CN1976699A

CN1976699A - Synthesis of epothilones, intermediates thereto, analogues and uses thereof

Info

Publication number: CN1976699A
Application number: CN 200580013480
Authority: CN
Inventors: 塞缪尔·J·丹尼斯海弗斯凯; 亚历克谢·芮弗金; 吉村文彦; 周廷潮; 安娜·埃斯特·加巴尔答欧特佳; 董华进; 吴开达; 马尔科姆·A·S·摩尔; 大卫·杜恩
Original assignee: Sloan Kettering Institute for Cancer Research
Current assignee: Memorial Sloan Kettering Cancer Center
Priority date: 2004-02-27
Filing date: 2005-02-28
Publication date: 2007-06-06

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, associated intermediate, analog synthetic and uses thereof

Background of invention

Epothilones (Epothilone) A and B (2a and 2b, sketch map 1) be from the cellulose degradation mycobacterium, the n cell toxin macrolide of separating in the sorangium cellulosum (sorangium cellulosum) (people Angew.Chem. such as H  fle, Int.Ed.Engl.1996,35,1567 and J.Antibiot.1996,49,560; Every piece of document is incorporated the present invention by reference into).Although their structure differs widely, Epothilones A and B have as paclitaxel (Taxol (Taxol) ^) the mechanism of action, this mechanism comprises growth (people Cancer Res.1995 such as Bollag, 55,2325 of the anticancer by the stable of tubulin polymerization and microtubule assembling (microtubuleassemblies); Incorporate the present invention by reference into).Although it has unquestionable clinical value, Taxol as the front chemotherapeutant ^It far not ideal medicament.But its dissolubility that is slightly soluble in water makes it must be by preparation vehicle Li Mofu (Cremophor) for example, and this causes himself danger and problem of management (people J.Allergy Clin.Immunol.1996 such as Essayan, 97,42; Incorporate the present invention by reference into).In addition, Taxol ^Be easy to inactivation (people J.Biol.Chem.1997 such as Giannakakou, 272,17118 by multi-drug resistant (MDR); Incorporate the present invention by reference into).Yet this has illustrated that also Epothilones A and B have kept remarkable effectiveness (the people Mol.Biol.Cell 1995,6,2137 such as Kowalski of anti-MDR tumor cell; Incorporate the present invention by reference into).In addition, for the prescription ability (formulability) of Epothilones, it may be useful that the water-soluble of increase is compared with paclitaxel.Though native compound, epothilone B (2b, EpoB, in the sketch map 1), be the effective Epothilones family member of natural product, but at least in the xenotransplantation mice, unfortunate have narrow troublesomely therapeutic index (people Angew.Chem.Int.Ed.Engl.1997 such as Su, 36,1093; People J.Org.Chem.1999 such as Harris, 64,8434; Every piece of document is incorporated the present invention by reference into).

2a R ₁=H, R ₂=CH ₃, Epothilones

A(EpoA)

2b R ₁=CH ₃, R ₂=CH ₃, Epothilones

1a R=Ph, paclitaxel (Taxol) B (EpoB)

1b R=t-Bu, Docetaxel (Taxotere) 2c R ₁=H, R ₂=CH ₂OH, Epothilones

E(EpoE)

2d R ₁=CH ₃, R ₂=CH ₂OH, the dust slope is mould

Plain F (EpoF)

Sketch map 1: taxanes (Taxoids) and Epothilones

In view of EpoB has limited therapeutic index, people study other epothilone analogs, and particularly 12, the 13-NSC-703147, the ability that improved treatment characteristic (therapeuticprofile) is provided is (referring to United States Patent (USP) 6,242,469,6,284,781,6,300,355,6,369,234,6,204,388,6,316,630; Every piece of document is incorporated the present invention by reference into).Experimental results show that 12 in the body of on various mouse models, implementing, 13-NSC-703147 B (3b in the sketch map 2, dEpoB) in the mice xenograft, have anti-various sensitivities and drug-fast people's tumor treatment and render a service (people Proc.Natl.Acad.Sci.U.S.A.1998 such as Chou, 95,9642 and 15798; Incorporate the present invention by reference into).Recently, these NSC-703147 compare other anticarcinogen the treatment advantage by comparative study by final certification (people Proc.Natl.Acad.Sci.U.S.A.2001 such as Chou, 98,8113; Incorporate the present invention by reference into).Because its impressive body internal characteristic, dEpoB have passed through the intravital toxicity evaluation of Canis familiaris L. in advance, and now carrying out human trial as cancer therapy drug.

3a R ₁=H, R ₂=CH ₃, deoxidation dust 4a R ₁=H, R ₂=CH ₃, dehydrogenase 35 a R ₁=H, R ₂=CH ₃, different

Slope mycin A (dEpoA) dEpoA (ddEpoA)-dEpoA

3b R ₁=CH ₃, R ₂=CH ₃, deoxidation 4b R ₁=CH ₃, R ₂=CH ₃, dehydrogenase 35 b R ₁=CH ₃, R ₂=CH ₃, different

Epothilone B (dEpoB) dEpoB (ddEpoB)-dEpoB

3c R ₁=H, R ₂=CH ₂OH, deoxidation 4c R ₁=H, R ₂=CH ₂OH, dehydrogenase 35 c R ₁=H, R ₂=CH ₂OH, different

Epothilone E (-)-Epothilone E. (dEpoE) dEpoE (ddEpoE) dEpoE

3d R ₁=CH ₃, R ₂=CH ₂OH, 4d R ₁=CH ₃, R ₂=CH ₂OH takes off 5d R ₁=CH ₃, R ₂=CH ₂OH,

The different dEpoF of NSC-703147 F (dEpoF) hydrogen dEpoF (ddEpoF) (ddEpoF)

3e R ₁=H, R ₂=NH ₂, demethylation 4e R ₁=H, R ₂=NH ₂, demethylation ammonia 5e R ₁=H, R ₂=NH ₂, demethylation

Amino-dEpoA (dadEpoA) base-ddEpoA amino-different-dEpoA

3f R ₁=CH ₃, R ₂=NH ₂, piptonychia 4f R ₁=CH ₃, R ₂=NH ₂, piptonychia 5fR ₁=CH ₃, R ₂=NH ₂, piptonychia

The basic amino of base amino-dEpoB (dadEpoB)-ddEpoB base amino-different-dEpoB

3g R ₁=CH ₂F, R ₂=CH ₃, 26-fluorine 4g R ₁=CH ₂F, R ₂=CH ₃, the 26-fluorine

-dEpoB -ddEpoB

3h R ₁=CF ₃, R ₂=CH ₃, 26-three 4h R ₁=CF ₃, R ₂=CH ₃, the 26-trifluoro

Fluoro-dEpoB-ddEpoB

Sketch map 2. various NSC-703147 analog

Consider describedly 12, the promising treatment effectiveness of 13-NSC-703147 is necessary to study other analog and other synthetic method, be used for synthetic existing Epothilones, NSC-703147 and analog thereof with and novel analog.Especially, in view of on the treatment effectiveness of this compounds, becoming interested, be necessary to develop can provide the chemical compound described in a large amount of aforesaid any Epothilones or NSC-703147 or the present invention methodology, be used for clinical trial and mass preparation.

Description of drawings

Fig. 1 represents 12-trifluoromethyl-9, and 10-dehydrogenation NSC-703147 D's is synthetic.

Fig. 2 represents to prepare 12-trifluoromethyl-9 by Epo A, the several method of 10-dehydrogenation NSC-703147 D.

Fig. 3 represents to prepare 9 to the C9-C10 position, 10-dehydrogenation-epoD by Epo 490 by making the C10-C11 double-bond isomerization.

Fig. 4 has described 9, and the various epothilone analogs of modification are arranged on the 10-position.

Fig. 5 represents to adopt 9, the locational closed loop metathesis reaction of 10-and carry out various 9,10-dehydrogenation-epothilone analogs synthetic.

Fig. 6 represents experimental result, and wherein the MX-1 xenograft of super large is implanted in the nude mice.With MX-1 tumor tissues (50mg) subcutaneous implantation in the 0th day.When the tumor size reached 960 ± 132mg (be about body weight 3.4%), 6hr intravenous perfusion Fu Dilong (Fludelone) 25mg/kg was as shown by arrows in D22, D25, D28, D31 and D34 administration Q3D * 5 at the 22nd day (D22).Subsequently 9 days intermittences, carry out treatment second round in D43, D46, D49 and D52.The tumor size is treated in matched group (●) and the Fu Dilong treatment group () (every group of n=5) at excipient and is changed.Continue to observe Q3D until D180, at this moment stop experiment (after D52 stops to treat 128 days).Fig. 6 A represents to be reduced and the therapeutic effect of expression by the tumor size.Fig. 6 B represents that nude mice (being selected from a mouse of matched group and treatment group separately) is at photo that D25, D31, D37, D43 and D52 took.When stopping, the D180 experiment do not observe recurrence.Fig. 6 C represents body weight change.

Fig. 7 T-cell lymphoblastic leukemia (CCRF-CEM/ Taxol) (drug resistance to Taxol is 44 times) of representing to choose is implanted the experimental result of nude mice.Be implanted in the nude mice so that the 50mg/ mice is subcutaneous at the 0th day tumor tissues CCRF-CEM/ Taxol (44 times of drug resistance outside the organism).In D8 begin with Fu Dilong 15mg/kg () (n=3) and 30mg/kg (△) (n=4) carry out 6hr-intravenous perfusion therapy, or D16 begins, Q2D * 3 and then dosage is brought up to 40mg/kg, Q2D * 3 (D22-26) and 60mg/kg then, Q2D * 3 (D28-40).Figure 10 A represents to be reduced and the therapeutic effect of expression by the tumor size.Figure 10 B represents body weight change.

Figure 11 represents to adopt 26-three fluoro-9, and 10-dehydrogenation-dEpoB and Taxol are to the treatment (intravenous perfusion in 6 hours, Q3D * 11) of nude mice with people's pulmonary carcinoma (A549/ Taxol) (is 44 times to the Taxol drug resistance).Figure 11 A represents to be reduced and the therapeutic effect of expression by the tumor size.Figure 11 B represents body weight change.

Figure 12 represents to adopt 9, and 10-dehydrogenation-dEpoB is to the treatment (intravenous perfusion in 6 hours, Q4D * 5, * 3) of nude mice with Human Lung Cancer A549/ Taxol (is 44 times to the Taxol drug resistance).Figure 12 A represents to be reduced and the therapeutic effect of expression by the tumor size.Body weight change before and after Figure 12 B represents to treat.

Figure 13 represents to adopt 26-three fluoro-9, and 10-dehydrogenation-dEpoB, dEpoB and Taxol are adopting oral formulations (PO, Q2D * 7, * 5) to treat the comparison that has in the human breast carcinoma MX-1 xenograft nude mice.Figure 13 A represents to be reduced and the therapeutic effect of expression by the tumor size.Figure 13 B represents body weight change.

Figure 14 is the various treatments of summary epothilone derivate and the table of pharmacokinetic parameter.

Figure 15 represents to adopt 26-three fluoro-9, and 10-dehydrogenation-dEpoB and dEpoB treatment have the heteroplastic nude mice of Human Lung Cancer (A549) (intravenous perfusion in 6 hours, (Q2D * 6) * 2, * 2).Figure 15 A represents to be reduced and the therapeutic effect of expression by the tumor size.Figure 15 B is illustrated in the body weight change after the treatment neutralization treatment.

Figure 16 is illustrated in the stability that the microtubule under the 10 μ M medicines forms.The microtubule that will form in the presence of 10 μ M Taxols is defined as 100%.The tubulin that derives from Medulla Bovis seu Bubali is the product of Sigma.The tubulin assembling test is implemented according to the description of manufacturer.Tubulin (1mg among the 200 μ l) is with buffer agent (0.1M MES, 1mM EGTA, the 0.5mM MgCl of 790 μ ₂, 0.1mM EDTA and 2.5M glycerol pH6.5) and with the medicine (ultimate density 10 μ M) of 10 μ l are cultivated.For assembling, implement down to cultivate 40 minutes and, implement down to cultivate 40 minutes at 4 ℃ for the dismounting of same sample at 35 ℃.Stable for microtubule, measure absorbance at the 350nm place.Solvent blank (DMSO) deducts from absorbance.

Figure 17 represents the effectiveness and relative therapeutic index of the external anticancer cell growth of Epothilones.

The cell cycle analysis of Figure 18 for determining by propidium iodide DNA dyeing.After treating with dEpoB (last figure) and Fu Dilong (figure below), cell cycle was ended and the blocking-up fully at 24 hours in the back 6 hours G2M stage of beginning for OPM-2 myeloma cell.Two kinds of medicines cause the pattern of same cell cycle termination.

Figure 19 represents that Annexin V fastens dyeing RPMI8226 myeloma cell.In the apoptotic cell, membrane phospholipid Phosphatidylserine (PS) translocates to the outer little page or leaf of cell membrane by the cell membrane endite, therefore PS is exposed to outside cellular environment in the early stage.Annexin V has the highly affine 35-36kD Ca to PS ⁺The phospholipids incorporate albumen that depends on, and utilize the PS that exposes to be attached on the cell.In conjunction with vital stain, 7-amino-D actinomycin D (7-AAD) for example, the dyeing dead cell, this test is used to identify viable apoptotic cell.Shown data show is in most cells or apoptosis or the death after 24 hours with dEpoB or Fu Dilong treatment.

Figure 20 represents with 24 hours the myeloma of Fu Dilong (125nM) treatment and the microphotograph of lymphoma cell line.Can see the feature circulus of the cell that terminates in G2/M.

Figure 21 represents the cell counting with the RPMI8226 myeloma cell of Fu Dilong and dEpoB (125nM) treatment.In different time points (1,2,4,8,24 hour),, and this cell continued to cultivate until 48 hours with the medicine eccysis.When noticing that being exposed to Fu Dilong is as short as 1 hour, tumor cell number promptly progressively reduces, and most cells experience apoptosis in the time of 24 hours simultaneously, and use dEpoB, and cell then still continues propagation.

Figure 22 represents RPMI8226 myeloma cell's alpha-tubulin dyeing.Fu Dilong has obviously stablized microtubule and has promptly increased the microtubule polymerization amount greatly in treatment early stage (treatment RPMI8226 myeloma cell 12 hours).After the Drug therapy 24 hours, the microtubule quality reduces and is destroyed, simultaneously cell experience apoptosis.

Figure 23 is the cell cycle analysis of being determined by propidium iodide DNA dyeing.Solid line is represented the value of G1 and G2, and the gray shade district is illustrated in the cell of S in the stage, and dotted lines goes out the master curve of double cell number, adopts Taxol especially obviously (the picture left above).Last row's explanation ovarian cancer cell line IGROV stops in the G2M stage after 24 hours with Fu Dilong (10nM), dEpoB (100nM) and Taxol (100nM) cultivation.Following row's explanation increases Fu Dilong concentration has increased stopping of HT-29 cell cycle.100nM cultivation with Fu Dilong causes a large amount of apoptosis after 24 hours, in comprising several cell lines of IGROV and HT-29, has hindered cell cycle analysis.

Figure 24 represents the Annexin V dyeing with 100nM Fu Dilong, Taxol or dEpoB cultivation Ovcar3 ovarian cancer cell line after 24 hours.The percentage ratio that provides at left lower quadrant is AnnexinV ⁺/ P1 ^-The percentage ratio of negative cells, this is with the apoptosis stages of cell is similar in early days.

Figure 25 is the cytospin (200 * amplification) with the HT-29 colon cancer cell of the painted treatment of HEMA3 after 24 hours.(a) control cells for the treatment of with solvent (DMSO).(b)-(d) expression increases

concentration

1,10 and the 100nM of Fu Dilong.(e) the HT-29 cell after using the dEpoB of 100nM and (f) the HT-29 cell behind the Taxol of using 100nM.All three kinds of medicines all produced same phenotype after 24 hours, obviously be the class ring structure of the nuclear that causes apoptosis.

Figure 26 is illustrated in the xenotransplantation of diffusivity people tumor and shifts among the human tumor xenograft model experimental design of the system of the effect of evaluation Fu Dilong and dEpoB.

Figure 27 represents cell proliferation and IC50 (concentration that 50% cell suppresses) test.Cell proliferation adopts 3 '-[1-(phenylamino-carbonyl)-3,4-tetrazolium ]-two (4-methoxyl group-6-nitro) benzene sulfonic acid sodium salt hydrates (XTT) and measures, and it measures the conversion of tetrazolium  chemical compound to first  by the mitochondrial dehydrogenase in the living cells.The amount of first  is with to be present in the test mixture quantity of living cells proportional.Each data is the meansigma methods of four independent measurements.(RPMI8226, CAG), the IC50 of Fu Dilong is about 7.6～36.67nM, and the IC50 of dEpoB is 36.67～61.34nM for myeloma cell line.For lymphoma system (SKIDLBCL and RL), the IC50 of Fu Dilong is about 60～80nM.

Figure 28 is illustrated in cell proliferation and the IC50 test on the normal stromal cell.Adopt as the identical method described in Figure 104.Human marrow-interstitial cell line, HS-27A and HS-5 do not go out by the E6/E7 gene, have the function of normal bone marrow matrix, and it has supported self renewal and the propagation of stem cell.For these substrate systems, the IC50 of Fu Dilong and dEpoB is about 90～100nM, and this and tumor cording have the population doubling time of equity.

Figure 29 represents the titration that the concentration cell cycle of Fu Dilong and dEpoB is stagnated.Adopt the CAG myeloma cell line.When concentration was 31.25nM, two kinds of medicines were all blocked cell cycle fully in G2M stage (data not shown that is lower than 31.25nM goes out).

Figure 30 represents the titration that the concentration cell cycle of Fu Dilong and dEpoB is stagnated.Adopt the RPMI8226 myeloma cell line.When concentration was 31.25nM, two kinds of medicines were all blocked cell cycle fully in G2M stage (data not shown that is lower than 31.25nM goes out).

Figure 31 represents the dyeing that Annexin V fastens CAG myeloma cell.In the apoptotic cell, membrane phospholipid Phosphatidylserine (PS) translocates to the cell membrane exite from the endite of cell membrane in the early stage, therefore PS is exposed to outside cellular environment.Annexin V has the highly affine 35-36kD Ca to PS ⁺The phospholipids incorporate albumen that depends on, and utilize the PS that exposes to be attached on the cell.Be incorporated into vital stain, 7-amino-D actinomycin D (7-AAD) for example, the cell that dyeing is dead, this test is used to identify viable apoptotic cell.Shown data show is with Fu Dilong or dEpoB treatment most cells or enter apoptosis or death after 24 hours.Notice that the X-axle is that Annexin dyeing and Y-axle are 7-ADD dyeing.

Figure 32 is the dna fragmentation test.The main biochemistry characteristics of apoptosis remove for nucleosome from chromatin.Nucleosome removes by the digestion of the Cobra venom endonuclease mediation of the DNA join domain that exposes between the nucleosome in chromatin and causes.Exempt from digestion owing to center on 180-200 the base of the DNA of histone nuclear on conformation, the nucleosome of this Cobra venom endonuclease mediation removes in agarose gel and can be viewed as dna ladder.Shown in data declaration after with Fu Dilong or dEpoB treatment RPMI8226 and CAG myeloma cell 24hr, detected typical dna ladder.This data declaration causes apoptosis by Epothilones by Guang winter peptidase (caspase) approach.

Figure 33 represents the alternate and the result behind Epothilones (20mg/kg) treatment diffusivity CAG xenotransplantation myeloma mice of body weight.Shown in the data show control mice dead and observe body weight in treatment after 20 days and significantly reduce in the time of about 30 days.Between mice for the treatment of with dEpoB and contrast, in the life-span, there is not the significance difference; Yet demonstrate the time-to-live of organizing equal significant prolongation than matched group and dEpoB with the mice of Fu Dilong treatment.The quantity of used mice is four in every group, and before injection CAG cell radiation 300Rad.

Figure 34 represents the treatment of diffusivity CAG xenotransplantation myeloma mice in the 4th week.With 10 * 10 ⁶Mouse tail vein is gone in individual CAG myeloma cell's intravenous injection with the transformation of Luc-eGFP-TK fusion gene, and when the 7th day of implanting to suitable myeloma cell by the bioluminescence image detection begin treatment.This figure shows the bioluminescence image of the mice of using contrast or dEpoB or Fu Dilong treatment respectively.

Figure 35 be illustrated in (N=4) average tumor photo emissions in the CAG xenotransplantation mice quantitatively.This figure shows that the mice ratio with the Fu Dilong treatment has significantly reduced tumor photo emissions with excipient or the independent mice for the treatment of with dEpoB.Tumor photo emissions and tumor load positive correlation.

Figure 36 represents the alternate and the result behind Epothilones (20mg/kg) treatment diffusivity CAG xenotransplantation myeloma mice of body weight.This figure showed the feature similar as Figure 33 at first 30 days; But unique survival winding of Fu Dilong treatment mice is strengthened by the Velcade (6.25 μ g/ mices, intravenous perfusion) of other 5 dosage.

Figure 37 is illustrated in the treatment of the 18th day diffusivity CAG xenotransplantation myeloma mice.This figure demonstrates the remarkable difference of image between each group, and this reflects the tumor load in its place group.

Figure 38 be illustrated in the 18th day average tumor photo emissions in the treatment of diffusivity CAG xenotransplantation myeloma mice quantitatively.This figure demonstrates the remarkable difference of image between each group, and this reflects the tumor load in its place group.

Figure 39 is illustrated in the treatment of the 40th day diffusivity CAG xenotransplantation myeloma mice.This figure demonstrates the difference of image, reflects three groups of tumor loads in the mice.

Figure 40 be illustrated in the 40th day average tumor photo emissions in the treatment of diffusivity CAG xenotransplantation myeloma mice quantitatively.This figure demonstrates the difference of image, reflects three groups of tumor loads in the mice.

Figure 41 represents with Fu Dilong and Velcade combined treatment CAG xenotransplantation myeloma mice.(20mg/kg, Q2d) and with Velcade (6.25 μ g/ mices, intravenous 3/w) were treated mice from the 35th day to 45 days from the 0th day to 28 days with Fu Dilong.At the 10th day begin treatment, and behind the 59th day Fu Dilong 12 dosage and Velcade 5 dosage photographic images.With compare at the 40th day image, in three mices two all have significantly reduced tumor load at femur with in no vertebral column.

Figure 42 is the Kaplan-Meier survival curve with heteroplastic seven the NOD/SCID mices of CAG myeloma cell.Control mice is dead and dead in 50 days with the mice of dEpoB treatment in 40 days after transplanting the CAG cell.Dead in 70 days except a mice, the mice of the useful Fu Dilong treatment of institute survived above 80 days.

Figure 43 represents apoptotic approach.

Figure 44 is illustrated in inductive time dependent Guang winter peptidase 3 processes of Epothilones among the CAG myeloma cell, and it shows the cutting form that has increased the Guang winter peptidase 3 of 17kD along with the time lengthening of Drug therapy.

Figure 45 represents the immunohistochemical dyeing with the CAG myeloma cell that carries out of Guang winter peptidase-3 antibody of cutting, and it demonstrates Cytoplasm and the location in nuclear week (showing low and high amplification) in apoptotic cell.

Figure 46 represents the immunohistochemical dyeing with the CAG myeloma cell that carries out of Guang winter peptidase-3 antibody of cutting, and it demonstrates Cytoplasm and the location in nuclear week (showing low and high amplification) in apoptotic cell.

Figure 47 shows that the activity of Guang winter peptidase 8 has increased after the Epothilones treatment, and this increase can suppress by the special inhibitor of Guang winter peptidase 8.

Figure 48 shows that the activity of Guang winter peptidase 8 has increased after the Epothilones treatment, and this increase can suppress by the special inhibitor of Guang winter peptidase 8.

Figure 49 shows that the activity of Guang winter peptidase 9 has increased after the Epothilones treatment.

Figure 50 is illustrated in KL and does not exist or exist down, cultivates the Annexin V dyeing of the CB CD34+ cell of 24h with dEpoB or Fu Dilong.Then with the medicine eccysis and implement Annexin V dyeing.On behalf of excipient matched group and hollow area, solid area represent medication therapy groups.Short time is exposed to the apoptosis that Epothilones does not have obviously to increase acyclic people CD34+ cell.

Figure 51 is illustrated in during colony forms, and Epothilones is to the influence of acyclic people CD34+ cell.By the progenitor cell that the colony formation of 2 weeks is estimated, significantly not different between matched group and medication therapy groups.

Figure 52 is illustrated in during colony forms, and Epothilones is to the influence of acyclic people CD34+ cell.By the progenitor cell that the colony formation of 2 weeks is estimated, significantly not different between matched group and medication therapy groups.

Figure 53 represents the therapeutic effect (body weight reduction) by Fu Dilong and the chemical sproof human T-cell's lymphoblastic leukemia of paclitaxel antiradiation drug (CCRT-CEM/ paclitaxel) xenograft.Although reduce body weight significantly, there is not toxicity death for all treatments.

Definition

Chemical compounds more of the present invention and particular functional group's definition also is described in greater detail in down.For the purposes of the present invention, the chemical element root is according to the periodic table of elements (CAS version, Handbook ofChemistry and Physics (Chemical Physics handbook), the 75th edition, inside front cover) and determining, and the particular functional group is described General Definition therein.In addition, vitochemical general principle, and particular functional group part and reactivity are described in " Organic Chemistry (organic chemistry) ", Thomas Sorrell, University Science Books, Sausalito:1999, its full content is incorporated the present invention by reference into.In addition, it will be recognized by those of ordinary skill in the art that synthetic method has been utilized the kinds of protect group as described in the present invention.Term " blocking group " as used in the present invention, the meaning is meant special functional moiety, for example O, S or N are sealed so that can optionally react on another response location in the polyfunctional compound temporarily.In preferred embodiments, blocking group optionally reacts substrate to provide protected with good yield, this substrate to plan to carry out reaction be stable; Blocking group should optionally be removed with good yield, and its reagent by preferred nontoxic other functional group of not attack of easy acquisition carries out; Blocking group forms can be easy to isolating derivant (more preferably not forming new chiral centre); And blocking group has minimum additional functional to avoid the reaction of other position.As describing in detail in the present invention, oxygen, sulfur, nitrogen and carbon blocking group can be utilized.Though typical blocking group describes in detail in the present invention, what will recognize is that the present invention is not meant to and limited by these blocking groups; More properly, various other equal blocking group can easily be determined and is applied in the method for the present invention with above-mentioned standard.In addition, in " Protective Groups in Organic Synthesis (blocking group in the organic synthesis) " third edition, Greene, T.W. and Wuts, P.G. compiles, John Wiley ﹠amp; Sons has described the kinds of protect group among the New York:1999, its full content is incorporated the present invention by reference into.

Be that chemical compound described in the present invention can replace with any amount of substituent group or funtion part with what recognize.Usually, no matter whether term " replacement " add term " randomly " in front, and be contained in the substituent group in the molecular formula of the present invention, is meant with specifying substituent group to replace to the hydrogen group in the fixed structure.When in any given structure, replacing when being selected from the substituent group of specifying group, can be identical or different in each locational substituent group more than one more than a position.As used in the present invention, term " replacement " is conceived to comprise the admissible substituent group of all organic compound.Aspect generalized, admissible substituent group includes acyclic and cyclic, side chain and the substituent group unbranched, isocyclic and heterocyclic, aromatic and non-aromatic of organic compounds.For the purposes of the present invention, hetero atom such as nitrogen can have the substituent group of any admissible organic compound described in hydrogen substituent group and/or the present invention, as long as it satisfies this heteroatomic quantivalence.In addition, the present invention is not meant to by any way and is limited by the allowed substituent group of organic compound.Preferably cause forming those of stable chemical compound by the combination of the substituent group of the present invention imagination and variant, described chemical compound is used for the treatment of, and for example proliferative disease includes but not limited to cancer.Term " stable ", as used among the present invention, preferably refer to such chemical compound, it has is enough to allow the stability that is produced and its integrity that keeps this chemical compound with the detected sufficiently long time and be preferred for being specified in sufficiently long time of the purpose among the present invention.

Term " aliphatic " as used among the present invention, comprises saturated and (promptly unbranched), side chain, cyclic or polycyclic aliphatic hydrocarbon undersaturated, straight chain, and it randomly replaces with one or more functional groups.It will be recognized by those of ordinary skill in the art that " aliphatic " is intended to include but not limited among the present invention: alkyl, thiazolinyl, alkynyl, cycloalkyl, cycloalkenyl group and cycloalkynyl radical part.Therefore, as used in the present invention, term " alkyl " comprises straight chain, side chain and cyclic alkyl.Similarly convention is applied to other general terms, for example " thiazolinyl ", " alkynyl " or the like.In addition, as used in the present invention, term " alkyl ", " thiazolinyl ", " alkynyl " etc. comprise replacement and unsubstituted group.In some embodiments, as used in the present invention, " low alkyl group " refers to that those have the alkyl of 1-6 carbon atom (cyclic, acyclic, that replace, unsubstituted, side chain or unbranched).

In some embodiments, used alkyl, thiazolinyl and alkynyl comprises 1-20 aliphatic carbon atom among the present invention.In other embodiments, used alkyl, thiazolinyl and alkynyl comprises 1-10 aliphatic carbon atom among the present invention.In some embodiments again, used alkyl, thiazolinyl and alkynyl comprises 1-8 aliphatic carbon atom among the present invention.In other embodiment, used alkyl, thiazolinyl and alkynyl comprises 1-6 aliphatic carbon atom among the present invention.In some embodiments again, used alkyl, thiazolinyl and alkynyl comprises 1-4 aliphatic carbon atom among the present invention.Therefore the aliphatic group that exemplifies includes but not limited to, for example, methyl, ethyl, n-pro-pyl, isopropyl, cyclopropyl ,-CH ₂-cyclopropyl, pi-allyl, normal-butyl, sec-butyl, isobutyl group, the tert-butyl group, cyclobutyl ,-CH ₂-cyclobutyl, n-pentyl, sec-amyl, isopentyl, tertiary pentyl, cyclopenta ,-CH ₂-cyclopenta, n-hexyl, Sec-Hexyl, cyclohexyl, CH ₂-cyclohexyl part or the like, it can have one or more substituent groups equally.Thiazolinyl includes but not limited to, for example, and vinyl, acrylic, cyclobutenyl, 1-methyl-2-butene-1-base etc.Typical alkynyl includes but not limited to acetenyl, 2-propynyl (propargyl), 1-propinyl etc.

Term " alkoxyl " or " alkylthio " refer to the alkyl as preceding definition as used in the present invention, are connected to parent molecular moiety by oxygen atom or by sulphur atom.In some embodiments, described alkyl comprises 1-20 aliphatic carbon atom.In other embodiments, described alkyl comprises 1-10 aliphatic carbon atom.In some embodiments again, used alkyl, thiazolinyl and alkynyl comprises 1-8 aliphatic carbon atom among the present invention.In other embodiment, described alkyl comprises 1-6 aliphatic carbon atom.In some embodiments again, described alkyl comprises 1-4 aliphatic carbon atom.The example of alkoxyl includes but not limited to, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, n-butoxy, tert-butoxy, neopentyl oxygen and positive hexyloxy.The example of alkylthio includes but not limited to, methyl mercapto, ethylmercapto group, rosickyite base, iprotiazem base, positive butylthio etc.

