CN1229339C

CN1229339C - Inhibiting angiogenesis and tumor growth

Info

Publication number: CN1229339C
Application number: CNB018097448A
Authority: CN
Inventors: D·L·博格; D·A·彻雷什
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2000-03-27
Filing date: 2001-03-27
Publication date: 2005-11-30
Anticipated expiration: 2021-03-27
Also published as: AU2001249499B2; MXPA02009504A; KR20020084258A; HUP0301797A2; SK14842002A3; JP2003528850A; PL358272A1; KR100767616B1; CZ20023509A3; KR20020091156A; CN1245967C; RU2002128751A; KR100776185B1; HUP0301621A2; CA2403871A1; CN1441777A; HUP0301621A3; CA2403630A1; WO2001072699A1; EP1272173A4

Abstract

Compounds which inhibit tumor growth and angiogenesis, of general formula (II) are provided. These compounds include glycyl lysine derivatives bound to a central aromatic linking core.

Description

The compound that suppresses vasculogenesis and tumor growth contains the medicinal preparations of this compound and the pharmaceutical applications of this compound

Invention field

The present invention relates to suppress the composition of vasculogenesis and tumor growth.More precisely, the present invention relates to integrin binding α _vβ ₃And blocking-up beta 2 integrin alpha _vβ ₃With the interactional composition of matrix metalloproteinase 2 (MMP2).The invention still further relates to the application beta 2 integrin alpha _vβ ₃Method with MMP2 bonded selective depressant inhibition vasculogenesis and tumor growth.

Background of invention

Vascular cell is invaded tissue and need be comprised the proteolytic enzyme of extracellular matrix structural remodeling and the cooperative interaction of numerous factors of discerning the cell adhesion molecule of this interim matrix.Nearest report points out that 72kDa matrix metalloproteinase 2 (MMP2) is a kind of crux participant in blood vessel generation and vasculogenesis.For example, Z such as Kitoh (J.Cell Sci., 109,953-8 (1996)) report, MMP2 and activator I type film-matrix metalloproteinase (MT1-MMP) thereof are almost only expressed by mesenchymal cell during fetal development synchronously, show that the particular substrate reconstruction only limits to these tissues.In addition, vasculogenesis and corresponding tumor growth reduce (referring to Itoh etc., Cancer Res., 58 1048-51 (1998)) in the MMP2 knock-out mice.What is interesting is Saftor etc. (Proc.Natl.Acad.Sci.U.S.A., 89,1557-61 (1992)) description taken in conjunction beta 2 integrin alpha _vβ ₃The generation that (itself is known vasculogenesis medium) induces MMP2 shows these two kinds of molecules cooperative interaction (seeing Bafetti etc. in addition, J.Biol.Chem., 273,143-9 (1998)) in the reconstructing blood vessel process relevant with vasculogenesis.In fact, Brooks etc. has proved MMP2 and beta 2 integrin alpha _vβ ₃Between direct interaction (Cell, 85,683-93 (1996)).Brooks etc. had proved again afterwards that the negative adjusting of MMLP2 depended on α in involvement of blood vessel and ripening process _vβ ₃Expression (Cell, 92,391-400 (1998)).

Can suppress vasculogenesis and the property followed inhibition tumor growth though put down in writing the natural inhibitor and the synthetic inhibitor of MMP (comprising MMP2), but the application of these strategies on clinical mode has limited success, mainly is because due to the toxic side effect of this class wide spectrum inhibitor.Generally speaking, in adult organism, owing in many processes, may need the MMP function, so the inhibition at enzyme functionally active position may have far-reaching influence to the various bioprocesss (for example wound healing) that participate in tissue reconstruction.In fact, put down in writing in clinical study and caused severe side effect with the various cancer types of wide spectrum MMP inhibitor for treating, comprise struvite tendonitis, polyarthritis and musculoskeletal pain syndrome, these diseases are dose limitation and still lasting usually after therapy discontinued.Known beta 2 integrin alpha _vβ ₃Limited distribution in adult organism, however people predict MMP2 and α _vβ ₃Between interaction be limited to neovascularization or cell invasion position, should correspondingly limit this class and treat xicity related effect.In fact, the reorganization non-catalytic carboxyl terminal Hemopexin structural domain (PEX) of MMP2, its mediation MMP2 and beta 2 integrin alpha _vβ ₃Combination, showing has angiogenesis inhibitor and anti-tumor activity in vivo.The big segmental potential utility of this albumen but need the actual solution of this problem with a plurality of shortcomings (for example mass production problem, FDA quality and Security Control Problem and antigenicity) promptings.

Therefore, need optionally suppress the MMP activity of tumor location and minimum degree suppresses the compound of the MMP at other position of body.

Summary of the invention

The invention provides the novel cpd that can be used as vasculogenesis and tumor growth inhibitor.The present invention also provides and suppresses MMP2 and beta 2 integrin alpha _vβ ₃Interactional method and inhibition contain beta 2 integrin alpha _vβ ₃The method of cell vasculogenesis.In addition, the invention provides by giving MMP2-α _vβ ₃Make the method for inhibitors to inhibitor tumor growth mutually.

The compounds of this invention is represented by following formula (I), and is comprised and the chemical glycyl lysine derivative that is connected of linking group:

G wherein ¹And G ²Be-NH-C (O)-O-R independently of one another ¹,-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹,-NH-C (O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹,-O-C (O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹,-O-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹Or-NH-C (O)-CH ₂-(C ₆H ₄)-X ¹Y ¹And Y ²Be OH, C independently of one another ₁-C ₄Alkyl, C ₁-C ₄Hydroxyalkyl, C ₁-C ₄Alkoxyl group, phenyl, benzyl or-NH ₂R ¹Be C ₁-C ₄Alkyl; X ¹Be halogen, nitro, C ₁-C ₄Alkyl, C ₁-C ₄Alkoxyl group or C ₁-C ₄Perfluoroalkyl; Z is-C ≡ C-,-C ₆H ₄-, cis-CH=CH-, trans-CH=CH-, cis-CH ₂-CH=CH-CH ₂-, trans-CH ₂-CH=CH-CH ₂-, 1,4-naphthyl, cis-1,3-cyclohexyl, anti-form-1,3-cyclohexyl, cis-1,4-cyclohexyl or anti-form-1,4-cyclohexyl; A is H or covalent linkage; M and n are the integer of 0 or 1 numerical value independently of one another; T is the integer of 0 or 1 numerical value; V is the integer of 1 or 2 numerical value; Condition is when A is H, and t is 0; When A was covalent linkage, t was 1; When m is 0, Y ¹Be C ₁-C ₄Hydroxyalkyl; When n is 0, Y ²Be C ₁-C ₄Hydroxyalkyl.These compounds and α _vβ ₃In conjunction with and suppress MMP2 and α _vβ ₃Interaction.

The preferred compound of structural formula (I) is represented with structural formula (II):

R wherein ²And R ³Be H, C independently of one another ₁-C ₄Alkyl, phenyl or benzyl; X ²And X ³Be halogen, nitro, C independently of one another ₁-C ₄Alkoxyl group, C ₁-C ₄Alkyl or C ₁-C ₄Perfluoroalkyl; A is H or covalent linkage; T is the integer of 0 or 1 numerical value; Condition is when A is H, and t is 0, and when A was covalent linkage, t was 1.When A is covalent linkage and t when being 1, described glycyl lysine derivative part can with the benzene linking group at the ortho position, a position or contraposition be connected.

When formula (I) compound and formula (II) compound are contained α _vβ ₃Cell the time, α _vβ ₃Be suppressed with combining of MMP2, thereby disturb the essential machine-processed of vasculogenesis.Thereby disturb vasculogenesis also can make tumour lack nutrition and suppress tumor growth by the vascularization that stops tumour.Therefore, the The compounds of this invention of inhibition vasculogenesis and tumor growth is the effective curative that is used for treating the patient who suffers from tumour or angiogenic disease.Because The compounds of this invention and α _vβ ₃In conjunction with, so also can be with these compound inflammation-inhibiting processes.

The compounds of this invention can be formulated in the suitable pharmaceutically acceptable matrix, can be used to treat tumour and other and comprises the medicinal compositions of the disease that does not need vasculogenesis to provide.

Aspect a method of the present invention, the medicinal compositions that contains formula (I) compound and formula (II) compound prepares by described compound is formulated in the pharmaceutically acceptable matrix.Can give tumour patient with the medicinal compositions of described active compound, to lower or the elimination tumor growth.Can be by injection or by infusion or give described active compound gradually at the appointed time by any other method parenteral that is suitable for particular dosage form.

The accompanying drawing summary

In the accompanying drawing:

Fig. 1 is with schematic view illustrating MMP2 and beta 2 integrin alpha _vβ ₃Interaction and the effect in vasculogenesis thereof and suppress MMP2 and α such as the antagonist of The compounds of this invention _vβ ₃Interaction.

Fig. 2 A be shown in solid phase in conjunction with inhibitor formula (I) compound in measuring to MMP2 and beta 2 integrin alpha _vβ ₃Interactional influence.

Fig. 2 B formula (I) compound and beta 2 integrin alpha _vβ ₃And α ₅β ₁Combination.

Fig. 2 C relatively The compounds of this invention and control compound in conjunction with α _vβ ₃Influence.

Fig. 2 D comparing amino acid residue RGD is to The compounds of this invention and α _vβ ₃Bonded influence and RGD are to bVN and α _vβ ₃The bonded influence.

Fig. 3 A illustrates β ₃Positive cell and β ₃Protease activity in the negative cells.

Fig. 3 B illustrates β ₃Positive cell and β ₃MMP2 combination in the negative cells.

Fig. 4 A is that chicken CAM organizes medium vessels to generate the microphotograph that suppresses.

Fig. 4 B is with the angiogenesis suppression action in the schematic view illustrating chicken CAM tissue.

Fig. 4 describes the MMP2 level in treated and the undressed cell.

Fig. 5 A is the microphotograph of growth of chicken CAM tissue tumor and vascular system.

Fig. 5 B is the microphotograph of chicken CAM tissue blood vessel density.

Fig. 5 C is with the tumor weight in the schematic view illustrating chicken CAM tissue.

Fig. 5 D is with the vascularization in the schematic view illustrating chicken CAM tissue.

Fig. 5 D is the microphotograph of chicken CAM tissue tumor cell density.

Detailed Description Of The Invention

MMP2 and beta 2 integrin alpha_vβ ₃Combination be an important machine in the angiogenesis System. The specificity of this binding interactions suppresses to cause the tumour medium vessels for example in growing tissue Form and reduce, thereby stop tumor growth. MMP2 and beta 2 integrin alpha_vβ ₃Interaction In Fig. 1, schematically illustrate. The Angiogenesis that one class is new and tumor growth inhibitor are hereinafter The small molecular antagonists of describing, described antagonist disturbs MMP2 and integrin specifically α_vβ ₃Combination, thereby a kind of important new treatment tool is provided.

