HK1089178A

HK1089178A - Synthesis of epothilones, intermediates thereto, analogues and uses thereof

Info

Publication number: HK1089178A
Application number: HK06111060.6A
Authority: HK
Inventors: 赛缪尔．J．达尼谢夫斯凯; 亚力克西．里夫金; 吉村文彦; 安娜．以斯贴．加瓦尔达．奥尔特加; 朝永新; 舒定朝; 董华金
Original assignee: 索隆-基特林癌症研究协会
Priority date: 2002-08-23
Filing date: 2003-08-22
Publication date: 2006-11-24

Abstract

The present invention provides compounds of formula (I): as described generally and in classes and subclasses herein. The present invention additionally provides pharmaceutical compositions comprising compounds of formula (I) and provides methods of treating cancer comprising administering a compound of formula (I).

Description

EPOTHILONEs (EPOTHILONEs), intermediates for the synthesis of EPOTHILONEs, analogues and uses thereof

Technical Field

The present invention provides compounds of formula (I): as generally described herein and described in classes and subclasses. The invention further provides pharmaceutical compositions comprising compounds of formula (I), and methods of treating cancer comprising administering a compound of formula (I).

Background

Epothilones (epothilones) a and B (2a and 2B, scheme 1) are naturally occurring cytotoxic macrolides that can be isolated from a cellulose degrading mycobacterium (mycobacterium) sorangium (H * fly et al, angle. chem., int. ed. engl. (1996, 35, 1567) and j.Antibilt. (1996, 49, 560), all of which are incorporated herein by reference. Although the structures of epothilones A and B are very different, they all have the same structure as paclitaxel (Taxol)^*) The same mechanism of action, which involves inhibiting the growth of tumor cells by polymerizing tubulin and stabilizing microtubule assembly (bolag et al, Cancer Res, (1995, 55, 2325), incorporated herein by reference). Although there is no question of Taxol^*It has clinical value as a first-line chemotherapeutic agent, but it is far from ideal. Their extremely low water solubility makes them dependent on formulation vehicles such as hydrogenated castor oil (Cremophore), which not only pose a risk in themselves but also cause regulatory problems (Essayan et al, j. allergyclin. immunol.1996, 97, 42; which is incorporated herein by reference). Furthermore, Taxol^*Susceptible to inactivation by multidrug resistance (MDR) (Giannakakou et al, J.biol.chem. (1997, 272, 17118); incorporated herein by reference). However, epothilones A and B have also been shown to retain significant anti-MDR tumor cell efficacy (Kowalski et al, mol. biol. cell (1995, 6, 2137); incorporated herein by reference). Furthermore, the increased water solubility of epothilones compared to paclitaxel facilitates formulation. Although the naturally occurring compound epothilone B (2B, EpoB in scheme 1) is an important member of the natural product epothilone family, it unfortunately has a narrow therapeutic index, at least in xenograft mice, which is a serious concern (Su et al, Angew. chem. int. ed. Engl.1997, 36, 1093; Harris et al, J.org. chem. (1999, 64, 8434); each of which is incorporated herein by reference).

1a R-ph paclitaxel (Taxol) 2a R₁＝H，R₂＝CH₃Epothilone A (EpoA)

1b R-t-Bu, docetaxel (Taxotere) 2b R₁＝CH₃，R₂＝CH₃Epothilone B (EpoB)

2c R₁＝H，R₂＝CH₂OH, epothilone E (EpoE)

2d R₁＝CH₃，R₂＝CH₂OH, epothilone F (EpoF)

Schematic diagram 1: taxanes (taxoids) and epothilones

The limited therapeutic index of EpoB has led to studies of the ability of other epothilone analogs, particularly 12, 13-desoxyepothilone, to provide improved therapeutic profiles (a)See alsoU.S. patent nos. 6,242,469, 6,284,781, 6,300,355, 6,369,234, 6,204,388, 6,316,630; each incorporated herein by reference). In vivo experiments performed on various mouse models demonstrated that 12, 13-deoxyepothilone B (3B, dEpoB in scheme 2) has therapeutic potential in mouse xenografts against a variety of sensitive and resistant human tumors (Chou et al, Proc. Natl. Acad. Sci. U.S. A.1998, 95, 9642 and 15798; which is incorporated herein by reference). More recently, the therapeutic advantages of these desoxyepothilones over other anticancer agents have been determined by a comprehensive comparison (Chou et al, Proc. Natl Acad. Sci. U.S.A. (2001, 98, 8113); incorporated herein by reference). Due to its unusual in vivo properties, dEpoB has been evaluated by toxicology in dogs, andis used as an anticancer medicine in human body tests.

3a R₁＝H，R₂＝CH₃Desoxyepothilone A (dEpoA) 4a R₁＝H，R₂＝CH₃dehydro-dEpoA (dEpoA) 5a R₁＝H，R₂＝CH₃iso-dEpoA

3b R₁＝CH₃，R₂＝CH₂Desoxyepothilone B (dEpoB) 4b R₁＝CH₃，R₂＝CH₃Dehydrogenation of dEpoB (dEpoB) 5b R₁＝CH₃，R₂＝CH₃iso-dEpoB

3c R₁＝H，R₂＝CH₂OH, desoxyepothilone E (dEpoE) 4c R₁＝H，R₂＝CH₂OH, deoxy-dEpoE (dEpoE) 5c R₁＝H，R₂＝CH₂OH, iso-dEpoE

3d R₁＝CH₃，R₂＝CH₂OH, desoxyepothilone F (dEpoF) 4d R₁＝CH₃，R₂＝CH₂OH, dehydro-dEpoF (dEpoF)5d R₁＝CH₃，R₃＝CH₂OH, iso-dEpoF (ddEpoF)

3e R₁＝H，R₂＝NH₂Demethylamino- (dEpoA) 4e R₁＝H，R₂＝NH₂demethylamino-ddEpoA 5e R₁＝H，R₂＝NH₂demethylamino-iso-dEpoA

3f R₁＝CH₃，R₂＝NH₂demethylamino-dEpoB (dadEpoB) 4f R₁＝CH₃，R₂＝NH₂demethylamino-ddEpoB 5f R₁＝CH₃，R₂＝NH₂demethylamino-iso-dEpoB

3g R₁＝CH₃，R₂＝CH₂26-fluoro-dEpoB- (dadEpoB) 4g R₁＝CH₃F，R₂＝CH₂26-fluoro-ddEpoB

3h R₁＝CF₃，R₂＝CH₂26 trifluoro-dEpoB- (dadEpoB) 4h R₁＝CF₃，R₂＝CH₂26-trifluoro-ddEpoB

Schematic diagram 2: various desoxyepothilone analogs

In view of the promising therapeutic utility of 12, 13-desoxyepothilones, it is desirable to investigate other analogs and other synthetic methods for synthesizing existing epothilones, desoxyepothilones and their analogs, and their novel analogs. More particularly, given the interest in the therapeutic utility of such compounds, it would also be desirable to develop methods that can provide large quantities of any of the epothilones or desoxyepothilones described above or herein for clinical testing and for large scale preparation.

Drawings

FIG. 1 is a table of the IC50 values for growth of epothilone against CCRF-CEM, CCRF-CEM/VBL and CCRF-CEM/paclitaxel cells. Cell growth inhibition was measured by XTT tetrazolium assay after 72 hours of cell growth culture as previously described (Scudiero et al, Cancer Res.46: 4827-4833, 1988; incorporated herein by reference). By using a computer program (Chou et al, adv. Enzyregul. (22: 27-55, 1984); Chou et al, CalcuSyn for Windows (Biosoft, Cambridge, UK), 1997); each incorporated herein by reference) IC50 values were determined from dose-potency relationships at six or seven concentrations of each drug, as previously described (Chou et al, proc. natl. acad. sci. usa (95: 15798-15802, 1998); incorporated herein by reference).

FIG. 2 is a drawing of trans-9, 10-dehydro-12, 13-deoxy EpoB¹H NMR spectrum.

FIG. 3 is a drawing of trans-9, 10-dehydro-12, 13-deoxy EpoB¹³C NMR spectrum.

FIG. 4 shows a schematic of the synthesis of 11-R and 14-R epothilones using LACDAC-ring closure olefin metathesis and illustrates some of the substitutions available via the synthesis of 9, 10-dehydroepothilone.

Figure 5 shows relative cytotoxicity data in vitro for various epothilone compounds and derivatives, including certain 9, 10-dehydro compounds (e.g., compound 7 in figure 5A and compounds 88 and 99 in figure 5B) against human leukemia cells.

FIG. 6 depicts an alternative synthetic method for the preparation of 9, 10-dehydroepothilone analogs. FIG. 6A shows a Macro-Stille method, sp³-sp³The coupling method and the beta-Suzuki method. FIG. 6B shows Julia olefin synthesis, Wadsworth-Emmons process, and Macro-Reformatosyk process. FIG. 6C depicts a McMurry coupling method and a lactam analog synthesis method.

Figure 7 shows various analogs of 9, 10-dehydro-12, 13-deoxyepob.

Figure 8 shows the therapeutic effect of 9, 10-dehydro-12, 13-deoxyEpoB and EpoB on nude mice bearing human breast cancer MX-1 xenografts (intravenous infusion, Q2D X3).

Figure 9 shows the stability of epothilone analogs in murine plasma. Epo1 is 12, 13-deoxy EpoB and Epo2 is 26-F₃-12, 13-deoxy EpoB, Epo3 being (E) -9, 10-dehydro-12, 13-deoxy EpoB and Epo4 being 26-F₃- (E) -9, 10-dehydro-12, 13-deoxy EpoB.

Figure 10 shows the therapeutic effect of epothilone analogs on nude mice bearing HCT-116 xenografts (intravenous infusion, Q2D × 7, n ═ 3). Arrows indicate drug administration. Epo3 is (E) -9, 10-dehydro-12, 13-deoxy EpoB.

FIG. 11 shows the efficacy and therapeutic index of various epothilone analogs against tumor cell growth in vitro compared to paclitaxel and vinblastine.

FIG. 12 is a table summarizing the effect of dEpoB, paclitaxel and 26-trifluoro-9, 10-dehydro-dEpoB against MX-1 xenografts in nude mice.

Figure 13 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dpob and 9, 10-dehydro-EpoB on tumor size in nude mice bearing MX-1 xenografts (6 hour intravenous infusion, Q2D x 6 and Q2D x 9, respectively).

Figure 14 shows the body weight change (6 hour infusion, Q2D x 6 and Q2D x 9, respectively) of nude mice with human breast cancer tumor MX-1 xenografts following treatment with 26-trifluoro-9, 10-dehydro-dpob and 9, 10-dehydro-EpoB.

FIG. 15 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dEpoB and 9, 10-dehydro EpoB on tumor size in nude mice bearing MX-1 xenografts (6 hour intravenous infusion, Q2D X6 and Q2D X9, respectively).

FIG. 16 shows the body weight change following treatment of nude mice bearing MX-1 xenografts of human breast cancer tumors with 26-trifluoro-9, 10-dehydro-dEpoB and 9, 10-dehydro-EpoB (6 hour intravenous infusion, Q2D X6 and Q2D X9, respectively).

FIG. 17 shows the therapeutic effect of 9, 10-dehydro-dEpoB on tumor size in nude mice bearing HCT-116 xenografts (intravenous infusion, Q2D X7).

FIG. 18 shows the effect of 9, 10-dehydro-dEpoB on tumor size in nude mice bearing human colon cancer HCT-116 xenografts (intravenous infusion, Q3D X5).

Figure 19 shows the effect of 9, 10-dehydro-dppob on tumor size in nude mice with a 549/paclitaxel xenograft (6 hour intravenous infusion, Q3D x 7).

Figure 20 shows the change in body weight of nude mice with a 549/paclitaxel xenograft after treatment with 26-trifluoro-9, 10-dehydro-dpob and 9, 10-dehydro-dpob (6 hour intravenous infusion, Q3D × 7).

FIG. 21 shows the effect of 26-trifluoro-9, 10-dehydro-dEpoB and 9, 10-dehydro-dEpoB on tumor size in nude mice bearing A549/paclitaxel xenografts (intravenous infusion, Q2D X7).

Figure 22 shows the change in body weight of nude mice with a 549/paclitaxel xenograft after treatment with 26-trifluoro-9, 10-dehydro-dpob and 9, 10-dehydro-dpob (6 hour intravenous infusion, Q2D x 7).

FIG. 23 shows the effect of 9, 10-dehydro-EpoB on tumor size in nude mice bearing human colon carcinoma HCT-116 tumor xenografts (6 hour intravenous infusion).

Figure 24 shows the change in body weight (6 hour intravenous infusion) of nude mice bearing human colon cancer HCT-116 tumor xenografts after treatment with 9, 10-dehydro-EpoB.

FIG. 25 shows the formation of microtubules from tubulin in the presence of various epothilone analogs at 37 ℃.

FIG. 26 shows the formation of microtubules from tubulin in the presence of various epothilone analogs at 4 ℃.

FIG. 27 shows the effect of 9, 10-dehydro-dEpoB and dEpoB on tumor size in nude mice bearing HCT-116 xenografts (intravenous infusion, Q2D X6).

Figure 28 shows the change in body weight of nude mice with HCT-116 xenografts after treatment with 9, 10-dehydro-dpob and dpob (intravenous infusion, Q2D x 6).

FIG. 29 shows the effect of 9, 10-dehydro-dEpoB on tumor size in nude mice bearing human colon cancer HCT-116 xenografts (intravenous infusion, Q3D X4).

FIG. 30 shows the change in body weight of nude mice with human colon cancer HCT-116 xenografts after treatment with 9, 10-dehydro-dEpoB (5mg/kg, intravenous infusion, X3D X4).

FIG. 31 is an IC on growth of epothilone analogs against CCRF-CEM cells₅₀Table of values.

FIG. 32 shows the in vitro metabolic stability of epothilone analogs.

The table shown in figure 33 details the therapeutic effect of various epothilone analogs on human tumor xenografts in mice (6 hour intravenous infusion).

FIG. 34 shows the effect of 9, 10-dehydro-EpoB on tumor size in nude mice bearing human colon cancer HCT-116 tumor xenografts (6 hour intravenous infusion, Q2D X7).

FIG. 35 shows the change in body weight after treatment of nude mice with human colon carcinoma HCT-116 tumor xenografts with 9, 10-dehydro-EpoB and oxazole-EpoD (6 hour infusion, Q2D X7).

FIG. 36 shows the effect of 26-trifluoro-9, 10-dehydro-dEpoB and 9, 10-dehydro-dEpoB on tumor size in nude mice bearing A549/paclitaxel xenografts (6 hour intravenous infusion, Q2D X4).

Figure 37 shows the effect of 9, 10-dehydro-dppob on tumor size in nude mice with a 549/paclitaxel xenograft (6 hour intravenous infusion, Q3D x 3).

Figure 38 shows the stability of epothilone analogs in 20% mouse plasma/PBS.

FIG. 39 shows the stability of epothilone analogs in 10% human liver S9/PBS.

FIG. 40 shows a stable chromatogram of EpoD in 10% human liver S9/PBS.

FIG. 41 is a table illustrating the effect of various epothilone analogs on microtubule polymerization in vitro in the absence of GTP at 37 ℃ (A) and the cytotoxicity of various epothilone analogs on human lung cell strain A549 (B).

FIG. 42 shows the stabilising effect of epothilones on microtubule formation at 35 ℃ and 4 ℃.

FIG. 43 shows the therapeutic effect of 9, 10-dehydro-dEpoB on nude mice bearing T human breast cancer (MX-1) xenografts (6 hour infusion, Q2D X5).

Figure 44 shows the change in body weight after treatment of nude mice with human breast cancer (MX-1) xenografts with 9, 10-dehydro-dpob (6 hour infusion, Q2D x 8).

Figure 45 shows the change in body weight of nude mice with HCT-116 xenografts after treatment with 9, 10-dehydro-dpob (intravenous infusion, Q2D x 7).

Figure 46 shows the therapeutic effect of 9, 10-dehydro-dppof, dppob and paclitaxel on tumor size in nude mice bearing human breast cancer (MX-1) tumor xenografts (6 hour intravenous infusion, Q2D x 6).

FIG. 47 shows the change in body weight of nude mice bearing human breast cancer (MX-1) tumor xenografts after treatment with 9, 10-dehydro-dEpoF, dEpoB, and paclitaxel (6 hour infusion, Q2D X6).

Figure 48 shows the therapeutic effect of 9, 10-dehydro-dppof and dpob on nude mice bearing human colon cancer HCT-116 xenografts (6 hour infusion, Q2D x 8).

FIG. 49 shows the change in body weight of nude mice with HCT-116 xenografts after treatment with 9, 10-dehydro-dEpoF and dEpoB (6 hour infusion, Q2D X8).

Figure 50 shows the therapeutic effect of 9, 10-dehydro-dppof and dppob in nude mice with paclitaxel-human lung cancer (a 549/paclitaxel) xenografts (6 hour infusion, Q2D x 5).

FIG. 51 shows the change in body weight after treatment of nude mice bearing paclitaxel-human lung carcinoma (A549/paclitaxel) xenografts with 9, 10-dehydro-dEpoF and dEpoB (6 hour infusion, Q2D X5).

FIG. 52 is a table comparing the in vitro tumor growth inhibition and relative therapeutic index of various epothilone analogs.

FIG. 53 shows the therapeutic effect of 9, 10-dehydro-dEpoB in nude mice bearing MX-1 xenografts (Q3D X9, 6 hour intravenous infusion).

FIG. 54 shows the change in body weight of nude mice with MX-1 xenografts after treatment with 9, 10-dehydro-dEpoB (Q3D X9, 6 hour intravenous infusion).

FIG. 55 shows the therapeutic effect of 9, 10-dehydro-epothilone B in nude mice bearing MX-1 xenografts (Q3D X9, 6 hour infusion).

FIG. 56 shows the change in body weight of nude mice bearing MX-1 xenografts after treatment with 9, 10-dehydro-epothilone B (Q3D X9, 6 hour intravenous infusion).

FIG. 57 shows the therapeutic effect of low dose of 26-trifluoro-dehydro-dEpoB in nude mice bearing MX-1 xenografts (6 hour intravenous infusion, Q2D X12).

FIG. 58 shows the change in body weight of nude mice with MX-1 xenografts after treatment with low dose 26-trifluoro-9, 10-dehydro-dEpoB (6 hour intravenous infusion, Q2D X12).

FIG. 59 shows the chemotherapeutic effect of epothilone analogs against human tumor xenografts in nude mice. Tumor tissue (40-50mg) was implanted subcutaneously at day 0. When the tumor size reaches about 100mm³Or begin treatment as shown above. All treatments indicated by arrows were performed via tail vein by 6 hour intravenous infusion using a microcatheter and programmable pump as described in the prior literature (Su, D. -S. et al, Angew. chem. Iht. Ed (1997, 36, 2093); Chou, T.C. et al, Proc. Natl, Acad. Sci. USA.1998, 95, 15798; each of which is incorporated herein by reference). Each dose group consisted of four or more mice. Assume 1mm³Tumor equals 1mg tumor tissue, body weight refers to total body weight minus tumor body weight. A. In comparison with the doses of Table 1 (20mg/Kg and 30mg/Kg), with a low dose of 25-trifluoro- (E) -9, 10-dehydro-12, 13-deoxy EpoB (10mg/Kg)A MX-1 xenograft for treating breast cancer. B. Treatment of MX-1 Large xenografts (50 mm) with 25-trifluoro- (E) -9, 10-dehydro-12, 13-deoxy EpoB (25mg/Kg) and dEpoB (30mg/Kg)³). C. Slow growing a549 lung cancer xenografts treated with 25-trifluoro- (E) -9, 10-dehydro-12, 13-deoxyepob (25mg/Kg) and dpeob (30 mg/Kg). D. A549/paclitaxel (44-fold in vitro anti-paclitaxel) xenografts treated with 25-trifluoro- (E) -9, 10-dehydro-12, 13-deoxy EpoB (20mg/Kg) and (E) -9, 10-dehydro-12, 13-deoxy EpoB (4 mg/Kg). For the deH-dEpoB treatment, the day's treatment was skipped due to a significantly rapid decrease in body weight at 28 days.

FIG. 60 depicts the synthesis of C-21 modified 9, 10- (E) -dehydro-epothilone. FIG. 60A shows the synthesis of 26-trifluoro-21-methylamino-9, 10- (E) -dehydro-12, 13-desoxyepothilone B. FIG. 60B is a schematic diagram of the synthesis used to prepare 26-trifluoro-21-amino-9, 10- (E) -dehydro-12, 13-desoxyepothilone B as an intermediate in the synthesis of 26-trifluoro-21-dimethylamino-9, 10- (E) -dehydro-12, 13-desoxyepothilone B.

FIG. 61 shows a table listing the IC's of C-21 modified epothilone anti-tumor cell lines CCRF-CEM and drug-resistant sub-lines thereof₅₀The value is obtained.

FIG. 62 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dEopB and paclitaxel in nude mice with human T-cell lymphoid leukemia CCRF-CEM xenografts (6 hours intravenous infusion, Q2D X8).

FIG. 63 shows the change in body weight of nude mice bearing human T-cell lymphoid leukemia CCRF-CEM xenografts following treatment with 25-trifluoro-9, 10-dehydro-dEopB and paclitaxel (6 hour intravenous infusion, Q2D X8).

FIG. 64 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dEopB and paclitaxel in nude mice bearing human T-cell lymphoid leukemia CCRF-CEM/paclitaxel xenografts (anti-paclitaxel) (6 hours intravenous infusion, Q2D X7,. times.5).

FIG. 65 shows the change in body weight of nude mice bearing human T-cell lymphoid leukemia CCRF-CEM/paclitaxel xenografts (anti-paclitaxel) after treatment with 26-trifluoro-9, 10-dehydro-dEopB and paclitaxel (6 hour intravenous infusion, Q2D X7,. times.5).

FIG. 66 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dEopB and paclitaxel in nude mice bearing human colon cancer HCT-116 xenografts (Q2D X4, × 2, 6 hours intravenous infusion).

FIG. 67 shows the change in body weight of nude mice with human colon cancer HCT-116 xenografts after treatment with 26-trifluoro-9, 10-dehydro-dEopB and paclitaxel (Q2D X4, × 2, 6 hr intravenous infusion).

FIG. 68 shows the therapeutic effect of 9, 10-dehydro-dEopB in nude mice with MX-1 xenografts (6 hour intravenous infusion).

FIG. 69 shows the change in body weight of nude mice with human breast cancer MX-1 xenografts after treatment with 9, 10-dehydro-EopB (6 hour intravenous infusion).

FIG. 70 shows the therapeutic effect of 9, 10-dehydro-EopB in nude mice with human T-cell lymphoid leukemia CCRF-CEM/paclitaxel xenograft (anti-paclitaxel) (6 hours intravenous infusion, Q3D X5,. times.2).

FIG. 71 shows the change in body weight of nude mice bearing human T-cell lymphoid leukemia CCRF-CEM/paclitaxel xenografts (anti-paclitaxel) after treatment with 9, 10-dehydro-EopB (6 hour intravenous infusion, Q3D X5,. times.2).

FIG. 72 shows the therapeutic effect of 26-trifluoro-dEopB and 26-trifluoro-9, 10-dehydro-dEopF in nude mice bearing breast cancer MX-1 xenografts (Q2D X11, intravenous infusion).

FIG. 73 shows the change in body weight of nude mice with breast cancer MX-1 xenografts after treatment with 26-trifluoro-dEopB and 26-trifluoro-9, 10-dehydro-dEopF (Q2D X11, intravenous infusion).

FIG. 74 shows the therapeutic effect of 9, 10-dehydro-dEopB in nude mice bearing breast cancer MX-1 xenografts (Q3D X9, 6 hour intravenous infusion).

FIG. 75 shows the change in body weight of nude mice with breast cancer MX-1 xenografts after treatment with 9, 10-dehydro-dEopB (Q3D X9, 6 hour intravenous infusion).

FIG. 76 shows the therapeutic effect of 26-trifluoro-9, 10-dehydro-dEopF in nude mice with breast lung cancer (MX-1) xenografts (6 hour intravenous infusion and intravenous injection).

FIG. 77 shows the change in body weight of nude mice with MX-1 xenografts after treatment with 26-trifluoro-9, 10-dehydro-dEopF (6 hour intravenous infusion and intravenous injection).

Definition of

The definition of certain compounds and specific functional groups of the present invention are also described in more detail below. For the purposes of the present invention, the chemical elements are determined according to the periodic table of the elements (CAS edition, Handbook of chemistry and Physics, 75 th edition, inner cover) and the specific functional groups are generally defined as described herein. In addition, the general principles of Organic Chemistry, as well as specific functional moieties and reactivities, are described in "Organic Chemistry" (Thomas Sorrell, University Science Books, Sausaltito: 1999, the entire contents of which are incorporated herein by reference). Moreover, those skilled in the art will appreciate that various protecting groups can be used with the synthetic methods described herein. The term "protecting group" as used herein means a group that temporarily blocks a particular functional moiety (e.g., O, S or N) so that a reaction can be selectively carried out at another reactive site of the polyfunctional compound. In a preferred embodiment, a protecting group reacts selectively with high efficiency to form a pair of protected substrates that are expected to react stably; the protecting group must be selectively removable with high efficiency by reagents that do not attack other functional groups and are readily available (preferably non-toxic); the protecting group forms a derivative which is easy to separate (more preferably does not generate a new chiral center); and the protecting group has minimal additional functionality to avoid other reactive sites. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be used. Exemplary protecting groups are described in detail herein, however, it will be understood that the invention is not intended to be limited to these protecting groups; rather, a variety of other equivalent protecting groups can be readily identified using the above criteria and used in the methods of the present invention. In addition, a variety of protecting Groups are described in "Protective Groups in Organic Synthesis" ("Protective Groups in Organic Synthesis" 3 rd edition, Greene, T.W. and Wuts, P.G. ed., John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference).

It will be appreciated that the compounds described herein may be substituted with any number of substituents or functional moieties. In general, the term "substituted" (whether preceded by the term "optionally") and substituents encompassed by the formulae herein, refers to the substitution of a specified substituent for a hydrogen radical in a given structure. When more than one position in a given structure is substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. The term "substituted" as used herein is intended to include all permissible substituents of organic compounds. Broadly, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of the present invention, a heteroatom such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein that satisfy the valence requirements of the heteroatom. Moreover, the present invention is not intended to be limited in any way by the permissible substituents of organic compounds. Combinations of substituents and variables contemplated by the present invention are preferably those that result in the formation of stable compounds useful, for example, in the treatment of proliferative diseases, including but not limited to cancer. The term "stable" as used herein is preferably a compound that has sufficient stability to permit manufacture and that maintains compound integrity for a sufficiently long period of time to be tested and for a sufficiently long period of time to be useful for the purposes described herein.

The term "aliphatic" as used herein includes saturated and unsaturated, straight-chain (i.e., unbranched), branched, cyclic, or polycyclic aliphatic hydrocarbons, which may optionally be substituted with one or more functional groups. It will be appreciated by those skilled in the art that "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, the term "alkyl" as used herein includes straight chain, branched chain and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. Also, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, the term "lower alkyl" is used herein to refer to those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 6 carbon atoms.

In certain embodiments, alkyl, alkenyl, alkynyl groups useful in the present invention contain 1 to 20 aliphatic carbon atoms. In certain other embodiments, alkyl, alkenyl, alkynyl groups useful in the present invention contain 1 to 10 aliphatic carbon atoms. In further embodiments, alkyl, alkenyl, alkynyl groups useful in the present invention contain 1 to 8 aliphatic carbon atoms. In further embodiments, alkyl, alkenyl, alkynyl groups useful in the present invention contain 1 to 6 aliphatic carbon atoms. In other embodiments, alkyl, alkenyl, alkynyl groups useful in the present invention contain 1 to 4 aliphatic carbon atoms. Thus, exemplary aliphatic groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH₂-cyclopropyl, allyl, n-butyl, second-butyl, isobutyl, third-butyl, cyclobutyl, -CH₂-cyclobutyl, n-pentyl, second-pentyl, isopentyl, third-pentyl, cyclopentyl, -CH₂-cyclopentyl, n-hexyl, second-hexyl, cyclohexyl, -CH₂Cyclohexyl moieties and the like, which in turn may have one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The terms "alkoxy" or "alkylthio" as used herein, refer to an alkyl group, as defined above, bonded to a parent molecular moiety through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl group contains 1 to 20 aliphatic carbon atoms. In certain other embodiments the alkyl group contains 1 to 10 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, alkynyl groups useful herein contain 1 to 8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1 to 6 aliphatic carbon atoms. In further embodiments, the alkyl group contains 1 to 4 aliphatic carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentyloxy, and n-hexyloxy. Examples of alkylthio include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

The term "alkylamino" refers to a group having the structure-NHR ', wherein R' is alkyl as defined herein. In certain embodiments, the alkyl group contains 1 to 20 aliphatic carbon atoms. In certain other embodiments the alkyl group contains 1 to 10 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, alkynyl groups useful herein contain 1 to 8 aliphatic carbon atoms. In further embodiments, the alkyl group contains 1 to 6 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1 to 4 aliphatic carbon atoms. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino, and the like.

Some examples of substituents for the above aliphatic (or other) moieties of the compounds of the present invention include (but are not limited to): an aliphatic group; a heteroaliphatic group; an aryl group; a heteroaryl group; an arylalkyl group; a heteroarylalkyl group; an alkoxy group; an aryloxy group; a heteroalkoxy group; a heteroaryloxy group; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; f; cl; br; i; -OH; -NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；-CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_X)₂；-OC(O)R_X；-OCO₂R_x；-OCON(R_X)₂；-N(R_X)₂；-S(O)₂R_X；-NR_x(CO)R_xWherein Rx as appearing independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic, or acyclic, and wherein any of the aryl and heteroaryl substituents described above and herein can be substituted or unsubstituted. Other examples of substituents that may generally be used are set forth in the specific examples shown in the examples described herein.

Generally, the terms "aryl" and "heteroaryl" are used herein to refer to stable mono-or polycyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated moieties, preferably having 3 to 14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the substituents described above, i.e., those described for the aliphatic moiety or for the other moieties described herein that result in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono-or bicyclic carbocyclic ring system having one or two aromatic rings, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl" as used herein refers to a cyclic aryl group having 5 to 10 ring atoms, wherein one ring atom is selected from S, O and N; zero, one or two ring atoms are an additional heteroatom independently selected from S, O and N; and the remaining ring atoms are carbon, which group is bonded to the rest of the molecule through any ring atom, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It is understood that aryl and heteroaryl groupsAryl (including bicyclic aryl) groups may be unsubstituted or substituted, wherein substitution includes independently replacing one, two or three hydrogen atoms thereon with any one or more of the following moieties, including but not limited to: an aliphatic group; a heteroaliphatic group; an aryl group; a heteroaryl group; an arylalkyl group; a heteroarylalkyl group; an alkoxy group; an aryloxy group; a heteroalkoxy group; a heteroaryloxy group; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; f; cl; br; i; -OH; -NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；-CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_X)₂；-OC(O)R_X；-OCO₂R_x；-OCON(R_X)₂；-N(R_X)₂；-S(O)₂R_X；-NR_x(CO)R_xWherein Rx as appearing herein each independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic, or acyclic, and wherein any of the aryl and heteroaryl substituents described above and herein can be substituted or unsubstituted. Other examples of substituents that may generally be used are set forth in the specific examples shown in the examples described herein.

