RU2276133C2 - Compounds, pharmaceutical composition and method for inhibition of tumor growth (variants) - Google Patents

Abstract

FIELD: organic chemistry, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to new compounds of the formula (I)

inhibiting tumor growth and angiogenesis wherein each R² and R³ represents independently hydrogen atom (H), (C₁-C₄)-alkyl; each X² and X³ represents independently (C₁-C₄)-perfluoroalkyl; A represents hydrogen atom (H) or a covalent bond; t represents a whole number 0 or 1 under condition that if A represents hydrogen atom (H) then t = 0, and if A represents a covalent bond then t = 1. Also, invention relates to a pharmaceutical composition and method for inhibition of the tumor growth.

EFFECT: valuable medicinal properties of composition.

32 cl, 3 sch, 5 dwg, 19 ex

Description

Field of Invention

The present invention relates to compositions that inhibit angiogenesis and tumor growth. In particular, the invention relates to compositions that bind to integrin α _v β ₃ and block the interaction of integrin α _v β ₃ and matrix metalloproteinase 2 (MMP2). The invention also relates to methods of inhibiting angiogenesis and tumor growth, which use selective integrin binding inhibitors α _v β ₃ and MMP2.

BACKGROUND OF THE INVENTION

The invasiveness of vascular cells in tissue requires a coordinated interaction of a large number of factors, including proteinases, which alter the structure of the extracellular matrix, as well as cell adhesion molecules that recognize this altered matrix. Recent reports have indicated that 72 kDa matrix metalloproteinase 2 (MMP2) plays a key role in the formation of new vessels and in angiogenesis. For example, Kitoh et al. (J. Cell Sci., 109, 953-8 (1996)) reported that the presence of MMP2 and its membrane-type activator, 1-matrix metalloproteinases (MT1-MMP), which are interconnected expressed by mesenchymal cells almost exclusively during embryonic development, indicates on specific rearrangements of bonds in the matrix of such tissues. In addition, angiogenesis and associated tumor growth are reduced in mice with the MMP2 gene turned off (see Itoh et al., Cancer Res., 58 1048-51 (1998)). An interesting fact, pointed to by Saftor et al. (Proc. Natl. Acad. Sci. USA, 89, 1557-61 (1992)), is that ligation of integrin α _v β ₃ , which is a known mediator of angiogenesis, induces the production of MMP2, which indicates a coordinated interaction of these two molecules in the time of vascular rearrangement associated with the formation of a blood vessel (see also Bafetti et al, J. Biol. Chem., 273, 143-9 (1998)). Indeed, Brooks et al (Cell, 85, 683-93 (1996)) demonstrated the direct interaction of MMP2 and integrin α _v β ₃ . More recently, Brooks et al showed that such downregulation of MMP2 during vascular invasion and formation is associated with the expression of α _v β ₃ (Cell, 92, 391-400 (1998)).

Although it has been confirmed that natural as well as synthetic MMP inhibitors, including MMP2 inhibitors, inhibit angiogenesis and simultaneously inhibit tumor growth, the introduction of such techniques into clinical use has been of limited success, mainly due to harmful side effects such as a wide range inhibition. Since the functioning of MMP as a whole may be necessary for many processes occurring in an adult organism, inhibition of the active site of enzymatic function can probably have a wide range of effects on effects in various biological processes, which include tissue remodeling, for example, during wound healing. In fact, at the clinical trial stage, it was confirmed that treatment of various types of cancer with broad-spectrum MMP inhibitors caused serious side effects, including tendon tissue inflammation, polyarthritis and muscle pain syndromes, which limited the dose and often persisted after treatment was stopped. However, with a limited distribution of integrin α _v β ₃ in the adult organism, it can be assumed that the targeted interaction of MMP2 and α _v β ₃ in areas in which new blood vessels form or in areas of cellular invasion may, accordingly, limit the effect of such toxicity associated with with treatment. Indeed, the recombinant non-catalytic hemopexin domain at the carboxy terminus of MMP2 (PEX), which mediates the binding of MMP2 to integrin α _v β ₃ , showed in vivo antiangiogenic and antitumor activity. The possible use of such a large protein fragment, accompanied by inconveniences (for example, problems of large-scale production, the FDA (US Food and Drug Administration) quality control and safety of release and antigenicity), suggests the need for a more convenient solution to this problem.

There is a need for chemical compounds that selectively inhibit MMP activity in areas of tumor growth, minimally inhibiting MMP in other areas of the body.

SUMMARY OF THE INVENTION

The present invention relates to new compounds used as inhibitors of angiogenesis and tumor growth. The present invention also relates to a method for inhibiting the interaction of MMP2 and integrin α _v β ₃ and a method for inhibiting angiogenesis in cells containing integrin α _v β ₃ . In addition, the present invention relates to a method of inhibiting tumor growth by introducing inhibitors of MMP2-α _v β ₃ interaction.

The compounds of the present invention are represented by formula (I) and include glycillisin derivatives chemically attached to a linking group:

where G ¹ or G ² each independently represents —NH — C (O) —O — R ¹ , —NH — C (O) —O— (CH ₂ ) _v - (C ₆ H ₄ ) —X ¹ , - NH-C (O) -NH- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ , -OC (O) -NH- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ , - OC (O) -O- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ or -NH-C (O) -CH ₂ - (C ₆ H ₄ ) -X ¹ ; Y ¹ and Y ² each independently represents OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or —NH ₂ ; R ¹ represents C ₁ -C ₄ alkyl; X ¹ represents halogen, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl; Z represents —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 represents H or a covalent bond; m and n each independently represents an integer having a value of 0 or 1; t is an integer having a value of 0 or 1; and v is an integer having a value of 1 or 2; provided that when A is H, then t is 0; when A is a covalent bond, then t is 1; when m is 0, then Y ¹ is C ₁ -C ₄ hydroxyalkyl; and when n is 0, then Y ² is C ₁ -C ₄ hydroxyalkyl. These compounds bind to α _v β ₃ and inhibit the interaction of MMP2 and α _v β ₃ .

Preferred compounds of structural formula (I) are represented by structural formula (II):

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl, phenyl or benzyl; X ² and X ³ each independently represents halogen, nitro, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl or C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; and t is an integer having a value of 0 or 1; provided that when A is H, then t is 0, and when A is a covalent bond, then t is 1. When A is a covalent bond and t is 1, then the glycillisine derivative groups can be attached to the benzene-binding group in ortho, meta or para position.

When the compounds of formulas (I) and (II) are introduced into cells containing α _v β ₃ , the binding of α _v β ₃ and MMP2 is inhibited, thus inhibiting the basic mechanism of angiogenesis. Violation of angiogenesis can also inhibit tumor growth by preventing vascularization of the tumor, thereby disrupting its nutrition. Compounds of the present invention that inhibit angiogenesis and tumor growth are thus useful therapeutic agents for treating patients with tumors and angiogenic disorders. Since the present compounds bind to α _v β ₃ , these compounds can also be used to suppress inflammation.

The compounds of the present invention may be formulated with a suitable pharmaceutically acceptable medium for the use of the pharmaceutical composition in the treatment of tumors and other disorders associated with unwanted angiogenesis.

In the method of the present invention, pharmaceutical compounds containing a compound of formula (I) and (II) are prepared by mixing the compound in a pharmaceutically acceptable matrix. Pharmaceutical compositions of the active compounds are administered to a patient with a tumor to reduce or stop tumor growth. The active compounds may be administered parenterally by injection or by gradual infusion over time, or by any other method suitable for a particular dosage form.

Brief Description of the Drawings

Figure 1 is a schematic drawing showing the interaction of MMP2 and integrin α _v β ₃ and its role in angiogenesis, as well as the inhibition of the interaction of MMP2 and α _v β _{3 by an} antagonist, such as the compounds of the present invention.

Fig. 2A graphically illustrates the effect of inhibitory compounds of formula (I) on the interaction of MMP2 and integrin α _v β ₃ in a solid phase binding assay.

2B shows the binding of compounds of formula (I) with integrin α _v β ₃ and α ₅ β ₁ .

FIG. 2C compares the effect of the compound of the present invention and the control compound on binding to α _v β ₃ .

2D shows a comparison of the effect of the amino acid residues of RGD on the binding of the present invention and α _v β ₃ with the effect of RGD on the binding of bVN and α _v β ₃ .

On figa graphically shows the proteinase activity in β ₃ positive cells and in β ₃ negative cells.

3B shows the binding of MMP2 and β ₃ positive cells and β ₃ negative cells.

Fig. 4A is a microscopic picture of the inhibition of angiogenesis in chicken CAM tissue.

4B graphically shows the inhibition of angiogenesis in chicken CAM tissue.

On figs shows the level of MMP2 in the treated and untreated cells.

Fig. 5A is a microscopic picture of tumor growth and vascularization in chicken SAM tissue.

Fig. 5B is a microscopic picture of blood vessel intensity in chicken CAM tissue.

On figs graphically presents the weight of the tumor in the tissue SAM chicken.

On fig.5D graphically presents the vascularization of the tissue SAM chicken.

Fig. 5D is a microscopic picture of the number of tumor cells in chicken CAM tissue.