Some compound of the present invention can have one or more asymmetric centers, and can To exist with the optically-active form. In such as the substituting group of alkyl, may exist other asymmetric The center. The racemic mixture of pure S-isomers and pure R-isomers, described isomers, with And their mixture comprises within the scope of the invention. Considered some compound of the present invention The chirality form, and specifically comprise within the scope of the invention.

Term " alkoxyl " refer to the oxygen atom that connects by ehter bond and with undefined, refer to The alkyl of sizing. The example of alkoxyl is methoxyl group, ethyoxyl, tert-butoxy etc. Art Language " alkyl " refers to specify the straight or branched carbon-based group of size. Typical alkyl be methyl, Ethyl, n-pro-pyl, isopropyl, normal-butyl, sec-butyl, isobutyl group, the tert-butyl group, 2-ethyl Hexyl, n-octyl, 2,4-dimethyl amyl group etc. Term " hydroxyalkyl " refers to be connected with hydroxyl , the alkyl of the appointment size of above definition. Example comprises methylol, 2-ethoxy, 3-hydroxyl Base-1-propyl group, 2-hydroxyl-1-propyl group, 4-hydroxyl butyl etc.

Term " perfluoroalkyl " refer to undefined specify size, carry and replace each The alkyl of the fluoro substituents of hydrogen, for example trifluoromethyl and pentafluoroethyl group.

Term " halogen " or " halogen " refer to bromine, chlorine, fluorine and iodine.

The compounds of this invention is represented by following formula (I), and comprises and the chemical glycyl lysine derivative that is connected of linking group:

G wherein¹And G²Be-NH-C (O)-O-R independently of one another¹、 -NH-C(O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹、-NH-C(O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹、 -O-C(O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹、-O-C(O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹Or-NH-C (O)-CH₂-(C ₆H ₄)-X ¹； Y ¹And Y²Be OH, C independently of one another₁-C ₄Alkyl, C₁-C ₄Hydroxyalkyl, C₁-C ₄Alkoxyl, phenyl, benzyl or-NH₂；R ¹Be C₁-C ₄Alkyl; X¹Be halogen, nitro, C₁-C ₄Alkyl, C₁-C ₄Alkoxyl or C₁-C ₄Perfluoroalkyl; Z is-C ≡ C-,-C₆H ₄-, cis-CH=CH-, trans-CH=CH-, cis-CH₂-CH＝CH-CH ₂-, trans-CH₂-CH＝CH-CH ₂-, Isosorbide-5-Nitrae-Naphthyl, cis-1,3-cyclohexyl, anti-form-1,3-cyclohexyl, cis-Isosorbide-5-Nitrae-cyclohexyl or anti-form-1,4-Cyclohexyl; A is H or covalent bond; M and n are the integer of 0 or 1 numerical value independently of one another; T is the integer of 0 or 1 numerical value; V is the integer of 1 or 2 numerical value; Condition is when A is H, T is 0; When A was covalent bond, t was 1; When m is 0, Y¹Be C₁-C ₄Hydroxyalkyl; When n is 0, Y²Be C₁-C ₄Hydroxyalkyl.

G preferably¹And G²For-NH-C (O)-O-(CH₂) _v-(C ₆H ₄)-X ¹，Y ¹And Y²Be OH, M and n are 1. X¹Be preferably C₁-C ₄Perfluoroalkyl most preferably is trifluoromethyl. In structure Preferred compound is by structural formula (II) expression in the scope of formula (I), and be included in the ortho position, The glycyl lysine derivative that position or contraposition are connected with the benzene linking group:

R wherein ²And R ³Be H, C independently of one another ₁-C ₄Alkyl, phenyl or benzyl; X ²And X ³Be halogen, nitro, C independently of one another ₁-C ₄Alkoxyl group, C ₁-C ₄Alkyl or C ₁-C ₄Perfluoroalkyl; A is H or covalent linkage; T is the integer of 0 or 1 numerical value; Condition is when A is H, and t is 0; When A was covalent linkage, t was 1.When A is covalent linkage and t when being 1, described glycyl lysine derivative part can with described benzene linking group at the ortho position, a position or contraposition be connected.

Preferred substituents X ²And X ³At CH with respect to benzyl ₂The 4-position of (being para-orienting group) is connected with the phenyl ring of described benzyl moiety.Preferred X ²And X ³Group is C ₁-C ₄Perfluoroalkyl is most preferably to trifluoromethyl.Preferred R ²And R ³Group is hydrogen and methyl.Substituent X ²And X ³Can be identical or different, substituent R ²And R ³Also can be identical or different.

Wherein A be covalent linkage and t be 1 be the compound of following compound 1 expression by a kind of activated especially compound in the compound family of formula (II) expression:

(compound 1)

Formula (I) compound and formula (II) compound can calm facile material synthesize with the synthesis step of medium number.For example, flow process 1 has shown the synthetic of compound 1, and the universal method of explanation production formula (II) compound, and wherein A is a covalent linkage, R ²And R ³Identical, X ²And X ³ Identical.Flow process 1 is synthesizing of Ming Dynasty style (II) compound furtherly, wherein X ²And X ³Be to trifluoromethyl R ²And R ³Be methyl or hydrogen.

Flow process 1.

In flow process 1, will trifluoromethyl-benzyl-alcohol and the reaction of two succinimdyl carbonates be formed the Acibenzolar intermediate, and then react with N-ε-BOC-lysine methyl ester, obtain compound 2 (yield 99%).Hydrolysis BOC protecting group subsequently with the coupling of BOC-glycine, obtains compound 3 (yield 96%).The BOC protecting group of acidolysis compound 3 obtains compound 4 (yield 99%).With 2 normal compounds 4 and the coupling of isophthaloyl dichloro, obtain compound 5 (yield 68%), be equivalent to formula (II), wherein R ²And R ³Be methyl, X ²And X ³For to trifluoromethyl, A is a covalent linkage.With lithium hydroxide hydrolysis compound 5, obtain compound 1 (yield 93%).

Having the synthetic of other R and the substituent formula of X (I) compound and formula (II) compound and can realize that described modification is conspicuous for the technician in synthetic chemistry field as the analogue of compound 1 by flow process 1 is made amendment.Use have except that to the substituting group the trifluoromethyl (for example with respect to described benzyl CH ₂Halogen in ortho position, a position or contraposition, nitro or other C ₁-C ₄Perfluoroalkyl) benzylalcohol or use have the protection Methionin ester of the ester group except that methyl, will obtain other compound of formula (II).

Have and remove 1, the synthesis example of formula (I) compound of the Z group beyond the 3-phenyl is as realizing by the isophthaloyl dichloro that replaces in the flow process 1 with other two acyls dichloro compound (for example two chloroformyl acetylenes, fumaryl dichloro, phthalyl dichloro).Perhaps, can not use described chloride of acid, and use corresponding acid, the coupling of described acid and suitable amine can realize by standard peptide coupling technology well known in the art.Skilled in the art will readily recognize that other chemical substance substituent that can prepare with the synthetic method that is shown in flow process 1 and flow process 2, but other compound of the described compound group shown in synthesis type (I) and the formula (II).Boger etc. are at J.Am.Chem.Soc., and 123, introduce the synthetic of related compound among the 1280-1288 (2001) and be applicable to the useful chemistry strategy of synthesis type (I) compound and formula (II) compound.

By method shown in the flow process 1 is made amendment, also can synthesis type (II) compound, wherein A is a covalent linkage, t is 1, perhaps R ²With R ³Difference, perhaps X ²With X ³Difference, perhaps R ²/ R ³And X ²/ X ³All different, described modification is conspicuous for those skilled in the art.For example; can be with 1 normal compound 4 and isophthaloyl dichloro or any suitable isophthaloyl halogenide or active ester reaction; the described second activation acyl group can be sequentially and the analogue of compound 4 (having different X groups, perhaps different R group, or both are all different) reaction.Equally, two kinds of analogues of compound 4 (have different X groups, perhaps different R group, or both are all different) can be sequentially and isophthaloyl dichloro or same activatory isophthalic acid ester reaction, obtain having other analogue of the compound 1 of different X and R group.

In following flow process 2, illustrate that with the example that synthesizes of compound 6 and compound 7 A wherein is that hydrogen, t are the synthetic of 0 formula (II) compound:

Flow process 2

In flow process 2, compound 4 and Benzoyl chloride reaction with flow process 1 obtain compound 6 (R ²=methyl; X ²=to trifluoromethyl) (yield 90%).With the ester group of lithium hydroxide hydrolysis 6, obtain compound 7 (R ²=H, X ²=to trifluoromethyl) (yield 88%).

To on described benzyl, having different R groups (other C for example ₁-C ₄Alkyl) and/or the analogue with the substituent compound 4 of different X carry out benzoylation, obtain other compound of the formula (II) of A=H and t=0.

In addition, explanation compound 8 (compound 6 and compound 7 non-activity analogue) is synthetic in flow process 2, and the benzoyl that is connected with the glycine unit of formula (II) in the compound 8 is partly replaced by the glycol ether acid amides.By compound 4 and diglycollic acid coupling, obtain compound 9 (yield 77%).

Following flow process 3 explanation compounds 12, it is the non-activity analogue of compound 1, its Chinese style (II) the trifluoromethyl carbobenzoxy-(Cbz) is replaced by benzamide.

Flow process 3

Aspect a method of the present invention, can be by described compound being formulated in the medicinal preparations that comes preparation formula (I) compound and formula (II) compound in the pharmaceutically acceptable carrier matrix.The medicinal compositions that will comprise described active formula (I) compound and formula (II) compound gives tumour patient, to lower or the elimination tumor growth.Described active compound can be by injection or infusion and parenteral gives gradually at the appointed time.Though need that organizing of treatment is the most common treats by intraperitoneal or subcutaneous administration, described active compound also can intraocular, in the intravenously, intramuscular, synovial membrane, in the chamber or transdermal administration, and can pass through the transmission of creeping motion type means.

Term used herein " gives " compound of the present invention or composition is meant system applies, for example need to adopt oral, parenteral approach to give, give by sucking spraying, give or topical administration contains the dosage forms unit preparation of conventional nontoxic pharmaceutically acceptable carrier, auxiliary material and solvent by nasal cavity, rectum or buccal approach.Term used herein " parenteral " comprises intravenously, intramuscular, intraperitoneal, breastbone is interior, subcutaneous and joint cavity injection and infusion techniques.