The term "cycloalkyl" as used herein specifically refers to a group having three to seven (preferably three to ten) carbon atoms. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, as in the case of other aliphatic, heteroaliphatic, or heterocyclic moieties, which may be optionally substituted with substituents including, but not limited to: an aliphatic group; a heteroaliphatic group; an aryl group; a heteroaryl group; an arylalkyl group; a heteroarylalkyl group; an alkoxy group; an aryloxy group; a heteroalkoxy group; a heteroaryloxy group; an alkylthio group; an arylthio group; a heteroalkylthio group; heteroaromatic compoundsA sulfur radical; f; cl; br; i; -OH; -NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；-CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_X)₂；-OC(O)R_X；-OCO₂R_x；-OCON(R_X)2；-N(R_X)₂；-S(O)₂R_X；-NR_x(CO)R_xWherein Rx as appearing independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic, or acyclic, and wherein any of the aryl and heteroaryl substituents described above and herein can be substituted or unsubstituted. Other examples of substituents that may generally be used are set forth in the specific examples shown in the examples described herein.

The term "heteroaliphatic" as used herein refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, for example, in place of a carbon atom. The heteroaliphatic moiety can be branched, unbranched, cyclic or acyclic and include saturated or unsaturated heterocycles, e.g., morpholinyl, pyrrolidinyl, and the like. In certain embodiments, a heteroaliphatic moiety may be substituted by independently replacing one or more hydrogen atoms thereon with a moiety including, but not limited to: an aliphatic group; a heteroaliphatic group; an aryl group; a heteroaryl group; an arylalkyl group; a heteroarylalkyl group; an alkoxy group; an aryloxy group; a heteroalkoxy group; a heteroaryloxy group; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; f; cl; br; i; -OH; -NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；-CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_X)₂；-OC(O)R_X；-OCO₂R_x；-OCON(R_X)₂；-N(R_X)₂；-S(O)₂R_X；-NR_x(CO)R_xWherein Rx as appearing independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic, or acyclic, and wherein any of the aryl and heteroaryl substituents described above and herein can be substituted or unsubstituted. Other examples of substituents that may generally be used are set forth in the specific examples shown in the examples described herein.

The terms "halo" and "halogen" as used herein refer to an atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

The term "haloalkyl" refers to an alkyl group as defined above having 1, 2, or 3 halogen atoms bonded thereto and may be exemplified by groups such as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term "heterocycloalkyl" or "heterocyclic" as used herein refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group, including, but not limited to, di-or tricyclic groups comprising a fused six-membered ring having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bond and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms are optionally oxidized, (iii) the nitrogen heteroatom is optionally quaternized, and (iv) any of the above heterocycles can be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuranyl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" is used and as used herein means a group in which 1, 2 or 3 hydrogen atoms are independently replaced by, but not limited toSubstituted heterocycloalkyl or heterocyclic group as defined above: an aliphatic group; a heteroaliphatic group; an aryl group; a heteroaryl group; an arylalkyl group; a heteroarylalkyl group; an alkoxy group; an aryloxy group; a heteroalkoxy group; a heteroaryloxy group; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; f; cl; br; i; -OH; -NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；-CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_X)₂；-OC(O)R_X；-OCO₂R_x；-OCON(R_X)₂；-N(R_X)₂；-S(O)₂R_X；-NR_x(CO)R_xEach R present therein_xIndependently include, but are not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic, or acyclic, and wherein any of the aryl and heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of substituents that may generally be used are set forth in the specific examples shown in the examples described herein.

"marking": the term "label" as used herein is intended to mean that a compound has attached at least one element, isotope, or chemical that enables detection of the compound. In general, markers fall into three categories: a) isotopic labels, which can be radioactive or heavy isotopes, including (but not limited to)²H、³H、³²P、³⁵S、⁶⁷Ga、^99mTc(Tc-99m)、¹¹¹In、¹²³I、¹²⁵I、¹⁶⁹Yb and¹⁸⁶re; b) an immunological marker, which may be an antibody or an antigen; and c) a coloured or fluorescent dye. It will be appreciated that the labels may be incorporated into the compound at any location that does not interfere with the biological activity or properties of the compound being detected.In certain embodiments of the invention, photoaffinity labels are used to directly explain intermolecular interactions in biological systems (e.g., to probe epothilone binding sites in tubulin dimers). Various known luminophores, most of which rely on the phototransformation of diazo, azide or diazomethane to nitrene or carbene (see, Bayley, h., photophered Reagents in Biochemistry and Molecular Biology (1983), Elsevier, amsterdam, the entire contents of which are incorporated herein by reference), may be used. In certain embodiments of the invention, the photoaffinity labels used are ortho-, meta-and para-azidobenzoyl groups substituted with one or more halogen moieties, including, but not limited to, 4-azido-2, 3, 5, 6-tetrafluorobenzoic acid.

"Polymer": the term "polymer" as used herein refers to a compound comprising an open, closed, linear, branched or crosslinked chain of the same or different repeating units (monomers). It will be appreciated that in certain embodiments, the term polymer refers to a biopolymer, which as used herein is intended to refer to polymeric materials found in nature or polymeric materials based on polymeric materials found in nature, including (but not limited to) nucleic acids, peptides, and the like. In certain other embodiments, the term polymer refers to synthetic polymers, such as biodegradable polymers or other polymeric materials. It is understood that solid polymeric carriers are also encompassed within the scope of the polymers of the present invention. The compounds of the present invention may be bound to a polymeric support and, therefore, certain synthetic modifications may be performed on the solid phase. The term "solid support" as used herein is intended to include, but is not limited to, pellets, disks, capillaries, hollow fibers, probes, alignment pins, solid fibers, cellulose beads, microporous-glass beads, silica gel, polystyrene beads optionally crosslinked with divinylbenzene, graft copolymerized beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N' -bis-acryloylethylenediamine, and glass beads coated with a hydrophobic polymer. Those skilled in the art will appreciate that the choice of a particular solid carrier is limited by the compatibility of the carrier with the reaction chemistry used. An exemplary solid support is Tentagel amino resin, which is a composite of 1) polystyrene beads crosslinked with divinylbenzene and 2) PEG (polyethylene glycol). Tentagel is a particularly useful carrier since it provides a versatile carrier for on-bead or off-bead assays, and it also has excellent swelling properties in toluene to water solvents.

Detailed Description

In recognition of the need to develop novel and effective cancer therapies, the present invention provides novel synthetic methods that enable the obtainment of macrocycles with a broad range of biological and pharmacological activities, as well as novel compounds having such activities, novel therapeutic compositions, and methods of using such compounds and compositions.

In certain embodiments, the compounds of the present invention are useful for treating cancer. Certain compounds of the invention exhibit cytotoxic or growth inhibitory effects on cancer cell lines, exhibit the ability to polymerize tubulin and stabilize microtubule assembly, and/or cause tumor shrinkage or disappearance in cancer cell xenograft models. In certain embodiments, side effects of the compounds, including toxicity to vital organs, nausea, vomiting, diarrhea, hair loss, weight gain, liver toxicity, skin disorders, and the like, are reduced or minimized. These compounds are also easier to formulate due to their increased water solubility, reduced toxicity, expanded therapeutic range, increased therapeutic efficacy, and the like.

Summary of the Compounds of the invention

The compounds of the present invention include compounds of general formulae (0) and (0') and pharmaceutically acceptable derivatives thereof as further defined below:

wherein R is₀Is a substituted or unsubstituted aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylAn alkenyl or heteroaryl alkynyl moiety; in certain embodiments, R₀Is an arylalkyl, arylalkenyl, heteroarylalkyl or heteroarylalkenyl moiety; in other embodiments, R₀Is a heteroarylalkenyl moiety; in certain embodiments, R₀Is a heteroarylalkyl moiety; in certain embodiments, R₀Is a 5-to 7-membered aryl or heteroaryl moiety; in further embodiments, R₀Is an 8 to 12 membered bicyclic aryl or heteroaryl moiety; in other embodiments, R₀Is a bicyclic moiety in which a phenyl ring is fused to a heteroaryl or aryl moiety; in further embodiments, R₀Is a bicyclic moiety in which a phenyl ring is fused to a thiazole, oxazole or imidazole moiety; in other embodiments, R₀Is a substituted or unsubstituted phenyl moiety;

R₃and R₄Independently is hydrogen; or a substituted or unsubstituted, linear or branched, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety (optionally substituted with one or more hydroxyl groups), a protected hydroxyl group, an alkoxy group, a carboxyl group, formaldehyde (carboxaldehydee), a linear or branched alkyl or cyclic acetal, fluorine, an amino group, a protected amino group, an amino group substituted with one or two alkyl or aryl moieties, an N-oximino group or an N-alkoxyimino group; in certain embodiments, R₃And R₄Independently hydrogen, fluorine or lower alkyl; in certain embodiments, R₃And R₄Independently is hydrogen or methyl; in other embodiments, R₃Is methyl and R₄Is hydrogen;

R₅and R₆Independently hydrogen or a protecting group; in certain embodiments, R₅And R₆Both are hydrogen;

x is O, S, C (R)₇)₂Or NR₇Wherein R is present₇Independently hydrogen or lower alkyl; in certain embodiments, X is O; in certain embodiments, X is NH;

y is O, S, NH, C(R₇)₂、CH₂、N(R₇) Or NH, in which R is present₇Independently hydrogen or lower alkyl; in certain embodiments, Y is O; in certain embodiments, Y is NH; in other embodiments, Y is CH₂；

Each R₈Independently is hydrogen; halogen, hydroxy, alkoxy, amino, dialkylamino, alkylamino, fluoro, cyano or substituted or unsubstituted, linear or branched, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl or heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl moiety (optionally substituted with one or more hydroxy groups), protected hydroxy, alkoxy, carboxy, formaldehyde, linear or branched alkyl or cyclic acetal, fluoro, amino, protected amino, amino substituted with one or two alkyl or aryl moieties, N-oximino or N-alkoxyimino; in certain embodiments, R₈Is hydrogen; in certain embodiments, R₈Is a hydroxyl group; in other embodiments, R₈Is fluorine; in still other embodiments, R₈Lower alkyl, such as methyl; in other embodiments, R₈is-CF₃、-CF₂H or-CFH₂(ii) a In certain embodiments, R₈Is perfluoro or fluoroalkyl; in further embodiments, R₈Is halo or perhaloalkyl;

R₉and R₁₀Independently is hydrogen; or a substituted or unsubstituted, linear or branched, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, aryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety (optionally substituted with one or more hydroxyl groups), protected hydroxyl, alkoxy, carboxyl, formaldehyde, a linear or branched alkyl or cyclic acetal, fluorine, amino, protected amino, amino substituted with one or two alkyl or aryl moieties, N-oximino or N-alkoxyimino group; in certain embodiments, R₉And R₁₀One of them is methyl; in some casesIn the examples, R₉And R₁₀Is methyl; in still other embodiments, R₉And R₁₀One of which is methyl and the other is hydrogen; in certain embodiments, R₉And R₁₀Is hydrogen; occurrence of R_BIndependently is hydrogen; halogen; -OR_B’；-SR_B’；-N(R_B’)₂(ii) a -C (O) or_B’-；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’-；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more hydrogens; halogen; -OR_B’-；-SR_B’-；-N(R_B’)2；-C(O)OR_B’-；-C(O)R_B’-；-CONHR_B’；-O(C＝O)R_B’-；-O(C＝O)OR_B’-；-NR_B’(C＝O)R_B-；N₃；N₂R_B’(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or is a radioactive label; in certain embodiments, R_BIs hydrogen,Methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, each unsubstituted or optionally substituted by one or more halogens, -OH, -or_B’、NH₂Or N (R)_B’)₂Or any combination thereof, wherein R is present_B’Independently hydrogen, alkyl, aryl or a protecting group, in certain embodiments, R_BIs hydrogen, methyl or ethyl, in other embodiments, R_BIs methyl, and in certain embodiments is-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B’、NHR_B’、N(R_B’)₂Or SR_B’(ii) a In still other embodiments, R_Bis-CF₃、-CH₂F or CHF₂(ii) a In certain embodiments, R_BIs perfluoro or fluoroalkyl; in other embodiments, R_BIs halo or perhaloalkyl;

occurrence of R_B’Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety;

m is 1, 2, 3, or 4, in certain embodiments m is 1 or 2, in other embodiments m is 1.

The compounds of the present invention include compounds of general formula (I) or (Γ) as further defined below and pharmaceutically acceptable derivatives thereof:

wherein R is₁Is hydrogen or lower alkyl; in certain embodiments, R₁Is methyl; in certain embodiments, R₁is-CF₃、-CF₂H or CH₂F; in other embodiments, R₁Is perfluoro or fluoroalkyl; in still other embodiments, R₁Is halo or perhaloalkyl;

R₂is a substituted or unsubstituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety; in certain embodiments, R₂Is a substituted or unsubstituted oxazole; in other embodiments, R₂Is substituted or unsubstituted thiazole;

R₃and R₄Each independently is hydrogen; or substituted or unsubstituted, linear or branched, cyclic or acyclicA cycloaliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl moiety (optionally substituted with one or more hydroxyl groups), a protected hydroxyl, alkoxy, carboxyl, formaldehyde, a linear or branched alkyl or cyclic acetal, fluorine, amino, a protected amino, an amino substituted with one or two alkyl or aryl moieties, an N-oximino, or an N-alkoxyimino; in certain embodiments, R₃And R₄Each independently is hydrogen, fluorine or lower alkyl; in other embodiments, R₃And R₄Each independently is hydrogen or methyl; in still other embodiments, R₃Is methyl and R₄Is hydrogen;

R₅and R₆Each independently is hydrogen or a protecting group; in certain embodiments, R₅And R₆Both are hydrogen;

x is O, S, C (R)₇)₂Or NR₇Wherein R is present₇Independently hydrogen or lower alkyl; in certain embodiments, X is O; in other embodiments, X is NH; occurrence of R_BIndependently is hydrogen; halogen; -OR_B’；-SR_B’；-N(R_B’)₂；-C(O)OR_B’；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more hydrogens; halogen; -OR_B’；-SR_B’；-N(R_B’)₂；-C(O)OR_B’；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone orAnalogs thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; in certain embodiments, R_BIs hydrogen,Methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl OR cyclohexyl, each unsubstituted OR optionally substituted by one OR more halogens, -OH, -OR_B’or-N (R)_B’)₂Or any combination thereof, wherein R is present_B’Independently hydrogen, alkyl, aryl or a protecting group, in other embodiments, R_BIs hydrogen, methyl or ethyl, in yet other embodiments, R_BIs methyl, in yet other embodiments, R_Bis-CF₃、-CH₂F or CHF₂。

In certain embodiments, the compounds of the present invention include compounds of formula (II) or (II') shown having a defined stereochemistry:

or

X, R therein₁、R₂、R₃、R₄、R₅、R₆、R_BAnd X are as defined above.

In certain embodiments, X is O. In other embodiments, X is NH. In other embodiments, X is CH₂。

In certain embodiments, R₂Is substituted or unsubstituted thiazole. In certain embodiments, R₂Is 2-methyl-thiazol-4-yl. In other embodiments, R₂Is 2-hydroxymethyl-thiazol-4-yl. In still other embodiments, R₂Is 2-aminomethyl-thiazol-4-yl. In other embodiments, R₂Is 2-thiomethyl-thiazol-4-yl.

In certain embodiments, R₂Is a substituted or unsubstituted oxazole. In certain embodiments, R₂Is 2-methyl-oxazol-4-yl. In other embodiments, R₂Is 2-hydroxymethyl-oxazol-4-yl. In still other embodiments, R₂Is 2-aminomethyl-oxazol-4-yl. In other embodiments, R₂Is 2-thiomethyl-oxazol-4-yl.

In certain embodiments, R_BIs hydrogen, methyl, ethyl, -CF₃、-CH₂F、-CF₂H. In certain embodiments, R_BIs methyl. In still other embodiments, R_Bis-CF₃. In certain embodiments, R_BIs hydrogen. In other embodiments, R_BIs ethyl.

Certain preferred compounds include (for example):

the compounds of the present invention include those specifically set forth above and described herein, and are illustrated in part by the various classes, subgenera, and species disclosed elsewhere herein.

It will be appreciated by those skilled in the art that asymmetric centers may be present in the compounds of the present invention. Thus, the compounds of the present invention and pharmaceutical compositions thereof may be in the form of a single enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present invention may be enantiomerically pure compounds. In certain other embodiments, a mixture of stereoisomers or diastereomers is provided.

It will be appreciated that certain classes and subclasses of the above compounds may exist in various isomeric forms. The present invention encompasses compounds in a single isomeric form substantially free of other isomers and alternatively in a mixture of various isomers (e.g., a racemic mixture of stereoisomers). Furthermore, unless otherwise specified, the present invention encompasses both (Z) and (E) double bond isomers. Thus, compounds of the present invention generally depicted by the structures (O), (O '), (I '), (II), and (II ') encompass structures wherein the double bond is (Z) or (E). In certain preferred embodiments, the double bond at the C12-C13 position is in the cis or Z configuration. In certain embodiments, the double bond at the C9-C10 position is in the trans or E configuration. In still other embodiments, the double bond at the C12-C13 position is in the cis or Z configuration and the double bond at the C9-C10 position is in the trans or E configuration. The present invention also encompasses tautomers of the specific compounds described above.

In addition, the invention provides pharmaceutically acceptable derivatives of the compounds of the invention, and methods of treating a patient using these compounds, pharmaceutical compositions thereof, or a combination of either with one or more additional therapeutic agents. The phrase "pharmaceutically acceptable derivative" as used herein means any pharmaceutically acceptable salt, ester, or salt of such ester of the compound, or any other adduct or derivative which, when administered to a subject, is capable of providing (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Thus, pharmaceutically acceptable derivatives include, inter alia, prodrugs. Prodrugs are derivatives of a compound, which usually have significantly reduced pharmacological activity, which comprise an additional moiety that is readily removed in vivo to yield the parent molecule as the pharmacologically active substance. An example of a prodrug is an ester that dissociates in vivo to yield a related compound. Prodrugs of various compounds and materials and methods for derivatizing the parent compounds to produce the prodrugs are well known and suitable for use in the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives are described in more detail below.

Compounds of the invention which are of particular interest are those having the following properties:

● exhibit cytotoxic or growth inhibitory effects on cancer cell lines maintained in vitro or in animal studies using a scientifically acceptable cancer cell xenograft model;

● exhibit an ability to polymerize tubulin and stabilize microtubule assembly;

● have minimal levels of toxicity to vital organs;

● cause tumor disappearance in a scientifically acceptable cancer cell xenograft model;

● cause tumor shrinkage in a scientifically acceptable cancer cell xenograft model;

● causing the tumor to disappear and the recurrence of the tumor to be delayed and/or not to recur after treatment is stopped in a scientifically acceptable cancer cell xenograft model;

● exhibit temporary and reversible weight loss and demonstrate therapeutic effects in a scientifically acceptable cancer cell xenograft model;

● exhibit enhanced water solubility over epothilone A, B, C or D or paclitaxel or alternatively exhibit sufficiently high solubility such that they can be formulated in an aqueous medium with a reduced proportion of chremophor; and/or

● exhibit superior treatment profiles (e.g., optimal safety and therapeutic efficacy) over epothilone B, epothilone D, or paclitaxel.

Various epothilone analogs described above have been prepared, characterized, and tested as exemplified herein. 9, 10-dehydro-epothilone analogs have been found to be useful in the treatment of cancer, and in particular, the compounds have been prepared and found to possess one or more of the desirable properties described above.

Synthesis method

The synthesis of certain epothilones, desoxyepothilones, and analogs thereof has been previously described (see, U.S. Pat. Nos. 6,242,469, 6,284,781, 6,300,355, 6,204,388, 6,316,630, and 6,369,234; U.S. patent application Nos. 09/797,027, 09/796,959, and 10/236,135; and PCT publications WO 99/01124, WO 99/43653, and WO 01/64650, the entire contents of which are incorporated herein by reference). Recognizing the need for improved or other synthetic methods to produce epothilones, desoxyepothilones, and analogs thereof on a large scale and with high efficiency, the present invention provides an efficient and patterned route for the synthesis of epothilones, desoxyepothilones, and analogs thereof. Although the synthesis of certain exemplary compounds is described in the examples herein, it will be appreciated that the method is generally applicable to the generation of analogs and combinations of each and every class of compounds described herein above.

In particular, the 9, 10-dehydroepothilone compounds of the invention can be prepared in a variety of ways using synthetic methods for the synthesis of epothilones. In certain embodiments, the compounds are prepared using a convergent synthetic route. For example, epothilones can be synthesized by preparing two or three intermediates which can be combined to obtain the desired compound. In one embodiment, one of the intermediates is an acyl moiety comprising 1 to 9 carbons, and the other intermediate comprises 10 to 15 carbons, and may also comprise a thiazole side chain. The two substantially equal portions of the epothilone may be first combined using an esterification reaction between C-1 and deoxy C-15. The macrocycle may then be closed using a carbon-carbon coupling reaction (e.g., Suzuki (Suzuki) coupling) or a ring closure displacement reaction. In one embodiment, the final ring closure step is accomplished using a ring closure displacement reaction to form a 9, 10-double bond and close the macrocycle. The ring-closing metathesis reaction can be accomplished using an organometallic catalyst, such as the Grubbs catalyst shown in scheme 8 below. In certain embodiments, the 9, 10-double bond is reduced or oxidized, or the 9, 10-double bond is further functionalized to make other epothilone derivatives.

In other embodiments, the final ring closure step is performed using a ring closure displacement reaction to form a 12, 13-double bond and close the macrocycle. In certain embodiments, the 12, 13-double bond is reduced or oxidized. In other embodiments, the macrocycle may be formed using a macrocyclic aldol condensation reaction or a macrocyclic lactonization reaction.

Certain exemplary synthetic methods of the compounds of the invention are provided in the figures and examples. It will be appreciated by those skilled in the art that various analogs and derivatives can be prepared using the synthetic procedures described herein. For example, one can use different protecting groups or different substituents on the 16-membered ring to accomplish the synthetic steps.

Pharmaceutical composition

The present invention also provides a pharmaceutical preparation comprising at least one of the compounds described above and herein, or a pharmaceutically acceptable derivative thereof, wherein the compounds inhibit the growth of or kill cancer cells, and in certain related embodiments, inhibit the growth of or kill multi-drug resistant cancer cells. In certain embodiments, the pharmaceutical formulation also comprises a solubilizing or emulsifying agent, such as hydrogenated castor oil (polyoxyl35 castor oil) or Solutol (polyethylene glycol 66012-hydroxystearate).

As described above, the present invention provides novel compounds having anti-tumor and anti-proliferative activities, and thus the compounds of the present invention can be used for the treatment of cancer. Thus, another aspect of the invention provides pharmaceutical compositions comprising any of the compounds described herein, and optionally a pharmaceutically acceptable carrier. In certain embodiments, the compositions optionally further comprise one or more additional therapeutic agents. In certain other embodiments, the additional therapeutic agent can be an anti-cancer agent, as discussed in more detail below.

It will also be appreciated that certain compounds of the invention may be in free therapeutic form or, if desired, in the form of pharmaceutically acceptable derivatives thereof. According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative, such as a prodrug, which upon administration to a patient in need thereof is capable of providing, directly or indirectly, a compound described herein or a metabolite or residue thereof.

The term "pharmaceutically acceptable salts" as used herein refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without excessive toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio. Various pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in J.pharmaceutical Sciences (66: 1-19(1977), which is incorporated herein by reference, by S.M.Berge et al. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or isolated by reaction of the free base functionality with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are the amino salts formed with inorganic acids (e.g., hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids) or with organic acids (e.g., acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids) or by using other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, phosphates, Picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Other pharmaceutically acceptable salts include, where applicable, non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

Furthermore, the term "pharmaceutically acceptable ester" as used herein refers to esters that hydrolyze in vivo and include those that break down readily in the human body to yield the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, naphthenic and alkenedioic acids, wherein each alkyl or alkenyl moiety preferably does not exceed 6 carbon atoms. Examples of specific esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.

Moreover, the term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, and the potentially zwitterionic forms of the compounds of the present invention. The term "prodrug" refers to a compound that is rapidly converted in vivo, e.g., by hydrolysis in blood, to yield the parent compound of the above formula. A comprehensive discussion is provided in Pro-drugs as Novel Delivery Systems of t.higuchi and v.stella (volume 14 of a.c.s.symposium Series) and Bioreversible Carriers in Drug Design (american pharmaceutical Association and Pergamon Press, 1987) edited by Edward b.roche, both incorporated herein by reference.

As noted above, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable carrier, which as used herein includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences (15 th edition, e.w. martin, Mack Publishing co., Easton, Pa., 1975) disclose various carriers for formulating Pharmaceutical compositions and well-known techniques for preparing the same. The use of any conventional carrier medium is contemplated within the scope of the present invention, except, for example, where the conventional carrier medium is incompatible with the anticancer compounds of the present invention by producing any undesirable biological effect or interacting in a deleterious manner with any of the other components of the pharmaceutical composition. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, carbohydrates, such as lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc powder; hydrogenated castor oil; solutol; excipients, for example, cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, for example, propylene glycol; esters, for example, ethyl oleate and ethyl laurate; agar; buffering agents, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer solution; the compositions may also contain ethanol and phosphate buffers and other non-toxic compatible lubricants (e.g., sodium lauryl sulfate and magnesium stearate) and coloring, releasing, coating, sweetening, flavoring and perfuming agents, preservatives and antioxidants, according to the judgment of the formulator.

Use of compounds and pharmaceutical compositions

The invention further provides methods for inhibiting tumor growth and/or tumor metastasis. In certain related embodiments, the invention provides a method of treating cancer by inhibiting tumor growth and/or tumor metastasis of tumor multi-drug resistant cancer cells. The method involves administering a therapeutically effective amount of the compound or a pharmaceutically acceptable derivative thereof to a patient (including but not limited to humans or animals) in need thereof. In certain embodiments, particularly with respect to treating cancer comprising multi-drug resistant cancer cells, the therapeutically effective dose is an amount sufficient to kill or inhibit the growth of the multi-drug resistant cancer cells. In certain embodiments, the compounds of the present invention are useful for treating solid tumors.

The compounds and pharmaceutical compositions of the invention are useful for treating or preventing any disease or condition, including proliferative diseases (e.g., cancer), autoimmune diseases (e.g., rheumatoid arthritis), and infectious diseases (e.g., bacteria, fungi, etc.). The compounds and pharmaceutical compositions can be administered to an animal, preferably a mammal (e.g., domesticated livestock, cats, dogs, mice, rats), and more preferably a human. The compounds of the pharmaceutical compositions can be administered to an animal using any method of administration. In certain embodiments, the compound or pharmaceutical composition can be administered parenterally.

In another aspect, according to the treatment methods of the present invention, tumor cells can be killed or growth inhibited by contacting the tumor cells with a compound or composition of the present invention, as set forth herein. Thus, in a further aspect, the invention provides a method of treating cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising a compound of the invention, in an amount and for a time necessary to achieve the desired result. In certain embodiments of the invention, a "therapeutically effective amount" of a compound or pharmaceutical composition of the invention is an amount effective to kill or inhibit the growth of tumor cells. In accordance with the methods of the present invention, the compounds and compositions can be administered in any amount and by any route of administration effective to kill or inhibit the growth of tumor cells. Thus, the expression "an amount effective to kill or inhibit the growth of tumor cells" as used herein refers to an amount of the agent sufficient to kill or inhibit the growth of tumor cells. The exact amount required will vary from subject to subject depending on the subject species, age and general condition, severity of infection, the particular anti-cancer agent, its mode of administration and the like. The anticancer compounds of the present invention are preferably formulated in unit dosage form for ease of administration and consistent dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of an anti-cancer agent suitable for use in the patient to be treated. It will be understood, however, that the total daily amount of the compounds and compositions of this invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed; the duration of treatment; drugs used in combination or concomitantly with the specific compound employed; and similar factors well known in the medical arts.

In addition, the pharmaceutical compositions of the present invention can be administered to humans and other animals in the following manner, depending on the severity of the infectious disease being treated, after formulating at the desired dosage with a suitable pharmaceutically acceptable carrier: oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (in the form of powders, ointments or drops), buccal (in the form of an oral or nasal spray), or similar administration. In certain embodiments of the invention, the compounds of the invention described herein may be formulated by conjugation with a water-soluble chelating agent or a water-soluble polymer, such as polyethylene glycol, poly (1-glutamic acid), or poly (1-aspartic acid), as set forth in U.S. patent No. 5,977,163, the entire contents of which are incorporated herein by reference. In certain embodiments, the compounds of the present invention may be administered orally or parenterally one or more times a day at dosage levels sufficient to deliver the following to achieve the desired therapeutic effect: from about 0.001mg/Kg to about 100mg/Kg of body weight of the subject, from about 0.01mg/Kg to about 50mg/Kg, preferably from about 0.1mg/Kg to about 40mg/Kg, preferably from about 0.5mg/Kg to about 30mg/Kg, from about 0.01mg/Kg to about 10mg/Kg, from about 0.1mg/Kg to about 10mg/Kg, and more preferably from about 1mg/Kg to about 25 mg/Kg. The desired dose may be delivered every other day, every third day, every week, every second week, every third week, or every fourth week. In certain embodiments, the desired dose may be delivered in multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, or ten administrations).

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, slurries and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic pharmaceutically acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that can be used are, inter alia, water, ringer's solution, U.S. p. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by inclusion of a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

To prolong the effect of the drug, it is generally desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials that are poorly water soluble. Thus, the rate of absorption of the drug depends on its rate of dissolution, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered drug may be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be prepared by forming microencapsule matrices of the drug in biodegradable polymers (e.g., polylactide-polyglycolide). The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly (orthoesters) and polyanhydrides. Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one inert and pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or calcium hydrogen phosphate) and/or: a) fillers or extenders, for example starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants, for example glycerol, d) disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarders, for example paraffin, f) absorption promoters, for example quaternary ammonium compounds, g) wetting agents, for example cetyl alcohol and glycerol monostearate, h) absorbents, for example kaolin and bentonite and i) lubricants, for example talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also include buffering agents.

Solid ingredients of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols. Tablets, dragees, capsules, pills, and granules of solid dosage forms can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may optionally include opacifying agents and may also be of a composition that they release the active ingredient(s) only (or preferentially) in a certain portion of the intestinal tract, optionally, in a delayed manner. Examples of embedding components that can be used include polymeric substances and waxes. Solid ingredients of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

The active compounds may also be in the form of microcapsules containing one or more of the above-mentioned excipients. Tablets, dragees, capsules, pills and granules of solid dosage forms can be prepared with coatings and envelopes such as enteric coatings, release controlling coatings and other coatings well known in the art of pharmaceutical formulation. In these solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. In addition to inert diluents, the dosage forms may also normally include additional substances other than inert diluents, such as tablet lubricants and other tablet aids (e.g., magnesium stearate and microcrystalline cellulose). In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also be of a composition which releases the active ingredient(s) only (or preferentially) in a certain part of the intestinal tract, optionally with a delay. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives and required buffers. Ophthalmic formulations, ear drops and eye drops are also contemplated within the scope of the present invention. Furthermore, the present invention encompasses the use of skin patches, which have the additional advantage of delivering a compound to the body in a controlled manner. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable vehicle. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

As described above, the compounds of the present invention can be used as anticancer agents, and thus can be used for treating cancer by dying tumor cells or inhibiting the growth of tumor cells. In general, the anticancer agents of the present invention are useful for the treatment of cancer and other proliferative diseases, including, but not limited to, breast cancer, brain cancer, skin cancer, cervical cancer, colon and rectal cancer, leukemia, lung cancer, melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer, to name a few. In certain embodiments, the anticancer agents of the present invention are effective against leukemia cells and melanoma cells, and thus may be useful in the treatment of leukemia (e.g., myelogenous, lymphocytic, promyelocytic, myelocytic, and lymphoblastic leukemias, whether acute or chronic forms) and malignant melanoma. In further embodiments, the anticancer agents of the present invention are effective against solid tumors and may also kill and/or inhibit the growth of multi-drug resistant cells (MDR cells). In certain embodiments, the anticancer agents of the present invention are effective against cancers that are resistant to or found to be clinically unresponsive to other well-known antineoplastic agents. In other embodiments, the anticancer agents of the present invention are effective against cancers that are resistant to other well-known anti-tumor microtubule stabilizing agents (e.g., paclitaxel).