Term " alkylamino " refers to have-group of NHR ' structure, and wherein R ' is alkyl as defined in the present invention.In some embodiments, described alkyl comprises 1-20 aliphatic carbon atom.In other embodiments, described alkyl comprises 1-10 aliphatic carbon atom.In some embodiments again, used alkyl, thiazolinyl and alkynyl comprises 1-8 aliphatic carbon atom among the present invention.In other embodiment, described alkyl comprises 1-6 aliphatic carbon atom.In some embodiments again, described alkyl comprises 1-4 aliphatic carbon atom.The example of alkylamino includes but not limited to, methylamino, ethylamino, isopropylamino or the like.

More substituent examples of the above-mentioned aliphatic of The compounds of this invention (and other) part include but not limited to, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyl, aryloxy group, assorted alkoxyl, heteroaryloxy, alkylthio group, arylthio, assorted alkylthio group, heteroarylthio, F, Cl, Br, I ,-OH ,-NO ₂,-CN ,-CF ₃,-CH ₂CF ₃,-CHCl ₂,-CH ₂OH ,-CH ₂CH ₂OH ,-CH ₂NH ₂,-CH ₂SO ₂CH ₃,-C (O) R _x,-CO ₂(R _x) ,-CON (R _x) ₂,-OC (O) R _x,-OCO ₂R _x,-OCON (R _x) ₂,-N (R _x) ₂,-S (O) ₂R _x,-NR _x(CO) R _x, wherein each R that occurs _xInclude but not limited to independently, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein above-mentioned and any aliphatic described here, assorted aliphatic, aralkyl or heteroarylalkyl substituent group can be that replace or unsubstituted, side chain or unbranched, ring-type or acyclic, and wherein above-mentioned and any aryl described here or heteroaryl substituent group can be to replace or unsubstituted.Common applicable substituent example is in addition illustrated by the particular in embodiments of the present invention.

Usually, term " aryl " and " heteroaryl " as used among the present invention, refer to have the stable monocycle of a preferred 3-14 carbon atom or multi-ring, heterocycle, the unsaturated part of multi-ring and many heterocycles, and wherein each all can be substituted or not replace.Substituent group includes but not limited to, any aforementioned substituent group, and promptly as disclosed substituent group that aliphatic portion is enumerated or the substituent group that other parts are enumerated among the present invention, this substituent group causes forming stable chemical compound.In some embodiments of the present invention, " aryl " refers to have the monocycle or the bicyclo-carbocyclic ring system of one or two aromatic rings, includes but not limited to phenyl, naphthyl, tetralyl, indanyl, indenyl etc.In some embodiments of the present invention, used term " heteroaryl " among the present invention refers to have the ring-type aromatic radical of five to ten annular atomses, and one of them annular atoms is selected from S, O and N; Zero, one or two annular atoms are the extra hetero atom that is independently selected from S, O and N; And remaining annular atoms is a carbon, this ring-type aromatic radical is connected to the other parts of molecule of the present invention, for example pyridine radicals, pyrazinyl, pyrimidine radicals, pyrrole radicals, pyrazolyl, imidazole radicals, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl group, oxadiazole base, thiophenyl, furyl, quinolyl, isoquinolyl or the like by any annular atoms.

To recognize that aryl and heteroaryl (comprising aryl bicyclic) can be unsubstituted or replace, wherein replace one, two, three or more hydrogen atoms comprising on it replace independently with any one or a plurality of as the lower part, include but not limited to: aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyl, aryloxy group, assorted alkoxyl, heteroaryloxy, alkylthio group, arylthio, the alkylthio group of mixing, heteroarylthio, F, Cl, Br, I ,-OH ,-NO ₂,-CN ,-CF ₃,-CH ₂CF ₃,-CHCl ₂,-CH ₂OH ,-CH ₂CH ₂OH ,-CH ₂NH ₂,-CH ₂SO ₂CH ₃,-C (O) R _x,-CO ₂(R _x) ,-CON (R _x) ₂,-OC (O) R _x,-OCO ₂R _x,-OCON (R _x) ₂,-N (R _x) ₂,-S (O ₂) R _x,-NR _x(CO) R _x, the R of each appearance wherein _xInclude but not limited to independently, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein any above-mentioned and aliphatic described here, assorted aliphatic, aralkyl or heteroarylalkyl substituent group can be that replace or unsubstituted, side chain or unbranched, cyclic or acyclic, and wherein any above-mentioned and aryl described here or heteroaryl substituent group can be that replace or unsubstituted.Common applicable substituent example is in addition illustrated by the particular in embodiments of the present invention.

Used term " cycloalkyl " refers in particular to and has three to seven among the present invention, the group of preferred three to ten carbon atoms.The cycloalkyl that is fit to includes but not limited to, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl or the like, as the situation in other aliphatic, assorted aliphatic or the heterocyclic moiety, its optional replacement includes but not limited to following group: aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyl, aryloxy group, assorted alkoxyl, heteroaryloxy, alkylthio group, arylthio, assorted alkylthio group, heteroarylthio, F, Cl, Br, I ,-OH ,-NO ₂,-CN ,-CF ₃,-CH ₂CF ₃,-CHCl ₂,-CH ₂OH ,-CH ₂CH ₂OH ,-CH ₂NH ₂,-CH ₂SO ₂CH ₃,-C (O) R _x,-CO ₂(R _x) ,-CON (R _x) ₂,-OC (O) R _x,-OCO ₂R _x,-OCON (R _x) ₂,-N (R _x) ₂,-S (O ₂) R _x,-NR _x(CO) R _x, the R of each appearance wherein _xInclude but not limited to independently, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein any above-mentioned and aliphatic described here, assorted aliphatic, aralkyl or heteroarylalkyl substituent group can be that replace or unsubstituted, side chain or unbranched, cyclic or acyclic, and wherein any above-mentioned and aryl described here or heteroaryl substituent group can be that replace or unsubstituted.Common applicable substituent example is in addition illustrated by the particular in embodiments of the present invention.

Term " assorted aliphatic " used among the present invention refers to aliphatic portion, comprises one or more oxygen, sulfur, nitrogen, phosphorus or silicon atom, for example, substitutes carbon atom.Assorted aliphatic portion can be side chain, unbranched, cyclic or acyclic and comprise saturated and undersaturated heterocycle for example morpholinyl, pyrrolidinyl etc.In some embodiments, the one or more hydrogen atoms of assorted aliphatic portion on it substitute independently with one or more parts, include but not limited to, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyl, aryloxy group, assorted alkoxyl, heteroaryloxy, alkylthio group, arylthio, assorted alkylthio group, heteroarylthio, F, Cl, Br, I ,-OH ,-NO ₂,-CN ,-CF ₃,-CH ₂CF ₃,-CHCl ₂,-CH ₂OH ,-CH ₂CH ₂OH ,-CH ₂NH ₂,-CH ₂SO ₂CH ₃,-C (O) R _x,-CO ₂(R _x) ,-CON (R _x) ₂,-OC (O) R _x,-OCO ₂R _x,-OCON (R _x) ₂,-N (R _x) ₂,-S (O ₂) R _x,-NR _x(CO) R _x, the R of each appearance wherein _xInclude but not limited to independently, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein any above-mentioned and aliphatic described here, assorted aliphatic, aralkyl or heteroarylalkyl substituent group can be that replace or unsubstituted, side chain or unbranched, cyclic or acyclic, and wherein any above-mentioned and aryl described here or heteroaryl substituent group can be that replace or unsubstituted.Common applicable substituent example is in addition illustrated by the particular in embodiments of the present invention.

The atom that used term " halo " and " halogen " refer to be selected from fluorine, chlorine, bromine and iodine among the present invention.

Term " haloalkyl " refers to alkyl as defined above, and it has one, two or three halogen atoms connect on it and be illustrated as group as chloromethyl, bromoethyl, trifluoromethyl or the like.

Term " Heterocyclylalkyl " or " heterocycle " used among the present invention refer to that 5 yuan, 6 yuan or 7 yuan of non-aromatic are encircled or multi-ring group, include but not limited to bicyclo-or three cyclic groups, comprise condensed hexatomic ring with one to three hetero atom (being independently selected from oxygen, sulfur and nitrogen), wherein (i) each 5 yuan of ring have 0 to 1 two key and have 0 to 2 two key with each 6 yuan of ring, (ii) nitrogen and sulfur heteroatom can be randomly oxidized, (iii) nitrogen heteroatom can randomly can be fused on the phenyl ring by quaternized and (iv) any above-mentioned heterocycle.Typical heterocycle includes but not limited to pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidyl, piperazinyl, oxazolidinyl, isoxazole alkyl, morpholinyl, thiazolidinyl, isothiazole alkyl and tetrahydrofuran base.In some embodiments, " Heterocyclylalkyl of replacement or heterocycle " group is available and as using among the present invention, it refers to Heterocyclylalkyl or heterocyclic group as defined above, be substituted by substitute on it one, two or three hydrogen atoms independently with following group, described group includes but not limited to, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyl, aryloxy group, assorted alkoxyl, heteroaryloxy, alkylthio group, arylthio, assorted alkylthio group, heteroarylthio, F, Cl, Br, I ,-OH ,-NO ₂,-CN ,-CF ₃,-CH ₂CF ₃,-CHCl ₂,-CH ₂OH ,-CH ₂CH ₂OH ,-CH ₂NH ₂,-CH ₂SO ₂CH ₃,-C (O) R _x,-CO ₂(R _x) ,-CON (R _x) ₂,-OC (O) R _x,-OCO ₂R _x,-OCON (R _x) ₂,-N (R _x) ₂,-S (O ₂) R _x,-NR _x(CO) R _x, the R of each appearance wherein _xInclude but not limited to independently, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein any above-mentioned and aliphatic described here, assorted aliphatic, aralkyl or heteroarylalkyl substituent group can be that replace or unsubstituted, side chain or unbranched, cyclic or acyclic, and wherein any above-mentioned and aryl described here or heteroaryl substituent group can be that replace or unsubstituted.Common applicable substituent example is in addition illustrated by the particular in embodiments of the present invention.

" labelling ": used term " labelling " is intended to represent that chemical compound has at least one element, isotope or the chemical compound that connect on it and makes this chemical compound to detect among the present invention.Usually, label typically is divided into three classes: a) isotopic label, and it can be radioactive or heavy isotope, includes but not limited to, ²H, ³H, ³²P, ³⁵S, ⁶⁷Ga, ^99mTc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb and ¹⁸⁶Re; B) immune marker, it can be antibody or antigen, it can be incorporated into enzyme (for example horseradish peroxidase) but go up to make detectable; And c) dyestuff colour developing, luminous, phosphorescence or fluorescence.With what recognize be, this label can be incorporated in the The compounds of this invention on any position, as long as the biological activity or the characteristic of detected chemical compound are not disturbed in this position.In some embodiments of the present invention, utilize the directly intermolecular interaction (for example, epothilones bind position in detection tubulin dimer) of explanation in living things system of photoaffinity labeling.Available various known luminescent substance, major part depend on diazonium compound, nitrine or Azimethylene. (diazirine) to the conversion of the light of nitrene or Cabbeen (referring to, Bayley, H., Photogenerated Reagents in Biochemistry and MolecularBiology (1983), Elsevier, Amsterdam.), its full content is incorporated the present invention by reference into.In some embodiments of the present invention, used photoaffinity label for the neighbour that partly replaces with one or more halogens-,-and right-azido benzoyl base, include but not limited to 4-azido-2,3,5, the 6-tetrafluorobenzoic aid.

" polymer ": used term " polymer " among the present invention " refer to comprise the compositions of chain, described chain can be a repetitive (monomer) that open, closed, straight chain, side chain or crosslinked, described repetitive can be identical or different.With what recognize be, term polymer refers to biopolymer in some embodiments, and used in the present invention biopolymer is intended to refer to natural polymeric material or based on those natural materials, includes but not limited to nucleic acid, peptide and analog thereof.In other embodiments, term polymer refers to synthetic polymer, biological example degradable polymer or other polymeric material.With what recognize is that the polymeric solid phase support also is included in the polymer of the present invention.Chemical compound of the present invention can be connected on the polymer support and therefore can carry out synthetic modification on solid phase.Term " solid support " used among the present invention refers to include but not limited to, polystyrene bead, graft copolymerization pearl, polyacrylamide pearl, the latex particle of granule, pan, capillary tube, doughnut, spicule, spicule, solid fiber, cellulose bead, Bio-Glas, silica gel, optional and divinylbenzene crosslink, choose wantonly and N, DMAA that N '-two-acryloyl ethylenediamine is crosslinked and the glass particle that applies with hydrophobic polymer.The selection that it will be recognized by those of ordinary skill in the art that specific solid support will be subject to the compatibility of support and the reactive chemistry that is utilized.Typical solid support is a Tentagel amino resins, by 1) with the polystyrene bead and 2 of divinyl benzene crosslinked) complex formed of PEG (Polyethylene Glycol).Tentagel is useful especially solid support, because it is the common support that is used for (off-bead) test under granule (on-bead) or the granule, and it also has the good swellability in the solvent from toluene to water.

The description of some embodiments of the present invention

Recognize the needs of development of new and effective cancer therapy, the novel method for synthesizing that the invention provides the macro ring that can obtain to have wide biological activity and pharmacologically active is learned, and have these active new compounds, novel therapeutic compositions and use these chemical compounds and method for compositions.

In some embodiments, The compounds of this invention is used for the treatment of cancer.Compound exhibits more of the present invention go out the cytotoxicity of cancerous cell line or growth inhibited effect, demonstrate the ability of polymerization tubulin and stabilize microtubules assembling, and/or cause dwindling or disappearing of in cancerous cell xenograft models tumor.In some embodiments, The compounds of this invention can have side effect minimizing or minimized, comprises critical organ toxicity, nausea,vomiting,diarrhea, alopecia, body weight reduction, weight increase, liver toxicity, dermatosis etc.The compounds of this invention also may be owing to the water solublity that increases, the toxicity of reduction, the therapeutic domain of increase, enhanced effect etc. are easy to make preparation.

The generality explanation of The compounds of this invention

Chemical compound of the present invention comprises chemical compound and its pharmaceutically acceptable derivates of general formula further defined below (0); With its pharmacy acceptable derivates:

R wherein ₀For replacing or unsubstituted aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, heteroarylalkyl, impure aromatic ene base or hetaryne base section; In some embodiments, R ₀Be aralkyl, arylalkenyl, heteroarylalkyl or impure aromatic ene base section; In other embodiments, R ₀Be the impure aromatic ene base section; In some embodiments, R ₀Be the heteroarylalkyl part; In other embodiments, R ₀Aryl or heteroaryl moieties for 5-7 unit; In some embodiments again, R ₀Be 8-12 unit's aryl bicyclic or heteroaryl moieties; In other embodiment, R ₀Be two loop sections, wherein phenyl ring is fused on heteroaryl or the aryl moiety; In other embodiments, R ₀Be the dicyclo part, wherein phenyl ring is fused on thiazole, oxazole or the imidazoles part; In some embodiments again, R ₀For replacing or unsubstituted phenyl moiety;

R ₃And R ₄Be hydrogen independently of one another; Or replace or not replacement, straight or branched, ring-type or acyclic aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl part, the optional replacement: the alkyl of hydroxyl, protected hydroxyl, alkoxyl, carboxyl, carboxaldehyde radicals, straight or branched or cyclic acetal, fluorine, amino, protected amino, the amino, N-oxyimino or the N-Alkoximino that partly replace with one or two alkyl or aryl with one or more following groups; In some embodiments, R ₃And R ₄Be hydrogen, fluorine or low alkyl group independently of one another; In other embodiments, R ₃And R ₄Be hydrogen or methyl independently of one another; In other embodiment, R ₃Be methyl and R ₄Be hydrogen;

R ₅And R ₆Be hydrogen or blocking group independently of one another; In some embodiments, R ₅And R ₆All be hydrogen;

X is O, S, C (R ₇) ₂Or NR ₇, the R under every kind of situation wherein ₇Be hydrogen or low alkyl group independently; In some embodiments, X is O; In other embodiments, X is NH;

Y is O, S, NH, C (R ₇) ₂, CH ₂, N (R ₇) or NH, the wherein R under every kind of situation ₇Be hydrogen or low alkyl group independently; Y is O in some embodiments; In other embodiments, Y is NH; In some embodiments again, Y is CH ₂

Each R ₈Be hydrogen independently; Halogen, hydroxyl, alkoxyl, amino, dialkylamino, alkylamino, fluorine, cyano group, or replace or do not replace, straight or branched, ring-type or acyclic aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl or heteroarylalkyl, the impure aromatic ene base, the hetaryne base section, the optional replacement: hydroxyl with one or more following groups, protected hydroxyl, alkoxyl, carboxyl, carboxaldehyde radicals, the alkyl of straight or branched or cyclic acetal, fluorine, amino, protected amino, the amino that partly replaces with one or two alkyl or aryl, N-oxyimino or N-Alkoximino; In some embodiments, R ₈Be hydrogen; In other embodiments, R ₈Be hydroxyl; In some embodiments again, R ₈Be fluorine; In other embodiment, R ₈Be low alkyl group methyl for example; In other embodiments, R ₈For-CF ₃,-CF ₂H or CFH ₂In other embodiments, R ₈Be fluoridized or fluorinated alkyl; In some embodiments again, R ₈Be halogenated or fully halogenated alkyl;

R ₉And R ₁₀Be hydrogen independently of one another; Or replace or not replacement, straight or branched, ring-type or acyclic aliphatic, assorted aliphatic, aryl, heteroaryl, aryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, heteroarylalkyl, impure aromatic ene base or hetaryne base section optional the replacement: the alkyl of hydroxyl, protected hydroxyl, alkoxyl, carboxyl, carboxaldehyde radicals, straight or branched or cyclic acetal, fluorine, amino, protected amino, the amino, N-oxyimino or the N-Alkoximino that partly replace with one or two alkyl or aryl with one or more following groups; In some embodiments, R ₉And R ₁₀One of be methyl; In other embodiments, R ₉And R ₁₀Be methyl; In some embodiments again, R ₉And R ₁₀One of be methyl, and another is a hydrogen; In other embodiments, R ₉And R ₁₀All be hydrogen;

A-B represents CR _A=CR _B-, C (R _A) ₂-C (R _B) ₂-or-C ≡ C-;

C-D represents CR _C=CR _D-,-C (R _C) ₂-C (R _D) ₂-or-C ≡ C-;

The R under every kind of situation wherein _ABe hydrogen independently; Halogen;-OR _{A '}-SR _{A '}-N (R _{A '}) ₂-C (O) OR _{A '}-C (O) R _{A '}-CONHR _{A '}-O (C=O) R _{A '}-O (C=O) OR _{A '}-NR _{A '}(C=O) R _{A '}N ₃N ₂R _{A '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{A '}-SR _{A '}-N (R _{A '}) 2;-C (O) OR _{A '}-C (O) R _{A '}-CONHR _{A '}-O (C=O) R _{A '}-O (C=O) OR _{A '}-NR _{A '}(C=O) R _{A '}N ₃N ₂R _{A '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties;

For the R under every kind of situation _BBe hydrogen independently; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl, or heteroaryl moieties; In some embodiments, R _BFor hydrogen,

Methyl, ethyl, propyl group, butyl, amyl group, hexyl, cyclopropyl, cyclobutyl, cyclopenta or cyclohexyl do not replace or the optional following group that replaces with one or more appearance separately: halogen ,-OH ,-OR _{B '}, NH ₂Or N (R _{B '}) ₂Or its combination in any, the wherein R under every kind of situation _{B '}Be hydrogen, alkyl, aryl or blocking group independently, in other embodiments, R _BBe hydrogen, methyl or ethyl, in other embodiment, R _BBe methyl, in other embodiments ,-CY ₃,-CHY ₂,-CH ₂Y, wherein Y is F, Br, Cl, I, OR _{B '}, NHR _{B '}, N (R _{B '}) ₂Or SR _{B '}In some embodiments again, R _BFor-CF ₃,-CH ₂F or CHF ₂In other embodiments, R _BBe perfluorinate or fluorinated alkyl; In some embodiments again, R _BBe halogenated or fully halogenated alkyl;

For the R under every kind of situation _CBe hydrogen independently; Halogen;-OR _{C '}-SR _{C '}-N (R _{C '}) ₂-C (O) OR _{C '}-C (O) R _{C '}-CONHR _{C '}-O (C=O) R _{C '}-O (C=O) OR _{C '}-NR _{C '}(C=O) R _{C '}N ₃N ₂R _{C '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{C '}-SR _{C '}-N (R _{C '}) ₂-C (O) OR _{C '}-C (O) R _{C '}-CONHR _{C '}-O (C=O) R _{C '}-O (C=O) OR _{C '}-NR _{C '}(C=O) R _{C '}N ₃N2R _{C '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties; In some embodiments, R _CBe halogen, alkyl, hydroxyl or amino; In other embodiments, R _CBe fluorine; In some embodiments again, R _CBe hydroxyl;

For the R under every kind of situation _DBe hydrogen independently; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl, or heteroaryl moieties; Perhaps

R wherein _A, R _B, R _COr R _DIn any two lump together and can form annulus and can pass through oxygen, sulfur, carbon or the nitrogen-atoms connection, perhaps any two adjacent R _A, R _B, R _COr R _DLump together, can form replacement of 3-6 unit or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl ring; In some embodiments, R _AAnd R _BLump together and form 3 yuan of rings that connect by oxygen, sulfur, carbon or nitrogen-atoms; In other embodiments, R _CAnd R _DLump together and form 3 yuan of rings that connect by oxygen, sulfur, carbon or nitrogen-atoms;

The R under every kind of situation wherein _{A '}, R _{B '}, R _{C '}Or R _{D '}Be hydrogen independently; Blocking group; Straight or branched, replacement or not replacement, ring-type or acyclic, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, arylalkenyl, sweet-smelling alkynyl or heteroarylalkyl, impure aromatic ene base, hetaryne base section.

The compounds of this invention comprises the chemical compound of general formula further defined below (I):

R wherein ₁Be hydrogen or low alkyl group; In some embodiments, R ₁Be methyl; In some embodiments, R ₁For-CF ₃,-CF ₂H or CH ₂F; In other embodiments, R ₁Be fluoridized or fluorinated alkyl; In some embodiments again, R ₁Be halogenated or fully halogenated alkyl;

R ₂For replace or unsubstituted aryl, heteroaryl, aralkyl or heteroarylalkyl part; In some embodiments, R ₂For replacing or not replacing the De oxazole; In other embodiments, R ₂For replacing or unsubstituted thiazole; With

A, B, C, D, R ₃, R ₄, R ₅, R ₆As above define with X.

In some embodiments, The compounds of this invention comprises the chemical compound with stereochemical general formula (II) as giving a definition:

Wherein A, B, C, D, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆As above define with X.

In some embodiments, The compounds of this invention comprises the chemical compound of general formula as follows (III):

Wherein Z be oxygen atom, sulphur atom ,-NR _Z-or-C (R _Z) ₂-; And A, B, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆As above define with X.In some preferred embodiments, Z is an oxygen.In other embodiments, Z is-NH-.In some embodiments again, Z is-CH ₂-.In other embodiments, R _ZBe hydrogen, alkyl, halogen or acyl group.In some embodiments, R _ZBe fluorine.

In some embodiments, The compounds of this invention comprises the chemical compound with stereochemical general formula (IV) as giving a definition:

Wherein A, B, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆, X and Z as above define.In some embodiments, R _BBe methyl.In other embodiments, R _BFor-CF ₃

In some embodiments, The compounds of this invention comprises logical formula V as follows or chemical compound (VI):

Wherein Z be oxygen atom, sulphur atom ,-NR _Z-or-C (R _Z) ₂-; And A, B, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆As above define with X.In some preferred embodiments, Z is an oxygen.In other embodiments, Z is-NH-.Z is-CH in some embodiments again ₂-.In other embodiments, R _ZBe hydrogen, alkyl, halogen or acyl group.In some embodiments, R _ZBe fluorine.

In some embodiments, The compounds of this invention comprises the chemical compound of general formula as follows (VII):

Wherein A, B, R _C, R _D, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆As above define with X.In some preferred embodiments, each R _CBe hydrogen, halogen or low alkyl group independently.In other embodiment, each R _DBe hydrogen, halogen or low alkyl group independently.

In some embodiments, X is O.In other embodiments, X is NH.In other embodiments, X is CH ₂

In some embodiments, R ₂Be that replace or unsubstituted thiazole.In some embodiments, R ₂Be 2-methyl-thiazole-4-base.In other embodiments, R ₂Be 2-methylol-thiazole-4-base.In some embodiments again, R ₂Be 2-aminomethyl-thiazole-4-base.In other embodiments, R ₂Be 2-thiol methyl-thiazole-4-base.

In some embodiments, R ₂For replace or do not replace the De oxazole.In some embodiments, R ₂Be 2-methyl-oxazoles-4-base.In other embodiments, R ₂Be 2-methylol-oxazoles-4-base.In some embodiments again, R ₂Be 2-aminomethyl-oxazoles-4-base.In other embodiments, R ₂Be 2-thiol methyl-oxazoles-4-base.

In some embodiments, R _BFor hydrogen, methyl, ethyl ,-CF ₃,-CH ₂F ,-CF ₂H.In some embodiments, R _BBe methyl.In some embodiments again, R _BFor-CF ₃In some embodiments, R _BBe hydrogen.In other embodiments, R _BBe ethyl.

Some preferred chemical compounds comprise, for example:

The compounds of this invention comprises that as above specifically illustrate and those chemical compounds described herein, and by in the present invention other local disclosed various types of, belong to and plant and partly illustrate.Do not wish to be bound by any concrete theory, chemical compounds more of the present invention are modified to force molecular conformation with the mode that carbon-carbon double bond is identical on the C9-C10 position on C9 and C10.Electronic effect, steric effect, hydrogen bond, dipole effect or their combination can be used to set up this conformation and force.For example, cyclic rings system oxirane, cyclopropyl and the aziridine conformation that can be used for forcing this molecule for example.In other embodiments, 4,5 or 6 yuan of rings are used to the effect that reaches same.This effect can also be by conjugated p-track system, for example is found in the system in ester, thioester or the amide and finishes.This effect also can be passed through delocalization π system, for example be found in the aromatic rings system those and finish.In other embodiments, utilize near the substituent steric effect in C9 and C10 position to force the same way as of molecule to force molecular conformation with carbon-carbon double bond on the C9-C10 position.

It will be recognized by those of ordinary skill in the art that asymmetric center can be present in the The compounds of this invention.Therefore, The compounds of this invention and pharmaceutical composition thereof can be the forms of single enantiomer, diastereomer or geometric isomer, perhaps can be the form of mixtures of stereoisomer.In some embodiments, The compounds of this invention is an enantiopure compound.The mixture of stereoisomer or diastereomer is provided in other embodiments.

Be that the class of some aforesaid compounds and subclass can exist with various isomeric form with what recognize.Inclusion compound of the present invention is as the individual isomer of essentially no other isomer with alternatively as various mixture of isomers, for example the racemic mixture of stereoisomer.In addition, the present invention comprises (Z) and (E) both double bond isomers, unless spell out in addition.Therefore, be described in usually The compounds of this invention in the structure of the present invention comprise those wherein double bond structure be (Z) or chemical compound (E).In some preferred embodiments, be cis or Z configuration at locational pair of key of C12-C13.In some embodiments, be trans or the E configuration at locational pair of key of C9-C10.In other embodiment, be cis or Z configuration at locational pair of key of C12-C13, and be trans or the E configuration at locational pair of key of C9-C10.The present invention also comprises the tautomer of aforesaid specific compound.

In addition, the invention provides The compounds of this invention pharmacy acceptable derivates and use described these chemical compounds, its pharmaceutical composition or one of them and one or more other therapeutic agents combinations and the method for treatment target.Used term " pharmaceutically acceptable derivates " refers to the salt of any pharmaceutically acceptable salt, ester or this ester of this chemical compound among the present invention, or any other addition product or derivant, it is when giving patient's medication, can provide (directly or indirectly) as other local chemical compound of describing among the present invention, or its metabolite or residual.Pharmaceutically acceptable derivates is therefore particularly including prodrug.Prodrug is the derivant of chemical compound, has significantly reduced pharmacologically active usually, and it comprises other part, and this part is easy in vivo remove and generates parent molecule as pharmacological active substance.The example of prodrug is an ester, and it can cut in vivo and generate the purpose chemical compound.The prodrug of all cpds and material and the being used to parent compound of deriving forms the method for prodrug, is known and can be suitable for the present invention.Some typical pharmaceutical compositions and pharmacy acceptable derivates will be discussed below of the present invention in detail.

Interested especially The compounds of this invention comprises:

● demonstrate cytotoxicity or growth inhibited effect to cancerous cell line, described cell line maintains in the zooscopy of acceptable cancerous cell heteroplastic transplantation model on external or the employing science;

● demonstrate the ability of polymerization tubulin and stabilize microtubules assembling;

● demonstrate the minimum toxic level of vitals;

● cause the disappearance of the tumor in the acceptable cancerous cell heteroplastic transplantation model on science;

● cause the contraction of the tumor in the acceptable cancerous cell heteroplastic transplantation model on science;

● cause on science the tumor in the acceptable cancerous cell heteroplastic transplantation model to disappear and stopping to delay after the treatment/or do not cause tumor recurrence;

● on science, demonstrate in the acceptable cancerous cell heteroplastic transplantation model in short-term and to reduce with reversible body weight and demonstrate therapeutic effect;

● demonstrate the water solublity that is better than Epothilones A, B, C or D or paclitaxel, but or demonstrate enough dissolubilities additionally or alternati with in the water-bearing media that is formulated in the Li Mofu that uses the reduction ratio; And/or

● demonstrate the treatment characteristic (for example effect of Zui Jia safety and healing) that is better than epothilone B, epothilone d or paclitaxel

The analog of various Epothilones described above is as institute of the present invention illustration and be produced, characterize and test.Have been found that 9,10-dehydrogenation-epothilone analogs can be used for treatment for cancer, and particularly chemical compound has been produced and has found that it has one or more above-named characteristics of wanting.

Synthetic method

Some Epothilones, NSC-703147 and analog thereof synthetic be described in the past (referring to, United States Patent (USP) 6,242,469,6,284,781,6,300,355,6,204,388,6,316,630 and 6,369,234; U.S. Patent application 09/797,027,09/796,959 and 10/236,135; With PCT open WO 99/01124, WO 99/43653 and WO 01/64650, full content is incorporated the present invention by reference into).Recognize and to improve or to replenish the synthetic method that effectively generates Epothilones, NSC-703147 and analog thereof in large quantities, the invention provides effective canonical path and be used for synthetic Epothilones, NSC-703147 and analog thereof.Although some exemplary compounds synthetic is described among the embodiment of this paper, is that this methodology can be common to the analog and the conjugate of generation each class as above described in the present invention and subclass with what recognize.

