CN104854112A

CN104854112A - Rafamycin analogs and methods for making same

Info

Publication number: CN104854112A
Application number: CN201280077424.7A
Authority: CN
Inventors: 王兵; 赵中
Original assignee: Creation Thing Pharmaceuticals Ltd Is Returned In Hangzhou
Current assignee: Creation Thing Pharmaceuticals Ltd Is Returned In Hangzhou
Priority date: 2012-11-30
Filing date: 2012-11-30
Publication date: 2015-08-19
Also published as: KR20150003156A; AU2012395673A1; WO2014082286A1; SG11201404432QA; WO2014082286A9; JP2016500112A; ZA201405746B; EP2809675A1; CA2863243A1; EP2809675A4; AU2012395673A8

Abstract

A semi-synthetic rapamycin analog with a triazole moiety or a pharmaceutically acceptable salt or prodrug thereof, is a broad-spectrum cytostatic agent and a m TOR inhibitor, and is useful in the treatment of various cancers, or tumors in organs such as kidney, liver, breast, head and neck, lung, prostate, and restenosis in coronary arteries, peripheral arteries, and arteries in the brain, immune and autoimmune diseases. Also disclosed are fungal growth-, restenosis-, post- transplant tissue rejection- and immune- and autoimmune disease- inhibiting compositions and a method of inhibiting cancer, fungal growth, restenosois, post-transplant tissue rejection, and immune and autoimmune disease in a mammal. One particular preferred application of such triazole-moiety containing rapamycin analog is in treating renal carcinoma, lung cancer, colon cancer, and breast cancers wherein potency of the drug, its half-life, tissue distribution properties, and its pharmacokinetic properties including bioavailability through oral and intravenous routes are essential to the clinical outcomes.

Description

Forms of rapamycin analogs and preparation method thereof

Background technology

Compound ciclosporin (ciclosporin A) is promoted widely since obtaining after organ transplantation and immunomodulating regimens application, and significantly improves the success ratio of transplantation.Recent studies have found that a few class has the macrocyclic compounds of potential immunoregulatory activity.On June 11st, 1986, the people such as Okuhara are at European patent 184, disclose the multiple macrocylc compound separated from Streptomycin sulphate Pseudomonas in 162, wherein contain immunosuppressor FK-506 (a kind of be separated the 23 ring macrolides obtained from streptomyces tsukubaensis side chain).

Other relevant natural products, as being separated FR-900520 and FR-900523 obtained from S.hygroscopicus yakushimnaensis, being distinguished from FK-506 and are that the alkyl substituents of C-21 is different.Another one is separated the analogue obtained from S.tsukubaensis, FR-900525, and the place being different from FK-506 is that proline(Pro) group instead of nipecotic acid group.The ciclosporin undesirable side effect relevant with FK-506 is as renal toxicity, people are made to continue to seek immunosuppressant compounds safely and effectively, wherein also include the invalid immunosuppressor of the effective whole body in a kind of local (U.S. Patent number 5,457,111).

Rapamycin is the rare microbiotic of the large ring of one three produced by water suction Streptomycin sulphate, finds that it to have in body/antibacterial activity in vitro, especially anti-candida albicans (U.S. Patent number 3,929,992 and U.S. Patent number 3,993,749)

Rapamycin separately (U.S. Patent number 4,885,171) or the molten chain bacterium of combined utilization (U.S. Patent number 4,401,653) demonstrates anti-tumor activity.1977, it is a kind of effective immunosuppressor that rapamycin shows in experimental allergy myelencephalon model (a kind of Multiple Sclerosis Model), adjuvant arthritis model (a kind of model of rheumatoid arthritis), and shows its formation that effectively can suppress IgE antibody-like

Within 1989, FASEB delivers the immunosuppressive action of rapamycin, has the ability of the histoincompatibility survival time after extending rodent organ transplantation.Transplantation Reviews, 1992,6,39-8 have delivered above and that other rapamycin is relevant biologically.

The monoesters of rapamycin and diester (31 and 42 bit esterified) derivative is effective anti-mycotic agent (U.S. Patent number 4,316,885) and is the water-soluble prodrug (U.S. Patent number 4,650,803) of rapamycin.

Once attempted modifying the number of chemical of rapamycin chemical structure, comprised introducing tetrazole, the monoesters of rapamycin and diester deriv (WO 92/05179), rapamycin 27-oxime (european patent number 467606); Rapamycin 42-oxo analog (U.S. Patent number 5,023,262); Dicyclo rapamycin (U.S. Patent number 5,120,725); Rapamycin dipolymer (U.S. Patent number 5,120,727); The silyl ether (U.S. Patent number 5,120,842) of rapamycin; And aromatic yl sulphonate and sulfamate (U.S. Patent number 5,177,203).Rapamycin (K.C.Nicolaou etc., J.Am.Chem.Soc., 199,11, the 4419-4420 of its naturally occurring enantiomeric form are synthesized recently; S.L.Schreiber, J.Am.Chem.Soc., 1993,115,7906-7907; S.L.Danishefsky, J.Am.Chem.Soc., 1993,115,9345-9346).The example of a nearest forms of rapamycin analogs is containing tetrazolium mycin analogue (U.S. Patent number 6,015,815).Oh group makes analogue have activity

Although these compounds after modifying for chemical structure have immunosuppressive activity and are particularly suitable for being applied on bracket coating by suppressing vascular smooth muscle migration and hyperplasia and anti-angiogenic restenosis effect, still need that there is potential enhancing broad spectrum anticancer active, as renal cell carcinoma, mammary cancer, incidence cancers etc., have better lipotropy and longer transformation period or have forms of rapamycin analogs more stable in antioxygenation and preparation in tissue and blood.Realizing one of these order calibration methods is by introducing triazolyl on rapamycin side chain, give its better lipotropy, better stability, better bioavailability and better tissue and Cell uptake rate, have better curative effect compared with the forms of rapamycin analogs after other known modifications or derivative.Rapamycin after structural modification has better curative effect to various cancer, and better specific aim also likely better reduces drug toxicity.

Summary of the invention

An object of the present invention is to provide a kind of novel semi-synthetic forms of rapamycin analogs, with the triazole group wanted on the 31C-position and/or 42C-position of rapamycin.

Accordingly, an aspect of of the present present invention is the compound that the following structural formula provided embodies.

The present invention is to provide on 42C-and the 31C-position of rapamycin on the other hand containing 2 substituent compounds.

Triazolyl in compound of the present invention can be introduced by multiple reaction mechanism, and the reaction wherein with typical representative is as follows:

A series:

Wherein A can be following a kind of structure:

B series:

Another object of the present invention is to provide the synthetic method that the raw material obtained by fermentation prepares this compound and useful intermediate.

Another object of the present invention is to provide containing the pharmaceutical preparation of at least one above-claimed cpd as activeconstituents.

Another object of the present invention is for various diseases provides therapeutic scheme, as vascular restenosis, and tissue rejection reaction after transplanting, immunity and autoimmune disorders, fungi and cancer.

In addition, the compounds of this invention can make oral tablet with pharmaceutically acceptable carrier, oral administration solid or liquid preparation, oral instant or sustained release preparation, intravenous form, parenteral dosage forms, ointment or solution.

The pharmaceutical preparation that the present invention comprises contain namely release or slowly-releasing as the compound in the present invention of activeconstituents all and pharmaceutically acceptable vehicle.

The medicine equipment also including the compounds of this invention that the invention further relates to.Medicine equipment example comprises drug-eluting coronary artery or periphery support, esophagus, uropoiesis, ovary or neural blood vessel support.

Accompanying drawing explanation

Fig. 1 is that the result of tumor of urethra Carbazole alkaloid experiment is drawn;

Fig. 2 is that the result of tumor of urethra Carbazole alkaloid experiment is drawn;

Fig. 3 is that the result of lung cancer A549 cell Inhibition test is drawn;

Fig. 4 is that the result of lung cancer A549 cell Inhibition test is drawn;

Fig. 5 is that the result of lung cancer A549 cell Inhibition test is drawn;

Fig. 6 is that the result of melanoma SK-MEL-28 Carbazole alkaloid experiment is drawn;

Fig. 7 is that the result of melanoma SK-MEL-28 Carbazole alkaloid experiment is drawn;

Fig. 8 is that the result of melanoma SK-MEL-28 Carbazole alkaloid experiment is drawn;

Fig. 9 is epidermal carcinoma A431 tumor models;

Figure 10 is epidermal carcinoma A431 tumor models;

Figure 11 is epidermal carcinoma A431 tumor models;

Figure 12 is glioblastoma U87MG tumor models;

Figure 13 is glioblastoma U87MG tumor models;

Figure 14 is glioblastoma U87MG tumor models;

Figure 15 is mankind's colorectal tumours HCT116 model experiment;

Figure 16 is mankind's colorectal tumours HCT116 model experiment;

Figure 17 is mankind's colorectal tumours HCT116 model experiment;

Figure 18 is mammary cancer MDA-MB-231 tumor model;

Figure 19 is mammary cancer MDA-MB-231 tumor model;

Figure 20 is mammary cancer MDA-MB-231 tumor model;

Figure 21 is mammary cancer MCD-7 tumor model;

Figure 22 is mammary cancer MCD-7 tumor model;

Figure 23 is mammary cancer MCD-7 tumor model;

Figure 24 is prostate cancer PC-3 tumor model;

Figure 25 is prostate cancer PC-3 tumor model;

Figure 26 is prostate cancer PC-3 tumor model;

Figure 27 shows the validity that the compounds of this invention is used for the treatment of HCT116.

Embodiment

term definition

The term " prodrug " adopted herein refers to non-activity in vitro, and can convert rapidly female drug compound of said structure in vivo to, as passed through hydrolysis in blood.Discussing in detail " bioreversible carrier in medicinal design " of seeing that " prodrug is as a kind of Novel Drug Delivery Systems " in A.C.S. seminar the 14th volume of T.Higuchi and V.Stella and Edward B.Roche in 1987 etc. compile about prodrug, these two sections of documents are incorporated herein all by reference as part herein.

The term " pharmaceutically acceptable prodrug " adopted herein refers to the described prodrug in the present invention, it is within the scope of reliable medical verification, to be applicable to the mankind and the mammiferous contact tissue such as low and without excessive toxicity, pungency, anaphylaxis, they have rational benefit/risk ratio, and can be effective to its desired use also in the conceived case with the compounds of this invention that zwitterionic form exists.The particularly preferred pharmaceutically acceptable prodrug of the present invention is the C31-hydroxyl prodrug ester in the present invention.

