KR100767616B1 - Pharmaceutical compositions for inhibiting tumor growth - Google Patents

Abstract

Angiogenesis, tumor growth, and the interaction of matrix metalloproteinase 2 (MMP2) with integrin α _v β ₃ are inhibited by inhibitor compounds of formula (I).

Formula I

In the above formula,

G ¹ and G ² are each independently -NH-C (O) -0-R ¹ , -NH-C (O) -O- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -NH -C (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -0- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ or -NH-C (O) -CH _2- (C ₆ H ₄ ) -X ³ ,

Y ¹ and Y ² are each independently —OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or NH ₂ ,

R ¹ is C ₁ -C ₄ alkyl,

X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy,

X ³ is halo, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl,

Z is -C≡C-, -C ₆ H _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, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl or trans-1,4-cyclohexyl,

A is H or a covalent bond,

m and n are each independently an integer that is a value of 0 or 1,

t is an integer that is a value of 0 or 1,

p, r, and v are each independently integers that are 1 or 2,

Provided that when A is H, t is 0; When A is a covalent bond, t is 1; when m is 0, Y ¹ is C ₁ -C ₄ hydroxyalkyl; When n is 0, Y ² is C ₁ -C ₄ hydroxyalkyl.

Integrin, angiogenesis, tumor, matrix metalloproteinase 2.

Description

Pharmaceutical compositions for inhibiting tumor growth

The present invention relates to a method of inhibiting angiogenesis and tumor growth. More particularly, the present invention optionally integrins (integrin) α _v bind to β ₃ integrin α _v to β ₃ and matrix metalloproteinase protein kinase 2; to block the interaction of the (matrix metalloproteinase 2 MMP2) using the compound A method of inhibiting angiogenesis and tumor growth.

Invasion of vascular cells into tissues requires a cooperative interaction of a number of factors, including an extracellular matrix structure as well as proteinases that reform cell adhesion molecules that recognize this transient matrix. Recently, it has been reported that 72 kDa matrix metalloproteinase 2 (MMP2) plays an important role in angiogenesis and angiogenesis. For example, Kito et al., J. Cell Sci., 109, 953-8 (1996) indicate that MMP2 and its activator membrane type 1-matrix metalloproteinase (MT1-MMP) It has been reported that, during embryonic development, it is almost exclusively regulated and expressed by mesenchymal cells, suggesting that there are special matrix remodeling constraints in these tissues. In addition, angiogenesis and corresponding tumor growth are reduced in MMP2 knockout mice (Itoh et al., Cancer Res., 58 1048-51 (1998)). Interestingly, Safter et al., Saftor et al., Proc. Natl. Acad. Sci. USA, 89, 1557-61 (1992), reported that the binding of integrin α _v β ₃ , a known mediator of angiogenesis, induces the production of MMP2, both of which are involved during angiogenesis associated with angiogenesis. It suggests coordinated interaction of molecules. Bafetti et al., J. Biol. Chem., 273, 143-9 (1998). Indeed, a direct interaction between MMP2 and integrin α _v β ₃ has been demonstrated by Brooks et al., Cell, 85, 683-93 (1996). Brooks et al. Later revealed that negative regulation of MMP2 during vascular invasion and maturation depends on the expression of α _v β ₃ (Cell, 92, 391-400 (1998)).

Although natural and synthetic inhibitors of MMPs, including MMP2, have been shown to inhibit angiogenesis and concomitantly inhibit tumor growth, primarily the harmful side effects of a broad range of inhibitors limit the conversion of these strategies to clinical modalities. There is this. In general, because many biological processes in adults require MMP action, inhibition of the active site of enzymatic action may have more impact on a variety of biological processes, including tissue remodeling (eg wound healing). Indeed, in clinical studies of several types of cancer, treatment with a broad range of MMP inhibitors has been shown to treat inflammatory tendonitis, polyarthritis, and muskoskeletal pain syndrome, which often limits doses and persists even after discontinuation. Causes serious side effects, including. However, the distribution of integrin α _v β ₃ in adults should be limited so that the interaction between MMP2 and α _v β ₃ on angiogenesis or cell invasion areas should be targeted to correspondingly limit the effects of the above-described treatment-related toxicity. You can expect. Indeed, the recombinant noncatalytic carboxy terminal hemopexin domain (PEX) of MMP2 that mediates MMP2 binding to integrin α _v β ₃ exhibits antiangiogenic and anticancer activity in vivo. The potential utility of such large protein fragments, along with the accompanying disadvantages (eg, mass production issues, FDA quality and safety control issues, and antigenicity), requires a more practical solution to the above problems.

Thus, there is a need for methods of inhibiting angiogenesis and tumor growth using chemical compounds that have minimal inhibition of MMP and selectively inhibit MMP activity at tumor growth sites in other regions of the body. There is also a need for a method of specifically binding to the MMP2 binding site of integrin α _v β ₃ .

Summary of the Invention

The present invention provides a method of inhibiting the interaction of integrin α _v β ₃ with MMP2 and a method of inhibiting angiogenesis in cells containing integrin α _v β ₃ . The present invention also provides a method of inhibiting tumor growth by administering an interaction inhibitor of MMP2-α _v β ₃ . The activity inhibitor compounds shown in Formula I below are in contact with integrin α _v β ₃ on the cells, thereby inhibiting the binding of MMP2 to α _v β ₃ . Inhibition of binding of MMPS to α _v β ₃ by the method of the present invention results in inhibition of tumor growth by inhibiting angiogenesis. In addition, α _v β ₃ is involved in inflammation, and thus the compounds of formula (I) used according to the method of the invention can also inhibit inflammatory phenomena.

