This invention relates to compounds which are vitronectin receptor antagonists and are useful for the treatment of cancer, retinopathy, cardiovascular disorders, such as atherosclerosis and restenosis, and diseases wherein bone resorption is a factor, such as osteoporosis.

Integrins are a superfamily of cell adhesion receptors, which are transmembrane glycoproteins expressed on a variety of cells. These cell surface adhesion receptors include gpIIb/IIIa, also known as the " fibrinogen receptor," and α_vβ₃, also known as the "vitronectin receptor." The fibrinogen receptor gpIIb/IIIa is expressed on the platelet surface, and it mediates platelet aggregation and the formation of a hemostatic clot at the site of a bleeding wound. Philips, et al., Blood., 1988, 71, 831. The vitronectin receptor α_vβ₃ is expressed on a number of cells, including endothelial, smooth muscle, osteoclast, and tumor cells, and, thus, it has a variety of functions. The α_vβ₃ receptor expressed on the membrane of osteoclast cells mediates the bone resorption process and contributes to the development of osteoporosis. Ross, et al., J. Biol, Chem., 1987, 262, 7703. The α_vβ₃ receptor expressed on human aortic smooth muscle cells stimulates their migration into neointima, which leads to the formation of atherosclerosis and restenosis after angioplasty. Brown et al., Cardiovascular Res., 1994, 28, 1815. Additionally, a recent study has shown that a α_vβ₃ antagonist is able to promote tumor regression by inducing apoptosis of angiogenic blood vessels. Brooks, et al., Cell, 1994, 79, 1157. Thus, agents that would block the vitronectin receptor would be useful in treating diseases mediated by this receptor, such as osteoporosis, atherosclerosis, restenosis and cancer.

The vitronectin receptor is known to bind to bone matrix proteins, such as osteopontin, bone sialoprotein and thrombospondin, which contain the tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al., Exp. Cell Res. 1991, 195, 368, disclose that RGD-containing peptides and an anti-vitronectin receptor antibody (23C6) inhibit dentine resorption and cell spreading by osteoclasts. In addition, Sato, et al., J. Cell Biol. 1990, 111, 1713 disclose that echistatin, a snake venom peptide which contains the RGD sequence, is a potent inhibitor of bone resorption in tissue culture, and inhibits attachment of osteoclasts to bone. Fisher, et al., Endocrinology 1993, 132, 1411, has further shown that echistatin inhibits bone resorption in vivo in the rat. Bertolini et al., J. Bone Min. Res., 6, Sup. 1, S146,252 have shown that cyclo-S,S-N^α- acetyl-cysteinyl- N^α- methyl-argininyl-glycyl-aspartyl-penicillamine inhibits osteoclast attachment to bone. EP 0 528 587 and EP 0 528 586 report substituted phenyl derivatives which inhibit osteoclast mediated bone resorption.

Alig et al., EP 0 381033, Hartman, et al., EP 0 540 334, Blackburn, et al., WO 93/08174, Bondinell, et al., WO 93/00095, Blackburn, et al., WO 95/04057, Egbertson, et al., EP 0 478 328, Sugihara, et al., EP 0 529 858, Porter, et al., EP 0 542 363, and Fisher, et al., EP 0 635 492 disclose certain compounds that are useful for inhibiting the fibrinogen receptor. WO 96/00730 discloses certain compounds that are vitronectin receptor antagonists. Nicobou et al., Bioorganic & Medicinal Chemistry (1998), 6, 1185-1208; discloses the design, synthesis and biological evaluation of nonpeptide integrin antagonists. Two such compounds are patent inhibitors of α_IIbβ₃ and are compound shaved in vivo inhibition of angiogenesis.

We have invented novel compounds that are antagonists at the vitronectin receptor, i.e., they have a high affinity for the vitronectin receptor, thereby making them useful for treating disorders or diseases mediated by the vitronectin receptor, e.g., cancer, retinopathy, artherosclerosis, vascular restenosis and osteoporosis. The compounds of our invention have the formula:

• wherein n, p, q and r are each independently selected from 0 or 1;
• a, b, c, and d each independently represents a carbon or nitrogen atom, with the proviso that no more than two of a, b, c, and d are nitrogen atoms;
• Y and Y¹ each independently represents 1-4 optional substituents selected from alkyl, alkoxy, halo, -CF₃, and -C(O)OH;
• R¹ is H, alkyl, aryl, aralkyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloalkylalkyl, heterocycloalkylalkyl, -NHR^A, -NHC(O)R^A, -NHSO₂R^A, NHC(O)NHR^A or -NHC(O)OR^A, R¹ being optionally substituted by 1-3 groups selected from halo, alkyl, -CF₃, -CN, -OR^B, -SR^B, -CO₂R^B, -C(O)R^B, -OC(O)R^B, -OC(O)OR^B and -SO₂R^B, and R^A and R^B are independently selected from H, alkyl, aryl, arallcyl, arylcycloallcyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloalkylalkyl or heterocycloalkylalkyl, with the proviso that when R¹ is alkyl, R¹ is not substituted with halo, the proviso that when R¹ is -NHSO₂R^A or -NHC(O)OR^A, R^A is not H, and the proviso that for -SO₂R^B or -OC(O)OR^B, R^B is not H;
• R² is H, alkyl, aryl, aralkyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloalkylalkyl, or heterocycloalkylalkyl, R² being optionally substituted by 1-3 groups selected from halo, alkyl, -CF₃, -CN, -OR^C, SR^C, -CO₂R^C, -C(O)R^C, -OC(O)R^C, -OC(O)OR^C and -SO₂R^C, wherein R^C is selected from H, alkyl, aryl, arallcyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloalkylalkyl or heterocycloalkylalkyl, with the proviso that when R² is alkyl, R² is not substituted with halo, and the proviso that for -SO₂R^C or -OC(O)OR^C, R^C is not H;
• R³ is H, alkyl, aralkyl, arylcycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaralkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C(O)R^D, -C(O)OR^D, -SO₂R^E, -C(O)NR^FR^G, -C(O)NR^FSO₂R^E, or -C(=S)NR^FR^G, wherein R^D, R^E, R^F and R^G are independently selected from H, alkyl, aryl, aralkyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl or heterocycloalkylalkyl, or R^F and R^G taken together complete a 5-7 member ring containing 0 to 1 oxygen or sulfur atoms, and 1 to 2 nitrogen atoms, R³ being optionally substituted by 1-3 groups selected from halo, alkyl, aryl, -CF₃, -CN, -OR^H, -SR^H, -CO₂R^H, -C(O)R^H, -OC(O)R^H, -OC(O)OR^H, -SO₂R^H and -NR^HR^H, wherein R^H is selected from H, alkyl, aryl, aralkyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloalkylalkyl or heterocycloalkylalkyl, with the proviso that when R³ is alkyl, R³ is not substituted with halo, the proviso that when R³ is -SO₂R^E, -C(O)NR^FSO₂R^E, or -CO(O)R^D, R^D and R^E are not H, and the proviso that for -SO₂R^H or -OC(O)OR^H, R^H is not H;
• R⁴ is H, alkyl, aralkyl, arylcycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaralkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, R⁴ being optionally substituted by 1-3 groups selected from halo, alkyl, -CF₃, -CN, -OR^J, -SR^J, -CO₂R^J, -C(O)R^J, -OC(O)R^J, -OC(O)OR^J and -SO₂R^J, wherein R^J is selected from H, alkyl, aryl, aralkyl, arylcycloalkyl, heteroaryl, cycloalkyl, heterocycloalkyl, heteroaralkyl, cycloallcylalkyl or heterocycloalkylalkyl, with the proviso that when R⁴ is alkyl, R⁴ is not substituted with halo, and the proviso that for -SO₂R^J or -OC(O)OR^J, R^J is not H;
• R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from H or C₁-C₃ alkyl;