DETAILED DESCRIPTION OF THE INVENTION

The binding of MMP2 and integrin α _v β ₃ is an important mechanism in the process of angiogenesis. Certain inhibitors of this binding lead to a decrease in vascularization of growing tissues, such as tumors, and, thus, to a slowdown in tumor growth. The interaction of MMP2 and integrin α _v β ₃ graphically shown in figure 1. A new class of angiogenesis and tumor growth inhibitors are low molecular weight antagonists described below, which, in particular, inhibit the binding of MMP2 and integrin α _v β ₃ , thus providing an important new treatment approach.

Some compounds of the present invention may have one or more centers of asymmetry and may be in optically active forms. Additional centers of asymmetry may be present in a substituent group, such as an alkyl group. Pure S-isomers and pure R-isomers, racemic mixtures of isomers and mixtures thereof are included in the scope of the present invention. The intended chiral forms of certain compounds of this invention are specifically within the scope of the present invention.

By the term “alkoxy” is meant an oxygen atom linked by an ether bond to an alkyl group of the indicated size as defined below. Examples of alkoxy groups are methoxy, ethoxy, tert-butoxy and the like. By the term “alkyl” is meant straight or branched chain carbon radicals of the indicated size. Representative alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 2-ethylhexyl, n-octyl, 2,4-dimethylpentyl and the like. By the term “hydroxyalkyl” is meant the above-defined alkyl group of the indicated size attached to the hydroxyl group. Examples include hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-1-propyl, 2-hydroxy-1-propyl, 4-hydroxybutyl and the like.

By the term "perfluoroalkyl" is meant an alkyl group of the indicated size as defined below which carries fluoro substituents in place of each hydrogen, for example trifluoromethyl and pentafluoroethyl.

The term "halogen" refers to bromine, chlorine, fluorine and iodine.

The compounds of the present invention are represented by formula (I) and include glycillisin derivatives chemically attached to a linking group:

where G ¹ or G ² each independently represents —NH — C (O) —O — R ¹ , —NH — C (O) —O— (CH ₂ ) _v - (C ₆ H ₄ ) —X ¹ , - NH-C (O) -NH- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ , -OC (O) -NH- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ , - OC (O) -O- (CH ₂ ) _v - (C ₆ H ₄ ) -X ¹ or -NH-C (O) -CH ₂ - (C ₆ H ₄ ) -X ¹ ; Y ¹ and Y ² each independently represents OH, C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ alkoxy, phenyl, benzyl or —NH ₂ ; R ¹ represents C ₁ -C ₄ alkyl; X ¹ represents halogen, nitro, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or C ₁ -C ₄ perfluoroalkyl; Z represents —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 represents H or a covalent bond; m and n each independently represents an integer having a value of 0 or 1; t is an integer having a value of 0 or 1; and v is an integer having a value of 1 or 2; provided that when A is H, then t is 0; when A is a covalent bond, then t is 1; when m is 0, then Y ¹ is C ₁ -C ₄ hydroxyalkyl; and when n is 0, then Y ² is C ₁ -C ₄ hydroxyalkyl.

Preferably, G ¹ and G ² is —NH — C (O) —O— (CH ₂ ) _v - (C ₆ H ₄ ) —X ¹ , Y ¹ and Y ² are OH, and m and n are 1. Preferably, X ¹ is C ₁ -C ₄ perfluoroalkyl, more preferably trifluoromethyl. Preferred compounds of structural formula (I) are represented by structural formula (II) and include glycine-lysine derivatives attached to the benzene linking group either in the ortho, or meta, or in the para position:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl, phenyl or benzyl; X ² and X ³ each independently represent halogen, nitro, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl or C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; and t is an integer having a value of 0 or 1; provided that when A is H, then t is 0, and when A is a covalent bond, then t is 1. When A is a covalent bond and t is 1, then the glycillisine derivative groups can be attached to the benzene linking group in the ortho, meta or para position.

Preferably, the substituents X ² and X ^{3 are} attached to the phenyl ring of the benzyl group at the 4-position relative to benzyl CH ₂ (i.e., para-substituent). Preferred X ² and X ³ groups are C ₁ -C ₄ perfluoroalkyl, more preferred are paratrifluoromethyl. Preferred R ² and R ³ groups are hydrogen and methyl. The substituents X ² and X ³ may be the same or different and the substituents R ² and R ³ may also be the same or different.

A particularly active member of the family of compounds represented by formula (II), where A is a covalent bond and t is 1, is a compound represented by compound 1 below:

(Compound 1)

The compounds of formulas (I) and (II) can be synthesized from already available substances with a small number of synthesis steps. For example, scheme 1 shows the synthesis of compound 1, and this scheme illustrates a general method for preparing compounds of formula (II), where A is a covalent bond, R ² and R ^{3 are the} same, and X ² and X ^{3 are the} same. Scheme 1 further illustrates the synthesis of compounds of formula (II), where X ² and X ³ are both paratrifluoromethyl, and R ² and R ³ are both methyl or hydrogen.

Scheme 1

Scheme 1 shows that p-trifluoromethylbenzyl alcohol reacts with disuccinimidyl carbonate to form an activated intermediate, which in turn reacts with N-ε-BOC-lysine methyl ester to give compound 2 in 99% yield. Hydrolysis of the BOC protecting group and subsequent binding to BOC-glycine gives compound 3 in 96% yield. Acid hydrolysis of the BOC protecting group of compound 3 results in compound 4 in 99% yield. The binding of two equivalents of compound 4 to isophthaloyl dichloride yields 68% of compound 5 corresponding to formula (II), where R ² and R ³ are methyl, X ² and X ³ are p-trifluoromethyl and A is a covalent bond. Hydrolysis of compound 5 with lithium hydroxide gives 93% yield of compound 1.

The synthesis of compounds of formula (I) and (II), which are similar to compound 1 but have other substituents R and X, can be obtained by modifying Scheme 1, and are readily understood by a person skilled in the art of chemical synthesis. The use of benzyl alcohol with a substituent other than p-trifluoromethyl (for example, halogen, nitro or other C ₁ -C ₄ perfluoroalkyl groups located either in the ortho or meta or para position relative to benzyl CH ₂ ), or the use of protected a lysine ester having an ester group other than methyl will provide other compounds of formula (II).

The synthesis of compounds of formula (I) having Z groups other than 1,3-phenyl is possible, for example, by substitution with other diacid dichlorides, such as bischlorocarbonylacetylene, fumaryl dichloride, phthaloyl dichloride, instead of isophthaloyl chloride according to Scheme 1. Alternatively, the corresponding acids can be used instead of acid chlorides, and the binding of the acid to the corresponding amines can be achieved by conventional peptide binding, well known in the art. Specialists in this field will easily pick up other chemicals as substituents, which may be in the synthesis methods illustrated in Schemes 1 and 2, for the synthesis of other members of the group of the compound represented by formulas (I) and (II). The synthesis of such compounds and useful chemical procedures for the synthesis of compounds of formula (I) and (II) are described by Boger et al. in J. Am. Chem. Soc. 123, 1280-1288 (2001).

The compounds of formula (II), where A is a covalent bond, t is 1 and either R ² different from R ³ or X ² different from X ³ , or both R ² / R ³ and X ² / X ³ different from each other, can be synthesized by modifying the method presented in scheme 1, which will be easily understood by a person skilled in the art. For example, one equivalent of compound 4 may interact with isophthaloyl dichloride or any other suitable isophthalic acid halide or active ester, and the second activated acid group may suitably interact with an analog of compound 4, which differs either in the X group or R group, or both of these in groups. Similarly, two analogs of compound 4, which differ by either an X group, an R group, or both of these groups, can suitably react with isophthaloyl dichloride or equivalent activated isophthalate to form additional analogs of compound 1 with different X and R groups.

The synthesis of compounds of formula (II), where A represents hydrogen and t is 0, is shown by the example of the synthesis of compounds 6 and 7 in scheme 2 below:

Scheme 2

In Scheme 2, compound 4 of scheme 1 is reacted with benzoyl chloride to form compound 6 (R ² = methyl; X ² = p-trifluoromethyl) in 90% yield. Hydrolysis of the ester group of compound 6 with lithium hydroxide afforded compound 7 (R ² = H, X ² = p-trifluoromethyl) in 88% yield.

Benzoylation of analogues of compound 4 with different R groups (for example, with other C ₁ -C ₄ alkyl groups) and / or with different X substituents on the benzyl group led to the formation of other compounds of formula (II) having A = H and t = 0.

Scheme 2 also shows the synthesis of compound 8, an inactive analog of compounds 6 and 7, in which the benzoyl group attached to the glycine unit of formula (II) is replaced by a diglycol amide group. Compound 9 was obtained in 77% yield by reacting compound 4 with diglycolic acid.

Scheme 3 below shows, by way of example, the synthesis of compound 12, an inactive analog of compound 1, in which the p-trifluoromethylbenzyloxycarbonyl group of formula (II) is substituted with benzoylamide.