It is also to be understood that the compounds and pharmaceutical compositions of the present invention may be used in combination therapy, i.e., the compounds and pharmaceutical compositions may be administered simultaneously, prior to, or after one or more other desired therapies or medical procedures. The particular combination of therapies (therapy or procedure) employed in a combination therapy should take into account the compatibility of the intended therapy and/or procedure and the desired therapeutic effect to be achieved. It will also be appreciated that the therapy used may achieve the desired effect on the same disease (e.g., the compound of the invention may be administered simultaneously with another anti-cancer agent), or it may achieve a different effect (e.g., control of any side effects).

For example, other therapies and anticancer agents that may be used in combination with the anticancer agents of the present invention include surgery, radiation therapy (gamma-radiation therapy, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioisotope therapy, to name a few), endocrine therapy, biological response modifiers (interferon, interleukin, and Tumor Necrosis Factor (TNF), to name a few), hyperthermia and cryotherapy, agents that attenuate any side effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (nitrogen mustards, chlorambucil, cyclophosphamide, Melphalan, ifosfamide), Methotrexate (antimetabolite), purine antagonists and pyrimidine antagonists (6-mercaptopurine, 5-fluorouracil, antimetabolite, purine antagonists, and pyrimidine antagonists (6-mercaptopurine, 5-fluorouracil), Cytarabine, difluocytosine), spindle poison (Vinblastine), Vincristine (Vincristine), Vinorelbine (Vinorelbine), paclitaxel, docetaxel), podophyllotoxin (podophyllotoxin) (epipodophyllotoxin (Etoposide), Irinotecan (Irinotecan), Topotecan (Topotecan)), antibiotics (Doxorubicin), Bleomycin (Bleomycin), Mitomycin (Mitomycin)), nitrosourea (Carmustine, Lomustine), inorganic ions (cisplatin, carboplatin), enzymes (asparaginase) and hormones (Tamoxifen), Leuprolide (Leuprolide), flulibamide (Flutamide) and Megestrol (Megestrol)), just to name a few. More complete discussion of the latest therapySee alsohttp: // www.nci.nih.gov/, http: htm and The Merck Manual (17 th edition, 1999),the entire contents of which are incorporated herein by reference.

In another aspect, the invention provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the invention, and in certain embodiments additional approved therapeutic agents for use as a combination therapy. Optionally, the container(s) may carry a notice in the form prescribed by a governmental agency regulating the manufacture, use and sale of pharmaceuticals, which notice reflects approval by the agency of the manufacture, use and sale of pharmaceuticals for human consumption.

Equivalents of the formula

The following representative examples are intended to help illustrate the invention, but are not intended to, and should not be construed to, limit the scope of the invention. Indeed, various modifications of the invention, as well as several other embodiments thereof, in addition to those shown and described herein will become apparent to those skilled in the art from the entirety of this document, including the examples below and the scientific references and patent documents cited herein. It should be further appreciated that the contents of the cited references are incorporated herein by reference to help clarify the state of the art. The following examples contain important additional information, exemplification and guidance principles suitable for practicing the various embodiments of the invention and their equivalents.

Examples of the invention

Example 1: synthesis of 9, 10-dehydro-12, 13-deoxyepothilone

This example illustrates the synthesis of trans-9, 10-dehydro-12, 13-desoxyepothilone B, 26-trifluoro-12, 13-desoxyepothilone B and the bioassay of these compounds.

Fluorinated derivatives of epothilones were prepared and tested with known increases in the pharmacokinetic and chemotherapeutic index of other fluorine substituted drugs (Ojima, I.; Inoue, T.; Chakravarty, S.; J. fluorineneChem.1999, 97; Newman, R.A.; Yang, J.; Finlay, M.R.V.; Cabral, F., Vourlousis, D.; Stephens, L.C; Troncoso, P.; Wu, X.; Lolottis, C.J.; Nicolaou, K.C; Navone, N.M. cancer mother Pharmacol.2001, 48, 319-326; each of which is incorporated herein by reference).

1[16]dEpoB 2 26-F₃-[16]dEpoB

To obtain compound 2, we sought to synthesize dEpoB (1, scheme 3) using the highly convergent method recently reported in our laboratories by first synthesizing epothilone 490(6, dehydrodeoxyEpoB) (Biswas, K.; Lin, H.; Njardarson, J.T.; Chappell, M.D., Chou, T.C., Guan, Y.; Tong, W.P., He, L.; Horwitz, S.B., Danishefy, S.J.Am. chem.Soc 2002, 124 (33); 9825 sk9832; Rivkin, A.; Njarrson, J.T.; Biswas, K.; Chou, T.C.; Danishjeffeny, S.J.Org.chem.2002, 67, 7737, 7740; each of which is incorporated herein by reference). In this synthesis, a pendant vinyl group is introduced into compound 4 by Stille stereospecific coupling of vinyl iodide precursor 3 with tri-n-butyl vinyl stannane. Deprotection after ring-closing displacement affords compound 6, which can then be converted to dEpoB (I) by regioselective diimide reduction.

Scheme 3 Synthesis of epothilone 490

Attention is first drawn to the synthesis of 15 (scheme 4). The alkylation reaction of previously reported lithium enolate 7(Chappell, M.D.; Stachel, S.J.; Lee, C.B.; Danishefsky, S.J. org.Lett. (2000, 2(11), 1633-; 1636; incorporated by reference) with iodide 8 (synthesized from a known alcohol 16 using TMSI in dichloromethane) produced 9 in 78% yield with high non-specular stereoselectivity (> 25: 1 de). Compound 9 becomes shown as 10 by three steps. Attempts have been made to add methylmagnesium bromide to the Weinreb amide bond of 10, but without success. The failure of the reaction is due to the presence of the iodoalkyl linkage. However, our goal can be achieved by changing the order of the two C-C bond formation steps. Thus, the desired ketone 11 can be obtained by adding methyl Grignard reagent (Grignard reagent) after reaction of 10 with vinyl tributyltin under Stille conditions. Subsequent deprotection of triethylsilyl ether after condensation of ketone 11 with phosphine oxide 12 yields fragment 13 in high yield. The resulting 13 was esterified with a C1-C10 acid fragment (Biswas, K.; Lin, H.; Njardarson, J.T.; Chappell, M.D., Chou, T.C., Guan, Y.; Tong, W.P., He, L.; Horwitz, S.B., Danischefsky, S.J.J.Am.Chem.Soc.2002, 124 (33); 9825-membered 9832; Rivkin, A.; Njardarson, J.T.; Biswas, K.; Chou, T.C.; Danischesky, S.J.J.Org.Chem.2002, 67, 7737-membered 7740; incorporated herein by reference), to give the desired 15 at 75% yield (FIG. 4).

Scheme 4. Synthesis of RCM precursor 15

Unfortunately, when attempting to carry out a ring closure displacement reaction of 15 with a second generation Grubbs catalyst in methylene chloride (Reviews: Grubbs, R.H.; Miller, S.J.; Fu, G.C.Acc.Chem.Res.1995, 28, 446; Tmka, T.M; Grubbs, R.H.Acc.Chem.Res.2001, 34, 18; Alkene Metathesis in Organic Chemistry (eds.: F ü rstner, A.; Springer, Berlin, 1998); F ü rstner, A.Angew.Chem.Ed.Engl.2000, 39, 3012; Schrock, R.R.Top. Organomet.Chem. (1998, 1, 1; each of which is incorporated herein by reference), the primary dimerization reaction (equation 1, 1) occurs. Since RCM is known to be well suited for the 5 → 6 related environment, we naturally attribute the failure in case 15 to C₁₂Upper trifluoro benzene radicalThe presence of a methyl group.

It is predicted that the adverse effect of the presence of the 26-trifluoro substituent on the desired reaction can be mitigated by adding a carbon spacer between the RCM reaction center and the trifluoromethyl group. Thus, we synthesized 19 (equation 2) by ring closure displacement of 18, which places the trifluoromethyl group in a 17-membered ring environment containing a ship-type (1, 4) -diene.

The synthetic procedure for 19 starts with the preparation of compound 21, 21 corresponding to the O-alkyl moiety of our proposed RCM substrate (scheme 5). Starting from allylation at 10, this time under the free radical reaction conditions shown (Keck, G.E.; Yates, J.B.J.Am.chem.Soc.1982, 104, 5829; review: Curran, D.P.Synthesis, 1988, part 1, page 417 and 439; part 2, page 489; each of which is incorporated herein by reference). After this conversion, the alkylated product is reacted with methyl magnesium bromide, thereby obtaining the desired ketone 20. This compound was condensed with phosphine oxide 12, followed by deprotection of triethylsilyl ether to afford 21 in high yield.

Scheme 5 Synthesis of alcohol fragment 21

(a) i) allyl tributyltin AIBN, benzene, 80 ℃, 3h 74%; ii)

MeMgBr，0℃，93％；(b)i)12，n-BuLi，THF，-78℃，30

min.，ii)20，-78℃ to rt.85％；iii)HOAc∶THF∶H₂O(3∶1∶1)，

98％；(c)TMSI，CH₂Cl₂，0℃，92％

Esterification of 21 with C1-C10 acid fragment 14 gave 75% yield of the proposed RCM precursor 18 (scheme 6). It is desirable in this case that a ring closure metathesis of 18 can be accomplished in methylene chloride using a second generation Grubbs catalyst. Like the 5 → 6 transition, this reaction provided only the trans isomer 22 in 57% yield. Finally, reductive cleavage of the trichloroethoxycarbonyl protecting group with zinc and acetic acid followed by deprotection of the TES ether with HF-pyridine yields the compounds at C₁₂The desired compound 19, containing a trifluoromethyl functionality, although in the context of the 17-membered ring series.

Scheme 6.27-F₃Synthesis of-ddEpoB (19)

The cytotoxic activity of the synthesized 19 was evaluated. As shown in the following Table 1-1, previously reported [17 ]]ddEpoB (23) and 27-F₃-[17]direct comparison of ddEpoB (19) shows that the novel perfluorinated compounds have equally high cytotoxic potency.

TABLE 1-1 for tumor cell lines^αIn vitro cytotoxicity (IC)₅₀)

Compound (I)	CCRF-CEM(IC₅₀(μM)^α)	CCRF-CEM/VBL(IC₅₀(μM)^α)
Compound (I)	CCRF-CEM(IC₅₀(μM)^α)	CCRF-CEM/VBL(IC₅₀(μM)^α)	27-F₃-[17]ddEpoB(19)	0.068	0.191
[17]ddEpoB(23)	0.040	0.126	27-F₃-[17]ddEpoB(19)	0.068	0.191
[17]ddEpoB(23)	0.040	0.126	[16]ddEpoB(6)	0.020	0.068

After 72 hours of inhibition^αXTT analysis. CCRF-CEM is a human T-cell acute lymphoblastic leukemia cell line. CCRF-CEM-_VBL100、CCRF-CEM/_VM1And CCRF-CEM-_TaxolCell lines all overexpress P-glycoprotein and display a multi-drug resistance phenotype against MDR-associated anti-cancer agents (Ojima, I.; Inoue, T.; Chakravarty, S.; J. fluorinene chem.1999, 97; Newman, R.A.; Yang, J.; Finlay, M.R.V.; Cabral, F., Vourlomis, D.; Stephens, L.C; Troncoso, P.; Wu X.; Loothtis, C.J.; Nicolaou, K.C.; Navone, N.M. Chemoterhermharma.2001, 48, 319-326; each of which is incorporated herein by reference).

Although trifluoromethyl isospecific volume substitution (isospecific stabilization) had little effect on overall cytotoxic activity, preliminary data on metabolic degradation studies in mouse plasma suggest: 19 is significantly more stable than the matrix 23. Epothilones 19 and 23 were exposed to nude mouse and human plasma, with 23 degrading within 30 minutes, whereas epothilone 19 remained largely intact. This finding is considered to be quite encouraging, as pharmacokinetic problems may be critical in the practical application of any epothilone as a drug.

Can pass through and 27-F₃-[17]Highly convergent methods related to the method used in the synthesis of ddEpoB (19) to complete 26-F₃Synthesis of-dEpoB (2). Thus, fragments of similar composition can serve as key building blocks (scheme 7). It is contemplated that the acyl moiety 25 may serve as the polyacrylate domain and the alkyl moiety 21 or 24 may be prepared as previously described at the outset. The combination of the two segments 21, (24) and 25 can be initiated by an esterification reaction and ended by a subsequent ring-closing metathesis reaction. Finally, breaking the protecting groups can provide the desired analogs 28 and 29. Chemically selective reduction of the 9, 10-olefins of 28 and 29 will provide dEpoB (1) and the desired 26-F₃-12, 13-deoxyEpoB (2).

FIG. 7 is a schematic view of

Trans-9, 10-dehydro-12, 13-deoxy EpoB (28)26-F₃-12, 13-deoxy EpoB (1)

Trans-9, 10-dehydro-12, 13-deoxy EpoB (29) 26-F₃-12, 13-deoxy EpoB (2)

The synthesis of 1 and 2 begins with the preparation of the acyl moiety 25. The previously reported ketone 30 was subjected to an aldol condensation reaction with 3l of an easily available aldehyde. When the "lithiation" 30 is deprotonated and reacted with 31, a smooth condensation reaction yields a mixture of 5.3: 1 aldol products 32 and 33. Most of diastereomer 32 was easily isolated by flash chromatography and protected as TBS silyl ether. The diisopropylacetal group is hydrolyzed under acid catalysis to obtain the ketoaldehyde 34, which creates conditions for the second aldol condensation reaction. After carrying out the "titanyl" tert-butyl ester process described above with the novel aldehyde 34 as coupling partner, the desired aldol product 35 is obtained with high diastereoselectivity (dr > 20: 1) and yield (86%). After the 35C 3 alcohol was protected with the TES silyl group, the deprotection of the benzyl ether was performed. The product primary hydroxyl group is oxidized to obtain the corresponding aldehyde, which is then converted into a terminal olefin by Wittig reaction (Wittig reaction) to obtain 36 in high yield. Finally, hydrolysis of the tert-butyl ester of 36 with TEOSTf yielded the acyl moiety 25 (82%) and the byproduct 37 (14%), which byproduct 37 can be converted to the acyl moiety 38 in high yield. 38 was identical to that previously obtained from other procedures in the dr sinha laboratory (Scripps).

Schematically shown in FIG. 8.

Esterification of allyl alcohols 21 and 24 with C1-C9 acid fragment 25 affords the corresponding RCM cyclized precursors 26 and 27, respectively (scheme 9).

Schematically shown in FIG. 9.

Ring closure metathesis of 26, 27 and 54 was then carried out in toluene using a second generation Grubbs catalyst, as in earlier studies, producing only trans isomers 39a, 40a and 55 and the corresponding by-products 39b, 40b and 56. Finally, the silyl ether was deprotected with HF-pyridine to obtain the desired compounds 28, 29 and 57. 28 are not identical to those previously obtained from the epothilone program in the dr. While believes that 28 has been synthesized, 12, 13E isomer 41 has been synthesized inadvertently, which explains why he observed poor biological activity. Therefore, we first synthesized 28 and tested the compound for anti-tumor activity.

Evaluation of total synthesis 28, 29 and 2 has been evaluated for various cell types to determine their antitumor efficacy. As shown in tables 1-2, all three compounds showed high cytotoxic activity against various sensitive and resistant tumor cell lines. A direct comparison of 28 with the previously reported dupob (1) indicates that the new compound has almost three times the potency.

TABLE 1-2 for tumor cell lines^αIn vitro cytotoxicity (IC)₅₀)

Tumor cell strain	IC₅₀(μM)^α
	IC₅₀(μM)^α				28	29	dEpoB(1)	57
	CCRF-CEMCCRF-CEM/VBL₁₀₀CCRF-CEM/Taxol	0.00140.00650.0017	0.00350.02100.0057	0.00360.0140.0057	28	29	dEpoB(1)	57	0.000510.01060.00073

After 72 hours of inhibition^αXTT analysis. CCRF-CEM is a human T-cell acute lymphoblastic leukemia cell line. CCRF-CEM-_VBL100、CCRF-CEM/_VM1And CCRF-CEM-_TaxolCell lines all over-express P-glycoprotein and display a multi-drug resistance phenotype against MDR-related anticancer agents (prie, g.; Thibonnet, j.; Abarbri, m.; Duch e, a.; Parrain, j. synlett (1998, 839); incorporated herein by reference).

To increase the overall yield of syntheses 28, 29 and 2, we decided to carry out the RCM reaction in the absence of thiazole-substituted olefin, thus avoiding the formation of undesirable by-products 39b and 40 b. Examples of previously reported silyl ethers of 42 and 20Deprotection affords hydroxyketones 43 and 44. Products hydroxyketones 43 and 44 and C₁-C₉The acid moiety 25 undergoes esterification to yield the corresponding RCM cyclized precursors 45 and 46, respectively (scheme 10). The ring closure metathesis reactions of 45 and 46 were then carried out in toluene using a second generation Grubbs catalyst, which produced only trans isomers 47 and 48 in high yield as in previous studies. The insertion of the thiazole moiety gave high yields of 39a, 40a and 55. Both silyl ethers were deprotected with HF-pyridine to give 28 and 29. Finally, selective reduction of C9-C10 alkenes affords the corresponding epothilones 1 and 2. The structure of 28 is strictly demonstrated by its conversion to 1 with high yield. The total synthesis of 1 is considerably simplified with respect to the previously practiced route. Thus, the use of 31, obtained from a chiral source and readily available, is certainly a great improvement over (S) -2-methyl-4-pentenal, which relies on the participation of a chiral auxiliary in its synthesis.

Schematic diagram 10

For compound 28, which is held under control and whose structure is well documented, we have surprisingly found that its spectral properties are not the same as those of compounds previously reported to be presumed to be the same entity. However, it is clear from a review of the past that 28 has not been previously prepared, and indeed, the entire family of (E) -9, 10-dehydroepothilones reported herein is a novel class of compounds.

Testing of synthetic analogs (2, 28 and 29) in a cell culture environment revealed that they were more potent in inhibiting various sensitive and MDR tumor cell lines than our clinical term dEpoB (1) (tables 1-3). It is noted that Epo3(28) is the first 12, 13-deoxyepothilone compound with substantially improved cytotoxicity relative to doepb (1).

TABLE 1-3 for tumor cell lines^αIn vitro cytotoxicity (IC)₅₀)

Compound (I)

CCRF-CEM(C)(μM)

C/VBL₁₀₀(μM)

C/paclitaxel

			(μM)
			(μM)	Epo1(1，dEpoB)Epo2(2)Epo3(28)Epo4(29)	0.00360.00410.00090.0035	0.0160.00800.00420.0210	0.00460.0180.00120.0057

After 72 hours of inhibition^αXTT analysis. CCRF-CEM is a human T-cell acuteA lymphotropic leukemia cell line. CCRF-CEM/VBL₁₀₀The cell lines were vinblastine resistant and CCRF-CEM/paclitaxel resistant to paclitaxel.

Epothilones 2, 28 and 29(Epo2-4) exhibited significant cytostatic effects against multiple drug resistant tumors facilitating the determination of plasma stability of these novel (E) -9, 10-congeners. For example, the recently described (E) -10, 11-dehydro-dEpoB (1, with CH at C-12) in terms of lactone opening₃Group) showed very poor plasma stability. This plasma instability inhibits the development of (E) -10, 11-dehydro-dppob. In contrast, a decrease in the degree of drug degradation to 1/7 was observed when 2, 28 and 29(Epo2-4) were exposed to murine plasma compared to dupob (1). From a pharmaceutical availability point of view, the stability constitutes a substantial improvement with respect to dppob (see figure 9).

The combination of cytotoxicity and plasma stability data prompted us to synthesize large amounts of 28(Epo3) to determine its in vivo efficacy in nude mice bearing human tumor xenografts. Epothilone 28(Epo3) had significantly improved potency over dmepob in inhibiting the growth of transplanted tumors (see figure 10). This improved potency and plasma stability greatly reduced the drug dose of 28(Epo3) in the context of xenografts (by an order of magnitude).

We found in earlier studies that epothilone B (12, 13-epoxide) was significantly more cytotoxic than its 12, 13-deoxy analogue (dpob). In any case, the oxygen scavenging compound is more desirable from the therapeutic index point of view. Recently, we reported the total synthesis of (E) -9, 10-dehydro-12, 13-desoxyepothilone B (28) by a stereoselective ring closure displacement method. The results show that unsaturation of E-9, 10 in normal Z-12, 13-alkenes (see Compound 1) greatly enhances in vitro potency. Rather, this can be transferred to the in vivo environment in xenografted mice. Moreover, compound 28 has many medical advantages over dupob (1). This allows the dose level of 28 to be reduced by an order of magnitude relative to 1 in xenograft experiments.

Therefore, we wanted to know if the inclusion of a C9-C10 alkene in epothilone B (51, EpoB) would change its biological properties in the same way.

(E) -9, 10-dehydro-dEpoB (28) (E) -9, 10-dehydro EpoB (49)

Epoxidation of 28 with 2, 2' -dimethyldioxirane (DMDO) at high chemoselectivity to the more substituted C12-C13 olefin gave 87% yield of (E) -9, 10-dehydroepothilone B (49) and its diastereoisomer (50) in a 1: 2.6 ratio. The stereochemistry of the epoxide is determined by selective diimide reduction of the C9-C10 double bond. Spectroscopic property testing of these products revealed that the minor product (49) was dEpoB. The preferential alpha-epoxidation at 28 is in sharp contrast to the highly stereoselective epoxidation of dEpoB, which occurs in the beta plane to form EpoB (Meng, D.; Bertinito, P.; Balog, A.; Su, D. -S.; Kamenecka, T.; Sorensen, E.J.; Danishefsky, S.J.J.J.Am.chem.Soc.1997, 119, 10073; incorporated herein by reference).

Schematic diagram 11

The activity of (E) -9, 10-dehydroepothilone B (51) against various types of cells was evaluated to determine its antitumor efficacy. As shown in tables 1-4, (E) -9, 10-dehydroepothilone B (49) demonstrates high cytotoxic activity against a variety of sensitive and resistant tumor cell lines. A direct comparison of 49 with EpoB (51) shows that the novel analogue has almost 3 times the potency of EpoB (51), making it one of the most potent epothilones reported to date. Interestingly, the α -epoxide series (50, 52) showed much lower activity than EpoB (51). The table below shows the in vivo findings for compound 49.

TABLE 1-4 for tumor cell lines^αIn vitro cytotoxicity (IC)₅₀)

Compound (I)	CCRF-CEM	CCRF-CEM/VBL	CCRF-CEM/Taxol
Compound (I)	CCRF-CEM	CCRF-CEM/VBL	CCRF-CEM/Taxol	1(dEpoB)2851(EpoB)4950	0.00360.00090.000620.000230.0134	0.0160.00420.00370.000320.0959	0.00460.00120.00110.000420.0802

52

0.083

0.4519

0.1507

After 72 hours of inhibition^αXTT analysis. CCRF-CEM is a human T-cell acute lymphoblastic leukemia cell line. All CCRF-CEM/VBL and CCRF-CEM/paclitaxel cell lines overexpress P-glycoprotein and display a multi-drug resistance phenotype against MDR-related anticancer agents.

Therapeutic efficacy of 9, 10-de-H-EpoB in nude mice bearing MX-1 xenografts (6 hr intravenous infusion, n ═ 4)

In summary, an efficient stereoselective total synthesis of 28(Epo3) was set forth above, and the synthesis of doepb (1) itself was carried out after performing the regioselective diimide reduction. Thus, the methods described herein can be used directly to prepare the corresponding trifluoro analogs 2 and 29(Epo 4). In addition, epoxidation of 28 can yield 49 and 50, which upon reduction with a regioselective imide yield epothilone B (51) and 52. The data reported above indicate the emergence of a most promising family of novel anti-cancer drugs that are suitable for possible advancement to human clinical settings evaluation after further intermediate evaluation. In addition, the novel synthesis method comprises a significant practical improvement in the total synthesis of dEpoB and epothilone B.

Experiment of

The general method comprises the following steps: unless otherwise stated, reagents from commercial suppliers were used without further purification. The following solvents were obtained from a solvent drying system (through a pre-packed alumina column) and used without further drying: tetrahydrofuran, dichloromethane, diethyl ether, benzene and toluene. All air and water sensitive reactions were carried out in a baked glass device under a positive pressure of prepurified argon. NMR (C)¹H and¹³C) spectra were recorded as a single note on a Broker AMX-400MHz or Bruker Advance DRX-500MHz reference CDCl₃(¹H is7.27ppm and¹³c77.0 ppm). Infrared spectra (IR) were obtained on a Perkin-Elmer FT-IR model 1600 spectrometer. The optical rotation was obtained on a JASCO model DIP-370 digital polarimeter at 22. + -. 2 ℃. Analytical thin layer chromatography was performed on e.merck silica gel 60F 254 plates. Compounds with no UV activity were visualized by immersing the plates in a solution of cerium ammonium molybdate or p-anisaldehyde and heating. Silica gel chromatography was performed on Davisil * (grade 1740, type 60A, 170-400 mesh) silica gel with the specified solvents.

Abbreviation and abbreviations

TES, triethylsilyl; TBS, dimethyl tert-butylsilyl; EDCI, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; HF-PY, hydrogen fluoride in pyridine; DMAP, 4-N, N-dimethylaminopyridine; DCM, dichloromethane; DMF, N-dimethylformamide; THF, tetrahydrofuran.

Compound 32: to a freshly prepared solution of LDA (11.6mmol) in THF (25mmol) at-78 deg.C was added dropwise a solution of ketone 30(2.4g, 10.4mmol) in THF (6.8 mL). After stirring at-40 ℃ for 0.5 h, the mixture was cooled to-90 ℃. A solution of aldehyde 31(1.38g, 7.72mmol) in THF (6.8mL) was added dropwise. After stirring at-90 ℃ for 35 minutes, saturated NH was added₄The reaction was quenched with aqueous Cl (15mL) and extracted with EtOAc (50 mL. times.3). The combined organic extracts were extracted with Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/EtOAc 15: 1 to 12: 1) to yield 32(2.09g, 66%) and isomer 33(0.39g, 12%), both as yellow oil. 32: [ alpha ] to]_D ²⁵13.1(c 1.22，CHCl₃) (ii) a IR (film) v3494, 2972, 2932, 1708, 1454, 1380, 1329, 1120, 1038, 998, 734cm^-1；¹H NMR(400MHz，CDCl₃)δ0.98(3H，d，J＝6.9Hz)，1.06(3H，d，J＝6.9Hz)，1.10(3H，d，J＝6.1Hz)，1.14(3H，d，J＝6.9Hz)，1.15(3H，s)，1.17(3H，d，J＝6.2Hz)，1.18(3H，s)，1.20(3H，d，J＝6.2Hz)，1.81-1.92(1H，m)，3.33(1H，qd，J＝7.0，2.2.Hz)，3.51(1H，dd，J＝8.9，6.3Hz)，3.64(1H，d，J＝1.8Hz)，3.66-3.71(2H，m)，3.78-3.86(2H，m)，4.51(1H，d，J＝12.0Hz)，4.54(1H，d，J＝12.0Hz)，4.58(1H，s)，7.25-7.35(5H，m)；¹³C NMR(100MHz，CDCl₃)δ10.0、14.3、20.5、21.3、21.9、22.5、23.5、23.6、36.4、42.1、54.1、69.8、71.2、72.8、73.3、73.4、103.8、127.6、127.7(2C)、128.5(2C)、138.9、221.6；C₂₄H₄₀O₅LRMS (ESI) calculation of Na [ M + Na ]⁺]431.3, Experimental value 431.4.

Compound 32a (not shown): to a cold (-40 ℃) solution of alcohol 32(1.01g, 2.47mmol) and 2, 6-lutidine (691 μ L, 5.93mmol) was added TBSOTf (681 μ L, 3.00mmol) and the mixture was heated to-20 ℃ over 3.5 hours. With saturated NaHCO₃The reaction was stopped with (10mL) aqueous solution. After extraction with hexane (50 mL. times.3), the combined organic extracts were extracted with Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/EtOAc 50: 1) to give 32a as a colourless oil (1.25g, 2.39mmol, 97%); [ alpha ] to]_D ²⁵-19.7(c 0.58，CHCl₃) (ii) a IR (film) v2966, 2931, 1696, 1455, 1378, 1320, 1255, 1091, 1044, 991, 873, 838, 773cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.08(6H, s), 0.89(9H, s), 0.99(3H, d, J ═ 7.0Hz), 1.04(3H, d, J ═ 7.0Hz), 1.07(3H, s), 1.14(3H, d, J ═ 6.1Hz), 1.17(3H, s), 1.17(3H, d, J ═ 6.0Hz), 1.20(3H, d, J ═ 6.2Hz), 1.76-1.85(1H, m), 3.21(1H, dd, J ═ 9.2, 7.3Hz), 3.32(1H, quintuple, J ═ 7.4Hz), 3.62(1H, dd, J ═ 9.2, 7.5, 3.5, 3H, 7.78, 7.3.78 Hz), 3.32(1H, dd, 7.4H, 7.4Hz), 3.62(1H, dd, 3.5, 7.7.7.4H, 7.7.4 Hz), 3.4H, 3.7.3.7.7.4H, 1.7.7.4 Hz), 1H, 1.7.7.7.3.3.3.3.7.4H, 1H, 1.3.7.7.3.7.7-7.37(5H，m)；¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.3、15.6、16.8、18.7、18.8、21.8、22.1、22.5、23.5、23.7、26.4(3C)、39.0、46.2、54.0、69.7、70.9、72.1、73.4、76.7、103.1、127.6、127.8(2C)、128.5(2C)、139.0、218.9；C₃₀H₅₄O₅LRMS (ESI) calculation of SiNa [ M + Na ]⁺]545.4, experimental value 545.4.

Compound 34: 32a (3.03g, 5.79mmol) was reacted with p-TsOH. H₂O (286mg) in aqueous THF (64mL, THF/H)₂O4: 1) was heated under reflux for 6.5 hours. The reaction mixture was cooled to room temperature and poured into saturated NaHCO₃(25mL) in aqueous solution. After extraction with EtOAc (100mL +50 mL. times.2), the combined organic layers were washed with brine, washed with Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/EtOAc 50: 1 to 30: 1) to give 34(2.37g, 5.64mmol, 98%) as a colorless oil: [ alpha ] to]_D ²⁵-25.8(c 0.515，CHCl₃) (ii) a IR (film) v2955, 2931, 1731, 1696, 1455, 1360, 1255, 1091, 1026, 873, 826, 767cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.06(3H, s), 0.07(3H, s), 0.90(9H, s), 0.95(3H, d, J ═ 7.1Hz), 1.03(3H, d, J ═ 7.0Hz), 1.28(3H, s), 1.33(3H, s), 1.73-1.82(1H, m), 3.16(1H, dd, J ═ 9.2, 6.1Hz), 3.28(1H, quintuple, J ═ 7.3Hz), 3.55(1H, dd, J ═ 9.2, 6.7Hz), 3.91(1H, dd, J ═ 7.8, 2.1Hz), 4.46(2H, s), 7.27-7.36(5H, m), 9.58(1H, s);¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.5、15.7、16.3、18.6、19.8、20.1、26.3(3C)、39.1、47.0、61.1、71.9、73.4、75.8、127.7、128.0(2C)、128.5(2C)、138.6、201.3、213.3；C₂₄H₄₀O₄LRMS (ESI) calculation of SiNa [ M + Na ]⁺]443.3, Experimental value 443.2.