In the above formula,

G ¹ and G ² are each independently -NH-C (O) -0-R ¹ , -NH-C (O) -O- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -NH -C (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -0- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ or -NH-C (O) -CH _2- (C ₆ H ₄ ) -X ³ ,

Y ¹ and Y ² are each independently —OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or -NH ₂ ,

R ¹ is C ₁ -C ₄ alkyl,

X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy,

X ³ is halo, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl,

Z is -C≡C-, -C ₆ H _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, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl or trans-1,4-cyclohexyl,

A is H or a covalent bond,

m and n are each independently an integer that is a value of 0 or 1,

t is an integer that is a value of 0 or 1,

p, r, and v are each independently integers that are 1 or 2,

Provided that when A is H, t is 0; When A is a covalent bond, t is 1; when m is 0, Y ¹ is C ₁ -C ₄ hydroxyalkyl; When n is 0, Y ² is C ₁ -C ₄ hydroxyalkyl.

Preferred compounds within the scope of formula (I) are represented by formula (II).                 

In the above formula,

R ² and R ³ are each independently H, C ₁ -C ₄ alkyl, phenyl, or benzyl,

X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy,

X ⁴ and X ⁵ are each independently halo, nitro, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl or C ₁ -C ₄ perfluoroalkyl,

A is H or a covalent bond,

p and r are each independently integers that are 1 or 2,

t is an integer that is a value of 0 or 1,

Provided that when A is H, t is 0; T is 1 when A is a covalent bond.

When A is a covalent bond and t is 1, the iminodiacetamide derivative residue may be attached to the benzene linking group in the ortho, meta or para position.                 

When contacting the compound of Formula I and Formula II and the cell containing the α _v β _3, α _v β ₃ The binding to the MMP2 is inhibited thereby blocking the essential mechanism in angiogenesis. Angiogenesis blockage can also prevent tumor angiogenesis, thereby inhibiting tumor growth by preventing the tumor from ingesting nutrients. Thus, the angiogenic and tumor growth inhibiting compounds of the present invention are useful therapeutics for the treatment of patients with tumors and angiogenic diseases. Since the compound of the present invention binds to α _v β ₃ , these compounds can also be used to suppress the inflammatory response.

The compounds of the present invention may be formulated in a suitable pharmaceutically acceptable matrix. Pharmaceutical compositions of the active compounds are administered to tumor patients to reduce or eliminate tumor growth. The active compound may be administered parenterally by injection or by gradual infusion over an extended period of time, or by any other method suitable for a particular dosage form.

In the accompanying drawings,

1 is a schematic diagram showing the interaction of integrin α _v β ₃ with MMP2 and its role in angiogenesis.

2 is described in Boger et al., Bioorg. Med. Chem, 6, 1347-1378 (1998), show the structural subunits A, B and C of the combinatorial library of 600 compounds.

3 graphically depicts the binding of a compound of 60 combination combinations with integrin α _v β ₃ competing with MMP2.

4 shows the binding of integrin α _v β ₃ with the mixture AxB10 and the ten independent components of A6B10C4.

5 shows the structure of analogs of A6B10C4.

6A graphically depicts the binding of analogs of Formula 6-26 with integrin α _v β ₃ and A6B10C4 (Compound 1) to compete with MMP2.

6B graphically depicts the binding of integrin α _v β ₃ to Formula 9 and Formula 19 compared to MMP2.

7 is a ^[14 C] - labeled compound 19 is specific to the α _v β ₃ binding and unlabeled 25 fold excess α _v β competitively may be substituted, but the excess amount of the compound from ₃ by the compound 19 in 9, not substituted by the RGD peptide or the c (RGDfV) peptide.

FIG. 8 shows that Compound 19 blocks binding of MMP2 to integrin α _v β ₃ but does not interfere with Vitronectin binding to integrin α _v β ₃ .

9 shows that Compound 19 does not directly inhibit active MMP2 proteolysis.

The binding of MMP2 to integrin α _v β ₃ is an important mechanism in angiogenesis. Specific inhibition of this binding interaction reduces angiogenesis in growth tissues such as tumors, thereby delaying tumor growth. The interaction of MMP2 with integrin α _v β ₃ is illustrated in FIG. 1. A new class of angiogenesis and tumor growth inhibitors can be an important therapeutic tool because they specifically bind to integrin α _v β ₃ in competition with MMP2 as described below.

Certain compounds of the present invention may include one or more asymmetric centers and may exist in optically active forms. Additional asymmetric centers may be present in substitution groups such as alkyl groups. Pure S- and pure R-isomers, racemic mixtures of isomers and mixtures thereof are included within the scope of the present invention. Chiral forms of certain compounds of the invention are also contemplated and are specifically included within the scope of the invention.

The term "alkoxy" means that the oxygen atom is connected to an alkyl group of a certain size as described below by ether linkage. Examples of alkoxy are methoxy, ethoxy, t-butoxy and the like. The term "alkyl" means a straight or branched carbon radical of a certain size. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, secondary-butyl, isobutyl, t-butyl, 2-ethylhexyl, n-octyl, 2,4-dimethylpentyl, and the like. . The term "hydroxyalkyl" means that an alkyl group of a certain size as described above is linked to a hydroxy group. Examples thereof include hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-1-propyl, 2-hydroxy-1-propyl, 4-hydroxybutyl and the like.

The term "perfluoroalkyl" means that there is a fluoro substituent at the hydrogen position of each alkyl group of a certain size, as described below, examples being trifluoromethyl and pentafluoroethyl.

The term "halo" or "halogen" means bromo, chloro, fluoro and iodo.

Compounds useful in the methods of the invention include iminodiacetamide derivatives represented by formula (I) and chemically attached to the linking group.

