor Volume
Dose
(g)
(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
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
MCF-7/Adr cell 3 × 106/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 mm3, respectively.
As evident from Table 15, desoxyepothilone B performs significantly better than taxol, 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 and adriamycin at half their lethal doses. Taxol 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 LD50 dosage for taxol (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, adriamycin, vinblastine and camptothecin, desoxyepothilone B showed superior chemotherapeutic effect against MDR xenografts.
TABLE 16
Extended Experiment of Desoxyepothilone B, Taxol in Nude Mice Bearing Human MX-1 Xenografta
Average Body Weight Change
Average Tumor
Dose
(g)
Tumor Disappearance
Disappear
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
2.10b
2/10
3/10
5/10
5/10
44(5/10)
0/10
Taxol
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
2/6(0/6)
38
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 on day 18, 20, 22, 24 and 26; Control group mice were sacrificed.
bIn one of the mice, a tumor reappeared on day 20.
As evident form Table 16, extended treatment of nude mice bearing human MX-1 xenographs were 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.
TABLE 17
Therapeutic Effects of Desoxyepothilone B, Taxol in Nude Mice Bearing Human MX-1 Xenograft.
Treatment Schedule
# Died of toxicity
Control
Day
8
10
12
14
16
18
20
Tumor Size
19 ± 2
78 ± 8
151 ± 15
372 ± 55
739 ± 123
1257 ± 184
1991 ± 331
Sacrificed (n = 10)
0/10
(mm3)
DESOXYEPOTHILONE B
Dose Schedule
35 mg/kg on day
40 mg/kg on day
No Treatment
Day
8
10
12
14
16
18
20
22
24
26
28
30
45
47
50
60
Tumor Size
Mouse 1
15
15
40
40
15
32
30
30
30
30
0
0
0
24
S*
—
0/10
Mouse 2
23
23
15
15
15
15
30
48
48
0
30
48
900
1200
S
—
Mouse 3
15
60
90
105
105
126
96
150
180
0
48
64
600
600
S
—
Mouse 4
21
38
38
0
0
10
8
8
8
8
0
0
0
0
0
0
Mouse 5
12
23
50
12
0
4
0
0
0
0
0
0
0
0
0
0
Mouse 6
15
40
32
8
8
8
8
12
12
12
12
30
120
120
S
—
Mouse 7
21
30
15
15
8
8
8
8
8
8
8
8
180
280
S
—
Mouse 8
20
48
70
15
15
8
8
0
0
0
0
0
0
8
S
—
Mouse 9
25
50
40
15
8
0
0
0
0
0
0
0
0
0
4
4
Mouse 10
20
38
38
38
38
25
25
25
0
0
15
15
100
100
S
—
TAXOL
Dose Schedule
5 mg/kg on day
5 mg/kg on day
Day
8
10
12
14
16
18
20
22
24
26
28
30
45
47
50
60
Tumor Size
17 ±
45 ±
54
128 ±
311 ±
596 ±
1114 ±
1930 ±
2285 ±
S ±
(n = 10)
2/10
2
7
13
42
115
151
346
569
597
Extended studies →
Extended observations →
Experiment ended
*S: Sacrificed due to tumor burden
TABLE 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 Ba
0.6 QD × 4
8
8
Desoxyepothilone B
25 QD × 4
6
0
aMice died of toxicity on day 5, 6, 6, 7, 7, 7, 7, 7
Su, Dai-Shi, Chou, Ting-Chao, Danishefsky, Samuel J., Meng, Dongfang, Bertinato, Peter, Kamenecka, Ted, Balog, Aaron, Savin, Kenneth A., Sorensen, Erik J.
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