• "alkyl" refers to straight or branched chain groups having 1 to 20 carbon atoms;
• "cycloalkyl" refers to non-aromatic carbocyclic ring or multi-carbocyclic ring system of from 3 to 20 carbon atoms;
• "cycloalkylalkyl" refers to groups having the formula cycloalkyl-R-, wherein R is alkyl;
• "heterocycloalkyl" refers to a cycloalkyl group, wherein one or more of the carbon atoms of such groups are replaced with a heteroatom selected from O, S and N;
• "heterocycloalkylalkyl" refers to groups having the formula hetcrocycloalkyl-R-, wherein R is alkyl;
• "aryl" refers to aromatic carbocyclic groups;
• "aralkyl" refers to groups having the formula aryl-R-, wherein R is alkyl;
• "heteroaryl" refers to aromatic carbocyclic groups, wherein one or more of the carbon atoms of such groups are replaced with a heteroatom selected from O, S and N;
• "heteroaralkyl" refers to groups having the formula heteroaryl-R-, wherein R is alkyl;
• "arylcycloalkyl" refers to groups having the formula aryl-R-, wherein R is cycloalkyl;

R¹ is preferably H, -NHR^A -NHC(O)R^A, NHC(O)OR^A,-NHC(O)NHR^A, or -NHSO₃R^A, R¹ is more preferably -NHC(O)OR^A. R¹ is most preferably,

• R² preferably H.
• R³ is preferably selected from H, alkyl, -C(O)R^D, -C(O)OR^D, -C(O)NR^FR^G, and -C(=S)NR^FR^G. R^D is preferably selected from phenyl, alkyl, aralkyl, arylcycloalkyl, cycloalkyl, and wherein R^D is optionally substituted by 1-3 substituents selected from alkoxy, halo, cycloalkyl, -S-CH₃, phenyloxy, -OC(O)CH₃, -C(O)OC₂H₅ and -N(OH₃)₂. R^F and R^G are preferably selected from H, alkyl, phenyl, cycloalkyl, and aralkyl, wherein R^F and R° are optionally substituted by alkoxy, halo or -CO₂R^H.
• R⁴ is preferably H or alkyl, most preferably H.
• R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each preferably H.

Preferably, the sum of n + p is 1.

Preferably, the sum of q + r is 1.

Preferably; a, b, c, and d are Carbon atoms.

Preferably, are positioned para relative to each other.

The following compounds, including biolabile esters, or pharmaceutically acceptable salts thereof, are particularly preferred:

Of the foregoing, and are particularly preferred.

Preferably, the compounds of the present invention are selected from those having affinities that are greater than 100 fold more specific for α_vβ₃ than for α_IIbβ₃.

As used herein, the following terms have the following meanings, unless defined otherwise:

• "Alkyl" refers to straight or branched hydrocarbon chain groups having 1 to 20 carbon atoms, preferably, 1 to 6 carbon atoms.
• "Alkoxy" refers to groups having the formula -OR, wherein R is alkyl.
• "Aryl" refers to carbocyclic groups having at least one aromatic ring.
• "Aralkyl" refers to groups having the formula aryl-R-, wherein R is alkyl.
• "Arylcycloalkyl" refers to groups having the formula aryl-R-, wherein R is cycloalkyl.
• "Arylalkoxy" refers to groups having the formula aryl-R-O-, wherein R is alkyl.
• "Carboxy" refers to a group having the formula -C(O)OH.
• "Carboxyalkyl" refers to groups having the formula, -R-C(O)OH, wherein R is alkyl.
• "Carbamoyl" refers to a group having the formula -C(O)NH₂.
• "Carbamoylalkyl" refers to groups having the formula -R-C(O)NH₂, wherein R is alkyl.
• "Cbz" refers to benzyloxycarbonyl.
• " CycloaDcyt" refers to a non-aromatic carbocyclic ring or multi-carbocyclic ring system of from 3 to 20 carbon atoms, preferably, 3 to 7 carbon atoms.
• "Cycloalkylalkyl" refers to groups having the formula cycloalkyl-R-, wherein R is alkyl.
• "Finoc" refers to 9-fluorenylmethoxycarbonyl.
• "Heteroaryl" refers to aromatic carbocyclic groups, wherein one or more of the carbon atoms of such groups are replaced with a heteroatom selected from O, S and N.
• "Heteroaralkyl" refers to groups having the formula heteroaryl-R-, wherein R is alkyl.
• "Heterocycloalkyl" refers to a cycloalkyl group, wherein one or more of the carbon atoms of such group is replaced with O, S, NH, or N-alkyl.
• "Hetetocycloalkylalkyl" refers to groups having the formula heterocycloalkyl-R-, wherein R is alkyl.
• "Halo" refers to a halogen substituent.

The term "biolablile ester" means a pharmaceutically acceptable, biologically degradable ester derivative of a compound of formula (I), that is a prodrug which, upon administration to a animal or human being, is converted in the body to a compound of formula (I). Biolabile esters of the invention are as defined previously.

The term "vitronectin - mediated disorder" refers to a disease state or malady which is caused or exacerbated by a biological activity of vitronectin receptors. Disorders mediated by the vitronectin receptor include, without limitation, cancer, retinopathy, artheroselerosis, vascular restenosis, and osteoporosis.

The term "effective amount" refers to an amount of vitronectin receptor antagonist compound sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without limitation, inhibiting the growth of undesired tissue or malignant cells, or increasing bone density. The precise effective amount for a subject will depend upon the subject's size and health, the nature and severity of the condition to be treated, and the like. The effective amount for a given situation can be determined by routine experimentation based on the information provided herein.

The following abbreviations are used for the solvents and reagents discussed herein: ethanol ("EtOH"); methanol ("MeOH"); acetic acid ("AcOH"); ethyl acetate ("EtOAc"); 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ("HBTU"); 1-hydroxybenzotriazole ("HOBt"); bromo-tris-pyrrolidino-phosphonium hexafluorophosphate ("PyBroP"); N,N-dimethylformamide (" DMF" ); trifluoroacetic acid ("TFA"); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (" EDCI" ); and diisopropylethylamine ("DIPEA"). In addition, "Ph" represents a phenyl group; "tBu" represents a -C(CH₃)₃ group; "OtBu" represents an -O-C(CH₃)₃ group, "n-Bu" or "Bu-n" represents an n-butyl group, "Et" represents an ethyl group, "Me" represents a methyl group, "Ac" represents an acetyl group, and "Boc" represents t-butoxycarbonyl.

The compounds of the invention have asymmetric carbon atoms, and therefore, all isomers, including enantiomers and diastereomers are within the scope of this invention. The invention includes d and l isomers in both pure form and in admixture, including racemic mixtures. Isomers can be prepared using conventional techniques, either by reacting chiral starting materials, or by separating isomers of compounds of formula (I).

Certain compounds of the present invention will be acidic in nature (e.g., those which have a carboxyl or phenolic hydroxyl group). These compounds form pharmaceutically acceptable salts with inorganic and organic bases. The salt may be prepared by treating a solution of the compound with the appropriate base. Non-limitative examples of such salts are sodium, potassium, calcium, aluminum, gold and silver salts, and salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, N-methylglucamine and the like.