Particularly, of the present invention 9,10-dehydrogenation epothilone compounds can prepare with the number of ways of the synthetic method that is used for synthetic Epothilones.In some embodiments, adopt the convergent synthesis approach to prepare these chemical compounds.For example synthetic Epothilones can form the chemical compound of wanting by preparing two or three intermediate and bonding them together.In one embodiment, one of intermediate is the acyl moiety that comprises 1-9 carbon, and another intermediate comprises 10-15 carbon and can also comprise the thiazole side chain.The intermediate of these two Epothilones about equally can utilize esterification and combine, and this esterification occurs in C1 and sloughs between the C15 of oxygen.Can utilize carbon carbon coupled reaction then, for example Suzuki coupling or closed loop metathesis reaction are closed into macro ring.In one embodiment, final closed loop step forms 9 by the closed loop metathesis reaction, and the two keys of 10-also are closed into macro ring and finish.The closed loop metathesis reaction is by using organo-metallic catalyst, for example is shown in the Grubbs catalyst of following sketch map 8 and finishes.In some embodiments, with this 9, the two keys of 10-are reduced or oxidation, perhaps can with this 9, the two keys of 10-are further functionalized to prepare other epothilone derivate.In some embodiments, by with Cabbeen or carbenoid reagent such as CH ₂N ₂Handle 9, the two keys of 10-and with this 9, the two keys of 10-are converted into the cyclopropyl part.

In some embodiments, described 9,10-dehydrogenation epothilone compounds by with two keys from 10,11-position (for example, Epo490) isomerization to 9,10-position and preparing.This isomerization can be by the transition metal such as the palladium catalysis that exist.

In other embodiments, final closed loop step forms 12 by adopting the closed loop metathesis reaction, and the two keys of 13-also are closed into macro ring and finish.In some embodiments, with this 12, two keys reduction of 13-or oxidation.In other embodiments, form macro ring with macro ring aldolisation or macrolide reaction.

The compounds of this invention is provided in drawings and Examples, and some are typically synthetic.Persons of ordinary skill in the art will recognize that and adopt the synthetic method that is described among the present invention can prepare multiple analog and derivant.For example, can realize a lot of synthesis steps with different blocking groups or the different substituent groups on 16 yuan of rings.

Pharmaceutical composition

The present invention also provides pharmaceutical preparation, its comprise at least a as mentioned above with chemical compound described here, or its pharmaceutically acceptable derivates, described chemical compound can anticancer growth or kill cancer cell, and in some embodiments, interested especially is that described chemical compound can suppress the growth of multi-drug resistant cancerous cell or kill them.In some embodiments, pharmaceutical preparation of the present invention also comprises solubilizing agent or emulsifying agent, but for example Li Mofu (CREMOPHORE EL) or Solutol (Polyethylene Glycol 660 12-hydroxy stearic acid esters).

As mentioned above, the invention provides and have anticancer and new compound antiproliferative activity, and therefore The compounds of this invention can be used for treatment for cancer.Therefore in another aspect of the present invention, provide pharmaceutical composition, wherein these compositionss comprise any one in the chemical compound described in the present invention, and the optional medicine acceptable carrier that comprises.In some embodiments, these compositionss randomly further comprise one or more other therapeutic agents.In other embodiments, as being described in further detail in the present invention, this other therapeutic agent is an anticarcinogen.

What also will recognize is that chemical compounds more of the present invention can exist with free form and be used for the treatment of, or suitable place, as the form of its pharmaceutically acceptable derivates.According to the present invention, pharmaceutically acceptable derivates includes but not limited to, the salt of pharmaceutically acceptable salt, ester, this ester or any other addition product or derivant, it is when giving the patient's medication that needs, can provide (directly or indirectly) as other local chemical compound of describing among the present invention, or its metabolite or residual, for example prodrug.

As used in the present invention, term " pharmaceutically acceptable salt " refers to those salt, it is suitable for contacting with the lower animal tissue with the people and not having unsuitable toxicity, zest, allergy etc., and has rational benefit/risk ratio in rational medical judgment scope.Pharmaceutically acceptable salt is known in the art.For example, the acceptable salt of pharmacy is described in detail in J.Pharmaceutical Sciences by people such as S.M.Berge, among the 66:1-19 (1977), incorporates the present invention by reference into.Described salt can original position generate in last separation of The compounds of this invention and purge process, or by free alkali functional group is separated preparation with suitable organic acid reaction.The example of the acceptable nontoxic acid-addition salts of pharmacy is the amino salt that forms with mineral acid or organic acid, wherein mineral acid is for example hydrochloric acid, hydrobromic acid, phosphoric acid, sulphuric acid and perchloric acid, organic acid is for example acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, perhaps by adopting available other method in this area, for example ion exchange.The acceptable salt of other pharmacy comprises adipate, alginate, Ascorbate, aspartate, benzene sulfonate, benzoate, disulfate, borate, butyrate, camphorate, camsilate, citrate, cipionate, digluconate, dodecane sulfonate, ethane sulfonate, formates, fumarate, gluceptate, glycerophosphate, gluconate, hernisulfate, enanthate, caproate, hydriodate, 2-hydroxyl-ethane sulfonate, Lactobionate, lactate, laruate, lauryl sulfate, malate, maleate, malonate, mesylate, the 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalates, palmitate, embonate (pamoate), pectate (pectinate), persulfate, 3-phenylpropionic acid salt, phosphate, picrate, Pivalate, propionate, stearate, succinate, sulfate, tartrate, rhodanate, tosilate, the hendecane hydrochlorate, valerate or the like.Typical alkali metal salt or alkali salt comprise sodium, lithium, potassium, calcium, magnesium or the like.The more acceptable salt of pharmacy comprises, in the time of suitably, nontoxic ammonium, quaternary ammonium and the amine cation that forms with the ion with anti-electric charge, described ion with anti-electric charge is halogen ion, hydroxide ion, carboxylic acid ion, sulfate ion, phosphate anion, nitrate ion, low alkyl group sulfonate ion and aryl sulfonic acid radical ion for example.

In addition, as used in the present invention, term " pharmaceutically acceptable ester " refers to the ester of hydrolysis in vivo, and is included in easy those esters that discharge parent compound or its salt that decompose in the human body.The ester group that is fit to comprises that for example, those are derived from the ester of the acceptable aliphatic carboxylic acid of pharmacy, particularly alkanoic acid, alkenoic acid, aphthenic acids and chain docosandioic acid, and wherein each alkyl or alkenyl part advantageously has no more than 6 carbon atoms.The example of concrete ester comprises formic acid esters, acetas, propionic ester, butyrate, acrylate and ethyl succinate.

In addition, " pharmaceutically acceptable prodrug " refers to those prodrug of The compounds of this invention as used in the present invention, it is in rational medical judgment scope, be applicable to and contact with the lower animal tissue with the people and do not have unsuitable toxicity, zest, allergy etc., and has a rational benefit/risk ratio, with effectiveness, and as long as, refer to zwitterionic form to the The compounds of this invention possibility to their desired use.Term " prodrug " refers in vivo conversion fast to form the as above parent compound of general formula, for example by hydrolysis in blood.At T.Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems, in the A.C.S.Symposium Series the 14th volume, with the BioreversibleCarriers in Drug Design that compiles at Edward B.Roche, American Pharmaceutical Association andPergamon Press, carried out in 1987 discussing completely, two pieces of documents are all incorporated the present invention by reference into.

As mentioned above, pharmaceutical composition of the present invention comprises pharmaceutically acceptable carrier in addition, it is as used in the present invention, comprise any and all solvent, diluent or other liquid vehicle, dispersion or suspension aids, surfactant, isotonic agent, thickening agent or emulsifying agent, antiseptic, solid binder, lubricant or the like, as the concrete form of medication that is suitable for wanting.Remington ' s Pharmaceutical Sciences, the 15th edition, E.W.Martin (MackPublishing Co., Easton, Pa., 1975) discloses variety carrier and the known technology of preparing thereof that is used to form pharmaceutical composition.With inconsistent all the conventional mounting mediums of anticancer compound of the present invention, for example produce any unwanted biological effect or with pharmaceutical composition in any other one or more components interact in deleterious mode, in addition, the expection of the purposes of this mounting medium is incorporated in the scope of the present invention.Some can be used as the examples of material of pharmaceutically acceptable carrier, and the judgement according to formulator includes but not limited to, saccharide is lactose, dextrose plus saccharose for example; Starch based is corn starch and potato starch for example; Cellulose and derivant thereof be sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate for example; Powdered tragakanta; Fructus Hordei Germinatus; Gelatin; Pulvis Talci; But Li Mofu; Solutol; Excipient is cocoa butter and suppository cured (suppository waxes) for example; Oils is Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, safflower oil, Oleum sesami, olive oil, Semen Maydis oil and soybean oil for example; Di-alcohols is propylene glycol for example; Esters is ethyl oleate and ethyl laurate for example; Agar; Buffer agent is magnesium hydroxide and aluminium hydroxide for example; Alginic acid; Apirogen water; Deng oozing salt; Ringer's mixture; Ethanol and phosphate buffered solution, and other nontoxic compatible lubricant for example sodium lauryl sulphate and magnesium stearate, and coloring agent, releasing agent, coating agent, sweeting agent, flavoring agent and aromatizing agent (perfuming agents), antiseptic and antioxidant also can be present in the present composition.

The purposes of chemical compound and pharmaceutical composition

The present invention further provides the method that is used to suppress tumor growth and/or neoplasm metastasis.In some interested especially embodiments,, the invention provides by suppressing tumor growth and/or neoplasm metastasis and treat method for cancer for the multidrug resistant cancerous cell of tumor.This method comprises the treatment The compounds of this invention of effective dose or its pharmacy acceptable derivates its object of needs (including but not limited to the human or animal) medication.In some embodiments, be used in particular for treating the cancer that comprises the multidrug resistant cancerous cell, described treatment effective dose is such amount: it is enough to kill or suppresses the multidrug resistant cancerous cell line.In some embodiments, The compounds of this invention is used for the treatment of solid tumor.

The compounds of this invention and pharmaceutical composition can be used for treatment or prevent any disease or symptom, comprise proliferative disease (for example cancer), autoimmune disease (for example rheumatoid arthritis) and infectious disease (for example infectious disease such as antibacterial, fungus).But The compounds of this invention and pharmaceutical composition medication be in animal, preferred mammal (for example domestic animal, cat, Canis familiaris L., mice, rat), and more preferably people.Can use the chemical compound of any administrated method to animal delivering drugs compositions.In some embodiments, The compounds of this invention or pharmaceutical composition are with parenterai administration.

More on the one hand, as described herein, according to Therapeutic Method of the present invention, the kill tumor cell or suppress their growth by tumor cell is contacted with The compounds of this invention or compositions.Therefore in still another aspect of the invention, be provided for treating method for cancer, it comprises is wanted result's amount and time medication to the object that needs it The compounds of this invention of treatment effective dose or the pharmaceutical composition that comprises The compounds of this invention to reach.In some embodiments of the present invention, " the treatment effective dose " of The compounds of this invention or pharmaceutical composition is such amount: it is kill cancer cell or anticancer growth effectively.The method according to this invention, chemical compound of the present invention and compositions can be with any amount and any administrations of effective kill cancer cell or anticancer growth.Therefore, statement as used in the present invention " the effectively amount of kill cancer cell or anticancer growth " refers to the reagent of the q.s of kill cancer cell or anticancer growth.Required definite amount will change with object is different, its depend on object species, age and comprehensive state, the seriousness of infection, concrete antitumor and anticancer agent, application method or the like.Anticancer compound of the present invention is preferably prepared to be easy to medication and dosage homogeneous with dosage unit form.Used statement " dosage unit form " refers to be suitable for treat the physically separated unit of patient's antitumor and anticancer agent among the present invention.Yet this will be interpreted as that the whole day consumption of The compounds of this invention and compositions will reasonably determined in the medical judgment scope by the attending doctor.The effective dosage level of particular treatment that is used for any concrete patient or organism will depend on various factors, comprise the seriousness of the disease of being treated and this disease; The activity of used specific compound; Used particular composition; Patient's age, body weight, comprehensive health state, sex and diet; The time of the medication of used specific compound, route of administration and drainage rate; The persistent period of treatment; The medicine that combines or use simultaneously with used specific compound; And in the known similar factor of medical domain.

In addition, after preparing with the dosage of wanting with suitable pharmaceutically acceptable carrier, pharmaceutical composition of the present invention can be to people and the administration as follows of other animal: oral administration, rectal administration, non-enterally administer, pond innerlich anwenden, intravaginal medication, intraperitoneal administration, local application's (as by powder, ointment or drop), through cheek medication such as oral cavity or nasal cavity spray or the like, it depends on the seriousness of the infection of being treated.In some embodiments of the present invention, chemical compound described in the present invention is prepared by combining with water-soluble chelator or water-soluble polymer, wherein said water-soluble polymer is for example: Polyethylene Glycol is as poly-(1-glutamic acid) or poly-(1-aspartic acid), as at United States Patent (USP) 5,977, described in 163 like that, its full content is incorporated the present invention by reference into.In some embodiments, The compounds of this invention can oral or non-intestinal with enough dosage level medications, described dosage level is enough to reach about 0.001mg/kg of object body weight every day to about 100mg/kg for the medicine of delivering, about 0.01mg/kg is to about 50mg/kg, preferred about 0.1mg/kg is to about 40mg/kg, preferred about 0.5mg/kg is to about 30mg/kg, about 0.01mg/kg is to about 10mg/kg, about 0.1mg/kg is to about 10mg/kg, and more preferably from about 1mg/kg is to about 25mg/kg, once a day or repeatedly, to obtain desirable therapeutic effects.Can with per two days once, per three days once, jede Woche once, every fortnight once, per three weeks once or every four stars phase the dosage of wanting once is provided.In some embodiments, can provide the dosage of wanting (for example, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten administrations) with multiple dosing.

Be used for oral and liquid dosage form parenterai administration and include but not limited to the acceptable Emulsion of pharmacy, microemulsion, solution, suspension, syrup and elixir.Except reactive compound, liquid dosage form for example also can comprise inert diluent commonly used in this area, water or other solvent, solubilizing agent and emulsifying agent be ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1 for example, the fatty acid ester of 3-butanediol, dimethyl formamide, oils (particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Semen Maydis oil, germ oil, olive oil, Oleum Ricini and Oleum sesami), glycerol, tetrahydrofurfuryl alcohol, Polyethylene Glycol and sorbitan, and their mixture.Except inert diluent, Orally administered composition also can comprise adjuvant for example wetting agent, emulsifying agent and suspending agent, sweeting agent, flavoring agent and aromatizing agent.Be used for the embodiment of parenterai administration at some, The compounds of this invention mixes with solubilizing agent, but for example Li Mofu, alcohols, oils, modification oils, glycols, polysorbate esters, cyclodextrin, polymer class, and their compositions.In some embodiments, The compounds of this invention and alcohol, but for example ethanol and Li Mofu (Oleum Ricini of polyethoxylated) mixing.

Injection, for example aseptic injection water or oily suspension, dispersant that can be fit to according to use known in the art or wetting agent and suspending agent are prepared.Aseptic injection also can be aseptic injectable solution, suspension or the emulsion in nontoxic non-intestinal acceptable diluent or solvent, for example solution in 1,3 butylene glycol.Spendable in these acceptable excipient and the solvent have water, Ringer's mixture, U.S.P. and an isotonic sodium chlorrde solution.In addition, aseptic not volatile oil is commonly used for solvent or suspension media.For this reason, available any non-stimulated fixed oil comprises synthetic monoglyceride or diglyceride.In addition, fatty acid for example oleic acid be used to injection.

Injection of the present invention can be sterilized in the following way: for example, keep filter by antibacterial and filter, or add sterilization solid composite form disinfectant, described disinfectant can be dissolved or dispersed in sterilized water or other aseptic injection medium before use.

In order to prolong drug effect, wish to delay from the absorption of the medicine of subcutaneous injection or intramuscular injection.This can finish by the liquid suspension that use has relatively poor water miscible crystallization or an amorphous material.So the infiltration rate of medicine depends on its dissolution rate, this may depend on crystallite size and crystal habit again.Perhaps, by in the oils excipient, medicine dissolution or suspension being reached the purpose that the medicament forms that delays parenterai administration absorbs.Injection storage storehouse dosage form, for example forms little encapsulation capsule substrate in polylactic acid-poly-glycolic acid lactide and prepares in biodegradable polymer by medicine.Depend on medicine to the ratio of polymer and the character of used concrete polymer, can control drug release speed.The example of biodegradable polymer comprises poe and polyanhydride.Storage storehouse injection is also caught medicine by liposome compatible with bodily tissue or microemulsion and is prepared.

The compositions that is used for rectum and vagina medicinal is preferably suppository, it can pass through The compounds of this invention and the non-irritating excipient or the carrier that are fit to, the for example cured mixing of cocoa butter, Polyethylene Glycol or suppository and preparing, described excipient or carrier at room temperature are solid, and be liquid under body temperature, and therefore fusing and discharge active medicine in rectum or vaginal canal.

The solid dosage forms of oral administration comprises capsule, tablet, pill, powder and granule.In these solid dosage formss, reactive compound and at least a inert, acceptable excipient of pharmacy or carrier mix, for example sodium citrate or calcium hydrogen phosphate and/or a) filler or extender starch for example, lactose, sucrose, glucose, mannitol and silicic acid, b) binding agent carboxymethyl cellulose for example, alginate, gelatin, polyvinylpyrrolidone, sucrose and Radix Acaciae senegalis, c) wetting agent glycerol for example, d) disintegrating agent agar--agar for example, calcium carbonate, potato starch or tapioca, alginic acid, some silicate and sodium carbonate, e) dissolve for example paraffin of delayer, f) absorption enhancer quarternary ammonium salt compound for example, g) for example monostearate spermaceti alcohol ester and glyceryl monostearate of wetting agent, h) for example Kaolin and bentonite and i of absorbent) lubricant Pulvis Talci for example, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate and composition thereof.Under the situation of capsule, tablet and pill, these dosage forms also comprise buffer agent.

The solid composite of similar type also can be used as the filler in soft filling and the hard gelatine capsule agent of filling, and the used excipient of described capsule is lactose and high molecular weight polyethylene glycol or the like.These solid dosage formss of tablet, dragee, capsule, pill and granule can be used at the known coating of pharmacy formulation art and shell and prepare, for example casing and other coating.These dosage forms can be chosen wantonly and comprise tranquilizer and also can be the compositions that only discharges one or more active components, or preferably, some part at intestinal randomly discharges in the time-delay mode.The example of available embedding composition comprises polymer material and wax.The solid composite of similar type also can be used as the filler in soft filling and the hard gelatine capsule agent of filling, and the used excipient of described capsule is as lactose and high molecular weight polyethylene glycol or the like.

Reactive compound also can with have one or more as above illustrated excipient in little packaging plastic cap dosage.These solid dosage formss of tablet, dragee, capsule, pill and granule can be used the known coating of pharmacy formulation art and shell and prepare, for example casing, controlled release coat and other coating.In this solid dosage forms reactive compound can with for example sucrose, lactose or the starch blending of at least a inert diluent.This dosage form also can comprise, as normal way, and except the other material of inert diluent, for example tablet lubricants and other sheet agent aid such as magnesium stearate and microcrystalline Cellulose.Under the situation of capsule, tablet and pill, described dosage form also can comprise buffer agent.These dosage forms can be chosen wantonly and comprise emulsifying agent and also can be the compositions that only discharges one or more active components, or preferably, in some part of intestinal, randomly the mode with time-delay discharges.The example of available embedding composition comprises polymer material and wax.

The dosage form that is used for part or percutaneous dosing of The compounds of this invention comprises ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant or patch.Described active component under aseptic condition with the pharmaceutically acceptable carrier that may need and any required antiseptic or buffer agent blending.Ophthalmic preparation, ear drop and eye drop are also expected to fall in the scope of the invention.In addition, the present invention relates to use transdermal patch, it has the additional advantage that the controlled release chemical compound is provided to health.This dosage form can prepare by The compounds of this invention is dissolved or dispersed in the suitable medium.Also can use absorption enhancer to increase the flux that The compounds of this invention passes skin.Can be by the speed controlling film being provided or controlling transdermal speed by dispersion The compounds of this invention in polymeric matrix or gel.

As discussed above, the The compounds of this invention useful asticancer agents, and therefore by making the growth of cancer cell death or anticancer be used for the treatment of cancer.Usually, the cancer that anticarcinogen of the present invention can be used for treating and other proliferative disease include but not limited to, breast carcinoma, the brain cancer, skin carcinoma, uterus carcinoma, colon and rectal cancer, leukemia, pulmonary carcinoma, melanoma, multiple myeloma, non-Hodgkin lymphoma, ovarian cancer, cancer of pancreas, carcinoma of prostate and gastric cancer only give some instances.In some embodiments, anticarcinogen of the present invention is effective to leukaemia and melanoma cells, and therefore (for example can be used for treating leukemia, bone marrow, lymphocytic, promyelocyte, myelocytic and lymphoblastic leukemia, no matter acute or chronic form) and malignant melanoma.In other embodiment, anticarcinogen of the present invention is effectively to solid tumor and can also kills multidrug resistant cell (MDR cell) and/or suppress its growth.In some embodiments, anticarcinogen of the present invention is to the chemical sproof cancer of other known antineoplastic agent or have been found that to the clinical nonreactive cancer of other known antineoplastic agents be effective.In other embodiments, for the chemical sproof cancer of other antitumor microtubule stabilizer (for example paclitaxel), anticarcinogen of the present invention is effective.

What also will recognize is, The compounds of this invention and pharmaceutical composition can be used to combined therapy, chemical compound promptly of the present invention and pharmaceutical composition can be simultaneously, prior to or medication subsequently in one or more other required therapy or medical procedures.The concrete therapeutic combination (therapeutics or rules) that is used for assembled scheme should be considered required therapeutics and/or rules and the compatibility of the required therapeutic effect that will reach.What also will recognize is, used therapy can reach the effect wanted (for example, The compounds of this invention can be simultaneously and another kind of anticarcinogen medication) for identical disease, or used therapy can obtain different effect (for example, controlling any adverse effect).

For example, can comprise surgical operation with other therapy or anticarcinogen of anticarcinogen applied in any combination of the present invention, radiotherapy (some of them example, γ-radiation, the neutron beam radiotherapy, the electron beam radiotherapy, proton therapeutic, brachytherapy and body radioactivity isotope, only give some instances), incretotherapy, biological respinse therapy (interferon, interleukin and tumor necrosis factor (TNT), only give some instances), overheated and cryotherapy, reduce the medicament (for example Bendectin) of any side reaction, chemotherapy drugs with other approval, include but not limited to alkylating agent (chlormethine, chlorambucil, cyclophosphamide, phenyalamine mustard, ifosfamide), antimetabolite (methotrexate), purine antagonist and pyrimidine antagonist (Ismipur, 5-fluorouracil, Cytarabile, gemcitabine (Gemcitabine)), spindle poisonous substance (vinblastine, vincristine, Herba Catharanthi Rosei, paclitaxel, Docetaxel (Docetaxel)), podophyllinic acid lactone (etioposide, Irinotecan, hycamtin), antibiotic (amycin, bleomycin, mitomycin), nitroso ureas (carmustine, chlorethyl cyclohexyl nitrosourea), inorganic ions (cisplatin, the Carboplatin), enzyme (asparaginase) and hormones (tamoxifen, bright dried meat Li Te, flutamide and megestrol), only give some instances.For the more fully discussion of up-to-date cancer therapy referring to http://www.nci.nih.gov/, the tabulation of the tumour medicine of FDA approval is at http://www.fda.gov/cder/cancer/druglistframe.htm, with The Merck Manual, in the 17 edition 1999, its full content is incorporated the present invention into by application.

On the other hand, the present invention also provides drug packages or test kit, it comprises one or more containers of filling with one or more compositions of pharmaceutical composition of the present invention, and in some embodiments, it comprises the therapeutic agent as combination treatment of other approval.Randomly accompany with such one or more containers, it can be bulletin with government organs' true-to-shape, manufacturing, use or the sale of described government organs standard drug products, wherein said bulletin have reflected the approval of government organs for production, use or the sale of people's medication.

Coordinate

Below the embodiment of the invention be intended to help to understand the present invention, and they are not meant to, and also they should be construed as limiting the invention.In fact, from full content of the present invention, comprise in the list of references of the science quoted among the following examples and the present invention and patent documentation, to those skilled in the art, except those illustrate in the present invention and describe those, various modifications of the present invention and its many further embodiments will be conspicuous.Be that the content of the document that those are cited is incorporated the present invention into by reference to help to understand prior art with what further recognize.The following examples comprise important additional information, illustration and guidance, and they can various embodiments of the present invention and coordinate and be suitable for practice of the present invention.

Embodiment

Embodiment 1:26-three fluoro-9,10-is trans-and the oral and non-enterally administer treatment of dehydrogenation-epothilone d implants the intravital people's tumor of nude mice

In the present embodiment, proved the microtubule stabilizer of 16 membered macrolide things of structural design, 26-three fluoro-9,10-is trans-dehydrogenation-epothilone d, and giving the perfusion of 6hr intravenous or when oral, tumor is being shunk, cause tumor to disappear, and reach the effect that does not recur for a long time.Mice with large scale tumor (Fig. 6) or Taxol resistant tumors xenograft (Fig. 7) is used to explanation can use 26-three fluoro-9, and 10-is trans-and dehydrogenation-epothilone d cures as single medicament single therapy.The therapeutic domain of curing comprises leukemia and breast carcinoma, colon cancer and pulmonary carcinoma (Fig. 6,9 and 13).Here the interior experiment of Bao Gao all chemotherapeutics bodies of laws is all adopted the people's tumor xenogeneic graft in the immunodeficiency nude mice and is carried out.This animal model was usually used in estimating antitumoral compounds before to the cancer patient clinical trial.

MX-1 and HCT-116 experiment carries out 5.5 and 6.5 months (Fig. 6 and 9) respectively.Tumor is all less than recurrence in two experiments, eliminated 3.8 and 5.2 months respectively stopping to treat the back tumor.For HCT-116 experiment (Fig. 9), paclitaxel and 26-three fluoro-9,10-is trans-and dehydrogenation-epothilone d all uses and all realized the tumor disappearance with 20mg/kg.Yet the paclitaxel treatment group recurs after stopping to treat back 1.1 months, and 26-three fluoro-9, and 10-is trans-and the animal tumor of dehydrogenation-epothilone d treatment disappears above 5.2 months.Suppose that the long twice time of tumor is 4 days (based on vehicle treatment contrast), the paclitaxel treatment of HCT-116 tumor causes 99.7% tumor suppression or the cell of 2.56-log to kill, yet when experiment stops, by 26-three fluoro-9,10-is trans-cell that dehydrogenation-epothilone d kills in MX-1 and HCT-16 experiment is respectively＞8.5-log and＞11.6log.Based on by treatment mice health, active state, weight recovery level before the treatment simultaneously if implement the treatment that prolongs or increase treatment cycle, estimates that this " healing " can guarantee to run through 2 year life-span of mice.Should obtain such result, although in fact this " healing " more is difficult to obtain in immunodeficient mouse than in immunocompetent mice is arranged.As far as our knowledge goes, this is the longest xenograft treatment research of implementing on nude mice in biomedical document, also is with the parenteral administration or with the longest the returning to one's perfect health of being reported in the oral administration for single anti-tumor agents.This is relevant with following statement: the chemical compound of finding the inhibition tumor growth is relatively easy, but finds that the chemical compound that tumor is dwindled is uncommon relatively.The compounds of this invention 26-three fluoro-9,10-is trans-dehydrogenation-epothilone d, it has realized making fully in all mices tumor to disappear, and recurrence does not reach 5.2 months, this discovery was never reported as far as our knowledge goes, this shows 26-three fluoro-9, and 10-is trans-and dehydrogenation-epothilone d is for the great potential of therapeutics progress.

But the other remarkable advantage of oral medication is the severe allergic reaction that can avoid using the Li Mofu prescription to cause.But use Li Mofu to cause that thorny anaphylaxis is known in Taxol, deoxidation-EpoB and 15-aza-Epo 13 preparations, this makes and must use antihistaminic and/or steroid to carry out pretreatment.

26-three fluoro-9,10-is trans-oral result of dehydrogenation-epothilone d and its in external mice plasma and in people's liver microsomes S9 fragment significant metabolic stability consistent.Described metabolic stability is owing to fluoridizing (Figure 14) in three of C-26 position in this Epothilones molecule.On the C9-C10 position, introduce two keys and also increased metabolic stability (Figure 14).26-three fluoro-9,10-is trans-and dehydrogenation-epothilone d is used for intravenous perfusion (20-30mg/kg, Q2D) and be used for oral administration (20mg/kg, QD or 30mg/kg Q2D), its optimal dose can be well absorbed and have good bioavailability in vivo near this chemical compound of explanation.

It should be noted that the invention process many parallel with Taxol (one of Chang Yong most important anticancer therapeutic agent clinically) carry out about the interior therapeutics of the anti-xenograft of Fu Dilong researchs (Figure 13,7,9,10,11 and 16).The important discovery of Fu Dilong is compared with Taxol, has shown that this chemical compound is used for the treatment of the promising potentiality of cancer.

Material and method

Chemical products: all Epothilones are synthetic like that as described in the present invention.Paclitaxel (Taxol ^) and vinblastine sulfate (VBL) available from Sigma.All these compound dissolutions are used in vitro tests in dimethyl sulfoxine, (except VBL is dissolved in saline).For studying in the body, but all Epothilones and paclitaxel all are dissolved in Li Mofu/ethanol (1: 1) vehicle and be used for utilizing the microtubular of autonomous Design and program-controlled pump to carry out the perfusion of 6hr intravenous (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Chou, 15798-15802 by the tail vein with the saline dilution then; People such as Chou (2001) Proc.Natl.Acad.Sci.USA 98,8113-8118; Every piece of document is incorporated the present invention by reference into).The medicine of oral administration prepares in the following way: compound dissolution in ethanol and be suspended in isopyknic tween 80, is diluted the saline of this suspension with 5 times of volumes before giving the nude mice medication.Use the animal feeding pin of the spherical band of 1ml syringe and standard #22 medicated cap to implement gavage (Popper﹠amp; Sons, Inc.New HydePark, NY).

Tumor and cell line: CCRF-CEM people's lymphoblast leukaemia and its vinblastine drug resistance subbreed (CCRF-CEM/VBL ₁₀₀, 720 times of drug resistance) derive from William doctor Beck of Chicago Illinois university, and CCRF-CEM/ Taxol (44 times of drug resistance) by six middle of the month with the CCRF-CEM cellular exposure in ever-increasing to lethal concentration (IC ₅₀-IC ₉₀) paclitaxel in and prepare.The drug resistance degree is shown among Figure 14.People's breast carcinoma (MX-1), human lung carcinoma cell (A549) and human colon carcinoma (HCT-116) derive from American type culture collection (ATCC, Rockville, MD).