So-called " pharmaceutically acceptable ", it is meant such salt, acid amides and ester: in rational medical judgment scope, be applicable to and contact tissue and lower animal tissue and do not have over-drastic toxicity, pungency, transformation reactions etc., and have rational interests/risk ratio, be effective to treat tumour and and angiogenesis-associated diseases.

Pharmacy acceptable salt is well-known in the art.For example S.M Berge etc. is at J.Pharmaceutical Sciences, and 66, the pharmacy acceptable salt of introducing in detail among the 1-19 (1977).Typical acid salt comprises hydrochloride, hydrobromate, vitriol, hydrosulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, lauroleate, borate, benzoate, lactic acid salt, phosphoric acid salt, tosylate, mesylate, Citrate trianion, maleate, fumarate, succinate, tartrate, ascorbate salt, glucose enanthate, Lactobionate, dodecyl sulfate etc.Typical an alkali metal salt or alkaline earth salt comprise sodium salt, calcium salt, sylvite, magnesium salts etc.

That term used herein " pharmaceutically acceptable carrier " is meant is nontoxic, the auxiliary of inert solid, semisolid or liquid filling agent, thinner, encapsulate capsule material or any kind.The part example that can be used as the described material of pharmaceutically acceptable carrier is sugar, for example lactose, dextrose plus saccharose; Starch, for example W-Gum and yam starch; Mierocrystalline cellulose and derivative thereof, for example sodium cellulose glycolate, ethyl cellulose and cellulose acetate; The powdery tragakanta; Fructus Hordei Germinatus; Gelatin; Talcum powder; Vehicle, for example theobroma oil and suppository wax; Oil, for example peanut oil, oleum gossypii seminis, Thistle oil, sesame oil, sweet oil, Semen Maydis oil and soya-bean oil; Di-alcohols, for example propylene glycol; Polyvalent alcohol, for example glycerine, sorbyl alcohol, N.F,USP MANNITOL and polyoxyethylene glycol; Ester class, for example ethyl oleate and Laurate ethyl; Agar; Buffer reagent, for example magnesium hydroxide and aluminium hydroxide; Alginic acid; Apirogen water; Isotonic saline solution; RingerShi solution; Ethanol and phosphate buffer solution and other used nontoxic compatible material in medicinal preparations.

Judgement according to the makers-up, in described composition, also can there be wetting agent, emulsifying agent and lubricant, for example sodium lauryl sulphate and Magnesium Stearate, and tinting material, releasing agent, Drug coating, sweeting agent, correctives and perfume compound, sanitas and antioxidant.The example of pharmaceutically acceptable antioxidant comprises water soluble antioxidant, for example xitix, cysteine hydrochloride, sodium bisulfite, Sodium Pyrosulfite, S-WAT etc.; Oil-soluble inhibitor, for example ascorbyl palmitate, butylated hydroxyanisol (BHA), Yoshinox BHT (BHT), Yelkin TTS, gallic acid propyl ester, alpha-tocopherol etc.; And metal chelator, for example citric acid, ethylenediamine tetraacetic acid (EDTA) (EDTA), sorbyl alcohol, tartrate, phosphoric acid etc.

" the treatment significant quantity " of medicine of the present invention or compound is meant with the q.s of the suitable interests that are suitable for any therapeutic treatment/risk than the described compound of treatment tumour and angiogenesis-associated diseases.Yet people know that the total daily dosage of The compounds of this invention and composition will be by the attending doctor through reasonably medical judgment decision.Any concrete patient's concrete treatment effective dose level depends on various factors, comprises the similar factor that the medicine of excretion rate, treatment time length, combined utilization of concrete composition, patient's age, body weight, general health situation, sex and diet, administration time, route of administration and employed particular compound of particular compound activity, use of severity, the use of the illness of being treated and illness or the medicine that uses simultaneously with used particular compound and medical field are known.

The present invention also provides the medicinal compositions of unit dosage, and described medicinal compositions comprises one or more The compounds of this invention and the conventional pharmaceutical carrier for the treatment of significant quantity.Injection formulations, for example sterile water for injection suspensoid or oily suspensoid can use suitable dispersion agent or wetting agent and suspension agent to prepare according to known technology.Aseptic injection preparation also can be solution, suspension or the emulsion of the sterile injectable in nontoxic parenteral acceptable diluent or solvent, for example 1,3 butylene glycol solution.Wherein operable described acceptable solvent and solvent are water, RingerShi solution, U.S.P and isotonic sodium chlorrde solution.In addition, sterile non-volatile oils class routine is used as solvent or suspension medium.For this reason, any bland nonvolatile oil be can use, synthetic glycerine monoesters or triglyceride comprised.

In addition, in described injection formulations, use lipid acid, for example oleic acid.Described injection formulations can perhaps mix sterilant for example by the membrane filtration of detention bacterium in facing with preceding available sterilized water or other aseptic injection medium dissolves or dispersive aseptic solid composite form, can make injection formulations aseptic.

For the effect of prolong drug, need slow down the drug absorption of subcutaneous injection or intramuscularly usually.Most common form is to realize by the crystallization of injection poorly water-soluble or the suspension of amorphous substance.The uptake rate of medicine depends on the dissolution rate of medicine so, and dissolution rate may depend on the physical condition of medicine, for example crystallographic dimension and crystal formation.The another kind of method that postpones drug absorption is that medicine gives with oil solution or oil suspension.Also can be by making for example polylactide-poly-glycollide formation micro-capsule skeleton of medicine and biodegradable polymer, preparation injection reservoir devices formulation.According to the ratio of medicine and polymkeric substance and the composition of described polymkeric substance, can control drug release speed.The example of other biodegradable polymer comprises poly--ortho ester and polyanhydrides.Also pharmaceutical pack can be embedded in the liposome or micro emulsion compatible preparation reservoir devices injection formulations with body tissue.

Can for example theobroma oil and polyoxyethylene glycol mix by making described medicine and suitable non-irritating excipient, preparation is used for the suppository of rectal administration, described vehicle is solid at normal temperatures, but is liquid under rectal temperature, therefore discharges described medicine in the internal rectum fusion.

The solid dosage that is used for oral administration can comprise capsule, tablet, pill, pulvis, prills and granule, in such solid dosage, and can for example sucrose, lactose or starch mix with at least a inert diluent with described active compound.According to conventional practice, such formulation can also comprise other material except that inert diluent, for example film-making lubricant and other film-making auxiliary agent, for example Magnesium Stearate and Microcrystalline Cellulose.If be capsule, tablet and pill, described formulation can also comprise buffer reagent.In addition, also available enteric coating and other control release clothing sheet prepare tablet and pill.This class vehicle of utilization such as lactose or toffee and high molecular weight polyethylene glycol etc., the solids composition of similar type also can be used as the weighting material of filling soft gelatin capsule and filling hard gelatin capsule.

That the liquid dosage form that is used for oral administration can comprise is pharmaceutically acceptable, contain this area inert diluent commonly used for example emulsion, microemulsion, solution, suspensoid, syrup and the elixir of water.Such composition can also comprise auxiliary material, for example wetting agent; Emulsifying agent and suspension agent; Sweeting agent, correctives and perfume compound.If desired, The compounds of this invention can be incorporated into slowly-releasing or targeted delivery of drugs system, for example polymer backbone, liposome and microsphere.Can perhaps in the aseptic solid composite form of facing, mix sterilant for example by the membrane filtration of detention bacterium, can make described preparation aseptic with preceding available sterilized water or some other aseptic injection medium dissolves.Described active compound also can be for having the microencapsulation form of aforesaid one or more vehicle.

Can with dressing and housing for example other Drug coating of knowing of enteric coating and medicine formulation art prepare tablet, dragee, capsule, pill and granule solid dosage.They can be chosen wantonly and comprise opalizer, and can be such composition: they optional with delayed mode only or preferably discharge described effective constituent at certain partial enteral.The example of operable embedding composition comprises polymer material and wax.The formulation that is used for topical administration or transdermal administration The compounds of this invention also comprises ointment, paste, missible oil, washing lotion, gelifying agent, pulvis, solution, sprays, inhalation or patch.Described effective constituent is mixed with buffer reagent that pharmaceutically acceptable carrier and any required sanitas maybe may need under aseptic condition.

Ophthalmic preparation, ear drop, Eye ointments, pulvis and solution have also been considered within the scope of the invention.Described ointment, paste, missible oil and gelifying agent remove and contain active ingredient beyond the region of objective existence of the present invention, can also contain vehicle, for example the fat of animal and plant, oil, wax, paraffin, starch, tragakanta, derivatived cellulose, polyoxyethylene glycol, silicone, bentonite, silicic acid, talcum powder and zinc oxide or their mixture.

Pulvis and sprays can also contain vehicle except that containing The compounds of this invention, for example the mixture of lactose, talcum powder, silicic acid, aluminium hydroxide, Calucium Silicate powder and Silon or these materials.In addition, sprays can also contain conventional propellent, for example chlorofluorocarbon.

Transdermal patch has to provide makes the attendant advantages of compound sustained release to body.By described compound being dissolved or disperseing, can prepare such formulation in appropriate medium.Also can use absorption enhancer to increase described compound and pass through skin.Rate controlling membranes that its speed can or be provided control or by making described compound in polymer backbone or gel, disperse to control.

With with the mode of drug-delivery preparation coupling with contain the composition of described active compound with the treatment significant quantity.The dosage and the time that gives depend on that host to be treated, host system utilize the ability of described effective constituent and the degree of required treatment effect.The accurate amount of the described effective constituent that need give depends on attending doctor's judgement, and all is distinctive for each is individual.

Herein disclosed is the suitable dose scope that is used for system applies, described dosage range depends on route of administration.Suitable dosage regimen also is transformable, but is generally after first administration, with one or more predetermined spaces by follow-up injection or other route of administration repeat administration.

The present invention also provides the medicinal compositions that can be used for implementing methods of treatment described herein.Described composition contains active compound mentioned above and pharmaceutically acceptable carrier.

Be used for the preparation that parenteral gives The compounds of this invention or composition and comprise the aseptic aqueous solution or non-aqueous solution agent, suspensoid and emulsion.Examples of non-aqueous is a for example sweet oil and injectable organic ester ethyl oleate for example of propylene glycol, polyoxyethylene glycol, vegetables oil.Aqueous carrier comprises water, alcoholic solution/aqueous solution, emulsion or suspension, comprises salt solution and buffering medium.Former solvent comprises sodium chloride solution, RingerShi glucose, glucose and sodium-chlor, lactic acid RingerShi or non-volatile oils.The intravenously solvent comprises liquid nutritional supplement, the electrolyte replenisher supplement of RingerShi glucose (for example based on) etc.Also can there be sanitas and other additive, for example antiseptic-germicide, antioxidant, sequestrant, rare gas element etc.