Compound 35: freshly prepared LDA (18mL in Et) at-78 deg.C₂0.5M solution of O, 9.0mmol) in Et₂To a solution of O (20mL) was added tert-butyl acetate (1.16mL, 8.61 mmol). After stirring for 50 minutes, CpTiCl (OR) was added dropwise over 65 minutes via syringe pump₂(100mL in Et₂0.1M solution in O, 10.0 mmol). Stirring was carried out for 20 minutes, the reaction mixture was heated to-30 ℃, stirred for 50 minutes and cooled again to-78 ℃.34 (2.42g, 5.75mmol) was dissolved in Et dropwise over 10 min₂O (9mL) and the resulting mixture was stirred at-78 ℃. After stirring for 2 hours, THF (5 MH) was used₂O, 37mL) was quenched and stirred at room temperature for 2 hours. After addition of water (40mL), the mixture was stirred for an additional 1 hour. Through Celite (Et)₂O wash) the precipitate formed was filtered off and the filtrate was washed with water (40 mL). Et for aqueous layer₂O (100 mL. times.2) extraction, washing the combined organic layers with brine (40mL), Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/EtOAc 10: 1) to give 35(2.65g, 4.94mmol, 86%) as a light yellow oil; [ alpha ] to]_D ²⁵-20.3(c 1.0，CHCl₃) (ii) a IR (film) v3523, 2957, 2930, 2856, 1732, 1700, 1472, 1368, 1252, 1152, 1091, 1042, 986, 834, 774cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.07(3H, s), 0.90(9H, s), 0.99(3H, d, J ═ 7.0Hz), 1.07(3H, d, J ═ 7.0Hz), 1.10(3H, s), 1.14(3H, s), 1.47(9H, s), 1.77-1.83(1H, m), 2.26(1H, dd, J ═ 16.0, 10.0Hz), 2.34(1H, dd, J ═ 15.9, 2.7Hz), 3.23(1H, dd, J ═ 9.2, 7.1Hz), 3.35(1H, d, J ═ 2.7Hz), -dt, 3.36(1H, quintuple, J ═ 7.0), 3.07(1H, 7H, s), 0.90(9H, s), 0.5 (7H, 7.7.7 Hz), 7.7H, 7.7.7.7H, 5(1H, 7.7Hz), 7.7.7H, 7H, 7.7H, 7J ═ 5.5 (7H, 7.7.7H, 7.7.7.7H, 7H, 7.7.7H, 7H;¹³C NMR(100MHz，CDCl₃)δ-3.5、-3.4、16.3、16.7、18.7、20.1、21.6、26.4(3C)、28.3(3C)、38.0、39.1、45.8、51.8、72.2、72.9、73.5、76.7、81.4、127.7、128.0(2C)、128.5(2C)、138.8、172.7、219.6；C₃₀H₅₂O₆LRMS (ESI) calculation of SiNa [ M + Na ]⁺]559.3, Experimental value 559.4.

Compound 35a (not shown): to a mixture of alcohol 35(10.2g, 8.9mmol) and imidazole (2.70g, 39.7mmol) in DMF (25mL) at 0 deg.C was added TESCl (3.3mL, 19.8mmol) and the mixture was stirred at room temperature for 2 h. Then saturated NaHCO₃The reaction was stopped with (50mL) aqueous solution. After extraction with hexane (500mL +120 mL. times.2), the combined organic extracts were washed with water (30 mL. times.2) and brine (30mL) in that order, and Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/EtOAc 40: 1) to give 35a (12.1g, 18.5mmol, 98%) as a colorless oil: [ alpha ] to]_D ²⁵-38.0(c 0.46，CHCl₃) (ii) a IR (thin film) v2955, 2877, 1733, 1697, 1456, 1367, 1298, 1251, 1155, 1099, 988, 835, 742cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.05(6H, s), 0.57-0.68(6H, m), 0.89(9H, s), 0.95(9H, t, J ═ 7.9Hz), 0.99(3H, d, J ═ 7.0Hz), 1.02(3H, d, J ═ 6.8Hz), 1.04(3H, s), 1.18(3H, s), 1.45(9H, s), 1.70-1.79(1H, m), 2.16(1H, dd, J ═ 17.0, 7.0Hz), 2.40(1H, dd, J ═ 17.0, 3.1Hz), 3.22(1H, dd, J ═ 9.1, 7.5Hz), 3.31(1H, quintuple, J ═ 6.9, 3.61(1H, 7.5Hz), 3.31(1H, 7.5H, 3.7.7.5H, 3.7.5H, 3.7.7.7.5 (3.7H, 3.7.7.7H, 3.7.7H, 3.7.7.7.7.7H, 3.7.7H, 3.7.7.7.7, 3.5Hz), 3.7.7.7.7, 3.7.7.7.7.7H, 3.;¹³C NMR(100MHz，CDCl₃)δ-3.5、-3.4、5.3(3C)、7.3(3C)、15.3、16.9、18.7、20.1、23.4、26.4(3C)、28.3(3C)、39.1、41.1、46.2、53.4、72.2、73.4、74.3、76.7、80.6、127.6、127.9(2C)、128.5(2C)、138.9、171.5、218.4；C₃₆H₆₆O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]673.4，Experimental value 673.5.

Compound 35b (not shown): to an agitated solution of 35a (4.37g, 6.72mmol) in THF (67mL) was added Pd/C (from Acros, 10% wt, 437mg) in H₂The mixture was stirred under an atmosphere. After stirring for 2.2 hours, the mixture was filtered through a pad of Celite, which was washed with THF (120 mL). The filtrate was concentrated and purified by flash column chromatography (SiO)₂hexane/EtOAc ═ 30: 1 to 10: 1) to give colorless oil 35b (3.53g, 6.28mmol, 94%); [ alpha ] to]_D ²⁵-16.1(c 0.62，CHCl₃) (ii) a IR (thin film) v3543, 2956, 1732, 1696, 1472, 1368, 1299, 1252, 1155, 1100, 988, 837, 775, 742cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.10(3H, s), 0.12(3H, s), 0.60-0.68(6H, m), 0.93(9H, s), 0.96(9H, t, J ═ 8.0Hz), 0.99(3H, d, J ═ 7.1Hz), 1.10(3H, d, J ═ 6.9Hz), 1.14(3H, s), 1.20(3H, s), 1.45(9H, s), 1.46-1.55(1H, m), 2.21(1H, dd, J ═ 17.2, 7.1Hz), 2.39(1H, dd, J ═ 17.2, 2.8Hz), 2.54(1H, t, J ═ 5.8, -OH), 3.30(1H, quintuple, J ═ 6.9, J ═ 9.8, 3H, J ═ 5.8Hz), 3.5H, J ═ 5.8, 5H, 5.8-H, 3.6, J ═ 4.6H, 3.6, 3H, 5, 1.6, 3.6, J ═ 4, 1H, 1, 1.6, 1H, 1, 1.6, 1;¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.5、5.3(3C)、7.2(3C)、16.0、16.1、18.6、20.0、23.4、26.4(3C)、28.3(3C)、40.0、40.9、46.9、53.7、64.8、73.3、78.1、80.9、171.7、218.5；C₂₉H₆₀O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]583.4, Experimental value 583.5.

Compound 35c (not shown): to a mixture of alcohol 35b (3.53g, 6.28mmol) and powdered MS4A (freshly activated, 2.50g) was addedCH₂Cl₂To a stirred mixture (32mL) was added NMO (1.17g, 10.0mmol) followed by TPAP (132mg, 0.377 mmol). After stirring at room temperature for 35 minutes. Passing through a silica gel column (Hexane/Et)₂O8: 1) to give 35c (3.34g, 5.98mmol, 95%) as a colorless oil; [ alpha ] to]_D ²⁵-69.6(c 0.25，CHCl₃) (ii) a IR (thin film) v2955, 2878, 1732, 1696, 1472, 1368, 1253, 1155, 1097, 989, 837cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.09(3H, s), 0.10(3H, s), 0.59-0.68(6H, m), 0.89(9H, s), 0.95(9H, t, J ═ 8.0Hz), 1.08(3H, s), 1.11(3H, d, J ═ 6.9Hz), 1.14(3H, d, J ═ 7.1Hz), 1.24(3H, s), 1.45(9H, s), 2.19(1H, dd, J ═ 17.0, 6.7Hz), 2.33(1H, qt, J ═ 7.1, 2.2Hz), 2.41(1H, dd, J ═ 17.0, 3.3Hz), 3.28(1H, quintuple, J ═ 7.5Hz), 4.07(1H, dd, 2.9H, 3.9H, dd, 3.3Hz), 3.9H, dd, 2.9H, 3.9H, dd, 3.9 Hz);¹³C NMR(100MHz，CDCl₃)δ-3.8、-3.5、5.3(3C)、7.2(3C)、12.6、15.6、18.5、20.5、23.3、26.2(3C)、28.3(3C)、41.1、46.9、51.1、53.5、74.0、76.5、80.7、171.1、204.3、218.0；C₂₉H₅₈O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁴]581.3, Experimental value 581.3.

Compound 36: MePPh in THF (40.0mL) was treated with t-BuOK (6.57mL of a 1.0M solution in THF, 6.57mmol) at 0 deg.C₃I (2.56g, 7.18 mmol). After stirring at 0 ℃ for 20 min, the resulting suspension was cooled to-78 ℃ and a solution of aldehyde 35c (3.34g, 5.98mmol) in THF (14mL) was added. After stirring at-78 ℃ for 15 minutes, the mixture was stirred at 0 ℃ for 15 minutes and at room temperature for 15 minutes. With saturated NH₄The reaction was stopped with aqueous Cl (20mL) and Et₂O (120mL +50 mL. times.2). The combined organic extracts were washed with brine (20mL), Na₂SO₄Dried and concentrated. By passingFlash column chromatography (SiO)₂About 80g, Hexane/Et₂O40: 1) to give 36 as a colorless oil (125.3mg, 0.225mmol, 78%); [ alpha ] to]_D ²⁵-33.6(c 0.250，CHCl₃) (ii) a IR (thin film) v2956, 2878, 1733, 1696, 1472, 1367, 1299, 1253, 1156, 1100, 988, 837, 774cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.08(3H, s), 0.60-0.68(6H, m), 0.93(9H, s), 0.96(9H, t, J ═ 8.0Hz), 1.04(6H, d, J ═ 7.0Hz), 1.09(3H, s), 1.20(3H, s), 1.45(9H, s), 2.08-2.15(1H, m), 2.29(1H, dd, J ═ 17.0, 7.0Hz), 2.41(1H, dd, J ═ 17.0, 3.1Hz), 3.08(1H, quintuple, J ═ 7.0Hz), 3.84(1H, dd, J ═ 7.0, 2.1), 4.32(1H, dd, 7.0, J ═ 5, 5H, 5J ═ 7.5, 10H, 1.06 Hz), 1.5J ═ 10.5, 1H, 1.5, 1, 5, 1H, 1.0, 1 Hz);¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.3、5.4(3C)、7.2(3C)、15.2、18.7、19.0、20.2、23.6、26.4(3C)、28.3(3C)、41.1、43.8、46.4、53.5、73.9、76.6、80.6、115.5、140.2、171.5、218.5；C₃₀H₆₀O₅Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]579.4, experimental value 579.4.

Compound 25: tert-butyl ester 36(4.87g, 8.74mmol) and 2, 6-lutidine (freshly distilled, 4.1mL, 35.0mmol) were dissolved in CH at 0 deg.C₂Cl₂To the solution in (58mL) was added TESOTf (4.0mL, 17.5 mmol). After stirring at 0 ℃ for 25 minutes, the mixture was stirred at room temperature for 3.2 hours. The mixture was washed with Et₂O (600mL) diluted sequentially with 5% KHSO₄(60 mL. times.2) aqueous solution and brine (60mL), Na₂SO₄Dried and concentrated. The residue was dried under high vacuum for 1.5 hours to give crude acid 25(6.30g, containing TESOH impurity). The crude product (6.30g) was dissolved in aqueous THF (87.5mL, THF/H)₂O6: 1) and saturated NaHCO₃(12.5mL) of the aqueous solution. After stirring at room temperature for 20 min, the resulting suspension was treated with Et₂O (500mL) diluted and treated with 5% KHSO₄The aqueous solution (55mL) was acidified. After separation, the aqueous layer was washed with Et₂O (100 mL. times.2) extraction and the combined organic layers were washed with brine (50 mL. times.2), Na₂SO₄Dried and concentrated. The residue was dried under high vacuum overnight to give the crude acid as a colorless oil (5.60g, containing TESOH impurity), which was used in the next reaction without further purification. Purification was performed by flash column chromatography (on silica gel, eluting with hexane/EtOAc-4/1).

[α]_D ²⁵-30.7(c 0.985，CHCl₃) (ii) a IR (film) v2956, 2936, 2879, 1712, 1472, 1417, 1303, 1253, 1107, 1046, 1003, 988, 872, 837, 775, 741cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.08(3H, s), 0.09(3H, s), 0.59-0.67(6H, m), 0.93(9H, s), 0.96(9H, t, J ═ 8.1Hz), 1.05(3H, d, J ═ 7.0Hz), 1.20(3H, s), 1.21(3H, s), 2.06-2.13(1H, m), 2.34(1H, dd, J ═ 16.4, 7.4Hz), 2.50(1H, dd, J ═ 16.4, 3.0Hz), 3.06(1H, quintuple, J ═ 7.3Hz), 3.87(1H, dd, J ═ 7.5, 1.8, 4.40(1H, 4H, 3.40, J ═ 10.5, 1H, 10.5, 1.8, 1H, 10.5, 10J ═ 10, 1H, 1.5, 10, 1.8, 1H, 10, 1.5, 1.8, 1H, 1.8, 1H, 1.5, 1.8, 1H, 1, 1.8, 1H, 1, 1.3.3, 1H, 1, 3;¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.3、5.3(3C)、7.1(3C)、15.6、18.7、19.1、19.2、24.1、26.4(3C)、39.8、43.6、46.4、53.5、73.7、76.6、115.6、140.0、177.9、218.7；C₂₆H₅₂O₅Si₂LRMS (ESI) calculation of Na [ M + Na ]¹]523.3, Experimental value 522.9.

Compound 45: the 3-O-TES-6-O-TBS protected acid 25 was dried by azeotropic distillation with benzene. Freshly dried alcohol 43(200mg, 1.19mmol) was dissolved in DCM (10mL) and cooled to 0 deg.CAt this point, solid DMAP (167mg, 1.37mmol) and solid EDCI (261mg, 1.37mmol) were added. After the reaction mixture was stirred at 0 ℃ for 15 minutes, a solution of acid 25(425mg, 0.85mmol) in DCM (2mL) was added dropwise. The cooling bath was removed and stirring was continued for 2 hours. The crude reaction mixture was diluted with DCM (10mL) and the mixture was purified by silica gel chromatography using 10% EtOAC/hexane as eluent to give the clear oily ester 45(380mg, 81% yield, two steps, starting from 36): [ alpha ] to]_D-15.1(c 1.2，CDCl₃) (ii) a IR (pure) 2955, 2932, 2877, 1743, 1732, 1694, 1474, 1461, 1417, 1380, 1360, 1295, 1252, 1169, 1094, 1043, 988.3, 912.9, 871.4, 836.5, 774.8, 741.6cm^-1；¹H NMR(500MHz，CDCl₃)0.08(3H, s), 0.60-0.68(6H, m), 0.93(9H, s), 0.95(9H, t, J ═ 8.0Hz), 1.04(3H, d, J ═ 6.9Hz), 1.05(3H, d, J ═ 6.9Hz), 1.10(3H, s), 1.25(3H, s), 1.69(3H, s), 2.08-2.15(2H, m), 2.16(3H, s), 2.38(1H, dd, J ═ 17.0, 7.0Hz), 2.48(2H, t, J ═ 6.5Hz), 2.57(1H, dd, J ═ 17.0, 2.7), 2.71-2.76(2H, 3H, t, J ═ 6.5Hz), 2.7H, 7, 7.7H, 7, 7.7H, 7H, 7, 7.7H, 7, 7.7H, 7H, 7.7H, 7H, 7.7, 7, 7.7, 7H, 7H, 7.7, 7.7.7, 7H, 7.7, 5.92(1H, dd, J ═ 15.7, 8.0 Hz);¹³C NMR(500MHz，CDCl₃)δ218.4、205.4、172.1、140.1、137.4、135.4、119.1、115.8、115.6、78.7、76.5、73.9、53.3、46.3、43.7、39.6、36.6、29.2、26.7、26.4、23.8、23.7、19.9、18.9、18.7、15.4、7.06、5.30、-3.29、-3.62；C₃₆H₆₆O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]673.4, Experimental value 673.5.

Compound 47: to a solution of compound 45(20mg, 0.031mmol) in dry toluene (60mL) was added one portion of tricyclohexylphosphine [1, 3-bis (2, 4, 6-trimethylphenyl) -4, 5-dihydroimidazol-2-yl ] at refluxSubunit (II)][ benzylidene group]Ruthenium (IV) dichloride (5.2mg, 0.0061mmol) was dissolved in a solution of dry toluene (2mL) and the mixture was heated for 10 min. The reaction mixture was immediately cooled in an ice bath, placed on silica and purified by silica gel chromatography with a gradient of 4-10% EtOAc/pentane as eluent to give compound 47 as an oil (15mg, 78% yield): [ alpha ] to]-28.6(c 1.2，CHCl₃) (ii) a IR (pure) 2955, 2933, 2878, 1745, 1731, 1695, 1471, 1462, 1380, 1361, 1251, 1159, 1104, 1080, 1019, 985.0, 876.1, 835.5, 774.7, 743.1, 670.1cm^-1；¹H NMR(500MHz，CDCl₃)0.07(3H，s)，0.10(3H，s)，0.59-0.68(6H，m)，0.91(9H，t，J＝8.0Hz)，0.93(9H，s)，1.04(3H，d，J＝7.0Hz)，1.10(3H，s)，1.11(3H，d，J＝7.0Hz)，1.17(3H，s)，1.71(3H，s)，2.21(3H，s)，2.27-2.32(1H)，2.38(1H，dd，J＝14.6、6.8Hz)，2.51-2.61(2H，m)，2.57(1H，dd，J＝15.5、3.3Hz)，2.93-3.1(3H，m)，3.94(1H，4J＝8.5Hz)，4.28(1H，dd，J＝8.6、3.0Hz)，5.04(1H，dd，J＝8.7、2.4)，5.16(1H，t，J＝7.5)，5.73(1H，^tdd，J＝12.8、9.94、6.9Hz)，5.92(1H，ddd，J＝18.0、10.3、7.8Hz)；¹³C NMR(125MHz，CDCl₃)δ215.9、204.8、171.3、140.0、132.7、129.2、118.6、79.1、78.2、75.4、54.0、48.2、41.7、40.3、35.0、29.2、26.6、26.5、23.5、22.8、20.6、18.8、17.5、14.3、7.19、5.53、-3.36；C₃₄H₆₂O₆Si₂LRMS (ESI) calculated 645.4, Experimental 645.4(M + Na)⁺)。

Compound 39 a: to a solution of Wittig reagent (19.1mg, 54.7. mu. mol) in THF (0.4mL) was added KHMDS (109. mu.L of a 0.5M solution in toluene, 54.7. mu. mol) at 0 ℃. The mixture was stirred at 0 ℃ for 0.5 h and then cooled to-78 ℃. To the mixture was added dropwise a solution of ketone 47(5.7mg, 9.12. mu. mol) in THF (0.3mL) and the resulting mixture was heated to-20 ℃ over 1.5 hours. For user to useAnd aqueous NH4Cl (2mL) to stop the reaction and extract with EtOAc (7 mL. times.3). The combined organic layers were washed with Na₂SO₄Dried and concentrated. By flash column chromatography (SiO)₂hexane/Et₂O10: 1) to yield 5.6mg of an inseparable E/Z olefin mixture (ElZ: 9: 1). By preparative TLC (Hexane/Et)₂O4: 1) to afford pure 39a as a colorless oil (5.0mg, 6.96 μmol, 76%); [ alpha ] to]_D ²⁵-41.5(c 0.715，CHCl₃) (ii) a IR (film) v2955, 2884, 1737, 1690, 1467, 1378, 1249, 1179, 1102, 1014, 979, 879, 826, 773cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.08(3H, s), 0.12(3H, s), 0.57(6H, q, J ═ 7.8Hz), 0.89(9H, t, J ═ 8.0Hz), 0.93(9H, s), 1.04(3H, s), 1.06(3H, d, J ═ 7.1Hz), 1.12(3H, s), 1.17(3H, d, J ═ 7.1Hz), 1.68(3H, s), 2.15(3H, d, J ═ 0.8Hz), 2.14-2.27(2H, m), 2.45(1H, dd, J ═ 14.0, 4.8Hz), 2.50(1H, dd, J ═ 14.9, 3.2Hz), 2.64-2.74(2H, m), 3.12 (3H, s), 3H, 7.5 (3H, J ═ 7.8Hz), 7H, 3H, 5J ═ 7.5 (3H, 7.8Hz), 3H, 7.5, 3.7H, 3.8, 3.5, 3.7H, 3.8 (ddh, 3.8 Hz), 3.7H, 5, 3.7.7H, 7, 7.7, 7.8, 7, 3.8, 7H, 7, 3.8, 3.7, 7, 3.7, 7, m), 5.69(1H, dd, J ═ 15.8, 8.2Hz), 6.57(1H, s), 6.96(1H, s);¹³C NMR(100MHz，CDCl₃)δ-3.3、-3.2、5.6(3C)、7.1(3C)、15.0、17.2、18.8、19.4、21.4、21.7、23.8、24.3、26.5(3C)、33.2、35.6、41.3、41.8、48.2、54.0、74.4、77.4、79.3、116.4、120.5、121.0、129.3、132.1、137.8、138.0、152.7、164.8、170.7、216.8；C₃₉H₆₈NO₅SSi₂LRMS (ESI) of [ M + H ] calculated value⁺]718.4, Experimental value 718.3.

Compound 28(Epo 3): to a solution of 39a (298.8mg, 0.416mmol) in THF (6.5mL) at 0 deg.C was added HF. pyridine (3.2mL) and the mixture was stirred at room temperature for 3 hours. TMSOMe (30mL) was added dropwise at 0 ℃ to stop the reaction. After concentration and drying under high vacuum, by flash column chromatography (SiO)₂hexane/EtOAc 1: 1) to afford 28 as a white solid (196.6mg, 0.402mmol, 97%); [ alpha ] to]_D ²⁵-96.6(c 0.235，CHCl₃) (ii) a IR (film) v3502, 2970, 2927, 1733, 1685, 1506, 1456, 1375, 1251, 1152, 1040, 977cm^-1；¹H NMR(400MHz，CDCl₃) δ 1.06(3H, s), 1.11(3H, d, J ═ 7.0Hz), 1.22(3H, d, J ═ 6.8Hz), 1.28(3H, s), 1.72(3H, s), 2.10(3H, s), 2.31-2.40(2H, m), 2.43(1H, dd, J ═ 16.0, 3.7Hz), 2.49(1H, dd, J ═ 16.0, 9.2Hz), 2.55-2.68(2H, m), 2.71(3H, s), 2.98(1H, dd, J ═ 14.4, 6.4Hz), 3.16(1H, quintuple, J ═ 6.2Hz), 3.76(1H, dd, 5.9, J ═ 4.4, 6.4Hz), 3.16(1H, 7H, 7.5, 7.8H, 7.8, 7H, 7.8H, 7.8Hz), 3.8H, 7.8, 7H, 1.8, 1H, 1.8, 15H, dd, 5H, 5H, 7.8, 7H, 1.8, 1H, 1.8, J ═ 7H, 1H, 1.8, 1H, 1.8, 7 Hz;¹³CNMR(100MHz，CDCl₃)δ15.1、16.0、17.7、19.2、19.5、22.5、23.6、32.0、35.0、39.6、40.3、44.8、53.3、71.8、75.6、78.3、116.1、119.6、120.5、129.9、131.3、137.5、138.2、152.2、165.0、170.7、218.8；C₂₇H₄₀NO₅LRMS (ESI) calculation of S [ M + H ]⁺]490.3, Experimental value 490.2.

dEpoB (1, Epo 1): 28(1.2mg, 2.5. mu. mol) and TrisNHNH were added at 50 ℃ to₂(29.3mg, 98. mu. mol) of ClCH₂CH₂Et was added to a Cl (0.7mL) solution₃N (13.7. mu.L, 98. mu. mol). By HPTLC (Hexane/EtOAc/CH)₂Cl₂1/1/2) the reaction was monitored. After stirring for 7 hours, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a pad of silica gel, which was washed with EtOAc. After concentration, preparative TLC (Hexane/EtOAc/CH) was used₂Cl₂1/1/2) purification residueThe residue, white solid 1(1.1mg, 2.2. mu. mol, 91%) was obtained.

The spectral data for 1 are identical to the reported data for dEpoB.

Compound 27: acid 25 and alcohol 24 were azeotroped with anhydrous benzene (5mL × 2) and dried under high vacuum before reaction. To alcohol 24(639mg, 2.63mmol) in CH at 0 deg.C₂Cl₂To the solution (13mL) were added EDCI (576mg, 3.09mmol) and DMAP (366mg, 3.09 mmol). To this mixture was added dropwise acid 25(1.11g, 1.88mmol) in CH over 16 minutes at 0 deg.C₂Cl₂(5mL +2mL wash). After stirring at 0 ℃ for 1.5 hours, the mixture was stirred at room temperature for 3.5 hours. After concentration, the residue is purified by flash column chromatography (SiO)₂hexane/EtOAc ═ 30: 1 to 20: 1) to give 27(1.20g, 1.61mmol, 86%, starting from tert-butyl ester) as a colourless oil; [ alpha ] to]_D ²⁴-25.1(c 1.30，CHCl₃) (ii) a IR (film) v2955, 2925, 2872, 1732, 1696, 1461, 1378, 1290, 1243, 1173, 1091, 985, 873, 773cm^-1；¹H NMR(400MHz，CDCl₃)δ0.06(3H，s)，0.06(3H，s)，0.58-0.66(6H，m)，0.92(9H，s)，0.95(9H，t，J＝8.0Hz)，1.02(3H，d，J＝6.5Hz)，1.03(3H，d，J＝6.5Hz)，1.07(3H，s)，1.21(3H，s)，1.67(3H，s)，2.07(3H，s)，2.05-2.12(1H，m)，2.30(1H，dd，J＝16.9、7.5Hz)，2.39(1H，dt，J＝14.8、6.7Hz)，2.49(1H，dd，J＝17.0、3.0Hz)，2.50(1H，dt，J＝14.8、6.7Hz)，2.70(3H，s)，2.74-2.30(2H，m)，3.07(1H，dd，J＝7.0Hz)，3.83(1H，dd，J＝7.1、2.0Hz)，4.35(1H，dd，J＝7.4、2.8Hz)，4.98-5.07(4H，m)，5.16(1H，brt，J＝7.0Hz)，5.23(1H，t，J＝6.9Hz)，5.74(1H，ddt，J＝16.7、10.2、6.5Hz)，5.91(1H，ddd，J＝17.8、10.5、7.8Hz)，6.50(1H，s)，6.95(1H，s)；¹³C NMR(100MHz，CDCl₃)δ-3.7、-3.3、5.3(3C)、7.2(3C)、14.8、15.2、18.7、18.9、19.4、20.3、23.6、23.7、26.4(3C)、31.7、36.7、40.1、43.8、46.4、53.3、74.2、76.5、79.6、115.5、115.6、116.5、120.5、121.3、135.8、136.1、137.4、140.2、152.9、164.7、171.5、218.4；C₄₁H₇₁NO₅SSi₂LRMS (ESI) of [ M + Na ] calculated⁺]768.5, Experimental value 768.5.

Compound 39 a: a solution of 27(26.9mg, 36.1. mu. mol) in toluene (70mL) was heated to reflux and treated with a solution of Grubbs catalyst (3.1mg, 3.61. mu. mol) in toluene (2 mL). The mixture was stirred for 25 min, cooled to 0 ℃ and filtered through a pad of silica gel, which was washed with hexane/EtOAc-2/1. The combined filtrates were filtered, concentrated and purified by flash column chromatography (SiO)₂hexane/Et₂O40: 1 to 5: 1) to give 39a (9.9mg, 13.8 μmol, 38%) as a colourless oil; [ alpha ] to]_D ²⁵-41.5(c 0.715，CHCl₃) (ii) a IR (film) v2955, 2884, 1737, 1690, 1467, 1378, 1249, 1179, 1102, 1014, 979, 879, 826, 773cm^-1；^]H NMR(400MHz，CDCl₃) δ 0.08(3H, s), 0.12(3H, s), 0.57(6H, q, J ═ 7.8Hz), 0.89(9H, t, J ═ 8.0Hz), 0.93(9H, s), 1.04(3H, s), 1.06(3H, d, J ═ 7.1Hz), 1.12(3H, s), 1.17(3H, d, J ═ 7.1Hz), 1.68(3H, s), 2.15(3H, d, J ═ 0.8Hz), 2.14-2.27(2H, m), 2.45(1H, dd, J ═ 14.0, 4.8Hz), 2.50(1H, dd, J ═ 14.9, 3.2Hz), 2.64-2.74(2H, m), 3.12 (3H, s), 3H, 7.5 (3H, J ═ 7.8Hz), 7H, 3H, 5J ═ 7.5 (3H, 7.8Hz), 3H, 7.5, 3.7H, 3.8, 3.5, 3.7H, 3.8 (ddh, 3.8 Hz), 3.7H, 5, 3.7.7H, 7, 7.7, 7.8, 7, 3.8, 7H, 7, 3.8, 3.7, 7, 3.7, 7, m), 5.69(1H, dd, J ═ 15.8, 8.2Hz), 6.57(1H, s), 6.96(1H, s);¹³C NMR(100MHz，CDCl₃)δ-3.3、-3.2、5.6(3C)、7.1(3C)、15.0、17.2、18.8、19.4、21.4、21.7、23.8、24.3、26.5(3C)、33.2、35.6、41.3、41.8、48.2、54.0、74.4、77.4、79.3、116.4、120.5、121.0、129.3、132.1、137.8、138.0、152.7、164.8、170.7、216.8；C₃₉H₆₈NO₅SSi₂LRMS (ESI) of [ M + H ] calculated value⁺]718.4, Experimental value 718.3.

Compound 28: to a solution of 39a (298.8mg, 0.416mmol) in THF (6.5mL) at 0 deg.C was added HF-pyridine (3.2mL) and the mixture was stirred at room temperature for 3 hours. TMSOMe (30mL) was added dropwise at 0 ℃ to stop the reaction and the mixture was stirred at room temperature for 3 hours. After concentration and drying under high vacuum, the residue is purified by flash column chromatography (SiO)₂hexane/EtOAc 1: 1) to afford 28 as a white solid (196.6mg, 0.402mmol, 97%);

[α]_D ²⁵-96.6(c 0.235，CHCl₃) (ii) a IR (film) v3502, 2970, 2927, 1733, 1685, 1506, 1456, 1375, 1251, 1152, 1040, 977cm^-1；¹H NMR(400MHz，CDCl₃) δ 1.06(3H, s), 1.11(3H, d, J ═ 7.0Hz), 1.22(3H, d, J ═ 6.8Hz), 1.28(3H, s), 1.72(3H, s), 2.10(3H, s), 2.31-2.40(2H, m), 2.43(1H, dd, J ═ 16.0, 3.7Hz), 2.49(1H, dd, J ═ 16.0, 9.2Hz), 2.55-2.68(2H, m), 2.71(3H, s), 2.98(1H, dd, J ═ 14.4, 6.4Hz), 3.16(1H, quintuple, J ═ 6.2Hz), 3.76(1H, dd, 5.9, J ═ 4.4, 6.4Hz), 3.16(1H, 7H, 7.5, 7.8H, 7.8, 7H, 7.8H, 7.8Hz), 3.8H, 7.8, 7H, 1.8, 1H, 1.8, 15H, dd, 5H, 5H, 7.8, 7H, 1.8, 1H, 1.8, J ═ 7H, 1H, 1.8, 1H, 1.8, 7 Hz;¹³C NMR(100MHz，CDCl₃)δ15.1、16.0、17.7、19.2、19.5、22.5、23.6、32.0、35.0、39.6、40.3、44.8、53.3、71.8、75.6、78.3、116.1、119.6、120.5、129.9、131.3、137.5、138.2、152.2、165.0、170.7、218.8；C₂₇H₄₀NO₅LRMS (ESI) calculation of S [ M + H ]⁺]490.3, Experimental value 490.2.