Animal: the nude mouse with nu/nu gene derives from NCI, Frederick, and MD also is used for the xenotransplantation of the somebody of institute tumor.Use 6 weeks above with body weight 20-22g or heavier male nude mouse.By using homemade microtubule and the limiter tube inculcated by perfusion administration in 6 hours in the tail cava vein.Have multichannel program-controlled Harvard PHD2000 syringe pump and be used to the intravenous perfusion.But inculcated volume in typical 6 hours is the Li Mofu/ethanol (1: 1) that contains every kind of medicine of 100 μ l in the 2.0ml saline.For oral administration, Fu Dilong and Taxol all are dissolved in the ethanol and with tween 80 and dilute 5 times.Taxol solution should use in 5min to avoid precipitation.Gross tumor volume is estimated by measure long * wide * high (or wide) with caliper.For the nude mice that has tumor, its body weight is meant that gross weight deducts the weight of tumor in the experimentation.All zooperies are all implemented according to the guideline of National Institute of Health Guide for the Care and Use of Animals with by the agreement of Memorial Sloan-Kettering Cancer Center ' s InstitutionalAnimal Care and Use Committee approval.

Cell toxicity test: for preparing the vitro cytotoxicity test, cell culture is 2-5 * 10 in initial density ⁴Every milliliter in individual cell.They are stored in 5%CO ₂In 37 ℃ the RPMI culture medium 1640 (GIBCO/BRL) of-moistening atmosphere, described RPMI culture medium 1640 comprises penicillin (100 units/mL), streptomycin (100 μ g/mL GIBCO/BRL) and 5% heat-inactivated FBS.For the solid tumor cell of in monolayer, growing (for example HCT-116 and A549), the cytotoxicity of medicine is by using lissamine rhodamine B method (people (1990) J.Natl.CancefInst.82 such as Skehan, 1107-1112 incorporates the present invention by reference into) on the microtitration plate of 96-hole, measure.For the cell (for example CCRF-CEM and subbreed thereof) that grows in the suspension, by using 2,3-is two-(2-methoxyl group-4-nitro-5-sulfophenyl)-5-N carboxanilides)-2H-terazodium hydroxide (XTT) microtubule method (people (1988) Cancer Res.48 such as Scudiero, 4827-4833; Incorporate the present invention by reference into) on the microtitration plate of 96-hole, measure in duplicate.For two kinds of methods, (Winooski VT) measures the absorbance in each hole for Power Wave XS, Bio-Tek with the micro plate plate reading machine.The relation data of dosage-effect is by 6 to 7 concentration of every kind of medicine, and is duplicate, by the program of using a computer (people (1997) CalcuSyn for Windows such as Chou (and Biosoft, Cambridge, U.K.); Wherein each all incorporates the present invention by reference into) analyze with middle effect figure.

Epothilones is in mice and the stability in people's liver S9 fragment: carry out stability study with full-automatic HPLC system, described HPLC system comprises Prospekt-2 (SparkHolland, Netherlands) sample preparation system and Agilent 1100 HPLC systems.In brief, Prospekt 2 selects the C8 extraction column and uses acetonitrile and water washing.The Agilent automatic sampler is set at 37 ℃, draws 20 μ l samples, and it is loaded on the extraction column, washes with water, and Prospekt 2 turns to mobile phase to flow by extraction column to analytical column then.(PA) Reliance Stable Bond C8 4 * 80mm and eluent are monitored at the 250nm place for MacMod, Chadds Ford to have guard column.Mobile phase is made up of 53 or 65% acetonitrile/0.1% formic acid, flow velocity 0.4ml/min, so the retention time of purpose chemical compound is about 6 minutes.Sample preparation comprises isopyknic blood plasma is joined among the PBS, obtains cumulative volume 400 μ l, filter, and this substrate (20mM) that adds 0.5-2 μ l is to obtain in HPLC analyzes at the about 30-50mAU in 250nm place.(Xeno Tech, Lenex KS), mix the S9 fragment of 20 μ l (400 μ g) and to carry out step as mentioned above then with the PBS of 280 μ l for people's liver microsomes S9 fragment of merging.Sampling period is by automatic sampler control and gather the peak area data to compare the rate of disappearance of parent compound.

The result

The structure-activity relation of external anti-human leukemia, Taxol drug resistance and vinblastine drug resistance leukaemia and solid tumor cell.According to IC ₅₀(in μ M) lists in the table of Figure 17 for the effectiveness of the anti-following cell growth of 12 kinds of typical Epothilones: the subbreed of human leukemia CCRF-CEM cell and their vinblastine drug resistance CCRF-CEM/VBL and taxol resistance CCRF-CEM/ Taxol.Also show the IC of two people's reality oncocyte systems simultaneously ₅₀Value: pulmonary carcinoma A549 and colon cancer HCT-116.This shows that deH-EpoB has replaced EpoB as the most virtuous known Epothilones.What also show is: i) at dEpoB, dEpoF or F ₃-dEpoF is last 9, and the 10-dehydrogenation is modified and always caused the remarkable increase renderd a service, has but reduced described effectiveness to a certain extent yet fluoridize at C-26 locational three, yet metabolic stability is but fluoridized greatly by three and increased (Figure 14); Ii) most of Epothilones is to vinblastine, and a kind of have the typical substrate of multiple drug resistance not have cross resistance to the P-glycoprotein, also not to the cross resistance of Taxol.Taxol is the good substrate of a kind of MDR phenotype, but the Taxol drug resistance also can produce by the sudden change in microtubule protein gene.15-azepine-EpoB is an exception, and 15-azepine-deH-EpoB demonstrates significant cross resistance to vinblastine and paclitaxel.DEpoF and derivant thereof demonstrate the cross resistance of some and vinblastine and paclitaxel are not had.The iii) almost same influence that is subject to Epothilones of leukemia CCRF-CEM cell and solid tumor A549 and HCT-116.Iv) F ₃-deH-dEpoB, deH-dEpoB and deH-EpoB, they are novel lead compound that the present invention is used for further pharmacological evaluation, have not a particle of the cross resistance to vinblastine, and paclitaxel is not had yet.

(people (1979) Nature (London) 277 such as Schiff, 665-667 in the research in early days; People such as Meng (1997) J.Am.Chem.Soc.119,2733-2734; Every piece of document is incorporated the present invention by reference into), sum up the Epothilones molecule and can be divided into three parts.Therefore, in the acyl moiety of C-1～8,, do not allow structural change according to vitro cytotoxicity and microtubule stabilisation ability.This side aryl moiety with the O-moieties of C-9～15 and C-15 is opposite, and wherein both structures of back can allow to change significantly (people (1996) Biochemistry 35 such as Haar, 243-250; People such as Kowalski (1997) J.Biol.Chem.272,2534-2541; Every piece of document is incorporated among the present invention by reference).

Have 12, the Epothilones of 13-epoxy radicals part, for example EpoB and deH-EpoB, although be the most virtuous medicament in the Epothilones series, they are stood in maximum only has the therapeutic index of being on duty mutually under the dosage.This hypothesis can illustrate with the treatment data that are shown in Figure 14 table.

The physicochemical properties of epothilone derivate, metabolisming property, pharmacological property and therapeutic outcome.The serial Epothilones of different nature that is mutually related has promoted the understanding to the factor at the treatment home to return to that contributes to leading chemical compound.Table among Figure 14 has been summed up the feature of nine kinds of epothilone derivates.Structure-cytotoxic external activity relation provides based on the initial assessment of rendeing a service.For example, 9, the new class that the 10-dehydrogenation is modified has increased in the body and external effectiveness greatly.In the chemical compound of these preliminary elections, be difficult to structure-microtubule stability is renderd a service and is associated, because this renders a service all very high (that is, being similar to the effectiveness of paclitaxel).Water-soluble and lipotropy play a part different in therapeutic effect and are important for the design of preparation.DeH-dEpoF, F3-deH-dEpoF and deH-dEpoB have the water solublity of remarkable increase with respect to other Epothilones.F ₃-deH-dEpoB is orally active, but this discovery reduced water solublity in pharmaceutical preparation necessity and can avoid using and cause Li Mofu hypersensitive.

External, deH-EpoB, deH-dEpoF, EpoB and deH-dEpoB have the IC of inferior nanomole in proper order with this ₅₀Yet, F ₃-deH-dEpoF, dEpoF, F ₃-deH-dEpoB, dEpoB and F ₃-dEpoB with this order, has IC ₅₀Scope be 1.3 to 9.3nM (Figure 17).DeH-EpoB and EpoB are the most virtuous known Epothilones in vitro and in vivo, but they do not produce best therapeutic index.Obviously, the epoxy radicals part on C-12～13 of EpoB and deH-EpoB contributes to the toxicity for the host greatly, reduces (and not dead) lower value obviously visible (Figure 14) by weight limit percentage ratio.On the contrary, F ₃-deH-dEpoB and dEpoB allow body weight to reduce peak and they all show the good curing result, for example eliminates tumor in all animals fully.Usually, with 6hr intravenous perfusion therapy, vitro efficacy and best therapeutic dose have shown good relatedness.Interesting especially is in listed Epothilones, F ₃-deH-dEpoB (Fu Dilong) has the wideest healing therapeutic dose scope (10-30mg/kg) (people (2003) Angew Chem.Int.Ed.Engl.42 such as Chou, 4762-4767 incorporates the present invention by reference into), fabulous metabolic stability and best overall therapeutic outcome (Figure 14).In addition, F ₃-deH-dEpoB provides the therapeutic effect of curing by oral administration route.In a word, the most virtuous Epothilones might not have better curative effect as anti-tumor agents, and F ₃-deH-dEpoB is used for the treatment of the leading drug candidate of learning development.

Treatment for super large MX-1 tumor xenogeneic graft.As shown in Fig. 6 A, 25mg/kg, 6hr intravenous perfusion Q3D * 5, tumor is implanted back D22 and is begun to use F ₃-deH-dEpoB causes significant tumor to shrink (＞97.4%) to the treatment MX-1 xenograft of body weight 3.4% greatly.In 9 day intermission of not treatment (D34-D43), the tumor size continues to shrink (＞99.3%) and has 2 mouse tumors to disappear in 5 mices that are studied, however the level (Fig. 6 B) of the body weight of treatment group before identical tempus intercalare all returns to treatment.Resume treatment at D43, Q3D * 4 cause disappearing at D50, D50 and D51 tumor in remaining 3 mices.Behind the D52 (promptly taking medicine the date at last), observed animal once in per three days, when the D165 test stops.All 5 animals all do not have tumor recurrence at D165 (promptly stopping to treat back 3.7 months).

A mice is shown among Fig. 6 B at the photo of this test nude mice of D25, D31, D37, D43 and D52 shooting from matched group and treatment group.

By oral administration by the healing treatment of cancer of Fu Dilong to the MX-1 xenograft.As shown in FIG. 13A, per two days orally give F ₃-deH-dEpoB 30mg/kg 7 times, cause the MX-1 tumor shrink and 4 mices in 2 tumors disappear.Two other dosage (dosage skip back) in 4 mices 4 all cause tumor to disappear.On the contrary, suppress the MX-1 growth of tumor with the oral medication of the Taxol that carries out under same dose and the identical progress but do not cause tumor to be shunk.In the end two days (D36) behind the potion Taxol, it causes tumor suppression 66.9%.F ₃-deH-dEpoB treatment is induced moderate but persistent body weight reduces maximum 15% body weight (Figure 13 B) that reduces.Very little body weight change is induced in the Taxol treatment, illustrates that the oral administration Taxol is obviously because drug metabolism inactivation or the bioavailability of difference rather than suitable treatment.Opposite with oral administration route, the present inventor's earlier report (people (2001) Proc.Natl.Acad.Sci.USA 98 such as Chou, 8113-8118; Incorporate the present invention by reference into), the MX-1 tumor can cause tumor to be shunk and the tumor disappearance with the 20mg/kg Taxol through 6hr intravenous perfusion therapy.

The healing therapeutic effect of the chemical sproof CCRF-CEM/ Taxol of the anti-Taxol of Fu Dilong xenograft.(people (2003) AngewClient.Int.Ed.Engl.42 such as Chou, 4762-4767 in the nearest report of the present inventor; Incorporate the present invention by reference into), the present inventor has illustrated F ₃-deH-dEpoB is at 20mg/kg, Q2D * 7, and the 6hr intravenous causes tumor growth to suppress fully when being poured in anti-Taxol drug resistance people pulmonary carcinoma A549/ Taxol (external to 44 times of drug resistance of paclitaxel), but does not reach vanishing tumor.The present inventor uses another kind of taxol resistance xenograft in the present invention, CCRF-CEM/ Taxol (external to 44 times of drug resistance of paclitaxel).As shown in Figure 7, Fu Dilong is in 30mg/kg Q2D * 7, and the perfusion of 6hr intravenous causes having in 4 mices 2 tumors to disappear.Other Q2D * 5 (dosage doubles) cause having in 4 mices 3 tumors to disappear, and final tumor suppression is 99.8% (Fig. 7 A).At the dosage that reduces is 15mg/kg, has only the 5th a day generation tumor disappearance after two cycle therapy in 4 mices.In the final tumor suppression of D34 is 98.8%.The parallel laboratory test of carrying out at 20mg/kg with Taxol has caused tumor growth to suppress but seldom or has not at all dwindled tumor.In the final tumor suppression of D34 is 75.6%.

F ₃Both all continue to reduce body weight-deH-dEpoB (at 15mg/kg and 30mg/kg) and Taxol (20mg/kg) in the period 1 therapeutic process of dabbling 7 treatments by the 6hr intravenous in per two days.Skip seance at D22 and cause in all mices weight increase immediately.Treatment second round of Q2D * 5 is same to continue to reduce body weight, but all are tested mices all less than deadly (Fig. 7 B).

Healing treatment by the anti-human colon carcinoma HCT-116 of Fu Dilong xenograft.Shown in Fig. 9 A, F ₃-deH-dEpoB at 20mg/kg and 30mg/kg or Taxol at 20mg/kg, Q2D * 4, in 6hr intravenous 3 cycles of perfusion, 4 all cause tumor to disappear in 4 mices.Implant back D9 begin treatment in tumor.Between first and second cycles, skip a dosage, and between the second and the 3rd circulation, skip two dosage at D27 and D29 at D17.D9-D37 is that 3 cycle therapy stages and D37-D200 are follow-up period.This Therapy lasted 200 days, it is for surpassing 1/4th of mice average life.

For F at 30mg/kg ₃-deH-dEpoB, tumor disappears at D21,23,33 and 41, and at 20mg/kg, tumor disappears at D31,35,41 and 45.F for two kinds of dosage ₃-deH-dEpoB, when the D200 experiment finished, 4 did not all have tumor recurrence in 4 mices.At 20mg/kg, tumor disappears at D33,33,41 and 45 for Taxol, this and F at 20mg/kg ₃Observed similar among the-deH-dEpoB.Yet in Taxol treatment group, at D71,75,81 and 81 tumor recurrences.

The treatment program that comprises tempus intercalare is represented by body weight reduction and the physical state of mice.For F at 30mg/kg ₃-deH-dEpoB treatment, maximum body weight are reduced to 30% and do not cause death, and this betides the second treatment cycle end (being D29) (Fig. 9 B).For the Taxol of 20mg/kg and the F of 20mg/kg ₃-deH-dEpoB, the reduction of body weight and recovery are in pattern and measure all similar.

Embodiment 2: determine Fu Dilong (F ₃-deH-dEpoB) the mechanism of action with and how to be different from the mechanism of action of EpoD; These different therapeuticss hints

The drug susceptibility of multiple myeloma of people (MM) and non-Hodgkin lymphoma (NHL) tumor cell line

MM account for all cancers 1% and malignant hematologic disease 10%, 15,500 newly-increased diagnosed SARS cases and＞15 were arranged, 000 example death in 2002.MM is incurable with the traditional chemical therapy for treating, and existence time limit median is about 3 years (people " Treatment of multiplemyeloma " Blood 2004 such as Barlogie; 103:20-32; Incorporate the present invention by reference into).Although the high dose chemotherapy with the hematopoietic stem cell carrier has increased the ratio that complete symptom goes down and do not have the incident life span, almost each patient can be recurred, and requires for rescuing pressing for of selection scheme.Paclitaxel has been used to treat multiple myeloma and non-Hodgkin lymphoma (people " Paclitaxel as the initial treatment of multiple myeloma:an EasternCooperative Oncology Group Study (E1A93) " Am.J.Clin.Oncol.1998 such as Miller; 21:553-556; People such as Jazirehi " Resveratrol modifies the expression of apoptoticregulatory proteins and sensitizes non-Hodgkin ' s lymphoma and multiplemyeloma cell lines to paclitaxel-induced apoptosis " Mol.Cancer Ther.2004; 3:71-84; Every piece of document is incorporated the present invention by reference into).Yet this application is but because its high toxicity and multi-drug resistant and restricted.The present inventor has estimated Fu Dilong and anti-lineup MM of dEpoB and NHL system, they use in many researchs recently that are used for estimating the NOD/SCID heteroplastic transplantation model of novel therapies, comprise 10-propargyl-10-denitrogenation assorted-aminopterin (PDX) (people such as Wang " Activity of a novel anti-folate (PDX, 10-propargyl-10-deazaaminopterin) against human lymphoma issuperior to methotrexate and correlates with tumor RFC-1geneexpression " Leukemia ﹠amp; Lymphoma 2003,44:1027-1035), anti-telomerase (people " Telomerase inhibition with an oligonucleotidetelomerase template antagonist:in vitro and in vivo studies in multiplemyeloma and lymphoma " Blood 2004 such as Wang; 103:258-66; Incorporate the present invention by reference into) and anti-VEGFR Mab (people " Targeting autocrine and paracrineVEGF receptor pathways regresses human lymphoma xenographs in vivo " Blood such as Wang is in the printing; Incorporate the present invention by reference into).Fu Dilong and dEpoB all can suppress myeloma and lymphoma cell propagation significantly.Myeloma cell line is very responsive to Fu Dilong with ultralow IC50 and dEpoB, and two kinds of NHL tie up to and are suppressed (table 2-1) when Fu Dilong dosage exceeds among the MM effective dose 5-10 times simultaneously.

The cell enlargement of anti-one group of normal person's tumor of table 2-1. Fu Dilong and dEpoB and normal cell population suppresses and IC50, by XTT bisazo salt (tetrazonium) test determination.

		IC50(nM)	IC50(nM)
		IC50(nM)	IC50(nM)	Tumor cell line	The histology	dEpoB	Fu Dilong
RPMI?8226	Myeloma	36.67±2.0	7.6±1.2	Tumor cell line	The histology	dEpoB	Fu Dilong
RPMI?8226	Myeloma	36.67±2.0	7.6±1.2	CAG	Myeloma	61.34±4.2	12.04±1.8
OPM-2	Myeloma	38.89±3.3	8.2±2.2	CAG	Myeloma	61.34±4.2	12.04±1.8
OPM-2	Myeloma	38.89±3.3	8.2±2.2	H929	Myeloma	42.75±4.5	9.2±1.9
MOLP-5	Myeloma	68.56±5.5	14.4±2.6	H929	Myeloma	42.75±4.5	9.2±1.9
MOLP-5	Myeloma	68.56±5.5	14.4±2.6	RL	Lymphoma		90±11	80±11
SKI-DLBCL	Lymphoma		72±9.8	RL	Lymphoma		90±11	80±11	60±4.2
SKI-DLBCL	Lymphoma		72±9.8	HS-27A	Bone marrow matrix	100±10	102±8		60±4.2
HS-5	Bone marrow matrix	100±8	96±7	HS-27A	Bone marrow matrix	100±10	102±8
HS-5	Bone marrow matrix	100±8	96±7	MRC-5	Embryo lung fibroblast (people)	8.2±4.3	7.4±2.7
HT-29	Colon cancer	7.2±2.2	4±1.7	MRC-5	Embryo lung fibroblast (people)	8.2±4.3	7.4±2.7
HT-29	Colon cancer	7.2±2.2	4±1.7	HCT-116	Colon cancer	7.5±3.1	3.6±1.3
MDA?MB435	Breast carcinoma	7.8±4.2	5.8±2.8	HCT-116	Colon cancer	7.5±3.1	3.6±1.3
MDA?MB435	Breast carcinoma	7.8±4.2	5.8±2.8	IGROV	Ovarian cancer	15±3.8	2±1.2
SKOV3	Ovarian cancer	13±4.7	1.6±0.5	IGROV	Ovarian cancer	15±3.8	2±1.2
SKOV3	Ovarian cancer	13±4.7	1.6±0.5	Ovcar-3	Ovarian cancer	14±3.6	1.1±0.4
Ovcar-4	Ovarian cancer	16±2.5	1.8±0.7	Ovcar-3	Ovarian cancer	14±3.6	1.1±0.4

Normal marrow stromal cell (HS-27A and HS-5 system) shows the relative drug resistance to these chemical compounds, shows that Fu Dilong and dEpoB have the safe treatment window (table 2-1) to MM.Fu Dilong has about 5 times of big effectiveness than dEpoB to MM cell line, and these two kinds of medicines all have the toxicity of equity to normal marrow stromal cell simultaneously.Human fetal lung fibroblast (MRC-5) is responsive to dEpoB and Flu, but for Flu, treating window clearly is significantly, even for these highstrung normal cells.Fu Dilong and dEpoB cause myeloma and lymphoma cell to stagnate (Figure 18) and inducing apoptosis of tumour cell (Figure 19 and 20) in the G2M stage.The present inventor has estimated the persistent period that causes the essential external drug exposure of myeloma cell's apoptosis.Pulsed exposures 1,2,4,8 and 24 hours, eccysis medicine and continuation subsequently cultivated and reached 48 hours.Cellular exposure causes all cells in 48hr dead (Figure 21) in Fu Dilong or dEpoB 24hr.Cultivate 4-8hr with dEpoB and delayed cell proliferation, being exposed to the identical persistent period of Fu Dilong has then reduced about 50% of initial cell quantity.Be exposed to dEpoB and reduced in 48hr in one hour but do not stop tumor cell proliferation, and use Flu, tumor quantity has reduced by 50% (Figure 21).Fu Dilong is the same with paclitaxel with dEpoB, all has after exposure soon, increases that microtubule fasolculus forms and the ability (Figure 22) that do not have the microtubule quality in this cell of slight modification in cancerous cell.Destroyed and the generation apoptosis at time (about 24hr) microtubule after a while.Fu Dilong and dEpoB for comparison available from the nascent CD138MM cell of parent marrow in, the present inventor illustrates Fu Dilong induced tumor apoptosis in 24hr, and dEpoB does not have.

Drug susceptibility to human solid tumor's cell line

The IC of Fu Dilong and dEpoB ₅₀Go up mensuration (table 2-1) in lineup's solid tumor system (colon, breast and ovary).In each example, the IC of Fu Dilong ₅₀All be lower than the IC of dEpoB ₅₀There is the IC of average out to 1.6nM in ovarian cancer system to the ovary cording of Fu Dilong sensitivity especially and 4/5 ₅₀, the IC of dEpoB Comparatively speaking ₅₀Be 16.5nM.These two kinds of medicines all cause tumor cell to stagnate (Figure 23) and rapid induction apoptosis (Figure 24 and 25) in the G2M stage.

RPMI-8226 myeloma cell ties up to dEpoB or Fu Dilong with * 10 full genomic expressions behind IC50 dosage treatment 6 and the 18hr separately

The comparison of full genomic expression (Affymetric chip AU133 2.0) and differential gene expression in the multiple myeloma cell line of RPMI-8226 people is with dEpoB or Fu Dilong treatment 6 or the 18hr (IC * 10 ₅₀Dosage) carry out after.The RPMI-08226 of Fu Dilong treatment and the untreated cell of contrast relatively in, in the time of 6 hours 5 genes just regulated and control (table 2-2) and during at 18hr 48 genes just regulated and control (table 2-3).JUN is all just regulated and control (+3.25 ,+2.64) at twice.When 6hr 3 genes by negative regulation (table 2-2) and when the 18hr 16 genes by negative regulation (table 2-3).HNRPD (allos nuclear nucleoprotein D analog) two times all by negative regulation (2.46 ,-3.25).The RPMI-08226 of dEpoB treatment and the untreated cell of contrast relatively in, in the time of 6 hours 21 genes just regulated and control (table 2-2) and during at 18hr 26 genes just regulated and control (table 2-3).JUN (+5.66 ,+3.73) and tubulin α 3 (+2.64 ,+2.30) were all just regulated and control two times.When 6hr 3 genes by negative regulation (table 2-2) and when the 18hr 16 genes by negative regulation (table 2-3).HNRPD two times all by negative regulation (4.92 ,-2.00).So the present inventor has compared the gene expression (table 2-2 and 2-3) that dEpoB is changed by Fu Dilong.

Table full genomic expression of 2-2. (Affymetrix chip-AU133 2.0) and the comparison of differential gene expression in RPMI-8226 (the multiple myeloma cell line of people).Control cells and 6 hours the comparison of cell (at 10 times of IC50 dosage) for the treatment of with Fu Dilong or dEpoB.

Fu Dilong 6hr	Multiple changes	NSC-703147 B 6hr	Multiple changes
Fu Dilong 6hr	Multiple changes	NSC-703147 B 6hr	Multiple changes	? JUNSKIL (class SKI) BASP1 (brain abundance symphysis signal) APP(amyloid precursor protein) HMGCS1(3-hydroxy-3-methylglutaric acid list acyl coenzyme synthase)	+3.25 +2.83 +2.83 ? +2.14 ? +200 ? ? ? ? ? ? ? ? ? ? ? ? ? ?	IFI27 (but interferon-' alpha ' induced protein) JUN G1P3 ^(but interferon-' alpha ' induced protein) IFITM1 ^(interferon-induced transmembrane protein 1) APP(amyloid precursor protein matter) CCL5 (Rantes chemotactic factor (CF)) TUBA3 (tubulin α 3) INSIG1 (insulin-induced gene) TRIM22 (containing tripartition motif 22) HLA-DPA1^(histocompatibility classification II DP α 1) IFIT1 ^(having the multiple interferon inducible protein of tetratricopeptide) HMGCS1(3-hydroxy-3-methylglutaric acid list acyl coenzyme A) MARCKS (being rich in meristoylated alanine PKC substrate) ABCG1 (ABC transports activity)	+18.38 +5.66 +5.28 +4.92 ? ? +2.83 +2.64 ? +2.64 +2.46 +2.30 +2.30 ? +2.30 ? +2.30 ? +2.14 ? +2.14

		MX1 ^(myxovirus drug resistance, interferon can be induced p78) OAS1 ^(2 ' 5 '-oligoadenylate synthetase) PLSCR1 (phosphatide translocase (phospholid scramblase)) STS (steoid sulfatase) DUSP4 (two special phosphate) AP1S1 (joint GAP-associated protein GAP compound 1) ARHE (the Ras homologue E of family)	+2.00 ? +2.00 +2.00 ? +2.00 +2.00 +2.00 +2.00 +2.00
			+2.00 ? +2.00 +2.00 ? +2.00 +2.00 +2.00 +2.00 +2.00	? HNRPD(allos nuclear nucleoprotein D). SUV39 H2. (inhibitive factor of raggle-taggle homologous chromosome). HIST1 (histone 1)	-2.46 ? -2.00 ? ? -2.00	? RNRPD(allos nuclear nucleoprotein D) HMOX1 (heme oxygenase) HASA1A (heat shock 70kDa albumen) MAT2A (methionine adenosyltransferase II α)	-4.92 ? -2.30 -2.14 ? -2.00

Underscore :-gene is just being regulated and control or negative regulation, in the cell of Fu Dilong or dEpoB treatment during 6hr.

^*But interferon induced gene.

Table full genomic expression of 2-3. (Affymetrix chip-AU133 2.0) and the comparison of differential gene expression in RPMI-8226 (the multiple myeloma cell line of people).Control cells and 18 hours the comparison of cell for the treatment of (at * 10 times of IC50 dosage) with Fu Dilong or dEpoB.

Fu Dilong 18hr	Multiple changes	NSC-703147 B 18hr	Multiple changes
Fu Dilong 18hr	Multiple changes	NSC-703147 B 18hr	Multiple changes	PRKDC (double-strand break reparation) fibrillin 2 REPS1 (the Eps zone that RALP1 is relevant) PKD1(polycystic kidney disease 1)	+21.11 ? +12.3 +9.85 ? +7.46	REPS1 (the Eps zone that RALBP1 is relevant) PKD1(polycystic kidney disease 1) JUN? ? SEMA3D(Ig sema zone)	+9.85 ? +5.6 +3.73 ? +3.73

? TRIO(Triple functn zone amidino groups NEF) FLJ20241 (NFkB activated protein) CCL3(chemotactic factor MIP-1 α) PCNX(Pecanex homologue) KIAA1025Albumen TRA2A(transformer 2 α, mRNA montage) GTF2H2 (general transcription factor IIH) SLC13A1 (sodium ion transport protein) PHC3(the special-shaped analog of poly-homology) SEMA3D(Ig sema zone) JUN? PRKCBP1(Protein kinase C combination) GA17 (dendritic cell albumen) SEC24D (the gene family D that SEC24 is relevant) SMA3(hydrolytic enzyme activities) ETS2 (homologue of v-ets oncogene) DNAJB1 (Hsp40 homologue subtribe B) CCT8 (chaperonin that contains TCP1) EIFA1(rotaring intertranslating start factor 4A-1) TCS2 (tuberous sclerosis 2) CD72 (cell adhesion) HSPA1A (heat shock protein 70 kDa 1A)

+4.29 ? +3.73 +3.48 +3.48 +3.25 +3.03 ? +3.03 ? +3.03 +2.83 +2.83 +2.64 +2.64 +2.64 ? +2.64 ? +2.46 +2.46 +2.30 +2.30 ? +2.30 ? +2.30 +2.30 +2.14 ?

? PRKCBP1(Protein kinase C combination) PCNX(Pecanex homology) GTF2H2 (general transcription factor IIH) KIAA1025Albumen TRIO(Triple functn zone guanyl-NEF) CCL3(chemotactic factor MIP-1 α) VEZATIN(transmembrane protein) TRA2A(transformer 2 α, mRNA montage) APRIN(androgen is induced proliferin inhi) KIAA0191Albumen TUBA3 (tubulin α 3) THEM1(transmembrane protein 1) PHC3(the special-shaped analog of poly-homology) SMA3(hydrolytic enzyme activities) AKAP9(PRKA kinases anchorin) INADL (PDZ, signal cascade) EIFA1(translation initiation factor 4A-1) GAPCENA (rab6GTPase activation) MGC10526 (sugar phosphotransferase) ATP8B1 (ATPase classification 1) RPL23(ribosomal protein L 2 3) RC3 (rabconnectin3)

+3.03 +3.03 +2.83 +2.83 +2.83 ? +2.64 +2.46 +2.46 ? +2.46 ? +2.46 +2.30 +2.30 +2.30 +2.30 +2.14 ? +2.14 ? +2.14 +2.14 ? +2.00 +2.00 +2.00 +2.00 ? ?