Formula I

In the above formula,

G ¹ and G ² are each independently -NH-C (O) -0-R ¹ , -NH-C (O) -O- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -NH -C (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -0- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ or -NH-C (O) -CH _2- (C ₆ H ₄ ) -X ³ ,

Y ¹ and Y ² are each independently —OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or -NH ₂ ,

R ¹ is C ₁ -C ₄ alkyl,

X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy,

X ³ is halo, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl,

Z is -C≡C-, -C ₆ H _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, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl or trans-1,4-cyclohexyl,

A is H or a covalent bond,

m and n are each independently an integer that is a value of 0 or 1,

t is an integer that is a value of 0 or 1,

p, r, and v are each independently integers that are 1 or 2,

Provided that when A is H, t is 0; When A is a covalent bond, t is 1; when m is 0, Y ¹ is C ₁ -C ₄ hydroxyalkyl; When n is 0, Y ² is C ₁ -C ₄ hydroxyalkyl.

Preferred compounds within the scope of formula (I) are represented by formula (II) and include iminodiacetamide derivatives attached to the benzene linking group to one of ortho, meta or para.

Formula II

Where

R ² and R ³ are each independently H, C ₁ -C ₄ alkyl, phenyl, or benzyl,

X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy,

X ⁴ and X ⁵ are each independently halo, nitro, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl or C ₁ -C ₄ perfluoroalkyl,

A is H or a covalent bond.

p and r are each independently integers that are 1 or 2,

t is an integer that is a value of 0 or 1,

Provided that when A is H, t is 0; T is 1 when A is a covalent bond.

Preferably, substituents X ¹ and X ² are attached at the 4-position of the phenyl ring relative to the CH ₂ group (ie para substituent). Preferably, at least one of X ¹ and X ² is fluoro, most preferably X ¹ and X ² are both para-fluoro. Preferably, r and p are two. X ⁴ and X ⁵ are preferably C ₁ to C ₄ perfluoroalkyl, most preferably para-trifluoromethyl. Preferred R ² and R ³ groups are hydrogen and methyl. Substituents X ² and X ³ may be the same or different, and substituents R ² and R ³ may be the same or different.

Compounds of formula (I) and formula (II) are described in Boger et al., Bioorg. Med. Chem, 6, 1347-1378 (1998)].

Particularly active members of the compound of formula (II) are compounds 19 of Scheme 1 when A is a covalent bond and t is 1.

Synthesis of Compound 19 is exemplified by the general method of preparing Formulas I and II described by Boger et al. Compound 19 was synthesized in three steps starting with commercially available N-ε-BOC-L-lysine methyl ester. Carbamate is reacted with 4- (trifluoromethyl) benzyl alcohol with N, N-diguccinimidyl carbonate and subsequently added to the activated product with an α-amino group providing intermediate compound 27 A yield of 99% was obtained. Subsequently, the N-BOC protecting group of this lysine derivative is removed (HCl) and free of iminodiacetic acid monoamide compound 28 using bromo tripyrrolidinophosphonium hexafluorophosphonate (PyBrOP, 74%). Coupling to the carboxylic acid functional group yielded diamide compound 29. After N-BOC protecting group removal (HCl), it was reacted with isophthaloyl dichloride to dimerize to complete the synthesis, yielding compound 19 with a yield of 60%. A small amount of radioactive isotope was incorporated to saponify two methyl esters (LiOH, 95%) to give dicarboxylic acid compound 30, followed by 1- (3- (dimethylamino) propyl) -3-ethylcarbodiimide hydrochloride [ ¹⁴ C] -Methanol mediated by (EDCI) and catalytic 4-dimethylaminopyridine (DMAP) was obtained to yield [ ¹⁴ C] -Compound 1 with a yield of 35%.

Pharmaceutical formulations of compounds of Formula I and Formula II may be prepared by formulating the compound in a pharmaceutically acceptable carrier matrix. Pharmaceutical compositions comprising the active compounds of Formula (I) and Formula (II) are administered to a host with tumors to reduce or inhibit tumor growth. The active compound can be administered parenterally by injection or by gradual infusion over an extended period of time. Treated tissue is very frequently treated by intraperitoneal or subcutaneous administration, but this active compound can also be administered intraocularly, intravenously, intramuscularly, intravitrealally, intraluminally or transdermally and can also be delivered by peristaltic means. .                 

"Administration" of a compound or composition of the invention herein is a dosage form unit dosage form comprising conventional, non-toxic pharmaceutically acceptable carriers, adjuvants and optionally vehicles, orally, or by inhalation spray, nasal, It is meant to be used parenterally or locally by the rectal or buccal route. As used herein, the term “parenteral” includes intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarticular injection or infusion techniques.

"Pharmaceutically acceptable" is suitable for use in contact with tissues of humans and lower animals within medically reasonable limits without causing unexpected toxicity, irritation, allergic reactions, and tumor and neovascularization. When used in the treatment of related diseases, it refers to salts, amides and esters and the like that are balanced with effective and reasonable benefits / damage ratios.

Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in S. M Berge, et al., J. Pharmaceutical Sciences, 66, 1-19 (1977). Representative acid addition salts include hydrochloride, hydrogen bromide, sulfate, hydrogen sulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, Methanesulfonate, citrate, maleate, fumarate, succinate, tartrate, ascorbate, glucoheptonate, lactobionate, lauryl sulfate salt and the like. Representative alkali or alkaline earth metal salts include sodium salts, calcium salts, potassium salts, magnesium salts, and the like.                 

The term "pharmaceutically acceptable carrier" as used herein means a nontoxic inert solid, semisolid or liquid filler, diluent, encapsulating material or any type of formulation aid. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; Starch such as corn starch and potato starch; Cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Powdered tragacanth; malt; gelatin; Talc; Excipients such as cocoa butter, and suppository waxes; Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; Glycols such as polypropylene glycol; Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; Esters such as ethyl oleate and ethyl laurate; Agar; Buffers such as magnesium hydroxide and aluminum hydroxide; Alginic acid; Pyrogenic material distilled water; Isotonic saline; Ringer's solution; Ethyl alcohol and phosphate buffers as well as other nontoxic materials suitable for use in pharmaceutical formulations.