Scheme 3

In an aspect of the method of the present invention, pharmaceutical preparations of the compounds of formulas (I) and (II) can be prepared by mixing the compound and a pharmaceutically acceptable carrier substance. Pharmaceutical compositions comprising the active compounds of formulas (I) and (II) are administered to the body with a tumor to reduce or stop tumor growth. Active compounds may be administered parenterally by injection or by gradual infusion over time. Although the exposed tissue is most often treated by intraperitoneal or subcutaneous administration, the active compounds can be administered intraocularly, intravenously, intramuscularly, intraarticularly, into the cavity, transdermally, or can also be delivered using peristaltic infusion pumps.

As used herein, the term “administration” of a compound or composition is understood to mean systemic administration by the oral, parenteral route, by inhalation spray, by the nasal, rectal or buccal route, or by topical application of the composition in the form of a dosage form containing the necessary conventional non-toxic pharmaceutically acceptable carriers , adjuvants or solvents. As used herein, the term “parenteral” refers to intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection or methods of infusion.

“Pharmaceutically acceptable” means those salts, amides and esters which, within medical terms, are suitable for interaction with human and lower animal tissues without non-specific toxicity, damage, allergic response and the like, and correspond to an acceptable benefit / risk ratio effective for their intended use in the treatment of tumors and angiogenic disorders.

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

As used herein, the term “pharmaceutically acceptable carriers” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating agent or adjuvant of any type. Some examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powder 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 and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethyl alcohol and phosphate buffered solutions, as well as other non-toxic combinable substances used in pharmacological compositions.

Moisturizing agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and flavoring agents, preservatives and antioxidants in accordance with pharmaceutical standards may also be included in the composition. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, 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; and metal chelate additives such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

By "therapeutically effective amount" of an agent or compound of this invention is meant a sufficient amount of a compound for the treatment of tumors and angiogenic disorders with a suitable benefit / risk ratio for therapeutic use. It is understood, however, that the total daily dose of the compounds and compositions of this invention will be determined by the attending physician within the medical standards. The level of specific therapeutically effective doses for any particular patient will depend on a number of factors, including the disorder the treatment is aimed at and the severity of the disorder; the activity of the specific compounds used; the particular composition used; age, body weight, general health, gender and nutrition of the patient; time of administration, route of administration and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or simultaneously with the specific compound used; and similar factors well known in the medical field.

The present invention also relates to pharmaceutical compositions in unit dosage form, which comprise a therapeutically effective amount of a compound (or compounds) of this invention in combination with conventional pharmaceutical carriers. Injectable preparations, for example, sterile injectable aqueous liquids or oily suspensions, may be prepared according to methods known in the art using suitable dispersing or moisturizing agents and suspending agents. The sterile preparation administered by injection may also be a sterile solution, administered by injection, suspension or emulsion in a non-toxic diluent or solvent suitable for parenteral administration, for example, as a solution of 1,3-butanediol. Acceptable solvents and vehicles include water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile solid oils are usually used as a solvent or suspending medium. For this purpose, any solid oil may be used, including synthetic mono- or diglycerides.

In addition, fatty acids such as oleic acid are used in injectable preparations. Compositions administered by injection can be sterilized, for example, by filtration through a bacteria-retaining filter, or by mixing sterile agents in the form of sterile solids that can be dissolved or diluted immediately before use in sterile water or other sterile injectable media.

In order to prolong the effect of the drug, it is often desirable to slow its absorption during subcutaneous or intramuscular injection. The easiest way to achieve this retardation is the introduction of a suspension of crystalline or amorphous substances with poor solubility in water. The absorption rate of a drug becomes dependent on its dissolution rate, which in turn depends on the physical state of the drug, for example, crystal size and crystalline form. Another approach to delay drug absorption is to administer the drug in the form of a solution or suspension in oil. Also, by molding the microcapsular substance of the drug and biodegradable polymers such as polylactide-polyglycolide, an injectable, sustained absorption form can be obtained. The release rate of the drug can be controlled depending on the ratio of drug to polymer and polymer composition. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides. A sustained absorption form administered by injection can also be obtained by incorporating the drug into liposomes or microemulsions compatible with body tissues.

Suppositories for rectal administration of the drug can be obtained by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycol, which is solid at ordinary temperature and liquid at rectal and thus will dissolve in the rectum, releasing the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, beads and granules. In each solid dosage form, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose and starch. Such dosage forms may also include, in normal use, additional substances in addition to inert diluents, for example, tabletting lubricants and other auxiliary substances, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, dosage forms may also include buffering agents. Tablets and pills may additionally be coated with enteric coatings and other coatings that control drug release. Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or maltose, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also include adjuvants, such as wetting agents; emulsifying and suspending agents; sweeteners, flavors and flavors. If desired, the compounds of the present invention can be incorporated into sustained release systems or targeted delivery systems such as polymer matrices, liposomes, and microspheres. They can be sterilized, for example, by filtration through a filter that traps bacteria, or by mixing sterilized agents in the form of sterile solid compounds that can be dissolved immediately before use in sterile water or some other sterile medium administered by injection. Active compounds can also be microencapsulated with one or more excipients, as described above.

Solid dosage forms in the form of tablets, dragees, capsules, pills and granules may be coated with coatings, such as enteric coatings and other coatings well known in the field of pharmaceutical compositions. They may optionally contain opacifying agents and may also be compositions that release only the active component (s), or, preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of submerged compositions that can be used include polymeric substances and waxes. Dosage forms for topical or transdermal administration of a compound of this invention include oils, pastes, creams, lotions, gels, powders, solutions, spray, inhalations or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers, if necessary.

Compositions for the eyes, ear drops, eye ointments, powders and solutions are also included in the scope of this invention. Ointments, pastes, creams and gels may contain, in addition to the active substance of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and spray may contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The spray may further comprise a commonly used propellant, such as chlorofluorocarbons.

Transdermal patches have the additional advantage of providing controlled release of the compound into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a representative medium. The absorption of vehicles can also be used to increase the flow through the skin. The speed can be controlled either by a speed-providing membrane or by dispersion of the compound into a polymer matrix or gel.

Compositions containing active compounds are administered in a manner compatible with the dosage form and in a therapeutically acceptable amount. The amount administered and the time of administration depend on the patient, the ability of the patient system to use the active substance and the degree of the desired therapeutic effect. A certain amount of the active substance required for administration depends on the prescription of the attending physician and is specific to each case.

Regions of suitable doses for systematic administration are described herein and depend on the route of administration. Suitable modes of administration may also be different, but usually include initial administration, then administration of repeated doses at one or more predetermined time intervals for subsequent injections or another route of administration.

The present invention also relates to pharmaceutical compositions that are used in conventional therapies described herein. The compositions contain the active compound described herein above, together with a pharmaceutically acceptable carrier.

Preparations for parenteral administration of the compounds or compositions of the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solutions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters that can be injected, such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saturated saline and a buffered medium. Parenteral vehicles include sodium chloride solution, Ringer's solution with dextrose, dextrose and sodium chloride solution, lactic acid Ringer's solution or solid oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's solution with dextrose) and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial additives, antioxidants, chelating agents, inert gases and the like.

Another aspect of the present invention relates to a method for inhibiting the interaction of MMP2 and α _v β ₃ and thus inhibiting angiogenesis in tumor tissue. The inhibition method comprises administering to the patient a composition comprising an amount of an angiogenesis inhibitory compound described hereinafter. The interaction of MMP2 and α _v β _{3 is} inhibited by the reaction of α _v β ₃ with the compound of the present invention.

Angiogenesis is the formation of a new vasculature from existing vessels of the body and it is necessary for tumor growth of more than 1-2 mm ³ . For the purposes of the present invention, angiogenesis is inhibited until the angiogenesis and symptoms of the disease caused by angiogenesis improve.

The dose level of the active compound for administration to a patient depends on the particular active compound and its effectiveness against a particular tumor or integrin. The person skilled in the art will easily determine the required dose for a particular active compound without unnecessary experiments. Sick can be any mammal. The dose should be sufficient to obtain the desired therapeutic effect, in which the angiogenesis and symptoms of the disease associated with angiogenesis are improved and usually the amount sufficient to reach the level of the active compound in the plasma is in the range from about 0.01 to about 100 micromoles (μM) preferably from about 0.2 to about 20 μM, more preferably from about 1 to about 10 μM. The dose should not be so large as to possess harmful similar actions. The dose per kilogram (kg) of body weight can vary from 1 to 20 mg per dose in one or more doses per day for one or several days or unlimitedly.

To inhibit angiogenesis, a therapeutically effective amount is equal to the amount of active compound sufficient to obtain moderate inhibition of angiogenesis in the treated tissue, i.e., an amount that inhibits angiogenesis or an amount that inhibits the interaction of MMP2 and α _v β ₃ . Inhibition of angiogenesis can be measured in situ using immunohistochemistry, as described herein, or by any other method known to a person skilled in the art.

The present invention also relates to pharmaceutical compositions used for the conventional methods of treatment described herein. The compositions contain the active compound described herein above, together with a pharmaceutically acceptable carrier.

The present invention also relates to a method for inducing apoptosis in tumor cells. This method involves administering to the patient a therapeutically effective amount of an active compound sufficient to initiate apoptosis of tumor cells.

For the purposes of the present invention, apoptosis of tumor cells is induced when an increase in apoptosis of tumor cells in a given tumor undergoing treatment is observed. Cell apoptosis can be measured by any of the methods described herein or well known in the art.