ZFY (zinc finger protein-Y-connects) AIP1 (atropine-1 interaction protein) RPL23(ribosomal protein L 2 3) THEM1(transmembrane protein-1) KIAA0191Albumen HSPA1B (heat shock protein 70 kDa 1B) VEZATIN(transmembrane protein) SERPINH1 (cystatin) MADH1 (mothers against decapentaplegic) BAX (Bc1-2 GAP-associated protein GAP) APRIN (androgen is induced proliferin inhi) APBB2 (amyloid precursor bindin)AKAP9(PRKA kinases anchorin) APG16L (APG16 autophagy 16-analog) CENPC1 (centromere protein C1)	+2.14 +2.14 +2.14 +2.14 +2.14 +2.00 ? +2.00 +2.00 ? +2.00 ? +2.00 +2.00 ? +2.00 ? +2.00 +2.00 ? +2.00
				? ZF(HCF-is in conjunction with transcription factor) Clorf29, (chromosome 1 open reading frame) FLJ20130, (hypothetical albumen) F11, (Stuart factor 1) G1P3, (but interferon-' alpha ' induced protein) HNRPD(allos nuclear nucleoprotein D-analog)	-6.50 -6.06 -5.66 ? -4.00 -3.73 -3.25 ?	? ZF(HCF-is in conjunction with transcription factor) cyclin E2 MARS(methionine-tRNA synzyme) POU4F1(Pou district transcription factor) NRTN(neuturin) PABPN1 poly-(A) is conjugated protein, nuclear)	-6.06 -3.03 -2.83 ? -2.46 -2.14 -2.00 ?

? CyclinE2 FLJ20045 (hypothetical albumen) CEB1 (cyclin E is in conjunction with ubiquitin ligase) FBXO5 (the unique albumen 5 of F box) NRTN(neuturin) DUSP6 (dual specificity phosphatase enzyme) POU4F1(Pou district transcription factor) MARS(methionine-tRNA synzyme) HDAC9 (histone deacetylase enzyme 9) CENPC1 (centromere protein C1)

-2.64 ? -2.46 -2.30 ? -2.30 -2.3 -2.14 -2.14 -2.00 -2.00 -2.00

? HNRPD(allos nuclear nucleoprotein D-analog)

-2.00 ? ? ? ? ? ? ? ? ? ? ?

Underscore :-gene is just being regulated and control or negative regulation, in the cell of Fu Dilong or dEpoB treatment during 18hr.

But interferon induced gene *.

At 6 hours, just regulating and control 5 genes by Fu Dilong, just regulating and control 21 by dEpoB, and the two is all just regulating and control 3 genes, simultaneously by 3 genes of Fu Dilong negative regulation,, and there is not the two all gene of negative regulation by 4 of dEpoB negative regulations.At 18 hours, just regulating and control 48 genes by Fu Dilong, just regulating and control 26 by dEpoB, and the two is all just regulating and control 18 genes, simultaneously by 16 genes of Fu Dilong negative regulation, by 7 of dEpoB negative regulations, and the two 6 gene of negative regulation all.Specifying this scheme to test enhanced antitumor efficacy that following hypothesis: Fu Dilong is better than dEpoB and former Epothilones is because except microtubule stability, other cancer target mechanism.Therefore the present inventor has determined which kind of gene just regulated and control and negative regulation by Flu difference ground, simultaneously with the MM cell of dEpoB-treatment relatively (table 2-4).

The table full genomic expression of 2-4. (Affymetrix chip-AU133 2.0) and differential gene expression are with the comparison among 6 or 18 hours the RPMI-8226 (the multiple myeloma cell line of people) of dEpoB or Fu Dilong (at * 10 times of IC50 dosage) treatment.Shown in those of gene Wei You Fu Dilong selectively changing.

6 hours gene differences	Multiple changes	18 hours gene differences	Multiple changes
6 hours gene differences	Multiple changes	18 hours gene differences	Multiple changes	The BCL3 Heme oxygenase	+ 3.25+2.30	HMT-1 (transmethylase-analog-1)	+ 11.3
IFI27 ^(derivable interferon (IFN) α) GIP3 ^(derivable IFN α) T RIM34 (containing the tripartition motif) IFTM1 ^(the inductive film of striding of IFN) HLA-II DP α ^AIM2 ^(in melanoma, lacking) PTPL (tyrosine phosphatase-analog) TRIM22 (containing the tripartition motif) CA4 (carbonic anhydrase) MX1 ^(myxovirus drug resistance, IFN is derivable).	-7.46 -6.06 -5.66 -4.00 -3.73 -2.64 -2.46 -2.46 -2.14 -2.00	IFI27 ^(derivable interferon (IFN) α) GIP3 ^(derivable IFN α) IFITM1 ^(the inductive film of striding of IFN) MX1 ^(myxovirus drug resistance, IFN is derivable) DUSP4 (two special phosphatase) PPAP2C (phosphatidic acid phosphatase) PLSCR1 ^(phospholipid translocase) IFIT1 ^(the inductive albumen tetra-tricopeptide of IFN repeats) OAS2 ^*(2 '-5 '-oligoadenylate synthetase-2) TRIM22 (containing tripartition motif-22) CEB1 (cyclin E is conjugated protein)	-5.28 -5.28 -3.25 -2.30 -2.14 -2.14 -2.00 -2.00 -2.00 -2.00 -2.00	The BCL3 Heme oxygenase	+ 3.25+2.30	HMT-1 (transmethylase-analog-1)	+ 11.3

Underscore :-negative gene regulation and control, in the cell of Fu Dilong or dEpoB treatment 6 with during 18hr.

^*But interferon induced gene.

At 6 hours, just regulating and control two genes, and, just regulating and control a gene at 18 hours.At 6 hours, negative regulation ten genes, and at 18 hours, negative regulation 11 genes, and at two time negative regulations four gene (IFI27,-7.46 ,-5.28), GIP3 (6.06 ,-5.28), IFTM1 (4.00 ,-3.25), MX-1 (2.00 ,-2.30).These are the derivable genes of all interferon.The gene expression figure of ovary 1A9 and 1A9PTX22 ovary xenograft is reported, be shown as IFN responsive genes (GIP3 after 24 hours with paclitaxel treatment, IFI27, IFITM1, IFII6, (people " Gene expressioncorrelating with response to paclitaxel in ovarian carcinoma xenografts " the Mol.Cancer Thera.2004 such as Bani of expression decreased ISG15); 3:111-121; Incorporate the present invention by reference into).Three (GIP3 in these genes, IFI27, IFITM1), at dEpoB rather than after 6 hours with the RPMI-8226 cell of Fu Dilong treatment, by overexpression consumingly, and these also are that it expresses the gene of distinguishing Fu Dilong and dEpoB gene map the most consumingly.The overexpression of IFN-responsive genes (for example, IFR9) with in the tumor cell line drug resistance of paclitaxel is linked together recently, and such probability has been supported in these discoveries: be the response that the medium of INF signal is involved in paclitaxel, rather than INF self (people such as Luker, " Overexpression of IRF9confers resistance to antimicrotubule agents in breast cancer cells " CancerRes.2001; 61:6540-7; Incorporate the present invention by reference into).In the chemical sproof development of antagonism microtubule agent, the report of this Novel IFN independent action of IRF9 accounts near half of breast carcinoma and uterus carcinoma (people such as Luker, " Overexpression of IRF9confers resistance toantimicrotubule agents in breast cancer cells " Cancer Res.2001; 61:6540-7; Incorporate the present invention by reference into).But the microarray analysis of the chemical sproof tumor of eEpoA or Taxol system shows in the EpoA resistant tumors most gene codes of in the Taxol resistant tumors, highly not expressed interferon induced gene (people " Geneexpression profiling of epothilone A-resistantcells " Novartis Found Symp.2002 such as Atadja; 243:119-32; Incorporate the present invention by reference into).Obviously, the present inventor finds that (just regulated and control, neither one changes (showing 2-2 to 2-4) by the Flu treatment in derivable and these genes for IFN for all genes of 8/21 (38%) after dEpoB treatment RPMI-8226.But IFN induced gene subsequently demonstrates in the 8226MM cell with the dEpoB treatment just to be regulated and control after 6 hours or 18 hours and do not change when treating with Fu Dilong.By the degree ordering that dEpoB is just regulating and control, they are 1.) interferon (IFN) α-derivable IFI 27 (increasing+18.83).But this belongs to the family of little interferon-' alpha '-induced gene, and its unknown function is for just being regulated and control (people " Interferon alpha-inducibleprotein 27 (IFI27) is upregulated in Psoriatic skin and certain epithelialcancers " J Invest.Dermatol.2004 such as Suomela in dermatosis, epithelial cancer and the wound repair of inflammation; 122:717-721; Incorporate the present invention by reference into).2.) but IFN α-induced protein (clone IFI-6-16) GIP3 (people " Gene expressioncorrelating with response to paclitaxel in ovarian carcinoma xenografts " Mol.Cancer.Thera.2004 such as Bani; 3:111-121; Incorporate the present invention by reference into) (increasing+5.28).3.) IFN-induces transmembrane protein 1, IFTM1 people such as () Bani (increasing+4.92).4.) mucosal virus drug resistance, IFN is derivable, MX1 (increasing+2.3 increase+2 in 6 hours with at 18 hours).This MX1 gene is given the optionally congenital drug resistance of influenza virus (Staeheli, Ad.Viral Res.38:147,1990; Incorporate the present invention by reference into).They also may as gtp binding protein take on basic cell function (Arnheiter H and Meier E. " Mx proteins:antiviral proteins by chance or necessity? " New Biol.1990; 90 851; Incorporate the present invention by reference into).5.) phospholipid translocase 1 gene PLSCR1.The response element that the control of this gene transcription is stimulated by the single IFN-that is positioned at first exon and regulation and control fully (people " Transcriptional control of the human plasma membrane phospholipidscramplase 1 gene is mediated by interferon-alpha " Blood 2000 such as Zhou; 95:2593-2599; Incorporate the present invention by reference into).PLSCR1 relates to the motion to cell surface of reconstruction membrane plasmapheresis phospholipid and Phosphatidylserine.It can help the film of striding fast of membrane plasmapheresis phospholipid to move, this can be in being exposed to the rising cell Ca ⁺⁺Injured and apoptotic cells in observe.Studies show that it can also make the nuclear transposition and can be used as transcription factor (people " PhospholipidScramblase 1 is imported into the nucleus by a receptor-mediated pathwayand interacts with DNA " Biochem.2004 such as Ben-Efraim; 43:35181; Incorporate the present invention by reference into).6.) has the multiple interferon inducible protein of tetracopeptide, IFITI (increasing+2.3).Its relevant with the whole body lupus erythematosus and protein-protein studies show that it can activate Rho albumen (people " Protein interaction for an interferon-inducible syste-3526mic lupusassociated gene, IFIT1 " Rheumatology 2003 such as Ye by interacting with the Rho/Rac guanine nucleotide exchange factor; 42:1155-63; Incorporate the present invention by reference into).7.) 2 '-5 ' oligoadenylate synthetase-2, OAS2 (increasing+2).(OAS1, OAS2, OAS3, interferon-induced gene OAS4) relate to stress and are important (Chebath, Nature 330:587,1987 for the antiviral activity of interferon in this gang; People J.Virol.66:5804 such as Meurs, 1992; Every piece of document is incorporated the present invention by reference into).OAS2 catalysis adenosine oligomer (2-5A) synthetic.So these oligomer have activated RNase L, hide endoribonuclease in most of mammalian cells, it can make RNA inactivation (the people Eur.J.Biochem.2004 such as Anderson of virus (picornoviruses is encephalomyocardis virus for example) and cell conversely again; 271:628-36; Incorporate the present invention by reference into).8.) histocompatibility class II DP α 1, HLA-DPA1 (increasing+2).MHC-class II is medium (people " the Molecular cloning of a new Interferon-induced factorthat represses human immunodeficiency virus Type 1long terminal repeatexpression " Biol.Chem.1995 such as Tissot of known IFN biological function; 270:14891-14898; Incorporate the present invention by reference into).

Sum up

The CAG myeloma cell line has shown many features of clinical multiple myeloma, and described multiple myeloma has graft in phenotype (CD38+CD138+CD45-) and the body.Set up xenotransplantation NOD-SCID myeloma mouse model by 10～1,500 ten thousand CAG cells of intravenous perfusion with the transformation of fusion HSV-TK-eGFP-luciferase gene.Therefore the whole animal imaging that is formed by the luciferase bioluminescence can be used for real-time, Noninvasive report and estimates tumor load and efficacy of drugs in the mice of work.The myeloma mice shows bone marrow infiltration and the molten bone infringement of pathology at tumor injection after 7 to 20 days.Behind the implantation tumour cell, mice is divided into vehicle control group, dEpoB treatment group and Fu Dilong treatment group randomly.The dosage of dEpoB and Fu Dilong is 20mg/kg, and these medicines are all by the intraperitoneal administration.The meansigma methods of dosage number of times: in first 30 days of treatment, being 10 times for dEpoB, is 12 times for Fu Dilong.In addition, initially with 3 in 7 mices of Fu Dilong treatment, the Velcade of five dosage of administration between 35 to 45 days (6.25 μ g/ Mus, intravenous perfusion).The result shows, estimates by the bioluminescence imaging, and compares with control mice with the mice of dEpoB treatment, significantly reduces with its tumor load of the myeloma mice of Fu Dilong treatment.All mice in control group are dead in 30 days after initial therapy, yet the time-to-live of the mice of Fu Dilong treatment is at least the twice of matched group.Matched group and do not have difference with the time-to-live between two groups of the mices of dEpoB treatment.With proteasome inhibitor Velcade combination, can significantly reduce to be arranged in the tumor of vertebrates gray columns and femur, this shows this combination even also can attack the myeloma cell under the bone marrow microenvironment protection.

The data of front show that the myeloma with the Epothilones treatment demonstrates typical apoptosis feature.Whether it be unclear that Guang winter peptidase involves in this process.At first, the present inventor has studied the activity of Guang winter peptidase 3 in the myeloma cell (a kind of effector Guang winter peptidase in Guang winter peptide enzymatic pathway) by the Western trace.With dEpoB or Fu Dilong treatment, the myeloma cell demonstrates the cracked Guang winter peptidase 3 that has increased 17kd, and this is active relevant with it.The antibody of the Guang winter peptidase 3 that is specific to cut-out has been adopted in the immunohistochemical dyeing of CAG myeloma cell, is used for being illustrated in the apoptotic cell Cytoplasm and the local feature in nuclear week.These data acknowledgement Guang winter peptidases are activated in the cell of Epothilones treatment.Secondly, the present inventor attempts having tested by fluorometric method the activity of Guang winter peptidase 8 and Guang winter peptidase 9 (the initiator Guang winter peptidase in Guang winter peptide enzymatic pathway).Equally, find that in the myeloma cell Guang winter peptidase 8 and Guang winter peptidase 9 activity all have been increased.The specific activity Guang winter peptidase 8 of the Guang winter peptidase 9 that attention increases is more outstanding.Cause

Guang winter peptidase

8 or 9 activatory signals just in active research.

Drug cell toxicity to hematopoietic stem cell is people's major concern always.From Cord blood, isolate people CD34+ stem cell and or with dEpoB or with Fu Dilong single culture 24 hours.Behind the eccysis medicine, carry out apoptosis test and the test of the origin in 2 week to estimate the influence of Epothilones to stem cell.Data show dEpoB of the present invention or Fu Dilong do not have remarkable toxicity to acyclic stem cell.

Embodiment 3:(E)-9,10-dehydrogenation Epothilones: the novel microtubule of a class that has fine foreground features in the Muridae heteroplastic transplantation model is stablized antitumor agent

The 26-F that comprises common 16 yuan of rings ₃-[16] dEpoB (2) synthetic can compile strategy by height finishes, and this strategy is for being applied to 27-F ₃Relevant strategy in-[17] ddEpoB (19) synthetic.This strategy is estimated to form E-9 by closed loop metathesis reaction as follows, 10-alkene (example that RCM is cyclized into complex structure greatly that passes through in the past is, referring to: Sinha, S.C.; Sun, J.Angew.Chem.Int.Ed.2002,41,1381; Incorporate the present invention by reference into).The present inventor estimates 28 and 29 E-9, the 26-F that the chemical selective reduction of 10-alkene will provide dEpoB (1) and want ₃-dEpoB (2).The precursor of this RCM is by preparing in conjunction with two fragments (21 or 24) and 123 by esterification.Coupling companion 123 is connected in the methylene spacer groups (seeing in the early stage approach of the present inventor, referring to chemical compound 18) of C8 secondary methyl by elimination and makes up.As above illustrated, comprise the discovery of external level of the Epothilones (referring to 18) of 17 yuan of rings that diene at interval arranges, emphasized needs for the corresponding research of the biological results of diene such in the 16 membered ring lactone structures of being familiar with.In view of the cis among the dEpoB among the present invention 12, the existence of 13-diene, such interval diene should comprise 9, the two keys of 10-.

9,10-dehydrogenation Epothilones and 26-F ₃The retrosynthesis of-dEpoB

With being easy to synthetic trifluoro allyl iodide and methacrylic iodine (being respectively 8 and 124) Wan Jiization oxazolidone 7 (people such as Lee, J.Am.Chem.Soc.2001,123,5249; Incorporate the present invention by reference into), allow in the absolute configuration that is fit to, to set up the three-dimensional center of C15 (Evans, D.A. with high diastereomeric excess (referring to product 9 or 125); Morrissey, M.M.; Dorow, R.L.J.Am.Chem.Soc.1985,107,4346; Paterson, I.; Bower, S.; Mcleod, M.D.Tetrahedron lett.1995,36,175; Every piece of document is incorporated the present invention by reference into).The latter is converted to corresponding Weinreb amide (people Tetrahedron Lett.1981 such as Nahm, 22,3815; People Synth.Commun.1982 such as Levin, 12,989; Every piece of document is incorporated the present invention by reference into), and be converted into 126 and 42 thus, then by nucleophilic methylate (MeMgBr) become olefination (people such as Lythgoe, Tetrahedron lett.1975,40,3863 with the Homer Wittig that is fit to; Lythgoe, Chem.Soc.Rev.1981,449; People Org.Chem.1983 such as Toh, 48,1414; People J.Org.Chem.1986 such as Baggiolini, 51,3098; Every piece of document is incorporated the present invention by reference into) be converted into 24 and 21.Although use this as chirality adminicle De oxazolidone by Evans and colleague (people J.Am.Chem.Soc.1985 such as Evans, 107,4346; People Tetrahedron Lett.1995 such as Paterson, 36,175; Every piece of document is incorporated the present invention by reference into) initiative, but its application by the glycolate of alkylation (rather than passing through hydroxylating) synthesizing optical definition is not developed as yet.

Fragment

21 and 24 synthetic

The synthetic of many propionic esters fragment 25 can realize that it has set up the relative configuration at C3, C6 and the three-dimensional center of C7 by the aldol reaction of two keys.First aldol reaction relates to the ethyl ketone 30 of Z-enolization and 31 reactions of Roche aldehyde (people Org.Chem.1976 such as Cohen, 41,3505; People Tetrahedron such as Nagaoka 1981,37,3873; People J.Am.Chem.Soc.1990 such as Roush, 112,6348; Every piece of document is incorporated the present invention by reference into) with provide want have the height non-enantiomer selectivity 32.Should synthesize by means of 31, its remarkable advantage is to be better than using former aldehyde, and former aldehyde need solve the source problem that comes of optical voidness initiation material.

Protection C7-alcohol provides the aldehyde of wanting 34 by the acetal hydrolysis subsequently and is that aldol reaction creates conditions for the second time.34 with the reaction of DAG (diketone glucose) the Ti-enolate of Duthaler (people Angew.Chem.Int.Ed.Engl.1989 such as Duthaler, 28,495; Incorporate the present invention by reference into) provide want have the height (＞95%) non-enantiomer selectivity hydroxyl tertiary butyl ester 35.Then the latter is converted into the acid of wanting 25.

Synthesizing of acid 25

Allyl alcohol

24,21 and C ₁-C ₉Acid fragment 25 combine by EDCI esterification rules, therefore RCM precursor 26 and 27 are provided respectively.26 and 27 closed loop metathesis reaction is by using RCM catalyst (people Tetrahedron Lett.1999 such as Scholl, 40,2247; People Acc.Chem.Res.1995 such as Grubbs, 28,446; People Acc.Chem.Res.2001 such as Trnka, 34,18; Alkene Metathesis in Organic Chemistry Ed.:F ü rstner, A; Springer, Berlin, 1998; F ü rstner, A.Angew.Chem.Int.Ed.Engl.2000,39,3012; Schrock, Top.Organomet.Chem.1998,1,1; Every piece of document is incorporated the present invention by reference into) in toluene, carry out.These reactions provide transisomer 39a and 40a really.Yet principal product but is 39b and 40b, and it is by significantly another RCM approach acquisition.These undesired RCM isomers are about 3: 1 above 39a that wants and the ratio of 40a.At last, with the HF-pyridine silyl ether of 39a and 40a is sloughed protection and cause forming the E-9 that wants, 10-dehydrogenation-Epothilones 28 (people J Am.Chem.Soc.2001 such as White, 123,5407; People J such as White; Am.Chem.Soc. (augment/revise) 2003,125,3190; Every piece of document is incorporated the present invention by reference into.The structure of the strictness proof of known compound 28, the present inventor is surprised to find its spectral quality and the spectrum of previously reporting of thinking the chemical compound with identical entity and inconsistent.The practical structures of this chemical compound had before thought 28, was identified again now.Review the past, 28 never be produced and, whole (E)-9 of in fact reporting among the present invention, 10-dehydrogenation Epothilones family all is new kind) and 29.According to plan, the latter reduces E-9 by imidodicarbonic diamide, 10-alkene and be converted into dEpoB (1) and 26-three fluoro-dEpoB (2) (people Tetrahedron Lett.1961 such as Corey, 347; People Org.React.1991 such as Pasto, 40,91; Every piece of document is incorporated the present invention by reference into).

By corresponding methodology, the present inventor has synthesized 9, and 10-dehydrogenation-dEpoF (57) vide infra.Imidodicarbonic diamide reduction E-9 optionally, 10-alkene has confirmed the structure of aforesaid various synthetic intermediates, therefore impels the distribution before evaluating in the document once more.

9,10-dehydrogenation-Epothilones synthetic

Synthetic analog (28,29 and 57), inspection in cell culture system (referring to [) demonstrates them various responsive things and the generation of MDR tumor cell line is better than the inhibitory action that the present drug candidate dEpoB (1) of the present inventor is had.The impressive cell growth inhibition that Epothilones 28,29 and 57 is had covers the scope of various resistant tumors, and this has impelled measures the plasma stability of these novel (E)-9,10 congeners.The present inventor remembers (E)-10, and 11-dehydrogenation-dEpoB (Epo490) has the plasma stability of non-constant.In fact, this just blood plasma unstability has hindered the further exploitation of Epo490 (6).On the contrary, when being exposed to Mus blood plasma with 28,29 and 57, the present inventor observes with dEpoB (1) and compares very slow drug degradation (factor is about 7).From drug utilization degree aspect, with respect to dEpoB (1), say nothing of epo490 (6), this stability has constituted substantial progress.Based on preliminary (E)-9,10-dehydro derivatives 28 and 29 cell culture and pharmacokinetic data are further used for them that research is suitable in the body.Such research is certainly than the availability of more emphasizing medicine in vitro study (people J.Am.Chem.Soc.2003 such as Rivkin, 125,2899; People Angew.Chem.Int.Ed.Engl.2003 such as Chou, 42,4761-4767; People Angew.Chem.Int.Ed.Engl.2003 such as Yoshimura, 42,2518-2521; Every piece of document is incorporated the present invention by reference into.For the example of nearest in addition effectively epothilone analogs referring to people Bioorg.Med.Chem.Lett.2000 such as Altmann, 10,2765; People Chem.Biol. such as Nicolaou, 2000,7,593; Every piece of document is incorporated the present invention by reference into).

This demand and in fact may need prepare 9 of number gram amount at last, the 10-dehydro derivatives is used for further research, impels the complete synthesis route of the present invention is carried out significant revaluing.Certainly, unique the most serious problem is that 39b, the 40b and 56 that the RCM reaction on 26,27 and 54 produces only is secondary product.The main path strictness that relates to the RCM reaction is limited to 26,27 and 54 O-moieties, causes mainly producing undesired 39b and 40b.Therefore, decision is attempted (videing infra) after the RCM postponed till in the introducing (passing through olefination) of thiazole.

The handlabilities of final number gram magnitudes as target of the present invention, have therefore been rebuild alkyl and the acyl group fragment that enters the RCM reaction.Chemical compound 86 as illustrated being easy to are synthesized.Use it for alkylation height enantiomeric excess De oxazolidone 7.Carry out subsequently the OTES group go the protection and the nucleophilic methylation, obtain chemical compound 90.This alpha-alcohol ketone is in the formation of the important ester for preparing all key RCM, as acyl acceptor.The possible weakness of making us obvious concern is, but such hydroxy-ketone is as the acyl acceptor partial racemizationization or change into the α-keto-alcohol of position isomery.

Accessible synthetic fragment 90

The serious problems of the synthetic in the past acid fragment 25 of the present inventor are very high and technical Duthaler chemistry (people Angew.Chem.Int.Ed.Engl.1989 such as Duthaler, 28,495 of needing of cost; Incorporate the present invention by reference into) on the C3 position, produce the S spatial chemistry want.In order to avoid this problem, need not any chirality adminicle and carry out aldol reaction so that 1: 1 mixture of corresponding beta-hydroxy ketone to be provided.The reagent of control institute's deutero-ketone (referring to chemical compound 69) asymmetric reduction, employing Noyori condition (people J.Am.Chem.Soc.1987 such as Noyori, 109,5856; Incorporate the present invention by reference into) on the C3 position, produced the S spatial chemistry of wanting with the excessive form of high antimer.Now available beta-hydroxy esters 70 was converted into acid 25 (people Proc.Natl.Acad.Sci.U.S.A.2001 such as Chou, 98,8113 according to former scheme in several steps; Incorporate the present invention into by application).

Accessible synthetic sour 25

Significantly, the hydroxy-ketone 43 and 44 that obtains is used C ₁-C ₉Acid fragment 25 esterifications provide corresponding RCM cyclisation precursor 45 and 46, and do not have obvious raceme on C15, lose the integrity of initial α keto-alcohol Cheng Jian in other words.45 and 46 closed loop metathesis reaction is by adopting RCM catalyst (people Tetrahedron Lett.1999 such as Scholl, 40,2247; People Acc.Chem.Res.1995 such as Grubbs, 28,446; People Acc.Chem.Res.2001 such as Trnka, 34,18; Alkene Metathesis in Organic Chemistry Ed.F ü rstner, A; Springer, Berlin, 1998; F ü rstner, A.Angew.Chem.Int.Ed.Engl.2000,39,3012; Schrock, Top.Organomet.Chem.1998,1,1; Every piece of document is incorporated the present invention by reference into) in toluene, carry out.This reacts, and is also uncomplicated owing to the double decomposition approach that substitutes now, and the complete transisomer 47 and 48 of high yield is provided.Introduce the thiazole part by Wittig reaction with high E/Z selectivity and high yield, obtain 28 and 29 after with two monosilane ether deprotections (people Tetrahedron Lett.2000 such as Hindupur, 2,7341; Incorporate the present invention by reference into.Attempt using the product of scheme introducing thiazole of Avery, the low and E/Z poor selectivity of productive rate) to obtain to want.This approach meets a large amount of synthetic standards of obeying.

The present inventor whether consider epothilone B (51, introduce C9-C10 alkene in EpoB) and will change its biological nature, with its 12, the identical direction of 13-deoxidation homologue situation.For this reason, the present inventor studied and adopted 2,2 '-dimethyldioxirane (DMDO) carries out epoxidation to 28.This is reflected on the more polysubstituted C12-C13 alkene and carries out with highly-solid selectively really.Obtain productive rate and be 87% ratio and be 1: 2.6 (E)-9,10-dehydrogenation epothilone B (49) and its have α-12, the diastereomer of 13-oxirane (structure is not shown).In vitro study 49 (it is established at C12 and C13 configuration, provides EpoB with its reduction) shows that it than parent compound EpoB (51) to the powerful 2-4 of being about of various cell lines doubly.Although chemical compound 49 is proved to be the most virtuous Epothilones in the program that the present inventor carries out, it is for the narrow therapeutic index of xenograft, with and be difficult to acquired (on seeing) and hindered it in preclinical further research.What is interesting is that non-natural α-oxirane does not almost have activity.

Accessible synthetic (E)-9,10-dehydrogenation Epothilones

Synthetic another advantage of aforesaid (second filial generation) that rebulids is: can various heterocycles be introduced by ketone intermediate 39a and 40a.This point is by 9, and 10-dehydrogenation-dEpoF's is synthetic and well outstanding.Wittig that ketone and He Shi De phosphonium salt ylide carry out reaction with high yield and high E/Z selectivity provide want 9,10-dehydrogenation-dEpoF chemical compound 57 and 59.In addition, the present inventor can be converted into 21-hydroxyl 59 derivant of 96 and 97 types effectively, is included in the C21 position and carries out aminofunctional, and a few step reactions are as described below.

(E)-9, the variation of the C21 of 10-dehydrogenation Epothilones

Aspect anti-various kinds of cell type, estimated the complete synthesis Epothilones homologue of the present invention, renderd a service with the antitumor of determining them.

The most outstanding feature of the present inventor's discovery is as follows: by with E-12,13 pairs of keys substitute C12-C13 β epoxide, can predict order of magnitude of about reduction (to the CCRF-CEM cell line of sensitivity relatively EpoB and dEpoB).What another can be expected is: comprise E-9,10 pairs of keys add Z-12,13 alkene, and its cytotoxicity that causes covering several cell lines significantly increases.

Another useful trend is found in 12-three fluoro-E-9,10-dehydrogenation-dEpoB (29) (Fu Dilong) with corresponding E-9, in the comparison of 10-dehydrogenation compound 28.With respect to (1), on C26, comprise three fluorine atoms and weakened cytotoxicity and be up to the factor 4.The effect that weakens of this 12-trifluoromethyl functional group also is found in and lacks 9, in the chemical compound of 10-unsaturated bond (relatively dEpoB (1) and 12-three fluoro-dEpoB (2)).

In view of these data and in view of these 9,10-dehydrogenation compound (comprising 12-trifluoro congener) is by the available of chemosynthesis, the present inventor begins to carry out testing in the body with the most promising chemical compound of the present invention.The inventor this described with some of chemical compound 29 (Fu Dilong) surprising especially with promising result, chemical compound 29 demonstrated the exciting probability that is used for further clinical evaluation.In the immunodeficiency nude mice, utilize people's tumor xenogeneic graft to carry out testing in the body.Although their shortcoming is arranged, this model in the oncology still is widely used in estimating (Fiebig, H.H.; Berger, D.P.; Preclinical Phase II trials.At Boven, among the The Nude Mouse in Oncology Research that E. and Winograd, B. edit, CRC Press, Boca Raton (1995), 318; Incorporate the present invention into by application) possible antitumor lead compound to be to carry out clinical research.