Another aspect of the present invention provides and suppresses MMP2 and beta 2 integrin alpha _vβ ₃Suppress the method for the vasculogenesis in the tumor tissues thereby interact.Described inhibition method comprises and gives the compound compositions mentioned above that described host comprises the vasculogenesis amount of suppression.By making α _vβ ₃Contact with The compounds of this invention, suppress MMP2 and α _vβ ₃Interact.

Vasculogenesis is to form the neovascularity network from existing host blood vessel, and tumor growth surpasses 1-2mm ³Need vasculogenesis.For purpose of the present invention,, then suppress vasculogenesis as long as the illness of vasculogenesis and vasculogenesis mediation is alleviated.

The dosage range that awards the described active compound of host depends on concrete active compound and the effectiveness thereof at specific tumors or integrin.Need not too much experiment, those skilled in the art can easily determine the accurate dosage of concrete active compound.Described host can be any Mammals.Described dosage should be even as big as producing required treatment effect, wherein the illness of vasculogenesis and vasculogenesis mediation is alleviated, and normally the blood plasma level of described active compound is enough to maintain the amount in the following scope: about 0.01 micromole-Yue 100 micromoles (μ M), the preferred about 20 μ M of about 0.2 μ M-, the more preferably from about about 10 μ M of 1 μ M-.Yet described dosage should be greatly to causing toxic side effect.The dosage of per kilogram (kg) body weight can change in the scope of every dose of 1-20mg, gives potion or multi-agent every day, continues one day or a couple of days or give for a long time.

In order to suppress vasculogenesis, described treatment significant quantity is the amount that is enough to produce the active compound of the vasculogenesis inhibition that can measure in the tissue of treatment, i.e. vasculogenesis amount of suppression or MMP2-α _vβ ₃The interaction amount of suppression.Can be by immunohistochemistry as herein described or by other method well known by persons skilled in the art, the inhibition of in-site detecting vasculogenesis.

In addition, the present invention also provides the medicinal compositions that can be used for implementing methods of treatment described herein.Described composition contains active compound defined above and pharmaceutically acceptable carrier.

The present invention also provides the method for inducing apoptosis of tumour cell.This method comprises that giving described host treats active compound significant quantity, that be enough to cause apoptosis of tumor cells.

For purpose of the present invention, increase if in the target tumour of treatment, observe apoptosis of tumor cells, then brought out apoptosis of tumor cells.By methods described herein or method well known in the art, can measure apoptosis of tumor cells.

Provide following non-limiting example that all respects of the present invention are described.

Material and method

Antibody cell and reagentCS-1 hamster melanoma cells and personnel selection β ₃CS-1 cell (the β of-integrin subunit transfection ₃The CS-1 cell) previous existing (Cell, 85, the 683-93 (1996) of describing; Cell, 92,391-400 (1998)).Put together the monoclonal antibody antibiotin mAb BN-34 of horseradish peroxidase (HRP) and anti-Actin muscle mAb AC-40 derive from Sigma (St.Louis, MO).Anti--von Willebrand Factor (vWF) polyclonal antibody (pAb) derives from DAKO (Glostrup, Denmark).Cyclic peptide cRGDfV and cRADfV and integrin-α _vβ ₃Provide by MerckKGaA (Darmstadt, Germany) generosity.Purifying proMMP2 and integrin-α _vβ ₃(Temecula CA) provides by Chemicon International.The active MMP2 of purifying derive from Calbiochem (La Jolla, CA).(Mountain View, CA) close friend provides Prostatropin (bFGF) by Scios.

Synthetic [ ¹⁴ C]-compoundBy improving a little, but use N-BOC-[1-to describing flow process (flow process 1) more than the unmarked material ¹⁴C]-glycine (55mCi/mmol, AmericanRadiolabeled Chemicals, St.Louis, MO) synthesize [ ¹⁴C]-compound 1 of mark.For the flow process of 4 steps, [ ¹⁴C]-total recovery of compound 1 is 25%.

Embodiment 1. solid phase integrins are in conjunction with mensuration

The purifying integrin is adsorbed spend the night (1-5 μ g/ml, 50 μ g/ holes) on microtiter well, use Caseinblocker (Pierce, Rockford, IL) sealing then.Under the situation that has or do not exist compound 1, compound 12, ring RGD or RAD peptide or independent buffering solvent, will be at binding buffer liquid (50mM Tris, pH8,150mM NaCl, 1mM MgCl ₂, 0.5mM MnCl ₂) in purifying biological elementization MMP2 (bMMP2 3-5nM) joins in each hole.Control wells does not add integrin.Biotinylation vitronectin (bVN, 1 μ g/ml) is as reference substance.Conjugated proteinly detect with HRP-antibiotin mAb, and in 450nm with 3,3 ', 5,5 '-tetramethyl biphenyl amine aqueous solution (TMB; The substrate of described peroxidase) (BioRad, Hercules CA) carry out quantitatively.

For assessing compound 1 direct integrin binding, with α _vβ ₃And α ₅β ₁(10 μ g/ml, 50 μ l/ holes) be coated on the Immulon-4 microtiter well (Dynatech Laboratories, Chantilly, VA) on, after being closed substantially, with increment gradually [ ¹⁴C]-compound 1 hatches together, adds the binding buffer liquid that 150 μ l contain 0.1% tween 20 then, the sucking-off all liquid.Separate each hole of exsiccant, be immersed in then BetaMat liquid scintillation mixture (ICNBiochemicals, Costa Mesa, CA) in for quantitatively.According to this binding curve, under the situation of the non-labelled compound 1 that has and do not exist 25 times of molar excess (75 μ l) or

compound

12 or 100 μ M ring RGD or RAD peptide, check [ ¹⁴C]-the sub-saturated concentration (3 μ M) of compound 1.Contrast is for bVN, uses as mentioned above and detects.

Embodiment 2.MMP2 cell in conjunction with measure and [ ³ H]-the collagen protein IV mensuration of degrading

CS-1 cell or β 3CS-1 cell were hatched 45 minutes in 37 ℃ in containing the adhesion buffering inoblast basic medium (FBM) of following composition, washing then joins in each hole of [3H]-collagen protein IV bag quilt: only containing active MMP2 of 4nM purifying or combination has 10 μ M compounds 1 or compound 12 and replenishes 0.5% bovine serum albumin (BSA), 0.4mM MnCl ₂With 10 μ g/ml aprotinins.With 50 μ l 0.414mCi/ml[ ³H]-(ICN Biochemicals, Costa Mesa's collagen protein IV CA) are spent the night each hole bag, and the radioactivity of thorough washing in the washing soln that is reclaiming reaches background values.Perhaps, under the situation that does not have MMP2, as above handle cell, perhaps described MMP2 solution directly is added in acellular each hole, in contrast.According to liquid flashing counter measuring, be discharged into the radioactivity of 50 μ l substratum by measurement, quantitative to the degraded of collagen protein IV.In order to estimate combining of biotinylation MMP2 and CS-1 cell, cell suspension in adhesion buffer, existing or not existing under the situation of 10 μ M compounds 1 or compound 12, is arised from 37 ℃ with 12n,MbM,MP2 one and hatched 45 minutes.Washed cell subsequently, cracking and processing are used for SDS-PAGE then, carry out immunoblotting with antibiotin mAb.

Embodiment 3. chicken chorioallantoic membrane (CAM) vasculogenesis are measured

Vasculogenesis is estimated (Cell, 85,683-93 (1996) substantially as previously mentioned; Cell, 92,391-400 (1998)).After the stimulation of 3 μ g/ml Prostatropins (bFGF), handle instar chicken embryo CAM on the 10th with 20 μ l, 3 μ M compounds 1 or compound 12.Induced back 3 days, quantitative with blind method evaluation to described CAM.The CAM of each group is merged, rub, do not contain EDTA (Boehringer with containing COMPLETE board proteinase inhibitor mixture, Mannheim, Germany) 50mM Tris, 150nM NaCl, 0.1%TritonX-100 extract, and analyze by the receptor method of signal recognition particle body then.

The receptor method of embodiment 4.SDS-PAGE, immunoblotting and signal recognition particle body

ImmunoblottingThe albumen of equivalent separates by SDS-PAGE under reductive condition, then electroblotting to the Immubilon-P film (Millipore, Bedford, MD) on.With described membrane closure, by hatching with the antigen-specific first antibody, the second antibody of puting together with HRP-is as required hatched then, detects fixing protein.(Southfield MI) manifests band for Lumigen, Inc. with chemical luminous substrate PS-3.

The receptor method of signal recognition particle bodyPrepare chicken CAM lysate as mentioned above, under the situation that does not have reductive agent or boil, on polyacrylamide gel, separate the protein of equal quantities with 0.2% gelatin embedding.Described gel washs with 2% Triton X-100 earlier, and water thorough washing then is at last at Collagenase damping fluid (50mM Tris 7.4,200mMNaCl, 10mM CaCl ₂) in 37 ℃ of overnight incubation.By with the described gel of 0.5% Coomassie blue stain, manifest the gelatin degrading activity.

Embodiment 5. tumor growths are measured

By transplanting 5 * 10 ⁶CS-1 cell and hatching 7 days allows primary tumor grow on the CAM of 9 age in days embryos.At this moment, the section of these tumours of 50mg uploaded at fresh 9 age in days CAM be commissioned to train fosterly, allow it implant at 24 o'clock, the test compound in the intravenous injection 100 μ l 100 μ MHankShi balanced salt solutions (HBSS) once then.Independent damping fluid is with comparing.Tumour was hatched 10 days altogether, gathered in the crops and did not have excessive attaching substratum tissue, measured weight in wet base then and handled for histology and use.

Embodiment 6. immunofluorescence assays

The CS-1 tumor biopsy of quick-frozen is fixed with 4% paraformaldehyde, changed processing thoroughly with 0.1% Triton X-100.Section is used anti--vWF pAb dyeing then with phosphate buffered saline(PBS) (PBS) sealing that contains 5% bovine serum albumin (BSA), and the anti-rabbit second antibody of puting together with Alexa 568-manifests.With MRC1024 focusing microscope (BioRad, Hercules, CA) analytic sample.Vessel density is quantitative to each 4 visual field of section and 4 tumours of every kind of condition with the 20X object lens.