The following non-limiting examples are presented to illustrate various aspects of the present invention.

Materials and methods

Cellular antibodies and reagents. CS-1 hamster melanoma cells and CS-1 cells transfected with human β ₃ -integrin unit (β ₃ CS-1 cells) (Cell, 85, 683-93 (1996); Cell, 92, 391-400 ( 1998)). Horseradish peroxidase (HRP) conjugated with monoclonal antibodies, antibiotic mAb BN-34 and antiactin mAb AC-40, were obtained from Sigma (St. Louis, MO). Polyclonal antibodies (pAb) against von Willebrand factor (vWF) were obtained from DAKO (Glostrup, Denmark). The cyclic peptides cRGDfV and cRADfV and integrin-α _v β ₃ were kindly provided by Merck KGaA (Darmstadt, Germany). Purified pro MMP2 and integrin-α _v β ₃ were obtained from Chemicon International (Temecula, CA). Purified active MMP2 was obtained from Calbiochem (La Jolla, CA). The main factor for fibroblast growth (bFGF) was kindly provided by Scios (Mountain View, CA).

Synthesis of [ ¹⁴ C] Compounds. ^[14 C] -labelled compound 1 was synthesized using slightly modified sequence of steps described earlier for the unlabeled substance (Figure 1), but using N-BOC- [1- ¹⁴ C] -glycine (55 mCi / mmol, American Radiolabeled Chemicals, St. Louis, MO). The total yield of [ ¹⁴ C] compound 1 in the four-step process was 25%.

Example 1. Solid-phase integrin binding assay.

Purified integrins were absorbed overnight onto the surface of the microtiter plates (1-5 μg / ml, 50 μg / plate), then the plates were blocked with casein (Pierce, Rockford, IL). Purified biotinylated MMP2 (bMMP2, 3-5 nM) was added to the plates in binding buffer (50 mM Tris, pH 8.150 mM NaCl, 1 mM MgCl ₂ , 0.5 mM MnCl ₂ ) with or without compound 1, compound 12, cyclic RGD or RAD peptides, or only buffer was added. No integrin was added to the control plates. Biotinylated vitronectin (bVN, 1 μg / ml) was used in accordance with the link. Protein binding was determined with HRP-antibiotic mAb and a solution at 450 nm was counted using a solution of 3.3 ', 5.5'-tetramethylbenzidine (TMB; substrate for peroxidase) (BioRad, Hercules, CA).

To evaluate the direct binding of integrin to compound 1, α _v β ₃ and α ₅ β ₁ (10 μg / ml, 50 μl / plate) were applied to Immulon-4 microtiter plates (Dynatech Laboratories, Chantilly, VA), which blocked accordingly and incubated with a titer of [ ¹⁴ C] -compound 1, then 150 μl of binding buffer containing 0.1% Tween-20 was added and the liquid was aspirated. The dried plates were separated and immersed in a BetaMax liquid scintillation mixture (ICN Biochemicals, Costa Mesa, CA) for quantification. The concentration of the substrate (3 μM) of the [ ¹⁴ C] compound 1 with or without a 25-fold molar excess (75 μl) of unlabeled compound 1 or compound 12, or 100 μM cyclic RGD or RAD peptides was evaluated by the binding curve. The control was bVN, used and determined as described above.

Example 2. Analysis of the degradation of the binding of MMP2 cells and [ ³ H] -collagen IV.

CS-1 cells or β ₃ CS-1 cells were incubated in adhesive buffer; basic fibroblast medium (FBM) supplemented with 0.5% bovine serum albumin (BSA), 0.4 mM MnCl ₂ and 10 μg / ml aprotinin; containing either 4 nM purified activated MMP2, or a mixture thereof with 10 μM of compound 1 or compound 12, for 45 minutes at 37 ° C, then washed and added to plates coated with [ ³ H] -collagen IV. The plates were coated overnight with 50 μl of 0.414 mCi / ml of [ ³ H] -collagen IV (ICN Biochemicals, Costa Mesa, CA) and washed thoroughly until the radioactivity in the background wash solutions was reached. Alternatively, the cells were treated as described above without the addition of MMP2, or the MMP2 solution was directly added to the plateless cells as controls. Collagen IV degradation was quantified by measuring radioactivity in 50 μl of culture medium, which was determined in a scintillation counter for liquids. To evaluate biotinylated MMP2 binding to CS-1 cells, the cells were suspended in adhesion buffer and incubated with 12 nM bMMP2 for 45 minutes at 37 ° C with or without 10 μM compound 1 or compound 12. Cells were washed, then lysed, performed SDS-PAGE and immunoblotting with antibiotic mAb.

Example 3. Analysis of angiogenesis in the chorionic allantoic membrane of chicken (CAM).

Angiogenesis was determined as previously described (Cell, 85, 683-93 (1996); Cell, 92, 391-400 (1998)). After stimulation with 3 μg / ml basic fibrobast growth factor (bFGF), the 10-day-old CAM of the chicken embryo was treated with 20 μl of 3 μM of compound 1 or compound 12. Three days after induction, CAM was quantified by a blind evaluation method. The CAMs of each group were combined, homogenized, and extracted with 50 mM Tris, 150 nM NaCl, 0.1% Triton X-100 containing a mixture of COMPLETE proteinase inhibitors without EDTA (Boehringer, Mannheim, Germany), then analysis was performed using zymography.

Example 4. SDS-PAGE, immunoblotting and zymography.

Immunoblotting. Identical amounts of protein were separated by SDS-PAGE with reducing agents and electro-blotted onto an Immobilon-P Millipore membrane, Bedford, MD). The membrane was blocked and transferred proteins were determined by incubation with antigen-specific primary antibodies, and then with HRP-conjugated secondary antibodies, as required. The band was visualized using the chemiluminescent substance PS-3 (Lumigen, Inc., Southfield, MI).

Zymography. Chicken CAM lysates were prepared as described above, and equal amounts of protein were separated in the absence of reducing agents or by boiling polyacrylamide gels with 0.2% gelatin. The gels were washed with 2% Triton X-100, then washed thoroughly with water, and then incubated overnight at 37 ° C in collagenase buffer (50 mM Tris 7.4, 200 mM NaCl, 10 mM CaCl ₂ ). Gelatin liquefaction activity was visualized by staining with 0.5% Coomassie blue gels.

Example 5. Analysis of tumor growth.

Primary tumors were grown in 9-day-old CAM tissue after implantation of 5x10 ⁶ CS-1 cells and incubation for 7 days. Thereafter, 50 mg sections of these tumors were subcloned into 9-day-old CAM tissues and left for implantation for 24 hours before a single intravenous (IV) injection of 100 μl of 100 μM test compound in Hank's balanced saline (HBSS). As a control, only a buffer was introduced. Tumors were incubated for a total of 10 days, excess stromal tissue was collected and removed, then fresh weight was determined and histology was performed.

Example 6. Immunofluorescence analysis

Instantly frozen sections of the CS-1 tumor were fixed with 4% paraformaldehyde and treated with 0.1% Triton X-100. Sections were blocked with 5% bovine serum albumin (BSA) in phosphate saline (PBS), stained with anti-vWF pAb and visualized with Alexa 568 conjugated to anti-rabbit secondary antibodies. Samples were analyzed on an MRC1024 confocal microscope (BioRad, Hercules, CA). Blood vessel density was quantified at a 20X magnification of four fields per section and four tumors per condition.

Example 7. (S) -Metyl 6 - (((tert-butyloxy) carbonyl) amino) -2 - ((4-trifluoromethyl) benzyloxycarbonyl) hexanoate (2).

A solution of N, N'-disuccinimidyl carbonate (5.38 g, 21 mmol) in acetonitrile (150 ml) was treated with 4- (trifluoromethyl) benzyl alcohol (2.87 ml, 21 mmol) and Et ₃ N (5.8 ml, 42 mmol) and stirred at 25 ° C. After 3 hours, the reaction mixture was added to a flask containing N-ε-BOC-lysine methyl ester (4.2 g, 14 mmol) in acetonitrile and stirred for another 3 hours. The solvent was evaporated and the residue was dissolved in CH ₂ Cl ₂ ( 250 ml) and washed with 10% aqueous HCl (2 × 200 ml) and saturated aqueous NaHCO ₃ (200 ml).

Flash chromatography (SiO ₂ , 3: 1 CH ₂ Cl ₂ / EtOAc) afforded 6.4 g (99%) of compound 2 as a pale yellow oil: [α] _D ²⁵ -8.9 (c 5.6, 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, 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). 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 , 463,2056 required).

Example 8. (S) -methyl 6- [2 - (((tert-butyloxy) carbonyl) amino) acetamido] -2 - [(4-trifluoromethyl) benzyloxycarbonyl] hexanoate (3).