Significantly, with the Fu Dilong treatment MX-1 xenograft of 30mg/kg dosage, it causes the tumor complete obiteration and did not have any recurrence (referring to Fig. 6 A) in back 2 months stopping to treat.The most important thing is that these successful treatments can or be finished (referring to Fig. 6 B and 10A) by the perfusion of 6 hours intravenouss or by oral administration.On the other hand, do not influence this tumor (referring to Fig. 6 B and 10A) by oral Taxol treatment MX-1 xenograft.Self-evident, if transfer in people's the clinical setting, it will be significantly useful obtaining Orally active.

Taxol resistant tumors xenograft (Fig. 7 A) and human colon carcinoma (HCT-116, Fig. 9 A) also can be cured by the intravenous perfusion with Fu Dilong.The experiment that breast carcinoma of choosing in nude mice (MX-1) and human colon carcinoma (HCT-116) xenograft carry out continues 6.0 and 6.6 months respectively.After stopping treatment, do not have tumor recurrence each comfortable 4.3 and 5.3 middle of the month in two experiments.For the HCT-116 experiment, under 20mg/kg, compare Taxol and Fu Dilong, the both makes the group of tumor disappearance Taxol treatment end recurrence in back 1.1 months in treatment, however the animal tumor that Fu Dilong treats disappeared above 5.3 months.

These results relate to research especially long and that run through whole treatment, and described research is adopted xenograft and remarkably reported with the medication of single antitumor agent parenteral or oral medication and the disease that disappears fully for a long time.

Experimental example:

General pharmacological method:

Tumor and cell line.CCRF-CEM people's lymphoblastic leukemia cell and the chemical sproof subbreed (CCRF-CEM/VBL of its vinblastine ₁₀₀, 720 times of drug resistance) derive from Chicago, William doctor Beck of Illinois university, and CCRF-CEM/ Taxol (external 44 times of drug resistance) by six middle of the month with the CCRF-CEM cellular exposure in ever-increasing to lethal concentration (IC ₅₀-IC ₉₀) paclitaxel in and prepare.People's breast carcinoma (MX-1), human lung carcinoma cell (A549) and human colon carcinoma (HCT-116) cell derive from American type culture collection (ATCC, Rockville, MD).

Animal.The nude mouse that has the nu/nu gene derives from NCI, Frederick, and MD also is used for the xenotransplantation of the somebody of institute tumor.Except pointing out female nude mice in addition, use above body weight 20-22g of 6 weeks or heavier male nude mouse.Use homemade microtubule and the limiter inculcated by perfusion administration in 6 hours in the tail cava vein.Have multichannel program-controlled HarvardPHD2000 syringe pump and be used to the intravenous perfusion.Inculcated volume in typical 6 hours every kind of medicine in can sharp good fortune/ethanol (1: 1) is contained 100 μ l in for 2.0ml saline.For oral administration, Fu Dilong and Taxol all are dissolved in the ethanol and with tween 80 and dilute 5 times.Taxol solution should use in 5min to avoid precipitation.Gross tumor volume is measured long * wide * high (or wide) and is estimated by caliper.For the nude mice that has tumor, its body weight is meant that gross weight deducts the weight of tumor in the experimentation.All zooperies are all implemented according to the guideline of National Institute of HealthGuide for the Care and Use of Animals with by the rules of MemorialSloan-Kettering Cancer Center ' s Institutional Animal Care and UseCommittee approval.

Cell toxicity test.For preparing the vitro cytotoxicity test, cell culture is 2-5 * 10 in initial density ⁴Every milliliter in individual cell.They are stored in 5%CO ₂In 37 ℃ the RPMI medium 1640 (GIBCO/BRL) of-moistening atmosphere, described RPMI medium 1640 comprise penicillin (100 units/mL), streptomycin (and 100 μ g/mL, GIBCO/BRL) and 5% heat-inactivated FBS.For the solid tumor cell of in monolayer, growing (for example HCT-116 and A549), the cytotoxicity of medicine is by using sulphonyl rhodamine B method (people J.Natl.Cancer Inst. (1990) 82 such as Skehan, 1107-1112 incorporates the present invention by reference into) on the microtitration plate of 96-hole, measure.For the cell (for example CCRF-CEM and subbreed thereof) that grows in the suspension, by using dimethoxy azoles sulphur (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide)-2H-terazodium hydroxide (XTT)) microtubule method (people Cancer Res. (1988) 48,4827 such as Scudiero; Incorporate the present invention by reference into) on 96 hole microtitration plates, measure in duplicate.For two kinds of methods, (Winooski VT) measures the absorbance in each hole for PowerWave XS, Bio-Tek with the microtitration plate plate reading machine.The relation data of dosage-effect is by 6 to 7 concentration of every kind of medicine, and is duplicate, by the program of using a computer (Chou, T.-C.﹠amp; Talalay, P.T.Adv.Enzyme Reg.1984,22,27; Chou, T.-C.﹠amp; Hayball, M.CalcuSyn for Windows (Biosoft, Cambridge, U.K.) (1997); Wherein every piece of document is incorporated the present invention by reference into) analyze with middle effect figure.

General chemical method: use the reagent that derives from commercial suppliers to be further purified, except as otherwise noted.Following solvents is obtained by dry solvent system (by the alumina column of pre-filling), and need not further dry and be used: oxolane, dichloromethane, ether, benzene and toluene.From calcium hydride, distill triethylamine, N, N-diisopropylethylamine, diisopropylamine, pyridine and 2,6-lutidines.All to the reaction of air and water sensitive all in flame-dried glass drying oven under the positive pressure of ultrapure nitrogen or argon and carry out.NMR ( ¹H and ¹³C) spectrum on Bruker AMX-400MHz or Bruker Advance DRX-500MHz as respectively the explanation record, with reference to CDCl ₃(for ¹H 7.27ppm and for ¹³C 77.1ppm).Infrared spectrum (IR) obtains on Perkin-Elmer FT-IR model 1600 spectrogrphs.Optical rotation obtains in the polarimeter of JASCO model DIP-370 numeral.Low resolution (electrojet) mass spectrum record on PESCIEX API 100.Analyzing thin layer chromatography carries out on E.Merck silica gel 60F254 plate.To ultraviolet do not have active chemical compound by silica gel plate being impregnated in alcoholic acid P-methoxybenzal-dehyde solution, alcoholic acid phosphomolybdic acid or the cerous molybdate ammonia or/and heating develop.The preparation of lamina chromatograph is at Whatman ^Use specified solvent on (LK6F silica gel 60A) TLC plate and carry out.Silica gel chromatography is at Davisil ^Use specified solvent to carry out on (1740 grades, 60A type, 170-400 order) silica gel.

Chemical shift is with the δZhi report with respect to chloroform (NMR is for proton δ 7.24 with for carbon δ 77.0).

At room temperature to 4,4, the 4-trifluoroacetic ethyl acetoacetate (24.0mL, (3: 1=V: V adds allyl bromide, bromoallylene (20.0mL to THF-water 0.164mol) in solution 320mL), 1.4 equivalent), add indium (powder ,-100 orders, 25g subsequently, 1.3 equivalent), and with resulting mixture stir 15h down at 48 ℃.This reactant mixture is cooled to room temperature,, and uses CH with HCl aqueous solution (400mL) cancellation of 2N ₂Cl ₂(400mL, 2 * 200mL) extractions.With the organic facies drying (MgSO that merges ₄), filter, and under vacuum, concentrate.The alcohol that flash chromatography (hexane → hexane-ether 10: 1 → 8: 1 → 6: 1 → 4: 1) provides is clarification grease (31.64g, 85% productive rate): 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, CDCl ₃) δ 5.82 (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, CDCl ₃) 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 spec m/z 227.0895[(M+H) ⁺C ₉H ₁₄O ₃F ₃Value of calculation: 227.0895].

This alcohol is volatile.After column chromatography, should thoroughly not concentrate by alcohol.Product that productive rate obtains by gross weight with by the NMR integration and the ratio between solvent are determined.

With alcohol (16.71g, 0.07386mol) and the mixture of pyridine (15.0mL, 2.5 equivalents) be cooled to-10 ℃ and in 11min, slowly handle (yellow mercury oxide) with thionyl chloride (11.3mL, 2.1 equivalents).The mixture of gained is warmed to 55 ℃ (heating the complex mixture that 17h obtains chlorizate down at 75 ℃) and stirs 12h.Reactant mixture is cooled to-5 ℃, water (200mL) cancellation is also used CH ₂Cl ₂(2 * 200mL, 2 * 150mL) extractions.The saturated NaHCO of organic facies that merges ₃(2 * 200mL) and saline (200mL) washing, dry (MgSO ₄) and under vacuum, concentrate.(pentane: ether 15: 1) ester that provides (11.90g, 77% productive rate) is yellow oil: IR (film) 2986 (w) to flash chromatography, 1731 (s), 1308 (s), 1265 (w), 1227 (m), 1197 (s), 1133 (s), 1025 (m), 920 (w), 896 (w) cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 6.36 (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, J=7.1Hz, 3H); ¹³C NMR (100MHz, CDCl ₃) δ 164.22,143.37 (q, J=29Hz), 132.71,123.21 (q, J=274Hz), 122.60 (q, J=6Hz), 117.32,60.85,30.54,13.85; High resolution mass spec m/z 209.0788[(M+H) ⁺C ₉H ₁₂O ₂F ₃Value of calculation: 227.0789].

This ester is volatile.Through after the column chromatography, this ester is not thoroughly concentrated.

To refrigerative (75 ℃) described ester (7.12g, CH 0.0342mol) ₂Cl ₂(120mL) in the solution, in 35min, add the CH of DIBAL-H (75mL, 2.2 equivalents) ₂Cl ₂Solution (1.0M) is warmed to room temperature with the solution of gained in 3h.Reactant mixture is cooled to 0 ℃, uses saturated NH ₄Cl (12mL) cancellation and at stirring at room 20min.Reactant mixture is diluted dry (MgSO with ether (200mL) ₄) and vacuum concentration.(pentane: ether 3: 1 → 1: 1) alcohol that provides (5.68g, 99%) is clarification grease: IR (film) 3331 (br s), 2929 (m) to flash chromatography, 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, CDCl ₃) δ 6.33 (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, CDCl ₃) δ 134.45 (q, J=6Hz), 133.38,127.97 (q, J=29Hz), 123.76 (q, J=271Hz), 116.25,57.87,29.79.

Alcohol 56 is volatile.Through after the column chromatography, thoroughly do not concentrate 56.

With refrigerative (0 ℃) alcohol (5.97g, CH 0.0358mol) ₂Cl ₂(50mL) solution PPh ₃(11.17g, 1.2 equivalents), imidazoles (3.55g, 1.5 equivalents) and I ₂(9.10g, 1.1 equivalents) are handled and (are added I at last ₂), (yellow muddy) mixture of gained is stirred 10min down at 0 ℃.The saturated Na of this reactant mixture ₂S ₂O ₃Saturated NaHCO ₃(1: 1=V: V, 200mL) solution cancellation and with pentane extraction (3 * 200mL).The saturated Na of organic facies that merges ₂S ₂O ₃Saturated NaHCO ₃(1: 1=V: V, 200mL) solution and saline (100mL) washing, dry (MgSO ₄) and under vacuum, concentrate.The iodide that flash chromatography (pentane) provides (6.69g, 68%) are pale red grease (preserving in-78 ℃ of household freezers): IR (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, CDCl ₃) δ 6.45 (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, J=6.1,1.4Hz, 2H); ¹³C NMR (100MHz, CDCl ₃) 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 spectrum m/z 298.7[(M+Na) ⁺C ₇H ₈F ₃INa value of calculation: 299.0].

This allyl iodide is volatile.Through after the column chromatography, this allyl iodide is not thoroughly concentrated.

α-hydroxyoxazolidinones.4-benzyl-3-hydroxyacetyl-oxazolidines-2-ketone (16.28g to refrigerative (78 ℃) TES protection; 1.92 in THF equivalent) (160mL) solution; in 51min, drip LHMDS (42mL; 1.73 THF solution (1.0M) equivalent) stirs 35min with resulting mixture down at-78 ℃.(6.69g, THF 24.2mmol) (10mL) solution-treated slowly are warmed to ambient temperature overnight with the mixture of gained to this reactant mixture with allyl iodide in 15min.The saturated NaHCO of this reactant mixture ₃(200mL) cancellation and with EtOAc extraction (3 * 200mL).The saturated NH of organic facies that merges ₄Cl (150mL), saline (150mL) washing, dry (MgSO ₄) and under vacuum, concentrate.Flash chromatography (hexane-EtOAc 6: 1 → 3: 1) provides alkylate mixture (13.6g), and it is used for the next step and needn't be further purified (not separating diastereomer in this stage).With the HOAc-water-THF solution of described alkylate (3: 1: 1=V: V: V, 200mL) at room temperature stir 4h.The reactant mixture vacuum concentration to remove HOAc, is used saturated NaHCO ₃(400mL) cancellation and with EtOAc extraction (3 * 200mL).With the organic facies drying (MgSO that merges ₄) and under vacuum, concentrate.Flash chromatography (hexane: EtOAc3: 1 → 2: 1) provide α-hydroxyoxazolidinones (7.55g, the productive rate in two steps is 81%) and be clarification grease: [α] _D ²⁰(25 ℃)-48.2 ° (c 1.08, CHCl ₃); IR (film) 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, CDCl ₃) δ 7.32 (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, CDCl ₃) 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 spec m/z 384.1421[(M+H) ⁺C ₁₉H ₂₁NO ₄F ₃Value of calculation: 384.1423].

The Alpha-hydroxy amide.(MeO) THF (100mL) suspension of NHMe.HCl (10.1g, 5.25 equivalents) is used AlMe under 0 ℃ ₃(50mL, handle 5.1 toluene solution equivalent) (2.0M) drips, and resulting settled solution is at room temperature stirred 34min, slowly join refrigerative (0 ℃) α-hydroxyoxazolidinones (7.55g then, 19.7mmol) THF (70mL) solution in (muddiness → clarification, faint yellow).Resulting mixture is warmed to room temperature and stirs 12h.Reactant mixture is cooled to 0 ℃,, stirs 25min under the room temperature, with EtOAc extraction (3 * 200mL) by aqueous tartaric acid solution (100mL) cancellation of slow adding 1N.With the organic facies drying (MgSO that merges ₄) and under vacuum, concentrate.The Alpha-hydroxy amide (5.12g, productive rate 97%) that flash chromatography (hexane: EtOAc 2: 1 → 1: 1) provides is clarification grease: [α] _D ²⁰(24 ℃)-57.2 ° (c 1.03, CHCl ₃); IR (film) 3432 (br s), 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, CDCl ₃) δ 6.32 (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, CDCl ₃) δ 173.43,133.68,130.59 (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 spec m/z 268.1161[(M+H) ⁺C ₁₁H ₁₇NO ₃F ₃Value of calculation: 268.1161].

Alpha-alcohol ketone.(4.87g adds the diethyl ether solution (3.0M) of MeMgBr (75mL, 12 equivalents) in THF 18.2mmol) (150mL) solution to refrigerative (0 ℃) Alpha-hydroxy amide.Behind the 5min, with the saturated NH of this reactant mixture ₄Cl (250mL) cancellation and with EtOAc extraction (5 * 200mL).With the organic facies drying (MgSO that merges ₄) and under vacuum, concentrate.The alpha-alcohol ketone that flash chromatography (hexane: EtOAc 4: 1 → 2: 1 → 1: 2) provides (2.16g, productive rate 53% are 73% based on the raw material meter productive rate that reclaims) is clarification grease, and provides raw material Alpha-hydroxy amide (1.30g, yield are 27%): [α] _D ²⁰(23 ℃)+58.5 ° (c 1.30, CHCl ₃); IR (film) 3460 (br s), 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, CDCl ₃) δ 6.21 (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, CDCl ₃) 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 spec m/z 223.0945[(M+H) ⁺C ₁₀H ₁₄NO ₂F ₃Value of calculation: 223.0946].

Even this reaction uses excessive MeMgBr can not carry out fully.

Carbonic acid 1-(2 benzyloxies-1-Methylethyl)-5,5-diisopropoxy-2,4,4-trimethyl-3-oxo pentyl ester 2,2,2-three chloro-ethyl esters.7-benzyloxy-5-hydroxyl-1 under 0 ℃, 1-diisopropoxy-2,2,4,6-tetramethyl-heptan-3-ketone (1.0g, 2.4mmol) and pyridine (0.8mL, CH 7.3mmol) ₂Cl ₂(10.0mL) add chloro-carbonic acid 2,2 in the solution, (668.0 μ L, 4.9mmol), the mixture with gained is warmed to room temperature to the 2-trichloro ethyl ester then.Behind the 1h, with reactant mixture with the saline cancellation and use CH ₂Cl ₂Extraction.With the organic layer that merges through MgSO ₄Dry and under reduced pressure concentrated.Crude product provides the product of wanting (1.285g, 92%) and is clarification grease by flash chromatography (gradient hexane to hexane/EtOAc 93: 7) purification: ¹H NMR (400MHz, CDCl ₃) 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=6.0Hz, 1H), and 7.29-7.35 (m, 5H); ¹³C NMR (100MHz, CDCl ₃) δ 11.9,15.0,18.8,21.4,21.7,22.3,23.2,23.4,35.7,42.5,53.4,53.9,69.4,70.9,71.4,73.3,81.3,94.7,103.4,127.5,127.6,128.2,138.2,154.0,215.6; IR (film, NaCl, cm ^-1) 2966,1760,1698,1247; LRMS (ESI) C ₂₇H ₄₁O ₇Cl ₃Na[M+Na ⁺] value of calculation 605.2, experiment value 605.2; [α] ²³ _D=-20.4 (c=1.0, CHCl ₃).

Carbonic acid 1-(2 benzyloxies-1-Methylethyl)-2,4,4-trimethyl-3,5-dioxo pentyl ester 2,2,2-three chloro-ethyl esters.To raw material (1.28g, 4: 1 THF/H 2.25mmol) ₂Add in O (25mL) solution p-TsOH (111.0mg, 0.6mmol).Behind heating 5h under 70 ℃, (0 ℃) saturated NaHCO that the reactant mixture impouring is cold ₃(12mL) extracts with EtOAc then in the aqueous solution.With the organic layer salt water washing that merges, through MgSO ₄Dry and under reduced pressure concentrated.Crude product is clarification grease through the product (793.2mg, 76%) that flash chromatography (gradient hexane to hexane/EtOAc 84: 16) purification provides: ¹H NMR (400MHz, CDCl ₃) δ 0.90 (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, 1H); ¹³CNMR (100MHz, CDCl ₃)-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; LRMS (ESI) C ₂₁H ₂₇O ₆Cl ₃Na[M+Na ⁺] value of calculation 503.0, experiment value 503.0; [α] ²³ _D=-18.5 (c=0.8, CHCl ₃).

9-benzyloxy-4,4,6,8-tetramethyl-3,5-dioxo-7-(2,2,2-trichlorine ethoxy carbonyl oxygen the base)-n-nonanoic acid tert-butyl ester.(1.17mmol is at Et to LDA under-78 ℃ ₂0.3M among the O) adds tert-butyl acetate (1.0mmol, 135.0 μ l) in the solution.Behind the 30min, in 15min, slowly add raw material (464.0mg, Et 1mmol) ₂O (2mL) solution.After stirring 1h, use saturated NH ₄Cl aqueous solution cancellation reaction also extracts with EtOAc then.The organic layer salt water washing that merges is through MgSO ₄Dry and under reduced pressure concentrated.Crude product is clarification grease with the product (1: 1 epimer mixture 461.4mg, 80%) that flash chromatography (gradient hexane to hexane/EtOAc 86: 14) purification provides: ¹H NMR (400MHz, CDCl3) δ 0.87 (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), and 2.27-2.31 (m, 2H), 3.22-3.27 (m, 3H), 3.39-3.48 (m, 5H), and 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, CDCl ₃) δ 11.9,12.7,14.9,15.2,18.7,19.3,21.4,21.6,28.0,35.6,37.4,41.7,42.0,51.8,51.9,71.3,71.3,72.5,73.0,73.3,73.3,80.6,81.2,81.3,94.6,127.5,127.7,127.8,128.3,138.0,138.1,154.0,154.1,172.3,172.4,216.0,216.3; IR (film, NaCl, cm ^-1) 3509,2975,1759,1707,1368,1248,1152; LRMS (ESI) C ₂₇H ₃₉O ₈Cl ₃Na[M+Na ⁺] value of calculation 619.1, experiment value 619.2.

To raw material (350.0mg, CH 0.6mmol) ₂Cl ₂Add in 0 ℃ of solution (10mL) Dess-Martin cross iodine alkane (periodinane) (398.0mg, 0.9mmol).This mixture is at room temperature stirred 1h be poured into sufficient 1: the 1 saturated Na of stirring then ₂S ₂O ₃/ saturated NaHCO ₃In the mixture.Layering behind the 30min.Water layer Et ₂O extraction three times.Merge organic extract, use saturated NaHCO ₃, the salt water washing, through MgSO ₄Dry and concentrated under vacuum.Crude product is clarification grease with the product (258.4mg, 74%) that flash chromatography (gradient hexane to hexane/EtOAc 91: 9) purification provides: ¹H NMR (400MHz, CDCl ₃) 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); ¹³CNMR (100MHz, CDCl ₃) 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; LRMS (ESI) C ₂₇H ₃₇O ₈Cl ₃Na[M+Na ⁺] value of calculation 617.1, experiment value 617.1; [α] ²³ _D=-49.1 (c=0.9, CHCl ₃).

9-benzyloxy-3-hydroxyl-4,4,6,8-tetramethyl-5-oxo-7-(2,2,2-trichlorine ethoxy carbonyl oxygen the base)-n-nonanoic acid tert-butyl ester.With (R)-RuBINAP catalyst (16.8mg, 10.0pmol) filing of containers lining (bomb liner).Add HCl (555 μ L, 0.2N in methanol), and with the ultrasonic 15sec of this mixture.(59.4mg, methanol 0.1mmol) (555 μ L) solution add and this mixture are transferred in the Parr device with raw material then.With this container H ₂Clean 5min, be forced into 1200psi then.Behind the 17h, reaction is got back under the atmospheric pressure and with the saturated NaHCO of its impouring ₃In the aqueous solution.Water layer extracts three times with EtOAc.The organic extract that merges is through MgSO ₄Dry and under reduced pressure concentrated.Crude product is clarification grease with the product (47.6mg, 80%) that flash chromatography (gradient hexane to hexane/EtOAc 88: 12) purification provides: ¹H NMR (400MHz, CDCl ₃) 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), 7.27-7.35 (m, 5H); ¹³C NMR (100MHz, CDCl ₃) 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; LRMS (ESI) C ₂₇H ₃₉O ₈Cl ₃Na[M+Na ₊] value of calculation 619.1, experiment value 619.2; [α] ²³ _D=-13.0 (c=0.4, CHCl ₃).

9-benzyloxy-4,4,6,8-tetramethyl-5-oxo-7-(2,2,2-trichlorine ethoxy carbonyl oxygen base)-3-(triethyl silicane oxygen base)-n-nonanoic acid tert-butyl ester.Under 0 ℃, in DMF (0.4mL) solution of raw material (37.6mg, 6.3 μ mol) and imidazoles (9.4mg, 13.8 μ mol), add TESCl (11.6 μ l, 69.3 μ mol).Behind the 3h, use saturated NaHCO ₃Aqueous solution dilutes this mixture.Water layer hexane extraction three times.The organic extract salt water washing that merges is through MgSO ₄Dry and under reduced pressure concentrated.Crude product is by flash chromatography (gradient hexane to hexane/EtOAc 93: 7) purification, with eluting order obtain product (22.9mg, 51%) and the raw material (12.9mg, 34%) that reclaims for clarifying grease: ¹H NMR (400MHz, CDCl ₃) 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 and 6.5Hz, 1H), 4.45 (s, 2H), 4.65 (d, J=11.9Hz, 1H), 4.79 (d, J=11.9Hz, 1H), 4.97 (dd, J=3.7 and 8.1Hz, 1H), 7.29-7.52 (m, 5H); ¹³C NMR (125MHz, CDCl ₃) 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; LRMS (ESI) C ₃₃H ₅₃O ₈SiCl ₃Na[M+Na ⁺] value of calculation 733.2, experiment value 733.3.[α] ²³ _D＝-16.1(c＝0.1，CHCl ₃)。

9-benzyloxy-3-(diethylmethyl silicon alkoxyl)-7-hydroxyl-4,4,6,8-tetramethyl-5-oxo-n-nonanoic acid tert-butyl ester.To 1 of raw material (22.9mg, 3.2 μ mol): add Zn (5.0mg, 7.8 μ mol, nanoscale) in 1THF/AcOH (1.4mL) solution.With the ultrasonic 15min of this mixture.Add more Zn (5.0mg, 7.8 μ mol, nanoscale), ultrasonic more subsequently 15min.This suspension filters by the celite pad, with the EtOAc washing for several times.The saturated NaHCO of filter liquor ₃, the salt water washing, through MgSO ₄Dry and concentrated under vacuum.By the short plug of silica gel, with 4: 1 eluting of hexane/EtOAc, the product that provides 17.1mg (99% yield) is a colorless oil with thick residue: ¹HNMR (400MHz, CDCl ₃) δ (m, 6H), 0.96 (t, J=7.9Hz, 9H), 0.97 (d, J=6.8Hz, 3H), 1.05 (d, J=6.8Hz, 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, CDCl ₃) 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; LRMS (ESI) C ₃₀H ₅₂O ₆SiCl ₃Na[M+Na ⁺] value of calculation 559.3, experiment value 559.3; [α] ²³ _D=-41.0 (c=0.4, CHCl ₃).

9-benzyloxy-7-(tert-butyl group dimethyl-silicon alcoxyl base)-3-(diethylmethyl silicon alkoxyl)-4,4,6,8-tetramethyl-5-oxo-n-nonanoic acid tert-butyl ester.Under-78 ℃ to raw material (4.1mg, 7.6 μ mol) and 2,6-lutidines (10.0 μ l, CH 43.5mmol) ₂Cl ₂(0.2mL) add in the solution TBSOTf (10.0 μ l, 85.8mmol).Behind the 2h, add more 2, the 6-lutidines (10.0 μ l, 43.5mmol) and TBSOTf (10.0 μ l, 85.8mmol).Behind the 6h with the saturated NaHCO of this mixture ₃The aqueous solution dilution.Water layer extracts three times with EtOAc.With the organic extract salt water washing that merges, through MgSO ₄Dry and under reduced pressure concentrated.Crude product is clarification grease with the product (5.4mg, 82%) that flash chromatography (gradient hexane to hexane/EtOAc 91: 9) purification provides.Spectroscopic data accords with the value of having reported well.

With acid and alcohol and anhydrous benzene (5mL * 2) azeotropic and dry under fine vacuum before reaction.Under 0 ℃ to alcohol (639mg, CH 2.63mmol) ₂Cl ₂(13mL) add in the solution EDCI (576mg, 3.09mmol) and DMAP (366mg, 3.09mmol).At the CH that in this mixture, drips acid (1.11g is equivalent to 1.88mmol) under 0 ℃ ₂C1 ₂(5mL+2mL flushing) is more than the solution 16min.After stirring 1.5h under 0 ℃, this mixture is at room temperature stirred 3.5h.After reactant mixture concentrated, residue was by hurried column chromatography (SiO ₂, hexane/EtOAc=30: 1 to 20: 1) and the ester (1.20g, 1.61mmol is by tert-butyl ester meter 86%) that provides of purification is colorless oil.

[α] _D ²⁴-25.1 (c 1.30, CHCl ₃); IR (film) ν 2955,2925,2872,1732,1696,1461,1378,1290,1243,1173,1091,985,873,773cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 0.06 (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), and 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, CDCl ₃) 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; LRMS (ESI) C ₄₁H ₇₁NO ₅SSi ₂Na[M+Na ⁺] value of calculation 768.5, experiment value 768.5; HRMSC ₄₁H ₇₂NO ₅SSi ₂[M+H ⁺] value of calculation 746.4670, experiment value 746.4680.

Toluene (70mL) solution of diene (26.9mg, 36.1 μ mol) is heated to refluxes and with toluene (2mL) solution-treated of Grubbs catalyst (3.1mg, 3.61 μ mol).This mixture is stirred 25min, be cooled to 0 ℃, filter by using hexane/EtOAc=2/1 washed silica gel pad.The filtrate that merges concentrated and by hurried column chromatography (SiO ₂, hexane/Et ₂O=40: 1 to 5: 1) purification provides product (9.9mg, 13.8 μ mol, 38%) and the cycloheptadiene (14.4mg, 22.3 μ mol, 62%) wanted and is colorless oil.

[α] _D ²⁵-41.5 (c 0.715, CHCl ₃); IR (film) ν 2955,2884,1737,1690,1467,1378,1249,1179,1102,1014,979,879,826,773cm ^-1 ¹HNMR (400MHz, CDCl ₃) 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), 2.72 (3H, s), 3.02 (1H, quintet, J=7.0Hz), 3.10 (1H, dd, J=14.4,7.3Hz), 3.96 (1H, d, J=8.7Hz), 4.43 (1H, dd, J=8.3,2.9Hz), 5.22 (1H, dd, J=9.8,5.7Hz), 5.33-5.42 (2H, m), 5.69 (1H, dd, J=15.8,8.2Hz), 6.57 (1H, s), 6.96 (1H, s); ¹³C NMR (100MHz, CDCl ₃) 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; LRMS (ESI) C ₃₉H ₆₈NO ₅SSi ₂[M+H ⁺] value of calculation 718.4, experiment value 718.3; HRMSC ₃₉H ₆₈NO ₅SSi ₂[M+H ⁺] 718.4357, experiment value 718.4355.

[α] _D ²⁶-38.5 (c 0.400, CHCl ₃); IR (film) ν 2955,2878,1741,1693,1472,1458,1385,1295,1253,1169,1098,988,871,837,775cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.07 (6H, s), 0.61-0.68 (6H, m), 0.93 (9H, s), 0.97 (9H, t, J=8.0Hz), 1.03 (3H, d, J=7.0Hz), 1.04 (3H, d, J=7.0Hz), 1.10 (3H, s), 1.21 (3H, s), 1.65 (3H, s), 1.75 (3H, s), 2.06-2.14 (1H, m), 2.31 (1H, dd, J=17.2,7.2Hz), 2.34-2.51 (2H, m), 2.49 (1H, dd, J=17.1,2.8Hz), 2.65-2.81 (2H, m), 3.07 (1H, quintet, J=7.0Hz), 3.84 (1H, dd, J=7.2,2.1Hz), 4.40 (1H, dd, J=7.2,2.8Hz), 4.98-5.09 (2H, m), 5.38-5.42 (1H, m), 5.65 (1H, t, J=5.9Hz), 5.93 (1H, ddd, J=17.9,10.1,7.8Hz); ¹³C NMR (100MHz, CDCl ₃) δ-3.6 ,-3.3,5.4 (3C), 7.3 (3C), 15.3,18.7,19.0,20.0,22.1,23.8,25.8,26.4 (3C), 31.3,32.3,40.0,43.8,46.3,54.0,72.5,73.8,76.5,115.6,119.8,125.6,136.5,140.1,140.6,171.9,218.5; LRMS (ESI) C ₃₅H ₆₄O ₅Si ₂Na[M+Na ⁺] value of calculation 643.4, experiment value 643.3; HRMSC ₃₅H ₆₄O ₅Si ₂Na[M+Na ⁺] value of calculation 643.4190, experiment value 643.4219.