The term " prodrug ester " adopted herein refers to any one-tenth ester group that can be hydrolyzed in physiological conditions.The ester group that the example of prodrug ester comprises ethanoyl, acetylize base, valeryl, pivalyl yloxymethyl, acetyl-o-methyl, indanyl etc. and obtained by the C31-hydroxyl coupling in natural or alpha-non-natural amino acid and the compounds of this invention.

The term " isomer " adopted herein refers to have identical chemical formula but have different chemical structures or the compound of optical texture.

But the term " epimer " adopted herein refers to have identical chemical formula the compound having different optical textures in specific position.For rapamycin, 42-Epi rapamycin refers to that its specific rotation is contrary compared with the rapamycin that zymotechnique is produced.

The term " 15-isomer " adopted herein refers to that forms of rapamycin analogs has 7 rings at its 15 compared with the rapamycin of the normal fermentation explained hereafter containing a six-ring.This conversion is otherwise known as " tautomerism ".15-isomer herein also refers to 15 tautomers of rapamycin.

the preparation of invention compound

Compound in the present invention and preparation method are able to better elaboration by following synthetic route chart.

Compound in the present invention prepares by multiple synthesis path.Compounds main in above-mentioned rapamycin patent is by being obtained by reacting at the coupled in common of 42-and/or 31-hydroxyl position, and its content is all included in by reference at this.

Invention example

The synthesis of rapamycin derivative.

The structure of the female medicine of rapamycin is as follows:

The synthetic route of the forms of rapamycin analogs series A in the present invention is as follows:

In the present invention, the similar synthetic route of other forms of rapamycin analogs is as follows:

example 1: the synthesis of compd A 1

Stir rapamycin (3g, 3.2mmol) and Cs ₂cO ₃dry DMF (90mL) solution of (3.2 grams, 9.6 mmoles), and add NaI (1.5 grams, 9.6 mmoles) and 3-propargyl bromide (1.2 grams, 9.6 mmoles), reaction mixture is at room temperature stirred 30 hours.After having reacted, add 300mL water, and be extracted with ethyl acetate (200mL x 3).Merge organic layer, use 300mL normal saline washing, and use anhydrous sodium sulfate drying.Concentrated, concentrated solution silica column purification (sherwood oil of the ethyl acetate of 50% to 100% is as elutriant) obtains compd A 1 (2.1g, 68%).

LCMS(m/z)ES-950(M-1) ^-。

the synthesis of compound A-13

To 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with 1-nitrine diamantane (100mg; in anhydrous THF (9mL) solution 0.6mmol); add DIPEA (100 μ L under nitrogen protection; 0.6mmol) with CuI (20mg, 0.1mmol).Reaction solution stirred overnight at room temperature.Then 20mL water is added, and with 20mL extraction into ethyl acetate 3 times.Merge organic layer, normal saline washing, rear Na ₂sO ₄dry.After concentrated, concentrated solution is crossed silica column purification (sherwood oil of the ethyl acetate of 25% to 50% is as elutriant) and is obtained white solid, purifies further obtain compound as white solid A3 (26mg, 10%) with preparative HPLC. ¹H NMR(300MHz,CDCl3)δ7.71(s,1H),6.74(m,1H),6.39-6.02(m,5H),5.62-5.36(m,5H)；LCMS(m/z)ES-1128(M-1) ^-。

example 3: the synthesis of compd A 4

At 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with 4-triazobenzene formic acid (100mg; in anhydrous THF solution (9mL) 0.6mmol); add DIPEA (100 μ L under nitrogen protection; 0.6mmol) with CuI (20mg, 0.1mmol).Stirring at room temperature reaction solution 3 hours, adds 20mL water and uses 20mL extraction into ethyl acetate 3 times.Merge organic layer, after normal saline washing, use Na ₂sO ₄dry.Concentrated, concentrated solution is crossed silica column purification (sherwood oil of the ethyl acetate of 5% to 10% is as elutriant) and is obtained white solid, and purifies with preparative HPLC further and obtain white solid chemical combination A4 (29mg, 12%). ¹H NMR(300MHz,CDCl ₃)δ8.27(m,1H),7.90(m,1H),7.73(m,1H),7.56(m,1H),6.74(m,1H),6.55-6.00(m,5H),5.60-5.36(m,5H)；LCMS(m/z)ES-1114(M-1) ^-。

example 4: the synthetic method of compound A-45

The preparation of intermediate 2

At 0 DEG C with syringe is slow must toward NaN ₃tf2O (7.3g, 25.8mmol) is added in the acetonitrile aaerosol solution (20mL) of (2.0g, 30.8mmol).Stir this solution at such a temperature 2 hours, cross and filter insolubles.Toward compound 1 (2.0g, 13mmol) at 0 DEG C, CuSO ₄(160mg, 1mmol), H ₂o (6mL) and Et ₃filtrate is instilled in N (3.6mL, 25.8mmol) mixed solution.Stirred at ambient temperature reaction mixture 6 hours.Then normal saline washing is used with EtOAc dilution mixture liquid.Organic layer Na ₂sO ₄dry be also evaporated to brown solid, rear silica gel chromatographic column (sherwood oil of the ethyl acetate of 30% to 50% is as elutriant) obtains white intermediate 2 (1.1g, 48%). ¹H NMR(300MHz,CDCl ₃)δ9.93(s,1H),7.63(m,2H),7.03(m,2H),2.02(s,3H)；LCMS(m/z)ES+177(M+1) ⁺.

At 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with N-(4-azido-phenyl) ethanamide; intermediate 2 (100mg; in anhydrous THF (9mL) 0.6mmol); add DIPEA (100 μ L under nitrogen protection; 0.6mmol) with CuI (20mg, 0.1mmol).Stirred at ambient temperature 4 hours.Then, add 20mL water, 20mL extraction into ethyl acetate 3 times.Merge organic layer, Na after normal saline washing ₂sO ₄dry.Concentrated, concentrated solution silicagel column (sherwood oil of the ethyl acetate of 30% to 100% is as elutriant) purifying obtains compound as white solid, obtains A5 (56mg, 25%) after preparative HPLC purifying. ¹H NMR(300MHz,CDCl ₃)δ8.13(m,1H),7.73(m,4H),6.74(m,1H),6.49-6.00(m,5H),5.65-5.37(m,5H)；LCMS(m/z)ES-1127(M-1) ^-.

example 5: the synthetic method of compd A 6

At 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg, 0.2mmol) and TMS-N ₃the t-BuOH (6mL) of (100mg, 0.9mmol) and H ₂na is added under nitrogen protection in O (6mL) solution ₂cO ₃(100mg, 1mmol), CuSO ₄(20mg, 0.13mmol) and sodium ascorbate (40mg, 0.2mmol).Stirring at room temperature reaction solution 3 hours.Add 20mL water and extract 3 times with 20mL EtOA.Merge organic layer, normal saline washing also uses Na ₂sO ₄dry.Concentrated rear silica column purification (sherwood oil of the ethyl acetate of 25% is as elutriant) obtains white compound A2 (189mg, 82%).A2 is dissolved in THF (10mL) solution of TBAF at 0 DEG C, and stirs 7 hours.Reaction mixture is layering in ethyl acetate aqueous solution.Aqueous phase 25mL ethyl acetate Cui Wu 3 times.Merge organic layer, normal saline washing also uses Na ₂sO ₄dry.After concentrated, concentrated solution through silica gel chromatography column purification (sherwood oil of the ethyl acetate of 5%-10% is as elutriant), and is purified with preparative HPLC further and is obtained compound as white solid A6 (38mg, 24%). ¹H NMR(300MHz,CDCl ₃)δ7.75-7.55(m,1H),6.76(m,1H),6.49-6.08(m,5H),5.53-5.35(m,3H)；LCMS(m/z)ES-1012(M-1+18) ^-.

example 6: the synthetic method of compd A 7

Prepare intermediate 5 and 6:

In the 100ml aqueous solution of 3 (5g, 41mmol), add NaN3 (5g, 83mmol), backflow is spent the night.100mlDCM is added after reaction mixture cool to room temperature.Be separated organic layer and use Na ₂sO ₄drying, filters.In filtrate, Et is added at 0 DEG C ₃n (5.05g, 50mmol) and TsCl (9.55g, 50mmol).Reaction mixture adds 100ml water after at room temperature stirring 4 hours.Be separated organic phase and use Na ₂sO ₄dry.Filter also vacuum concentration and obtain crude product.Colorless oil intermediate 5 (5.7g, 58%) is obtained by chromatography (10% ethyl acetate petroleum ether is as elutriant). ¹HNMR(300MHz,CDCl ₃)δ7.81(d,2H),7.39(d,2H),4.14(m,2H),3.48(m,2H),2.43(s,3H)；LCMS(m/z)ES+242(M+1)+.

0 DEG C, at intermediate 5 (1g, 4.1mmol) and Cs ₂cO ₃morpholine (0.71g, 8.2mmol) is added in the 30mL anhydrous DMF solution of (2.8g, 8.2mmol).Room temperature for overnight, reaction mixture layering in 50mL EtOAc and 60mL water.Organic phase Na ₂sO ₄dry.Filter also vacuum concentration and obtain crude product.Colorless oil intermediate 6 (0.4g, 72%) is obtained with silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 50% is as elutriant).LCMS(m/z)ES+157(M+1) ⁺.