A solution of compound 2 (2.7 g, 5.8 mmol) in CH ₂ Cl ₂ (3 ml) was treated with 4 N. HCl-dioxane (10 ml) and stirred for 20 min at 25 ° C. The solvent and excess acid were removed under reduced pressure, and the crude hydrochloride salt was dissolved in DMF (50 ml), treated with N - ((tert-butyloxy) carbonyl) glycine (1.0 g, 5.8 mmol), 1- (3 hydrochloride) - (dimethylamino) propyl) -3-ethylcarbodiimide (EDCI) (1.2 g, 6.4 mmol) and i-Pr ₂ NEt (2.0 ml, 11.6 mmol) and stirred for 12 h at 25 ° C. The reaction mixture was diluted with EtOAc (400 ml) and washed with 10% aqueous HCl (3 x 250 ml) and saturated aqueous NaHCO ₃ (250 ml), dried (Na ₂ SO ₄ ) and evaporated to give 2.89 g (96%) of the compound 3 as a white foamy solid: [α] _D ²⁵ -10,4 (c 2,5, CH ₃ OH).

¹ H NMR (CDCl ₃ , 400 MHz) 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). ¹³ C NMR (CDCl ₃ , 100 MHz) δ 174.5, 172, 158.2, 158.1, 142.7, 130.8 (q, J = 31.8 Hz), 128.8, 126, 3, 125.5 (q, J = 269.7 Hz), 80.5, 66.5, 55.3, 52.7, 44.6, 39.8, 32.1, 29.8, 24 , 0.

IR (film) V _max 3320, 2932, 1721, 1692, 1326 cm ^-1 ; FABHRMS (NBA-CsI) m / z 652.1234 (M ⁺ + Cs ⁺ , C ₂₃ H ₃₂ F ₃ N ₃ O ₇ , 652.1247 required).

Example 9. Hydrochloride of 6- [2- (amino) acetamido] -2 - [(4-trifluoromethyl) benzyloxycarbonyl] hexanoate (4).

A solution of compound 3 (350 mg) in CH ₂ Cl ₂ (2 ml) was treated with 4 N. HCl-dioxane (5.0 ml) and stirred at 25 ° C. After 0.5 h, the solvent and excess acid were removed under reduced pressure to obtain 300 mg (99%) of compound 4 as a pale yellow oil: [α] _D ²⁵ -10.4 (c 3.0, CH ₃ OH).

¹ H NMR (CD ₃ OD, 400 MHz) δ 7.65 (d, J = 8.2 Hz, 2H), 5.18 (d, J = 13.3 Hz, 1H), 5.16 (d, J = 13.3 Hz, 1H), 4.16 (m, 1H), 3.70 (s, 3H), 3.65 (s, 2H), 3.21 (t, J = 7.1 Hz, 2H), 1.84 (m, 1H), 1.68 (m, 1H), 1.54 (m, 2H), 1.42 (m, 2H). ¹³ C NMR (CD ₃ OD, 100 MHz) δ 174.5, 167.1, 158.2, 142.8, 130.8 (q, J = 31.8 Hz), 128.8, 126.3 125.5 (q, J = 270.1 Hz), 66.5, 55.4, 52.7, 41.5, 40.2, 32.1, 29.6, 24.1.

IR (film) V _max 3317, 2954, 1718, 1684, 1530, 1327 cm ^-1 ; MALDIFTMS (DHB) m / z 442.1586 (M ⁺ + Na ⁺ , C ₁₈ H ₂₄ F ₃ N ₃ O ₅ , 442.1566 required).

Example 10. N, N ¹ bis [(5- (S) - (methoxycarbonyl) -5 [((4-trifluoromethyl) benzyloxycarbonyl) amino] pentyl) carboxamidomethyl] benzene-1,3-dicarboxamide (5)

A solution of compound 4 (2.05 g, 4.0 mmol) in CH ₂ Cl ₂ (3.0 ml) was treated with 4 N. HCl-dioxane (10.0 ml) and stirred for 20 min at 25 ° C. The solvent and excess acid were removed under reduced pressure, and the crude hydrochloride salt was suspended in DMF (40 ml), treated with isophthaloyl dichloride (400 mg, 2.0 mmol) and i-Pr ₂ NEt (1.4 ml, 8.0 mmol) and stirred for 1 h at 25 ° C. The reaction mixture was diluted with EtOAc (400 ml) and washed with 10% aqueous HCl (3 × 200 ml) and 5% aqueous Na ₂ CO ₂ (200 ml), dried (Na ₂ SO ₄ ) and evaporated. Flash chromatography (SiO ₂ , 1: 4.5: 4.5 MeOH / CH ₂ Cl ₂ / EtOAc) afforded 1.30 g (68%) of compound 5 as a yellow powder: [α] _D ²⁵ -6.4 (c 2.1, CH ₃ OH).

¹ H NMR (CDCl ₃ , 400 MHz) 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). ¹³ C NMR (CDCl ₃ , 100 MHz) 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) V _max 3305, 2951, 1716, 1651, 1538 cm ^-1 ; FABHRMS (NBA-CsI) m / z 1101.2398 (M ⁺ + Cs ⁺ , C ₄₄ H ₅₀ F ₆ N ₆ O ₁₂ , 1101.2445 required).

Example 11. N, N ¹ bis - [(5- (S) -carboxy-5 - [((4-trifluoromethyl) benzyloxycarbonyl) amino] pentyl) carboxamidomethyl] benzene-1,3-dicarboxamide (1)

A solution of compound 5 (0.95 g, 0.98 mmol) in THF-MeOH (8.0 ml, 3: 1) was treated with LiOH · H ₂ O (165 mg, 3.9 mmol) in H ₂ O (2, 0 ml) and stirred at 0 ° C. After 2 hours, the reaction was quenched by the addition of 10% aqueous HCl (20 ml) and the mixture was extracted with EtOAc (3 × 50 ml). The organic layers were collected and washed with saturated aqueous NaCl (50 ml), dried (Na ₂ SO ₄ ) and evaporated to give 0.86 g (93%) of compound 1 as a white powder: [α] _D ²⁵ -0.6 (c 3.2, CH ₃ OH).

¹ H NMR (CD ₃ OD, 400 MHz) δ 8.40 (m, 1H), 8.03 (dd, J = 1.8, 7.8 Hz, 2H), 7.62 (d, J = 8 , 1 Hz, 4H), 7.53 (t, J = 6.1 Hz, 1H), 7.51 (d, J = 8.1 Hz, 4H), 5.16 (d, J = 13.3 Hz, 2H), 5.13 (d, J = 13.3 Hz, 2H), 4.12 (m, 2H), 4.00 (s, 4H), 3.22 (t, J = 6.6 Hz, 4H), 1.85 (m, 2H), 1.70 (m, 2H), 1.55 (m, 4H), 1.37 (m, 4H). ¹³ C NMR (CD ₃ OD, 100 MHz) 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) V _max 3334, 2933, 1718, 1646, 1631, 1528 cm ^-1 ; MALDIFTMS (DHB) m / z 963.2953 (M ⁺ + Na ⁺ , C ₄₂ H ₄₆ F ₆ N ₆ O ₁₂ , 963.2976 required).

Example 12. [ ¹⁴ C] connection (1)

A solution of [1- ¹⁴ C] -glycine (American Radiolabeled Chemicals, 1,0 mCi, 55 mCi / mmol, 0.018 mmol) in 0.1N. HCl was placed in a 4 ml tube and the solvent was removed under a stream of N ₂ . The resulting residue was treated with a solution of NaHCO ₃ (4.6 mg, 0.054 mmol) in H ₂ O (0.25 ml) and a solution of di-tert-butyl dicarbonate (10.5 ml, 0.045 mmol) in THF (0.25 ml) and stirred at 25 ° C. After 12 hours, the solution became homogeneous, it was diluted with H ₂ O (1.0 ml) and washed with diethyl ether (2 × 1.0 ml). The aqueous solution was then acidified by adding 10% aqueous HCl (0.5 ml) and extracted with ethyl acetate (4 × 1.0 ml). The combined extracts were dried (Na ₂ SO ₄₎ and evaporated under a stream of N ₂ to give 2.9 mg (92%) of [1- ¹⁴ C] -N-BOC-glycine as a white film.

A solution of compound 2 (50 mg, 0.11 mmol) in CH ₂ Cl ₂ (1 ml) was treated with 4 N. HCl-dioxane (1 ml) and stirred for 1 h at 25 ° C. The reaction mixture was diluted with ethyl acetate (25 ml) and washed with 10% aqueous Na ₂ CO ₃ (25 ml) and saturated aqueous NaCl (25 ml), dried (Na ₂ SO ₄ ) and evaporated to give lysine with the removed protective group as a colorless film . A portion of this free amine (4.5 mg, 0.013 mmol) in DMF (0.2 mL) was added to 4 ml vial containing [1- ¹⁴ C] -N-BOC-glycine (1.5 mg, 0.0085 mmol), treated with i-Pr ₂ NEt (2 ml) and a solution of EDCI (5.0 mg, 0.026 mmol) in methylene chloride (0.1 ml) and stirred for 3 h at 25 ° C. The reaction mixture was diluted with EtOAc (2.0 ml), washed with 10% hydrochloric acid (3 x 1.0 ml), saturated aqueous Na ₂ CO ₃ (1.0 ml) and saturated aqueous NaCl (1.0 ml), and evaporated. Preparative thin layer chromatography (PTLC) (SiO ₂ , EtOAc / CHCl ₃ 1: 1) gave 1.8 mg (41%) of the [ ¹⁴ C] compound 3 as a white film.