At the discretion of the formulator, wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coatings, sweeteners, flavors, fragrances, preservatives and antioxidants, may be used in the composition. have. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium sulfite, and the like; Oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; Metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

A "therapeutically effective amount" of an agent or compound of the invention means an amount of the compound sufficient to treat tumors and angiogenesis related diseases at a reasonable benefit / damage ratio applicable to any medical treatment. However, it will be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the appropriate medical judgment. Particular therapeutically effective levels for a particular patient may include the severity of the disease and disorder to be treated; Activity of the specific compound employed; The specific composition employed; The age, body weight, medical condition, sex and regimen of the patient; The period of administration, route of administration, and rate of excretion of the specific compound employed; Duration of treatment; Drugs used in combination or coincidental with the specific compound employed; And various factors including those well known in the medical arts.

The present invention also provides pharmaceutical compositions in unit dosage form containing a therapeutically effective amount of a compound (or compounds) of the invention in admixture with a conventional pharmaceutical carrier. Injectables, for example, sterile injectable aqueous or oily suspensions may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectables may be sterile injectable solutions, suspensions or emulsions in nontoxic parenterally acceptable carriers or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. And isotonic sodium chloride aqueous solution. In addition, sterile cured oils are conventionally employed as a solvent or suspending medium. Any blended hardening oil can be used for this purpose including synthetic mono- or diglycerides.

Fatty acids such as oleic acid are also used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter or by incorporation of a sterilant in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable media immediately prior to use. have.

In order to sustain the effect of the drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. The most common way to do this is by injecting a suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug depends on the rate of dissolution of the drug, in other words the physical state of the drug (eg crystal size and crystalline form). Another way to delay absorption of the drug is to administer the drug as a solution or suspension in oil. Injection depot forms may also be prepared by forming microcapsule matrices of biodegradable polymers such as polylactide-polyglycolide and drugs. Depending on the ratio of drug to polymer and the composition of the polymer, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly-orthoesters and polyanhydrides. Depot injections can also be prepared by entrapping the drug in microemulsions or liposomes suitable for body tissue.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with suitable non-irritating excipients (eg, cocoa butter and polyethylene glycol) that are solid at ordinary temperatures but liquid at rectal temperature and will therefore melt in the rectum to release the drug.                 

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, prills, and granules. In such solid dosage forms the active compound may be mixed with one or more inert diluents (eg sucrose, lactose or starch). Such dosage forms may also generally include additive materials other than inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage form may comprise a buffer. Tablets and pills can further be prepared using enteric coatings and other controlled release coatings. Solid compositions of a similar type may be prepared using excipients such as lactose or lactose, high molecular weight polyethylene glycols, and the like as fillers in soft and hard filled gelatin capsules.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, including inert diluents commonly used in the art, such as water. Such compositions may include auxiliaries such as wetting agents; Emulsifying and suspending agents; Sweeteners, flavors and fragrances may also be included. If desired, the compounds of the present invention may be incorporated into sustained or targeted delivery systems such as polymer matrices, liposomes and microspheres. They can be sterilized, for example, by filtration through a bacterial retention filter or by incorporation of a sterilizing agent in the form of a sterile solid composition which can be dissolved in sterile water or other sterile injectable media immediately prior to use. The active compound may be microencapsulated with one or more excipients as described above.

Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may optionally comprise an opacifying agent and may preferably be a composition which releases only the active ingredient in any delayed manner to a particular part of the intestine. Embedding compositions that can be used include polymeric substances and waxes. Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. . Under sterile conditions, the active ingredient may be mixed with a pharmaceutically acceptable carrier and optionally with the required preservatives or buffers.

Also contemplated ophthalmic formulations, ear drops, eye ointments, powders and solutions within the scope of the present invention. Ointments, pastes, creams and gels may be used in addition to the active ingredients of the present invention in animal and vegetable fats, oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, talc And excipients such as zinc oxide or mixtures thereof.

Powders and sprays may include, in addition to the compounds of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures thereof. Sprays may further comprise conventional propellants, such as chlorofluorohydrocarbons.

Transdermal patches have another advantage of being able to modulate compound delivery to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the absorption of the compound through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Compositions comprising the active compound can be administered in a therapeutically effective amount in a manner suitable for dosage formulation. Dosage and time of administration depend on the host to be treated, the dose of host systemic system in which the active ingredient is available, and the degree of therapeutic effect desired. The exact amount of active ingredient required at the time of administration depends on the judgment of the practitioner and depends on the individual.

Suitable dosage ranges for systemic application are described herein and depend on the route of administration. Suitable dosage regimens can vary, but are determined by initial administration, followed by a dose repeated at one or more predetermined intervals by subsequent injection or other route of administration.

The invention also provides pharmaceutical compositions useful for practicing the methods of treatment described herein. The composition comprises a pharmaceutically acceptable carrier with the compound described above.

Formulations for parenteral administration of a compound or composition of the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, sodium chloride, lactated Ringer's solution or hardened oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, Ringer's dextrose system), and the like. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, inert gases and the like may also be present.

Another aspect of the invention provides a method of inhibiting angiogenesis in tumor tissue by inhibiting the interaction of MMP2 with α _v β ₃ . Inhibition methods include administering to a host a composition comprising an angiogenesis inhibitory amount of the preferred compound described above. The interaction of MMP2 with α _v β ₃ is inhibited by contacting the compounds of the present invention with α _v β ₃ .

Angiogenesis is the formation of neovascular nets from host vessels that already exist, and in the case of tumor growth requires 1 to 2 mm 3 or more. For the purposes of the present invention, angiogenesis is suppressed to alleviate angiogenesis and disease symptoms mediated by angiogenesis.