At 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with 4-(2-Azidoethyl) morpholine; intermediate 6 (100mg; in anhydrous THF (9mL) solution 0.6mmol), nitrogen protection adds DIPEA (100 μ L; 0.6mmol) with CuI (20mg, 0.1mmol).Stirred at ambient temperature reaction solution 3 hours.Then, add 20mL water and use 20mL extraction into ethyl acetate 3 times.Merge organic layer, normal saline washing also uses Na ₂sO ₄dry.Concentrated, concentrated solution silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 30% is as elutriant) obtains compound as white solid and purifies by preparation liquid phase further obtaining compound as white solid A7 (45mg, 20%). ¹H NMR(300MHz,CDCl ₃)δ7.89(m,1H),6.72(m,1H),6.44-6.05(m,5H),5.60-5.37(m,5H)；LCMS(m/z)ES-1107(M-1)-.

example 7: the synthesis of compd A 9

40-oxygen (the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with 2-nitrine ethanol (100mg; DIPEA (100 μ L are added under nitrogen protection in anhydrous THF (9mL) solution 1.2mmol); 0.6mmol) with CuI (20mg, 0.1mmol).Reaction solution at room temperature stirs and spends the night.Then, add 20ml water and use 20ml extraction into ethyl acetate 3 times.Merge organic layer, normal saline washing also uses anhydrous Na ₂sO ₄dry.After concentrated, concentrated solution obtains solid chemical compound and is further purified through preparation liquid phase obtaining white compound A9 (26mg, 11%) through silica gel chromatography column purification (the methanol dichloromethane solution of 5%-10% is as elutriant). ¹H NMR(300MHz,CDCl ₃)δ8.03-7.78(m,1H),6.70(m,1H),6.46-6.00(m,5H),5.61-5.39(m,5H)；LCMS(m/z)ES-1038(M-1) ^-.

example 8: the synthesis of compd A 10

The preparation of intermediate 8

40%HBr (10mL) solution of compound 7 (1.3g, 12.7mmol) at room temperature stirs 1 hour, adds 20ml water, rear 20mL extraction into ethyl acetate 3 times.Merge organic layer and use normal saline washing, Na ₂sO ₄dry.After concentrated, concentrated solution obtains white intermediate 8 (0.7g, 31%) through silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 50% is as elutriant).LCMS(m/z)ES+183(M+1) ⁺.

Prepare intermediate 9

Intermediate 8 (0.7g, 3.9mmol), NaN ₃dMSO (16mL) solution of (1.13g, 15mmol) stirs 2 days at 80 DEG C.Add 20mL water and use 20mL extraction into ethyl acetate 3 times.Organic layer after normal saline washing merges, Na ₂sO ₄dry.Concentrated, concentrated solution obtains white solid intermediate 9 (0.25g, 45%) through silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 50%-100% is as elutriant).LCMS(m/z)ES+146(M+1)+.

Prepare compd A 10

At 40-oxygen-(the third-2-alkynyloxy group) rapamycin A1 (200mg, 0.2mmol) with 2-(azido-methyl) 2-methylpropane-1, the t-BuOH (6mL) of 3-diol intermediates 9 (100mg, 0.7mmol) and H ₂na is added under nitrogen protection again in O (6mL) solution ₂cO ₃(100mg, 1mmol), CuSO ₄(20mg, 0.13mmol) and sodium ascorbate solution (40mg, 0.2mmol).Hybrid reaction also at room temperature stirs 6 hours.Add 20ml water and extract 3 times with 20ml ethyl acetate solution.Merge organic layer, normal saline washing also uses Na ₂sO ₄dry.After concentrated, concentrated solution also obtains compound as white solid A10 (15mg, 7%) through preparation liquid phase purifying further through silica gel chromatographic column (the methanol dichloromethane solution of 5%-10% is as elutriant) purifying.

¹H NMR(300MHz,CDCl ₃)δ7.76(m,1H),6.69(m,1H),6.55-6.00(m,5H),5.63-5.33(m,5H),LCMS(m/z)ES-1096(M-1) ^-.

example 9: the synthesis of compd A 12

The preparation of intermediate 11

The aqueous solution (10ml) of LiOH (0.9g, 39mmol) is added in the mixing solutions of past compound 10 (1g, 7.8mmol) and MeOH/THF (10mL/10mL).Gained solution is at room temperature stirred 3 hours.Adjust the acidity of mixed solution to PH=4 with 2N HCl, be extracted with ethyl acetate (25mL × 2).Merge organic layer and concentrate under vacuo, obtaining colorless oil intermediate 11 (0.7g, 91%). ¹H NMR(300MHz,CDCl ₃)δ2.34(s,2H)；LCMS(m/z)ES+102(M+1)+.

Prepare compd A 12

At 40-oxygen (the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) and in the t-BuOH (6mL) of 2-nitrine acid intermediates 11 (100mg, 1mmol) and H2O (6mL) add Na under nitrogen protection ₂cO ₃(100mg, 1mmol), CuSO ₄(20mg, 0.13mmol) and sodium ascorbate (40mg, 0.2mmol).Stirred at ambient temperature reaction solution 2 hours.Add 20ml water and use 20ml extraction into ethyl acetate 3 times.Merge organic layer, normal saline washing also uses Na ₂sO ₄dry.Concentrated, concentrated solution is through silica gel chromatography column purification (the methanol dichloromethane solution of 5%-20% is as elutriant) and obtain compound as white solid A12 (15mg, 7%) through preparation liquid phase purifying further.

¹H NMR(300MHz,CDCl ₃)δ7.89(m,1H),6.72(m,1H),6.49-6.08(m,5H),5.60-5.35(m,5H)；LCMS(m/z)ES-1052(M-1) ^-.

example 10: the synthesis of compound 13

The preparation of intermediate 13

0 DEG C time with syringe is slow must toward NaN ₃tf is added in (2.0g, 30.8mmol) stirred suspension in acetonitrile (20mL) ₂o (7.3g, 25.8mmol).This mixed reaction solution is continued at this temperature stirring 2 hours.Insolubles is gone out in filtration, will instill compound 12 (2.0g, 10mmol), CuSO when 0 DEG C in filtrate ₄(160mg, 1mmol), in the mixing solutions of water (6mL) and Et3N (3.6mL, 25.8mmol).Stirring at room temperature reaction mixture 6 hours.Use diluted ethyl acetate mixed solution, normal saline washing.Organic layer Na ₂sO ₄drying is also evaporated to brown solid compound, silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 30% to 50% is as elutriant) is used to obtain brown solid compound afterwards, brown compound intermediate 13 (0.4g, 17%) is obtained further across preparation colleges and universities liquid phase purifying.1H NMR(300MHz,CDCl ₃)δ6.94(m,4H),3.19(m,4H),2.60(m,4H),2.36(s,3H)；LCMS(m/z)ES+218(M+1)+.

The preparation of compd A 13

At 40-oxygen (the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) and in the t-BuOH (6mL) of 1-(4-azido-phenyl)-4-methylpiperazine intermediate 13 (100mg, 0.5mmol) and water (6mL) add Na under nitrogen protection ₂cO ₃(100mg, 1mmol), CuSO ₄(20mg, 0.13mmol) and sodium ascorbate (40mg, 0.2mmol).This mixed solution of stirring at room temperature 3 hours.Then, in acetic acid ethyl acetate extract (20mL x 3), 20ml water is added.Merge organic layer, normal saline washing is also dry with Na2SO4.Concentrated, concentrated solution silica gel chromatography column purification (methanol dichloromethane of 5%-20% is as elutriant) also obtains white oil compd A 13 (33mg, 14%) through efficient preparation liquid phase purifying further. ¹h NMR (300MHz, CDCl ₃) δ 8.11 (m, 1H), 7.71 (m, 2H), 7.06 (m, 2H), 6.70 (m, 1H), 6.45-6.00 (m, 5H), 5.66-5.36 (m, 5H); LCMS (m/z) ES-1168 (M-1) ^-.

example 11: the synthesis of compd A 14

At 40-oxygen (the third-2-alkynyloxy group) rapamycin A1 (200mg; 0.2mmol) with 1-(azido methyl)-4-fluorobenzene (100mg; anhydrous THF (9mL) 0.6mmol) adds DIPEA (100 μ L under nitrogen protection; 0.6mmol) with CuI (20mg, 0.1mmol).Stirred at ambient temperature reaction also 3 hours, after add 20ml water and with EtOAc (20mL x 3) extraction.Merge organic layer, normal saline washing, uses Na ₂sO ₄dry.After concentrated, concentrated solution silica gel chromatography column purification (the ethyl acetate petroleum ether solution of 30%-100% is elutriant) obtains compound as white solid, compound as white solid A14 (32mg, 14%) is obtained further through preparation liquid phase purifying.1H NMR(300MHz,CDCl ₃)δ7.58(m,1H),7.26(m,2H),7.06(m,2H),6.75(m,1H),6.50-6.00(m,5H),5.60-5.36(m,5H)；LCMS(m/z)ES-＝1102(M-1).

example 12: the synthesis of compd A 15

The preparation of intermediate 15

Compound 14 (3g, 48mmol) with Et3N (5g, add DMAP (0.6g, 5mmol) in DCM (100mL) solution 50mmol) and dropwise instill TBDPS-Cl (4.4g, 16mmol) at 0 DEG C and stir and spend the night.Add 100ml water and extract with DCM (80mL x 3).Merge organic layer and normal saline washing, Na ₂sO ₄dry.After concentrated, obtain colorless oil intermediate 15 (1.7g, 12%) with silica gel chromatography column purification (30% ethyl acetate petroleum ether is as elutriant).1H NMR(300MHz,CDCl ₃)δ7.63(m,4H),7.36(m,6H),3.74(m,2H),3.66(m,2H),1.04(s,9H)；LCMS(m/z)ES+301(M+1)+

Prepared by intermediate 16

Tf is added at 0 DEG C in DCM (40mL) solution of intermediate 15 (1.7g, 5.7mmol) and DIPEA (1.5g, 11.4mmol) ₂o (1.7g, 6mmol) and at room temperature stir spend the night.Add 50ml water and extract with DCM (40mL x 3).Organic layer merges, and normal saline washing also uses Na ₂sO ₄dry.After concentrated, enriched material silica gel chromatography column purification (10% ethyl acetate petroleum ether is as elutriant) obtains colorless oil intermediate 16 (1.5g, 63%).1H NMR(300MHz,CDCl ₃)δ7.63(m,4H),7.36(m,6H),4.58(m,2H),3.95(m,2H),1.04(s,9H)；LCMS(m/z)ES+433(M+1)+.