Compound 26: acid 25 and alcohol 21 were azeotroped with dry toluene (5mL × 2) and dried under high vacuum prior to reaction. To alcohol 21(240mg, 0.756mmol) in CH at 0 deg.C₂Cl₂To a solution (5mL) were added EDCI (192.7mg, 1.01mmol) and DMAP (122.8mg, 1.01 mmol). To the mixture was added dropwise acid 25(314.6mg, 0.628mmol) in CH over 15 minutes at 0 deg.C₂Cl₂(2mL +1mL wash) solution. After stirring at 0 ℃ for 2 hours, the mixture was stirred at room temperature for a further 2 hours. After concentration, the residue is purified by flash column chromatography (SiO)₂hexane/EtOAc 20.1 to 15: 1) to give 26(340.1mg, 0.425mmol, 68% based on acid) as a colorless oil; [ alpha ] to]_D ²⁴-27.5(c 0.28，CHCl₃) (ii) a IR (film) v2956, 2878, 1740, 1692, 1472, 1378, 1317, 1253, 1174, 1118, 988, 915, 872, 837, 775cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.06(6H, s), 0.57-0.65(6H, m), 0.92(9H, s), 0.94(9H, t, J ═ 7.9Hz), 1.02(3H, d, J ═ 6.9Hz), 1.03(3H, d, J ═ 6.8Hz), 1.07(3H, s), 1.22(3H, s), 2.07-2.10(1H, m), 2.09(3H, s), 2.31(1H, dd, J ═ 16.9, 7.3Hz), 2.51(1H, dd, J ═ 16.8, 3.0Hz), 2.49-2.65(2H, m), 2.71(3H, s), 2.96-2.99(2H, m), 3.06(1H, quintuple, 7.92, 7.65 (1H, 7.5H, 7.5 (1H, 7.5Hz), 2.5H, 7.5 (1H, 5H, 7.5H, ddd, J ═ 17.8, 9.9, 7.8Hz), 6.19(1H, t, J ═ 7.0Hz), 6.51(1H, s), 6.97(1H, s); c₄₁H₆₈F₃NO₅SSi₂LRMS (ESI) calculation of Na [ M + Na ]⁺]822.4, Experimental value 822.4.

Compound 40a (RCM by 26): will 26 (57)6mg, 72.0. mu. mol) in toluene (142mL) was heated to reflux and treated with a solution of Grubbs catalyst (6.1mg, 7.20. mu. mol) in toluene (2 mL). The mixture was stirred for 28 min, cooled to 0 ℃ and filtered through a pad of silica gel rinsed with hexane/EtOAc-2/1 (300 mL). The combined filtrates were concentrated and purified by flash column chromatography (SiO)₂hexane/Et₂O40: 1 to 15: 2) to give 40a (12.0mg, 15.5 μmol, 22%) as a colourless oil;

IR (film) v2955, 2884, 1743, 1690, 1472, 1320, 1173, 1114, 1038, 1008, 873, 832, 773cm^-1；¹H NMR(400MHz，CDCl₃)δ0.09(3H，s)，0.12(3H，s)，0.55(6H，q，J＝7.7Hz)，0.88(9H，t，J＝8.0Hz)，0.96(9H，s)，1.01(3H，s)，1.06(3H，d，J＝7.1Hz)，1.12(3H，s)，1.20(3H，d，J＝7.1Hz)，2.07-2.17(1H，m)，2.19(3H，s)，2.38(1H，dd，J＝14.3、3.5Hz)，2.39-2.49(1H，m)，2.50(1H，dd，J＝14.3、7.3Hz)，2.73(3H，s)，2.77-2.91(2H，m)，2.96-3.09(2H，m)，3.98(1H，dd，J＝8.9Hz)，4.54(1H，dd，J＝7.3、3.4Hz)，5.28-5.38(1H，m)，5.63(1H，dd，J＝9.6、2.3Hz)，5.77(1H，dd，J＝15.9、8.5Hz)，6.21-6.28(1H，m)，6.60(1H，s)，6.99(1H，s)；C₃₉H₆₅F₃NO₅SSi₂LRMS (ESI) of [ M + H ] calculated value⁺]772.4, Experimental value 772.4.

Compound 29: HF-pyridine (12.5mL) was slowly added to a solution of 40a (1.78g, 2.31mmol) in THF (25mL) at 0 deg.C, and the mixture was stirred at room temperature for 4 hours. The reaction was stopped by adding TMSOMe (80mL) dropwise over 10 min at 0 ℃. The mixture was stirred vigorously at room temperature for 2.5 hours. After concentration and drying under high vacuum for 2 hours, the residue is purified by flash column chromatography (SiO)₂About 50g, hexanes/EtOAc 1: 1) to afford 29(1.20g, 2.21mmol, 96%) as a colorless powder;

[α]_D ²⁵-54.6(c 0.28，CHCl₃) (ii) a IR (film) v3478, 2974, 2929, 1736, 1689, 1449, 1381, 1318, 1247, 1169, 1113, 1039, 983, 867, 736cm^-1；¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.8Hz), 1.37(3H, s), 2.04(1H, brd, J ═ 3.8Hz, -OH), 2.12(3H, s), 2.25-2.33(1H, m), 2.38(1H, dd, J ═ 15.3, 3.0Hz), 2.48(1H, dd, J ═ 15.4, 9.8Hz), 2.54-2.61(1H, m), 2.66-2.76(1H, m), 2.71(3H, s), 2.96(1H, dd, J ═ 16.5, 4.5Hz), 3.02(1H, dd, J ═ 16.5, 4.5Hz), 3.5 (1H, 3.6, 6.5H, 7.6H, 7.5H, 7H, 7.6H, 7.5H, 7H, 7.6H, 7H, 6H, 5H, 7H, 5H, 7H, dd, J15.8, 5.8Hz), 6.24(1H, t, J7.2 Hz), 6.64(1H, s), 7.00(1H, s);¹³C NMR(100MHz，CDCl₃)δ15.1、16.1、17.7、18.5、19.3、22.5、28.8、31.1、39.6、39.7、45.0、53.7、71.4、75.3、76.8、116.7、120.2、124.3[q，¹J(C，F)＝273.4Hz]、127.9、130.2[q，³J(C，F)＝6.0Hz]、130.6[q，²J(C，F)＝28.4Hz]、132.5、136.7、152.0、165.4、170.2、218.4；C₂₇H₃₇F₃NO₅LRMS (ESI) calculation of S [ M + H ]⁴]544.2, Experimental value 544.1.

Compound 2: to 29(1.22mg, 2.24. mu. mol) and TrisNHNH at 50 deg.C₂(26.7mg, 89.6. mu. mol) of ClCH₂CH₂Et was added to a solution of Cl (1mL)₃N (12.5. mu.L, 89.6. mu. mol). By HPTLC (Hexane/EtOAc/CH)₂Cl₂1/1/2) the reaction was monitored. After stirring for 6.5 hours, the TrisNHNH was added again₂(26.7mg, 89.6. mu. mol) and Et₃N (12.5. mu.L, 89.6. mu. mol) was added to the mixture. After stirring for 14 h, the mixture was cooled to room temperature, diluted with EtOAc and passed over silicaThe pad was filtered and the pad was rinsed with EtOAc. After concentration, the residue was purified by preparative TLC (Hexane/EtOAc/CH)₂Cl₂1/1/2) to yield 2 as a white solid (1.16mg, 2.13 μmol, 94%);

¹H NMR(400MHz，CDCl₃)δ1.03(3H，d，J＝7.0Hz)，1.08(3H，s)，1.19(3H，d，J＝6.8Hz)，1.25-1.35(2H，m)，1.37(3H，s)，1.42-1.55(2H，m)，1.65-1.82(2H，m)，2.10(3H，d，J＝0.8Hz)，2.21-2.47(2H，m)，2.27(1H，dd，J＝14.2、2.6Hz)，2.48(1H，dd，J＝14.3、10.8Hz)，2.70(3H，s)，2.70-2.28(1H，m)，3.02(1H，d，J＝2.0Hz，-OH)，3.19(1H，qd，J＝6.9、2.2Hz)，3.65(1H，d，J＝6.2Hz，-OH)，3.69-3.72(1H，m)，4.34(1H，ddd，J＝10.8、6.2、2.6Hz)，5.28(1H，dd，J＝10.2、2.2Hz)，6.12(1H，dd，J＝10.2、5.2Hz)，6.61(1H，s)，6.98(1H，s)；C₂₇H₃₉F₃NO₅LRMS (ESI) calculation of S [ M + H ]⁺]546.3, Experimental value 546.2.

Compound 54: acid 25 and alcohol 53 were azeotroped with dry toluene (3mL × 2) and dried under high vacuum prior to reaction. To alcohol 53(68.0mg, 0.173mmol) in CH at 0 deg.C₂Cl₂To a solution (1.3mL) were added EDCI (37.8mg, 0.197mmol) and DMAP (24.1mg, 0.197 mmol). To the mixture was added dropwise acid 25(72.6mg, 0.123mmol) in CH over 5 minutes at 0 deg.C₂Cl₂(0.7mL) solution. After stirring at 0 ℃ for 1 hour, the mixture was stirred at room temperature for 2 hours. After concentration, the residue is purified by flash column chromatography (SiO)₂hexane/EtOAc ═ 30: 1) to give 54(99.5mg, 0.114mmol, 92% from tert-butyl ester) as a colourless oil; [ alpha ] to]_D ²⁵-23.4(c 0.56，CHCl₃) (ii) a IR (film) v2955, 2931, 2880, 1735, 1696, 1506, 1472, 1386, 1362, 1294, 1254, 1174, 1104, 988, 878, 776, 742cm^-1；¹H NMR(400MHz，CDCl₃) δ 0.06(3H, s), 0.14(6H, s), 0.63(6H, q, J ═ 8.0Hz), 0.92(9H, s), 0.94(9H, t, J ═ 8.0Hz), 0.97(9H, s), 1.02(3H, d, J ═ 6.6Hz), 1.05(3H, d, J ═ 6.5Hz), 1.07(3H, s), 1.21(3H, s), 1.67(3H, s), 2.06(3H, d, J ═ 0.8Hz), 2.05-2.14(1H, m), 2.30(1H, dd, J ═ 16.9, 7.5Hz), 2.33-2.53(2H, m), 2.50(1H, dd, 7.5H, 7.7.5H, 7.7.7H, 7.7 (7H, 7J ═ 4.7, 7.7H, 7.7, 7H, 7.7H, 7J ═ 4,7, 7.7, 7H, 7J ═, m), 5.16(1H, t, J ═ 7.2Hz), 5.24(1H, t, J ═ 6.9Hz), 5.74(1H, ddt, J ═ 16.6, 10.0, 6.5Hz), 5.91(1H, ddd, J ═ 17.6, 9.9, 7.7Hz), 6.50(1H, s), 7.06(1H, s);¹³C NMR(100MHz，CDl₃)δ-5.2(2C)、-3.7、-3.3、5.3(3C)、7.2(3C)、14.7、15.2、18.5、18.7、18.9、20.3、23.6、23.7、26.0(3C)、26.4(3C)、31.7、36.7、40.1、43.8、46.4、53.3、63.4、74.2、76.5、79.6、115.5、115.6、116.6、120.5、121.3、135.8、136.1、137.4、140.1、153.0、171.5、172.2、218.4；C₄₇H₈₆NO₆SSi₃LRMS (ESI) of [ M + H ] calculated value⁺]876.6, Experimental value 876.5.

Compound 55: a solution of 54(69.7mg, 79.5. mu. mol) in toluene (158mL) was heated to reflux and treated with a solution of Grubbs catalyst (6.7mg, 7.95. mu. mol) in toluene (2 mL). The mixture was stirred for 11 min, cooled to 0 ℃ and filtered through a pad of silica gel rinsed with hexane/EtOAc-3/1 (280 mL). The combined filtrates were concentrated and purified by flash column chromatography (SiO)₂hexane/Et₂O20: 1 to 15: 1) to give 55(18.4mg, 21.7 μmol, 27%) as a colourless oil; [ alpha ] to]_D ²⁴-40.4(c 0.26，CHCl₃) (ii) a IR (film) v2955, 2930, 2879, 1740, 1694, 1472, 1387, 1362, 1253, 1200, 1107, 1007, 838, 776, 742cm^-1；¹H NMR(400MHz，CDCl₃)δ0.08(3H，s)，0.12(3H，s)，0.15(6H，s)，0.57(6H，q，J＝7.9Hz)，0.88(9H，t，J＝8.0Hz)，0.95(9H，s)，0.97(9H，s)，1.04(3H，s)，1.06(3H，d，J＝7.1Hz)，1.12(3H，s)，1.17(3H，d，J＝7.0Hz)，1.69(3H，s)，2.06-2.30(2H，m)，2.14(3H，s)，2.45(1H，dd，J＝15.6、3.6Hz)，2.50(1H，dd，J＝14.9、3.1Hz)，2.63-2.75(2H，m)，2.97-3.06(1H，m)，3.10(1H，dd，J＝14.6、7.7Hz)，3.97(1H，d，J＝8.5Hz)，4.44(1H，dd，J＝8.4、2.9Hz)，4.97(2H，s)，5.22(1H，dd，J＝8.7、5.2Hz)，5.33-5.44(2H，m)，5.70(1H，dd，J＝15.6、8.1Hz)，6.57(1H，s)，7.07(1H，s)；C₄₅H₈₂NO₆SSi₃LRMS (ESI) of [ M + H ] calculated value⁺]848.5, Experimental value 848.5.

Compound 57: to a solution of 55(61.8mg, 72.8. mu. mol) in THF (2mL) was added HF-pyridine (1mL) at 0 deg.C, and the mixture was stirred at room temperature for 3.2 hours. The reaction was stopped by adding TMSOMe (15mL) dropwise at 0 ℃. The mixture was stirred at room temperature for 2 hours. After concentration and drying under high vacuum, the residue is purified by flash column chromatography (SiO)₂hexane/EtOAc 1: 3) to afford 57 as a white solid (32.4mg, 64.1 μmol, 88%);

[α]_D ²⁵-108.4(c 0.285，CHCl₃) (ii) a IR (film) v3422, 2968, 2919, 2729, 1689, 1449, 1377, 1252, 1152, 1064, 978cm^-1；¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 6.9Hz), 1.22(3H, d, J ═ 6.8Hz), 1.32(3H, s), 1.72(3H, s), 2.08(3H, s), 2.31-2.40(3H, m), 2.43(1H, dd, J ═ 15.5, 3.5Hz), 2.49(1H, dd, J ═ 15.5, 9.5Hz), 2.55-2.67(2H, m), 2.95(1H, dd, J ═ 14.6, 6.3Hz), 3.13(1H, quintuple, J ═ 6.6Hz), 3.34(1H, brs, -OH), 3.75(1H, dd, 6.6, J ═ 4.6, 4.4H, -4, 4.06H, -OH), 3.06(1H, 4.06 — H, 3.06), 3.06.0Hz)，4.92(2H，s)，5.18(1H，t，J＝6.9Hz)，5.33(1H，dd，J＝8.0、2.5Hz)，5.52(1H，dd，J＝15.8、6.4Hz)，5.59(1H，ddd，J＝15.8、6.6、5.0Hz)，6.63(1H，s)，7.13(1H，s)；¹³C NMR(100MHz，CDCl₃)δ15.3、16.3、17.8、19.2、22.8、23.7、31.9、35.1、39.7、40.2、45.0、53.4、61.8、71.7、75.8、78.1、116.7、119.0、120.5、130.0、131.2、137.6、138.9、152.5、170.0、170.7、218.7；C₂₇H₃₉NO₆LRMS (ESI) calculation of SNa [ M + Na ]⁺]528.2, Experimental value 528.0.

Compound 46: crude acid 25(4.65g, 7.27mmol) and alcohol 44(2.18g, 9.84mmol) were azeotroped with anhydrous benzene and dried under high vacuum before reaction. To alcohol 44(2.18g, 9.84mmol) in CH at 0 deg.C₂Cl₂To a solution (65mL) were added EDCI (2.09g, 10.9mmol) and DMAP (1.33g, 10.9 mmol). To this mixture was added dropwise crude acid 25(4.65g, 7.27mmol) in CH over 20 minutes at 0 deg.C₂Cl₂(20mL +5mL rinse) solution. After stirring at 0 ℃ for 40 minutes, the mixture was stirred at room temperature for a further 4 hours. After concentration, the residue is purified by flash column chromatography (SiO)₂About 160g, hexanes/EtOAc ═ 20: 1) to afford 46(4.85g, 6.87mmol, 94% from tert-butyl ester) as a colorless oil;

¹H NMR(400MHz，CDCl₃) δ 0.08(3H, s), 0.60(6H, q, J-7, SHz), 0.93(9H, s), 0.94(9H, t, J-8.0 Hz), 1.04(3H, d, J-7.0 Hz), 1.11(3H, s), 1.23(3H, s), 2.05-2.14(1H, m), 2.17(3H, s), 2.40(1H, dd, J-16.9, 7.0Hz), 2.59(1H, dd, J-17.0, 3.6Hz), 2.56-2.64(2H, m), 2.90-3.01(2H, m), 3.06(1H, dd, 7.5H, 7.7H, 7.5, 7.7H, 7H, 7.5, 7.7H, 7.5H, 7.5H, 7.5, 7.7.7.7.7.5H, 7.7.7.7.7.7.7.8.8、10.5、7.8Hz)，6.21(1H，td，J＝7.2、1.5Hz)；C₃₆H₆₃F₃O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]727.4, Experimental value 727.3.

Compound 48: a solution of 46(510.0mg, 0.723mmol) in toluene (500mL) was heated to reflux and treated with a solution of Grubbs catalyst (92.1mg, 0.109mmol) in toluene (10 mL). The mixture was stirred at reflux for 17 minutes and immediately cooled to 0 ℃ and maintained at 0 ℃ before filtration through a pad of silica gel. A second batch of diene (510.0mg, 0.723mmol) was treated simultaneously in the same manner. The combined reaction mixture was filtered through a pad of silica gel (100g) which was rinsed with hexane/EtOAc 3/1 (1.4L). The combined filtrates were concentrated and purified by flash column chromatography (SiO)₂About 65g, hexane/Et₂O10: 1 to 5: 1) to give 48(742.4mg, 1.10mmol, 76%) as a colourless oil;

¹H NMR(400MHz，CDCl₃)δ0.08(3H)，0.10(3H，s)，0.60(6H，q，J＝7.8Hz)，0.93(9H，s)，0.94(9H，t，J＝7.8Hz)，1.03(3H，d，J＝7.1Hz)，1.08(3H，s)，1.13(3H，d，J＝7.0Hz)，1.17(3H，s)，2.26(3H，s)，2.25-2.34(1H，m)，2.64(1H，dd，J＝15.5、5.0Hz)，2.68-2.75(2H，m)，2.76(1H，dd，J＝15.6，6.4Hz)，2.85(1H，dd，J＝15.6，5.7Hz)，2.97(1H，dq，J＝8.3、6.9Hz)，3.04(1H，dd，J＝15.6、6.3Hz)，3.92(1H，dd，J＝8.3、1.2Hz)，4.36(1H，t，J＝5.3Hz)，5.30-5.39(2H，m)，5.58(1H，dd，J＝15.5、8.0Hz)，6.13(1H，bit，J＝7.2Hz)；¹³C NMR(100MHz，CDCl₃)δ-3.6、-3.6、5.4(3C)、7.0(3C)、17.5、18.5、19.0、21.6、23.5、26.3(3C)、26.5、28.6、29.1、41.0、42.3、47.3、54.1、74.2、76.8、77.7、124.0[¹J(C，F)＝273.7Hz]，126.0、128.7[³J(C，F)＝5.9Hz]、132.2[²J(C，F)＝28.1Hz]、133.8、170.5、204.1、216.1；C₃₄H₅₉F₃O₆Si₂LRMS (ESI) calculation of Na [ M + Na ]⁺]699.4, Experimental value 699.4.

Compound 40a (wittig reaction by ketone 48):

ketone 48 was azeotroped with benzene and then dried under high vacuum for 0.5 hours. To a solution of Wittig salt (907mg, 2.59mmol) in THF (19mL) at 0 deg.C over 5 minutes was added t-BuOK (2.4mL of a 1.0M solution in THF, 2.43mmol) dropwise. The mixture was stirred at 0 ℃ for 0.5 h and then cooled to-78 ℃. To this mixture was added dropwise a solution of ketone 48(1.10g, 1.62mmol) in THF (13mL) over 10 minutes, and the mixture was heated to-20 ℃ over 2 hours. With saturated NH₄The reaction was quenched with aqueous Cl (15mL) and extracted with EtOAc (50 mL. times.3). The combined organic layers were washed with brine (20mL), Na₂SO₄Dried and concentrated. The residue was purified by flash chromatography (SiO)₂hexane/Et₂O20: 1 to 10: 1) to give the desired 16(E) -isomer 40a (940mg, 1.22mmol, 75%) and the undesired 16(Z) -isomer 40b (140.9mg, 0.182mmol, 11%) as colorless oil;

[α]_D ²⁶-17.1(c 0.14，CHCl₃)；₁H NMR(400MHz，CDCl₃)δ0.09(3H，s)，0.12(3H，s)，0.55(6H，q，J＝7.7Hz)，0.88(9H，t，J＝8.0Hz)，0.96(9H，s)，1.01(3H，s)，1.06(3H，d，J＝7.1Hz)，1.12(3H，s)，1.20(3H，d，J＝7.1Hz)，2.07-2.17(1H，m)，2.19(3H，s)，2.38(1H，dd，J＝14.3、3.5Hz)，2.39-2.49(1H，m)，2.50(1H，dd，J＝14.3、7.3Hz)，2.73(3H，s)，2.77-2.91(2H，m)，2.96-3.09(2H，m)，3.98(1H，dd，J＝8.9Hz)，4.54(1H，dd，J＝7.3、3.4Hz)，5.28-5.38(1H，m)，5.63(1H，dd，J＝9.6、2.3Hz)，5.77(1H，dd，J＝15.9、8.5Hz)，6.21-6.28(1H，m)，6.60(1H，s)，6.99(1H，s)；¹³C NMR(100MHz，CDCl₃)δ-3.4、-3.3、5.5(3C)、7.0(3C)、14.6、17.1、18.7、19.4、19.9、21.3、24.8、26.4(3C)、29.6、32.8、42.0、42.1、48.2、54.1、73.4、76.9、77.8、117.0、121.6、124.3[¹J(C，F)＝273.5Hz]、127.2、130.6[²J(C，F)＝28.2Hz]、130.8[³J(C，F)＝6.1Hz]、133.2、136.5、152.3、165.0、170.1、217.1；C₃₉H₆₅F₃NO₅SSi₂HRMS (ESI) of (a) calculated value [ M + H⁺]772.4074, Experimental value 772.4102.

[α]_D ²⁵62.7(c 0.33，CHCl₃)；1H NMR(400MHz，CDCl₃)δ0.09(3H，s)，0.13(3H，s)，0.49(6H，q，J＝7.8Hz)，0.85(9H，t，J＝7.8Hz)，0.97(9H，s)，0.99(3H，s)，1.06(3H，d，J＝7.1Hz)，1.11(3H，s)，1.20(3H，d，J＝7.1Hz)，2.00(3H，s)，2.03-2.13(1H，m)，2.35(1H，dd，J＝14.3、3.0Hz)，2.46(1H，dd，J＝14.3、7.8Hz)，2.41-2.50(1H，m)，2.73(3H，s)，2.71-2.90(2H，m)，2.98-3.12(2H，m)，3.99(1H，d，J＝9.2Hz)，4.56(1H，dd，J＝7.7、2.8Hz)，5.33(1H，ddd，J＝15.6、8.9、4.1Hz)，5.82(1H，dd，J＝15.6、8.4Hz)，6.29(1H，s)，6.33-6.40(1H，m)，6.94(1H，m)，7.09(1H，brd，J＝8.4Hz)；¹³C NMR(100MHz，CDCl₃)δ-3.2、-3.2、5.5(3C)、7.0(3C)、17.2、18.7、19.3、19.6、20.0、22.3、24.9、26.4(3C)、29.7、32.9、41.9、42.0、48.6、54.0、72.2、73.3、77.0、116.7、120.7、124.5[¹J(C，F)＝273.3Hz]、127.9、129.7[²J(C，F)＝28.0Hz]、131.9[³J(C，F)＝6.1Hz]、132.9、136.6、152.1、165.4、170.2、217.4；C₃₉H₆₅F₃NO₅SSi₂H of (A) to (B)RMS (ESI) calculated value [ M + H⁺]772.4, Experimental value 772.4.

Compound 58 (wittig reaction by ketone 48):

ketone 48 was azeotroped with benzene (5mL × 2) and then dried under high vacuum for 0.5 hour. To a solution of Wittig salt (1.19g, 2.27mmol) in THF (18mL) was added t-BuOK (2.2mL of a 1.0M solution in THF, 2.20mmol) dropwise over 5 minutes at 0 ℃. The mixture was stirred at 0 ℃ for 20 minutes and then cooled to-78 ℃. To this mixture was added dropwise a solution of the ketone (1.06g, 1.51mmol) in THF (10mL +2mL rinse) over 10 minutes and the mixture was heated to-20 ℃ over 2 hours. With saturated NH₄The reaction was quenched with aqueous Cl (15mL) and extracted with EtOAc (50 mL. times.3). The combined organic layers were washed with brine (20mL), Na₂SO₄Dried and concentrated. The residue was purified by flash column chromatography (SiO)₂About 65g, hexane/Et₂O30: 1 to 20: 1) to give the desired 16(E) -isomer 58(1.01g, 1.11mmol, 74%) and the undesired 16(Z) -isomer 58a (154.5mg, 0.182mmol, 11%), both as colorless oil;

¹H NMR(400MHz，CDCl₃)δ0.09(3H，s)，0.12(3H，s)，0.15(6H，s)，0.55(6H，q，J＝7.8Hz)，0.87(9H，t，J＝8.0Hz)，0.96(9H，s)，0.97(9H，s)，1.01(3H，s)，1.06(3H，d，J＝7.1Hz)，1.12(3H，s)，1.20(3H，d，J＝7.1Hz)，2.07-2.16(1H，m)，2.18(3H，d，J＝1.0Hz)，2.38(1H，dd，J＝14.4、3.3Hz)，2.34-2.46(1H，m)，2.49(1H，dd，J＝14.4、7.4Hz)，2.78-2.90(2H，m)，2.97-3.09(2H，m)，3.98(1H，d，J＝8.9Hz)，4.54(1H，dd，J＝7.3、3.3Hz)，4.97(2H，s)，5.33(1H，ddd，J＝15.8、8.6、4.9Hz)，5.63(1H，dd，J＝9.6、2.4Hz)，5.78(1H，dd，J＝15.8、8.2Hz)，6.22-6.27(1H，m)，6.60(1H，s)，7.09(1H，s)；¹³C NMR(100MHz，CDCl₃)δ-5.3(2C)、-3.4、-3.3、5.5(3C)、7.0(3C)、14.6、17.1、18.4、18.7、19.8、21.3、24.8、25.9(3C)、26.4(3C)、29.6、32.9、42.0、42.1、48.2、54.1、63.4、73.4、76.9、77.8、117.2、121.7、124.3[q，¹J(C，F)＝273.6Hz]、127.2、130.7[q，²J(C，F)＝27.5Hz]、130.8[q，²J(C，F)＝6.2Hz]、133.2、136.4、152.6、170.1、172.4、217.1；C₄₅H₇₈F₃NO₆SSi₃LRMS (ESI) calculation of Na [ M + Na ]⁺]924.5, experimental 924.5.

¹H NMR(400MHz，CDCl₃)δ0.07(3H，s)，0.13(3H，s)，0.16(6H，s)，0.48(6H，q，J＝7.8Hz)，0.84(9H，t，J＝7.9Hz)，0.97(18H，s)，0.98(3H，s)，1.06(3H，d，J＝7.1Hz)，1.11(3H，s)，1.20(3H，d，J＝7.2Hz)，2.00(3H，s)，2.03-2.11(1H，m)，2.33(1H，dd，J＝14.1、2.8Hz)，2.43(1H，dd，J＝14.0、7.8Hz)，2.40-2.48(1H，m)，2.76-2.89(2H，m)，2.97-3.10(2H，m)，3.99(1H，d，J＝93Hz)，4.57(1H，dd，J＝7.8、2.6Hz)，4.95(1H，d，J＝14.6Hz)，5.00(1H，d，J＝14.6Hz)，5.33(1H，ddd，J＝15.6、9.1、3.8Hz)，5.82(1H，dd，J＝15.6、8.3Hz)，6.30(1H，s)，6.32-6.38(1H，m)，7.04(1H，s)，7.11(1H，dd，J＝11.0，2.3Hz)；C₄₅H₇₈F₃NO₆LRMS (ESI) calculation of SNa [ M + Na ]⁺]924.5, experimental 924.5.

Compound 59:

HF pyridine (11mL) was added slowly to a solution of 58(1.04g, 2.25mmol) in THF (22mL) at 0 deg.C, and the mixture was stirred at room temperature for 4.3 h. The reaction was stopped by the addition of TMSOMe (75mL) dropwise over 10 min at 0 ℃. At room temperatureThe mixture was then stirred vigorously for 4.2 hours. After concentrated to dryness under high vacuum for 1 hour, the residue is purified by flash column chromatography (SiO)₂About 25g, hexanes/EtOAc 3: 4 to 1: 2) to afford 59(615.7mg, 1.00mmol, 96%) as a colorless solid;

[α]_D ²⁵-57.7(c 1.20，CHCl₃)；¹H NMR(400MHz，CDCl₃) δ 1.04(3H, s), 1.12(3H, d, J ═ 6.9Hz), 1.25(3H, d, J ═ 6.8Hz), 1.36(3H, s), 1.90(1H, d, J ═ 6.6Hz, OH), 2.08(3H, s), 2.23-2.32(1H, m), 2.34(1H, dd, J ═ 15.7, 2.4Hz), 2.49(1H, dd, J ═ 15.7, 10.1Hz), 2.59-2.69(2H, m), 2.95-3.01(2H, m), 3.04(1H, quintuple, J ═ 6.8Hz), 3.72(1H, td, J ═ 7.0, 3.0), 3.78(1H, J ═ 4, t, 7.78, t ═ 4.6.6H, 5, t ═ 4.6.6.5 Hz), 1H, 5J ═ 4.6.6.6H, 5, 1H, 5, 1H, 5, 1H, 1, 5, 1H, 5, 1H, 5, 1H, 5, J ═ 6, 5, j15.9, 5.0Hz), 6.28(1H, t, J6.7 Hz), 6.73(1H, s), 7.16(1H, s); c₂₇H₃₇F₃NO₆LRMS (ESI) calculation of SNa [ M + H ]⁺]560.2, experimental value 560.1.

Compounds 49 and 50: 28(12.2mg, 24.9. mu. mol) of CH₂Cl₂The solution (1.25mL) was cooled to-78 deg.C and treated with a cold solution of DMDO (-78 deg.C, 0.06M in acetone, 914. mu.L, 54.8. mu. mol). The mixture was heated to-50 ℃ and stirred at-50 ℃ for 2.7 hours. Excess DMDO was quenched by addition of dimethyl sulfide (117. mu.L) at-50 ℃ and the mixture was stirred at this temperature for 0.5 h. The solvent was removed in vacuo. Purification by preparative thin layer chromatography (hexanes/EtOAc ═ 1/2) gave β -epoxide 49(3.0mg, 5.93 μmol, 24%) and α -epoxide 50(7.9mg, 15.6 μmol, 63%) as colorless solids.