Certain compounds of the invention will be basic in nature, and may form pharmaceutically acceptable salts with organic and inorganic acids. Non-limitative examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those in the art. The salt is prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt.

It may be desirable when providing the compounds of the invention for oral administration to use the compounds of formula (I) in the form of a biolabile ester. The suitability of any particular ester-forming group can be assessed by conventional in vivo animal or in vitro enzyme hydrolysis studies. Thus, desirably, for optimum effect, the ester should only be hydrolysed after absorption is complete. Accordingly, the ester should be resistant to premature hydrolysis by digestive enzymes before absorption, but should be productively hydrolysed by, for example, gutwall, plasma or liver enzymes. In this way, the active acid is released into the bloodstream following oral absorption of the prodrug.

Suitable biolabile esters may include alkyl, alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl and alkoxycarbonyloxyalkyl esters, including cycloalkyl and aryl substituted derivatives thereof, aryl esters and cycloalkyl esters, wherein said alkyl, alkanoyl or alkoxy groups may contain from 1 to 8 carbon atoms and be branched-chain or straight-chain, said cycloalkyl groups may contain from 3-7 carbon atoms and said cycloalkanoyl groups from 4-8 carbon atoms wherein both are optionally benzo-fused, and said aryl and aroyl groups include substituted phenyl, naphthyl or indanyl ring systems. Preferably, the biolabile esters of the invention are C₁-C₄ alkyl esters. More preferably, they are methyl, ethyl and pivaloyloxymethyl esters.

Biolabile esters may be obtained from the acids of formula (I) by standard reactions well known to persons skilled in the art. For example, aryl and alkyl esters can be synthesized via activation of a carboxylic acid group of (I) in a variety of ways, such as by forming the acyl chloride, followed by reaction with the required phenol or alcohol. Alternatively, alkyl esters are obtainable by alkylation of a suitable alkali, or alkaline earth, metal carboxylate salt of a compound of formula (I).

The compounds of the present invention may be prepared according to the following reaction scheme (Scheme I):

In Scheme 1, which depicts a solid phase preparation of compounds wherein at least one of q or r is 1, compound 2 is attached by conventional means to a polymeric resin 3 (e.g., a cross-linked polystyrene or a polyethylene glycol/polystyrene copolymer) through a cleavable acid labile linker, L, having an -OH or -Cl group, e.g., Wang, Sasrin and chlorotrityl resin, to form resin compound 4. For example, the attachment to the resin may be carried out by reacting compound 2 with the resin 3 (Cl-form) in the presence of DIPEA in an organic solvent, e.g., DMF or methylene chloride. The Fmoc group of compound 4 is removed by conventional means, e.g., by treating with piperidine in DMF at 0° to 80°C, and acylated with benzoyl chloride 5 to form amide 6. The acylation is preferably carried out in an organic solvent (e.g., methylene chloride or DMF) at 0° to 80°C in the presence of a tertiary amine, preferably DIPEA. Amide 6 is reacted with benzimidazole-amine 7 in a displacement reaction to produce compound 8. The displacement reaction is preferably carried out by shaking the reactants in DMF for an extended period, preferably 1-2 days. For compounds in which the R³ group is not H, such compounds may be made by subjecting compound 8 to conventional reactions to add the R³ substituent to form compound 9. For example, depending on the desired substituent, compound 8 may be reacted with a carboxylic acid, an acyl chloride, acyl anhydride, isocyanate, carbamoyl chloride, isothiocyanate, alkyl halide, alkyl sulfonate, or epoxide, or alternatively, compound 8 may be subjected to reductive alkylation with an aldehyde or ketone. Compound 10 is formed by cleavage from the linker and the resin portion of compound 9 by conventional means, e.g., by treating with dilute TFA in methylene chloride at ambient temperature for 10 to 60 minutes. If desired, compound 10 may be converted to a biolabile ester by standard esterification methods.

Compounds wherein q and r are both 0 may be prepared according to the solid phase synthesis shown in Scheme 2, below.

In Scheme 2, compound 4, prepared as described in Scheme 1, is treated with piperidine in DMF at 0° to 80°C, and acylated with benzoyl chloride 11 to form amide 12. The acylation is preferably carried out in an organic solvent (e.g., methylene chloride or DMF) at 0° to 80°C in the presence of a tertiary amine, preferably DIPEA. Amide 12 is subsequently reacted with a benzimidazole 13 to form compound 14, and if desired, reacted with a suitable reagent to add the R³ group under the conditions described for Scheme 1 to form compound 15. Compound 16 is formed by cleavage from the linker and resin portion of compound 15 under the conditions described in Scheme 1. If desired, compound 16 may be converted to a biolabile ester by standard esterification methods. The starting compounds and reagents used in the foregoing schemes are either commercially available or may be prepared by methods well-known to those skilled in the art.

Those skilled in the art will recognize that reactive groups in the foregoing reaction schemes (e.g., carboxyl, amino, hydroxy) may be protected if desired or necessary with conventional protecting groups that can be subsequently removed by standard procedures. See, e.g., McOmie, Protecting groups In Organic Chemistry, Plenum Press, N.Y., 1973, and Greene and Wuts, Protecting Groups In Organic Synthesis, 2nd Ed., John Wiley & Sons, N.Y. 1991.

As an alternative to solid phase synthesis, the compounds of the present invention may be prepared by solution synthesis, employing appropriate protective groups for reactive groups. Particularly useful for carboxy protection are t-butyl esters, although other groups such as allyl and benzyl are also suitable. Intermediate esters may be converted to the acids by appropriate deprotection methods.

For preparing pharmaceutical compositions from the compounds of this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 70 percent active ingredient. Suitable solid carriers are known in the art, e.g. magnesium carbonate, magnesium stearate, talc, sugar, lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral inj ection.

Liquid form preparations may also include solutions for intranasal administration.

Opthalmic preparations may be formulated using commercially available vehicles such as Sorbi-care® (Allergan) or Neodecadron® (Merck, Sharp & Dohme).

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

Preferably, the pharmaceutical preparation is in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may be varied or adjusted from 0.01 mg to 1000 mg, more preferably from 0.1 mg to 200 mg, most preferably from 5 mg to 100 mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

The amount and frequency of administration of the compounds of the invention will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended dosage regimen is oral administration of from 0.02 mg to 4,000 mg/day, preferably 0.2 mg to 800 mg/day, most preferably 10 mg to 400 mg/day in two to four divided doses to block tumor growth.

The following examples illustrate the foregoing invention, although such examples should not be construed as limiting the scope of the invention. Alternative mechanistic pathways and analagous structures within the scope of the invention will be apparent to those skilled in the art.

In the Examples below, the "funnel apparatus" is a sintered glass funnel for agitating the contents with nitrogen and removal of the solvent by filtration. Where resins are "washed" with solvent, e.g., (20 mL x 5), the resin in solvent (20 mL) is agitated for 2 minutes in a funnel apparatus, and solvent is removed by filtration (draining), and this sequence is repeated 4 additional times.

For the Examples below, "AA" refers to depending on the particular compounds used from the preparative examples. "-U-" refers to -CH₂-, -CH₂-CH₂- , or -CH(CH₃)- , depending on the particular compounds used from the preparative examples.