Prepared by intermediate 17

In toluene (30mL) solution of rapamycin (400mg, 0.43mmol) and DIPEA (278mg, 2.15mmol), at room temperature add intermediate 16 (0.93g, 2.15mmol), 80 DEG C are stirred 2 hours.Add 50ml water, 30ml extraction into ethyl acetate 3 times.Merge organic layer, 0.5N HCl rinses, NaHCO3 and salt water saturation, Na ₂sO ₄dry.Concentrated, concentrated solution is crossed silica gel chromatographic column (25%-40% ethyl acetate petroleum ether solution is as elutriant) and is obtained colorless solid intermediate 17 (280mg, 53%).LCMS(m/z)ES-1194(M-1)-.

the preparation of compd A 15

In the THF solution of intermediate 17 (280mg, 0.23mmol), 2mlHF pyridine solution is added, stirring at room temperature 4 hours at 0 DEG C.Add 20ml water, EtOAc (20mL x 3) extracts.Merge organic layer, 0.5N HCl rinses, and uses NaHCO ₃with salt water saturation, Na ₂sO ₄dry.Concentrated, concentrated solution obtains compound as white solid compd A 15 (34mg, 15%) with preparative liquid chromatography column purification after crossing silica gel chromatography column purification (30%-100% ethyl acetate petroleum ether solution is as washing fluid) further.1H NMR(300MHz,CDCl ₃)δ6.40-6.00(m,5H),5.53-5.25(m,4H),4.83(s,1H),4.13(m,1H)；LCMS(m/z)ES-957(M-1)-.

the synthesis of rapamycin derivative B in present invention

The preparation of rapamycin derivative B series is prepared according to following synthetic route:

Above synthetic route display, in the rapamycin derivative example of some B series, R and B ' has following chemical structure:

example 13: the synthesis of compound B-11

In anhydrous DCM (150mL) solution of rapamycin (5g, 5.5mmol) and 2,6-di-t-butyl-4-picoline, add Trifluoromethanesulfonic anhydride when 0 DEG C, 400 DEG C time, stir 5 hours.Water is added in mixed reactant, and with DCM extraction (200mL × 2), with water cooling, Na ₂sO ₄drying, vacuumizes drying.Crude product obtains compound as white solid B1 (1.5g, 30%) through chromatographic column (ethyl acetate petroleum ether of 25% is elutriant).LCMS(m/z)ES-937(M-H)-.

example 14: the synthesis of compd B 2

VC sodium salt (63mg, 0.32mmol) is added, CuSO in the methanol-water solution (4mL/2mL) of compound B-11 (150mg, 0.16mmol) and SM1 (27mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filtering mixt, concentrated, and obtains yellow solid compound B2 (33.2mg, 21%) .1H NMR (300MHz, CDCl with chromatography (0-2% methanol dichloromethane is elutriant) ₃) δ 7.83 (m, 1H), 6.37-6.01 (m, 4H), 5.40-5.31 (m, 4H); LCMS (m/z) ES-993 (M-H)-.

example 15: the synthesis of compd B 3

VC sodium salt (63mg, 0.32mmol) is added, CuSO in the methanol-water solution (4mL/2mL) of compound B-11 (150mg, 0.16mmol) and SM1 (40mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, and concentrated filtrate also crosses chromatography (0-2% methanol dichloromethane is elutriant), obtains compound as white solid B3 (20.7mg, 13%).

¹H NMR(300MHz,CDCl ₃)δ8.55(s,1H),7.89(m,1H),6.39-6.02(m,4H),5.46-4.83(m,4H)；LCMS(m/z)ES-1020(M-H)-.

example 16: the synthesis of compd B 4

At 0 DEG C, (100mL) slowly (more than 1 hour) instillation SM1 (2g, 16.4mmol) in the DCM solution of compound 1 (2g, 32.8mmol).Enriched mixture, concentrated solution is crossed chromatography (0-2% methanol dichloromethane is elutriant) and is obtained yellow oily intermediate 2 (0.9g, 54%).LCMS(m/z)ES+100(M+H)+.

VC sodium salt (63mg, 0.32mmol), CuSO is added in the methanol-water solution (4mL/2mL) of compound B-11 (150mg, 0.16mmol) and intermediate 2 (48mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Mixture stirs and spends the night, filtering mixt, and concentrated filtrate is also crossed chromatography (0-2% methanol dichloromethane is elutriant) and obtained white solid chemical combination B4 (17.1mg, 11%).

¹H NMR(300MHz,CDCl ₃)δ8.40(s,1H),6.39-6.02(m,4H),5.37-4.94(m,4H)；LCMS(m/z)ES-1036(M-H)-.

example 17: the synthesis of compd B 5

In THF (40mL) solution of compound 1 (2g, 19.0mmol), when 0 DEG C, add K ₂cO ₃(5.2g, 38mmol) and SM1 (2.2g, 19mmol).Stirred at ambient temperature 4 hours, filter mixed liquor, concentrated filtrate is also crossed chromatography (0-3% methanol dichloromethane is elutriant) and is obtained yellow oily intermediate 2 (1.2g, 44%).

¹H NMR(300MHz,CDCl ₃)δ3.65(m,4H),3.49(m,2H),2.75(m,4H),2.22(m,1H).

VC sodium salt (63mg, 0.32mmol), CuSO is added in the methanol-water solution (4mL/2mL) of compound B-11 (150mg, 0.16mmol) with intermediate 2 (69mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, and mixing filtrate also crosses chromatography (0-2% methanol dichloromethane is elutriant), obtains white solid mixture B5.

¹H NMR(300MHz,CDCl ₃)δ7.92(s,1H),6.39-5.99(m,4H),5.45-4.84(m,4H)；LCMS(m/z)ES-1080(M-H)-.

example 18: the synthesis of compound B-26

Compound 1 (0.5g, 5.8mmol)) THF (40mL) solution in, add K when 0 DEG C ₂cO ₃(1.6g, 11.6mmol) and SM1 (0.69g, 5.8mmol).Stirred at ambient temperature 4 hours, filter mixed liquor, concentrated filtrate is also crossed chromatography (3% methanol dichloromethane is elutriant) and is obtained yellow oily intermediate 2 (0.3g, 42%).LCMS(m/z)ES+125(M+H)+.

VC sodium salt (63mg, 0.32mmol), CuSO is added in compound B-11 (150mg, 0.16mmol) and the methanol-water solution (4mL/2mL) of intermediate 2 (60mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, and mixing filtrate also crosses chromatography (0-2% methanol dichloromethane is elutriant), obtains white solid mixture B6.

¹H NMR(300MHz,CDCl ₃)δ7.81(s,1H),6.40-6.02(m,4H),5.45-4.81(m,4H)；LCMS(m/z)ES-1062(M-H)-.

example 19: the synthesis of compd B 7

Compound 1 (0.5g, 5.8mmol)) THF (40mL) solution in, add K when 0 DEG C ₂cO ₃(1.4g, 10mmol) and SM1 (0.6g, 5mmol).Stirred at ambient temperature 4 hours, filter mixed liquor, concentrated filtrate is also crossed chromatography (3% methanol dichloromethane is elutriant) and is obtained yellow oily intermediate 2 (0.5g, 72%).LCMS(m/z)ES+139(M+H)+.

VC sodium salt (63mg, 0.32mmol), CuSO is added in compound B-11 (150mg, 0.16mmol) and the methanol-water solution (4mL/2mL) of intermediate 2 (60mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, and concentrated mixing filtrate also crosses chromatography (2% methanol dichloromethane is elutriant), obtains white solid mixture B7 (12mg, 7%).

¹H NMR(300MHz,CDCl ₃)δ8.39(s,1H),7.76(s,1H),6.37-6.01(m,4H),5.41-4.78(m,4H)；LCMS(m/z)ES-1075(M-H)-.

example 20: the synthesis of compd B 9

VC sodium salt (63mg, 0.32mmol), CuSO is added in compound B-11 (150mg, 0.16mmol) and the methanol-water solution (4mL/2mL) of SM1 (50mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, and concentrated mixing filtrate also crosses chromatography (2% methanol dichloromethane is elutriant), obtains white solid mixture B9 (37.1mg, 22%).

¹H NMR(300MHz,CDCl ₃)δ9.37-7.89(m,5H),6.39-6.01(m,4H),5.42-4.99(m,4H)；LCMS(m/z)ES-1040(M-H)-.

example 21: the synthesis of compound B-11 1

Trimethylsilyl acetylene (2g, 20mmol) is added, CuI (191mg, 1mmol) and Pd (PPh under protection at nitrogen in the dioxan (20mL) of SM1 (2.1g, 10mmol) ₃) ₂cl ₂(730mg, 1mmol), then dropwise adds Et ₃n (10g, 100mmol).Stir at 100 DEG C and spend the night, shrend, extract with EtOAc (50mL x 2).Merge organic layer, Na ₂sO ₄drying, the concentrated crude product obtaining intermediate 1.

THF (20mL, the 20mmol) solution of the crude product TBAF of intermediate 1 at room temperature dissolves 2 hours, shrend, and extracts with EtOAc (50mL x 2).Merge organic layer, Na ₂sO ₄drying, and chromatography obtains yellow solid compound 2 (0.9g, 58%) excessively.

¹H NMR(300MHz,DMSO-d ₆)δ7.96(d,2H),7.62(d,2H),4.48(s,1H),3.87(s,3H).

LiOH (0.312g, the 12.52mmol) aqueous solution (10mL) is added in methyl alcohol (10mL) solution of compound 2 (0.5g, 3.13mmol).Stir 2 hours, HCL solution (2N) extraction is gone out, and EtOAc (30mL X 3) extracts.Merge organic layer, Na ₂sO ₄drying, concentrates and obtains yellow intermediate 3 (0.35g, 77%).

VC sodium salt (63mg, 0.32mmol), CuSO is added in the methanol-water solution (6mL/3mL) of compound B-11 (150mg, 0.16mmol) and intermediate 3 (70mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, pH value to 3 ~ 4 of unity mixture, filters, and concentrated filtrate also obtains compound as white solid B11 (15.5mg, 9%) with chromatography (1.5% methanol dichloromethane solution is elutriant).

¹H NMR(300MHz,CDCl ₃)δ8.19(m,3H),7.98(d,2H),6.77-6.11(m,4H),5.49-4.51(m,4H)；LCMS(m/z)ES-1083(M-H)-。

example 22: the synthesis of compound B-11 2

NaOH (2.4g, the 20mmol) aqueous solution and 3-bromine third-1-alkynes (2.4g, 20mmol) is added in the solution in propylene glycol (20mL) of SM1.Stir 4 hours at 70 DEG C, concentrated mixed solution, filters, and washing filter cake, drying obtains yellow solid intermediate 1 (2g, 37%).

LCMS(m/z)ES+234(M+Na) ⁺.

Toward DCM (4mL) solution of instillation oxalyl chloride (1.1g, 8.6mmol) in the DMF solution (8mL) of intermediate 1 (1g, 4.3mmol) at 0 DEG C.Room temperature for overnight, shrend, DCM (30mL x 3) extracts.Merge organic layer, Na2SO4 is dry, concentrated, crosses chromatographic column (the ethyl acetate petroleum ether solution of 0-10% is as elutriant) and obtains yellow solid intermediate 2 (0.35g, 77%).In ammoniacal liquor (5mL), add intermediate 2 (0.35g, 1.5mmol), stirring at room temperature mixed solution 1 hour, shrend, EtOAc (20mL x 2) extracts.Merge organic layer, Na ₂sO ₄drying, concentrated, cross chromatographic column (ethyl acetate petroleum ether of 0-50% is as elutriant) and obtain yellow solid intermediate 3 (0.2g, 20%).

