JP2002500162A - Vitronectin receptor antagonist - Google Patents

Abstract

(57) [Summary] Formula (I) which is a vitronectin receptor antagonist and is useful for treating osteoporosis: Or a pharmaceutically acceptable salt thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to vitronectin receptor inhibiting, inflammation, cancer and cardiovascular disorders such as atherosclerosis and restenosis, and diseases caused by bone resorption,
For example, it relates to pharmaceutically active compounds useful for treating osteoporosis.

BACKGROUND OF THE INVENTION Integrins are a superfamily of cell adhesion receptors that are transmembrane glycoproteins expressed on various cells. These cell surface adhesion receptors are gpI
Ib / IIIa (fibrinogen receptor) and α _v β ₃ (vitronectin receptor). The fibrinogen receptor gpIIb / IIIa is expressed on the platelet surface and mediates platelet aggregation and formation of hemostatic clots at the bleeding wound site. Philips et al., Blood., 1988, 71, 831. Vitronectin receptor α _V β ₃
It is expressed on many cells, including endothelium, smooth muscle, osteoclasts and tumor cells, and thus has various functions. The α _v β ₃ receptor expressed on the membrane of osteoclasts mediates the adhesion of osteoclasts to the bone matrix, a key step in the process of bone resorption. Ross et al.
, J. Biol. Chem., 1987, 262, 7703. A disease characterized by excessive bone resorption is osteoporosis. Α _V β ₃ Receptors Expressed on Human Aortic Smooth Muscle Cells Mediate Their Translocation to Neointima, a Process That Can Cause Restenosis After Percutaneous Coronary Angioplasty I do. Brown et al., Cardiovascular Res., 1994, 28,
1815. In addition, Brooks et al., In Cell, 1994, 79, 1157, showed that α _v β ₃ antagonists can promote tumor regression by causing angiogenesis in apoptosis of blood vessels. Thus, agents that block the vitronectin receptor would be useful in treating diseases such as osteoporosis, restenosis and cancer.

[0003] The vitronectin receptor is currently known to refer to three types of integrins, designated α _v β ₁ , α _v β ₃ and α _v β ₅ . Horton et al., Int. J. Exp. Pathol.,
1990, 71, 741. α _V β ₁ binds to fibronectin and vitronectin. α _V β ₃ is used for fibrin, fibrinogen, laminin, thrombospondin,
It binds to a variety of ligands including vitronectin, von Willebrand factor, osteopontin and bone sialoprotein I. α _V β ₅ binds vitronectin. The vitronectin receptor α _V β ₅ has been shown to be involved in cell adhesion of various cell types including microvascular endothelial cells (Davis et al., J. Cell. Biol.
., 1993, 51, 206), confirming its role in angiogenesis. Brooks et al., Scie
nce, 1994, 264, 569. This integrin is expressed on blood vessels in human wound granulation tissue, but not in normal skin.

[0004] The vitronectin receptor is a tripeptide Arg-Gly-Asp (or RG).
D) It is known to bind to bone matrix proteins containing motifs. Thus, Horton et al., Exp. Cell Res. 1991, 195, 368 disclose that RGD-containing peptides and anti-vitronectin receptor antibody (23C6) inhibit dentin resorption and cell spreading by osteoclasts. I have. Further, Sato et al., J. Cell
Biol. 1990, 111, 1713 states that exstatin, a snake venom peptide containing the RGD sequence, is a potent inhibitor of bone resorption in tissue culture and inhibits the adhesion of osteoclasts to bone. Is disclosed.

It has now been found that certain compounds are potent inhibitors of the α _v β ₃ and α _v β ₅ receptors. In particular, they have found that such compounds are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor.

SUMMARY OF THE INVENTION [0006] The present invention has the pharmacological activity of vitronectin receptor inhibition and is attributed to inflammation, cancer and cardiovascular disorders such as atherosclerosis and restenosis, and bone resorption. Includes compounds of formula (I) that are useful for treating certain diseases, for example, osteoporosis.

[0007] The present invention is also a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutical carrier. The present invention is also a method of treating a disease mediated by a vitronectin receptor. In certain embodiments, the compounds of the invention are useful for treating atherosclerosis, restenosis, inflammation, cancer and diseases caused by bone resorption, such as osteoporosis.

DETAILED DESCRIPTION The present invention includes novel compounds that are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor. The novel compounds include a benzazepine nucleus in which a nitrogen-containing substituent is present on the aromatic six-membered ring of benzazepine and an aliphatic substituent containing an acidic group is present on the seven-membered ring of benzazepine. The benzazepine ring system interacts favorably with the vitronectin receptor, and the substituent side chains on the 6- and 7-membered ring also orient the substituent side chains such that they interact favorably with the receptor. Conceivable. Approximately 12 to 14 intervening covalent bonds via the shortest intramolecular route form an acidic group on the aliphatic substituent of the seven-membered ring of benzazepine and a nitrogen-containing substituent on the aromatic six-membered ring of benzazepine. It is preferably present between nitrogen.