"2-chlorotrityl resin, chloride form" refers to wherein represents the resin (polymer) portion. " CTR" refers to 2-chlorotrityl resin. Thus, for example, N²-Cbz-L-2,3-diaminopropionic acid on 2-chlorotrityl resin refers to

Add 2-(aminomethyl)bcnzimidazole, dihydrochloride, hydrate (18.50 g) to a solution of potassium hydroxide (9.50 g) in methanol (400 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and concentrate the filtrate in vacuo. Extract the residue with EtOAc (5 x 500 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (9.60 g).

Combine 3-amino-3-phenylpropionic acid (3.70 g, 22.4 mmol) and NaHCO₃ (8.42 g, 100 mmol) in acetone (50 mL) and water (50 mL). Cool in an ice-bath. Add Fmoc-O-hydroxysuccinimide (9.40 g, 28.0 mmol), and stir the resulting mixture for 3 hours while the ice melts. Concentrate the mixture in vacuo, and extract the aqueous portion with EtOAc. Wash the EtOAc solution with 5% glacial acetic acid in water (3 x 300 mL), 5% NaHCO3 solution (3 x 300 mL) and brine (3 x 300 mL). Concentrate the dried (MgSO₄) EtOAc solution in vacuo to give the title compound (contains Fmoc-O-hydroxysuccinimide) as a white foam which is used in Step 2.

Reference: W. M. Kazmierski, Int. J. Pep. Prot. Res., 45, 242 (1995).

Step 2a. To a solution of DIPEA (1.6 mL) in DMF (10 mL), add the crude product (Preparation 2, Step 1) (0.64 g). Add 2-chlorotrityl resin, chloride form (2.00 g, 0.65 mmol/g). Agitate the resulting mixture for 30 minutes. Add MeOH (0.44 mL), agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (30 mL x 5) and then CH₂Cl₂ (30 mL x 5) to give 3-Fmoc-amino-3-phenylpropionic acid on 2-chlorotrityl resin.

Step 2b. Wash the resin (Preparation 2, Step 2a) with DMF (20 mL x 5). Add 20% piperidine in DMF (30 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times. To determine loading level, combine filtrates in 100 mL volumetric flask, and add DMF to 100 mL (Solution A). Dilute Solution A (0.2 mL) to 100 mL in a volumetric flask. UV absorbance at 301 nM: 0.374 $$0.374\times \mathrm{concentration}/7800$$ $$0.374\times 20,000/7800=0.959\mathrm{mmol}/2\mathrm{g}\left(\mathrm{mmol}/\mathrm{g}\right)$$

Place resin (Preparation 2, Step 2b) (2.00 g, 0.959 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (1.84 mL, 10.6 mmol), followed by 4-chloromethylbenzoylchloride (1.89 g, 9.6 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x 3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin (Preparation 2, Step 3) (2.00 g, 0.479 mmol) and 2-(aminomethyl)benzimidazole (9.6 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine N²-Cbz-L-2,3-diaminopropionic acid (2.66 g, 11.27 mmol) and NaHCO3 (4.21 g, 50 mmol) in acetone (25 mL) and water (25 mL). Cool in an ice-bath. Add Fmoc-O-hydroxysuccinimide (4.70 g, 14.0 mmol), and stir the resulting mixture for 3 hours while the ice melts. Concentrate the mixture in vacuo, and extract the aqueous portion with EtOAc. Wash the EtOAc solution with 5% glacial acetic acid in water (3 x 125 mL), 5% NaHC03 solution (3 x 100 mL) and brine (3 x 100 mL). Concentrate the dried (MgSO₄) EtOAc solution in vacuo to give the title compound (contains Fmoc-O-hydroxysuccinimide) as a white foam (5.12 g) which is used in Step 2.

Reference: W. M. Kazmierski, Int. J. Pep. Prot. Res., 45, 242 (1995).

Step 2a. To a solution of DIPEA (1.47 mL) in DMF (30 mL), add the crude product of Preparation 3, Step 1 (1.5 g). Add 2-chlorotrityl resin, chloride form (2.0 g, 0.65 mmol/g). Agitate the resulting mixture for 30 minutes. Add MeOH (0.86 mL), and agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (30 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give N³-Fmoc-N²-Cbz-L-2,3-diaminopropionic acid on 2-chlorotrityl resin.

Step 2b. Wash the resin (Preparation 3, Step 2a) with DMF (20 mL x 5). Add 20% piperidine in DMF (30 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times. Determine the loading as in Preparation 2. Measure UV absorbance at 301 nM: 0.391 $$0.391\times \mathrm{concentration}/7800$$ $$0.391\times 20,000/7800=1.0026\mathrm{mmol}/2\mathrm{g}\left(0.501\mathrm{mmol}/\mathrm{g}\right)$$

Place resin (Preparation 3, Step 2b) (1.00 g, 0.50 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (0.96 g, 5.5 mmol), followed by 3-chloromethylbenzoyl chloride (0.95 g, 5 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x 3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin from Preparation 3, Step 3 (1.00 g, 0.5 mmol) and 2-(aminomethyl)benzimidazole (5 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 3) to give the title resin.

Place resin (Preparation 3, Step 2b) (1.00 g, 0.50 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (0.96 g, 5.5 mmol,) followed by 4-chloromethylbenzoyl chloride (0.95 g, 5 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x 3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin from Step 1 (1.0 g, 0.5 mmol) and 2-(aminomethyl)benzimidazole (5.00 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine N²-Cbz-D-2,3-diaminopropionic acid (1.3 g, 5.6 mmol) and NaHCO₃ (2.10 g, 25 mmol) in acetone (15 mL) and water (15 mL). Cool in an ice-bath. Add Fmoc-O-hydroxysuccinimide (2.35 g, 7.0 mmol), and stir the resulting mixture for 3 hours while the ice melts. Concentrate the mixture in vacuo, and extract the aqueous portion with EtOAc. Wash the EtOAc solution with 5% glacial acetic acid in water (3 x 60 mL), 5% NaHC03 solution (3 x 50 mL) and brine (3 x 50 mL). Concentrate the dried (MgSO₄) EtOAc solution in vacuo to give the title compound (contains Fmoc-O-hydroxysuccinimide) as a white foam which is used Step in 2.

Reference: W. M. Kazmierski, Int. J. Pep. Prot. Res., 45, 242 (1995).

Step 2a. To a solution of DIPEA (0.8 mL) in DMF (10 mL), add the crude product from Preparation 5, Step 1 (0.81 g). Add 2-chlorotrityl resin, chloride form (1.00 g) ( 0.65 mmol/g). Agitate the resulting mixture for 30 minutes. Add MeOH (0.4 mL), and agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (30 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give N³-Fmoc-N²-Cbz-D-2,3-diaminopropionic acid on 2-chlorotrityl resin.

Step 2b. Wash the resin from Preparation 5, Step 2a with DMF (20 mL x5). Add 20% piperidine in DMF (30 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times. Determine the loading as in Preparation 2. Measure UV absorbance at 301 nM: 0.154 $$0.154\times \mathrm{concentration}/7800$$ $$0.154\times 20,000/7800=0.394\mathrm{mmol}/\mathrm{g}$$

Place resin (Preparation 5, Step 2b) (1.00 g, 0.394 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (0.75 mL, 4.33 mmol) followed by 4-chloromethylbenzoyl chloride (0.75 g, 3.94 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x 3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin (Preparation 5, Step 3) (1.00 g, 0.394 mmol) and 2-(aminomethyl)benzimidazole (5.00 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Step 1a. To a solution of DIPEA (1.60 mL) in DMF (10 mL), add (N³-Fmoc- N^2-Boc-L-2,3-diaminopropionic acid) (0.72 g). Add the 2-chlorotrityl resin, chloride form (2.00 g) (0.65 mmol/g). Agitate the resulting mixture for 30 minutes. Add MeOH (0.8 mL), agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (30 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give N³-Fmoc-N²-Boc-L-2,3-diaminopropionic acid on 2-chlorotrityl resin.