¹H NMR(300MHz,DMSO-d ₆)δ7.77(d,2H),7.24(s,2H),7.14(d,2H),4.90(d,2H),3.63(m,1H).

VC sodium salt (63mg, 0.32mmol) is added, CuSO in the methanol-water solution (6mL/3mL) of compound B-11 (150mg, 0.16mmol) and intermediate 3 (101mg, 0.48mmol) ₄(51mg, 0.32mmol), and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, concentrates and obtains compound as white solid B12 (13.4mg, 7.3%) through chromatography (methanol dichloromethane of 0 ~ 2.5% is as elutriant).

¹H NMR(300MHz,CDCl ₃)δ7.88(m,3H),7.12(m,2H),6.39-6.01(m,4H),5.42-4.63(m,4H)；LCMS(m/z)ES-1148(M-H)-.

example 23: the synthesis of compound B-11 3

Trimethylsilyl acetylene (2g, 20mmol) is added under nitrogen protection, CuI (191mg, 1mmol) and Pd (PPh in dioxane (20mL) solution of compound S M1 (2.3g, 10mmol) ₃) ₂cl ₂(730mg, 1mmol), and instill Et3N (10g, 100mmol).Stir at 100 DEG C and spend the night, shrend, ethyl acetate (50mL x 2) extracts.Merge organic layer, Na ₂sO ₄drying, the concentrated crude product obtaining intermediate 1.

The crude product of intermediate 1 stirring at room temperature 2 hours in the THF solution of TBAF, shrend with EtOAc (50mL x 2) extraction.Merge organic layer, Na ₂sO ₄dry chromatography of also crossing obtains yellow solid intermediate 2 (1.5g, 84%).

¹H NMR(300MHz,DMSO-d ₆)δ7.82(d,2H),7.68(d,2H),7.46(s,2H),4.45(s,1H).

VC sodium salt (63mg, 0.32mmol) is added, CuSO in the methanol-water solution (6mL/3mL) of compound B-11 (150mg, 0.16mmol) and intermediate 2 (87mg, 0.48mmol) ₄(51mg, 0.32mmol), and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, concentrates and cross chromatography (methanol dichloromethane of 0 ~ 1.5% is as elutriant) to obtain compound as white solid B13 (48.7mg, 27%). ¹H NMR(300MHz,CDCl ₃)δ8.21(m,1H),7.93(m,4H),6.37-6.00(m,4H),5.44-5.30(m,5H),LCMS(m/z)ES-1118(M-H)-.

example 24: the synthesis of compound B-11 4

At 0 DEG C, aqueous sodium hydroxide solution (1.6g, 40mmol) is added, 3-propargyl bromide (2.4g, 20mmol) in the methanol solution (20mL) of SM1 (2.3g, 20mmol).Stirred at ambient temperature compound 4 hours, with hydrochloric acid soln (2N) cancellation, and regulates PH to 3-4, and EtOAc (50mL*5) extracts, concentrated, Na ₂sO ₄dry chromatographic column (the methanol dichloromethane solution of 0-3% is elutriant) of also crossing obtains clear crystal intermediate 1 (1.2g, 40%).LCMS(m/z)ES-154(M+H)+.

VC sodium salt (63mg, 0.32mmol) is added, CuSO in the methanol-water solution (4mL/2mL) of compound B-11 (150mg, 0.16mmol) and intermediate 1 (73mg, 0.48mmol) ₄(51mg, 0.32mmol) and Na ₂cO ₃(51mg, 0.48mmol).Stirring is spent the night, filter mixed liquor, concentrates and cross chromatography (methanol dichloromethane of 2% is as elutriant) to obtain compound as white solid B14 (12.6mg, 7.2%).1H NMR(300MHz,CDCl ₃)δ8.17(m,1H),6.39-5.99(m,4H),5.56-4.87(m,4H)；LCMS(m/z)ES-1090(M-H)-。

the enzymic activity of forms of rapamycin analogs in the present invention

MTOR is a kind of serine/threonine protein kitase, has various kinds of cell reaction regulatory function and comprises Growth of Cells, increment, migration, existence and protein synthesis.MTOR kinase activity is regulated by multiple upstream signal transduction path, the polytype related to cancer of its insufficiency of accommodation.The substrate of the Green Fluorescent Protein of we the anti-phosphorylation 4E-BP1 antibody test phosphorylation mTOR that uses terbium to mark now, TR-FRET method may be used for the screening of mTOR inhibitors in vitro.

Material: damping fluid moiety: 1M HEPES pH7.5, GIBCO, Cat#15630; 1M MgCl2, Sigma, Cat#M1028; 0.5M EDTA, GIBCO, Cat#15575; DTT, Sigma Cat#43819; EGTA, Sigma Cat#E3889; Triton X100, Sigma, Cat#T8787; BSA CALBIOCHEM Cat#126575.

Enzyme, substrate and detection reagent: mTOR:Invitrogen, Cat#PV4753; GFP-4E-BP1:Invitrogen, Cat#PV4759; FKBP12:SinoBiological, Cat#10268-H08E; ATP:Sigma Cat#A26209; Tb-anti-p4E-BP1:Invitrogen, Cat#PV4755; TR-FRET Dilution buffer Invitrogen, Cat#PV3574.

Base plate: compound prepares base plate: 384 hole transparent panels, Corning cat#3657; Analysis plates: black lower volume 384 hole enzyme plate (Greiner Bio-One, Cat#784076).

Process: the preparation of compound concentration gradient solution:

On enzyme plate (healthy and free from worry 3674) with 100%DMSO to compound from 11 different concns scopes, 100uM to 1.7nM (100 μMs, 33 μMs, 11 μMs, 3.7 μM, 1.2 μMs, 411nM, 137nM, 46nM, 15nM, 5nM, 1.7nM) carry out 3 times amount serial dilutions.Then ddH2O dilutes 100%DMSO compound solution to 10 times, and therefore compound concentration is the DMSO solution of 10%.

The typical experimental program of restraining effect measuring the mTOR of the rapamycin derivative in the present invention is as follows:

Assay protocol: measure clause:

Pipette 0.5 μ l 10%DMSO compound solution on the enzyme plate of black lower volume 384 hole, (Greiner Bio-One, Frickenhausen, Germany, cat#784076); Add damping fluid [the 50mM HEPES/NaOHpH 7.5 of 2 μ l mTOR, 5mM MgCl2,1.0mM dithiothreitol, 1mM EGTA, 0.01% (v/v) Triton-X100 (Sigma), 0.01% (w/v] (mTOR, the ultimate density of 0.3125ng/ μ l=> in 5 μ l damping fluids is 0.125ng/ μ l) in assay plate, compound-enzyme mixture 22 DEG C cultivate 15 minutes, make compound be attached in advance enzyme swash reaction start before enzyme on.

Enzyme swashs reaction from adding 2.5 μ l ATP solution (ATP, ultimate density in 200 μMs=>5 μ l test fluid is 10 μMs) and substrate (ultimate density in 0.8 μM=>5 μ l test fluid is 0.4 μM) start in damping fluid, mixture hatch at 22 DEG C after 18 minutes terminate.

Add the 30mM EDTA (EDTA of 5ul, the ultimate density of 30mM=> in 10 μ l test fluid is 15mM) and 4nM Tb-chelate mark anti--4E-BP1 [pT46] phospho-AB [Invitrogen Cat#PV4755] (Tb-traget antibody, the ultimate density of 4nM=> in 10 μ l test fluid is is 2nM) in TR-FRET dilution buffer, terminate reaction, final mixture hatches 1 hour 22 DEG C time, forms the antibody of phosphorylated substrate and Tb-chelate mark.

The amount of phosphorylated substrate is evaluated by the resonance energy changing into GFP from terbium inner complex.Therefore, at envision2104 multiple labeling microwell plate tester (Perkin-Elmer) upper mensuration 340nm Fluorescence emission values, the Fluorescence emission values at rear mensuration 495nm and 520nm place.520nm and 495nm place transmitted value is for calculating the amount of phosphorylated substrate.(enzyme reaction unrestraint=0% suppresses in data normalization process, every otherly not suppress containing mensuration component=100% of enzyme), (equation (1)) IC is calculated with 4 parameter fittings of IDBS xlfit software (ID business solution company, Britain) ₅₀, equation (1):

Y＝Bottom+(TOP-Bottom)/(1+10^((LogIC50-X)*hillslope))

In above equation, Y is enzymic activity percentage ratio, and X is test compounds log concentration value.IC50 is the compound concentration reaching the maximum suppression of half.

In the present invention, the test result of the mTOR retarding effect of forms of rapamycin analogs/derivative is as follows:

inhibition tumor cell is studied

Cell proliferating determining

The impact of different compound on intracellular activity is used luminescence method cell viability detection kit measures ATP, carrys out quantitative assay cytoactive.ATP is living cells metabolize index.Universal test method is: add in containing the culturing cell of serum single reagent ( reagent), cell or separation and Culture medium need not be cleaned.96 holes and 384 porocyte culture plates add detection reagent mixing, and in 10 minutes, the cell count of systems measurement is low to moderate 15, every hole.

Prepared by reagent

By the culture medium culturing various kinds of cell containing the dual anti-10%FBS of 1% penicillin/streptomycin, and containing following suitable additive: DMEM substratum (Gibco, article No.: 11995073) for cultivating intestinal bacteria cancer cells HCT116, breast cancer cell MCF-7 and MDA-MB-231 melanocytoma SK-MEL-28, A549 and epidermis squamous cell carcinoma A431; RPMI-164 substratum is (containing 2Mm L-glutaminate, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM HEPES, the Sodium.alpha.-ketopropionate (Gibco, article No. #72400-120) of 1.0mM) for cultivating U87/MG and 786-O kidney cancer cell; F-12K mixed culture medium (Gibco, article No. #21127) is for cultivating prostate cancer cell PC-3.

Instrument

The multiple labeling microplate reader Envison214 of Perkin Elmer company

Cell culture condition

Above 9 kinds of cells all cultivated for 9 generations with 3000/hole in porous culture plate.