Compound 49:¹H NMR(400MHz，CDCl₃) δ 1.03(3H, s), 1.11(3H, d, J ═ 7.0Hz), 1.14(3H, d, J ═ 6.9Hz), 1.34(3H, s), 1.36(3H, s), 2.00(1H, ddd, J ═ 15.1, 7.3, 4.0Hz), 2.14(1H, dt, J ═ 15.1, 5.2Hz), 2.14(3H, s), 2.21(1H, dd, J ═ 14.6, 8.0Hz), 2.33(1H, dd, J ═ 14.7, 4.8Hz), 2.47(1H, dd, J ═ 13.8, 3.3Hz), 2.59(1H, dd, J ═ 13.8, 9.4), 2.73(3H, 3.73, 7, 7.7H, 7, 3.7, 7H, 7.7H, 3.7H, 7, 7.7H, 7H, 7.7, 3.7H, 7H, 7.7H, 7, 3.7H, 7H, 3.7H, 7H, 7.7H, 7.7.7H, 7H, 3.7H, 7H, 3.7H, 5H, 7H, 3.7H, 5H, 5.64(1H, dd, J ═ 15.7, 5.6Hz), 6.94(1H, s), 7.01(1H, s); c₂₇H₄₀NO₆LRMS (ESI) calculation of S [ M + H ]⁺]506.3, experimental value 506.3.

Compound 50:¹H HMR(400MHz，CDCl₃)δ1.00(3H，s)，1.04(3H，d，J＝6.9Hz)，1.12(3H，d，J＝7.0Hz)，1.35(3H，s)，1.35(3H，s)，1.87(1H，dt，J＝15.0、9.2Hz)，2.03(1H，dd，J＝13.9、9.2Hz)，2.13(3H，s)，2.13-2.19(1H，m)，2.36(1H，dd，J＝13.9、3.4Hz)，2.39(1H，dd，J＝12.2、2.1Hz)，2.42-2.51(1H，m)，2.49(1H，dd，J＝12.4、10.9Hz)，2.69(1H，d，J＝2.7Hz)，2.72(3H，s)，3.06(1H，dd，J＝9.7、3.1Hz)，3.54(1H，qd，J＝7.0、2.0Hz)，3.76-3.80(1H，m)，4.07-4.14(1H，m)，4.31(1H，d，J＝4.1Hz)，5.52(1H，dd，J＝15.5、8.7Hz)，5.60(1H，ddd，J＝15.1、9.4、3.4Hz)，5.71(1H，d，J＝8.4Hz)，6.63(1H，s)，6.99(1H，s)；C₂₇H₃₉NO₆LRMS (ESI) calculation of SNa [ M + Na ]⁺]528.2, Experimental value 528.2.

Compound 52: 50(1.7mg, 3.4. mu. mol) and TrisNHNH at 50 ℃ in a single reaction vessel₂(40.1mg, 0.134mmol) in ClCH₂CH₂Et was added to a solution of Cl (0.8mL)₃N (18.7. mu.L, 0.134 mmol). The reaction was monitored by HPTLC (hexane/EtOAc ═ 1/2). After stirring for 4 hours, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a pad of silica gel, which was rinsed with EtOAc. After concentration, the residue was purified by preparative TLC (hexane/EtOAc ═ 1/2) to give white solid 52(1.2mg, 2.4 μmol, 70%).

¹H NMR(400MHz，CDCl₃)δ0.95(3H，d，J＝7.1Hz)，1.04(3H，s)，1.11(3H，d，J＝7.0Hz)，1.28(3H，s)，1.37(3H，s)，1.35-1.44(1H，m)，1.45-1.59(4H，m)，1.71-1.82(2H，m)，1.86(1H，dt，J＝15.3、9.5Hz)，2.10(1H，dd，J＝15.3、3.6Hz)，2.13(3H，s)，2.40(1H，dd，J＝12.5、2.5Hz)，2.49(1H，dd，J＝12.5、11.0Hz)，2.74(3H，s)，2.80(1H，brs，OH)，3.07(1H，dd，J＝10.3、3.3Hz)，3.34(1H，qd，J＝7.0、1.0Hz)，3.89(1H，brs，OH)，4.03-4.09(1H，m)，4.12-4.17(1H，m)，5.69(1H，d，J＝9.1Hz)，6.63(1H，s)，7.00(1H，s)；C₂₇H₄₁NO₆LRMS (ESI) calculation of SNa [ M + Na ]⁺]530.3, Experimental value 530.2.

Compound 51: 49(0.7mg, 1.38. mu. mol) and TrisNHNH at 50 ℃ in a reactor₂(20.6mg, 69. mu. mol) in ClCH₂CH₂Et was added to a solution of Cl (0.4mL)₃N (9.6. mu.L, 69. mu. mol). The reaction was monitored by HPTLC (hexane/EtOAc ═ 1/2). After stirring for 6 hours, the mixture was cooled to room temperature, diluted with EtOAc and filtered through a pad of silica gel, which was rinsed with EtOAc. After concentration, the residue was purified by preparative TLC (hexane/EtOAc ═ 1/2)White solid 51(0.5mg, 0.985. mu. mol, 71%) was obtained. The spectral data for 51 is identical to the reported data for EpoB.

Example 2: alternative synthesis method for synthesizing epothilone intermediates

The following examples provide methods for preparing various intermediates in the synthesis of epothilone analogs.

Optimization of 9, 10-dehydroepothilone synthesis

Example 1:

example 3: noyori reduction

Example 4: alternative synthesis of key diketones

Example 5:

method 1 silyl transfer-decarboxylation

Method 2 decarboxylation-incorporation of silyl groups

Example 6: evans auxiliary method for synthesizing 2-hydroxy ketone

Example 7: synthesis of 2-hydroxyketone by Kowalsky-Sharpless method

Test of

Carbonic acid l- (2-benzyloxy-l-methylethyl) -5, 5-diisopropyloxy-2, 4, 4-trimethyl-3-oxopentyl ester 2, 2, 2-trichloroethyl ester (32a)

7-benzyloxy-5-hydroxy-1, 1-diisopropoxy-2, 2,4, 6-tetramethyl-heptan-3-one 32(1.0g, 2.4mmol) and pyridine (0.8mL, 7.3mmol) were dissolved in CH at 0 deg.C₂Cl₂(10.0mL) to a solution was added 2, 2, 2-trichloroethyl chloroformate (668.0. mu.L, 4.9mmol), and the mixture was warmed to room temperature. After 1 hour, the reaction mixture was quenched with brine and then CH was added₂Cl₂And (4) extracting. The combined organic layers were over MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 93: 7) to afford clear oil 32a (1.285g, 92%):¹HNMR(400MHz，CDCl₃) δ 1.03-1.09(m, 12H), 1.15(d, J ═ 1.8Hz, 3H), 1.17(d, J ═ 1.9Hz, 3H), 1.19-1.21(m, 6H), 1.97-2.11(m, 1H), 3.2(dd, J ═ 6.2 and 9.0Hz, 1H), 3.54(dd, J ═ 4.8 and 9.1Hz, 1H), 3.57-3.60(m, 1H), 3.82(qd, J ═ 3.6 and 5.9Hz, 2H), 4.47(s, 2H), 4.57(s, 1H), 4.72(d, J ═ 11.9Hz, 1H), 4.81(d, J ═ 11.9Hz, 1H), 5.08(t, J ═ 6, 7.7H), 7.35(m, 1H);¹³C NMR(100MHz，CDCl₃)δ11.9、15.0、18.8、21.4、21.7、22.3、23.2、23.4、35.7、42.5、53.4、53.9、69.4、70.9、71.4、73.3、81.3、94.7. 103.4, 127.5, 127.6, 128.2, 138.2, 154.0, 215.6; IR (film, NaCl, cm)^-1)2966、1760、1698、1247；C₂₇H₄₁O₇Cl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]605.2, experimental value 605.2; [ alpha ] to]²³ _D＝-20.4(c＝1.0，CHCl₃。

Carbonic acid l- (2-benzyloxy-l-methylethyl) -2, 4, 4-trimethyl-3, 5-dioxopentyl ester 2, 2, 2-trichloroethyl ester (67)

To 32a (1.28g, 2.25mmol) in 4: 1 THF/H₂To a solution of O (25mL) was added p-TsOH (111.0mg, 0.6 mmol). After heating at 70 ℃ for 5 hours, the reaction mixture was poured into cold (0 ℃) saturated NaHCO₃Aqueous (12mL) and then extracted with EtOAc. The combined organic layers were washed with brine, MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/BtOAc 84: 16) to afford 67 as a clear oil (793.2mg, 76%):¹H NMR(400MHz，CDCl₃) □ 0.90.90 (d, J ═ 5.8Hz, 3H), 1.0(d, J ═ 6.9Hz, 3H), 1.24(s, 6H), 1.97-2.04(m, 1H), 3.24(dd, J ═ 4.8 and 9.2Hz, 1H), 3.34(m, 1H), 3.42(dd, J ═ 5.8 and 9.2Hz, 1H), 4.35(d, J ═ 11.9Hz, 1H), 4.39(d, J ═ 11.9Hz, 1H), 4.64(d, J ═ 11.9Hz, 1H), 4.69(d, J ═ 11.9Hz, 1H), 4.96(t, J ═ 6.0Hz, 1H), 7.19-7.28(m, 5H), 9.49(s, 49H);¹³C NMR(100MHz，CDCl₃) -12.0, 14.8, 19.5, 19.6, 35.4, 43.3, 60.9, 71.1, 73.3, 80.37, 94.5, 127.7, 127.8, 128.3, 137.9, 154.1, 201.0, 210.1; IR (film, NaCl, cm)^-1)2973、2880、1758、1701、1453、1380、1248；C₂₁H₂₇O₆Cl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]503, 0, experimental value 503.0; [ alpha ] to]²³ _D＝-18.5(c＝0.8，CHCl₃)。

9-benzyloxy-4, 4, 6, 8-tetramethyl-3, 5-dioxo-7- (2, 2, 2-trichloroethoxycarboxyloxy) -nonanoic acid tert-butyl ester (69)

LDA (1.17mmol, 0.3M in Et) at-78 deg.C₂O) to a solution of tert-butyl acetate (1.0mmol, 135.0 μ L). After 30 min, 67(464.0mg, 1mmol) was slowly added to Et over 15 min₂O (2 mL). After stirring for 1 hour, saturated NH was used₄The reaction was quenched with aqueous Cl and then extracted with EtOAc. The combined organic layers were washed with brine, MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 86: 14) to afford 68 as a clear oil (1: 1 epimer mixture, 461.4mg, 80%):¹H NMR(400MHz，CDCl₃)δ0.87(d，J＝5.3Hz，3H)，0.89(d，J＝5.5Hz，3H)，1.02-1.10(m，18H)，1.38(s，18H)，1.97-2.2(m，2H)，2.27-2.31(m，2H)，3.22-3.27(m，3H)，3.39-3.48(m，5H)，4.03-4.06(m，1H)，4.11-4.14(m，1H)，4.38-4.45(m，4H)，4.58-4.73(m，4H)，4.97(t，J＝5.8Hz，1H)，5.02(t，J＝5.8Hz，1H)，7.18-7.27(m，10H)；¹³C NMR(100MHz，CDCl₃) δ 11.9, 12.7, 14.9, 15.2, 18.7, 19.3, 21.4, 21.6, 28.0, 35.6, 37.4, 41.7, 42.0, 51.8, 51.9, 71.3, 72.5, 73.0, 73.3, 80.6, 81.2, 81.3, 94.6, 127.5, 127.7, 127.8, 128.3, 138.0, 138.1, 154.0, 154.1, 172.3, 172.4, 216.0, 216.3; IR (film, NaCl, cm)^-1)3509、2975、1759、1707、1368、1248、1152；C₂₇H₃₉O₈Cl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]619.1, Experimental 619.2.

To the solution of 68(350.0mg, 0.6mmol) in CH₂Cl₂(10mL) in a 0 ℃ solution was added Dess-Martinperiodinane (398.0mg, 0.9 mmol). The mixture was stirred at room temperature for 1 hour and then poured to a well stirred 1: 1Saturated Na₂S₂O₂Saturated NaHCO₃In the mixture of (1). After 30 min the layers were separated and the aqueous layer was treated with Et₂O extraction three times, combined organic extracts with saturated NaHCO₃Brine wash, MgSO₄Dried and concentrated under vacuum. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 91: 9) to afford 69(258.4mg, 74%) as a clear oil:¹H NMR(400MHz，CDCl₃) δ 0.80(d, J ═ 6.9Hz, 3H), 0.87(d, J ═ 6.9Hz, 3H), 1.13(s, 3H), 1.19(s, 3H), 1.23(s, 9H), 2.04-2.12(m, 1H), 3.09-3.28(m, 5H), 4.23(s, 2H), 4.48(d, J ═ 11.9Hz, 1H), 4.55(d, J ═ 11.9Hz, 1H), 4.79(dd, J ═ 4.6 and 7.3Hz, 1H), 7.04-7.13(m, 5H);¹³C NMR(100MHz，CDCl₃) δ 11.7, 14.6, 20.7, 21.5, 27.9, 35.5, 42.2, 43.4, 63.3, 71.3, 73.3, 79.9, 81.5, 90.5, 94.5, 127.6, 127.7, 128.2, 138.0, 154.0, 166.2, 202.9, 210.0; IR (film, NaCl, cm)^-1)2977、1758、1697、1368、1248、1154；C₂₇H₃₇O₈Cl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]617.1, Experimental value 617.1; [ alpha ] to]²³ _D＝-49.1(c＝0.9，CHCl₃。

9-benzyloxy-3-hydroxy-4, 4, 6, 8-tetramethyl-5-oxo-7- (2, 2, 2-trichloroethoxycarbonyloxy) -nonanoic acid tert-butyl ester (70)

To a bomb (bombliner) was added (R) -RuBINAP catalyst (16.8mg, 10.0. mu. mol), to which was added HCl (555. mu.L, 0.2N in MeOH), and the mixture was sonicated for 15 seconds. A solution of 69(59.4mg, 0.1mmol) in MeOH (555. mu.L) was then added and the mixture was transferred to a Parr apparatus. The container is H₂Purged for 5 minutes and then pressurized to 1200 psi. After 17 hours, the reaction was brought to atmospheric pressure and poured into saturated NaHCO₃In aqueous solution. The aqueous layer was extracted three times with EtOAc. The combined organic extracts were extracted with MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 88: 12) to afford a clear oil 70 (by¹H NMR analysis judged dr > 20: 1) (47.6mg, 80%):¹H NMR(400MHz，CDCl₃) δ 1.06(d, J ═ 6.9Hz, 3H), 1.11(d, J ═ 6.8Hz, 3H), 1.14(s, 3H), 1.18(s, 3H), 1.47(s, 9H), 2.05-2.12(m, 1H), 2.35-2.40(m, 1H), 3.31-3.37(m, 2H), 3.51-3.54(m, 2H), 4.11-4.14(m, 1H), 4.46(s, 2H), 4.72(d, J ═ 11.9Hz, 1H), 4.80(d, J ═ 11.9Hz, 1H), 5.05(dd, J ═ 5.0 and 6.7Hz, 1H), 727-7.35(m, 5H);¹³C NMR(100MHz，CDCl₃) δ 12.0, 15.0, 19.3, 21.7, 28.0, 35.6, 37.5, 41.7, 51.8, 71.3, 73.0, 73.3, 80.6, 81.3, 94.7, 127.5, 127.7, 128.3, 138.2, 154.1, 172.4, 216; IR (film, NaCl, cm)^-1)3849、2974、2879、1758、1701、1454、1368、1248、1152、926、734；C₂₇H₃₉O₈Cl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]619.1, Experimental 619.2; [ alpha ] to]²³ _D＝-13.0(c＝0.4，ooCHCl₃)。

9-benzyloxy-4, 4, 6, 8-tetramethyl-5-oxo-7- (2, 2, 2-trichloroethoxycarbonyloxy) -3- (triethylsilanyloxy) -nonanoic acid tert-butyl ester (71)

TESC1 (11.6. mu.L, 69.3. mu. mol) was added to a solution of 70(37.6mg, 6.3. mu. mol) and imidazole (9.4mg, 13.8pmol) in DMF (0.4mL) at 0 ℃. After 3 hours, the mixture was taken up with saturated NaHCO₃And (5) diluting the aqueous solution. The aqueous layer was extracted three times with hexane. The combined organic extracts were washed with brine, MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 93: 7) to afford 71(22.9mg, 51%) according to the elution order, and recovered as a clear oil 70(12.9mg, 34%). 7:¹H NMR(400MHz，CDCl₃) δ 0.66(q, J ═ 7.9Hz, 6H), 0.96(t, J ═ 7.9Hz, 9H), 1.01(s, 3H), 1.05(d, J ═ 5.2Hz, 3H), 1.07(d, J ═ 5.3Hz, 3H), 1.35(s, 3H), 1.44(s, 9H), 2.05-2.11(m, 2H), 2.50(dd, J ═ 3.5 and 17.2Hz, 1H), 3.35(dd, J ═ 5.9 and 9.0Hz, 1H), 3.49(dd, J ═ 4.0 and 9.0Hz, 1H), 3.53(dd, J ═ 3.8 and 6.7Hz, 1H), 4.18(dd, J ═ 3.5, 6.5, and 9.0Hz, 1H), 3.79 (dd, J ═ 4.5, 7H), 7.7H, 1H, 7.65 (dd, 7H), 7.7H), 7H, 7.18 (dd, 7H), 7.65 (dd, 7.5, 7H), 7.7H, 7H, and 7H);¹³C NMR(125MHz，CDCl₃) δ 5.3, 7.3, 10.9, 14.9, 21.3, 22.6, 28.4, 35.9, 41.1, 42.7, 53.7, 71.9, 73.7, 75.7, 80.1, 80.9, 95.1, 127.9, 128.0, 128.7, 138.6, 154.3, 171.7, 215.7; IR (film, NaCl, cm)^-1)2956、2876、1732、1694、1456、1366、1257、1154、1098、988、835、774、741；C₃₃H₅₃O₈SiCl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]733.2, Experimental value 733.3. [ alpha ] to]²³ _D＝-16.1(c＝0.1，CHCl₃)。

9-benzyloxy-3- (diethylmethylsilanylalkoxy) -7-hydroxy-4, 4, 6, 8-tetramethyl-5-oxo-nonanoic acid tert-butyl ester (71a)

To a solution of 71(22.9mg, 3.2. mu. mol) in 1: 1 THF/AcOH (1.4mL) was added Zn (5.0mg, 7.8. mu. mol, nm). The mixture was sonicated for 15 minutes. Zn (5.0mg, 7.8. mu. mol, nanoscale) was again added, followed by additional sonication for 15 minutes. The suspension was filtered through a pad of celite, which was washed several times with EtOAc. The filtrate is saturated NaHCO₃Brine wash, MgSO₄Dried and concentrated under vacuum. The crude residue was eluted with 4: 1 hexanes/EtOAc through a short silica gel plug to yield 17.1mg (99% yield) of 71a as a colorless oil:¹H NMR(400MHz，CDCl₃)δ(m，6H)，0.96(t，J＝7.9Hz，9H)，0.97(d，J＝6.8Hz，3H)，1.05(d，J6.8Hz, 3H), 1.11(s, 3H), 1.26(s, 3H), 1.44(s, 9H), 1.84-1.90(m, 1H), 2.21(dd, J ═ 6.7 and 17.0Hz, 1H), 2.36(dd, J ═ 6.7 and 17.0Hz, 1H), 3.24-3.29(m, 1H), 3.44-3.52(m, 2H), 3.67(dd, J ═ 3.9 and 8.9Hz, 1H), 4.36(dd, J ═ 3.5 and 6.5Hz, 1H), 4.50(d, J ═ 12.0Hz, 1H), 4.54(d, J ═ 12.0Hz, 1H), 7.32-7.36(m, 5H);¹³C NMR(100MHz，CDCl₃) δ 5.0, 6.9, 9.7, 13.9, 20.2, 21.8, 28.0, 36.3, 40.8, 41.5, 53.7, 72.5, 72.9, 73.2, 73.6, 80.7, 127.4, 127.5, 128.2, 138.6, 171.0, 221.4; IR (film, NaCl, cm)^-1)3502、2959、2875、1731、1683、1456、1366、1154、1098、996、739；C₃₀H₅₂O₆SiCl₃LRMS (ESI) calculation of Na [ M + Na ]⁺]559.3, Experimental value 559.3; [ alpha ] to]²³ _D＝-41.0(c＝0.4，CHCl₃)。

9-benzyloxy-7- (tert-butyldimethylsilyloxy) -3- (diethylmethylsilylalkyloxy) -4, 4, 6, 8-tetramethyl-5-oxo-nonanoic acid tert-butyl ester (36)

71a (4.1mg, 7.6. mu. mol) and 2, 6-lutidine (10.0. mu.L, 43.5mmol) in CH at-78 deg.C₂Cl₂To a solution (0.2mL) was added TBSOTf (10.0. mu.L, 85.8 mmol). After 2h, 2, 6-lutidine (10.0. mu.L, 43.5mmol) and TBSOTf (10.0. mu.L, 85.8mmol) were added again. After 6 hours, the mixture was taken up with saturated NaHCO₃And (5) diluting the aqueous solution. The aqueous layer was extracted three times with EtOAc. The combined organic extracts were washed with brine, MgSO₄Dried and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient: hexanes to hexanes/EtOAc 91: 9) to afford 36 as a clear oil (5.4mg, 82%). The spectral data are consistent with the reported values.

Alcohol 83 to a solution of ethyl 4, 4, 4-trifluoroacetoacetate (24.0mL, 0.164mol) in THF-water (3: 1 ═ V: V, 320mL) were added allyl bromide (20.0mL, 1.4 equivalents) and indium (powder, -100 mesh, 25g, 1.3 equivalents) at room temperature, and the resulting mixture was stirred at 48 ℃ for 15 hours. The reaction mixture was cooled to room temperature, quenched with 2N HCl (400mL) in water and quenched with CH₂Cl₂(400mL, 2X 200mL) was extracted. The combined organics were dried (MgSO)₄) Filtered and concentrated in vacuo. Flash chromatography (Hexane- > Hexane-Ether 10: 1- > 8: 1- > 6: 1- > 4: 1) afforded alcohol 83(31.64g, 85% yield) as a clear oil: IR (film) 3426(br m), 2986(m), 1713(s), 1377(m), 1345(m), 1301(m), 1232(m), 1173(s), 1095(m), 1023(m), 927(m) cm^-1；¹H NMR(400MHz，CDCl₃)δ5.82(m，1H)，5.15(m，3H)，4.17(m，2H)，2.59(m，1H)，2.58(d，J＝3.4Hz，2H)，2.29(dd，J＝14.2、8.6Hz，1H)，1.24(t，J＝7.2Hz，3H)；¹³C NMR(100MHz，CDCl₃) δ 172.08, 130.89, 125.65(q, J ═ 280Hz), 120.27, 73, 79(q, J ═ 28Hz), 61.55, 38.97, 35.65, 13.82; high resolution Mass Spectrometry M/z 227.0895[ (M + H)⁺；C₉H₁₄O₃F₃The calculated value of (a): 227.0895]。

Ester 84. A mixture of alcohol 83(16.71g, 0.07386mol) and pyridine (15.0mL, 2.5 equiv.) was cooled to-10 deg.C and slowly treated with thionyl chloride (11.3mL, 2.1 equiv.) over 11 minutes. The resulting mixture was heated to 55 ℃ and stirred for 12 hours. The reaction mixture was cooled to-5 ℃, quenched with water (200mL) and quenched with CH₂Cl₂(2X 200mL, 2X 150 mL). The combined organics were washed with saturated NaHCO₃Washed (2X 200mL) with brine (200mL) and dried (MgSO₄) And concentrated in vacuo. Flash chromatography (pentane: ether 15: 1) afforded 84 as a yellow oil (11.90g,77% yield): IR 2986(w), 1731(s), 1308(s), 1265(w), 1227(m), 1197(s), 1133(s), 1025(m), 920(w), 896(w) cm^-1；¹H NMR(400MHz，CDCl₃)δ6.36(s，1H)，5.79(ddt，J＝16.9、10.2、6.6Hz，1H)，5.15(dd，J＝17.1、1.5Hz，1H)，5.08(dd，J＝10.0、1.4Hz，1H)，4.22(q，J＝7.1Hz，2H)，3.44(d，J＝6.5Hz，2H)，1.29(t，7＝7.1Hz，3H)；¹³C NMR(100MHz，CDCl₃) δ 164.22, 143.37(q, J ═ 29Hz), 132.71, 12321(q, J ═ 274Hz), 122.60(q, J ═ 6Hz), 117.32, 60.85, 30.54, 13.85; high resolution Mass Spectrometry M/z 209.0788[ (M + H)⁺；C₉H₁₂O₂F₃The calculated value of (a): 209.0789]。

Alcohol 85 to ester 84(7.12g, 0.0342mol) in CH₂Cl₂(120mL) in cold (-75 ℃ C.) solution DIBAL-H (75mL, 2.2 equiv.) was added to CH₂Cl₂(1.0M) and the resulting mixture was heated to room temperature over 3 hours. The reaction mixture was cooled to 0 ℃ with saturated NH₄The reaction was stopped with Cl (12mL) and stirred at room temperature for 20 min. The reaction mixture was diluted with ether (200mL) and dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (3: 1- > 1: 1 pentane: ether) afforded alcohol 85(5.68g, 99%) as a clear oil: IR (film) 3331(br s), 2929(m), 1642(m), 1445(m), 1417(w), 1348(s), 1316(s), 1217(s), 1175(s), 1119(s), 1045(m), 985(s), 921(m), 831(w) cm^-1；¹H NMR(400MHz，CDCl₃)δ6.33(td，J＝6.1、1.6Hz，1H)，5.75(ddt，J＝17.2、10.0、6.2Hz，1H)，5.07(m，2H)，4.29(ddd，J＝6.3、4.3、2.1Hz，2H)，2.95(d，J＝6.2Hz，2H)；¹³C NMR(100MHz，CDCl₃)δ134.45(q，J＝6Hz)，133.38，127.97(q，J＝29Hz)，123.76(q，J＝271Hz)，116.25，57.87，29.79。

Iodide 86. alcohol 85(5.97g, 0.0358mol) in CH₂Cl₂(50mL) of a cold (0 ℃ C.) solution with PPh₃(11.17g, 1.2 equiv.), imidazole (3.55g, 1.5 equiv.) and I₂(9.10g, 1.1 eq.) and the resulting mixture was stirred at 0 ℃ for 10 minutes. The reaction mixture was saturated with Na₂S₂O₃-saturated NaHCO₃(1: 1. V: V, 200mL) to stop the reaction and extract with pentane (3X 200 mL). The combined organics were saturated with Na₂S₂O₃-saturated NaHCO₃(1: 1 ═ V: V, 200mL) and brine (100mL) were washed and dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (pentane) afforded iodide 86 as a light red oil (6.69g, 68%): (IRI film) 3083(w), 2982(w), 1636(w), 1558(w), 1456(w), 1367(w), 1317(s), 1216(m), 1181(s), 1151(s), 1120(s), 989(m), 921(m), 896(m) cm^-1;¹H NMR(400MHz，CDCl₃)δ6.45(td，J＝8.9、1.5Hz，1H)，5.79(ddt，J＝16.8、10.3、6.2Hz，1H)，5.12(m，2H)，3.85(ddd，J＝8.9、2.9、1.4Hz，2H)，3.00(dt，7＝6.1、1.4Hz，2H)；¹³C NMR(100MHz，CDCl₃) δ 132.42, 131.64(q, J ═ 6Hz), 129.63(q, J ═ 29Hz), 123.64(q, J ═ 272Hz), 117.00, 29.32, -4.27; low resolution Mass Spectrometry M/z 298.7[ (M + Na)⁺；C₇H₈F₃Calculated value of INa: 299.0]。

α -Hydroxyoxazolidinone 88 to a cold (-78 ℃ C.) solution of TES protected 4-benzyl-3-hydroxyacetyl-oxazolidin-2-one 7(16.28g, 1.92 equivalents) in THF (160mL) was added dropwise a solution of LHMDS (42.0mL, 1.73 equivalents) in THF (1.0M) over 51 minutes and the resulting mixture was stirred at-78 ℃ for 35 minutes. The reaction mixture was treated with a solution of iodide 86(6.69g, 24.2mmol) in THF (10mL) and the resulting mixture was addedSlowly warm to room temperature overnight. The reaction mixture was saturated NaHCO₃(200mL) to stop the reaction and extract with EtOAc (3X 200 mL). Saturated NH for combined organics₄Cl (150mL), brine (150mL) and dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (Hexane-EtOAc 6: 1- > 3: 1) afforded a mixture of monoalkylated product (13.6g), which was used in the next reaction without further purification. A solution of these alkylated products in HOAc-water-THF (3: 1 ═ V: V, 200mL) was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo to remove HOAc and saturated NaHCO₃(400mL) the reaction was stopped and extracted with EtOAc (3X 200 mL). The combined organics were dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (Hexane: EtOAc 3: 1- > 2: 1) afforded α -hydroxyoxazolidinone 88(7.55g, 81% yield over two steps) as a clear oil: [ alpha ] to]_D ²⁵-48.2(c 1.08，CHCl₃) (ii) a IR (films) 3486(br s), 3030(m), 2983(s), 2925(m), 1790(s), 1682(s), 1481(m), 1393(m), 1360(m), 1217(m), 1171(m), 1113(m), 992(m), 919(m), 847(w) cm^-1；¹H NMR(400MHz，CDCl₃)δ7.32(m，3H)，7.17(m，2H)，6.33(td，J＝7.2、1.5Hz，1H)，5.77(ddt，J＝16.6、10.1、6.2Hz，1H)，5.08(m，3H)，4.74(ddt，J＝4.8、3.7、4.4Hz，1H)，4.33(dd，J＝8.6、8.6Hz，1H)，4.26(dd，J＝9.2、3.4Hz，1H)，3.42(br d，J＝6.4Hz，1H)，3.24(dd，J＝13.5、3.4Hz，1H)，2.99(m，2H)，2.79(dd，J＝13.5、9.4Hz，1H)，2.70(m，1H)，2.50(m，1H)；¹³C NMR(125MHz，CDCl₃) δ 173.93, 153.05, 134.43, 133.64, 129.98(q, J ═ 6Hz), 129.82(q, J ═ 28Hz), 129.29, 120.01, 127.58, 124.00(q, J ═ 272Hz), 116.34, 69.60, 67.31, 54.95, 37.78, 32.29, 29.84; high resolution Mass Spectrometry M/z 384.1421[ (M + H)⁺；C₁₉H₂₁NO₄F₃The calculated value of (a): 384.1423]。

Alpha-hydroxyamide 89 to a 0 ℃ solution of (MeO) NHMe HCl (10.1g, 5.25 equiv.) in THF (100mL) was added dropwise AlMe₃(50mL, 5.1 equiv.) of toluene (2.0M) and the resulting clear solution stirred at room temperature for 34 minutes before being added to a cold (0 ℃ C.) solution of α -hydroxyoxazolidinone 88(7.55g, 19.7mmol) in THF (70 mL). The resulting mixture was warmed to room temperature and stirred for 12 hours. The reaction mixture was cooled to 0 ℃, quenched by the slow addition of 1N aqueous tartaric acid (100mL), stirred at rt for 25 min, and extracted with EtOAc (3 × 200 mL). The combined organics were dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (Hexane: EtOAc 2: 1- > 1: 1) afforded α -hydroxyamide 89(5.12g, 97% yield) as a clear oil: [ alpha ] to]_D ²⁵-57.2(c 1.03，CHCl₃) (ii) a IR (film) 3432(brs), 3084(w), 2980(m), 2943(m), 1652(s), 1464(m), 1373(m), 1318(m), 1214(m), 1171(m), 1112(m), 991(m), 919(m), 818(w) cm^-1；¹H NMR(400MHz，CDCl₃)δ6.32(td，J＝7.3、1.5Hz，1H)，5.74(ddt，J＝16.9、10.3、6.1Hz，1H)，5.05(m，2H)，4.43(dd，J＝7.6、3.5Hz，1H)，3.70(s，3H)，3.35(br s，1H)，3.24(s，3H)，2.94(d，J＝6.1Hz，2H)，2.59(m，1H)，2.36(m，1H)；¹³C NMR(100MHz，CDCl₃) δ 173.43, 133.68, 13059(q, J ═ 6Hz), 129.25(q, J ═ 28Hz), 124.05(q, J ═ 271Hz), 116.17, 67.57, 61.44, 32.56, 32.38, 29.75; high resolution Mass Spectrometry M/z 268.1161[ (M + H)⁺；C₁₁H₁₇NO₃F₃The calculated value of (a): 268.1161]。