React through Wittig: under 0 ℃, in THF (0.4mL) solution of Wittig reagent (19.1mg, 54.7 μ mol), add KHMDS (toluene solution of 109 μ l 0.5M, 54.7 μ mol).This mixture is stirred 0.5h down at 0 ℃ be cooled to-78 ℃ then.In this mixture, drip THF (0.3mL) solution of ketone (5.7mg, 9.12 μ mol), the mixture that obtains is warmed to-20 ℃ at 1.5h.Use saturated NH ₄Cl aqueous solution (2mL) cancellation should be reacted and be extracted (7mL * 3) with EtOAc.The organic layer that merges is through Na ₂SO ₄Dry and concentrated.Residue is by hurried column chromatography (SiO ₂, hexane/Et ₂O=10: 1) purification provides the inseparable alkene mixture (E/Z=9: 1) of 5.6mg.This mixture is passed through preparation TLC (hexane/Et ₂O=4: 1) to provide the pure isomer of wanting (5.0mg, 6.96 μ mol, 76%) be colorless oil to purification.

¹H NMR (400MHz, CDCl ₃) δ 0.08 (3H, s), 0.12 (3H, s), 0.51 (6H, q, J=7.9Hz), 0.86 (9H, t, J=7.9Hz), 0.97 (9H, s), 1.01 (3H, s), 1.06 (3H, d, J=7.1Hz), 1.12 (3H, s), 1.18 (3H, d, J=7.1Hz), 1.69 (3H, s), 1.97 (3H, s), 2.10-2.18 (1H, m), 2.24-2.31 (1H, m), 2.38-2.59 (3H, m), 2.68-2.78 (1H, m), 2.72 (3H, s), 2.98-3.14 (2H, m), 3.97 (1H, d, J=9.0Hz), 4.45-4.48 (1H, m), 5.29-5.41 (2H, m), 5.73 (1H, dd, J=15.6,8.3Hz), 6.30 (1H, s), 6.73 (1H, d, J=8.7Hz), 6.56 (1H, s); LRMS (ESI) C ₃₉H ₆₈NO ₅SSi ₂[M+H ⁺] value of calculation 718.4, experiment value 718.1.

(298.8mg adds HF pyridine (3.2mL) in THF 0.416mmol) (6.5mL) solution, this mixture is at room temperature stirred 3h to the silyl ether in plastic tube under 0 ℃.Drip TMSOMe (30mL) cancellation reaction down at 0 ℃, this mixture is at room temperature stirred 3h.It is dry under fine vacuum to concentrate the back, and residue is with hurried column chromatography (SiO ₂, hexane/Et ₂O=1: 1) to provide alcohol (196.6mg, 0.402mmol, 97%) be colorless solid to purification.

[α] _D ²⁵-96.6 (c 0.235, CHCl ₃); IR (film) ν 3502,2970,2927,1733,1685,1506,1456,1375,1251,1152,1040,977cm ^-1 ¹H NMR (400MHz, CDCl ₃) 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, quintet, J=6.2Hz), 3.76 (1H, dd, J=5.9,3.2Hz), 4.30 (1H, dd, J=9.2,3.7Hz), 5.18 (1H, brt, J=7.3Hz), 5.32 (1H, dd, J=8.4,2.5Hz), 5.63 (1H, dd, J=15.7,6.4Hz), 5.60 (1H, ddd, J=15.7,6.9,5.1Hz), 6.60 (1H, s), 6.98 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 15.1,16.0,17.7,19.2,19.5,22.5,23.6,32.0,35.0,39.6,40.3,44.8,53.3,71.8,75.6,78.3,116.1,119.6,120.5,129.9,131.3,137.5,138.2,152.2,165.0,170.7,218.8; LRMS (ESI) C ₂₇H ₄₀NO ₅S[M+H ⁺] value of calculation 490.3, experiment value 490.2; HRMS C ₂₇H ₄₀NO ₅S[M+H ⁺] value of calculation 490.2627, experiment value 490.2602.

Under 50 ℃ to alkene (1.2mg, 2.5 μ mol) and TrisNHNH ₂The ClCH of (29.3mg, 98 μ mol) ₂CH ₂Add Et in Cl (0.7mL) solution ₃N (13.7 μ L, 98 μ mol).Reaction is by HPTLC monitoring (hexane/EtOAc/CH ₂Cl ₂=1/1/2).After stirring 7h, this mixture is cooled to room temperature, filters with the EtOAc dilution and by the silicagel pad of washing with EtOAc.After concentrating, residue is by preparation TLC (hexane/EtOAc/CH ₂Cl ₂=1/1/2) to provide reductive product (1.1mg, 2.2 μ mol, 91%) be white solid to purification.The spectroscopic data of this chemical compound is identical with the spectrum of the dEpoB that has reported.

With acid and alcohol and anhydrous benzene (5mL * 2) azeotropic and dry under fine vacuum before reaction.Under 0 ℃ to alcohol (10: 1 mixture of isomers, 240mg, CH 0.756mmol) ₂Cl ₂(5mL) add in the solution EDCI (192.7mg, 1.01mmol) and DMAP (122.8mg, 1.01mmol).Under 0 ℃, in 15min, in mixture, drip acid (314.6mg, CH 0.628mmol) ₂Cl ₂(2mL+1mL flushing) solution.After stirring 2h under 0 ℃, this mixture is at room temperature stirred 2h.After concentrating, carefully with residue with hurried column chromatography (SiO ₂, hexane/EtOAc=20: 1 to 15: 1) and to give ester output (340.1mg, 0.425mmol is based on acid meter 68%) be colorless oil to purification.

[α] _D ²⁴-27.5 (c 0.28, CHCl ₃); IR (film) ν 2956,2878,1740,1692,1472,1378,1317,1253,1174,1118,988,915,872,837,775cm ^-1 ¹HNMR (400MHz, CDCl ₃) 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, quintet, J=7.1Hz), 3.83 (1H, dd, J=7.3,2.1Hz), 4.35 (1H, dd, J=7.2,3.0Hz), 4.98-5.12 (4H, m), 5.30 (1H, t, J=6.7Hz), 5.76 (1H, ddt, J=16.7,10.2,6.2Hz), 5.92 (1H, ddd, J=17.8,9.9,7.8Hz), 6.19 (1H, t, J=7.0Hz), 6.51 (1H, s), 6.97 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ-3.7 ,-3.4,5.2 (3C), 7.1 (3C), 14.7,15.2,18.6,18.9,19.3,19.9,23.8,26.3 (3C), and 30.1,31.2,40.0,43.7,46.3,53.3,73.9,76.5,77.9,115.5,116.5,117.0,121.5,124.1[q, ¹J (C, F)=273.4Hz], 129.6[q, ²J (C, F)=28.5Hz], 130.5[q, ³J (C, F)=6.1Hz], 133.6,136.3,140.1,152.4,164.8,171.3,218.3; LRMS (ESI) C ₄₁H ₆₈F ₃NO ₅SSi ₂Na[M+Na ⁺] value of calculation 822.4, experiment value 822.4; HRMS C ₄₁H ₆₉F ₃NO ₅SSi ₂[M+H ⁺] value of calculation 800.4387, experiment value 800.4374.

Toluene (142mL) solution of diene (57.6mg, 72.0 μ mol) is heated to refluxes and with toluene (2mL) solution-treated of Grubbs catalyst (6.1mg, 7.20 μ mol).This mixture is stirred 28min, be cooled to 0 ℃, filter by the silicagel pad of washing with hexane/EtOAc=2/1 (300mL).The filtrate that merges concentrated and by hurried column chromatography (SiO ₂, hexane/Et ₂O=40: 1 to 15: 2) purification provides product (12.0mg, 15.5 μ mol, 22%) and the cycloheptadiene (29.2mg, 43.3 μ mol, 60%) wanted and is colorless oil.

[α] _D ²⁶-17.1 (c 0.14, CHCl ₃); IR (film) ν 2955,2884,1743,1690,1472,1320,1173,1114,1038,1008,873,832,773cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.09 (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, CDCl ₃) 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), and 29.6,32.8,42.0,42.1,48.2,54.1,73.4,76.9,77.8,117.0,121.6,124.3[q, ¹J (C, F)=273.5Hz], 127.2,130.6[q, ²J (C, F)=28.2Hz], 130.8[q, ³J (C, F)=6.1Hz], 133.2,136.5,152.3,165.0,170.1,217.1; LRMS (ESI) C ₃₉H ₆₅F ₃NO ₅SSi ₂[M+H ⁺] value of calculation 772.4, experiment value 772.4; HRMS C ₃₉H ₆₅F ₃NO ₅SSi ₂[M+H ⁺] value of calculation 772.4074, experiment value 772.4102.

[α] _D ²⁶-35.6 (c 0.365, CHCl ₃); IR (film) ν 2956,2878,1737,1693,1472,1458,1305,1279,1252,1173,1116,988,871,775cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.08 (6H, s), 0.64 (6H, q, J=7.8Hz), 0.93 (9H, s), 0.96 (9H, t, J=7.8Hz), 1.04 (6H, d, J=7.0Hz), 1.10 (3H, s), 1.22 (3H, s), 1.70 (3H, s), and 2.05-2.14 (1H, m), 2.32 (1H, dd, J=17.0,7.1Hz), 2.50 (1H, dd, J=17.0,3.0Hz), 2.51-2.63 (2H, m), 2.87 (1H, dd, J=18.4,6.7Hz), 2.90-3.02 (1H, m), 3.07 (1H, quintet, J=7.1Hz), 3.85 (1H, dd, J=7.2,2.0Hz), 4.39 (1H, dd, J=7.1,2.9Hz), 4.98-5.08 (2H, m), 5.51 (1H, dd, J=8.1,3.8Hz), 5.67 (1H, t, J=5.9Hz), 5.93 (1H, ddd, J=17.8,10.5,7.8Hz), 6.29 (1H, t, J=5.5Hz); ¹³C NMR (100MHz, CDCl ₃) δ-3.7 ,-3.4,5.3 (3C), 7.1 (3C), 15.3,18.7,18.9,19.6,21.3,23.9,24.1,26.3 (3C), and 30.3,40.0,43.7,46.3,53.4,70.9,73.7,76.5,115.5,123.4,123.8[q, ¹J (C, F)=272.2Hz], 129.1[q, ³J (C, F)=6.1Hz], 131.5[q, ²J (C, F)=28.8Hz], 138.1,140.0,171.7,218.5; LRMS (ESI) C ₃₅H ₆₁F ₃O ₅Si ₂Na[M+Na ⁺] value of calculation 697.4, experiment value 697.4; HRMS C ₃₅H ₆₁F ₃O ₅Si ₂Na[M+Na ⁺] value of calculation 697.3907, experiment value 697.3892.

(1.78g slowly adds HF pyridine (12.5mL) in THF 2.31mmol) (25mL) solution, this mixture is at room temperature stirred 4h to the silyl ether in plastic tube under 0 ℃.Under 0 ℃, in 10min, react with dripping TMSOMe (80mL) cancellation.This mixture of vigorous stirring 2.5h at room temperature.Concentrate and behind high vacuum dry 2h, with residue by hurried column chromatography (SiO ₂～50g, hexane/EtOAc=1: 1) purification, providing glycol (1.20g, 2.21mmol, 96%) is colorless solid.

[α] _D ²⁵-54.6 (c 0.28, CHCl ₃); IR (film) ν 3478,2974,2929,1736,1689,1449,1381,1318,1247,1169,1113,1039,983,867,736cm ^-1 ¹HNMR (400MHz, CDCl ₃) 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=16.3,6.5Hz), 3.11 (1H, quintets, J=6.7Hz), 3.19 (1H, brs ,=OH), 3.74 (1H, brs), 4.35 (1H, brd, J=9.5Hz), 5.42 (1H, dd, J=6.2,4.1Hz), 5.60 (1H, ddd, J=15.8,5.6,4.5Hz), 5.66 (1H, dd, J=15.8,5.8Hz), 6.24 (1H, t, J=7.2Hz), 6.64 (1H, s), 7.00 (1H, s); ¹³CNMR (100MHz, CDCl ₃) δ 15.1,16.1,17.7,18.5,19.3,22.5,28.8,31.1,39.6,39.7,45.0,53.7,71.4,75.3,76.8,116.7,120.2,124.3[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; LRMS (ESI) C ₂₇H ₃₇F ₃NO ₅S[M+H ⁺] value of calculation 544.2, experiment value 544.1; HRMS C ₂₇H ₃₇F ₃NO ₅S[M+H ⁺] value of calculation 544.2345, experiment value 544.2346.

Under 50 ℃ to glycol (1.22mg, 2.24 μ mol) and TrisNHNH ₂The ClCH of (26.7mg, 89.6 μ mol) ₂CH ₂Add Et in Cl (1mL) solution ₃N (12.5 μ L, 89.6 μ mol).Reaction is by HPTLC monitoring (hexane/EtOAc/CH ₂Cl ₂=1/1/2).After stirring 6.5h, in mixture, add TrisNHNH again ₂(26.7mg, 89.6 μ mol) and Et ₃N (12.5 μ L, 89.6 μ mol).After stirring 14h, this mixture is cooled to room temperature, filters with the EtOAc dilution and by the silicagel pad of washing with EtOAc.After concentrating, residue is with preparing TLC (hexane/EtOAc/CH ₂Cl ₂=1/1/2) to provide reductive product (1.16mg, 2.13 μ mol, 94%) be white solid to purification.

[α] _D ²⁴-75.1 (c 0.35, CHCl ₃); IR (film) ν 3483,2968,1337,1685,1466,1381,1322,1247,1168,1113,1010,833,736cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 1.03 (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), and 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), and 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); ¹³CNMR (100MHz, CDCl ₃) δ 13.0,15.9,16.0,17.7,19.1,23.0,25.6,26.2,31.3,32.3,37.4,39.8,41.6,53.9,72.3,73.6,77.7,116.2,119.9,124.3[q, ¹J (C, F)=274.4Hz], 129.8[q, ³J (C, F)=6.1Hz], 132.6[q, ²J (C, F)=27.8Hz], 138.3,151.7,165.4,170.2,220.7; LRMS (ESI) C ₂₇H ₃₉F ₃NO ₅S[M+H ⁺] value of calculation 546.3, experiment value 546.2; HRMS C ₂₇H ₃₉F ₃NO ₅S[M+H ⁺] value of calculation 546.2501, experiment value 544.2496.

With acid and alcohol and anhydrous benzene (3mL * 2) azeotropic and dry under fine vacuum before reaction.Under 0 ℃ to alcohol (68.0mg, CH 0.173mmol) ₂Cl ₂(1.3mL) add in the solution EDCI (37.8mg, 0.197mmol) and DMAP (24.1mg, 0.197mmol).Under 0 ℃, in 5min, in mixture, drip acid (72.6mg, CH 0.123mmol) ₂Cl ₂(0.7mL) solution.After stirring 1h under 0 ℃, this mixture is at room temperature stirred 2.5h.After concentrating, with residue with hurried column chromatography (SiO ₂, hexane/EtOAc=30: 1) purification is a colorless oil to ester output (99.5mg, 0.114mmol is by tert-butyl ester meter 92%).

[α] _D ²⁵-23.4 (c 0.56, CHCl ₃); IR (film) ν 2955,2931,2880,1735,1696,1506,1472,1386,1362,1294,1254,1174,1104,988,878,776,742cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 0.06 (3H, s), 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), and 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, J=16.9,2.7Hz), 2.76-2.80 (2H, m), 3.07 (1H, quintet, J=7.0Hz), 3.83 (1H, dd, J=7.0,2.2Hz), 4.35 (1H, dd, J=7.4,2.8Hz), 4.97 (2H, s), 4.97-5.07 (4H, 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, CDCl ₃) 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; LRMS (ESI) C ₄₇H ₈₆NO ₆SSi ₃[M+H ⁺] value of calculation 876.6, experiment value 876.5; HRMS C ₄₇H ₈₆NO ₆SSi ₃[M+H ⁺] value of calculation 876.5484, experiment value 876.5482.

Toluene (158mL) solution of diene (69.7mg, 79.5 μ mol) is heated to refluxes and with toluene (2mL) solution-treated of Grubbs catalyst (6.7mg, 7.95 μ mol).This mixture is stirred 11min, be cooled to 0 ℃, filter by the silicagel pad of washing with hexane/EtOAc=3/1 (280mL).The filtrate that merges concentrated and by hurried column chromatography (SiO ₂, hexane/Et ₂O=20: 1 to 15: 1) purification provides product (18.4mg, 21.7 μ mol, 27%) and the cycloheptadiene (28.3mg, 45.5 μ mol, 57%) wanted and is colorless oil.

[α] _D ²⁴-40.4 (c 0.26, CHCl ₃); IR (film) ν 2955,2930,2879,1740,1694,1472,1387,1362,1253,1200,1107,1007,838,776,742cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.08 (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); ¹³C NMR (125MHz, CDCl ₃) δ-5.2 ,-3.3 ,-3.2,5.6,7.2,7.3,15.0,17.2,18.5,18.8,21.4,23.9,24.4,26.0,26.5,33.3,35.6,41.4,41.8,48.2,54.0,63.5,74.4,78.1,79.3,116.6,120.6,121.0,129.3,132.1,137.8,137.9,153.0,170.7,172.3,216.8; LRMS (ESI) C ₄₅H ₈₂NO ₆SSi ₃[M+H ⁺] value of calculation 848.5, experiment value 848.5; HRMS (ESI) C ₄₅H ₈₂NO ₆SSi ₃[M+H ⁺] value of calculation 848.5171, experiment value 848.516l.

Under 0 ℃, in THF (2mL) solution of the silyl ether in plastic tube (61.8mg, 72.8 μ mol), add HF pyridine (1mL), this mixture is at room temperature stirred 3.2h.Drip TMSOMe (15mL) cancellation reaction down at 0 ℃.At room temperature stir this mixture 2h.Concentrate and after high vacuum dry, with residue by hurried column chromatography (SiO ₂, hexane/EtOAc=1: 3) purification, providing triol (32.4mg, 64.1 μ mol, 88%) is white solid.

[α] _D ²⁵-108.4 (c 0.285, CHCl ₃); IR (film) ν 3422,2968,2919,2729,1689,1449,1377,1252,1152,1064,978cm ^-1 ¹H NMR (400MHz, CDCl ₃) 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, quintet, J=6.6Hz), 3.34 (1H, brs ,-OH), 3.75 (1H, dd, J=6.6,2.4Hz), 4.06 (1H, brs,-OH), 4.33 (1H, dd, J=9.4,3.0Hz), 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); ¹³CNMR (100MHz, CDCl ₃) δ 15.3,16.3,17.8,19.2,22.8,23.7,31.9,35.1,39.7,40.2,45.0,53.4,61.8,71.7,75.8,78.1,116.7,119.0,120.5,130.0,131.2,137.6,138.9,152.5,170.0,170.7,218.7; LRMS (ESI) C ₂₇H ₃₉NO ₆SNa[M+Na ⁺] value of calculation 528.2, experiment value 528.0; HRMS C ₂₇H ₄₀NO ₆S[M+H ⁺] value of calculation 506.2576, experiment value 506.2552.

Will thick acid (4.65g is equivalent to 7.27mmol) and alcohol (2.18g, 9.84mmol) and the anhydrous benzene azeotropic, dry 20min under the fine vacuum before reaction then.Under 0 ℃ to alcohol (2.18g, CH 9.84mmol) ₂Cl ₂(65mL) add in the solution EDCI (2.09g, 10.9mmol) and DMAP (1.33g, 10.9mmol).Under 0 ℃, in 20min, in mixture, drip the CH of thick acid (4.65g is equivalent to 7.27mmol) ₂Cl ₂(20mL+5mL flushing) solution.After stirring 40min under 0 ℃, this mixture is at room temperature stirred 4h.After concentrating, with residue with hurried column chromatography (SiO ₂～160g, hexane/EtOAc=20: 1) purification is a colorless oil to ester output (4.85mg, 6.87mmol is from tert-butyl ester meter 94%).

[α] _D ²⁵-22.7 (c 0.26, CHCl ₃); IR (film) ν 2958,2936,2800,1748,1732,1693,1473,1416,1360,1317,1296,1254,1174,1119,989,916,872,838,776cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 0.08 (3H, s), 0.08 (3H, s), 0.60 (6H, q, J=7.8Hz), 0.93 (9H, s), 0.94 (9H, t, J=8.0Hz), 1.04 (3H, d, J=7.0Hz), 1.04 (3H, d, J=7.0Hz), 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), and 2.90-3.01 (2H, m), 3.06 (1H, quintets, J=7.0Hz), 3.85 (1H, dd, J=7.3,2.0Hz), 4.38 (1H, d, J=7.0,3.4Hz), 4.97-5.14 (5H, m), 5.75 (1H, ddt, J=16.0,9.9,6.2Hz), 5.92 (1H, ddd, J=17.8,10.5,7.8Hz), 6.21 (1H, td, J=7.2,1.5Hz); ¹³C NMR (100MHz, CDCl ₃) δ-3.7 ,-3.4,5.2 (3C), 7.1 (3C), 15.4,18.7,18.9,19.5,23.9,26.3 (3C), and 26.6,28.5,30.0,39.8,43.7,46.3,53.3,73.6,76.5,77.1,115.6,117.8,124.0[q, ¹J (C, F)=273.5Hz], 129.2[q, ³J (C, F)=6.1Hz], 130.6[q, ²J (C, F)=28.7Hz], 133.4,140.0,171.8,204.6,218.4; LRMS (ESI) C ₃₆H ₆₃F ₃O ₆Si ₂Na[M+Na ⁺] value of calculation 727.4, experiment value 727.3; HRMS C ₃₆H ₆₄F ₃O ₆Si ₂[M+H ⁺] value of calculation 705.4194, experiment value 705.4193.

(510.0mg, toluene 0.723mmol) (500mL) solution are heated to and reflux and with Grubbs catalyst (92.1mg, toluene 0.109mmol) (10mL) solution-treated with diene.This mixture is being stirred 17min and is being cooled to 0 ℃ and remain on 0 ℃ before filtering by silicagel pad immediately under the reflux state.Second batch of diene (510.0mg, 0.723mmol) same processing simultaneously.The reactant mixture that merges filters by the silicagel pad (100g) of washing with hexane/EtOAc=3/1 (1.4L).The filtrate that merges concentrated and by hurried column chromatography (SiO ₂～65g, hexane/Et ₂O=10: 1 to 5: 1) to provide macrolide (742.4mg, 1.10mmol, 76%) be colourless amorphous grease to purification.

[α] _D ²⁵-7.5 (c 0.12, CHCl ₃); IR (film) ν 2956,2979,1748,1732,1695,1472,1415,1384,1252,1170,1119,1018,986,876,835cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.08 (3H, s), 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), and 5.30-5.39 (2H, m), 5.58 (1H, dd, J=15.5,8.0Hz), 6.13 (1H, brt, J=7.2Hz); ¹³C NMR (100MHz, CDCl ₃) δ-3.6 ,-3.6,5.4 (3C), 7.0 (3C), 17.5,18.5,19.0,21.6,23.5,26.3 (3C), and 26.5,28.6,29.1,41.0,42.3,47.3,54.1,74.2,76.8,77.7,124.0[q, ¹J (C, F)=273.7Hz], 126.0,128.7[q, ³J (C, F)=5.9Hz], 132.2[q, ²J (C, F)=28.1Hz], 133.8,170.5,204.1,216.1; LRMS (ESI) C ₃₄H ₅₉F ₃O ₆Si ₂Na[M+Na ⁺] value of calculation 699.4, experiment value 699.4; HRMS C ₃₄H ₆₀F ₃O ₆Si ₂[M+H ⁺] value of calculation 677.3881, experiment value 677.3892.

Through Wittig reaction: with ketone and benzene (5mL * 2) azeotropic dry 0.5h under fine vacuum then.Under 0 ℃ in 5min to Wittig salt (907mg, drip in THF 2.59mmol) (19mL) solution t-BuOK (the THF solution of the 1.0M of 2.4mL, 2.43mmol).Under 0 ℃, this mixture is stirred 0.5h and be cooled to-78 ℃ then.(1.10g, THF 1.62mmol) (13mL) solution are warmed to-20 ℃ with the mixture that obtains in 2h to drip ketone in 10min in this mixture.Use saturated NH ₄Cl aqueous solution (15mL) cancellation should be reacted and be extracted (50mL * 3) with EtOAc.The organic layer that merges is with saline (20mL) washing, through Na ₂SO ₄Dry and concentrated.Residue is with hurried column chromatography (SiO ₂, hexane/Et ₂O=20: 1 to 10: 1) purification provides 16 (E)-isomers (940mg, 1.22mmol, 75%) and undesired 16 (the Z)-isomers (140.9mg, 0.182mmol, 11%) wanted and is colourless amorphous grease.

[α] _D ²⁵62.7 (c 0.33, CHCl ₃); IR (film) ν 2955,2878,1743,1692,1472,1379,1320,1253,1169,1114,1007,956,877,835,775cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.09 (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, CDCl ₃) 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), and 29.7,32.9,41.9,42.0,48.6,54.0,72.2,73.3,77.0,116.7,120.7,124.5[q, ¹J (C, F)=273.3Hz], 127.9,129.7[q, ²J (C, F)=28.0Hz], 131.9[q, ³J (C, F)=6.1Hz], 132.9,136.6,152.1,165.4,170.2,217.4; LRMS (ESI) C ₃₉H ₆₅F ₃NO ₅SSi ₂[M+H ⁺] value of calculation 772.4, experiment value 772.4; HRMS C ₃₉H ₆₅F ₃NO ₅SSi ₂[M+H ⁺] value of calculation 772.4074, experiment value 772.4044.

CH with deH-dEpoB (12.2mg, 24.9 μ mol) ₂Cl ₂(1.25mL) solution is cooled to-78 ℃ and handle with refrigerative DMDO solution (78 ℃, 0.06M in the acetone, 914 μ l, 54.8 μ mol).This mixture is warmed to-50 ℃ and stir down 2.7h at-50 ℃.Excessive DMDO passes through to add dimethyl sulfide (117 μ l) and cancellation down at-50 ℃, and this mixture is stirred 0.5h under this temperature.Remove under the vacuum and desolvate.(hexane/EtOAc=1/2) purification provides beta epoxide thing (3.0mg, 5.93 μ mol, 24%) and α-epoxide (7.9mg, 15.6 μ mol, 63%), is colorless solid with the preparation thin layer chromatography.

[α] _D ²⁵-78.5 (c 0.33, CHCl ₃); IR (film) ν 3454,2974,2928,1734,1689,1450,1379,1250,1152,1061,978,735cm ^-1 ¹H NMR (400MHz, CDCl ₃) 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=13.8,9.4Hz), 2.73 (3H, s), 2.77 (1H, brs, OH), 2.93 (1H, dd, J=7.3,4.8Hz), 3.34 (1H, qd, J=6.9,3.8Hz), 3.75-3.82 (1H, m), (4.12-4.24 2H, m comprise OH), 5.54 (1H, ddd, J=15.7,7.4,5.0Hz), and 5.54-5.60 (1H, m), 5.64 (1H, dd, J=15.7,5.6Hz), 6.94 (1H, s), 7.01 (1H, s); ¹H NMR (500MHz, CD ₂Cl ₂) δ 0.91 (3H, s), 1.01 (3H, d, J=6.9Hz), 1.03 (3H, d, J=6.9Hz), 1.22 (3H, s), 1.27 (3H, s), 1.96-2.02 (1H, m), 2.04 (3H, d, J=0.7Hz), 2.16-2.23 (2H, m), 2.33 (1H, dd, J=14.2,3.1Hz), 2.30-2.35 (1H, m), 2.44 (1H, dd, J=14.4,10.3Hz), 2.69 (3H, s), 2.77 (1H, t, J=5.9Hz), 3.24 (1H, qd, J=6.9,4.5Hz), 3.63 (1H, t, J=4.1Hz), and 4.18-4.26 (1H, m), 5.37 (1H, t, J=4.5Hz), 5.48 (1H, dtd, J=15.7,6.7,0.5Hz), 5.58 (1H, dd, J=15.7,6.2Hz), 6.58 (1H, s), 7.00 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 14.4,16.3 (2C), 19.3,19.7,21.6,22.6,31.8,35.9,38.7,39.6,44.1,52.8,60.8,61.8,74.0,75.7,75.9,116.5,119.6,124.3,135.8,136.2,152.1,165.2,170.8,221.5; LRMS C ₂₇H ₄₀NO ₆S[M+H ⁺] value of calculation 506.3, experiment value 506.3; HRMS (ESI) C ₂₇H ₄₀NO ₆S[M+H ⁺] value of calculation 506.2576, experiment value 506.2566.

[α] _D ²⁵-53.9 (c 0.700, CHCl ₃); IR (film) ν 3460,2976,2928,1735,1688,1506,1451,1378,1252,1186,1151,1087,1042,976,879,735cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 1.00 (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, q d, J=7.0,1.8Hz), 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); ¹³CNMR (100MHz, CDCl ₃) δ 13.7,15.3,15.7,18.5,19.4,21.2,22.4,32.5,35.5,39.1,43.4,43.8,51.9,61.3,64.8,73.5,75.9,76.4,116.7,120.1,124.3,137.5,137.7,152.3,165.2,171.0,222.3; LRMS (ESI) C ₂₇H ₃₉NO ₆SNa[M+Na ⁺] value of calculation 528.2, experiment value 528.2; HRMS C ₂₇H ₄₀NO ₆S[M+H ⁺] value of calculation 506.2576, experiment value 506.2583.

Under 50 ℃ to epoxide (0.7mg, 1.38 μ mol) and TrisNHNH ₂The ClCH of (20.6mg, 69 μ mol) ₂CH ₂Add Et in Cl (0.4mL) solution ₃N (9.6 μ L, 69 μ mol).Reaction is by HPTLC monitoring (hexane/EtOAc=1/2).After stirring 6h, this mixture is cooled to room temperature, filters with the EtOAc dilution and by the silicagel pad of washing with EtOAc.After concentrating, (it is white solid that the purification of hexane/EtOAc=1/2) provides reductive product (0.5mg, 0.985 μ mol, 71%) to residue with preparing TLC.

The spectroscopic data of this chemical compound is identical with the spectrum of the dEpoB that has reported.