Cell culture medium and culture condition:

Get out compound and cell culture condition: often kind of compound all uses 100%DMSO solution dilution to 10mM storing solution, 2Mm is diluted to again with 100%DMSO, 10 final concentration point (2000 are diluted to subsequently with continuous 5 times of serum-free cell culture medium, 400,80,16,3.2,0.64,0.128,0.0256,0.00512,0.00102 μM), then add 0.5%DMSO (without compound) respectively as maximum dose level control group, 10 μMs of rapamycins are lowest dose level control group.

In each cell culture plate well, add the compound that 0.5 μ l dilutes rear different concns, compound has final 10 concentration (10,2,0.4,0.08,0.016,0.0032,0.00064,0.000128,0.0000256 .00000512 μM).Cell is put into 37 DEG C of incubators and is cultivated 72 hours.In order to ensure the reliability of experiment, the concentration gradient of each compound needs to do 2 parts (Table 1) experiment, and the mensuration of each compound also needs to do 2 times.

Cell reading

Cell cultures, after 72 hours, adds the CellTiter Glo of 50uL in each culture hole, earthquake device shakes 5 minutes, and places 10 minutes to room temperature.By microplate reader analysis of cells quantity.

Data analysis

Cell survival rate is measured by many labels microplate reader.Following formula calculates compound each concentration gradient cell survival rate %: cell inhibitory rate %=100-100* (measurement group-low dosage control group)/(high dosage control group-low dosage control group), wherein test group, low dose group, high dose group is respectively test compounds reading, minimum value, maximum value.

The IC50 value of each test compounds is calculated by formula 2:

Wherein X, Y are given value, and IC50, hillslope, Top and Bottom 4 parameters are produced automatically by analysis software.Y is inhibiting rate, and X is the concentration of compound, the compound concentration of IC50 required for suppression 50% cell survival.Slope is fitting of a curve slope, usually near 1.

All experimental datas are all analyzed by IDBS XLfit5 (ID Business Solutions Ltd., UK).

experimental result and conclusion

The effect of all compounds to often kind of test cancer cells model is all presented in following chart.Curve in chart is lower, and the effect of compound to cancer cells is higher.From the data shown by following each figure, obviously, the chemical combination of all B series shows the different effect to test cancer cells.Some B series compound is very effective, the titer level reached in nanometer concentration range had.

Tumor of urethra Carbazole alkaloid is studied: Fig. 1 and Fig. 2.

A549 lung carcinoma cell suppresses research: Fig. 3, Fig. 4 and Fig. 5.

Melanoma SK-MEL-28 Carbazole alkaloid is studied: Fig. 6, Fig. 7 and Fig. 8.

Epidermal carcinoma A431 tumor models: Fig. 9, Figure 10 and Figure 11.

Glioblastoma U87MG tumour cell is studied: Figure 12, Figure 13 and Figure 14.

Human colorectal tumour HCT116 model research: Figure 15, Figure 16 and Figure 17.

Mammary cancer MDA-MB-231 tumor model: Figure 18, Figure 19 and Figure 20.

Mammary cancer MCD-7 tumor model: Figure 21, Figure 22 and Figure 23.

Prostate cancer PC-3 tumor model: Figure 24, Figure 25 and Figure 26.

adm derivative is to the study on the efficiency of human colon cancer cell (HCT116) model

Object: the object of this research evaluates A15 clinical precursor internal therapy study on the efficiency (positive control) and B series lead compound by oral administration, to the validity delaying or eliminate subcutaneous HCT-116 human colon cancer cell model.

Animal: Balb/c nude mice, female, in 6-8 week, body weight is about 18-20 gram, quantity 70, purchases from VitalRiver Laboratory Animal Technology Co.Ltd..

Tumor inoculation: with PBS (3x106) solution of the HCT-116 tumour cell of 0.1ml, carries out subcutaneous right rib inoculation to each mouse, modeling.When the size of tumour cell about arrives ~ 150mm ³in time, starts its treatment.The testing scheme often organized and the design of size of animal are as following table:

Grouping and treatment

Sequence number

Quantity

Group

Dosage (mg/kg)

Administering mode

Dosage

Treatment plan

1

10

Vehicle

-

p.o.

10μl/g

QD x 21

2	10	A15	9	p.o.	10μl/g	QD x 21
							3	10	B	3	p.o.	10μl/g	QD x 21
4	10	B	9	p.o.	10μl/g	QD x 21
							5	10	B	18	p.o.	10μl/g	QD x 21

Remarks: n-size of animal; Dosage-according to Mouse Weight is with 10 μ l/g administrations; If Mouse Weight alleviates >15%, then need to adjust treatment plan.

Animal divides into groups: before experiment starts, to all mouse weights, measures gross tumor volume.The validity for the treatment of plan can be affected due to gross tumor volume size and guarantee the comparable baseline between group, according to mouse tumor size, random group distribution being carried out to it.

Research terminal: primary endpoint sees that can compound suppress mouse tumor increase or be cured.With slide calliper rule, two-dimensional measurement is carried out to tumor size 2 times weekly, with following formulae discovery volume (mm3): V=0.5a x b ², a and b is respectively the long diameter of tumour and short diameter.Gross tumor volume is for calculating T-C and T/C value.It is that treatment group tumors arrives expection size (as 500mm that T-C calculates with T ³) Median Time (number of days is unit), C controls tumour at the time middle finger (number of days is unit) of formed objects.T/C value (%) is antitumous effect availability indexes, T and C is the average-volume of certain day for the treatment of group and control group respectively.Weight and the photo of tumor cell tissue is collected at the end of experiment.

Experimental endpoints: the terminal of this experiment is that the volume of control group tumour cell reaches 600-1000mm ³.When the deterioration of mouse state continuance or comatose state, euthanasia is performed to mouse.When animal demonstrate serious puzzlement and/or pain time should carry out humanity to it and put to death.When laboratory animal occurs that following situation should implement euthanasia to it:

During the weight of animals decline >20%;

Animal feed or water inlet difficulty;

When the gross tumor volume of the mouse in treatment control group reaches 2000mm ³time tackle all euthanizing animals of all groups.

Data analysis: relatively adopt independently Samples T tests between two groups, adopts One-way ANOVA for the comparison between three groups or more groups.If there is significant F significant difference (ratio for the treatment of variance and error variance), multiple comparisons program should be adopted after variance analysis.By the synergy between LSD or Dunnett ' s T3 method processing data.The data that SPSS 17.0 software analysis is all.P<0.05 is considered to have significant significant difference.

Sum up: as shown in the table, treat after 22 days, 9mg/kg/day (Afinitor from Novartis) the positive control dosage group of compd A 15 significantly can suppress SubQ HCT116 transplantability model of colon cancer, inhibiting rate 63% (* P<0.05), consistent with the result of reported literature.Treat after 22 days, compared with blank group, the inhibiting rate of 3,9, and 18mg/kg/day dosage groups to tumour of compd B is respectively 42%, 57% and 64% (* *, P<0.01).Do not observe significant drug toxicity.These data show, compd B is to suppressing colon cancer cell unusual effect in body.

In Figure 27, number from top to bottom, Article 1 curve (Diamond spot) represents blank group (vehicle), the 3mg/Kg treatment group that Article 2 curve (triangle form point) is compd B 7; Article 3 curve (purple fork-shaped point) represents the 9gm/Kg treatment group of B7, Article 4 (pink is square) represents the 9mg/Kg treatment group of Afinitor, and bottom that line (blue fork-shaped) represents the 18mg/Kg treatment group of B7.

methods for the treatment of

Compound in the present invention, comprises but is not limited to concrete case study on implementation, has immunomodulatory and anti-tumor activity to Mammals (especially the mankind).As immunosuppressor, the compound in the present invention can be used for treatment and epidemic prevention disease mediated, after one's own heart, kidney, liver, marrow, skin, cornea, lung, pancreas, four limbs, muscle, nerve, duodenum, small intestine, the transplant rejection of the organs such as islet cells or tissue rejection; The inhibition opposing host disease caused by bone marrow transplantation; Autoimmune disorder as rheumatoid arthritis, systemic lupus erythematous, Hashimoto thyroiditis, multiple sclerosis, myasthenia gravis, type i diabetes, uveitis, irritated encephalomyelitis, glomerulonephritis etc.Other application comprise treatment and the prevention of inflammation, proliferative skin disorders skin immunization disease mediated, as the dermatitis of psoriasis, allergic dermatitis, contact dermatitis and eczema, seborrheic dermatitis, lichen planus, pemphigus, BP, urticaria, angioedema, vascular inflammation disease, erythema, skin increases addicted to daybreak eosinophile, lupus erythematosus, acne and alopecia areata; Various eye disease (autoimmune disorder and other), as conjunctivitis, vernal conjunctivitis, the clear Te Shi of shellfish is sick, keratitis, herpetic keratitis, keratoconus, malnutritive epithelialis cornea, the uveitis that walleye is relevant and ocular pemphigus etc.In addition, reversible obstructive airways disease, comprise the diseases such as asthma (such as, bronchial asthma, allergic asthma, inherent asthma, external asthma and dust), especially chronic or inveteracy asthma (such as, asthma and respiratory tract overreaction), bronchitis, allergic rhinitis etc. are the specific aim indications of the compounds of this invention.The ischemic disease that mucous membrane and vascular inflammation cause as stomach ulcer, blood vessel injury and thrombosis.In addition the disease such as intimal smooth muscle cells hyperplasia, restenosis angiemphraxis, particularly biological---or mechanical vascular injury, also can be treated by the compounds of this invention or prevent.Other diseases that can be treated comprise ischemic enteropathy, inflammatory bowel, necrotizing enterocolitis, enteritis/allergy as celiaca, rectitis, Eosinophilic Gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; Sacred disease as polymyositis, Guillain Barre syndrome, Meniere (having another name called endolymphatic hydrops), polyneuritis, polyneuritis, Ge-Ba two generation syndromes and radiculopathy; Endocrinopathy, as hyperthyroidism and Exophthalmus goiter; Hemopathy, as pure red cell aplasia, aplastic anemia, undergrown anaemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, granulopenia, pernicious anemia, megaloblastic anemia and red corpuscle occur can not; Osteopathia is as osteoporosis; Respiratory tract disease is as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; Tetter is as ordinary in dermatomyositis, leukodermia, ordinary type ichthyosis, photoallergic susceptibility and cutaneous T cell lymphoma, circulation system disease, as arteriosclerosis, atherosclerosis, aortitis syndromes, and polyarteritis nodosa and myocardosis; Collagen diseases, as scleroderma, wegener granulomatosis and sjogren syndrome; Obesity; Eosinophilic fasciitis; The gums such as periodontal disease pathology, periodontium, alveolar bone and black ossea dentis; Nephrotic syndrome is as glomerulonephritis; Male sex's alopecia areata or alopecia, prevent epilation or provide hair to sprout and/or promote that hair generates and the growth of hair; Muscular dystrophy; Pyoderma and Sezary syndrome; Addison's disease; A Disenshi disease, the organ (after one's own heart, liver, kidney and digestive tube) of such as organ damage as ischemical reperfusion injury occurs in preservation, transplants or ischemic disease (such as, thrombosis and myocardial infarction); Intestinal tract disease, as endotoxic shock medicine or radiation-induced colitis and colitis; Kidney diaseases, as ischemic acute renal insufficiency and chronic renal insufficiency; The pulmonary disorder that toxinosis pulmonary oxygen or medicine (such as, paracort and bleomycin) cause is as lung cancer, pulmonary emphysema; The eye diseases such as cataract, arc-welder's disease, retinitis pigmentosa, sex change, senile macular degeneration SMD, vitreal scar and corneal alkali burn; Dermatitis is as erythema multiforme, linear IgA ballou dermatitis and cement dermatitis; With other as gingivitis, periodontitis, septicemia, pancreatitis, the disease caused by environmental pollution (such as, atmospheric pollution), aging, carcinogenesis, metastatic carcinoma and hypobaropathy; The disease caused by histamine or leukotriene-C4; Behcet's disease is as sick in intestines, blood vessel or neural Behcet's, and Behcet's disease also affects oral cavity, skin, eye, vulva, epididymis, lung, kidney etc.In addition, compound of the present invention may be useful treatment and prevention of liver disease, as immunological disease (such as, chronic auto-immune hepatopathy is as lupoid hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partially hepatectomized, acute severe hepatitis (the such as downright bad toxin caused, viral hepatitis, impact or anoxic), b viral hepatitis, license/hepatitis B, liver cirrhosis (such as alcoholic cirrhosis) and liver failure, as fulminant hepatic failure, late-onset liver failure and chronic-acute attack liver failure (acute hepatic failure chronic hepatic diseases).In addition, they can also strengthen chemotherapy effect, therefore can be used for various disease, as cytomegalovirus infection, particularly infect blood cytomegalovirus, anti-inflammatory is movable, hardening and fibrotic disease are as ephrosis, scleroderma, pulmonary fibrosis, arteriosclerosis, congestive heart failure, ventricular hypertrophy, tissue adhesion, scar, apoplexy, myocardial infarction and injury and ischemia and reperfusion, etc.