When administering an active compound to a host, the range of doses depends on the particular active compound and its efficacy on a particular tumor or integrin. One skilled in the art will readily be able to determine the appropriate dose for a particular active compound without unnecessary experimentation. The host can be any mammal. The dose should be capable of sufficiently exhibiting the desired therapeutic effect in which angiogenesis and disease symptoms mediated by angiogenesis are alleviated, and generally the level of plasma active compound is from about 0.01 to about 100 μM, preferably from about 0.2 to about 20 μM. More preferably about 1 to about 10 μM. However, it should not be high enough to cause side effects. Upon administration of one or more doses per day for one or several days or for an indefinite period, the dose per kg of body weight may vary from 1 to 20 mg per dose.

When inhibiting angiogenesis, the therapeutically effective amount is an amount of active compound sufficient to inhibit melanogenesis in the tissue to be treated to a measurable level, i.e., angiogenesis inhibition or MMP2-α _v β ₃ interaction inhibition. Inhibition of angiogenesis can be measured in situ by immunohistochemistry as described herein or by other methods known to those skilled in the art.

The invention further provides pharmaceutical compositions useful for practicing the methods of treatment described herein. The composition comprises a pharmaceutically acceptable carrier in combination with the active compound described above.

The invention also provides a method of inducing apoptosis in tumor cells. The method comprises administering to the host a therapeutically effective amount of an active compound sufficient to initiate apoptosis of tumor cells.

For the purposes of the present invention, when apoptosis of tumor cells is observed in treated target tumors, apoptosis of tumor cells is induced. Apoptosis of tumor cells can be measured by the methods described herein or by methods well known in the art.

The following non-limiting examples are provided to describe various aspects of the present invention.

Substances and Methods

Antibody Cells and Reagents: CS-1 cells and CS-1 hamster melanoma cells infected with human β ₃ -integrin subunit (β ₃ CS-1 cells) are as previously described. Cell, 85, 683- 93 (1996); Cell, 92, 391-400 (1998). Horseradish peroxidase (HRP) -conjugated monoclonal antibody anti-biotin mAb BN-34 and anti-actin mAb AC-40 were obtained from Sigma (Sigma, St. Louis, MO). Anti-von Willebrand Factor (vWF) polyclonal antibodies (pAb) were obtained from Dakosa (DAKO, Glostrup, Denmark). Cyclic peptides cRGDfV and cRADfV, and integrin α _v β ₃ were obtained from Merck KGaA (Merck KGaA, Darmstadt, Germany). Purified proMMP2 and integrin α _v β ₃ are provided by Chemicon International (Tmecula, Calif.). Purified active MMP2 was obtained from Calbiochem, La Jolla, Calif. Basic fibroblast growth factor (bFGF) is provided by Scios, Mountain View, CA.

Example 1 Solid Phase Integrin Binding Assay

Purified integrins were adsorbed overnight on microtiter wells (1-5 μg / ml, 50 μg / well) before blocking with caseinblocker (Pierce, Rockford, IL). Under test cyclic compound RGD or RAD peptides, or buffer vehicle alone in the presence or absence of, binding buffer (50mM Tris, pH 8, 150mM NaCl, 1mM MgCl 2, 0.5mM MnCl 2) of the purified biotinylated MMP2 (bMMP2, 3-5 nM) was added to the wells. Control wells lack integrins. Biotinylated Vitronectin (bVN, 1 μg / ml) was used as reference material. Bound proteins were detected with HRP-anti-biotin mAb and 3,3 ', 5,5'-tetramethylbenzidine solution [TMB; Substrates of peroxidase; Source: BioRad, Hercules, Calif., USA.

To assess direct integrin binding by Compound 19, add 150 μL of binding buffer containing 0.1% Tween-20 and titrate [ ¹⁴ C] -Compound 19 to substantially block wells and incubate before aspirating all liquids. Α _v β ₃ and α ₅ β ₁ (10 μg / ml, 50 μl) in one Imulon-4 microtiter well (Source: Dynatech Laboratories, Chantilly, Va.) / Well). Dried wells for quantification were separated and immersed in BetaMax liquid scintillation cocktail (Source: ICN Biochemicals, Costa Mesa, CA, USA). From this binding curve, the saturation concentration of [ ¹⁴ C] -Compound 19 (3 μM) in the presence or absence of 25-fold molar excess (75 μl) of unlabeled Compound 19 or Compound 9, or 100 μM cyclic RGD or RAD peptide was determined. Investigate. BVN was used as a control and detected as described above.

Example 2. MMP2 cell-binding and [ ³ H]-Collagen IV Degradation Assay

CS-1 cells or β ₃ CS-1 cells comprise 4 nM of purified active MMP2 alone or 10 μM of compound supplemented with 0.5% calf serum albumin (BSA), 0.4 mM MnCl ₂ and 10 μg / ml aprotinin 19 or Compound 9 was incubated for 45 minutes at 37 ° C. in adherent buffer fibroblast basal medium (FBM), then washed and added to [ ³ H] -collagen IV-coated wells. The wells were coated with 50 μl of 0.414 mCi / ml [ ³ H] -collagen IV [Source: Icy Biochemicals] overnight and carefully washed until the radioactivity in the recovered washes reached the background radioactivity. In addition, cells were treated as described above in the absence of MMP2, or MMP2 solution was added directly to the cell free wells as a control. Collagen IV degradation was quantified by measuring the radioactivity released in 50 μl of culture medium as measured by a liquid scintillation counter. To assess the binding of biotinylated MMP2 to CS-1 cells, the cells were suspended in adhesion buffer and incubated with 12 nM bMMP2 for 45 minutes at 37 ° C. in the presence or absence of 10 μM Compound 19 or Compound 9. Cells were then washed, then lysed, treated to undergo SDS-PAGE and immunoblotted with anti-biotin mAbs.