Under 50 ℃ to epoxide (14.0mg, 27.7 μ mol) and TrisNHNH ₂(165mg, ClCH 0.554mmol) ₂CH ₂Add Et in Cl (3.3mL) solution ₃N (77.0 μ L, 0.554mmol).Reaction is by HPTLC monitoring (hexane/EtOAc=1/2).After stirring 6h, this mixture is cooled to room temperature, filters with the EtOAc dilution and by the silicagel pad of washing with EtOAc.After concentrating, (it is colorless solid that the purification of hexane/EtOAc=1/2) provides reductive product (12.3mg, 24.2 μ mol, 87%) to residue with preparing TLC.

[α] _D ²⁴-13.8 (c 0.61, CHCl ₃); IR (film) ν 3475,2971,2875,1735,1689,1456,1382,1253,1181,1151,1056,980,884,735cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.95 (3H, d, J=7.1Hz), 1.04 (3H, s), 1.11 (3H, d, J=7.0Hz), 1.28 (3H, s), 1.37 (3H, s), 1.25-1.44 (2H, m), 1.45-1.59 (2H, m), 1.71-1.82 (3H, 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=7.0,0.5Hz), 3.89 (1H, brs, OH), 4.03-4.09 (1H, m), and 4.12-4.17 (1H, m), 5.69 (1H, d, J=9.1Hz), 6.63 (1H, s), 7.00 (1H, s); ¹³CNMR (100MHz, CDCl ₃) δ 12.9,15.4,16.3,18.8,19.3,21.6,22.0,23.0,31.5,32.1,33.6,38.6,38.9,42.6,51.7,62.6,65.5,71.2,74.5,76.3,116.6,119.9,138.0,152.2,165.2,170.6,222.7; LRMS (ESI) C ₂₇H ₄₁NO ₆SNa[M+Na ⁺] value of calculation 530.3, experiment value 530.2; HRMS C ₂₇H ₄₂NO ₆S[M+H ⁺] value of calculation 508.2733, experiment value 508.2754.

With ketone and benzene (5mL * 2) azeotropic dry 0.5h under fine vacuum then.Under 0 ℃ in 5min to Wittig salt (1.19g, drip in THF 2.27mmol) (18mL) solution t-BuOK (the THF solution of the 1.0M of 2.2 L, 2.20mmol).Under 0 ℃, this mixture is stirred 20min and is cooled to-78 ℃ then.(1.06g, THF 1.51mmol) (10mL+2mL flushing) solution is warmed to-20 ℃ with the mixture that obtains in 2h to drip ketone in 10min in this mixture.Use saturated NH ₄Cl aqueous solution (15mL) cancellation should be reacted and be extracted (50mL * 3) with EtOAc.The organic layer that merges is with saline (20mL) washing, through Na ₂SO ₄Dry and concentrated.Residue is with hurried column chromatography (SiO ₂～65g, hexane/Et ₂O=30: 1 to 20: 1) purification provides 16 (E)-isomers (1.01g, 1.11mmol, 74%) and undesired 16 (the Z)-isomers (154.5mg, 0.182mmol, 11%) wanted and is colourless amorphous grease.

[α] _D ²⁴-19.0 (c 0.10, CHCl ₃); IR (film) ν 2954,2930,2880,1744,1692,1472,1381,1321,1252,1171,1114,1038,1006,837,776cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.09 (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, CDCl ₃) 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, ³J (C, F)=6.2Hz], 133.2,136.4,152.6,170.1,172.4,217.1; LRMS (ESI) C ₄₅H ₇₈F ₃NO ₆SSi ₃Na[M+Na ⁺] value of calculation 924.5, experiment value 924.5; HRMS C ₄₅H ₇₉F ₃NO ₆SSi ₃[M+H ⁺] value of calculation 902.4888, experiment value 902.4887.

[α] _D ²⁶65.7 (c 1.76, CHCl ₃); IR (film) ν 2955,2931,2879,1743,1692,1472,1380,1321,1253,1170,1113,1007,836,776cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 0.07 (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), and 2.97-3.10 (2H, m), 3.99 (1H, d, J=9.3Hz), 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); ¹³C NMR (100MHz, CDCl ₃) δ-5.3 (2C) ,-3.2 ,-3.2,5.5 (3C), 7.0 (3C), 17.2,18.4,18.8,19.3,19.8,22.4,25.1,25.9 (3C), 26.5 (3C), 29.7,33.0,41.9,42.1,48.6,54.0,63.5,72.1,73.3,76.9,117.0,120.8,124.5[q ¹J (C, F)=273.5Hz], 127.9,129.7[q, ²J (C, F)=27.6Hz], 131.9[q, ³J (C, F)=6.1Hz], 132.9,136.4,152.4,170.1,172.9,217.5; LRMS (ESI) C ₄₅H ₇₈F ₃NO ₆SNa[M+Na ⁺] value of calculation 924.5, experiment value 924.5.

(1.04g slowly adds HF pyridine (11mL) in THF 2.25mmol) (22mL) solution, this mixture is at room temperature stirred 4.3h to the silyl ether in plastic tube under 0 ℃.Under 0 ℃, in 10min, drip TMSOMe (75mL) cancellation reaction.This mixture of vigorous stirring 4.2h at room temperature.Concentrate and behind high vacuum dry 1h, with residue by hurried column chromatography (SiO ₂～25g, hexane/EtOAc=3: 4 to 1: 2) purification, providing triol (615.7mg, 1.00mmol, 96%) is colourless powder.

[α] _D ²⁵-57.7 (c 1.20, CHCl ₃); IR (film) ν 3441,2974,2932,1734,1685,1507,1456,1374,1318,1248,1169,1112,1054,982,888,737cm ^-1 ¹HNMR (400MHz, CDCl ₃) 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, d d, J=15.7,10.1Hz), 2.59-2.69 (2H, m), 2.95-3.01 (2H, m), 3.04 (1H, quintet, J=6.8Hz), 3.72 (1H, td, J=7.0,3.0Hz), 3.78 (1H, d, J=5.7Hz, OH), 4.38 (1H, ddd, J=10.1,5.7,2.4Hz), 4.90 (2H, d, J=6.1Hz), 5.10 (1H, t, J=6.1Hz, OH), 5.44 (1H, t, J=4.7Hz), 5.60 (1H, dd, J=15.9,4.4Hz), 5.66 (1H, dd, J=15.9,5.0Hz), 6.28 (1H, t, J=6.7Hz), 6.73 (1H, s), 7.16 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 16.0,16.5,17.4,17.5,22.9,28.5,30.3,39.0,39.6,45.6,54.0,60.9,70.6,75.6,75.7,116.8,119.2,124.2[q, ¹J (C, F)=273.6Hz], 127.9,129.8[q, ²J (C, F)=28.4Hz], 130.3[q, ³J (C, F)=5.9Hz], 131.2,137.0,152.2,169.8,170.0,218.3; LRMS (ESI) C ₂₇H ₃₇F ₃NO ₆SNa[M+H ⁺] value of calculation 560.2, experiment value 560.1; HRMS C ₂₇H ₃₇F ₃NO ₆S[M+H ⁺] value of calculation 560.2294, experiment value 560.2299.

Under 0 ℃, in THF (1mL) solution of the silyl ether in plastic tube (42.8mg, 55.4 μ mol), slowly add HF pyridine (0.5mL), and this mixture is at room temperature stirred 4.3h.Under 0 ℃, in 10min, drip TMSOMe (3.2mL) cancellation reaction.This mixture of vigorous stirring 1.5h at room temperature.Concentrate and behind high vacuum dry 1h, with residue by hurried column chromatography (SiO ₂, hexane/EtOAc=1: 1) purification, providing glycol (23.6mg, 43.4 μ mol, 78%) is colorless oil.

[α] _D ²⁵31.6 (c 1.00, CHCl ₃); IR (film) ν 2955,2878,1743,1692,1471,1379,1320,1253,1169,1114,1007,877,835,741cm ^-1 ¹HNMR (400MHz, CDCl ₃) δ 1.04 (3H, s), 1.11 (3H, d, J=6.9Hz), 1.20 (3H, d, J=6.9Hz), 1.30 (3H, s), 1.93 (3H, brs), 2.22 (1H, d, J=4.3Hz, OH), 2.25-2.33 (1H, m), and 2.38-2.41 (2H, m), 2.51-2.59 (2H, m), 2.70 (3H, s), 2.80-2.90 (1H, m), 2.94 (1H, dd, J=15.6,4.7Hz), 3.06 (1H, dd, J=15.6,7.4Hz), 3.19 (1H, quintet, J=6.6Hz), 3.71-3.76 (1H, m), 4.26-4.32 (1H, m), 5.57 (1H, ddd, J=15.8,7.2,5.0Hz), 5.67 (1H, dd, J=15.8,6.8Hz), 6.27 (1H, s), 6.33 (1H, dd, J=7.6,6.3Hz), 6.76 (1H, dd, J=8.3,2.9Hz), 6.94 (1H, s); ¹³CNMR (100MHz, CDCl ₃) δ 14.5,17.9,19.2,19.5,19.8,22.2,28.7,32.4,39.8 (2C), 44.7,53.3,71.9,74.1,75.1,117.0,120.4,124.4[q, ¹J (C, F)=272.7Hz], 128.4,130.1[q, ²J (C, F)=28.9Hz], 131.5[q, ³J (C, F)=5.9Hz], 133.0,136.9,152.2,165.5,170.7,218.5; LRMS (ESI) C ₂₇H ₃₆F ₃NO ₅SNa[M+Na ⁺] value of calculation 566.2, experiment value 566.3.

Under 0 ℃ to alcohol (18.9mg, 33.8 μ mol) and Et ₃N (18.8 μ L, CH 0.135mmol) ₂Cl ₂(1mL) add TsCl (12.9mg, 67.5 μ mol) in the solution, add DMAP (2.1mg, 16.9 μ mol) subsequently.After stirring 1.5h under the room temperature, this mixture is filtered by silicagel pad (EtOAc flushing).After concentrating, (purification of hexane/EtOAc=1/1) provides tosylate (8.5mg, 11.9 μ mol, 35%) and chloride (4.3mg, 7.44 μ mol, 22%) is colourless powder with preparing TLC for residue.

¹H NMR (400MHz, CDCl ₃) δ 1.06 (3H, s), 1.12 (3H, d, J=7.0Hz), 1.23 (3H, d, J=6.7Hz), 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, quintet, J=6.8Hz), 3.70-3.75 (1H, m), 4.30 (1H, ddd, J=9.3,6.1,3.2Hz), 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); LRMS (ESI) C ₃₄H ₄₂F ₃NO ₈S ₂Na[M+Na ⁺] value of calculation 736.2, experiment value 736.3.

IR (film) ν 3494,2975,2935,1734,1689,1319,1248,1170,1113,1040,979,738cm ^-1 ¹H NMR (400MHz, CDCl ₃) 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), 2.25-2.35 (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, quintet, J=6.4Hz), 3.70-3.76 (1H, m), 4.32 (1H, ddd, J=9.2,5.9,3.1Hz), 4.85 (2H, s), 5.43 (1H, dd, J=6.0,4.4Hz), 5.59 (1H, ddd, J=15.9,6.4,4.5Hz), 5.66 (1H, dd, J=15.9,6.1Hz), 6.23 (1H, t, J=6.8Hz), 6.63 (1H, s), 7.20 (1H, s); LRMS (ESI) C ₂₇H ₃₅ClF ₃NO ₅SNa[M+Na ⁺] value of calculation 600.2, experiment value 600.2.

Under 0 ℃, in THF (1mL) solution of triol (50.4mg, 90.1 μ mol), add (PhO) ₂PON ₃(27.2 μ l, 0.126mmol).Behind the 5min, (16.2 μ l 0.108mmol), stir 2h with this mixture down at 0 ℃, at room temperature stir 20.5h then to add DBU.This mixture extracts (three times) with EtOAc dilution and water (2mL) cancellation with EtOAc, and the organic layer of merging is through Na ₂SO ₄Dry.After concentrating, residue is passed through hurried column chromatography (SiO ₂, hexane/EtOAc=3: 2) purification, providing azide (45.6mg, 78 μ mol, 87%) is colourless powder.

[α] _D ²⁴-60.3 (c 0.345, CHCl ₃); IR (film) ν 3492,2975,2931,2105,1732,1688,1319,1248,1169,1113,982,733cm ^-1 ¹H NMR (400MHz, CDCl ₃) 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=16.0,4.2Hz), 3.03 (1H, dd, J=16.1,6.7Hz), 3.11 (1H, quintets, J=6.8Hz), and 3.71-3.76 (1H, m), 4.31 (1H, ddd, J=9.2,5.9,3.2Hz), 4.65 (2H, s), 5.43 (1H, dd, J=6.0,4.3Hz), 5.58 (1H, ddd, J=15.8,6.4,4.6Hz), 5.66 (1H, dd, J=15.8,6.1Hz), 6.23 (1H, t, J=7.3Hz), 6.63 (1H, s), 7.18 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 15.4,15.9,17.8,18.6,22.8,28.7,30.9,39.5,39.7,45.1,51.3,53.5,71.5,75.4,76.8,118.2,119.6,122.7[q, ¹J (C, F)=273.6Hz], 127.9,130.0[q, ³J (C, F)=6.1Hz], 130.6[q, ²J (C, F)=27.9Hz], 132.3,137.2,153.1,163.9,170.0,218.3; LRMS (ESI) C ₂₇H ₃₅F ₃N ₄O ₅SNa[M+Na ⁺] value of calculation 607.2, experiment value 607.2; HRMS C ₂₇H ₃₆F ₃N ₄O ₅S[M+H ⁺] value of calculation 585.2359, experiment value 585.2344.

In DMF (0.4mL) solution of tosylate (8.9mg, 12.5 μ mol), add NaN ₃(12.2mg, 0.188mmol).At room temperature stir 21h, the saturated NH of this mixture ₄Cl (aqueous solution) cancellation is with EtOAc extraction (three times).After concentrating, (hexane/EtOAc=1: 1) to provide azide (6.9mg, 11.8 μ mol, 94%) be colourless powder to purification to residue with preparing TLC.

In THF (0.6mL) solution of azide (21.0mg, 35.9 μ mol), add PMe ₃(1.0M in THF, 43.1 μ L, 43.1 μ mol).Behind the 2min, add entry (0.1mL) and mixture is at room temperature stirred 3h.Add more PMe ₃(1.0M in THF, 7.2 μ L, 7.2 μ mol), and at room temperature mixture is stirred 1.5h.The NH of adding 28% ₄OH aqueous solution (54.5 μ L).After stirring 1h under the room temperature, mixture is directly used preparation TLC (CH ₂Cl ₂/ MeOH=100: 7.5) to provide amine (15.9mg, 28.5 μ mol, 79%) be colourless powder to purification.

[α] _D ²⁶-64.2 (c 0.815, CHCl ₃); IR (film) ν 3504,3363,2975,2931,1733,1688,1450,1383,1318,1248,1169,1113,1054,984,736cm ^-1 ¹HNMR (400MHz, CDCl ₃) 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), and 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), and 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, quintet, J=6.8Hz), 3.74 (1H, dd, J=6.7,3.5Hz), 4.18 (2H, s), 4.34 (1H, dd, J=9.8,2.9Hz), 5.43 (1H, dd, J=6.0,4.3Hz), 5.55-5.64 (1H, m), 5.67 (1H, dd, J=15.9,5.8Hz), 6.24 (1H, brt, J=7.3Hz), 6.66 (1H, s), 7.10 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 15.3,16.1,17.7,18.2,22.6,28.7,30.9,39.4,39.7,43.9,45.1,53.8,71.2,75.3,76.6,116.8,120.1,124.2[q, ¹J (C, F)=273.5Hz], 127.8,130.2[q, ³J (C, F)=6.1Hz], 130.4[q, ²J (C, F)=28.6Hz], 132.2,136.6,152.3,170.1,172.7,218.4; LRMS (ESI) C ₂₇H ₃₈F ₃N ₂O ₅S [M+H ⁺] value of calculation 559.2, experiment value 559.2; HRMSC ₂₇H ₃₈F ₃N ₂O ₅S[M+H ⁺] value of calculation 559.2454, experiment value 559.2440.

CH to the amine that stirred (15.9mg, 28.5 μ mol) ₃(37% aqueous solution, 31.4 μ L 0.143mmol), add NaBH subsequently to add HCHO in CN (0.78mL) solution ₃CN (1.0M in THF, 85.5 μ L.85.5μmol)。20min at room temperature stirs the mixture.Add AcOH (1), 40min stirs the mixture under the room temperature.Mixture is directly used preparation TLC (CH ₂Cl ₂/ MeOH=100: 8) to provide dimethylamine (15.6mg, 26.6 μ mol, 93%) be colourless powder to purification.

[α] _D ²⁴-49.9 (c 0.74, CHCl ₃); IR (film) ν 3424,2974,1729,1689,1468,1318,1247,1169,1112,754cm ^-1 ¹H NMR (400MHz, CDCl ₃) δ 1.05 (3H, s), 1.12 (3H, d, J=6.9Hz), 1.23 (3H, d, J=6.8Hz), 1.33 (3H, s), 2.17 (3H, s), 2.24-2.35 (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 comprise OH), 2.68-2.77 (1H, m), 2.80 (3H, s), 2.81 (3H, s), and 2.92-3.06 (2H, m), 3.10 (1H, quintets, J=6.8Hz), and 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, J=15.7,6.4Hz), 6.22 (1H, brt, J=7.2Hz), 6.64 (1H, s), 7.30 (1H, s); ¹³C NMR (100MHz, CDCl ₃) δ 15.3,15.8,17.8,18.8,22.3,28.8,30.9,39.6,39.6,45.2,49.7,49.7,53.4,61.5,71.7,75.4,77.4,119.2,120.2,124.2[q, ¹J (C, F)=273.5Hz], 127.8,129.9[q, ³J (C, F)=6.2Hz], 130.7[q, ²J (C, F)=28.4Hz], 132.4,137.6,154.2,157.2,170.0,218.3; LRMS (ESI) C ₂₉H ₄₂F ₃N ₂O ₅S[M+H ⁺] value of calculation 580.2, experiment value 580.2.

Fusing point; Two equal not re-crystallizations of sample, but pass through SiO ₂Purification.

Claims

1. the chemical compound of following formula and pharmacy acceptable derivates thereof:

R wherein ₁Be hydrogen or low alkyl group;

R ₂For replacing or unsubstituted aryl, heteroaryl, aralkyl or heteroarylalkyl part;

R ₅And R ₆Be hydrogen or blocking group independently of one another;

X is O, S, C (R ₇) ₂Or NR ₇, the R under every kind of situation wherein ₇Be hydrogen or low alkyl group independently;

A-B represents CR _A=CR _B-, C (R _A) ₂-C (R _B) ₂-or-C ≡ C-;

C-D representative-CR _C=CR _D-,-C (R _C) ₂-C (R _D) ₂-or-C ≡ C-;

The R under every kind of situation wherein _ABe hydrogen independently; Halogen;-OR _{A '}-SR _{A '}-N (R _{A '}) ₂-C (O) OR _{A '}-C (O) R _{A '}-CONHR _{A '}-O (C=O) R _{A '}-O (C=O) OR _{A '}-NR _{A '}(C=O) R _{A '}N ₃N ₂R _{A '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{A '}-SR _{A '}-N (R _{A '}) ₂-C (O) OR _{A '}-C (O) R _{A '}-CONHR _{A '}-O (C=O) R _{A '}-O (C=O) OR _{A '}-NR _{A '}(C=O) R _{A '}N ₃N ₂R _{A '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties;

For every kind of situation, R _BBe hydrogen independently; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties;

For every kind of situation, R _CBe hydrogen independently; Halogen;-OR _{C '}-SR _{C '}-N (R _{C '}) ₂-C (O) OR _{C '}-C (O) R _{C '}-CONHR _{C '}-O (C=O) R _{C '}-O (C=O) OR _{C '}-NR _{C '}(C=O) R _{C '}N ₃N ₂R _{C '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{C '}-SR _{C '}-N (R _{C '}) ₂-C (O) OR _{C '}-C (O) R _{C '}-CONHR _{C '}-O (C=O) R _{C '}-O (C=O) OR _{C '}-NR _{C '}(C=O) R _{C '}N ₃N ₂R _{C '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties;

For every kind of situation, R _DBe hydrogen independently; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties; Perhaps

R wherein _A, R _B, R _COr R _DIn any two lump together and can form annulus and can pass through oxygen, sulfur, carbon or the nitrogen-atoms connection, perhaps any two adjacent R _A, R _B, R _COr R _DGroup lumps together, and can form replacement of 3-6 unit or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl ring;

The R under every kind of situation wherein _{A '}, R _{B '}, R _{C '}Or R _{D '}Be hydrogen independently; Blocking group; Straight or branched, replacement or not replacement, ring-type or acyclic, aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, arylalkenyl, sweet-smelling alkynyl or heteroarylalkyl, impure aromatic ene base or hetaryne base section.

2. the chemical compound of following formula and its pharmacy acceptable derivates:

Wherein X is O, S, C (R ₇) ₂Or NR ₇, the R under every kind of situation wherein ₇Be hydrogen or low alkyl group independently;

Y is O or S;

R ₅And R ₆Be hydrogen or blocking group independently of one another;

R ₈Be independently hydrogen, halogen ,-OR ₉,-SR ₉,-N (R ₉) ₂,-CZ ₃,-CHZ ₂,-CH ₂Z, wherein Z is F, Br, Cl, I, OR _{B '}, NHR _{B '}, N (R _{B '}) ₂Or SR _{B '}-(CV ₂) _nOR ₉,-(CV ₂) _nN (R ₉) ₂,-(CV ₂) _nSR ₉The R of ,-(C=O) ₉,-O (C=O) R ₉The OR of ,-(C=O) ₉,-O (C=O) OR ₉-NH (C=O) R ₉,-NH (C=O) OR ₉The NHR of ,-(C=O) ₉, or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl part, the optional following group that replaces with one or more appearance: halogen ,-OR ₉,-SR ₉,-N (R ₉) ₂,-(CV ₂) _nOR ₉,-(CV ₂) _nN (R ₉) ₂,-(CV ₂) _nSR ₉The R of ,-(C=O) ₉,-O (C=O) R ₉The OR of ,-(C=O) ₉,-O (C=O) OR ₉-NH (C=O) R ₉,-NH (C=O) OR ₉The NHR of ,-(C=O) ₉, or ring-type or acyclic, straight or branched, replacement or unsubstituted aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl or heteroarylalkyl part,

The R under every kind of situation wherein ₉Be hydrogen independently; Blocking group; Ring-type or acyclic, straight or branched, replacement or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties; Or Epothilones, NSC-703147 or its analog; Polymer; Saccharide; The photoaffinity label; Or radioactive marker;

Wherein the V under every kind of situation is hydrogen, halogen, hydroxyl, sulfo-, amino, alkylamino or protected hydroxyl, sulfo-or amino independently; T under every kind of situation is 0,1 or 2 independently; N under every kind of situation is 0-10 independently;

A-B represents CR _A=CR _B-, C (R _A) ₂-C (R _B) ₂-or-C ≡ C-;

C-D representative-CR _C=CR _D-, C (R _C) ₂-C (R _D) ₂-or-C ≡ C-;

For every kind of situation, R _BBe hydrogen independently; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl, or heteroaryl moieties;

For every kind of situation, R _DBe hydrogen independently; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; Halogen;-OR _{D '}-SR _{D '}-N (R _{D '}) ₂-C (O) OR _{D '}-C (O) R _{D '}-CONHR _{D '}-O (C=O) R _{D '}-O (C=O) OR _{D '}-NR _{D '}(C=O) R _{D '}N ₃N ₂R _{D '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl, or heteroaryl moieties; Perhaps

3. the chemical compound of claim 2, wherein A-B is-CH=C (R _B)-.

4. the chemical compound of claim 2, wherein A-B is

5. the chemical compound of claim 2, wherein R _BBe methyl.

6. the chemical compound of claim 2, wherein R _BFor-CF ₃

7. claim 2,3 or 4 chemical compound, wherein R ₈Be methyl.

8. claim 2,3 or 4 chemical compound, wherein R ₈For-CH ₂OH.

9. claim 2,3 or 4 chemical compound, wherein R ₈For-CH ₂NH ₂

10. claim 2,3 or 4 chemical compound, wherein C-D is Wherein Z is O, S, NR _ZOr C (R _Z) ₂, R wherein _ZBe hydrogen, halogen, lower acyl or low alkyl group.

11. the chemical compound of claim 10, wherein Z is NH.

12. the chemical compound of claim 10, wherein Z is CH ₂

13. the chemical compound of claim 10, wherein Z is O.

14. the chemical compound of claim 10, wherein Z is S.

15. claim 2,3 or 4 chemical compound, wherein C-D is Or Wherein Z is O, S, NR _ZOr C (R _Z) ₂, R wherein _ZBe hydrogen, halogen, lower acyl or low alkyl group.

16. the chemical compound of claim 15, wherein Z is NH.

17. the chemical compound of claim 15, wherein Z is CH ₂

18. the chemical compound of claim 2, wherein Y is S.

19. the chemical compound of claim 2, wherein Y is O.

20. claim 2,3 or 4 chemical compound, wherein C-D is-C (R _C) ₂-C (R _D) ₂-.

21. the chemical compound of claim 20, the wherein R under every kind of situation _CAnd R _DBe selected from hydrogen, low alkyl group, hydroxyl, halogen and lower alkoxy.

22. the chemical compound of claim 20, the wherein R under every kind of situation _CAnd R _DBe selected from hydrogen and methyl.

23. the chemical compound of claim 20, wherein each R _CBe methyl and each R _DBe hydrogen.

24. the chemical compound of claim 20, wherein each R _CBe hydrogen and each R _DBe methyl.

25. the chemical compound of claim 20, one of them R _CBe hydrogen and another R _CBe methyl.

26. the chemical compound of claim 20, one of them R _DBe hydrogen and another R _DBe methyl.

27. the chemical compound of claim 1 is selected from the chemical compound of following formula:

28. the chemical compound of claim 1 is selected from the chemical compound of following formula:

29. the chemical compound of claim 1 is selected from the chemical compound of following formula:

30. the chemical compound of claim 1 is selected from the chemical compound of following formula:

31. the chemical compound of claim 1 is selected from the chemical compound of following formula:

32. the chemical compound of claim 1 is selected from the chemical compound of following formula:

33. be used for the treatment of the pharmaceutical composition of cancer, it comprises the chemical compound and the acceptable excipient of pharmacy of claim 1.

34. the pharmaceutical composition of claim 33, but further comprise Li Mofu.

35. the pharmaceutical composition of claim 33, but further comprise Li Mofu and ethanol.

36. the pharmaceutical composition of claim 33, wherein said chemical compound is suspended in 1: but Li Mofu/ethanol of 1.

37. the pharmaceutical composition of claim 33 further comprises other cytotoxic agent.

38. be used for the treatment of the pharmaceutical composition of cancer, it comprises: claim 1 chemical compound or the acceptable salt of its pharmacy of treatment effective dose; With pharmaceutically acceptable carrier or diluent, the treatment effective dose of wherein said chemical compound is such amount: it is enough to send the every kg object of about chemical compound of 0.001 to about 40mg body weight.

39. be used for the treatment of the pharmaceutical composition of cancer, it comprises claim 1 chemical compound; With the acceptable excipient of pharmacy; Wherein said pharmaceutical composition is suitable for the object oral administration.

40. be used for the treatment of the pharmaceutical composition of cancer, it comprises claim 2 chemical compound; With the acceptable excipient of pharmacy; Wherein said pharmaceutical composition is suitable for the object oral administration.

41. the pharmaceutical composition of claim 40, wherein R ₈For-CH ₂OH.

42. the pharmaceutical composition of claim 40, wherein R _BFor-CHF ₂,-CH ₂F or-CF ₃

43. the pharmaceutical composition of claim 40, wherein C-D is-CH=CH-, and wherein this pair key is an anti-configuration.

44. the pharmaceutical composition of claim 40, wherein said chemical compound as shown in the formula:

45. the pharmaceutical composition of claim 40, wherein said compositions are solid form.

46. the pharmaceutical composition of claim 40, wherein said compositions are liquid form.

47. the treatment method for cancer, it comprises that chemical compound with the claim 1 of treatment effective dose is to its object administration of needs.

48. the method for claim 47, wherein the treatment effective dose of chemical compound is such amount, and it is enough to send the chemical compound every kg body weight of about 0.001mg to about 40mg.

49. the method for claim 47, wherein the treatment effective dose of chemical compound is such amount, and it is enough to send the chemical compound every kg body weight of about 0.1mg to about 25mg.

50. the treatment method for cancer, it comprises the following formula: compound of treatment effective dose to its object oral administration of needs:

51. prepare the method for following formula: compound:

R wherein ₁Be hydrogen or low alkyl group;

R ₅And R ₆Be hydrogen or blocking group independently of one another;

X is O, S, C (R ₇) ₂Or NR ₇, the R under every kind of situation wherein ₇Be hydrogen or low alkyl group independently; With

For every kind of situation, R _BBe hydrogen independently; Halogen;-OR _{B '}-SR _{B '}-N (R _{B '}) ₂-CY ₃,-CHY ₂,-CH ₂Y, 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl or heteroaryl moieties; Or be Epothilones, NSC-703147 or its analog; Or be polymer; Saccharide; Photoaffinity label or radioactive marker; The R under every kind of situation wherein _{B '}Be hydrogen independently; Blocking group; Straight or branched, replacement or not replacement, ring-type or acyclic aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, heteroarylalkyl, impure aromatic ene base or hetaryne base section;

It is in the presence of metal and Reducing agent with the following formula: compound isomerization:

52. the method for claim 51, wherein said metal are transition metal.

53. the method for claim 51, wherein said metal are palladium.

54. the method for claim 51, wherein said Reducing agent is selected from LiAlH ₄, NaBH ₄And NaBH ₃CN.

55. prepare the method for following formula: compound:

R wherein ₁Be hydrogen or low alkyl group;

R ₅And R ₆Be hydrogen or blocking group independently of one another;

For every kind of situation, R _BBe hydrogen independently; Halogen;-OR _{B '}-SR _{B '}-N (R _{B '}) ₂-CY ₃,-CHY ₂,-CH ₂Y, 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the aliphatic of ring-type or acyclic, straight or branched, assorted aliphatic, aryl or heteroaryl, the optional replacement: hydrogen with one or more following groups; 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 '}N ₃N ₂R _{B '}Cyclic acetal; Or the replacement of ring-type or acyclic, straight or branched or unsubstituted aliphatic, assorted aliphatic, aryl, or heteroaryl moieties; Or be Epothilones, NSC-703147 or its analog; Or be polymer; Saccharide, photoaffinity label or radioactive marker; The R under every kind of situation wherein _{B '}Be hydrogen independently; Blocking group; Straight or branched, replacement or not replacement, ring-type or acyclic aliphatic, assorted aliphatic, aryl, heteroaryl, aralkyl, arylalkenyl, sweet-smelling alkynyl, heteroarylalkyl, impure aromatic ene base or hetaryne base section;

It is with following formula: compound and Cabbeen or carbenoid reagent reacting:

56. the method for claim 55, wherein Cabbeen is CH ₂N ₂