Step 1b. Wash the resin (Preparation 6, Step 1) with DMF (20 mL x 5). Add 20% piperidine in DMF (30 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times. Determine the loading as in Preparation 2. Measure UV absorbance at 301 nM: 0.216 $$0.216\times \mathrm{concentration}/7800$$ $$0.216\times 20,000/7800=0.276\mathrm{mmol}/\mathrm{g}$$

Place resin (Preparation 6, Step 1b) (2.00 g, 0.55 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (1.05 g, 6.08 mmol) followed by 4-chloromethylbenzoyl chloride (1.04 g, 5.52 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin (Preparation 6, Step 2) (2.00 g, 0.55 mmol) and 2-(aminomethyl)benzimidazole (5.00 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine o-phenylenediamine (10.8 g, 100 mmol) and β-alanine (13.4 g, 150 mmol) in 6N HCl (100 mL). Heat at reflux 25 hours, allow to cool, and chill at -15°C. Filter the solid and wash with cold 6N HCl, then cold EtOH. Dissolve the solid in 80% EtOH (125 mL), treat with decolorizing charcoal, and concentrate in vacuo to 40 g. Warm while adding EtOH (80 mL). Allow to cool, filter, and wash with EtOH to obtain the product as plates.

Add the product (Preparation 7, Step 1a) (7.18 g) to a solution of potassium hydroxide (3.45 g) in methanol (120 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and concentrate the filtrate in vacuo. Extract with EtOAc (3 x 500 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (3.33 g).

Shake the resin (Preparation 4, Step 1) (0.8 g, 0.4 mmol) and 2-[2-(aminoethyl)]benzimidazole (3.25 g) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine o-phenylenediamine (10.8 g, 100 mmol) and d,1-alanine (13.4 g, 150 mmol) in 6N HCl (100 mL) Heat at reflux 75 hours, allow to cool, and chill at -15°C. Filter to remove 2.4 g solid. Decolorize the filtrate with charcoal, concentrate in vacuo to 30 g, and dilute with 95% EtOH (90 mL). Chill at -15°C, filter and wash with cold 90% EtOH to obtain the title compound as a white powder.

Add the product (Preparation 8, Step 1a) (6.99 g) to a solution of potassium hydroxide (3.36 g) in methanol (120 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and concentrate the filtrate in vacuo. Extract the residue with EtOAc (3 x 500 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (4.23 g).

Shake the resin (Preparation 4, Step 1) (1.0 g, 0.5 mmol) and 2-(1-aminoethyl)benzimidazole (4.20 g) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine o-phenylenediamine (10.8 g, 100 mmol) and sarcosine (13.4 g, 150 mmol) in 6N HCl (100 mL). Heat at reflux 90 hours, allow to cool, and concentrate in vacuo to 45 g. Add EtOH (50 mL) and chill at -15°C. Filter the solid and wash with cold 90% EtOH. Dissolve in 80% EtOH (150 mL) and decolorize with charcoal. Concentrate in vacuo to 28 g, warm with 95% EtOH (160 mL), allow to cool, and filter to provide colorless rods.

Add the product (Preparation 9, Step 1 a) (2.33 g) to a solution of potassium hydroxide (1.21 g) in methanol (50 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and concentrate the filtrate in vacuo. Extract the with EtOAc (400 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (1.28 g).

Shake the resin (Preparation 4, Step 1) (0.30 g, 0.15 mmol) and 2-(methylaminomethyl)benzimidazole (1.25 g) in DMF (20 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

To L-Asparagine (10 g), add sodium hydroxide (3.4 g) and dioxane/water (50 mL / 50 mL). Cool resulting solution in an ice bath and add benzenesulfonyl chloride (10.6 mL), sodium hydroxide (3.4 g), and water (80 mL). Stir for 3 hours. Add water (200 mL) and extract with EtOAc. Acidify the aqueous solution to pH 3 with concentrated HCl and cool to give a precipitate. After 1 hour collect the solid and dry it in vacuo at 40° C to give the title compound.

Prepare a solution of sodium hydroxide (10.5 g) in water (50 g), cool, and add bromine (2.5 mL). Add product from Step la (10 g) and sodium hydroxide (2.9 g) in water (35 mL) and stir for 30 minutes. Heat at 90° C for 30 minutes and cool in an ice bath. Adjust to pH 7 with concentrated HCl. Collect the title compound as a white solid, mp 203-206° C.

Combine N²-benzenesulfonyl-L-2,3-diaminopropionic acid (2.92 g, 12.0 mmol) and NaHCO3 (4.57 g) in acetone (40 mL) and water (40 mL). Cool in a ice-bath. Add Fmoc-O-hydroxysuccinimide (4.97 g, 19.2 mmol), and stir the resulting mixture for 3 hours while the ice melts. Add additional NaHCO3 (1.5 g), acetone (40 mL) and water (40 mL), and dioxane (80 mL) and stir for 20 hours. Concentrate the mixture in vacuo, and extract the aqueous portion with EtOAc. Wash the EtOAc solution with 5% glacial acetic acid in water (3 x 300 mL), 5% NaHCO3 solution (3 x 300 mL) and brine (3 x 300 mL). Concentrate the dried (MgSO₄) EtOAc solution in vacuo to give the title compound (contains Fmoc-O-hydroxysuccinimide) as a light yellow solid which is used in Step 2.

Reference: W. M. Kazmierski, Int. J. Pep. Prot. Res., 45, 242 (1995).

Step 2a. To a solution of DIPEA (1.60 mL) in DMF (10 mL), add (N³-Fmoc- N^2-benzenesulfonyl-L-2,3-diaminopropionic acid) (0.787 g). Add the 2-chlorotrityl resin, chloride form (2.00 g) (0.65 mmol/g). Agitate the resulting mixture for 30 minutes. Add MeOH (0.8 mL), agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (30 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give N³-Fmoc-N²-benzenesulfonyl-L-2,3-diaminopropionic acid on 2-chlorotrityl resin.

Step 2b. Wash the resin (Preparation 10, Step 2a) with DMF (20 mL x 5). Add 20% piperidine in DMF (30 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times. Determine the loading as in Preparation 1. Measure UV absorbance at 301 nM: 0.389 $$0.389\times \mathrm{concentration}/7800$$ $$0.389\times 20,000/7800=0.9958/2\mathrm{g}\left(0.498\mathrm{mmol}/\mathrm{g}\right)$$

Place resin (Preparation 10, Step 2b) (1.00 g, 0.498 mmol) in CH₂Cl₂ (5 mL) in a vial, and treat with DIPEA (0.95 mL, 5.48 mmol) followed by 4-chloromethylbenzoyl chloride (0.94 g, 4.98 mmol). Seal vial and place on a shaker for 2.5 hours. Transfer resin to funnel apparatus. Wash the resin with CH₂Cl₂ (20 mL x 3), DMF (20 mL x3) and then CH₂Cl₂ (20 mL x 3) to yield title resin.