Example 3. Synthesis of Compound 27

A solution of N, N'-diisuccinimidyl carbonate (95.38 g, 21 mmol) in acetonitrile (150 mL) was added 4- (trifluoromethyl) benzyl alcohol (2.87 mL, 21 mmol) and triethylamine (Et ₃ N; 5.8 ml, 42 mmol) and stirred at 25 ° C. After 3 hours, this solution was added to a flask containing N-ε-BOC-lysine methyl ester in acetonitrile (4.2 g, 14 mmol) and stirred for a further 3 hours. The solvent was evaporated and the residue was dissolved in CH ₂ Cl ₂ (250 mL) and washed with 10% aqueous HCl solution (2 × 200 mL) and saturated aqueous NaHCO ₃ solution (200 mL). Flash chromatography (SiO ₂ , 3: 1 CH ₂ Cl ₂ / EtOAc) afforded 6.4 g (99%) of compound 27 as a pale yellow oil.

[α] _D ²⁵ -8.9 ^© 5.55, CH ₃ OH; ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.57 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 5.70 (d, J = 7.9 Hz), 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); ¹³ C NMR (CDCl ₃ , 100 MHz) δ 172.9, 156.2, 155.8, 140.4, 130.1 (q, J = 32.0 Hz), 127.8, 125.3, 122.9 (q, J = 270.0 Hz), 79.05, 65.8, 53.7, 52.3, 39.8, 31.7, 29.5, 28.4, 22.2; IR (film) v _max 3357, 2952, 1790, 1745, 1524 cm ⁻¹ ; FABHRMS (NBA-NaI) m / z 463.2044 (M + H ⁺ , C ₂₁ H ₂₉ F ₃ N ₂ O ₆ requires 463.2056)

Example 4. Synthesis of Compound 29

A solution of compound 27 (2.2 g, 48.8 mmol) in CH ₂ Cl ₂ (3 mL) was treated with HCl-dioxane (10 mL) 4N and stirred at 25 ° C. for 20 min. The solvent and excess acid are removed under reduced pressure, the crude hydrochloride salt is dissolved in DMF (40 mL) and N-((tert-butyloxy) carbonyl) -N '-(2- (4-fluoro) Phenyl) ethyl) iminodiacetic monoamide (Compound 28) (1.68 g, 4.8 mmol), PyBrOP (3.3 g, 7.1 mmol) and diisopropylethylamine (i-Pr ₂ NEt; 5.0 mL, 29 mmol) And stirred at 25 ° C. for 1 h. The reaction mixture was diluted with EtOAc (400 mL) and washed with 10% aqueous HCl solution (2 × 300 mL) and saturated aqueous NaHCO ₃ solution (300 mL). Flash chromatography (SiO ₂ , 1: 1 CH ₂ Cl ₂ / EtOAc) afforded 2.47 g (74%) of compound 29 as a white foam-like solid.

[a] _D ²⁵ -7.1 ^© 4.50, CH ₃ OH; ¹ H NMR (CDCl ₃ , 400 MHz) δ 8.23 and 7.59 (m, together 1 H), 7.58 (d, J = 8.1 μs, 2H), 7.43 (m, 2H), 7.13 (m, 2H), 7.06 and 6.78 (m, together 1H), 6.94 (m, 2H), 5.70 (dd, J = 12.9 and 8.2 Hz, 1H), 5.11 (m, 2H), 4.31 (m, 1H), 3.85-3.72 (m, 4H ), 3.71 (s, 3H), 3.49 (m, 2H), 3.22 (m, 2H), 2.79 (m, 2H), 1.81-1.39 (m, 6H), 1.38 (s, 9H); ¹³ C NMR (CDCl ₃ , 100 MHz) δ 172.8, 170.0, 169.9, 155.8, 154.8, 161.4 (d, J = 242.7 Hz), 140.2, 134.4, 130.1 (d, J = 33.4 Hz), 130.0, 127.7, 125.3 (q, J = 3.0 μs), 123.8 (q, J = 299.9 μs), 115.0, 81.2, 65.7, 53.9, 53.3, 52.2, 40.8, 38.6, 34.4, 31.5, 28.0, 22.4; IR (film) v _max 3267, 2935, 1708, 1657, 1151 cm <^-1>; FABHRMS (NBA-CsI) m / z 831.2026 (M + Cs ⁺ , C ₃₃ H ₄₂ F ₄ N ₄ O ₈ requires 831.1993)

Example 5. Synthesis of Compound 19

A solution of compound 29 (50 mg, 0.075 mmol) in CH ₂ Cl ₂ (1 mL) was treated with 4N HCl-dioxane (1 mL) and stirred at 25 ° C. for 1 h. Solvent and excess acid are removed under N ₂ vapor, crude hydrochloride salt is suspended in CH ₂ Cl ₂ (1 mL), isophthaloyl dichloride (7.6 mg, 0.038 mmol) and i-Pr ₂ NEt (0.05) ML, 0.3 mmol) and stirred at 25 ° C. for 12 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with 10% aqueous hydrochloric acid solution (3 x 30 mL), saturated aqueous NaHCO ₃ solution (30 mL) and saturated aqueous NaCl solution (30 mL). Flash chromatography (SiO ₂ , 1: 4.5: 4.5 MeOH / CH ₂ Cl ₂ / EtOAc) gave 19.30 mg (60%) of compound as a white powder.

¹ H NMR (CD ₃ OD, 400 MHz) δ7.62 (m, 4H), 7.52 (m, 4H), 7.42 (m, 4H), 7.19 (m, 4H), 6.96 (m, 4H), 5.14 ( m, 4H), 4.16 (m, 2H), 4.13 (m, 2H), 4.08 (m, 2H), 3.99 (m, 4H), 3.68 (s, 6H), 3.45-3.35 (m, 4H), 3.25 -3.11 (m, 4H), 2.82-2.70 (m, 4H), 1.82 (m, 2H), 1.69 (m, 2H), 1.60-1.34 (m, 8H); IR (film) v _max 3291, 2936, 1725, 1651, 1326 cm <^-1>; FABHRMS (NBA-CsI) m / z 1459.4015 (M + Cs ⁺ , C ₆₄ H ₇₀ F ₈ N ₈ O ₁₄ requires 1459.3938).