The [ ¹⁴ C] compound 3 (1.8 mg, 3.5 mmol) contained in the 4 ml tube was treated with 4 N HCl-dioxane (0.25 ml) and the reaction mixture was stirred for 0.5 h at 25 ° C. The solvent and excess acid were evaporated under a N ₂ stream and the resulting crude hydrochloride salt was treated with isophthaloyl dichloride (355 mg, 0.0018 mmol) in CH ₂ Cl ₂ (0.1 ml), and i-Pr ₂ NEt (2.4 ml, 0.014 mmol) in CH ₂ Cl ₂ (0.05 ml). After 3 hours at 25 ° C, the reaction mixture was directly purified using PTLC (SiO ₂ , MeOH / CHCl ₃ / EtOAc 1: 9: 9) to obtain 1.2 mg (71%) of the [ ¹⁴ C] compound 5.

A solution of [ ¹⁴ C] Compound 5 (1.2 mg) in THF-MeOH (0.2 ml, 1: 1) was treated with a solution of LiOH · H ₂ O (0.4 mg) in H ₂ O (0.05 ml ) at 0 ° C and stirred for 1 h. The reaction mixture was diluted with methanol (1.0 ml), treated with a Dowex 50WX-8 cation exchange resin (200 mg) and stirred for 1 min. The mixture was then filtered through cotton and the filtrate was evaporated to give 1.1 mg (94%) of [ ¹⁴ C] compound 1 with a relative activity of approximately 110 mCi / mmol. This compound and all its synthetic intermediates were identical when compared by thin layer chromatography (TLC) relative to the corresponding unlabeled material.

Example 13. (S) -methyl 6- [2- (benzoylamino) acetamido] -2 - [(4-trifluoromethyl) benzyloxycarbonyl) hexanoate (6).

A solution of compound 3 (44 mg, 0.085 mmol) in CH ₂ Cl ₂ (1.0 ml) was treated with 4 N. HCl-dioxane (1.0 ml) and stirred for 1 h at 25 ° C. The solvent and excess acid were removed under a N ₂ stream, and the crude hydrochloride salt was suspended in CH ₂ Cl ₂ (0.8 ml), treated with i-Pr ₂ NEt (30 μl, 017 mmol) and benzoyl chloride (11 μl, 0.094 mmol) and stirred for 25 ° C. After 2 hours, the reaction mixture was directly purified by flash chromatography (SiO ₂ , 1: 9: 9 MeOH / CH ₂ Cl ₂ / EtOAc) to give 40 mg (90%) of compound 6 as a white powder: [α] _D ²⁵ -2.7 (c 0.70, CHCl ₃ ).

¹ H NMR (CD ₃ OD, 400 MHz) 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, 3.8 Hz, 1H), 3.69 (s, 3H), 3.21 (t, J = 5.6 Hz, 2H ), 1.81 (m, 1H), 1.69 (m, 1H), 1.53 (m, 2H), 1.42 (m, 2H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ 172.8, 169.5, 168.4, 156.3, 142.3, 132.9, 131.4, 129.4 (q, J = 32, 2 Hz), 128.0, 127.1, 126.7, 126.3 (q, J = 269.0 Hz), 124.8, 65.1, 53.6, 51.6, 42.6 , 38.4, 30.6, 28.1, 22.2.

IR (film) V _max 3303, 2923, 1713, 1651, 1533 cm ^-1 ; FABHRMS (NBA-CsI) m / z 656.0961 (M + Cs, C ₂₅ H ₂₅ F ₃ N ₃ O ₆ , 656.0985 required).

Example 14. (S) -6- [2- (benzoylamino) acetamido] -2- (4-trifluoromethyl) benzyloxycarbonyl] hexanoic acid (7)

A solution of compound 6 (17 mg, 0.032 mmol) in THF-MeOH (0.4 ml, 3: 1) was treated with LiOH · H ₂ O (2.0 mg, 0.49 mmol), dissolved in H ₂ O (0, 1 ml) and stirred for 2 hours at 0 ° C. The reaction was quenched by the addition of concentrated aqueous HCl (5 μl), diluted with EtOAc (30 ml) and washed with water (2 × 15 ml). Drying (Na ₂ SO ₄ ) and evaporation gave 14.5 mg (88%) of compound 7 as a white powder: [α] _D ²⁵ + 2.2 (c 0.6, CH ₃ OH).

¹ H NMR (CD ₃ OD, 400 MHz) δ 7.86 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 7.53 (m, 1H), 7.43 (m, 2H), 5.17 (d, J = 13.2 Hz, 1H), 5.14 (d, J = 13.2 Hz, 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). ¹³ C NMR (CD ₃ OD, 125 MHz) δ 175.9, 171.7, 170.5, 158.4, 142.9, 135.1, 132.9, 130.9 (q, J = 32 , 4 Hz), 129.5, 128.9, 128.5, 126.3, 125.7 (q, J ~ 269.0 Hz), 66.5, 55.4, 44.1, 40, 1, 32.3, 29.9, 24.2.

IR (film) V _max 3318, 2935, 1713, 1644, 1538, 1326 cm ^-1 ; MALDIFTMS m / z 532.1672 (M ⁺ + Na ⁺ , C ₂₄ H ₂₆ F ₃ N ₃ O ₆ , 532.1671 required).

Example 15. (S) -Metyl 6- [2 - [(2 - ((carboxymethyl) methoxy) -acetamido] -2 - [(4-trifluoromethyl) benzyloxycarbonyl] hexanoate (8).

A solution of compound 3 (57 mg, 0.11 mmol) in CH ₂ Cl ₂ (1 ml) was treated with 4 N. HCl-dioxane (2 ml) and stirred for 1 h at 25 ° C. The solvent and excess acid were removed under N ₂ , the residue was dissolved in DMF (1 ml) and treated with diglycolic acid (16 mg, 0.12 mmol), EDCI (23 mg, 0.12 mmol) and i-Pr ₂ NEt (42 μl, 0.24 mmol) and stirred at 25 ° C. After 4 hours, the reaction mixture was poured into a separatory funnel containing EtOAc (50 ml), washed with 10% aqueous HCl (3 x 30 ml) and saturated aqueous NaCl (30 ml), dried (Na ₂ SO ₄ ) and evaporated to give 45 mg (77%) of the monoacid of compound 8 as a white solid: [α] _D ²⁵ - 7.5 (c 1.8, CH ₃ OH).

¹ H NMR (CD ₃ OD, 400 MHz) δ 7.65 (d, J = 5.2 Hz, 2H), 7.54 (d, J = 5.3 Hz, 2H), 5.18 (d, J = 9.0 Hz, 1H), 5.15 (d, J = 9.0 Hz, 1H), 4.42 (s, 2H), 4.16 (m, 1H), 4.11 (s, 2H), 3.87 (s, 2H), 3.70 (s, 3H), 3.20 (t, J = 4.5 Hz, 2H), 1.81 (m, 1H), 1.68 ( m, 1H), 1.53 (m, 2H), 1.39 (m, 2H). ¹³ C NMR (CD ₃ OD, 100 MHz) δ 174.5, 173.7, 172.5, 171.2, 158.2, 142.6, 130.9 (q, J = 33.2 Hz) , 128.2, 126.2, 125.4 (q, J = 270.1 Hz), 71.3, 69.0, 66.5, 55.3, 52.7, 42.9, 39, 9, 32.0, 29.7, 23.9.

IR (film) V _max 3315, 2931, 1725, 1661, 1538, 1326 cm ^-1 ; MALDIFTMS m / z 558.1686 (M ⁺ + Na ⁺ , C ₂₂ H ₂₃ F ₃ N ₃ O ₉ , 558.1673 required).

Example 16: (S) -methyl 2- (benzoylamino) -6 - (((tert-butyloxy) carbonyl) amino) hexanoate (9).

A solution of N-ε-BOC-lysine methyl ester hydrochloride (5.05 g, 17 mmol) in CH ₂ Cl ₂ (100 ml) was treated with benzoyl chloride (2.0 ml, 17 mmol) and i-Pr ₂ NEt (5.9 ml , 34 mmol) and stirred at 25 ° C. After 1 h, the reaction mixture was washed with 10% aqueous HCl (100 ml), dried (Na ₂ SO ₄ ) and evaporated to a white powder, which was crystallized from CH ₂ Cl ₂ -hexane to obtain 4.8 g (78%) of compound 9 in as a white crystalline solid with a melting point of 108-109 ° C; [α] _D ²⁵ -13.2 (c 5.8, CH ₃ OH).

¹ H NMR (DMSO-d ₆ , 400 MHz) δ 8.72 (d, J = 7.4 Hz, 1H), 7.89 (m, 2H), 7.55 (m, 1H), 7.48 (m, 2H), 6.88 (t, J = 5.2 Hz, 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). ¹³ C NMR (DMSO-d ₆ , 100 MHz) δ 172.9, 166.7, 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) V _max 3335, 2933, 1740, 1690, 1647, 1534 cm ^-1 ; MALDIFTMS (DHB) m / z 387.1895 (M ⁺ + Na ⁺ , C ₁₉ H ₂₅ N ₂ O ₅ , 387.1890 required).