Embodiment 7. (S)-methyl 6 (((tert.-butoxy) carbonyl) amino)-2-((4-trifluoromethyl) benzyloxy carbonyl Base) capronate (2)

With N, N '-two succinimdyl carbonate (5.38g, acetonitrile 21mmol) (150ml) solution with 4-(trifluoromethyl) benzylalcohol (2.87ml, 21mmol) and Et ₃(5.8ml 42mmol) handles N, stirs down in 25 ℃.After 3 hours, with reaction mixture be added to be equipped with N-ε-BOC-lysine methyl ester (4.2g, in the flask of acetonitrile solution 14mmol), restir 3 hours.With solvent evaporation, resistates is dissolved in CH ₂Cl ₂(250ml), use 10% aqueous hydrochloric acid (2 * 200ml) and saturated sodium bicarbonate aqueous solution (200ml) washing then.Through flash chromatography (SiO ₂, 3: 1CH ₂Cl ₂/ EtOAc), obtain 6.4g (99%) light yellow oil 2:[α] _D ²⁵-8.9 (c 5.6, CH ₃OH); ¹H NMR (CDCl ₃, 400MHz) δ 7.57 (d, J=8.1Hz, 2H), 7.39 (d, J=8.1Hz, 2H), 5.70 (d, J=7.9Hz, 1H), 5.13 (m, 2H), 4.71 (m, 1H), 4.28 (m, 1H), 3.67 (s, 3H), 3.03 (m, 2H), 1.78 (m, 1H), 1.64 (m, 1H), 1.46-1.32 (m, 4H), 1.35 (s, 9H); ¹³C NMR (CDCl ₃, 100MHz) δ 172.9,156.2,155.8,140.4,130.1 (q, J=32.0Hz), 127.8,125.3,122.9 (q J=270.0Hz), 79.05,65.8,53.7,52.3,39.8,31.7,29.5,28.4,22.2; IR (film) ν _Max3357,2952,1790,1745,1524cm ^-1FABHRMS (NBA-NaI) m/z 463.2044 (M+H ⁺, C ₂₁H ₂₉F ₃N ₂O ₆Theoretical value 463.2056).

Embodiment 8. (S)-methyl 6-[2-(((tert.-butoxy) carbonyl) amino) kharophen]-the 2-[(4-trifluoro Methyl) capronate (3) carbobenzoxy-(Cbz))

With compound 2 (2.7g, CH 5.8mmol) ₂Cl ₂(3ml) solution is handled with 4N HCl-diox (10ml), stirs 20 minutes down in 25 ℃.Removal of solvent under reduced pressure and excessive acid, the crude salt hydrochlorate is dissolved among the DMF (50ml), with N-((tert.-butoxy) carbonyl) glycine (1.0g, 5.8mmol), 1-(3-(dimethylamino) propyl group)-3-ethyl-carbodiimide hydrochloride (EDCI) (1.2g, 6.4mmol) and i-Pr ₂(2.0ml 11.6mmol) handles NEt, stirs 12 hours down in 25 ℃.Reaction mixture is with EtOAc (400ml) dilution, with 10% aqueous hydrochloric acid (3 * 250ml) and saturated sodium bicarbonate aqueous solution (250ml) wash drying (Na ₂SO ₄) and evaporation, obtaining 2.89g (96%) is white foam shape solid compound 3:[α] _D ²⁵-10.4 (c2.5, CH ₃OH); ¹HNMR (CDCl ₃, 400MHz) δ 7.59 (m, 2H), 7.45 (m, 2H), 6.19 (m, 1H), 5.51 (m, 1H), 5.13 (m, 2H), 4.32 (m, 2H), 3.74 (s, 3H), 3.73 (m, 2H), 3.26 (m, 2H), 1.81-1.39 (m, 6H), 1.44 (s, 9H); ¹³C NMR (CD ₃OD, 100MHz) δ 174.5,172,158.2,158.1,142.7,130.8 (q, J=31.8Hz), 128.8,126.3,125.5 (q, J=269.7Hz), 80.5,66.5,55.3,52.7,44.6,39.8,32.1,29.8,24.0; IR (film) ν _Max3320,2932,1721,1692,1326cm ^-1FABHRMS (NBA-CsI) m/z652.1234 (M+Cs ⁺, C ₂₃H ₃₂F ₃N ₃O ₇Theoretical value 652.1247).

Embodiment 9.6-[2-(amino) kharophen]-the 2-[(4-trifluoromethyl)-carbobenzoxy-(Cbz)) capronate salt Hydrochlorate (4)

CH with compound 3 (350mg) ₂Cl ₂(2ml) solution is handled with 4N HCl-diox (5.0ml), stirs down in 25 ℃.0.5 after hour, removal of solvent under reduced pressure and excessive acid obtain 300mg (99%) and are the compound 4:[α of light yellow oil] _D ²⁵-10.4 (c3.0, CH ₃OH);

¹H NMR (CD ₃OD, 400MHz) δ 7.65 (d, J=8.2Hz, 2H), 5.18 (d, J=13.3Hz, 1H), 5.16 (d, J=13.3Hz, 1H), 4.16 (m, 1H), 3.70 (s, 3H), 3.65 (s, 2H), 3.21 (d, J=7.1Hz, 2H), 1.84 (m, 1H), 1.68 (m, 1H), 1.54 (m, 2H), 1.42 (m, 2H); ¹³C NMR (CD ₃OD, 100MHz) δ 174.5,167.1,158.2,142.8,130.8 (q, J=31.8Hz), 128.8,126.3,125.5 (q, J=270.1Hz), 66.5,55.4,52.7,41.5,40.2,32.1,29.6,24.1; IR (film) ν _Max3317,2954,1718,1684,1530,1327cm ^-1MALDIFTMS (DHB) m/z 442.1586 (M+Na ⁺, C ₁₈H ₂₄F ₃N ₃O ₅Theoretical value 442.1566).

Embodiment 10.N, N ¹ -two [(5-(S)-(methoxycarbonyl)-5[((4-trifluoromethyl)-carbobenzoxy-(Cbz)) ammonia Base] amyl group) the formamido-methyl] benzene-1,3 diformamide (5)

With compound 4 (2.05g, CH 4.0mmol) ₂Cl ₂(3.0ml) solution is handled with 4N HCl-diox (10.0ml), stirs 20 minutes down in 25 ℃.Removal of solvent under reduced pressure and excessive acid are suspended in the crude salt hydrochlorate among the DMF (40ml), with the isophthaloyl dichloro (400mg, 2.0mmol) and i-Pr ₂(1.4ml 8.0mmol) handles NEt, stirs 1 hour down in 25 ℃.Reaction mixture is with EtOAc (400ml) dilution, with 10% aqueous hydrochloric acid (3 * 200ml) and 5% saturated aqueous sodium carbonate (200ml) washing, drying (Na ₂SO ₄) and evaporation.Through flash chromatography (SiO ₂, 1: 4.5: 4.5 MeOH/CH ₂Cl ₂/ EtOAc), obtain 1.30g (68%) and be the compound 5:[α of yellow powder] _D ²⁵-6.4 (c 2.1, CH ₃OH): ¹H NMR (CDCl ₃, 400MHz) δ 8.29 (m, 2H), 8.11 (m, 2H), 7.87 (m, 4H), 7.53 (m, 2H), 7.39 (m, 2H), 6.88 (m, 2H), 5.94 (m, 2H), 5.08 (m, 4H), 4.30 (m, 2H), 4.01 (m, 4H), 3.70 (s, 6H), 3.23 (m, 4H), 1.77 (m, 2H), 1.67 (m, 2H), 1.51 (m, 4H), 1.37 (m, 4H); ¹³C NMR (CD ₃OD, 100MHz) δ 174.6,171.5,169.3,158.3,142.7,135.3,131.6,30.8 (q, J=32.2), 129.8,128.8,127.6,126.3,125.5 (q, J=269.2), 66.5,55.4,52.7,44.1,40.0,32.1,29.8,24.1; IR (film) ν _Max3305,2951,1716,1651,1538cm ^-1FABHRMS (NBA-CsI) m/z 1101.2398 (M+Cs ⁺, C ₄₄H ₅₀F ₆N ₆O ₁₂Theoretical value 1101.2445).

Embodiment 11.N, N ¹ -two-[(5-(S)-carboxyl-5-[((4-trifluoromethyl) carbobenzoxy-(Cbz)) amino]-penta Base) formamido-methyl] benzene-1,3-diformamide (1)

With compound 5 (0.95g, THF-MeOH 0.98mmol) (8.0ml, 3: 1) solution LiOHH ₂O (165mg, H 3.9mmol) ₂O (2.0ml) handles, and stirs down in 0 ℃.After 2 hours, by adding the 10%HCl aqueous solution (20ml) quencher reactant, (3 * 50ml) extract mixture with EtOAc.Organic layer is merged, with the saturated NaCl aqueous solution (50ml) washing, dry (Na ₂SO ₄) and evaporation, obtain 0.86g (93%) and be the compound 1 of white powder:

[α] _D ²⁵-0.6 (c3.2, CH ₃OH); ¹H NMR (CD ₃OD, 400MHz) δ 8.40 (m, 1H), 8.03 (dd, J=1.8,7.8Hz, 2H), 7.62 (d, J=8.1Hz, 4H), 7.53 (t, J=6.1Hz, 1H), 7.51 (d, J=8.1Hz, 4H), 5.16 (d., J.=13.3Hz, 2H), 5.13 (d, J=13.3Hz, 2H), 4.12 (m, 2H), 4.00 (s, 4H), 3.22 (t, J=6.6Hz, 4H), 1.85 (m, 2H), 1.70 (m, 2H), 1.55 (m, 4H), 1.37 (m, 4H); ¹³C NMR (CD ₃OD, 100MHz) δ 175.9,171.6,169.4,158.4,135.4,131.6,30.5 (q, J=31.8), 129.8,128.8,127.7,126.3,125.6 (q, J=268.7); 66.5,53.3,44.2,40.1,32.2,29.8,24.2; IR (film) ν _Max3334,2933,1718,1646,1631,1528cm ^-1MALDIFTMS (DHB) m/z 963.2953 (M+Na ⁺, C ₄₂H ₄₆F ₆N ₆O ₁₂Theoretical value 963.2976).

Embodiment 12.[ ¹⁴ C]-compound (1)

With [1- ¹⁴C]-glycine (American Radiolabeled Chemicals, 1.0 mCi, 55mCi/mmol, 0.1N HCl solution 0.018mmol) is transferred in the 4ml phial, removes under nitrogen gas stream and desolvates.The resistates NaHCO of gained ₃(4.6mg, H 0.054mmol) ₂(10.5ml, THF 0.045mmol) (0.25ml) solution-treated stir down in 25 ℃ for O (0.25ml) solution and di-tert-butyl dicarbonic acid ester.After 12 hours, solution becomes gets evenly, uses H ₂O (1.0ml) dilution is with ether (2 * 1.0ml) washings.By adding the 10%HCl aqueous solution (0.5ml), make acidified aqueous solution then, with ethyl acetate (4 * 1.0ml) extractions.With the extraction liquid drying (Na that merges ₂SO ₄), under nitrogen gas stream, evaporate, obtain 2.9mg (92%) and be [the 1-of white membranoid substance ¹⁴C]-the N-BOC-glycine.