Shake the resin (Preparation 10, Step 3) (1.00 g, 0.55 mmol) and 2-(aminomethyl)benzimidazole (5.00 g) (Preparation 1) in DMF (25 mL) in a sealed vial for 44 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine N²-n-butoxycarbonyl-L-2,3-diaminopropionic acid (18.0 g, 88.2 mmol) and NaHC03 (38.2 g) in acetone (400 mL) and water (400 mL). Cool in a ice-bath. Add Fmoc-O-hydroxysuccinimide (42.7 g, 126.6 mmol), and stir the resulting mixture for 3 hours while the ice melts. Continue stirring overnight for 20 hours. Concentrate the mixture in vacuo, and extract the aqueous portion with EtOAc. Wash the EtOAc solution with 5% glacial acetic acid in water (3 x 100 mL), 5% NaHC03 solution (8 x 100 mL) and brine (3 x 100 mL). Concentrate the dried (MgSO₄) EtOAc solution in vacuo. Chase with heptane, dry in vacuum oven overnight, and then transfer to a large dish and dry in a stream of air (to remove AcOH) to give the title compound (contains Fmoc-O-hydroxysuccinimide) as a light yellow solid which is used Step 2.

Reference: W. M. Kazmierski, Int. J. Pep. Prot. Res., 45, 242 (1995).

Step 2a. Add Dissolve (N³-Fmoc- N²-n-butoxycarbonyl-L-2,3-diaminopropionic acid) (16.7 g) in DMF (100 mL), warm, add DMF (50 mL), and filter. Add DIPEA (14 mL), and then add the 2-chlorotrityl resin, chloride form (15.00 g) (0.65 mmol/g). Agitate the resulting mixture for 45 minutes. Add MeOH, agitate the mixture for 10 minutes, and drain. Wash the resin with DMF (100 mL x 5) and then CH₂Cl₂ (100 mL x 5) to give N^3-Fmoc-N²-n-butoxycarbonyl-L-2,3-diaminopropionic acid on 2-chlorotrityl resin.

Step 2b. Wash the resin (Preparation 11, Step 2a) with DMF (100 mL x 5). Add 20% piperidine in DMF (100 mL), agitate for 15 minutes, and collect the filtrate. Repeat two times and then DMF (2 x 100 mL). Determine the loading as in Preparation 1 (dilute the filtrate to 1000 mL (solution A); take 1 mL and dilute to 100 mL) Measure UV absorbance at 301 nM: 0.794 $$0.794\times \mathrm{concentration}/7800$$ $$0.794\times 10,000/7800=10.18\mathrm{mmol}/15\mathrm{g}\left(0.67\mathrm{mmol}/\mathrm{g}\right)$$

Place resin (Preparation 11, Step 2b) (15.00 g, 10 mmol) in CH₂Cl₂ (50 mL) in a vial, and treat with DIPEA (12.3 mL, 70 mmol) followed by 4-chloromethylbenzoyl chloride (11.5 g, 60 mmol). Gently sparge for 4 hours. Wash the resin with CH₂Cl₂ (100 mL x 3), NMP (100 mL x3) and then CH₂Cl₂ (100 mL x 5) to yield title resin.

Shake the resin (Preparation 11, Step 3) (15.00 g, 10 mmol) and 2-(aminomethyl)benzimidazole (80.85 g) (Preparation 1) in NMP (500 mL) in a sealed vial for 24 hours. Transfer resin to funnel apparatus, and wash the resin with NMP (100 mL x 3) and then CH₂Cl₂ (100 mL x 5) to give the title resin.

Combine N-methyl-o-phenylenediamine (12.2 g, 100 mmol) and glycine (11.3 g, 150 mmol) in 6N HCl (100 mL). Heat at reflux 90 hours, allow to cool, and concentrate in vacuo to 60 g. Add EtOH (50 mL) and chill at -15°C. Filter the solid and wash with cold 90% EtOH. Dissolve the blue solid in water (30 mL), add EtOH (100 mL) and treat with decolorizing charcoal. Wash the solid with 2:1 EtOH-water and concentrate filtrates in vacuo to 33 g. Add water (15 mL) and warm while adding EtOH (150 mL). Allow to cool, filter, and wash with 90% EtOH to obtain the product as blue flakes. Process the filtrate to obtain a second crop.

Add the product (Preparation 12, Step 1a) (10.3 g) to a solution of potassium hydroxide (5.20 g) in methanol (200 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and concentrate the filtrate in vacuo. Extract the with EtOAc (400 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (5.60 g).

Shake the resin (Preparation 4, Step 1) (1.50 g, 0.60 mmol) and 1-methyl-2-(aminomethyl)benzimidazole (5.00 g) in DMF (25 mL) in a sealed vial for 18 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Combine 4-chloro-o-phenylenediamine (14.3g, 100mmmol) and glycine (11.3g, 150 mmol) in 6N HCl (100 mL). Heat at reflux 72 hours, allow to cool, add EtOH (30 mL), and chill at -15° C. Filter and wash with 3:10 EtOH-water, then water. Combine the filtrates, concentrate in vacuo to 50 g, and dilute with EtOH (75 mL). Chill at -15° C, filter and wash with cold 90% EtOH. Dry to obtain solid (18.8 g). Take up in 2:1 EtOH-water (120 mL), treat with decolorizing charcoal, concentrate in vacuo to 29 g, add water (6 mL), and heat while adding EtOH (120 mL). Boil to 100 mL, add EtOH (50 mL), boil to 125 mL, and allow to cool. Collect the solid and wash with 95% EtOH. Dry to obtain the title compound as a light orange powder.

Add the product (Preparation 12, Step 1a) (10.3 g) to a solution of potassium hydroxide (5.20 g) in methanol (200 mL). Stir the resulting mixture at room temperature for 30 minutes, filter, and conccntrate the filtrate in vacuo. Extract the with EtOAc (400 mL) and filter. Concentrate the filtrate in vacuo to give the title compound as a white solid (7.62 g).

Shake the resin (Preparation 4, Step 1) (1.50 g, 0.60 mmol) and 1-methyl-2-(aminomethyl)benzimidazole (5.00 g) in DMF (25 mL) in a sealed vial for 18 hours. Transfer resin to funnel apparatus, and wash the resin with DMF (25 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Place the resin (0.16 g, -0.07 mmol) in CH₂Cl₂ (4 mL) in a vial, and treat with DIPEA (0.77 mmol), followed by acetic anhydride (0.70 mmol). Seal the vial and place it on a shaker for 2 hours at room temperature. Place the resin in a funnel apparatus, and wash the resin with CH₂Cl₂ (15 mL x 3), DMF (15 mL x 5), and then CH₂Cl₂ (20 mL x 5) to give a diacylated product. Wash the resin with DMF (15 ml x 5), and then treat the resin with 20% piperidine in DMF (30 mL) with agitation for 1.5 hours. Wash the resin with DMF (15 mL x 5) and then CH₂Cl₂ (20 mL x 5) to yield monoacetylated resin.

Using the same method, prepare the following compound

Place the resin (0.16 g, -0.07 mmol) in CH₂Cl₂ (4 mL) in a vial, and treat with DIPEA (0.77 mmol), followed by acid chloride or chloroformate (0.70 mmol). Seal the vial and place it on a shaker for 2 hours at room temperature. Place the resin in a funnel apparatus, and wash the resin with CH₂Cl₂ (15 mL x 3), DMF (15 mL x 5) and then CH₂Cl₂ (20 mL x 5) to give a diacylated product. Wash the resin with DMF (15 ml x 5), and then treat the resin with 20% piperidine in DMF (30 mL) with agitation for 1.5 hours. Wash the resin with DMF (15 mL x 5) and then CH₂Cl₂ (20 mL x 5) to yield monoacylated resin.