Example 17. (S) -methyl 2- (benzoylamino) -6- [2 - (((tert-butyloxy) carbonyl) amino] acetamido) hexanoate (10).

A solution of compound 9 (4.4 g, 12.1 mmol) in CH ₂ Cl (5.0 ml) was treated with 4 N. HCl-dioxane (10.0 ml) and stirred for 3 hours at 25 ° C. The solvent and excess acid were removed under reduced pressure, the residue was dissolved in DMF (150 ml), treated with N - ((tert-butyloxy) carbonyl) glycine (2.2 g, 12.1 mmol), PyBrOP (7.0 g, 15 mmol) and i-Pr ₂ NEt (8.4 ml, 48.4 mmol) and stirred at 25 ° C. After 12 h, the reaction mixture was diluted with EtOAc (500 ml), washed with 10% aqueous HCl (3 x 250 ml), 5% aqueous Na ₂ CO ₃ (250 ml) and saturated aqueous NaCl (250 ml), dried (Na ₂ SO ₄ ) and evaporated. Flash chromatography (SiO _2, EtOAc) gave 4.6 (90%) of compound 10 as a colorless oil: [α] _D ²⁵ -10.3 (c 0,30, CH ₃ OH).

¹ H NMR (CDCl ₃ , 400 MHz) δ 7.84 (m, 2H), 7.51 (m, 1H), 7.44 (m, 2H), 6.91 (d, J = 7.6 Hz , 1H), 6.35 (br t, J = 5.3 Hz, 1H), 5.22 (m, 1H), 4.76 (m, 1H), 3.76 (s, 3H), 3 70 (br t, J = 6.3 Hz, 4H), 3.26 (m, 2H), 1.95 (m, 1H), 1.84 (m, 1H), 1.57-1, 30 (m, 4H); 1.41 (s, 9H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ 173.0, 169.7, 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) V _max 3318, 2954, 1718, 1647, 1535, 1491 cm ^-1 ; MALDIFTMS m / z 444.2108 (M + Na ⁺ , C ₂₁ H ₃₁ N ₃ O ₆ , 444.2110 required).

Example 18. N, N'-bis [N - (- 5- (S) - ((benzoyl) amino) -5- (methoxycarbonyl) pentyl) carboxamidomethyl] benzene-1,3-dicarboxamide (11).

A solution of compound 10 (4.4 g, 10.4 mmol) in CH ₂ Cl ₂ (3 ml) was treated with 4.0 N. HCl-dioxane (20 ml) and stirred for 1 h at 25 ° C. The solvent and excess acid were removed under reduced pressure, the residue was dissolved in CH ₂ Cl ₂ (50 ml), treated with Et ₂ N (5.8 ml, 42 mmol) and isophthaloyl dichloride (1.06 g, 5.2 mmol) and stirred at 25 ° C. After 16 hours, the reaction mixture was washed with 10% aqueous HCl (50 ml) and evaporated to give a yellow oil. Flash chromatography (SiO ₂ , 5: 5: 2 EtOAc / CH ₂ Cl ₂ / MeOH) afforded 2.5 g (62%) of compound 11 as a white foamy solid: [α] _D ²⁵ M-8.0 ( c 4.8, CH ₃ OH).

¹ H NMR (CD ₃ OD, 500 MHz) 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 (two s, 4H); 3.72 (s, 6H), 3.22 (m, 4H), 1.99-1.83 (m, 4H), 1.59-1.40 (m, 8H). ¹³ C NMR (CD ₃ OD, 100 MHz) δ 174.2, 171.5, 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) V _max 3304, 2950, 1738, 1650, 1644, 1538 cm ^-1 ; MALDIFTMS m / z 795.3332 (M + Na ⁺ , C ₄₀ H ₄₅ N ₆ O ₁₀ , 795.3330 required).

Example 19. N, N'-bis- [N - (- 5- (S) - ((benzoyl) amino) -5- (carboxy) pentyl) carboxamidomethyl] benzene-1,3-dicarboxamide (12).

A solution of compound 11 (1.1 g, 1.4 mmol) in MeOH-THF (20 ml, 1: 1) was treated with LiOH · H ₂ O (240 mg, 5.7 mmol), dissolved in H ₂ O (10 ml ) and stirred for 1 h at 0 ° C. After 1 h, the reaction solvent was evaporated, the residue was redissolved in H ₂ O (20 ml) and cooled to 0 ° C. Concentrated aqueous HCl (0.47 ml, 5.7 mmol HCl) was added and the solid precipitated was filtered and washed with water (50 ml) to obtain 0.78 g (73%) of compound 12 as a white powder: [ α] _D ²⁵ -2.6 (c 0.35, CH ₃ OH).

¹ H NMR (CD ₃ OD, 400 MHz) 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 (two s, 4H), 3.26 (m, 4H), 1.99-1.83 (m, 4H); 1.63-1.45 (m, 8H). ¹³ C NMR (CD ₃ OD, 100 MHz) δ 175.5, 171.9, 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) V _max 3280, 2923, 1718, 1641, 1536 cm ^-1 ; MALDIFTMS (DHB) m / z 767.3005 (M + Na ⁺ , C ₃₈ H ₄₄ N ₆ O ₁₀ , 767.3017 required).

Results and discussion

Compound 1 disrupts the RGD-dependent interaction between integrin α _v β ₃ and MMP2. Recent observations that the carboxy-terminal hemopexin-like domain of MMP2 can affect the binding of MMP2 and integrin α _v β ₃ and thus block angiogenesis have prompted the authors to search for organic inhibitors of this interaction that could be used in treatment. To determine a specific inhibitor of the interaction of MMP2 and integrin α _v β ₃ , studies of solid-phase receptor binding with immobilization of integrins and biotinylated MMP2 were carried out. It was found that the binding of purified MMP2 is completely dependent on RGD in this system, which is confirmed by the insufficient effect of cRGDfV on the binding of MMP2 and integrin α _v β ₃ , even if this peptide inhibited the interaction of α _v β ₃ with its extracellular matrix ligand, vitronectin (VN; figa). It is important that the interaction of MMP2, and not VN, was completely inhibited by compound 1, which showed the specificity of compound 1 with respect to the interaction of MMP2 and α _v β ₃ . In addition, compound 1 did not inhibit the binding of MMP2 and tissue metalloproteinase 2 inhibitor (TIMP2), confirming that the effect of this compound 1 has a limited effect on the interaction of MMP2 and integrin α _v β ₃ , and that indicates a difference between the binding sites of the MMP2 PEX domain TIMP2 and integrin α _v β ₃ . It is important to note that neither the control compound nor the cRADfV control peptide affected the binding of MMP2 and integrin α _v β ₃ (Fig. 2A).

Compound 1 binds directly to integrin α _v β ₃ rather than MMP2. For further consideration of the mechanism of action of compound 1, an additional solid-phase analysis of receptor binding was performed with the immobilization of α _v β ₃ and [ ¹⁴ C] -labeled compound 1, or biotinylated VN as a control. As shown in FIG. 2B, in the solid phase binding assay, compound 1 binds directly to integrin α _v β ₃ . This interaction was dose dependent, saturable, and was specific, demonstrating minimal interaction of compound 1 and integrin α ₅ β ₁ unrelated to α _v β ₃ control (FIG. 2B). Undoubtedly, at high concentrations of the compound (data not shown), slight binding to integrin α ₅ β ₁ was observed. In addition, if microtiter plates covered MMP2, no binding to compound 1 was observed (data not shown), which suggests that the observed effects in the analysis of MMP2 / integrin binding α _v β _{3 are} due to the binding of compound 1 and integrin α _v β ₃ . Importantly, this interaction is inhibited in the presence of a 25-fold molar excess of the unrelated compound 1 of compound 12 (FIG. 2C). In addition, the interaction of compound 1 and integrin α _v β ₃ is associated with RGD, which was shown by the inability of the cRGDfV peptide to inhibit the interaction of radiolabeled compound 1 and integrin α _v β ₃ , even if cRGDfV completely inhibited the binding of bVN and integrin transferred to the same system (Fig. .2D). The cRADfV control peptid is shown as a control for binding inhibition specificity. Thus, compound 1 bound to α _v β ₃ shows comparable specificity, selectivity and low sensitivity for inhibition of RGD binding to MMP2.

Interestingly, in the solid-phase analysis, compound 6, a compound of formula (II), where A is hydrogen, n is 0, X ¹ is p-trifluoromethyl and R ¹ is methyl, had a slightly lower activity compared to compound 1, whereas compound 8 containing no phenyl groups attached to glycine units was inactive. This result shows that the phenyl ring plays a significant role in the inhibitory activity of the inhibitor compounds of formula (II) and, in addition, shows that at least one substituted glycynlisine unit is necessary for the antiangiogenic activity of inhibitors of this class.