Alpha-hydroxy ketone 90 to a cold (0 ℃) solution of alpha-hydroxy amide 89(4.87g, 18.2mmol) in THF (150mL) was added a solution of MeMgBr (75mL, 12 equivalents) in diethyl ether (3.0M). After 5 minutes, the reaction mixture was washed with saturated NH₄The reaction was stopped with Cl (250mL) and extracted with EtOAc (5X 200mL)And (6) taking. The combined organics were dried (MgSO)₄) And concentrated in vacuo. Flash chromatography (Hexane: EtOAc 4: 1- > 2: 1- > 1: 2) afforded alpha-hydroxyketone 90(2.16g, 53% yield, 73% based on recovered starting material) and starting material alpha-hydroxyamide 89(1.30g, 27% yield) as a clear oil: [ alpha ] to]_D ²⁵+58.5(c 1.30，CHCl₃) (ii) a IR (film) 3460(brs), 3085(w), 2984(m), 2926(m), 1716(s), 1679(m), 1641(m), 1417(m), 1361(m), 1319(s), 1247(m), 1216(s), 1172(s), 1113(s), 1020(m), 994(m), 968(w), 919(m) cm^-1；¹H NMR(500MHz，CDCl₃)δ6.21(t，J＝7.0Hz，1H)，5.75(ddt，J＝16.7、10.4、6.2Hz，1H)，5.07(m，2H)，4.26(dt，J＝7.1，4.5Hz，1H)，3.51(d，J＝4.7Hz，1H)，2.96(d，J＝6.1Hz，2H)，2.66(m，1H)，2.42(m，1H)，2.19(s，3H)；¹³C NMR(100MHz，CDCl₃) δ 208.53, 133.43, 129.80(q, J ═ 28Hz), 129.76(q, J ═ 6Hz), 123.85(q, J ═ 271Hz), 116.32, 75.36, 31.22, 29.81, 25.11; high resolution Mass Spectrometry M/z 223.0945[ (M + H)⁺；C₁₀H₁₄NO₂F₃The calculated value of (a): 223.0946]。

Example 8: catalytic asymmetric oxidation process

Example 9: synthesis of 21-amino-26-trifluoro- (E) -9, 10-dehydro-dEpoB

Compound 98:

to a solution of 59(50.4mg, 90.1. mu. mol) in THF (1mL) at 0 deg.C was added (PhO)₂PON₃(27.2. mu.L, 126. mu. mol). After stirring at 0 ℃ for 5 min, DBU (16.2. mu.L, 108. mu. mol) was added. After stirring at 0 ℃ for 2 hours, the mixture was stirred at room temperature for 20.5 hours. The reaction mixture was diluted with EtOAc and water (2mL) was added to stop the reaction. After separation, the aqueous layer was extracted with EtOAc (three times) and the combined organic layers were extracted with Na₂SO₄And (5) drying. After concentration, the residue was dried under high vacuum for 10 minutes to remove DBU. By flash column chromatography (SiO)₂hexane/EtOAc 3: 2) to afford azide 98(45.6mg, 78.0 μmol, 87%) as a colorless solid;¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.8Hz), 1.33(3H, s), 2.01(1H, d, J ═ 5.5Hz, OH), 2.17(3H, s), 2.25-2.35(1H, m), 2.41(1H, dd, J ═ 15.5, 3.2Hz), 2.49(1H, dd, J ═ 15.5, 9.5Hz), 2.54-2.60(1H, m), 2.66(1H, d, J ═ 6.0Hz), 2.65-2.76(1H, m), 2.96(1H, dd, J ═ 16.0, 4.2), 3.03H, J ═ 6.0Hz), 3.6H, J ═ 6.5, 3.6H, 3.5, 3.7H, 7.5, 7.6.6H, 3.5H, 3.5, 5, 3.6.6.6.6.6.6.6, 7, 6, 7, 5.66(1H, dd, J ═ 15.8, 6.1Hz), 6.23(1H, t, J ═ 7.3Hz), 6.63(1H, 3), 7.18(1H, s); c₂₇H₃₅F₃N₄O₅LRMS (ESI) calculation of SNa [ M + Na ]⁺]607.2, experimental value 607.2.

Compound 96:

to a solution of azide 98(21.0mg, 35.9. mu. mol) in THF (0.6mL) was added PMe₃(1.0M in THF, 43.1. mu.L, 43.1. mu. mol). After stirring at room temperature for 2 minutes, water (0.1mL) was added and the mixture was stirred at room temperature for 3 hours. Addition of PMe₃(1.0M in THF, 7.2. mu.L, 7.2. mu. mol) and the mixture in the chamberStir at room temperature for 1.5 hours. To this mixture was added 28% NH₄OH (aq) (54.5. mu.L). After stirring for 1 hour, the mixture was passed directly through preparative TLC (CH)₂Cl₂MeOH 100: 7.5) to afford amine 96 as a colorless solid (15.9mg, 28.5 μmol, 79%);

¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.8Hz), 1.34(3H, s), 2.12(3H, d, J ═ 0.7Hz), 2.24-2.35(1H, m), 2.39(1H, dd, J ═ 15.4, 3.0Hz), 2.49(1H, dd, J ═ 15.4, 9.8Hz), 2.54-2.63(1H, m), 2.66-2.76(1H, m), 2.97(1H, dd, J ═ 16.2, 4.2Hz), 3.03(1H, dd, J ═ 16.3, 6.5Hz), 3.10(1H, quintuple, J ═ 16.3, 6.5Hz), 3.6.5 (3.5H, 3.6H, J ═ 6.8, 3.5H, 3.5, 3.6.5 Hz), 3.6H, J ═ 3.5, 3.5H, 3.5 (1H, 3.9.5H, dd, 3.6, J ═ 3.6, 7, 3.6, 3.5H, 3.6, 3.9.6, 3.5, 3.6, 3.9.6, 3.9, 3.6, 6.66(1H, s), 7.10(1H, s); c₂₇H₃₈F₃N₂O₅LRMS (ESI) calculation of S [ M + H ]⁺]559.2, Experimental value 559.2.

Compound 97:

to amine 96(15.9mg, 28.5. mu. mol) in CH₃CN (0.78mL) solution 37% HCHO (aq) (31.4. mu.L, 0.143mmol) was added followed by NaBH₃CN (1.0M in THF, 85.5. mu.L, 85.5. mu. mol), and the mixture was stirred at room temperature for 20 minutes. AcOH (1 drop) was added and the mixture was stirred at room temperature for 40 minutes. The mixture was passed directly through preparative TLC (CH)₂Cl₂MeOH: 8) to give product 97 as a colorless solid (15.6mg, 26.6 μmol, 93%);

¹H NMR(400MHz，CDCl₃)δ1.05(3H，s)，1.12(3H，d，J＝6.9Hz)，1.23(3H，d，J＝6.8Hz)，1.33(3H，s)，2.17(3H，s)，2.24-235(1H, m), 2.43(1H, dd, J ═ 15.7, 3,6Hz), 2.49(1H, dd, J ═ 15.6, 9.1Hz), 2.55-2.64(2H, m, including OH), 2.68-2.77(1H, m), 2.80(3H, s), 2.81(3H, s), 2.92-3.06(2H, m), 3.10(1H, quintuple, J ═ 6.8Hz), 3.69-3.76(1H, m), 4.25-4.34(1H, m), 4.33(2H, s), 5.42(1H, t, J ═ 5.5Hz), 5.57(1H, dt, J ═ 15.8, 6.3Hz), 5.66(1H, dd, 7.7, 6.6, 7, J ═ 6.64, 7H, 7.6.6, 7H, 7.64, 1H, 6.6.6.6 Hz), 5.5.57 (1H, 7, 7.6.6.6.6.6 Hz); c₂₉H₄₂F₃N₂O₅LRMS (ESI) calculation of S [ M + H ]⁺]580.2, Experimental value 580.2.

Compounds 94 and 95:

reaction mixture of 59(18.9mg, 33.8mol) and Et at 0 deg.C₃N (18.8. mu.L, 0.135mmol) in CH₂Cl₂To a solution (1mL) were added TsCl (12.9mg, 67.5. mu. mol) and DMAP (2.1mg, 16.9. mu. mol). After stirring at room temperature for 1.5 h, the mixture was diluted with EtOAc and filtered through a pad of silica gel (EtOAc rinse). After concentration, the residue was purified by preparative TLC (hexane/EtOAc ═ 1: 1) to give tosylate 94(8.5mg, 11.9 μmol, 35%) and chloride 95(4.3mg, 7.44 μmol, 22%); both are colorless solids;

¹H NMR(400MHz，CDCl₃) δ 1.06(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.1Hz), 1.33(3H, s), 1.99(1H, d, J ═ 5.5Hz), 2.10(3H, s), 2.25-2.34(1H, m), 2.41(1H, dd, J ═ 15.5, 3.3Hz), 2.47(3H, s), 2.48(1H, dd, J ═ 15.7, 9.4Hz), 2.51-2.63(1H, m), 2.63(1H, d, J ═ 6.1Hz, OH), 2.64-2.75(1H, m), 2.91-3.05(2H, m), 3.10(1H, J ═ 6.1Hz), 3.70H, 3.75 (3H, m), 3.10(1H, J ═ 6.70H, 3.70 Hz), 3.75(1H, 3.75H, 3.9, 3.5Hz), 3.5H, J ═ 3.9.9.7 (1H, d, J ═ 6.8, 3.)，5.32(2H，s)，5.41(1H，dd，J＝5.8、4.5Hz)，5.57(1H，ddd，J＝15.8、6.4、4.6Hz)，5.65(1H，dd，J＝15.8、6.0Hz)，6.21(1H，t，J＝7.1Hz)，6.59(1H，s)，7.18(1H，s)，7.37(2H，d，J＝8.1Hz)，7.84(2H，d，J＝8.3Hz)；C₃₄H₄₃F₃NO₈S₂LRMS (ESI) calculation of Na [ M + Na ]⁺]736.2, experimental value 736.3.

¹H NMR(400MHz，CDCl₃) δ 1.06(3H, s), 1.12(3H, d, J ═ 6.9Hz), 1.23(3H, d, J ═ 6.7Hz), 1.34(3H, s), 2.00(1H, d, J ═ 5.6Hz, OH), 2.15(3H, s), 225-2.35(1H, m), 2.41(1H, dd, J ═ 15.5, 3.2Hz), 2.49(1H, dd, J ═ 15.5, 9.4Hz), 2.53-2.62(1H, m), 2.69(1H, d, J ═ 6.1Hz, OH), 2.66-2.76(1H, m), 2.92-3.05(2H, m), 3.11(1H, quintuple, J ═ 6.4, 3.70 (3H, 70H, 3.76, 3.6H, 5J ═ 6.6.6, 5H, 5H, 5, 5.6.6, 5H, 5H, 5, 5.6.6.6, 5H, 5H, 5H, 5, 3.6.6.6.6.6.6H, 5, 6.1Hz), 6.23(1H, t, J ═ 6.8Hz), 6.63(1H, s), 7.20(1H, s); c₂₇H₃₅ClF₃NO₅LRMS (ESI) calculation of SNa [ M + Na ]⁴]600.2, experimental value 600.2.

Compound 99:

to a solution of 59(6.9mg, 12.3. mu. mol) in CH₂Cl₂(0.4mL) solution with activated MnO₂(26.8 mg, 0.308mmol, commercially available from Acros). After stirring vigorously at room temperature for 4 hours, the mixture was filtered through a pad of celite, which was rinsed with EtOAc. After concentration, the residue was purified by preparative TLC (hexane/EtOAc 1: 1) to give aldehyde 99(2.7mg, 4.84 μm) as a colorless solidol，39％)；

¹H NMR(400MHz，CDCl₃) δ 1,06(3H, s), 1.13(3H, d, J ═ 7.2Hz), 1.24(3H, d, J ═ 6.9Hz), 1.35(3H, s), 1.96(1H, d, J ═ 5.6Hz, OH), 2.22(3H, d, J ═ 0.7Hz), 2.25-2.35(1H, m), 2.44(1H, dd, J ═ 15.4, 3.5Hz), 2.46(1H, d, J ═ 5.9Hz, OH), 2.51(1H, dd, J ═ 15.7, 9.3Hz), 2.57-28(1H, m), 2.68-2.79(1H, m), 2.96-3.03(2H, m), 3.10(1H, 6.6H, J ═ 6.7, 5H, 3.7, 5H, 5.5H, 5H, 5J ═ 5, 5H, 5, j ═ 6.6Hz), 6.72(1H, s), 7.57(1H, d, J ═ 0.9Hz), 10.01(1H, d, J ═ 1.2 Hz).

Compound 100:

aldehyde 99(4.6mg, 8.25. mu. mol) in CH at 0 deg.C₃CN (0.5mL) solution to which MeNH was added₂(2.0M in THF, 41.3. mu.L, 41.3. mu. mol). After stirring at 0 ℃ for 15 minutes, NaBH was added₃CN (1.0M in THF, 25. mu.L, 25. mu. mol). After stirring at 0 ℃ for 0.5 h, AcOH (3 drops) was added. After stirring at 0 ℃ for 2 hours, 28% NH was added₄OH (aq) (40 μ L) and the mixture was stirred at rt for 10 min. The mixture was passed directly through preparative TLC (CH)₂Cl₂MeOH ═ 100: 9) twice to give 100 as a colorless solid (2.4mg, 4.19 μmol, 51%);

¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.8Hz), 1.34(3H, s), 2.13(3H, s), 2.25-2.34(1H, m), 2.39(1H, dd, J ═ 15.3, 3.0Hz), 2.49(1H, dd, J ═ 15.3, 9.7Hz), 2.56(3H, s), 2.54-2.64(1H, m), 2.66-2.75(1H, m), 2.89(1H, d, J ═ 5.1Hz), 2.94-3.05(2H, m), 3.11(1H, quintuple, J ═ 6.8Hz), 3.74(1H, J ═ 6.6, J ═ 6.5Hz)，4.08(2H，s)，4.34(1H，dd，J＝9.6、2.9Hz)，5.43(1H，dd，J＝6.2、4.1Hz)，5.56-5.63(1H，m)，5.66(1H，dd，J＝15.9、5.7Hz)，6.24(1H，t，J＝7.3Hz)，6.66(1H，s)，7.11(1H，s)；C₂₈H₄₀F₃N₂O₅LRMS (ESI) calculation of S [ M + H ]⁺]573.3, experimental value 573.3.

Example 10: epothilone analogs for the eradication of xenograft tumors to a non-recurrent state

By a combination of chemical synthesis, molecular modeling and spectroscopic analysis, we found that the introduction of an E-9, 10-double bond (see compound 28 below) can increase drug potency by a factor of about 10 in xenograft assays of drug-resistant MX-1 tumors (a.rivkin et al, j.am.chem.soc.2003, 125, 2899; incorporated herein by reference). Based on the correlation of in vitro and in vivo experiments against MX-1 tumor types, it is evident that: 28 are inherently more cytotoxic than 2 b. Yet another factor of influence is that the lactone moiety in the 9, 10-dehydro series is significantly more stable in mouse and human plasma than the 9, 10-dehydro congeners. The sum of these two complementary effects enables 28 to achieve complete inhibition of tumors in various xenografts at 3mg/Kg (compared to 30mg/Kg of 1).

12, 13-DeoxyaPoB (1) E) -9, 10-dehydro-12, 13-DeoxyaPoB (28)

6-F₃-12, 13-deoxy EpoB (2) 26-F₃- (E) -9, 10-dehydro-12, 13-deoxo

EpoB(29)

At the time of treatment suspension, palpable tumors reappear in some parts of the animal. Thus, at least at present, total synthesis 28 has not fully achieved a highly favorable effective therapeutic index and strict criteria for eliminating tumors to a non-recurrent state.

These findings have focused attention on the replacement of the three hydrogens of the 26-methyl group of 28 with three fluorine atoms. Incorporation of these fluorine atoms at this position increases the stability of the 12, 13-double bond to oxidation (Smart, B.E.J. fluorine Chem. (2001, 109, 3); incorporated herein by reference). Previous experiments have attenuated partial cytotoxicity by placing polar groups in the C12-C13 double bond region (a. rivkin et al, j.am. chem. soc. (2003, 125, 2899); incorporated herein by reference). In the present disclosure, we have reported this finding through the total chemical synthesis of 9, 10-dehydro-26-trifluoroepothilone, focusing particularly on the unique biological properties of the parent structure 29.

We carefully studied dEpoB (30mg/Kg), paclitaxel (20mg/Kg) and F₃-deH-dEpoB (29, 20 and 30mg/Kg) therapeutic efficacy against human breast cancer MX-1 xenografts in tumor elimination and recurrence, and these results are shown in Table 10-1. Each dose group consisted of four or more nude mice. Body weight refers to total body weight minus tumor weight. All three compounds caused tumor disappearance. Mice with 5/10 (dpeob), 2/7 (paclitaxel) and 0/4 (compound 29) relapsed 10 days after treatment suspension. Prolonged observation after discontinuation of treatment with 29 at 20mg/Kg showed that the tumor had disappeared for a long time until 2 of 4 mice had recurred on day 27. It is clear that treatment with 29 at a dose of 30mg/Kg allows complete disappearance of the tumor without any recurrence more than two months after the cessation of treatment.

TABLE 10-1.dEpoB, paclitaxel and F₃-deH-dEpoB therapeutic efficacy against MX-1 xenografts in nude mice^[a]

Medicine	Dosage (mg/Kg)	Body weight change (%)		No tumors appeared after Q2D X66 hours of intravenous infusion	Re-development of tumors on day 10 after administration
		Body weight change (%)				Day 4 after the stop of administration	Day 8 after the stop of administration
		dEpoB (1) paclitaxel F₃-deH-dEpoB(29)	30202030			Day 4 after the stop of administration	Day 8 after the stop of administration	-25.3±2.1-23.9±2.1-22.4±0.6-27.1±2.7	-9.1±4.1-8.7±0.7-7.3±0.7-17.4±5.5	10/107/74/44/4	5/102/70/4^[b]0/4^[b]

^[a]Human breast cancer MX-1 xenograft tissue 50mg was implanted subcutaneously on day 0. Treatment with Q2D x 6 hour intravenous infusion started on day 8 and stopped on day 18.

^[b]Palpable tumors reappeared in 2/4-day 27 mice after treatment was discontinued. No further tumors appeared during days 28 to 64 after treatment discontinuation.

^[c]No further treatment was given during 64 days after cessation of treatment at the end of the trialThe tumor appeared again.

Reducing the dose of agent 29 to 10mg/Kg (Q2D) also caused the disappearance of MX-1 tumors, but 9 doses were required to achieve this result (FIGS. 57, 58 and 59A). As an additional requirement, chemotherapy is delayed until the tumor size reaches 0.5g (about 2.3% of body weight). 4/4 mice were treated with 29 doses of 25mg/Kg (Q2D X7) to eliminate tumors. In contrast, for dEpoB, a 30mg/Kg (Q2D X8) dose was required to eliminate the tumor in 3/4 mice. However, unlike in the case of 29, the disappearance of the surface that appears after treatment with dEpoB recurs over time. (FIG. 59B).

The fact that agent 29 completely inhibited the growth of human breast cancer MX-1 xenografts, shrunk the tumor and allowed it to disappear for as long as 64 days was impressive. Furthermore, following cure with 29(20mg/kg or 30mg/kg Q2D x 6,6 hour intravenous infusion, table 1, above), the xenograft body weight returned to pre-treated control levels within 12 to 18 days after cessation of treatment. This finding indicates that no vital organs were damaged. At a low therapeutic dose of 10mg/Kg Q2D × 12 (fig. 59B), the maximum weight loss was only 12%, while the body weight increased 6% during the last three administrations. Body weight was returned to pre-treated control levels for only three days after treatment cessation. Table 1 above shows that these animals were still viable with weight loss as high as 27%. The therapeutic safety margin achieved herein is quite broad for a therapeutic cancer therapeutic.

The therapeutic efficacy of 29 anti-human lung cancer xenografts (a549) and paclitaxel anti-human lung cancer a 549/paclitaxel xenografts was also evaluated (fig. 59C and 59D). Slow-growing lung cancer xenograft a549 was treated with 29(25mg/kg, Q2D x 6, twice at eight day intervals), which resulted in 99.5% tumor suppression, with 4 tumors eventually eradicated completely after two more administrations (fig. 59C). Interestingly, the mice lost 35% of body weight without any lethality and rapidly returned body weight to near the pre-treated control level after treatment was discontinued (fig. 59C). In contrast, a parallel study with dEpoB (30mg/Kg, Q2D X6) inhibited 97.6% of tumors, but failed to eradicate the tumors. In an additional experiment (fig. 59D) with 29(20mg/Kg dose) anti-a 549/paclitaxel xenografts, tumor growth was completely inhibited and tumors eventually decreased by 24.4% compared to the pre-treated control group. In this study, body weight was lost up to 24%, however, body weight returned to 90% of the pretreatment control group after discontinuation of drug treatment. In a comparative study of (E) -9, 10-dehydro-dEpoB (28, 4mg/Kg per group), 41.6% of tumor growth was inhibited.

Corresponding data for analysis of factors conferring a significant therapeutic index on compound 29, and comparative data relating to closely related congeners, are provided in table 10-2. It can be noted that the inherent cytotoxicity decreases by one complete order of magnitude from EpoB (2b) to dpeob (1). For 9, 10-dehydro-dEpoB (28), the reduction was about 60% recovery. For 29, this intrinsic cytotoxicity was partially lost, which was at least about 1.8 times the cytotoxicity of the benchmark compound, dppob, in the cells.

We note that of these 12, 13-dehydroepothilones, 29 currently exhibited the best stability in mouse plasma and also the most stable in human liver S9 plasma. It was also noted that in the 2-position of the 12, 13-dehydroisomer, it contained a 26-trifluoro structure which decreased lipophilicity and slightly increased water solubility (Table 10-2 below). It appears that a great advantage of 29 is the improvement in serum stability and bioavailability.

TABLE 10-2 Properties of dEpoB derivatives

Compound (I)	Cytotoxic efficacy IC₅₀(nM)^[a]	When not deadMaximum weight loss of%	Stability half life		Solubility in Water (μ g/mL)	Lipophilic octanol/water partition constant (POW)	Q2D 6 therapeutic dosing regimen (mg/Kg) for 6 hour intravenous injection	Relative therapeutic index at MTD^[b]
			Stability half life						Mouse plasma (min)	Human liver S9 part (hr)
			EpoB(2b)	0.53±0.2					Mouse plasma (min)	Human liver S9 part (hr)	15	57	15.8	ND	ND	0.6-0.8	+++
dEpoB(1)	5.6±2.8	32	EpoB(2b)	0.53±0.2	46±7	1.0±0.1	9.4	4.4	25-30	++++	15	57	15.8	ND	ND	0.6-0.8	+++
dEpoB(1)	5.6±2.8	32	deH-dEpoB(28)	0.90±0.40	46±7	1.0±0.1	9.4	4.4	25-30	++++	29	84±6	4.9+0.7	27	3.3	3-4	++++
F₃-dEpoB(2)	9.3±5.2	22	deH-dEpoB(28)	0.90±0.40	66±7	1.6±0.4	8	4.1	15-20	++	29	84±6	4.9+0.7	27	3.3	3-4	++++

F₃-deH-dEpoB(29)

3.2±0.3

33

212±88

10.5±2.3

20

3.3

10-30

+++++

^[a]IC₅₀The value is IC for CCRF-CEM leukemia cells₅₀The value is obtained. These values are in the range of two trials. All values were obtained from seven concentration points; ND was not determined.

^[b]Relative Therapeutic Index (TI) classification at MTD (maximum tolerated dose):

+ 25 to 50% of tumor growth is inhibited.

Between +50 and 100% of the tumor growth is inhibited.

The +++ tumor shrinks but no disappearance of the tumor appears.

The tumors disappeared in some or all nude mice, some within one week after treatment was discontinued

The body weight of the mice is slowly recovered and/or the old disease recurs.

Tumors disappeared, body weight recovered rapidly and/or there was no recurrence in all nude mice.

Chou, T.C. et al (Proc. Natl. Acad Sci. USA.1998, 95, 15798 and 2001, 98, 8113) investigated therapeutic trials of epothilones against human xenografts (e.g., MX-1) in nude mice.

All agents 1-2 and 28-29 were first discovered by total synthesis. The actual synthesis of 1 has been described in the previous literature (Rivkin et al, J.Am.chem.Soc. (2003, 125, 2899); White et al, J.Am.chem.Soc. (2001, 123, 5407); Yoshimura et al, Angew.chem. (2003, 42, 2518); Rivkin et al, J.org.chem.2002, 124, 7737; each of which is incorporated herein by reference). The first discovery-level approaches to obtain 28 and 29 have also been described. Selective reduction of the 9, 10-double bond of 29 gives 2. The significant results obtained from the xenograft study described above for the most promising compound 29 at present clearly indicate that more detailed toxicological and pharmacokinetic studies of compound 29 in higher animals are required and thus suitable further use in human clinical trials. These prospects change the nature of the synthetic challenge entirely from preparing a trial sample to preparing multi-gram quantities of the novel epothilone agents. The principal modifications to the previous approaches originally conceived and demonstrated in the background of the invention have been achieved. In particular, our new scheme achieves significant simplification in the stereoselective design of carbons 3 and 26. Alcohol 32 was prepared as previously described (Rivkin et al, J.Am.chem.Soc. (2003, 125, 2899); incorporated herein by reference). It should be noted that in the novel synthesis, stereocenters 6, 7 and 8 are all derived from readily available ketones 30 and aldehydes 31. After alcohol protection and acetal hydrolysis, the corresponding aldehyde was condensed with tert-butyl acetate to obtain a monoaldehyde-like product. Since the condensation reaction is not controlled with respect to stereoisomerism, a remedy is required and has been achieved. This 1: 1 mixture of C3 epimers was oxidized to afford ketone 69. After a very successful Noyori reduction (Noyori et al, J.Am.chem.Soc. (1987, 109, 5856); incorporated herein by reference) was carried out under the conditions shown, alcohol 70 was obtained. The preparation of acid 25 is then completed in several additional simple steps as shown.

Scheme 12 Synthesis of acyl moieties 25

Reagents and conditions: (a) (i) Troccl, pyridine, 92%; (ii) p-TsOH. H₂O, 76%; (m) LDA, tert-butyl acetate, THF, 80%; (iv) Dess-Martin periodinane, 74%; (b) noyori catalyst (10 mol%), MeOH/HCl, H₂1200psi, 80%. (c) (i) TESCl, imidazole, 77%; (ii) zn, AcOH, THF, 99%; (iii) TBSOTf, 2, 6-lutidine, 82%; see Rivkin et al J.am.chem.Soc.2003, 125, 2899 for the remaining steps.

A new simple and easily scalable method for synthesizing 90 has also been developed (fig. 13). The synthesis starts with the reaction of a commercially available trifluoroketoester 82 with allylindium bromide. The key step in the synthesis is the position-specific and stereoselective dehydrogenation of the resulting tertiary alcohol to yield the product 84 (65% overall yield of the two steps). The steric control of this reaction derives from the "dipole effect", in which a strong electron-withdrawing CF₃With CO₂The Et group is optimally present in trans relative to the double bond formed. The desired iodide 86 is obtained in two steps from 84. The previously reported alkylation of lithium enolate 7 with iodide 86 in THF gave 88 in 81% yield with high diastereoselectivity (> 25: 1 de). After deprotection of the secondary alcohol, the indicated 90 is obtained in three steps from compound 88.

Scheme 13 Synthesis of alkyl moiety 17

Reagents and conditions: (a) (i) allyl bromide, In, THF-water (3: 1)48 ℃, 85%; SOCl₂Pyridine 55 ℃, 77%; (b) (i) DIBAL-H, CH₂Cl₂-78 ℃ to room temperature, 99%; (ii) i is₂，PPh₃Imidazole, CH₂Cl₂74%; (c) (i) LHMDS, THF, -78 ℃ to room temperature; (ii) HOAc-THF-H₂O (3: 1), two-step yield 81%; (d) (i) AlMe₃MeONHMe, THF, 0 ℃ to room temperature, 97%; (ii) MeMgBr, THF, 0 ℃, 53% (73% borsm)

With 25 and 90 obtained by facile chemical reactions on hand, the route to preparation 29 is known from previous pharmaceutical formulation reports developed at the discovery stage (a. rivkin et al, j.am. chem. soc.2003, 125, 2899; incorporated herein by reference). The critical 25 ring closure Metathesis reaction was carried out in toluene with a second generation Grubbs catalyst (Grubbs, R.H.; Miller, S.J.; Fu, G.C.Acc.Chem.Res.1995, 28, 446; Trnka, T.M.; Grubbs, R.H.Acc.Chem.Res.2001, 34, 18; Alkene Metathesis in Organic Chemistry editors: F ü rstner, A.; Springer, Berlin, (1998)); fur tner, a.angelw.chem.int.ed.engl.2000, 39, 3012; schrock, r.r.top.organomet.chem.1998, 1, 1; each of which is incorporated herein by reference). The reaction only yielded the trans isomer 48 in 71% yield. After insertion of the thiazole moiety by the method shown in scheme 14, two silyl protecting groups were removed with HF-pyridine to obtain 29, which was then converted to 2 in high yield by reduction of 9, 10-alkene. Epothilones of gram order of structure novelty have been made by total synthesis in a laboratory environment at the research institute scale.

Scheme 14 Synthesis of 26-CF₃Last step of- (E) -9, 10-dehydro-dEpoB (29)

Reagents and conditions: (a) EDCI, DMAP, CH₂Cl₂，25，From 0 ℃ to room temperature, 86%, starting from tert-butyl ester; (b) grubb's catalyst, toluene, 110 ℃,20 min, 71%; (c) (i) KHMDS, 101, THF, -78 ℃ to-20 ℃, 70%; (ii) HF-pyridine, THF, 98%.

Experiment of

The general method comprises the following steps: unless otherwise stated, reagents from commercial suppliers were used without further purification. Dichloromethane was obtained from a solvent drying system (through a pre-packed alumina column) and used without further drying. All air and water sensitive reactions were carried out in a baked glass device under a positive pressure of prepurified argon. NMR (C)¹H and¹³C) the spectra were recorded as a single note on BrokerAMX-400MHz or Bruker Advance DRX-500MHz, reference CDCl₃(¹H is 7.27ppm and¹³c77.0 ppm) or CD₂Cl₂(¹H is 5.32ppm and¹³c was 53.5 ppm). Infrared spectra (IR) were obtained on a Perkin-Elmer FT-IR model 1600 spectrometer. The optical rotation was obtained on a JASCO model DIP-370 digital polarimeter. Analytical thin layer chromatography was performed on e.merck silica gel 60F 254 plates. Compounds with no UV activity were visualized by immersing the plates in a solution of cerium ammonium molybdate or p-anisaldehyde and heating. In Whatman^*Preparative thin layer chromatography was performed on (LK6F silica gel 60A) TLC plate with the specified solvent.