Example 6. Synthesis of Compound 30

A solution of compound 19 (13 mg, 0.01 mmol) in tert-butanol (0.3 mL) was treated with LiOH.H ₂ O (0.91 mg, 0.22 mmol) dissolved in H ₂ O (0.15 mL), followed by 2 at 0 ° C. Stir for hours. The reaction mixture was then quenched with HCO ₂ H (1 mL), diluted with EtOAc (10 mL) and washed with saturated aqueous NaCl solution (2 × 10 mL). Drying (Na ₂ SO ₄ ) and evaporation gave 12 mg (95%) of compound 30 as a white powder.

¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.54 (br s, 2H), 8.63 (m, 1H), 8.43 (m, 2H), 8.30 (m, 1H), 7.74 (m, 4H), 7.69 (m, 2H), 7.57 (m, 4H), 7.40 (m, 4H), 7.24 (m, 4H), 7.09 (m, 4H), 5.15 (m, 4H), 4.14 (m, 2H), 4.02 -3.87 (m, 8H), 3.31 (m, 4H), 3.208 (m, 4H), 2.74 (m, 4H), 1.71 (m, 2H), 1.62 (m, 2H), 1.50-1.34 (m, 8H ); IR (film) v _max 3287, 2928, 1705, 1659, 1320 cm <^-1>; MALDIHRMS m / z 1321.4493 (M + Na ⁺ , C ₆₂ H ₆₆ F ₈ N ₈ O ₁₄ requires 1321.4468).

Example 7. ¹⁴ C] -Synthesis of Compound 19

A solution of compound 27 (1.7㎎, 1.3mmol) and EDCI (2.0㎎, 10.3mmol) in _{DMF (20㎖) CH 2 Cl 2} (57mCi / mmol, 5.2mmol 14 CH 3 OH) of ¹⁴ CH ₃ OH solution was 0.3 Treated with 35 mL of DMAP stock solution in mL and CH ₂ Cl ₂ and stirred at 0 ° C. for 4 h. The reaction mixture was then diluted with EtOAc (3 L), washed with 10% aqueous hydrochloric acid solution (3 x 3 mL) and saturated aqueous NaHCO ₃ (3 mL) and dried over Na ₂ S0 ₄ . Purification on PTLC (SiO ₂ , 2: 3: 3 EtOH / CHCl ₃ EtOAc) gave 0.6 mg (35%) of [ ¹⁴ C] -Compound 19 as a white film. This material is identical to the corresponding unlabeled dimethylester compound 1 by ¹ H NMR and HPLC. Inactivity is about 104 Ci / mmol.

¹ H NMR (CD ₃ OD, 400 MHz) δ7.62 (m, 4H), 7.52 (m, 4H), 7.42 (m, 4H), 6.96 (m, 4H), 5.14 (m, 4H), 4.16 ( m, 2H), 4.13 (m, 2H), 4.08 (m, 2H), 3.99 (m, 4H), 3.68 (s, 6H), 3.45-3.35 (m, 4H), 3.25-3.11 (m, 4H) 2.82-2.70 (m, 4H), 1.82 (m, 2H), 1.69 (m, 2H), 1.60-1.34 (m, 8H).

Results and discussion

Combinatorial libraries comprising compounds of formula (I) and formula (II) together with their synthesis methods are described in Boger et al., Bioorg. Med. Chem, 6, 1347-1378 (1998). Boger et al. Describe the preparation of a combinatorial library of 60 mixtures of each of the 10 compounds, wherein independent compounds in the mixtures represent three subunits coupled together as shown in FIG. 2. Include. Subunits of compounds are designated A, B, C. The library consisted of six different A units (A1-A6), ten different B units (B1-B10), and ten different C linking groups (C ₁ -C ₁₀ ). Each A unit was coupled with each B unit to form 60 different AB compounds. The independent AB compounds are then coupled with a mixture of 10 different C linking groups to form 60 mixtures of 10 compounds, each designated AxBy, where x and y are each independent A incorporated into the compound of the mixture. And B subunits. Subunits of A, B and C of the combinatorial library of compounds are shown in FIG. 2.

Evaluation of the 60 mixtures described above in a competitive integrin α _v β ₃ binding assay competing with MMP2 showed that several mixtures inhibited the binding of integrin α _v β ₃ to MMP2. The results of the evaluation assay are shown in FIG. 3. In particular the active mixtures included A1B6, A1B7, A1B8, A4B1, A5B4, A5B5, A5B6, A5B10 and A6B10. The most active mixture is A6B10, so 10 independent compounds of the mixture are synthesized individually and investigated in the same assay, the results of which are shown in FIG. 4. Independent assays A6B10C1 to A6B10C10 in the assay were active at 3 μM concentration.

Similar compounds (2-26) of A6B10C4 (Compound 1) shown in FIG. 5 were also evaluated. The results of the binding assay of Compounds 2-26 are shown in Figure 6A. Compounds 8, 9 and 23 all inhibited the binding of MMP2 to integrins.

Active MMP2 / integrin-α _v -β ₃ binding inhibitors of the invention include Formula I and Formula II.

Compound 19 was examined in detail to determine its specific target and define its biological properties. Since the binding assays have shown a lack of antagonist activity, despite the similarities and similar physical properties (eg solubility and hydrophobicity) of their overall structure, benzoyl amide compound 9 has been selected as a suitable negative control for many of these studies. . Compound 19 showed that inhibition of binding of MMP2 to integrins is concentration dependent as shown in FIG. 6B.