With compound 2 (50mg, CH 0.11mmol) ₂Cl ₂(1ml) solution is handled with 4N HCl-diox (1ml), stirs 1 hour down in 25 ℃.Reaction mixture is used 10%Na with ethyl acetate (25ml) dilution ₂CO ₃The aqueous solution (25ml) and the saturated NaCl aqueous solution (25ml) washing, dry (Na ₂SO ₄) and evaporation, obtain de-protected glycine into colourless membranoid substance.(4.5mg, the part of DMF 0.013mmol) (0.2ml) solution joins 4ml [1-is housed with this unhindered amina ¹⁴C]-(1.5mg in phial 0.0085mmol), uses i-Pr to the N-BOC-glycine ₂(5.0mg, methylene dichloride 0.026mmol) (0.1ml) solution-treated stirred 3 hours down in 25 ℃ for NEt (2ml) and EDCI.Reaction mixture with ethyl acetate (2.0ml) dilution, with 10% hydrochloric acid (3 * 1.0ml), saturated Na ₂CO ₃The aqueous solution (1.0ml) and the saturated NaCl aqueous solution (1.0ml) washing, evaporation then.Through preparative thin-layer chromatography (PTLC) (SiO ₂, EtOAc/CHCl ₃1: 1), obtain 1.8mg (41%) for white membranoid substance [ ¹⁴C]-compound 3.

4ml be equipped with [ ¹⁴C]-compound 3 (1.8mg, handled with 4N HCl-diox (0.25ml), in 25 ℃ of following reaction stirred 0.5 hour by 3.5mmol) phial.Evaporating solvent and excessive acid under nitrogen gas stream, the crude salt hydrochlorate of gained isophthaloyl dichloro (355mg, CH 0.0018mmol) ₂Cl ₂(0.1ml) solution and i-Pr ₂NEt (2.4ml, CH 0.014mmol) ₂Cl ₂(0.05ml) solution-treated.In 25 ℃ after following 3 hours, reaction mixture is directly by PTLC purifying (SiO ₂, MeOH/CHCl ₃/ EtOAc 1: 9: 9), obtain 1.2mg (71%) [ ¹⁴C]-compound 5.

Will [ ¹⁴C]-THF-MeOH (0.2ml, 1: 1) the solution LiOHH of compound 5 (1.2mg) ₂The H of O (0.4mg) ₂O (0.05ml) solution was handled and is stirred under 0 ℃ 1 hour.Reaction mixture was handled and is stirred 1 minute with Dowex 50WX-8 acidic cation-exchange resin (200mg) with methyl alcohol (0.1ml) dilution.Allow mixture filter then by velveteen, evaporated filtrate, obtain 1.1mg (94%) relative activity be about 110mCi/mmol [ ¹⁴C]-compound 1.By thin-layer chromatography (TLC) relatively, this compound and all synthetic intermediate thereof are identical with corresponding unmarked material.

Embodiment 13. (S)-methyl 6-[2-(benzamido) kharophen]-the 2-[(4-trifluoromethyl) benzyl The oxygen carbonyl) capronate (6)

With compound 3 (44mg, CH 0.085mmol) ₂Cl ₂(1.0ml) solution is handled with 4N HCl-diox (1.0ml), stirs 1 hour down in 25 ℃.Under nitrogen gas stream, remove and desolvate and excessive acid, the hydrochloride raw product is suspended in CH ₂Cl ₂(0.8ml), use i-Pr ₂NEt (30 μ l, 017mmol) and Benzoyl chloride (11 μ l 0.094mmol) handle, and stir down in 25 ℃.After 2 hours, reaction mixture is directly passed through purification by flash chromatography (SiO ₂, 1: 9: 9MeOH/CH ₂Cl ₂/ EtOAc), obtain 40mg (90%) and be the compound 6:[α of white powder] _D ²⁵-9.7 (c0.70, CHCl ₃); ¹H NMR (CD ₃OD, 400MHz) δ 7.86 (m, 2H), 7.64 (m, 2H), 7.53 (m, 3H), 7.45 (m, 2H), 5.16 (m, 2H), 5.16 (dd, J=7.3,3.8Hz, 1H), 3.69 (s, 3H), 3.21 (t, J=5.6Hz, 2H), 1.81 (m, 1H), 1.69 (m, 1H), 1.53 (m 2H), 1.42 (m, 2H); ¹³C NMR (CDCl ₃, 100MHz) δ 172.8,169.5,168.4,156.3,142.3,132.9,131.4,129.4 (q, J=32.2Hz), 128.0,127.1,126.7,126.3 (q, J-269.0Hz), 124.8,65.1,53.6,51.6,42.6,38.4,30.6,28.1,22.2; IR (film) ν _Max3303,2923,1713,1651,1533cm ^-1FABHRMS (NBA-CsI) m/z 656.0961 (M+Cs, C ₂₅H ₂₅F ₃N ₃O ₆Theoretical value 656.0985).

Embodiment 14. (S)-6-[2-(benzamido) kharophen]-the 2-[(4-trifluoromethyl) the benzyloxy carbonyl Base] caproic acid (7)

(17mg, THF-MeOH 0.032mmol) (0.4ml, 3: 1) solution is with being dissolved in H with compound 6 ₂The LiOHH of O (0.1ml) ₂(2.0mg 0.49mmol) handles O, stirs 2 hours down in 0 ℃.By adding the dense HCl aqueous solution (5 μ l) quencher reaction mixture, with EtOAc (30ml) dilution, water (2 * 15ml) washings.Dry (Na ₂SO ₄) and evaporation, obtain 1.45mg (88%) and be the compound 7:[α of white powder] _D ²⁵+ 2.6 (c 0.6, CH ₃OH); ¹H NMR (CD ₃OD, 400MHz) δ 7.86 (d, J=7.2Hz, 2H), 7.62 (d, J=8.2Hz, 2H), 7.54 (d, J=8.2Hz, 2H), 7.53 (m, 1H), 7.43 (m, 2H), 5.17 (d, J=13.2Hz, 1H), 5.14 (d, J-13.2Hz, 1H), 4.14 (m, 1H), 4.04 (s, 2H), 3.25 (m, 2H), 1.88 (m, 1H), 1.71 (m, 1H), 1.52 (m, 2H), 1.44 (m, 2H); ¹³C NMR (CD ₃OD, 125MHz) δ 175.9,171.7,170.5,158.4,142.9,135.1,132.9,130.9 (q, J=32.4Hz), 129.5,128.9,128.5,126.3,125.7 (q, J-269.0Hz), 66.5,55.4,44.1,40.1,32.3,29.9,24.2; IR (film) ν _Max3318,2935,1713,1644,1538,1326cm ^-1MALDIFTMS m/z 532.1672 (M+Na ⁺, C ₂₄H ₂₆F ₃N ₃O ₆Theoretical value 532.1671).

Embodiment 15. (S)-methyl 6-[2-[(2-((carboxymethyl) methoxyl group) kharophen] kharophen]- The 2-[(4-trifluoromethyl) carbobenzoxy-(Cbz)] capronate (8)

With compound 3 (57mg, CH 0.11mmol) ₂Cl ₂(1ml) solution is handled with 4N HCl-diox (2ml), stirs 1 hour down in 25 ℃.Under nitrogen gas stream, remove and desolvate and excessive acid, resistates is dissolved among the DMF (1ml), with diglycollic acid (16mg, 0.12mmol), EDCI (23mg, 0.12mmol) and i-Pr ₂(42 μ l 0.24mmol) handle NEt, stir down in 25 ℃.After 4 hours, reaction mixture is inclined to the separating funnel that EtOAc (50ml) is housed, with the 10%HCl aqueous solution (3 * 30ml) and the saturated NaCl aqueous solution (30ml) washing, dry (Na ₂SO ₄) and evaporation, obtain 45mg (77%) and be the monoprotic acid compound 8:[α of white solid] _D ²⁵-7.5 (c1.8, CH ₃OH); ¹H NMR (CD ₃OD, 400MHz) δ 7.65 (d, J=5.2Hz, 2H), 7.54 (d, J=5.3Hz, 2H), 5.18 (d, J=9.0Hz, 1H), 5.15 (d, J=9.0Hz, 2H), 4.42 (s, 2H), 4.16 (m, 1H), 4.11 (s, 2H), 3.87 (s, 2H), 3.70 (s, 3H), 3.20 (t, J=4.5Hz, 2H), 1.81 (m, 1H), 1.68 (m, 1H), 1.53 (m, 2H), 1.39 (m, 2H); ¹³C NMR (CD ₃OD, 100MHz) δ 174.5,173.7,172.5,171.2,158.2,142.6,130.9 (q, J=33.2Hz), 128.2,126.2,125.4 (q, J=270.1Hz), 71.3,69.0,66.5,55.3,52.7,42.9,39.9,32.0,29.7,23.9; IR (film) ν _Max3315,2931,1725,166l, 1538,1326cm ^-1MALDIFTMS m/z 558.1686 (M+Na ⁺, C ₂₂H ₂₃F ₃N ₃O ₉Theoretical value 558.1673).

Embodiment 16. (S)-methyl 2-(benzamido)-6-(((tert.-butoxy) carbonyl) amino) caproic acid Ester (9)

With N-ε-BOC-lysine methyl ester hydrochloride (5.05g, CH 17mmol) ₂Cl ₂(100ml) solution with Benzoyl chloride (2.0ml, 17mmol) and i-Pr ₂(5.9ml 34mmol) handles NEt, stirs down in 25 ℃.After 1 hour, reaction mixture washs with the 10%HCl aqueous solution (100ml), dry (Na ₂SO ₄) and be evaporated to from CH ₂Cl ₂-hexane crystalline white powder obtains 4.8g (78%) and is 108-109 ℃ of the compound 9:mp of white crystalline solid; [α] _D ²⁵-13.2 (c5.8), CH ₃OH); ¹H NMR (DMS0-d ₆, 400MHz) δ 8.72 (d, J=7.4Hz, 1H), 7.89 (m, 2H), 7.55 (m, 1H), 7.48 (m, 2H), 6.88 (t, J-5.2Hz, 1H), 4.42 (m, 1H), 3.64 (s, 3H), 2.93 (m, 2H), 1.81 (m, 2H), 1.41 (m, 4H), 1.37 (s, 9H); ¹³C NMR (DMSO-d ₆, 100MHz) δ 172.9,166.7, and 155.6,133.8,131.5,128.3,127.6,77.4,52.8,51.9,40.8,30.2,29.2,28.3,23.2; IR (film) ν _Max3335,2933,1740,1690,1647,1534cm ^-1MALDIFTMS (DHB) m/z387.1895 (M+Na ⁺, C ₁₉H ₂₅N ₂O ₅Theoretical value 387.1890).