In addition, compound of the present invention has FK-506 antagonist properties.Therefore, compound of the present invention can be used for treatment immunosuppression or immunosuppression relative disease.As acquired immune deficiency syndrome (AIDS), cancer, fungi infestation, senile dementia, wound (comprising wound healing, operation and shock) chronic bacterial infection and some central nervous system disorder.The immunosuppressant disease treatment be treated may be caused by the compound of the large ring of excessive use immunosuppression, as 12-(2-cyclohexyl-1-methylvinyl)-13,19,21,27-tetramethyl-11,28-dioxa4-azatricyclo [22.3.1.0.sup.4.9] octacos-18-ene is as FK-506 or rapamycin derivative.Patient recognize forget drug administration and overdose supplements time, this may cause severe side effect.

The compounds of this invention, including, but not limited to concrete case, has anti-tumor activity to Mammals (especially the mankind).Compound of the present invention is as cancer therapy drug, can be used for treatment brain and neurovascular tumour, head and neck cancer, mammary cancer, lung cancer, mesothelioma, lymph cancer, cancer of the stomach, kidney, renal cell carcinoma, liver cancer and liver cirrhosis, ovarian cancer, adenoma endometrioides ovarii, carcinoma of testis, skin carcinoma, melanoma, neuroendocrine tumor, spleen tumor, pancreatic cancer, blood proliferative disease is as Huo Qijin cancer, lymphoma, leukemia, out of contior hyperplasia etc. that cancer cells confusion causes.

The compounds of this invention, can mix as Eudragit with conventional pharmaceutical excipient, and the vehicle of Xylo-Mucine and extraction or synthesis is naturally as the effective formula of medicine of formation.Formulation containing the compounds of this invention can make immediate release dosage form according to needs of medical treatment, slow release formulation, or fixed-point injection formulation.The compounds of this invention also can share with medicine equipment, as support, and foley's tube, the validity to improve medicine equipment such as shaping apparatus.The compounds of this invention be the chief component of medical preparation as local injection formula, or ancillary component is if medicine equipment is in conjunction with low molecule or coating of high molecular polymer, paint coatings or weighting material.

When the compounds of this invention treats vascular restenosis together with foley's tube or holder device, rapamycin plays its therapeutic efficiency by the mammiferous rapamycin target protein of suppression or mTOR, also can bind with FKBP acceptor and play curative effect.

When for above or other treatment, the compounds of this invention for the treatment of significant quantity or can exist with pharmacy acceptable salt, ester or prodrug forms and reaches result for the treatment of with neat compounds.Or compound of the present invention can in conjunction with one or more pharmaceutically acceptable vehicle." the treatment significant quantity " of the compounds of this invention refers to that compound is when being applied to any therapeutic treatment, with the amount being enough to disease therapy of rational benefit/risk value.But the compounds of this invention and other total consumptions every day containing compound component are decided by attending doctor within the scope of reliable medical science judgement.Therapeutic dose for especial patient depends on and many factors, comprises treated illness and the severity of illness; The activity of the particular compound adopted; Concrete drug regimen formula; The age of patient, body weight, general health situation, sex and diet; Administration time, route of administration, and the discharge rate of the specific compound used; The time length for the treatment of; With share and other the well-known factor of medical field similar of other drug.Such as, lower than required dosage begin treatment during the beginning that those of ordinary skill in the art know, then increase dosage gradually and reach required result for the treatment of.

The compounds of this invention is used for the mankind or the every TDD of lower mammal is about 0.01-20mg/kg/day.When for oral administration, preferred dose is 0.001 ~ 3mg/kg/day.If needed, effective per daily dose can administration several times, now, and single dose or unitized dose composition required dosage every day.Local using dosage scope is 0.001-10mg/kg/day, and it depends on use position.When this compound is used for local antiangiogenic and vascular plaque, using dosage is 1mg/mm stent length ~ 100mg/mm stent length.

drug regimen

The drug regimen of the compounds of this invention comprises pharmaceutically acceptable carrier or excipient, can be used for oral, rectum, enteron aisle, brains, vagina, locally (pulvis, ointment agent, drops or scalp patch) administration or as oral or nasal spray.Term " pharmaceutically acceptable carrier " refers to nontoxic solid, semi-solid or liquid filling agent, thinner, the material of any type of encapsulating or formulation auxiliary agents.Term used herein " parenteral " refers to mode of administration, and this pattern comprises intravenously, intramuscular, intraperitoneal, in breastbone, and subcutaneous and intra-articular injection and infusion.

The parenteral injection drug regimen of the compounds of this invention comprises pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion liquid, suspension or emulsion, and sterilized powder is made into aseptic injectable solution or dispersion before the use.Suitable water-based and non-aqueous carrier, thinner, solvent or vehicle comprise water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose and suitable mixture thereof are as vegetables oil (as sweet oil), and injectable organic ester is as ethyl oleate etc.By using coating material as Yelkin TTS, the particle diameter needed for maintaining in dispersion liquid or use tensio-active agent are to form suitable mobility.

These compositions also can comprise adjuvant, as sanitas, and wetting agent, emulsifying agent and dispersion agent.By adding antiseptic-germicide and anti-mycotic agent with prophylaxis of microbial, as added p-Hydroxybenzoate, butylene-chlorohydrin, phenol, Sorbic Acid etc.Also suitable isotonic agent can be added as sugar, sodium-chlor etc.Also can add as required and postpone absorbing material if aluminum monostearate and gelatin are to realize the Prolonged absorption of injectable drug form.

In some cases, in order to prolong drug curative effect, needing slows down delays the absorption of medicine from subcutaneous or intramuscularly.By the suspension of the crystal or amorphous substance that utilize poorly water-soluble to realize medicine delayed absorption.Drug absorption rate depends on its dissolution rate in formulation, and dissolution rate is then depend on crystalline form and crystalline form size.In addition, the medicine delay of administered parenterally absorbs and realizes by medicine is dissolved in or is suspended in oiliness carrier.

Injectable depot formulation can form microencapsule matrices by medicine in biodegradable polymer is as polylactide-polyglycolide.According to medicine and the ratio of polymkeric substance and the character Drug controlled release speed of specific polymkeric substance.The example of other biological degradable polymer comprises poly-(ortho ester) and poly-(acid anhydride).Depot is long-acting penetrate preparation also by pharmaceutical pack is embedded in the liposome of tissue compatible or micro emulsion in prepare.

Injection sterilizing, as by filtering bacterium device for trapping, or when aseptic solid composite, can carry out sterilizing being dissolved in by disinfectant before use or being dispersed in sterilized water or other sterile injectable medium.

Oral dosage form comprises capsule, tablet, pill, pulvis and granule.In these solid dosages, the pharmaceutically acceptable vehicle of active compound and at least one inertia or carrier are mixed, such as Trisodium Citrate or Si Liaodengji dicalcium phosphate feed grade and/or a) weighting agent or extender, as starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid, b) tackiness agent is as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) wetting Agent for Printing Inks is as glycerine, d) disintegrating agent is as agar, calcium carbonate, potato or tapioca (flour), alginic acid, some silicate and sodium carbonate, e) solution retarding agents is as paraffin, f) absorption enhancer is as quaternary ammonium compound, g) wetting agent is as hexadecanol and Zerol, h) absorption agent, such as kaolin and wilkinite, and i) lubricant as talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, Sodium Lauryl Sulphate BP/USP, and their mixture.Except capsule, tablet and pill, this formulation also comprises buffer reagent.

Also vehicle can be used in similar solid dosage formula as the soft matrix of lactose or toffee and high molecular weight polyethylene glycol etc., the weighting agent in semi-solid or hard gelatin capsule or liquid filled capsule.

Solid dosage as tablet, lozenge, capsule, other dressing that pill and granule also can be known with dressing and shell such as enteric coating and other drug formulation art.They also optionally can contain opalizer, also may be the methods that release of active ingredients is released in a certain position with sustained release fashion in enteron aisle.Such as, containing polymkeric substance and wax in embedding composition formula.