Radioisotopes ( ¹⁴ C) were incorporated into compound 19 in ester substituents (inactivation about 104 mCi / mmol). After incubation with immobilized α _v β ₃ (3 μM) and subsequent washing, it was found that this compound attached to integrins as demonstrated in FIG. 7. Incubation in the presence of a 25-fold molar excess of cooled compound 19 significantly reduced the amount of binder observed, whereas incubation in the presence of a 25-fold molar excess of (cooled) control compound 9 resulted in [ ¹⁴ C] -compound 19 There was no effect on binding. In a similar experiment measuring the interaction of [ ¹⁴ C] -Compound 19 with immobilized MMP2, no binding was observed. These results suggest that the development of antagonist activity observed in the MMP2-α _v -β ₃ binding assay results from the specific binding of compound 19 to α _v β ₃ . The nature of the interaction of compound 19-α _v -β ₃ is independent from the integrin site that recognizes the Arg-Gly-Asp sequence. Cyclic RGD peptide cyclo (Arg-Gly-Asp-D-Phe-Val) has no effect on the binding of [ ¹⁴ C] -Compound 19 (FIG. 7). Indeed, as shown in FIG. 8, Compound 19 did not inhibit the binding of Vitronectin, a typical high affinity ligand of α _v β ₃ to integrins, and the binding site of Compound 19 was associated with the site to which RGD-ligand binds. It is consistent with the idea of being different

In addition, compound 19 was studied in a cell assay measuring endothelial cells' ability to degrade protein matrix (the major step in angiogenesis) using MMP2. It is already known that interfering with the binding of MMP2 to α _v β ₃ inhibits [ ³ H] -collagen IV degradation. α _v β ₃ CS-1 melanoma cells transfected with is, β ₃ negative CS-1 cells to more than that (α _v is β ₃ Insufficient) break down, a fixed ^[3 H] - collagen IV much more It was found to degrade. As shown in FIG. 9, treatment of these cells with Compound 19 significantly reduced the increase in matrix degradation, consistent with the inability of MMP2 cells to bind to integrin surfaces. However, Compound 19 does not directly inhibit the proteolytic activity of MMP2 because purified (active) enzymes in the absence of cells can degrade [ ³ H] -collagen IV to a similar degree in the presence or absence of compound 19. It was.

These results support the suggestion that compounds of formula I block tumor cells' ability to degrade EMC proteins using MMP2 in a manner similar to PEX. The compounds of formula (I) do not interfere with the binding of α _v β ₃ to their typical RGD ligands, nor do they function as direct proteinase inhibitors.

The foregoing description and examples are presented to illustrate the invention and are not intended to limit the invention. Other variations within the spirit and scope of the invention may be possible and may be readily apparent to those skilled in the art.

Claims (37)

A pharmaceutical composition for inhibiting tumor growth by inhibiting the interaction of matrix metalloproteinase 2 (MMP2) with integrin α _v β ₃ in a host cell, comprising a compound of formula (I). Formula I

In the above formula, G ¹ and G ² are each independently -NH-C (O) -0-R ¹ , -NH-C (O) -O- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -NH -C (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -NH- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ , -OC (O) -0- (CH ₂ ) _V- (C ₆ H ₄ ) -X ³ or -NH-C (O) -CH _2- (C ₆ H ₄ ) -X ³ , Y ¹ and Y ² are each independently —OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or -NH ₂ , R ¹ is C ₁ -C ₄ alkyl, X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy, X ³ is halo, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl, Z is -C≡C-, -C ₆ H _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, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl or trans-1,4-cyclohexyl, A is H or a covalent bond, m and n are each independently an integer that is a value of 0 or 1, t is an integer that is a value of 0 or 1, p, r, and v are each independently integers that are 1 or 2, Provided that when A is H, t is 0; When A is a covalent bond, t is 1; when m is 0, Y ¹ is C ₁ -C ₄ hydroxyalkyl; When n is 0, Y ² is C ₁ -C ₄ hydroxyalkyl. The agent of claim 1, wherein G ¹ and G ² are —NH—C (O) —O— (CH ₂ ) _V — (C ₆ H ₄ ) —X ³ , A is a covalent bond and v is 1 Pharmaceutical composition. The pharmaceutical composition of claim 2, wherein X ³ is trifluoromethyl. The pharmaceutical composition of claim 2, wherein Y ¹ and Y ² are OH, m is 1 and n is 1. 4. The pharmaceutical composition of claim 2, wherein at least one of X ¹ and X ² is para-fluoro. The pharmaceutical composition of claim 2, wherein m is 1, n is 1 and at least one of R ² and R ³ is methyl. The pharmaceutical composition of claim 2, wherein R ² and R ³ are H, m and n are 1, X ¹ and X ² are fluoro and X ³ is trifluoromethyl. A pharmaceutical composition for inhibiting tumor growth by inhibiting interaction of matrix metalloproteinase 2 (MMP2) with integrin α _v β ₃ in a host cell, comprising a compound of Formula II. Formula II

In the above formula, R ² and R ³ are each independently H, C ₁ -C ₄ alkyl, phenyl, or benzyl, X ¹ and X ² are each independently halo or C ₁ -C ₄ alkoxy, X ⁴ and X ⁵ are each independently halo, nitro, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl or C ₁ -C ₄ perfluoroalkyl, A is H or a covalent bond, p and r are each independently integers that are 1 or 2, t is an integer that is a value of 0 or 1, Provided that when A is H, t is 0; T is 1 when A is a covalent bond. The pharmaceutical composition of claim 8, wherein at least one of X ⁴ and X ⁵ is C ₁ -C ₄ perfluoroalkyl and A is a covalent bond. The pharmaceutical composition of claim 8, wherein at least one of R ² and R ³ is H and A is a covalent bond. The pharmaceutical composition of claim 8, wherein at least one of R ² and R ³ is methyl and A is a covalent bond. The pharmaceutical composition of claim 8, wherein X ⁴ and X ⁵ are trifluoromethyl, R ² and R ³ are each methyl, and A is a covalent bond. The compound of claim 8, wherein A is a covalent bond, R ² and R ³ are methyl, X ⁴ and X ⁵ are trifluoromethyl, X ¹ and X ² are fluoro, m is 1 and n is 1 pharmaceutical composition. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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