Acid chlorides and chloroformates used:

Place the resin (0.16 g, ~0.07 mmol) in CH₂Cl₂ (4 mL) in a vial, and treat with DIPEA (0.70 mmol) followed by carboxylic acid (0.35 mmol) and PyBroP (0.35 mmol). Seal the vial and place it on a shaker for 1.5 hours at room temperature. Place the resin in a funnel apparatus and wash the resin with CH₂Cl₂ (15 mL x 3), DMF (15 mL x 5), and then CH₂Cl₂ (20 mL x 5) to give a diacylated product. Wash the resin with DMF (15 ml x 5), and then treat the resin with 20% piperidine in DMF (30 mL) with agitation for 1.5 hours. Wash the resin with DMF (15 mL x 5) and then CH₂Cl₂ (20 mL x 5) to yield monoacylated resin.

Acids used:

Place the resin (0.16 g, ~0.107 mmol) in DMF (5 mL) in a vial and add isocyanate (0.22 mmol). Seal the vial and place it on a shaker for 2-2.5 hours at room temperature. Place the resin in a funnel apparatus and wash the resin with CH₂Cl₂ (15 mL x 3), DMF (15 mL x 5), and then CH₂Cl₂ (20 mL x 5) to give the title resin.

Isocyanates used:

Isothiocyanate used:

Treat the resins from Preparations 2-13 or Examples 1-4 (~0.16 g) with CH₂Cl₂:TFA:H₂O (99:0.95:0.05) (10 mL) at room temperature for 15 minutes and filter. Repeat this one time. Combine the filtrates, and concentrate on a Speed Vac. Add heptane (1 mL) and concentrate in Speed Vac. Dry the products in a vacuum oven at 40° C for 20 hours to yield the following products listed in Tables 1 to 8, below (HPLC condition: Vydac column (218TP5405): 5-95% MeCN-H₂O (0.1% TFA) gradient over 10 minutes, at 1 mL/min. UV detection 254 nM): Example Benzoyl Substituent R³ MS m/e [M+H]⁺ HPLC Retention Time, min 5-1 para 502 3.91 5-2 para 640 3.95 5-3 para 544 4.13 5-4 para 650 5.45 5-5 para 574 4.15 5-6 meta 502 4.02 5-7 meta 640 6.10 5-8 meta 544 4.24 5-9 meta 650 5.58 5-10 meta 574 4.29 Example R³ MS m/e [M+H]⁺ HPLC Retention Time, min 5-11 572 5.84 5-12 598 5.99 5-13 572 5.77 5-14 626 6.85 5-15 612 6.43 5-16 634 6.52 5-17 630 6.36 5-18 678 6.33 5-19 636 5.77 5-20 574 6.43 5-21 652 6.57 5-22 646 6.58 5-23 589 5.68 5-24 587 4.92 5-25 588 5.49 5-26 588 5.50 5-27 621 6.22 5-28 627 6.40 5-29 635 6.19 5-30 585 5.72 5-31 665 6.95 5-32 665 6.46 5-33 516 5.33 5-34 601 6.06 5-35 655 6.72 5-36 699 6.94 5-37 671 6.54 5-38 697 6.83 5-39 587 5.65 5-40 679 7.10 5-41 573 5.43 5-42* 615 5.38 5-43 573 5.37 5-44 719 6.49 5-45* 631 5.57 5-46* 667 6.38 5-47* 693 6.53 5-48* 705 6.45 5-49* 639 6.31 5-50* 645 5.91 5-51* 673 6.46 5-52* 721 6.84 5-53* 687 6.77 5-54* 645 5.78 5-55* 643 6.93 5-56* 619 5.97 *Purified by Preparative TLC Example R³ MS m/e [M+H]⁺ HPLC Retention Time, min 5-57 H 502 5.22 5-58 574 5.30 5-59 634 6.50 Example R³ MS mle [M+H]⁺ HPLC Retention Time, min 5-60 H 468 4.13 5-61 510 4.84 5-62 540 4.85 5-63 600 6.29 5-64 592 6.64 Example R³ MS m/e [M + H]⁺ HPLC Retention Time, min 5-65 H 429 4.19 5-66 471 4.93 5-67 501 4.91 5-68 561 6.32 5-69 553 6.69 Example R³ MS m/e [M + H]⁺ HPLC Retention Time, min 5-70 H 516 4.94 5-71 558 5.25 5-72 588 5.30 5-73 648 6.50 5-74 640 6.78 Example R³ MS m/e [M + H]⁺ HPLC Retention Time, min 5-75 H 516 5.36 5-76 558 5.23 5-77 588 5.24 5-78 648 6.52 5-79 640 6.84 Example R³ MS m/e [M + H]⁺ HPLC Retention Time, min 5-80* 579 5.3 5-81* 632 6.23 *Purified by Preparative TLC Example R³ MS mle [M+H]⁺ HPLC Retention Time, min 5-82 * 593 6.17 5-83 * 587 6.01 5-84* 553 5.45 5-85* 567 5.91 5-86* 597 5.54 5-87* 671 6.25 5-88* 605 6.16 5-89* 611 5.74 5-90* 639 6.30 5-91* 659 6.46 *Purified by Preparative TLC Example R³ MS m/e [M + H]⁺ HPLC Retention Time, min 5-92* 641 6.32 5-93* 601 5.67 * Purified by Preparative TLC Example R³ MS m/e [M+H]⁺ HPLC Retention Time, min 5-94* 661 6.69 5-95* 621 5.98 * Purified by Preparative TLC

Dissolve N³-[4-[(benzimidazol-2-yl)methyl][[( ethoxycarbonylmethyl)amino]-carbonyl]aminomethylbenzoyl]-N²-Cbz-1-2,3-diaminopropionic acid (5-45) (2.60 g, 4.1 mmol) in MeOH (12 mL) and slowly add 1N NaOH (40 mL). After 3 hours, slowly add 1N HCl (40 mL) and then add 1N HCl to pH 6.5 to give a white precipitate. Decant water and wash with water (2x10 mL). Dry title compound (Table 8-4) in vacuum oven.

Dissolve N³-[4-[(benzimidazol-2-yl)methyl][[(ethoxycarbonylmethyl)amino]-carbonyl]aminomethylbenzoyl]-N²-(n-butoxycarbonyl)-L-2,3-diaminopropionic acid (5-86) (0.432 g, 0.72 mmol) in MeOH (5 mL) and slowly add 1N NaOH (4 mL). After 3 hours, evaporate the MeOH under a steam of nitrogen. Slowly add 1N HCl (4 mL) and then add 1N HCl to pH 6.5 to give a white gum. Decant water dry title compound (Table 8-4) in vacuum oven. Example Q MS m/e [M + H]⁺ HPLC Retention Time, min 5-96 Cbz 603 5.22 5-97 n-BO₂C 569 4.98

Add 3M HCl in MeOH (5 mL) to N³-[4-[(benzimidazol-2-yl)methyl][[(cyclohexyl)amino]carbonyl] amino-methylbenzoyl]-N²-(n-butoxycarbonyl)-L-2,3-diaminopropionic acid (0.155 g) in MeOH (20 mL) and heat under reflux for 7 hours. The reaction mixture was concentrated in vacuo. MeOH was added and concentrated in vacuo to give the title compound as a white solid. MS m/e [M + H] 641: HPLC retention time: 6.77 min.