Active constituents of medicine also may be microencapsulation form, if needed, mentions suitable vehicle containing more than one or more.

Oral liquid dosage forms, comprises pharmaceutically useful emulsion, solution, suspensoid, syrup and elixir.Except active compound components, liquid dosage form can containing inert diluent conventional in this area as water or other solvent, solubilizing agent and emulsifying agent, such as ethanol, Virahol, ethyl-carbonate, ethyl acetate, benzylalcohol, peruscabin, propylene glycol, 1,3 butylene glycol, dimethyl formamide, oil (particularly Oleum Gossypii semen, peanut oil, Semen Maydis oil, germ oil, sweet oil, Viscotrol C and sesame oil), glycerine, tetrahydrofurfuryl alcohol, polyoxyethylene glycol and fatty acid ester Sorbitol Powder, and their mixture.

Besides inert diluents, in oral dose formulation, can also adjuvant be contained, as wetting agent, emulsifying agent and suspending agent, sweeting agent, seasonings and perfume compound etc.

Suspension agent, in addition to the active compound, can contain dispersion agent, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and Isosorbide Dinitrate, Microcrystalline Cellulose, inclined aluminium hydroxide, wilkinite, aga agar and tragacanth gum, and their mixture.

Topical modes comprises skin, the local application of lung or ocular surface.Topical formulations comprises can be pressed the sucked dry powder maybe can not pressed.Can not in the formula of pressurized powder, active pharmaceutical ingredient be by pharmaceutically can be large to as the inert support component carrier Homogeneous phase mixing of 100 microns with particle diameter.Suitable inert support comprises sugar, as lactose.In theory, effective particle diameter of activeconstituents of 95% is between 0.01to 10 microns.Topical pharmaceutical formulations on skin also comprises ointment, creme, lotion and gel etc.

In addition, also as pressurized in the gas such as nitrogen, liquefied gas auxiliary agent by pressurized gas in formula.In liquefied propellant, active medicine component is not dissolved in its any carrier.This pressurized component also can contain tensio-active agent.Tensio-active agent can be the nonionogenic tenside of liquid or solid or the anion surfactant of solid.The solid anion tensio-active agent of preferred use sodium-salt form.

The another kind of form of topical be used for the treatment of eyes immunologically mediated disease as autoimmune disorder, supersensitivity or inflammatory conditions, and the treatment of corneal transplantation.Chemical combination of the present invention is combined with pharmaceutically acceptable eye carrier, keeping there are enough duration of contact with eye surface as made this compound, making compound penetration to cornea and interior region, as anterior chamber, back room, vitreum, aqueous humor, vitreous humor, cornea, iris, lens, on choroid/retina and sclera.Its pharmaceutically acceptable eye carrier is as ointment, vegetables oil or encapsulating material.

The preferred dosage form formula of rectum or vagina administration mode is suppository or indwelling enema, mix with non-stimulated tax row agent or carrier by the compounds of this invention, as being solid-state under normal temperature but be liquid theobroma oil under body temperature, polyoxyethylene glycol or suppository beeswax etc., therefore can discharge active compound in rectum or vaginal canal.

Compound of the present invention also can liposomal form administration.Known in the industry, liposome is derived by phosphatide or other liposomes usually.Liposome is by the list disperseed in an aqueous medium or multilayer hydration Formation of liquid crystals.Any liposome that can be formed, nontoxic, on physiology, acceptable and metabolizable lipid all can use.Except containing except compound of the present invention, this formula also may containing certain stablizer, and sanitas, composes row agent etc.Preferred liposome is that phosphatide that is natural or that synthesize and phosphatidylcholine (Yelkin TTS) synthesize.Disclosed in the synthetic method of liposome is in this area, see document Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33et seq.

The compounds of this invention also can share with one or more immunosuppressor.Immunosuppressor in the scope of the invention comprises IMURAN ^@azathioprine sodium, brequinar sodium, SPANIDIN ^@tri hydrochloride gusperimus, mizoribine, CELLCEPT ^@mycophenlate mofetil, NEORAL ^@, (registered trademark is SANDIMMUNE to ciclosporin A ^@under sell the ciclosporin A of different ingredients), PROGRAF ^@tacrolimus (also referred to as FK-506), sirolimus and RAPAMUNE ^@leflunomide (also referred to as HWA-486), glucocorticosteroid, as prednisolone and its derivative, Antybody therapy agent is as orthoclone (OKT3) and Zenapax ^@, Anti-thymocyte serum therapeutical agent, as thymus gland sphaeroprotein (thymoglobulins).

Local drug delivery medicine carrying apparatus such as coating stent of medicine or other implanting devices have following advantage: namely by the supporting role of support prevention blood vessel elasticity retraction, reinvent while medicine in order to prevent inner membrance new/hyperplasia or restenosis reduce inflammation and thrombosis.Mode/the unit of this topical or the coronary stent containing compound also have extra therapeutic action, as the administering mode by medicine carrying apparatus has higher Drug content in tissues than systemic administration approach.In addition can also reduce system toxicity by topical modes but have higher Drug content in tissues.Replace being administered systemically by carried stent topical modes, single surgical procedure just can ensure that patient has better compliance.Other benefit as can be reduced the dosage of medicine by medicine carrying mechanotherapy, thus is reduced its drug toxicity and reduces the formation of vascular restenosis, inflammation and thrombus.Local stent treatment therefore by improving antiangiogenic, anti-angiogenic inflammation, antithrombotic and improve the treatment benefit (validity/toxicity) of apparatus or medicine.

Should know that aforesaid detailed description and embodiment are only illustrative, and should not be construed as limitation of the scope of the invention, scope of the present invention only sells claims of annex and the restriction of equivalent thereof.In industry, those skilled in the art are easy to make various changes and modifications published case study on implementation.Do not deviating from or exceeding under the spirit and scope of the present invention, the change and the modification that include, but are not limited to relate to structural formula of compound, substituting group, derivative, intermediate, synthesis path, pharmaceutical formulation of the present invention and/or use method of the present invention can carried out.

Claims

1. a compound as chemical structural formula I or its pharmaceutically useful salt or prodrug,

Wherein A is selected from:

A) hydrogen, alkyl and substituted alkyl, thiazolinyl and substituted alkenyl, alkynyl and substituted alkynyl, cycloalkyl and substituted cycloalkyl, Heterocyclylalkyl and substituted heterocycle alkyl; Wherein substituting group is selected from hydroxyl, alkylsulfonyl, carbonyl, amino, cyano group, halogen, alkoxyl group, aryl and heteroaryl, and

B) aryl and substituted aryl, heteroaryl and substituted heteroaryl; Wherein substituting group is selected from hydroxyl, halogen, amino, carbonyl, cyano group, nitro, alkylsulfonyl, alkyl, alkoxyl group, cycloalkyl and Heterocyclylalkyl.

2. compound as claimed in claim 1, wherein this compound is selected from:

3. a compound as chemical structural formula II or its pharmaceutically useful salt or prodrug,

Wherein B is selected from:

B) aryl and substituted aryl, heteroaryl and substituted heteroaryl; Wherein substituting group is selected from hydroxyl, halogen, amino, carbonyl, cyano group, nitro, alkylsulfonyl, alkyl, alkoxyl group, cycloalkyl, Heterocyclylalkyl.

4. compound as claimed in claim 3, wherein this compound is selected from:

5. one comprises the pharmaceutical preparation of one of compound described in claim 1-4 and pharmaceutical excipient.

6. pharmaceutical preparation as claimed in claim 5, wherein this pharmaceutical preparation is applicable to by oral, and in nose, intravenously, through skin, parenteral, subcutaneous, intramuscular, the mode of intraocular or peritonaeum is to Mammals administration.

7. pharmaceutical preparation as claimed in claim 6, wherein said Mammals refers in particular to people.

8. a method for Therapeutic cancer, comprises one of compound as described in claim 1-4 of the experimenter's administering therapeutic effective dose needing this to treat.

9. method as claimed in claim 8, wherein said cancer is selected from: brain and neural blood vessel knurl, incidence cancer, mammary cancer, lung cancer, mesothelioma, lymphoma, cancer of the stomach, kidney, renal cell carcinoma, liver cancer and liver cirrhosis, ovarian cancer, endometriosis of ovary, carcinoma of testis, skin carcinoma, melanoma, neural and all internal secretion cancers, spleen cancer, carcinoma of the pancreas, blood proliferative disorders, as Huo Qijin cancer, lymphoma, leukemia and any Cancerous disease caused by uncontrolled cell proliferation.

10. treatment or the disease mediated method of epidemic prevention, comprise one of experimenter's compound as described in claim 1-4 using dose therapeutically effective for the treatment of to needs.

11. methods as claimed in claim 10, wherein said immunologically mediated disease is selected from: heart, kidney, liver, marrow, skin, cornea, lung, pancreas, four limbs, muscle, neural, duodenum, small intestine, pancreas or islet cells and the versus-host disease caused by bone marrow transplantation.

12. methods as claimed in claim 10, wherein said immunologically mediated disease is rheumatoid arthritis, systemic lupus erythematous, Hashimoto thyroiditis, multiple sclerosis, myasthenia gravis, type i diabetes, uveitis, allergic encephalomyelitis or glomerulonephritis.

13. methods as claimed in claim 10, wherein said immune-mediated disease is the graft versus host disease (GVH disease) caused by bone marrow transplantation.

14. to produce with one of compound described in claim 1-4 and are used for the medicine of Therapeutic cancer or immune-mediated disease.

15. purposes as claimed in claim 14, wherein said cancer is selected from: brain and neural blood vessel knurl, incidence cancer, mammary cancer, lung cancer, mesothelioma, lymphoma, cancer of the stomach, kidney, renal cell carcinoma, the liver cirrhosis selected in the group of liver cancer and liver, ovarian cancer, endometriosis of ovary, carcinoma of testis, skin carcinoma, melanoma, neural and all internal secretion cancers, spleen cancer, carcinoma of the pancreas, blood proliferative disorders, as Huo Qijin cancer, lymphoma, leukemia and any Cancerous disease caused by uncontrolled cell proliferation; Wherein said immunologically mediated disease is selected from: heart, kidney, liver, marrow, skin, cornea, lung, pancreas, four limbs, muscle, neural, duodenum, small intestine, pancreas or islet cells and the versus-host disease caused by bone marrow transplantation.