Chemicals all epothilones are synthesized internally (c.r. harris, s.j. danishefsky, j.org. chem.1999, 64, 8434; d.s.su et al, angelw.chem.iht.ed.engl.1997, 36, 2093; Smart, b.e.j.fluorinine chem.2001, 109, 3; f.yoshimura et al, angelw.chem.2003, 42, 2518; Rivkin et al, j.org. chem. (2002, 124, 7737) each incorporated herein by reference). Taxol (Taxol)^*) And vinblastine sulfate (VBL) were purchased from Sigma. All of the compounds were dissolved in dimethylsulfoxide for in vitro analysis (VBL in saline). For in vivo studies, all epothilones and paclitaxel were dissolved in hydrogenated castor oil/ethanol (1: 1) vehicle, then diluted with saline and mounted via tail mounted using a specially designed microcatheterIntravenous infusion was performed for 6 hours (T. -C.Chou et al, Proc. Natl. Acad. Sci. USA.2001, 98, 8113-.

CCRF-CEM human lymphoid leukemia cells were obtained from Dr.William Beam of University of Illinois (Chicago). Human breast cancer (MX-1) and human lung cancer cells (A549) were obtained from American Type Culture Collection (ATCC, Rockville, Md.). Paclitaxel-resistant a 549/paclitaxel cells (44-fold resistant) were formed using the methods described above (t. -c. chou et al, proc. natl acadsi. usa.2001, 98, 8113-.

Athymic nude mice with nu/nu gene were obtained from NCI (Frederick, MD) and used for all human tumor xenografts. Male nude mice aged 6 weeks or more and having a body weight of 20 to 22g or more are used. Drugs were administered by 6 hour tail intravenous infusion using self-made infusion microcatheters and storage tubes (containment tubes) (T. -C.Chou et al, Proc. Natl. Acad. Sci. USA.2001, 98, 8113-. Intravenous infusion was performed using a multi-channel programmable Harvard PHD2000 syringe pump. A typical 6 hour infusion volume of each drug dissolved in hydrogenated castor oil/ethanol (1: 1) is 100ml in 2.0ml saline. Tumor volume was estimated by measuring length x width x height (or width) with calipers. For nude mice with tumors during the course of the experiment, body weight refers to the total weight minus the weight of the tumor. All animal studies were conducted according to guidelines in the National institutes of health Guide for the Care and Use of Animals and protocols approved by the institutional animal Care and Use committee of the schlon-katherin Cancer-commemorative Center (the medical slope-committing Cancer Center).

In preparation for in vitro cytotoxicity assays, 2 to 5X 10⁴Cells were cultured at an initial density of cells/ml. The cells were cultured in RPMI Medium 1640(GIBCO/BRL) at 37 ℃ in 5% CO₂Maintained under humid atmosphere, RPMI vehicle 1640 contained penicillin (100 units/mL), streptomycin (100. mu.g/mL, GIBCO/BRL) and 5% heat-inactivated FBS. For solid tumor cells (e.g., A549) grown in monolayers, drug cytotoxicity was determined by sulforhodamine B (sulforhodamine B) method in 96-well microtiter plates (P.Skehan et al, J.Natl cancer. Inst.1990, 82, 1107-1112; incorporated herein by reference). For cells grown in suspension (e.g., CCRF-CEM and sublines thereof), cytotoxicity was determined in 96-well microtiter plates using 2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -5-anilino-formyl) -2H-tetrazolium hydroxide (XTT) microculture (D.A. Scudiero et al, Cancer Res. (1988, 48, 4827-4833); incorporated herein by reference), were determined twice in the same manner. For both methods, absorbance was measured for each well using a microplate reader (Power Wave XS, Bio-Tek, Winooski, VT). Dose-effect relationship data (T. -C.Chou, M.Hayball.CalcuSynfor Windows, Multiple-drug dose effect analyzer and manual. Biosoft, Cambridge Place, Cambridge, UK (1997); incorporated herein by reference) were analyzed from 6 to 7 concentration points per drug by a computer program using median-effect curves, determined twice in the same manner.

Stability of epothilones in mouse and human liver S9 sections A full-automatic HPLC system consisting of Prospekt-2(Spark Holland, Netherlands) sample preparation system and an Agilent 1100 HPLC system was used for stability studies. Briefly, Prospekt2 picked a C8 extraction tube and washed with acetonitrile and water. An Agilent autosampler set at 37 ℃ takes 20 μ l of sample, loads it into the tube, washes with water, then Prospekt-2 directs the mobile phase stream through the extraction tube onto an analytical column (reliability Stable Bond C8 with guard column, 4 x 80mm (MacMod, chads Ford, PA)) and monitors the eluent at 250 nm. The mobile phase consisted of 53 or 65% acetonitrile/0.1% formic acid with a flow rate of 0.4ml/min, so that the retention time of the compound under investigation was about 6 minutes. Sample preparation involved adding an equal volume of plasma to PBS to a total volume of 300-. For the collected human liver microsome S9 fraction (Xeno Tech, Lenex, KS), 20. mu.l (400. mu.g) of the S9 fraction was mixed with 280. mu.l of PBS and the study was continued as described above. The sampling period was controlled by an autosampler and peak area data was collected to compare the rate of disappearance of the parent compound.

Determination of octanol-water partition constant (POW) the partition constant of octanol-water was estimated using the HPLC method. An Agilent 1100 HPLC system with an eclipse XDB C18 column (4.6X 250mM) was used with a mobile phase of 60% acetonitrile/40% 25mM potassium phosphate buffer (pH 7.4) at a flow rate of 0.8ml/min and the eluate was monitored at 250 nm. The standard solutions used were benzyl alcohol, acetophenone, benzophenone, naphthalene, diphenyl ether and dibenzyl ether known as POW of 1.1, 1.7, 3.2, 4.2 and 4.8, respectively. Time zero was estimated using sodium dichromate, and was 2.5 minutes, and the retention times of these standard solutions were 3.9, 5.4, 10.6, 14.18.7, and 19.8 minutes, respectively. By the formula k ═ t_rt-t_o)/t_oThe k value is calculated. linear regression of log k against logPOW to obtain r²Straight line 0.966. The plot was used to estimate POW values for epothilone analogs.

Spectroscopic data for 29 (26-trifluoro- (F) -9, 10-dehydro-dpob):

[α]_D ²⁵-54.6(c 0.28，CHCl₃) (ii) a IR (film) v3478, 2974, 2929, 1736, 1689, 1449, 1381, 1318, 1247, 1169, 1113, 1039, 983, 867, 736cm^-1；¹H NMR(400MHz，CDCl₃) δ 1.05(3H, s), 1.12(3H, d, J ═ 7.0Hz), 1.23(3H, d, J ═ 6.8Hz), 1.37(3H, s), 2.04(1H, brd, J ═ 3.8Hz, -OH), 2.12(3H, s), 2.25-2.33(1H, m), 2.38(1H, dd, J ═ 15.3 and 3.0Hz), 2.48(1H, dd, J ═ 15.4 and 9.8Hz), 2.54-2.61(1H, m), 2.66-2.76(1H, m), 2.71(3H, s), 2.96(1H, dd, J ═ 16.5 and 4.5Hz), 3.02(1H, dd, J ═ 16.5 and 3.6H, 3.5), 3.7H, 19, 7H, 7, 7.5 (7H, 7Hz), br ═ H, 3.9.8 Hz),4.35(1H, brd, J ═ 9.5Hz), 5.42(1H, dd, J ═ 6.2 and 4.1Hz), 5.60(1H, ddd, J ═ 15.8, 5.6 and 4.5Hz), 5.66(1H, dd, J ═ 15.8 and 5.8Hz), 6.24(1H, t, J ═ 7.2Hz), 6.64(1H, s), 7.00(1H, s);¹³C NMR(100MHz，CDCl₃)δ15.1，16.1，17.7，18.5，19.3，22.5，28.8，31.1，39.6，39.7，45.0，53.7，71.4，75.3，76.8，116.7，1202，124.3[q，¹J(C，F)＝273.4Hz]，127.9，130.2[q，³J(C，F)＝6.0Hz]，130.6[q，²J(C，F)＝28.4Hz]，132.5，136.7，152.0，165.4，170.2，218.4；C₂₇H₃₇F₃NO₅LRMS (ESI) calculation of S [ M + H ]⁺]544.2, test value 544.1.

Example 11: in vitro study

Typical tests involve testing at 2 to 5X 10⁴Cells are cultured at an initial density of cells/ml (e.g., CCRF-CEM). The cells were cultured in RPMI Medium 1640(GIBCO/BRL) at 37 ℃ in 5% CO₂Maintained under humid atmosphere, RPMI medium 1640 contained penicillin (100 units/mL), streptomycin (100. mu.g/mL, GIBCO/BRL) and 5% heat-inactivated fetal bovine serum. For cells grown in suspension (e.g., CCRF-CEM and sublines thereof), cytotoxicity was determined in 96-well microtiter plates using 2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -5-anilino-formyl) -2H-tetrazolium hydroxide (XTT) microculture tetrazolium assay and was determined twice in the same manner. For both methods, absorbance was measured for each well using a microplate reader (Power Wave XS, Bio-Tek, Winooski, VT). Each test requires 6 or 7 concentration points of the test drug. Dose-effect relationship data were analyzed using median-effect curves.

CCRF-CEM human T cells, acute lymphocytic leukemia cells, their teniposide (teniposide) -resistant sublines (CCRF-CEM/VM)₁) And vinblastine-resistant sublines (CCRF-CEM/VBL)₁₀₀) Are all available from W.T. Beck (University of Illinois, Chicago, II).

In a typical experiment, as outlined above, certain aspects of the inventionThe compounds (e.g., 9, 10-dehydro-EpoD) exhibited activity in CCRF-CEM cell lines and in anti-paclitaxel CCRF-CEM cell lines. Certain of these compounds exhibit an IC in the range of 0.0015 to about 0.120 for CCRF-CEM cell lines₅₀. Certain other compounds exhibit an IC of 0.0015 to about 10.5₅₀. Certain of these compounds also exhibit an IC in the range of 0.011 to about 0.80 against CCRF-CEM/paclitaxel resistant cell lines₅₀And certain other compounds exhibit an IC in the range of 0.011 to about 13.0 μ M₅₀. In certain embodiments, 26F-EpoD exhibits activity in the 0.0015 μ M range for CCRF-CEM cell lines and in the 0.011 μ M range for CCRF-CEM/paclitaxel resistant cell line cell lines (fig. 11).

Example 12: in vivo studies

Athymic nude mice with nu/nu gene are commonly used for tumor xenografts. Inbred swiss mice were obtained from Charles River Laboratories. Most experiments used male mice that were 8 weeks old or more and weighed 22g or more. The drug was administered by 6 hour intravenous infusion into the tail vein. Each mouse was restrained in a perforated Falcon polypropylene tube restraint for drug administration. Tumor volume was estimated by measuring length x width x height (or width) with calipers. Intravenous infusion was performed using a multichannel programmable Harvard PHD2000 syringe pump (Harvard apparatus). All animal studies were conducted according to guidelines in the U.S. health agency "rules of care and use for animals" and protocols approved by the committee for laboratory animal care and use of the semen-kelin commemorative cancer center. To meet the committee's humane treatment policy for animals bearing tumors, the tumors were euthanized when they reached > 10% of the total weight of the mice.

As shown in figure 8, 9, 10-dehydro-EpoB was tested in nude mice with human breast cancer MX-1. In general, 9, 10-dehydro-EpoB can be formulated as follows: 9, 10-dehydro-EpoB was dissolved in ethanol and hydrogenated castor oil (1: 1) was added at a concentration of 20 mg/ml. The solution was diluted with saline for intravenous infusion. The diluted solution was used for intravenous infusion over 1 hour. Then, the tumor size and body weight were measured with doses of 10mg/Kg, 20mg/Kg and 30mg/Kg after 15 days. Tumor size and body weight can also be measured with dosing regimens of 0.4mg/Kg Q3D x 2, 0.5mg/KgQ3D x 2, and 0.6mg/Kg Q3D x 5 (see fig. 33, 34, 55, and 56). A dosing regimen of once every three days was used to reduce toxicity. Additional efficacy studies on 9, 10-dehydro-EpoB are shown in FIGS. 70 and 71 (CCRF-CEM/paclitaxel Q3D X5) and FIGS. 23 and 24(HCT-116, Q2D X7).

The compound 9, 10-dehydro-12, 13-desoxyepothilone B (iso-490 epothilone) was three times more potent than dEpoB. It has been demonstrated that 9, 10-dehydro-12, 13-desoxyepothilone D inhibits tumor growth after two to three infusions at 10mg/Kg or 20mg/Kg, each administered every other day. Better results were obtained in mice with two 6 hour intravenous infusions (once every other day) of 9, 10-dehydro-12, 13-desoxyepothilone B at a dose of 30 mg/kg. 9, 10-dehydro-dEpoB was also administered by intravenous infusion at 5mg/kg, Q3D X9, 6 hours, and tumors were eliminated in nude mice bearing MX-1 xenografts, no mice died and only modest weight loss (FIGS. 74 and 75). This appears to have been achieved by administering epothilone analogs every other day to reduce toxicity (see FIGS. 53 and 54). In conclusion, 9, 10-dehydro-12, 13-desoxyepothilone B has reduced toxicity, more effective tumor growth inhibition and better serum stability compared to other epothilones. Other treatment studies are shown in figures 17 and 18(HCT-116, Q2D x 5 and Q3D x 5); FIGS. 19 and 20 (A549/paclitaxel, Q3D X7); and FIGS. 21 and 22 (A549/paclitaxel, Q2D X7).

When 9, 10-dehydro-Epo B was administered at a dose of 0.4 to 0.6mg/Kg at a frequency of once every three days for 9 to 11 times (6 hour intravenous infusion), it resulted in tumor shrinkage and disappearance in mice with transplanted human breast cancer MX-1 xenografts (fig. 68 and 69). Tumor growth was inhibited but tumors were not reduced by 8 administrations at daily intervals. When 9, 10-dehydro-Epo B was administered at a frequency of 9 times every other day, the transplanted tumors continued to shrink moderately from day two to day 8, while the body weight recovered very slowly from 76% to 82% of the control group. On day ten, 1/4 disappeared the tumor. When 9, 10-dehydro-EpoB was administered to nude mice bearing HCT-116 xenografts at a dose of 0.6mg/kg (Q2W X6, 6 hour infusion), four mice died of toxicity within three days after the sixth dose. anti-CCRF-CEM/paclitaxel tumors were arrested by administration of 9, 10-dehydro-EpoB at a dosing regimen of 0.6mg/kg (Q3D X5,. times.2) (FIGS. 70 and 71).

As shown in the figure, 26-trifluoro-9, 10-dehydro-12, 13-deoxy-epothilone B (F) was injected into a nude mouse model with human breast cancer MX-1 xenograft₃-deH-dEpoB) at 20mg/kg and 30mg/kg (Q2D X6, 6 hour infusion). The data also show that: 30mg/kgQ2D X6 is the approximate maximum permissible dose. The 26-trifluoro-9, 10-dehydro-12, 13-deoxy-epothilone B, infused at 20mg/kg, Q2D X6, 6 hours, reduced and disappeared tumors in all four nude mice bearing human breast cancer MX-1 xenografts. No tumors reappeared on day 20 after treatment discontinuation. On day 27 after cessation of treatment, 2/4 mice reappeared tumors. No more tumors appeared during days 28-64 after the treatment was stopped. In contrast, dEpoB at 30mg/kg in the same mouse model allowed the tumor to disappear in 7 mice; however, tumors reappeared in 2/5 mice on day 8 after treatment discontinuation. The administration of 26-trifluoro-9, 10-dehydro-12, 13-deoxy-epothilone B by intravenous infusion at 20mg/kg, Q2D × 6,6 hours caused a temporary reduction in body weight of the mice of up to 26%. This weight loss did not lead to death, indicating that there was no serious toxicity to the vital organs. Two days after the last treatment, body weight began to recover. On day 16 post-treatment, body weight was restored to 109% of the pretreated control group, indicating complete elimination of toxicity (if any). In contrast, administration of dEpoB at 30mg/kg resulted in a 31% reduction in body weight, but was not lethal.

Tumor disappearance appeared 2 to 3 days earlier when 26-trifluoro-9, 10-dehydro-12, 13-deoxy-epothilone B was administered by intravenous infusion at 30mg/kg, Q2D X6, 6 hours than when it was administered at a dose of 20 mg/kg. Body weight loss was 27% at this higher dose and continued for 4 days without causing death, which demonstrated no severe toxicity to vital organs. Four days after the last treatment at 30mg/kg, body weight began to recover. At day 16 post-treatment, body weight was restored to 98% of the pretreatment control group, again demonstrating reversibility of toxicity. Treatment with 26-trifluoro-9, 10-dehydro-dEpoB at 20mg/kg and 30mg/kg resulted in the disappearance of all tumors and at 30mg/kg dose, with no recurrence after 62 days. Tumor disappearance was also achieved after 9 administrations at a dose of 10mg/kg and three additional administrations (FIG. 57). Only minor weight loss was observed with the administration of 26-trifluoro-9, 10-dehydro-dpob at 10mg/kg (figure 58). Continued treatment did not see further weight loss.

FIG. 59 summarizes 26-F₃The effect of 9, 10-deH-dEpo B (and other epothilones) against MX-1 xenografts, A. low dose; B. anti-large tumor; C. anti-a 549 lung cancer xenografts; anti-paclitaxel resistant lung cancer a 549/paclitaxel xenografts.

FIG. 61 shows the in vitro potency of C-21 modified epothilones against CCRF-CEM, CCRF-CEM/VBL and CCRF-CEM/paclitaxel.

FIG. 62 shows 26-F₃Therapeutic efficacy of 9, 10-deH-dEpoB (15mg/kg and 30mg/kg) and paclitaxel (20mg/kg) (Q2D X8, 6 hours intravenous infusion) against human T-cell lymphoid leukemia CCRF-CEM xenografts. Similar weight loss was observed in the three groups of treatments used (figure 63).

With 26-F₃Treatment of CCRF-CEM/paclitaxel xenograft (anti-paclitaxel) with 15mg/kg of-9, 10-deH-dEpo B achieved 1/3 tumor disappearance and 30mg/kg achieved 3/4 tumor disappearance. The same treatment with 20mg/kg paclitaxel only partially inhibited tumor growth and did not achieve tumor shrinkage (fig. 64). The change in body weight during this trial is shown in figure 65.

With 26-F₃Treatment of HCT-116 xenografts of human colon carcinoma with 9, 10-deH-dEpo B (20mg/kg) achieved similar effects to paclitaxel (20 mg/kg). However, 30mg/kg of F₃Better therapeutic effect was obtained with deH-dEpo B, 2/4 tumor disappeared after 5 doses (FIG. 66). The change in body weight during this test is shown in figure 67.

In different doses (5 to 30mg/kg)F₃The therapeutic effect of-9, 10-dehydro-dEpoF (6 hour intravenous infusion and intravenous injection) against MX-1 xenografts is shown in FIGS. 76 and 77.

In conclusion, 9, 10-dehydro modification, 26-trifluoro modification or both of these modifications on dEpoB resulted in an increase in cytotoxicity in vitro of 1.5 to 5 fold and an increase in half-life in plasma of mice in vitro of 2 to 5 fold. The antitumor efficacy and toxicity of 9, 10-dehydro-epothilone were evaluated by using a human solid tumor xenograft model in nude mice and at the maximum tolerated dose by the tail vein using the Q2D × 5-9, 6 hour intravenous infusion technique. The ability to achieve complete tumor growth inhibition, tumor shrinkage and disappearance was further studied to determine the recurrence rate and cure rate after treatment cessation. Although the most potent epothilones 9, 10-dehydro-EpoB are known to be extremely potent in vitro, they exhibit a narrow therapeutic safety margin in vivo. 4mg/kg of 9, 10-dehydro-dEpoB, 0.4mg/kg of 9, 10-dehydro-EpoB and 3mg/kg of 21-hydroxy-9, 10-dehydro-dEpoB strongly inhibited tumor growth over a long period of time and achieved shrinkage of some tumors and disappearance of some tumors. In all mice tested, 30mg/kg of dEpoB, 20mg/kg of 26-trifluoro-9, 10-dehydro-dEpoB and 20mg/kg of paclitaxel showed strong inhibition of tumor growth and achieved tumor shrinkage and disappearance (human breast cancer MX-1 xenograft). Compared to either dEpoB or paclitaxel, 26-trifluoro-9, 10-dehydro-dEpoB achieved long-term cure without tumor recurrence and showed that body weight returned equally rapidly to pretreatment control levels.

Example 13: synthesis of cyclopropyl-epothilone analogs

Claims

1. A compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₂is a substituted or unsubstituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety;

R₅and R₆Are each independently hydrogen or a protecting group;

x is O, S, C (R)₇)₂Or NR₇Wherein R is present₇Are all independently hydrogen or lower alkyl;

each occurrence of R_BIndependently is hydrogen; halogen; -OR_B’；-SR_B’；-N(R_B’)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B’、NHR_B’、N(R_B’)₂Or SR_B’；-C(O)OR_B’；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety; and

m is 1, 2, 3 or 4.

2. A compound having the formula:

wherein

X is O, S, C (R)₇)₂Or NR₇In which each occurrence of R₇Independently hydrogen or lower alkyl;

R₅and R₆Each independently is hydrogen or a protecting group;

R_Bis hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)0R_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety;

R₈independently of one another hydrogen, halogen, -OR₉、-SR₉、-N(R₉)₂、-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′；、NHR_B′、N(R_B′)₂Or SR_B′；-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-0(C＝0)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following groups: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety,

wherein each occurrence of R₉Independently is hydrogen; a protecting group; a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; a polymer; a carbohydrate; a photoaffinity label; or a radioactive label;

wherein each occurrence of V is independently hydrogen, halogen, hydroxy, sulfur, amino, alkylamino, or protected hydroxy, sulfur, or amino; each occurrence of t is 0, 1 or 2; and each occurrence of n is independently 0 to 10.

3. The compound of claim 2, wherein R_BIs methyl.

4. The compound of claim 2, wherein R_Bis-CF₃。

5. The compound of claim 2, 3 or 4, wherein R₈Is methyl.

8. The compound of claim 2, 3 or 4, wherein R₈is-CH₂OH。

9. The compound of claim 2, 3 or 4, wherein R₈is-CH₂NH₂。

10. A compound having the formula:

wherein

R₅and R₆Each independently is hydrogen or a protecting group;

R_Bis hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linearOr a branched aliphatic, heteroaliphatic, aryl or heteroaryl group, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety;

R₈independently of one another hydrogen, halogen, -OR₉、-SR₉、-N(R₉)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-0(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following groups: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety,

11. The compound of claim 10, wherein R_BIs methyl.

12. The compound of claim 10, wherein R_Bis-CF₃。

13. The compound of claim 10, 11 or 12, wherein R₈Is methyl.

14. The compound of claim 10, 11 or 12, wherein R₈is-CH₂OH。

15. The compound of claim 10, 11 or 12, wherein R₈is-CH₂NH₂。

16. A compound having the formula:

wherein R is₅And R₆Independently hydrogen or a protecting group;

R₈independently of one another hydrogen, halogen, -OR₉、-SR₉、-N(R₉)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV²)_nSR⁹、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-0(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following groups: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV²)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety,

wherein each occurrence of V is independently hydrogen, halogen, hydroxy, sulfur, amino, alkylamino, or protected hydroxy, sulfur, or amino;

each occurrence of R_BIndependently is hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a Ring (C)A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety.

17. The compound of claim 16, wherein R_BIs methyl.

18. The compound of claim 16, wherein R_Bis-CF₃。

19. The compound of claim 16, 17 or 18, wherein R₈Is methyl.

20. The compound of claim 16, 17 or 18, wherein R₈is-CH₂OH。

21. The compound of claim 16, 17 or 18, wherein R₈is-CH₂NH₂。

22. A compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₂is a substituted or unsubstituted aryl, heteroaryl, arylalkyl or heteroarylalkyl moietyDividing;

R₅and R₆Independently hydrogen or a protecting group;

each occurrence of R_BIndependently is hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety.

23. A compound having the formula:

wherein

R_BIs hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)0R_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently hydrogen, alkyl, aryl or a protecting group; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety;

R₅and R₆Independently hydrogen or a protecting group;

R₈independently of one another hydrogen, halogen, -OR₉、-SR₉、-N(R₉)₂、-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following groups: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety,

wherein each occurrence of V is independently hydrogen, halogen, hydroxy, sulfur, amino, alkylamino, or protected hydroxy, sulfur, or amino; each occurrence of t is independently 0, 1 or 2; and each occurrence of n is independently 0 to 10.

24. The compound of claim 23, wherein R_BIs methyl.

25. The compound of claim 23, wherein R_Bis-CF₃。

26. The compound of claim 23, 24 or 25, wherein R₈Is methyl.

27. The compound of claim 23, 24 or 25, wherein R₈is-CH₂OH。

28. The compound of claim 23, 24 or 25, wherein R₈is-CH₂NH₂。

29. A compound having the formula:

wherein

R_BIs hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety;

R₅and R₆Independently hydrogen or a protecting group;

R₈independently of one another hydrogen, halogen, -OR₉、-SR₉、-N(R₉)₂、-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B′、NHR_B′、N(R_B′)₂Or SR_B′；-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-0(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety, optionally substituted with one or more of the following groups: halogen, -OR₉、-SR₉、-N(R₉)₂、-(CV₂)_nOR₉、-(CV₂)_nN(R₉)₂、-(CV₂)_nSR₉、-(C＝O)R₉、-O(C＝O)R₉、-(C＝O)OR₉、-O(C＝O)OR₉；-NH(C＝O)R₉、-NH(C＝O)OR₉、-(C＝O)NHR₉Or a cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl moiety,

30. The compound of claim 29, wherein R_BIs methyl.

31. The compound of claim 29, wherein R_Bis-CF₃。

32. The compound of claim 29, 30 or 31, wherein R₈Is methyl.

33. The compound of claim 29, 30 or 31, wherein R₈is-CH₂OH。

34. The compound of claim 29, 30 or 31, wherein R₈is-CH₂NH₂。

35. A compound having the formula:

36. a compound having the formula:

37. a compound having the formula:

38. a compound having the formula:

a compound having the formula:

38b. a compound having the formula:

39. a trans-9, 10-dehydro-cis-12, 13-deoxyepothilone analog.

40. A trans-9, 10-dehydro-cis-12, 13-dehydro epothilone analog wherein the analog is characterized by IC in a CCRF-CEM cell line₅₀Less than 0.01.

41. A trans-9, 10-dehydro-cis-12, 13-dehydro epothilone analog wherein the analog is characterized by IC in a CCRF-CEM cell line₅₀Less than 0.05.

42. A trans-9, 10-dehydro-cis-12, 13-dehydro epothilone analog wherein the analog is characterized by IC in a CCRF-CEM cell line that is primary resistant to paclitaxel₅₀Less than 0.01.

43. A trans-9, 10-dehydro-cis-12, 13-dehydro epothilone analog wherein the analog is characterized by IC in a CCRF-CEM cell line that is primary resistant to paclitaxel₅₀Less than 0.05.

44. A pharmaceutical composition comprising a trans-9, 10-dehydro-cis-12, 13-dehydro epothilone analog and a pharmaceutically acceptable excipient.

45. A pharmaceutical composition for the treatment of cancer comprising a compound of any one of claims 1 to 32 and a pharmaceutically acceptable excipient.

46. The pharmaceutical composition of claim 44 or 45, further comprising hydrogenated castor oil.

47. The pharmaceutical composition of claim 44 or 45, further comprising hydrogenated castor oil and ethanol.

48. The pharmaceutical composition of claim 44 or 45, wherein the compound is suspended in 1: 1 hydrogenated castor oil/EtOH.

49. The pharmaceutical composition of claim 44 or 45, further comprising an additional cytotoxic agent.

50. A pharmaceutical composition for treating cancer, comprising:

a therapeutically effective amount of a compound of any one of claims 1 to 43, or a pharmaceutically acceptable salt thereof; and

a pharmaceutically acceptable carrier or diluent for the carrier or diluent,

wherein the therapeutically effective dose of the compound is an amount sufficient to deliver from about 0.001 to about 40mg of the compound per Kg of patient body weight.

51. A method of treating cancer, comprising:

administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 32.

52. The method of claim 51, wherein the therapeutically effective dose of the compound is an amount sufficient to deliver from about 0.001mg to about 40mg of the compound per Kg of patient body weight.

53. The method of claim 51, wherein the therapeutically effective dose of the compound is an amount sufficient to deliver from about 0.1mg to about 25mg of the compound per Kg of patient body weight.

54. A process for preparing a compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₅and R₆Independently hydrogen or a protecting group;

m is 1, 2, 3 or 4, the method comprising the steps of:

subjecting a compound having the formula:

55. a process for preparing a compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₅and R₆Independently hydrogen or a protecting group;

each occurrence of R_BIndependently is hydrogen; halogen; -OR_B’；-SR_B’；-N(R_B’)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B’、NHR_B’、N(R_B’)₂Or SR_B’；-C(O)OR_B’；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or a cyclic or acyclic, linear or branched substituted or unsubstituted aliphatic, heteroaliphatic, aryl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic radical, heteroaliphatic radical, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroaryl radicalAn alkynyl moiety; and

m is 1, 2, 3 or 4, the method comprising the steps of:

subjecting a compound having the formula:

56. the method of claim 54, wherein a ring-closing metathesis reaction conditions include a Grubbs catalyst.

57. The process of claim 565, wherein said Grubbs catalyst is tricyclohexylphosphine [1, 3-bis (2, 4, 6-trimethylphenyl) -4, 5-dihydroimidazol-2-ylidene ] [ benzylidene ] ruthenium (IV) chloride.

58. A process for preparing a compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₅and R₆Independently hydrogen or a protecting group;

m is 1, 2, 3 or 4, the method comprising the steps of:

reducing a compound having the formula:

59. a process for preparing a compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₅and R₆Independently hydrogen or a protecting group;

m is 1, 2, 3 or 4, the method comprising the steps of:

oxidizing a compound having the formula:

60. a process for preparing a compound having the formula:

wherein R is₁Is hydrogen or lower alkyl;

R₅and R₆Independently hydrogen or a protecting group;

each occurrence of R_BIndependently is hydrogen; halogen; -OR_B’；-SR_B’；-N(R_B’)₂；-CY₃、-CHY₂、-CH₂Y, wherein Y is F, Br, Cl, I, OR_B’、NHR_B’、N(R_B’)₂Or SR_B’；-C(O)OR_B’；-C(O)R_B’；-CONHR_B’；-O(C＝O)R_B’；-O(C＝O)OR_B’；-NR_B’(C＝O)R_B’；N₃；N₂R_B’(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched aliphatic, heteroaliphatic, aryl or heteroaryl, optionally substituted with one or more of the following: hydrogen; halogen; -OR_B′；-SR_B′；-N(R_B′)₂；-C(O)OR_B′；-C(O)R_B′；-CONHR_B′；-O(C＝O)R_B′；-O(C＝O)OR_B′；-NR_B′(C＝O)R_B′；N₃；N₂R_B′(ii) a A cyclic acetal; or cyclic or acyclic, linear or branched, substituted or unsubstituted aliphatic, heteroaliphatic, aromaticA phenyl or heteroaryl moiety; or an epothilone, desoxyepothilone or analog thereof; or a polymer; a carbohydrate; a photoaffinity label; or a radioactive label; wherein each occurrence of R_B′Independently is hydrogen; a protecting group; a linear or branched, substituted or unsubstituted, cyclic or acyclic aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl or heteroarylalkynyl moiety; and

m is 1, 2, 3 or 4, the method comprising the steps of:

reacting a phosphine oxide or Wittig reagent having the structure:

condensation with a ketone having the structure:

wherein R 'and R' are independently C_1-8Linear or branched chain alkyl, or a substituted or unsubstituted phenyl, aryl, alkoxy or aryloxy group; and

x is a counterion such as chloride or bromide.