Embodiment 17. (S)-methyl 2-(benzamido)-6-[2-(((tert.-butoxy) carbonyl) amino) second Amido] capronate (10)

With compound 9 (4.4g, CH 12.1mmol) ₂Cl (5.0ml) solution is handled with 4N HCl-diox (10.0ml), stirs 3 hours down in 25 ℃.Removal of solvent under reduced pressure and excessive acid are dissolved in resistates among the DMF (150ml), with N-((tert.-butoxy) carbonyl) glycine (2.2g, 12.1mmol), PyBrOP (7.0g, 15mmol) and i-Pr ₂(8.4ml 48.4mmol) handles NEt, stirs down in 25 ℃.After 12 hours, with reaction mixture with EtOAc (500ml) dilution, with the 10%HCl aqueous solution (3 * 250ml), 5%Na ₂CO ₃The aqueous solution (250ml) and the saturated NaCl aqueous solution (250ml) washing, dry (Na ₂SO ₄) and evaporation.Through flash chromatography (SiO ₂, EtOAc), obtain 4.6 (90%) and be the compound 10:[α of colorless oil] _D ²⁵-10.3 (c0.30, CH ₃OH); ¹H NMR (CDCl ₃, 400MHz) δ 7.84 (m, 2H), 7.51 (m, 1H), 7.44 (m, 2H), 6.91 (d, J-7.6Hz, 1H), 6.35 (brt, J-5.3Hz, 1H), 5.22 (m, 1H), 4.76 (m, 1H), 3.76 (s, 3H), 3.70 (brt, J-6.3Hz, 4H), 3.26 (m, 2H), 1.95 (m, 1H), 1.84 (m, 1H), 1.57-1.30 (m, 4H), 1.41 (s, 9H); ¹³C NMR (CDCl ₃, 100MHz) δ 173.0,169.7, and 167.3,156.5,133.7,131.8,128.6,127.2,81.8,52.5,52.3,44.3,38.7,32.0,28.9,28.3,22.4; IR (film) ν _Max3318,2954,1718,1647,1535,1491cm ^-1MALDIFTMS m/z 444.2108 (M+Na ⁺, C ₂₁H ₃₁N ₃O ₆Theoretical value 444.2110).

Embodiment 18.N, N '-two [N-(5-(S)-((benzoyl) amino)-5-(methoxycarbonyl) amyl group) first Amide methyl] benzene-1,3-diformamide (11)

With compound 10 (4.4g, CH 10.4mmol) ₂Cl ₂(3ml) solution is handled with 4.0N HCl-diox (20ml), stirs 1 hour down in 25 ℃.Removal of solvent under reduced pressure and excessive acid are dissolved in CH with resistates ₂Cl ₂(50ml), use Et ₂(5.8ml, 42mmol) (1.06g 5.2mmol) handles N, stirs down in 25 ℃ with the isophthaloyl dichloro.After 16 hours, reaction mixture washs and is evaporated to yellow oil with 10% aqueous hydrochloric acid (50ml).Through flash chromatography (SiO ₂, 5: 5: 2 EtOAc/CH ₂Cl ₂/ MeOH), obtaining 2.5g (62%) is white foam shape solid compound 11:[α] _D ²⁵-8.0 (c4.8, CH ₃OH); ¹H NMR (CD ₃OD, 500MHz) δ 8.37 (s, 1H), 8.00 (m, 2H), 7.84 (m, 4H), 7.52 (m, 3H), 7.44 (m, 4H), 4.58 (m, 2H), 3.99 (2s, 4H); 3.72 (s, 6H), 3.22 (m, 4H), 1.99-1.83 (m, 4H), 1.59-1.40 (m, 8H). ¹³C NMR (CD ₃OD, 100MHz) δ 174.2,171.5, and 170.2,169.0,135.0,134.8,132.8,131.5,129.4,128.4,127.6,54.3,52.7,44.2,40.1,31.7,29.8,24.3; IR (film) ν _Max3304,2950,1738,1650,1644,1538cm ^-1MALDIFTMS m/z 795.3332 (M+Na ⁺, C ₄₀H ₄₅N ₆O ₁₀Theoretical value 795.3330).

Embodiment 19.N, N '-two [N-(5-(S)-((benzoyl) amino)-5-(carboxyl) amyl group) methane amide Ylmethyl] benzene-1,3-diformamide (12)

(1.1g, MeOH-THF 1.4mmol) (20ml, 1: 1) solution is with being dissolved in H with compound 11 ₂The LiOHH of O (10ml) ₂(240mg 5.7mmol) handles O, stirs 1 hour down in 0 ℃.After 1 hour, evaporation reaction solvent, resistates are dissolved in H again ₂Among the O (10ml), and allow it be cooled to 0 ℃.Add the dense HCl aqueous solution (0.47ml, 5.7mmol HCl), precipitated solid is filtered, water (50ml) washing obtains 0.78g (73%) and is the compound 12:[α of white powder] _D ²⁵-2.6 (c0.35, CH ₃OH); ¹H NMR (CD ₃OD, 400MHz) δ 8.38 (m, 1H), 8.02 (m, 2H), 7.84 (m, 4H), 7.55-7.48 (m, 3H), 7.42 (m, 4H), 4.56 (m, 2H), 4.00 and 3.99 (2s, 4H), 3.26 (m, 4H), 1.99-1.83 (m, 4H), 1.63-1.45 (m, 8H); ¹³C NMR (CD ₃OD, 100MHz) δ 175.5,171.9, and 170.4,169.2,135.0,134.9,132.8,131.7,129.8,129.5,128.5,127.7,54.4,44.0,40.2,31.7,29.6,24.3; IR (film) ν _Max3280,2923,1718,1641,1536cm ^-1MALDIFTMS (DHB) m/z 767.3005 (M+Na ⁺, C ₃₈H ₄₄N ₆O ₁₀Theoretical value 767.3017).

Result and discussion

Compound 1 destroys beta 2 integrin alpha _vβ ₃And the RGD dependent/non-dependent between the MMP2 is mutual EffectNearest observation shows that MMP2 and beta 2 integrin alpha can be disturbed in the carboxyl terminal Hemopexin-like structure territory of MMP2 _vβ ₃Combination and therefore block vasculogenesis, thereby make us seek this interactional organic inhibitor, described organic inhibitor may be more suitable for giving in therapeutic.For identification of M MP2 and beta 2 integrin alpha _vβ ₃Between the interactional specific inhibitor of associativity, carry out solid phase receptor in conjunction with mensuration with immobilization integrin and biotinylation MMP2.Though the cRGDfV peptide suppresses α _vβ ₃With its extracellular matrix part vitronectin (VN; Fig. 2 A) interaction, but the combination of discovery purifying MMP2 in this system is complete and RGD is irrelevant, and its evidence is that cRGDfV is to MMP2 and beta 2 integrin alpha _vβ ₃Bonded is effect not.Importantly, compound 1 is eliminated the combination of MMP2 fully, but does not eliminate the combination of VN, and 1 couple of MMP2 of compound and α are described _vβ ₃Between to interact be specific.In addition, the combination between the tissue depressant of MMP2 and metalloprotease 2 (TIMP2) can not suppress by combined thing 1, and supported following argument: the effect of described compound 1 only limits to MMP2 and beta 2 integrin alpha _vβ ₃Between binding interactions, MMP2 PEX structural domain and beta 2 integrin alpha on the TIMP2 are described _vβ ₃Combining site between specificity.Be important to note that control compound and control peptide cRADFV do not disturb MMP2 and beta 2 integrin alpha _vβ ₃In conjunction with (Fig. 2 A).

Change and contain thing 1 and beta 2 integrin alpha _vβ ₃Directly in conjunction with and do not combine with MMP2.In order further to illustrate the mechanism of action of compound 1, use immobilization α _vβ ₃[ ¹⁴C]-compound 1 of mark and biotinylation VN in contrast carry out other solid phase receptor in conjunction with mensuration.Fig. 2 B as seen, at solid phase receptor in conjunction with in measuring, compound 1 and beta 2 integrin alpha _vβ ₃Directly combination.This interaction is a dose-dependently, saturable and specific, and compound 1 and incoherent contrast beta 2 integrin alpha 5 β are described ₁Minimum degree interact (Fig. 2 B).In fact, under the compound of higher concentration, observe and beta 2 integrin alpha ₅β ₁Negligible combination (data not shown).In addition, coated when the microtiter well as MMP2, do not observe the combination (data not shown) of compound 1, show at described MMP2/ beta 2 integrin alpha _vβ ₃In conjunction with the effect that observes in measuring is because compound 1 and beta 2 integrin alpha _vβ ₃In conjunction with what cause.Importantly, this interacts and is suppressed by the non-labelled compound 1 of 25 times of molar excess, and is not suppressed (Fig. 2 C) by relevant control compound 12.In addition, can not suppress radiolabeled compound 1 and beta 2 integrin alpha according to the cRGDfV peptide _vβ ₃Interaction confirmed, even cRGDfV eliminates combining of bVN and immobilization integrin fully in identical systems, illustrates that compound 1 is with RGD dependent/non-dependent mode and beta 2 integrin alpha _vβ ₃In conjunction with (Fig. 2 D).Confirm that control peptide cRADfV is described in conjunction with suppressing specific contrast.Therefore, with respect to MMP2 combine compound 1 and α _vβ ₃Lack the susceptibility that RGD is suppressed in conjunction with having suitable specificity, selectivity.

What is interesting is, in described solid phase in conjunction with in measuring, compound 6 promptly wherein A be that hydrogen, n are 0, X ¹For to trifluoromethyl and R ¹Be active lower slightly than compound 1 of formula (II) compound of methyl, and the compound 8 that lacks with described glycine unit link coupled phenyl is non-activities.This result shows that described phenyl ring is the active basic structural feature of inhibition of inhibitor formula (II) compound, and illustrates that at least one substituted glycyl lysine units is essential for the anti-angiogenesis activity of such inhibitor.