The following assay procedure, which is a competition radioligand binding assay, was carried out to determine the activity of the foregoing compounds as α_vβ₃ antagonists. The competitive assay procedure described in Kumar, et. al., "Biochemical Characterization Of The Binding Of Echistatin To Integrin α_vβ₃ Receptor", Journal Of Pharmacology And Experimental Therapeutics, Vol. 283, No. 2, pp. 843-853 (1997) was employed. Thus, binding of ¹²⁵I-echistatin (radiolabeled by the chloramine-T method to a specific activity of 2000 Ci/mmol (Amersham, Chicago, Il.)) to α_vβ₃ receptor (purified from human placenta), both prepared as described in Kumar, et al., was competed by the compounds prepared in the foregoing examples. Purified α_vβ₃ receptor was coated onto Microlite-2 plates at a concentration of 50 ng/well. ¹²⁵I-echistatin was added to the wells to a final concentration of 0.05 nM in binding buffer (50 µl/well) in the presence of the competing test compound. The competing test compounds were employed at serially diluted concentrations ranging from 1 pM to 100 nM. After a 3 hour incubation at room temperature, the wells were washed, and radioactivity, reflecting binding by ¹²⁵ I-echistatin to α_vβ₃ receptors, was determined with Top Count (Packard). Each data point is an average of values from triplicate wells.

Specific binding of ¹²⁵I-echistatin was calculated as the difference between the amount of ¹²⁵I-echistatin bound in the absence (total binding) and the amount of ¹²⁵I-echistatin bound in the presence of a 200-fold molar excess of unlabeled echistatin (nonspecific binding). The efficacy of the test compounds for inhibiting specific binding of ¹²⁵I-echistatin to α_vβ₃ receptors was determined by plotting a graph of specific binding (y-axis) as a function of test compound concentration (x-axis). The concentration of test compound required to inhibit 50% of the specific binding (IC₅₀) was determined from the plot. The IC₅₀ may be directly converted mathematically to Ki, which is a measure of the receptor binding affinity of the compounds under the defined assay conditions.

To measure the relative affinity of the test compounds for α_vβ₃ receptors versus affinity for α_llbβ₃ receptors, similar competitive assays were carried out using purified α_llbβ3 receptor and ¹²⁵I-echistatin (iodinated using the lactoperoxidase method). The specificity index, which is a measure of the relative binding affinity for α_vβ₃, versus α_llbβ₃, may be determined by dividing the IC₅₀ value for α_llbβ₃ by the IC₅₀ value for α_vβ₃.

The α_vβ₃ IC₅₀ values determined by the foregoing assay for the compounds identified in the preceding examples, and the specificity index (IC₅₀ α_llbβ₃/ IC₅₀ αvβ₃) are summarized in the tables below. Example IC₅₀, nM 5-1 5.4 5-2 6.5 5-3 1.9 5-4 2.9 5-5 0.42 5-6 ~500 5-7 ~ 500 5-8 ~500 5-9 ~500 5-10 ~500 Example IC₅₀, nM SPECIFICITY α_IIbβ₃ / α_vβ₃ 5-11 3.4 70 5-12 5.6 87 5-13 3.1 38 5-14 5.8 117 5-15 4.3 95 5-16 4.4 899 5-17 4.6 76 5-18 4.4 82 5-19 8.9 34 5-20 7.2 15 5-21 4.4 41 5-22 6.4 26 5-23 5.5 22 5-24 5.3 14 5-25 4.5 52 5-26 3.5 91 5-27 1.56 481 5-28 0.65 1136 5-29 4.3 144 5-30 2.3 296 5-31 6.0 80 5-32 6.5 151 5-33 17 128 5-34 2.3 421 5-35 8.1 240 5-36 11.7 129 5-37 5.3 203 5-38 13.6 49 5-139 3.1 223 5-40 5.7 288 5-41 2.8 19 5-42 1.8 39 5-43 2.2 585 5-44 19.6 66.3 5-45 2.2 1333 5-46 2.7 1065 5-47 2.2 1252 5-48 1.0 2028 5-49 4.2 504 5-50 3.0 805 5-51 1.1 1210 5-52 4.0 448 5-53 3.8 483 5-54 2.0 413 5-55 2.5 620 5-56 1.6 514 Example IC₅₀, nM Specificity α_llbβ₃ / α_vβ₃ 5-57 1293 1.43 5-58 118 46 5-59 129 65 Example IC₅₀, nM Specificity α_llbβ₃/ α_vβ₃ 5-60 25 90 5-61 17 324 5-62 30 419 5-63 20 293 5-64 16 499 Example IC₅₀, nM Specificity α_11bβ₃ / α_vβ₃ 5-65 9,227 0.4 5-66 9,468 0.4 5-67 7,373 0.5 5-68 6,630 0.4 5-69 10,514 0.4 Example IC₅₀ nM Specificity α_IIβ₃ / α_vβ₃ 5-70 413 0.2 5-71 266 0.3 5-72 559 0.5 5-73 170 0.7 5-74 238 0.6 Example IC₅₀, nM Specificity α_IIbβ₃ / α_vβ₃ 5-75 306 10.2 5-76 50 6.7 5-77 49 12.4 5-78 21 33.9 5-79 33 19.0 Example IC₅₀, nM Specificity α_llbβ₃ / α_vβ₃ 5-80 4.4 11 5-81 8.2 8.0 5-82 2.2 1259 5-83 1.8 1689 5-84 3.6 712 5-85 1.8 1223 5-86 3.3 793 5-87 2.0 1569 5-88 2.0 1657 5-89 3.3 1094 5-90 2.6 1697 5-91 2.8 1183 5-92 7.6 174 5-93 6.7 124 5-94 3.8 399 5-95 1.7 775 5-96 3.4 528 5-97 4.3 2600

The following are examples of pharmaceutical dosage forms which contain a compound (i.e., "active compound") of the invention. The scope of the invention in its pharmaceutical composition aspect is not to be limited by the examples provided.

No. Ingredients mg/tablet mg/tablet 1. Active compound 100 5 2. Lactose USP 122 40 3. Corn Starch, Food Grade, 30 25 as a 10% paste in Purified Water 4. Corn Starch, Food Grade 45 25 5. Magnesium Stearate 3 5 Total 300 100

Mix Item Nos. 1 and 2 in a suitable mixer for 10-15 minutes. Granulate the mixture with Item No. 3. Mill the damp granules through a coarse screen (e.g., 1/4", 0.63 cm) if necessary. Dry the damp granules. Screen the dried granules if necessary and mix with Item No. 4 and mix for 10-15 minutes. Add Item No. 5 and mix for 1-3 minutes. Compress the mixture to appropriate size and weigh on a suitable tablet machine.

No. Ingredient mg/capsule mg/capsule 1. Active compound 100 5 2. Lactose USP 106 45 3. Corn Starch, Food Grade 40 45 4. Magnesium Stearate NF 7 5 Total 253 100

Mix Item Nos. 1, 2 and 3 in a suitable blender for 10-15 minutes. Add Item No. 4 and mix for 1-3 minutes. Fill the mixture into suitable two-piece hard gelatin capsules on a suitable encapsulating machine.

While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.