Cell-mediated degradation of collagen IV by MMP2 is blocked by compound 1. Prior to inhibition of cell-mediated degradation of collagen IV in the α _v β ₃ -dependent method, prevention of binding of MMP2 and integrin α _v β ₃ to melanoma cells was shown. Thus, it was evaluated whether expressing or non-expressing α _v β ₃ melanoma cells can use activated MMP2 to degrade immobilized [ ³ H] collagen IV. Importantly, no cells endogenously produced a noticeable amount of MMP2. While both types of cells were capable of some degree of degradation of the main collagen, only B3-transfected CS-1 cells were able to utilize exogenous MMP2, showing significantly greater release of substrate radioactivity into the culture medium after pre-incubation with purified MMP2 (Fig. 3A). Such an increase in substrate degradation in response to the effect of MMP2 was significantly reduced when compound 1 was added, while when compound 12 was added, no significant effect was observed (Fig. 3A). It is important that the effect of compound 1 on cell-mediated degradation of collagen was not due to a direct inhibition of MMP2 activity, since purified active MMP2 in the absence of cells could destroy immobilized [ ³ H] collagen IV regardless of the presence or absence of compound 1 or compound 12. For this to show that the reduction of cell-mediated collagen degradation as shown in Figure 3A, was a result of inhibiting MMP2 interaction of compound 1 and integrin α _v β ₃ on the cell surface, CS-1 cells and their _v β ₃ -nesuschy equivalent was evaluated in analyzing the binding of biotinylated MMP2. As expected, β ₃ -negative CS-1 cells were able to bind some level of MMP2, however their binding ability did not decrease in the presence of any of the compounds (Fig. 3B). In contrast, β ₃ CS-1 cells bound a significantly larger amount of MMP2, and this increase in MMP2 binding was specifically suppressed by compound 1. In fact, when determining the amount in lanes, as shown by anti-actin mAb staining, compound 1 effectively reduced the binding of MMP2 and β ₃ CS -1 cells to the level observed in the absence of α _v β ₃ (i.e., parenteral CS-1 cells) (Fig. 3B, lane 2).

Compound 1 disrupts in vivo angiogenesis without suppressing MMP2 activity. Suppression of the interaction of α _v β ₃ / MMP2 with exogenous administration of the recombinant MMP2 PEX domain was shown before experiments with a decrease in angiogenesis in animal models. Thus, the effects of compound 1 on the intensity of factor-induced angiogenesis in 10-day chicken CAM were evaluated. The introduction of compound 1 into CAM, stimulated by the main fibroblast growth factor (bFGF), almost completely stopped the development of new blood vessels in response to such a stimulus (Figs. 4A and 4B), while control compounds 12 were not effective in this regard. It is important that the cessation of angiogenic infiltration in response to compound 1 was not associated with a suppression of MMP2 activity, since equivalent levels of active MMP2 (62 kDa) were determined in the treated and untreated tissue of CAM embryos (Fig. 4C). This completely contradicted the effect of the exogenous MMP2 PEX domain, which suppressed the activity of MMP2 in this system. These data are consistent with the view that compound 1 specifically affects the binding of MMP2 and integrin α _v β ₃ without directly affecting the activity of MMP2. Undoubtedly, the observed total levels of MMP2 in CAM lysates were also not affected by treatment with compound 1, excluding a strong effect on the expressed level of MMP2 in angiogenic tissues (Fig. 4C). These results suggest that the anti-angiogenic effects of compound 1 probably cause suppression of the binding of MMP2 and integrin α _v β ₃ on cell surfaces, as shown in FIG. 3B. These data also show that fully activated MMP2 is not used for angiogenesis in such a system, except for binding to integrin α _v β ₃ on the cell surface.

Compound 1 inhibits tumor growth in vivo. It has been shown that a violation of angiogenesis inhibits tumor growth in a large number of systems. As a result, blocking the invasive properties of endothelial cells by inhibiting the suppression of MMP angiogenesis and also tumor growth in animal models. In fact, it has been shown that a number of MMP inhibitors can act as anti-angiogenic agents in humans. Thus, this study evaluated whether the growth of an α _v β ₃ -negative tumor was sufficiently inhibited by inhibiting angiogenesis associated with blocking the interaction of MMP2 / α _v β ₃ . The use of an α _v β ₃ negative tumor made it possible to evaluate the effect of compound 1 on selective α _v β ₃ vascularization. As shown in FIG. 5A, with a single intravenous (IV) administration of compound 1, the growth of transplanted α _v β ₃ -negative CS-1 melanoma chicken CAM tumors significantly slowed down, and the tumor weight also decreased (FIG. 5B). It is likely that this effect was not due to the direct effect of compound 1 on tumor cells, such as the α _v β ₃ negative melanoma cells used in this study. In fact, the compound cultured with cells in vitro did not affect their growth (data not shown). In tumors treated with compound 1, a decrease in the surface of vascular formation was clearly observed (Fig. 5A), as well as a decrease in the total density of blood vessels (Fig. 5C). It is important that such a decrease in tumor vascularization was associated with significant cell death within the tumor mass, even when the control tumors showed a 6-fold increase in mass 10 days before the study.

The above detailed description and examples are provided for illustrative purposes only and are not limited. Other variations within the scope of the present invention are also possible and will be readily apparent to those skilled in the art.

Data on the activity of compounds 1, 5, 6 and 7

Inhibition of MMP2 (% inhibition of the binding of MMP2 to integrin α _v β ₃ in comparison with the control (no inhibition)):

Compound Inhibition of MMP2 at a concentration of 1 μm one fifty% 5 59% 6 58% 7 46%

The binding of MMP2 to integrin is necessary for angiogenesis (blood vessel formation), therefore, a compound that inhibits the binding of MMP2 also inhibits angiogenesis. Angiogenesis inhibitors are used to treat tumor diseases, since they prevent the formation of new blood vessels necessary for tumor growth.

In addition, for compound 1, cytotoxicity data are available on two cell lines: human leukemia cell line (L1210) and mouse leukemia cell line (CCRF-CEM). In the case of human leukemia, the IC ₅₀ (the concentration at which 50% of the cells die) for compound 1 was 100 μM. In the case of leukemia in mice, the IC _{50 was} also 100 μM. A value of 100 μM is considered as a good level of toxicity for cell lines.

Claims (32)

1. Compound structure:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1. 2. The compound of claim 1, wherein at least one of X ² and X ³ is para-trifluoromethyl and A is a covalent bond. 3. The compound according to claim 1, where at least one of R ² and R ³ represents H and A represents a covalent bond. 4. The compound according to claim 1, where at least one of R ² and R ³ represents methyl and A represents a covalent bond. 5. The compound according to claim 1, where X ² and X ³ each represents para-trifluoromethyl, R ² and R ³ each is methyl and A is a covalent bond. 6. The compound according to claim 1, where R ² represents H and A represents H. 7. The compound according to claim 1, where R ² represents methyl and A represents H. 8. The compound according to claim 1, where X ² represents para-trifluoromethyl, R ² represents H and A represents H. 9. The compound according to claim 1 of the structure:

10. A pharmaceutical composition having the properties to inhibit angiogenesis and tumor growth, containing a compound of the structure:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1; with a pharmaceutically acceptable carrier. 11. The pharmaceutical composition of claim 10, where A is a covalent bond. 12. The pharmaceutical composition according to claim 11, where X ² and X ³ each represents trifluoromethyl. 13. The pharmaceutical composition according to claim 11, where R ² and R ³ each represents N. 14. The pharmaceutical composition of claim 11, wherein X ² and X ³ are para-trifluoromethyl, R ² is H, and A is H. 15. The pharmaceutical composition according to p. 10, containing a compound of structure:

with a pharmaceutically acceptable carrier. 16. A method of inhibiting tumor growth in the body, which includes the introduction, if necessary, of such inhibition, a therapeutically effective amount of a compound represented by the formula:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1. 17. The method of inhibiting tumor growth according to clause 16, where A is a covalent bond. 18. The method of inhibiting tumor growth according to 17, where X ² and X ³ each represents trifluoromethyl. 19. The method of inhibiting tumor growth according to 17, where R ² and R ³ each represents N. 20. The method of inhibiting tumor growth according to clause 16, where a therapeutically effective amount of a compound represented by the formula is administered:

21. A method of inhibiting angiogenesis in tumor tissue, which comprises introducing into the tumor tissue an effective amount of a compound represented by the formula for inhibiting angiogenesis:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1. 22. The method according to item 21, where A is a covalent bond. 23. The method according to item 22, where X ² and X ³ each represents trifluoromethyl. 24. The method according to item 22, where R ² and R ³ each represents N. 25. A method of inducing apoptosis in tumor cells, which comprises introducing into the tumor cells a therapeutically effective amount of a compound represented by the formula:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1. 26. The method according A.25, where A is a covalent bond. 27. The method according to p, where X ² and X ³ each represents trifluoromethyl. 28. The method according to p, where R ² and R ³ each represents N. 29. A method of inhibiting the interaction of MMP2 and integrin α _v β ₃ in body cells, which comprises contacting integrin with an interaction inhibiting amount of a compound represented by the formula:

where R ² and R ³ each independently represents H, C ₁ -C ₄ alkyl; X ² and X ³ each independently represents C ₁ -C ₄ perfluoroalkyl; A represents H or a covalent bond; t is an integer having a value of 0 or 1; provided that when A represents H, then t is 0, and when A is a covalent bond, then t is 1. 30. The method according to clause 29, where A is a covalent bond. 31. The method according to clause 30, where X ² and X ³ each represents trifluoromethyl. 32. The method according to clause 30, where R ² and R ³ each represents N.

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