Cell-mediated property collagen protein IV by MMP2 combined thing 1 blocking-up of degradingThe previous beta 2 integrin alpha that stops on MMP2 and the melanoma cells that illustrated _vβ ₃In conjunction with the collagen protein IV of mediation capable of inhibiting cell with α _vβ ₃The dependency mode is degraded.Therefore, our evaluation expression or lack α _vβ ₃Melanoma cells whether can utilize activation MMP2 degraded immobilization [ ³H] collagen protein IV.Importantly, but cell does not produce the endogenous MMP2 of detection limit.The basic collagen albumen of certain level has only B although two kinds of cell types all can be degraded ₃The CS-1 cell of-transfection can utilize exogenous MMP2, and behind confirmation and the purifying MMP2 preincubate, obviously more radioactive substrates is released to (Fig. 3 A) in the substratum.MMP2 handles combined thing 1 inclusion of enhanced degradation of substrates and eliminates specifically, and the effect of compound 12 can ignore (Fig. 3 A).Importantly, the influence of the collagen protein of compound 1 pair cell mediation degraded is because active direct inhibition causes to MMP2 because the active MMP2 that does not have purifying under the situation of cell still can degrade immobilization [ ³H] collagen protein IV, and whether irrelevant with the existence of compound 1 or compound 12.In order to prove that the cell-mediated collagen protein degraded that observes among Fig. 3 A reduces is MMP2 and cell surface beta 2 integrin alpha _vβ ₃The result that the combined thing 1 that interacts suppresses, has detected the CS-1 cell and has carried α in conjunction with in measuring at biotinylation MMP2 _vβ ₃Counterpart.With the expection the same, described β ₃-negative CS-1 cell can be in conjunction with the MMP2 of certain level, yet any described compound that its binding ability can not be existed is eliminated (Fig. 3 B).On the contrary, β ₃The CS-1 cell is in conjunction with obvious more substantial MMP2, and this enhanced MMP2 suppresses specifically in conjunction with combined thing 1.In fact, according to proving that with anti-Actin muscle mAb dyeing when calibrating the band of application of sample, compound 1 makes MMP2 and β ₃The combination of CS-1 cell is reduced to effectively and does not have α _vβ ₃The time level (former generation CS-1 cell) (Fig. 3 B, band 2) that observed.

Compound 1 destroys intravital vasculogenesis, and does not suppress the MMP2 activation.Before shown α _vβ ₃The interact inhibition of being used reorganization MMP2 PEX structural domain of/MMP2 reduces vasculogenesis in the animal model exogenously.Therefore, we have detected the influence of vasculogenesis of the growth factor-induced of 1 pair 10 age in days chicken of compound CAM.Compound 1 is applied to the generation of almost completely eliminating on the CAM that stimulates with Prostatropin (bFGF) the neovascularity of this stimulation responses (Fig. 4 A and 4B), and control compound 12 is invalid on the one hand at this.Importantly, elimination that 1 pair of angiogenic of compound soaks into and MMP2 activatory suppress irrelevant, because detect the active MMP2 (62kDa) (Fig. 4 C) that equates level in deriving from CAM tissue treated and undressed chicken embryo.This effect with external source MMP2 PEX structural domain is opposite fully, the described activation that acts on inhibition MMP2 in this system.These data and compound 1 disturb MMP2 and beta 2 integrin alpha specifically _vβ ₃Combination and directly not influence MMP2 activatory design consistent.In fact, the aggregate level of the MMP2 that observes in the CAM lysate is not subjected to the influence of compound 1 processing yet, has got rid of the potential effect (Fig. 4 C) of MMP2 expression level in described angiogenic tissue.These results show that shown in Fig. 3 B, the angiogenesis inhibitor effect of compound 1 may be owing to suppress the beta 2 integrin alpha of MMP2 and cell surface _vβ ₃In conjunction with due to.These data illustrate also that in this system activatory MMP2 is used for vasculogenesis fully, unless the beta 2 integrin alpha of itself and cell surface _vβ ₃Coupling.

Compound 1 is eliminated intravital tumor growthShown that destroying vasculogenesis in numerous systems suppresses tumor growth.Therefore, the tumor growth that suppresses vasculogenesis and animal model by the invasion and attack characteristic that suppresses MMP blocking-up endotheliocyte.In fact, shown that many MMP inhibitor have the prospect that generates medicine as anti-people's blood vessel.Therefore, we estimate and observed MMP2/ α in this research _vβ ₃Whether the inhibition of the vasculogenesis that the blocking-up that is used as mutually is relevant is enough to suppress α _vβ ₃Negative growth of tumor.Use α _vβ ₃Negative tumour makes can 1 couple of blood vessel α of assessing compound _vβ ₃Selectively acting.Shown in Fig. 5 A, the α that on chicken CAM, transplants _vβ ₃Negative CS-1 melanoma growth of tumor is blocked effectively by an intravenously (IV) injection compound 1, and tumor weight also reduces (Fig. 5 B).May this effect not, because used melanoma cells lacks beta 2 integrin alpha in this mensuration because the direct effect of 1 pair of described tumour of compound causes _vβ ₃In fact, their growth in vitro is not subjected to the influence (data not shown) of cultivating altogether with described compound.Vascular surface system (Fig. 5 A) and and the obvious minimizing of total vessel density (Fig. 5 C) also see the tumour of having handled with compound 1.Importantly, this minimizing of tumor vessel system is relevant with effective cell death in the described tumor mass, and the while is at 6 times of the internal reference tumor weight increase of 10 day time of described mensuration.

The explanation of front and embodiment are illustrative, rather than restrictive.Other variation within the spirit and scope of the present invention is possible to those skilled in the art and itself is conspicuous.

Claims

1. the compound of a following structure:

G wherein ¹And G ²Independently be-NH-C (O)-O-R separately ¹,-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹,-NH-C (O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹,-O-C (O)-NH-(CH ₂) _v-(C ₆H ₄)-X ¹,-O-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹Or-NH-C (O)-CH ₂-(C ₆H ₄)-X ¹Y ¹And Y ²Independent separately is OH, C ₁-C ₄Alkyl, C ₁-C ₄Hydroxyalkyl, C ₁-C ₄Alkoxyl group, phenyl, benzyl or-NH ₂R ¹Be C ₁-C ₄Alkyl; X ¹Be halogen, nitro, C ₁-C ₄Alkyl, C ₁-C ₄Alkoxyl group or C ₁-C ₄Perfluoroalkyl; Z is-C ≡ C-,-C ₆H ₄-, cis-CH=CH-, trans-CH=CH-, cis-CH ₂-CH=CH-CH ₂-, trans-CH ₂-CH=CH-CH ₂-, 1,4-naphthyl, cis-1,3-cyclohexyl, anti-form-1,3-cyclohexyl, cis-1,4-cyclohexyl or anti-form-1,4-cyclohexyl; A is H or covalent linkage; M and n independently are the integer of 0 or 1 numerical value separately; T is the integer of 0 or 1 numerical value; V is the integer of 1 or 2 numerical value; Condition is when A is H, and t is 0; When A was covalent linkage, t was 1; When m is 0, Y ¹Be C ₁-C ₄Hydroxyalkyl; When n is 0, Y ²Be C ₁-C ₄Hydroxyalkyl.

2. the compound of claim 1, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

3. the compound of claim 2, wherein X ¹Be trifluoromethyl.

4. the compound of claim 2, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.

5. the compound of a following structure:

R wherein ²And R ³Independent separately is H, C ₁-C ₄Alkyl, phenyl or benzyl; X ²And X ³Independent separately is halogen, nitro, C ₁-C ₄Alkoxyl group, C ₁-C ₄Alkyl or C ₁-C ₄Perfluoroalkyl; A is H or covalent linkage; T is the integer of 0 or 1 numerical value; Condition is when A is H, and t is 0; When A was covalent linkage, t was 1.

6. the compound of claim 5, wherein X ²And X ³In at least one is to trifluoromethyl, A is a covalent linkage.

7. the compound of claim 5, wherein R ²And R ³In at least one is H, A is a covalent linkage.

8. the compound of claim 5, wherein R ²And R ³In at least one is a methyl, A is a covalent linkage.

9. the compound of claim 5, wherein X ²And X ³Respectively do for oneself to trifluoromethyl R ²And R ³The methyl of respectively doing for oneself, A is a covalent linkage.

10. the compound of claim 5, wherein R ²Be H, A is H.

11. the compound of claim 5, wherein R ²Be methyl, A is H.

12. the compound of claim 5, wherein X ²For to trifluoromethyl, A is H.

13. the compound of a following structure:

14. medicinal preparations that is used to suppress tumor growth that in pharmaceutically acceptable carrier, comprises the compound of following structure:

15. the medicinal preparations of claim 14, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

16. the medicinal preparations of claim 15, wherein X ¹Be trifluoromethyl.

17. the medicinal preparations of claim 15, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.

18. the medicinal preparations of claim 15, wherein A is H.

19. medicinal preparations that is used to suppress tumor growth that in pharmaceutically acceptable carrier, comprises the compound of following structure:

20. following formula: compound is used for suppressing the purposes of the medicine of host's tumor growth in vivo in preparation:

21. the purposes of claim 20, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

22. the purposes of claim 21, wherein X ¹Be trifluoromethyl.

23. the purposes of claim 21, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.

24. according to the purposes of claim 20, the administration of wherein said medicine comprises in intraperitoneal, subcutaneous, intravenously, transdermal, the synovial membrane, intramuscular or oral administration.

25. following formula: compound is used for suppressing the purposes of the medicine of host's tumor growth in vivo in preparation:

26. according to the purposes of claim 25, the administration of wherein said medicine comprises in intraperitoneal, subcutaneous, intravenously, transdermal, the synovial membrane, intramuscular or oral administration.

27. following formula: compound is used to suppress the purposes of the medicine that the tumor tissues medium vessels generates in preparation:

28. the purposes of claim 27, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

29. the purposes of claim 28, wherein X ¹Be trifluoromethyl.

30. the purposes of claim 28, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.

31. according to the purposes of claim 27, the administration of wherein said medicine comprises in intraperitoneal, subcutaneous, intravenously, transdermal, the synovial membrane, intramuscular or oral administration.

32. the purposes of following formula: compound in the medicine of preparation inducing apoptosis of tumour cell:

33. the purposes of claim 32, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

34. the purposes of claim 33, wherein X ¹Be trifluoromethyl.

35. the purposes of claim 33, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.

36. according to the purposes of claim 32, the administration of wherein said medicine comprises in intraperitoneal, subcutaneous, intravenously, transdermal, the synovial membrane, intramuscular or oral administration.

37. following formula: compound suppresses MMP2 and host cell beta 2 integrin alpha in preparation _vβ ₃Purposes in the interactional medicine:

38. the purposes of claim 37, wherein G ¹And G ²For-NH-C (O)-O-(CH ₂) _v-(C ₆H ₄)-X ¹, A is a covalent linkage, v is 1.

39. the purposes of claim 38, wherein X ¹Be trifluoromethyl.

40. the purposes of claim 38, wherein Y ¹And Y ²Be OH, m is 1, and n is 1.