The present invention provides a compound of formula (I):

Embedded image Or a pharmaceutically acceptable salt thereof. This compound is represented by (S)-
8- [3- (4-pyridin-2-ylamino) -1-propyloxy] -3-oxo-2- (2,3,4-trifluorobenzyl) -2,3,4,5-tetrahydro-1H −
2-benzoazepine-4-acetic acid.

The compounds of formula (I) inhibit the binding of vitronectin and other RGD-containing peptides to the vitronectin receptor. Inhibition of the vitronectin receptor on osteoclasts inhibits bone resorption of osteoclasts and is useful in treating diseases in which bone resorption is associated with a pathology, such as osteoporosis and osteoarthritis.

[0011] In another aspect, the present invention provides a compound of the formula (1) that causes an increase in osteocalcin release.
This is a method for stimulating bone formation, which comprises administering the compound represented by I). Increased bone production has clear benefits in conditions where there is a deficiency in calcified bone mass or where bone remodeling is desired, such as fracture healing and fracture prevention. Diseases and metabolic disorders that result in loss of bone structure will also benefit from such treatment. For example, hyperparathyroidism, Paget's disease, malignant hypercalcemia, osteolytic lesions caused by bone metastases, bone loss due to fixation or sex hormone deficiency, Behcet's disease, osteomalacia, ossification Excessive disease and osteopetrosis can benefit from administration of the compounds of the present invention.

In addition, because the compounds of the present invention inhibit the vitronectin receptor on many different types of cells, they are useful in treating inflammatory disorders such as rheumatoid arthritis and psoriasis, and cardiovascular diseases such as It may be useful in treating atherosclerosis and restenosis. The compounds of formula (I) of the present invention include, but are not limited to, thromboembolism, asthma, allergy, adult respiratory distress syndrome, graft versus host disease, organ transplant rejection, septic shock, eczema, contact It is useful for treating or preventing other diseases including dermatitis, inflammatory bowel disease and other autoimmune diseases. The compounds of the present invention are also useful for wound healing.

The compounds of the present invention are also useful for treatment, including prevention of angiogenic disorders.
As used herein, the term "angiogenic disorder" includes conditions involving abnormal neovascularization. If the growth of new blood vessels causes or contributes to a condition associated with the disease, inhibiting angiogenesis will reduce the deleterious effects of the disease. One example of such a disease target is diabetic retinopathy. Where growth of new blood vessels is required to maintain growth of harmful tissue, inhibition of angiogenesis reduces blood supply to the tissue, thereby reducing tissue volume based on blood supply requirements. Will contribute. For example, tumor growth where neovascularization is continuously required for tumor growth and establishment of solid tumor metastasis. Thus, the compounds of the present invention inhibit tumor tissue angiogenesis, thereby preventing tumor metastasis and tumor growth.

Thus, according to the method of the present invention, inhibition of angiogenesis using a compound of the present invention
It can improve the symptoms of the disease and, in some cases, treat the disease.

[0015] Another therapeutic target for the compounds of the present invention is an eye disease characterized by neovascularization. Such eye diseases include corneal neovascular disorders, such as corneal transplants, herpetic keratitis, syphilitic keratitis, pterygium and neovascular pannus associated with contact lens use. Additional eye diseases also include age-related macular degeneration, putative ocular histoplasmosis, retinopathy of prematurity, and neovascular glaucoma.

The present invention further provides tumor growth comprising administering a compound of formula (I) and an anti-tumor agent, such as topotecan and cisplatin, stepwise or physically in combination. A method for inhibiting is provided.

The present invention also includes prodrugs of the compounds of the present invention. Prodrugs are considered to be covalently bonded carriers that release the active parent drug of formula (I) in vivo. Thus, in another aspect of the invention, a compound of formula (II), which is also an intermediate in the preparation of a compound of formula (I):

Embedded image Or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention, a compound of formula (III):

Embedded image Or a pharmaceutically acceptable salt thereof.

The compounds of the present invention are described herein using abbreviations and symbols commonly used in the peptide and chemical arts. In general, amino acid abbreviations are referred to as Eur. J. Bioche.
m., 158, 9 (1984) according to the IUPAC-IUB Joint Commission on Biochemical Nomenclature for biochemical nomenclature.

As used herein, C _1-6 alkyl means an optionally substituted alkyl group of 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-alkyl. Includes butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl and hexyl and simple aliphatic isomers thereof.

Certain specific reagents are abbreviated herein. DCC indicates dicyclohexylcarbodiimide, DMAP indicates dimethylaminopyridine, DIEA indicates diisopropylethylamine, and EDC indicates 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. HOBt indicates 1-hydroxybenzotriazole, THF indicates tetrahydrofuran, and DIE
A represents diisopropylethylamine, DEAD represents diethyl azodicarboxylate, PPh ₃ represents triphenylphosphine, DIAD represents diisopropyl azodicarboxylate, DME represents dimethoxyethane, and DMF represents DMF. , Dimethylformamide, NBS, N-bromosuccinimide, Pd / C, palladium-carbon catalyst, PPA, polyphosphoric acid, DPPA, diphenylphosphoryl azide, BOP, benzotriazole -1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate, HF indicates hydrofluoric acid, TEA indicates triethylamine, TFA indicates trifluoroacetic acid, PCC indicates pyridinium chlorochromate Show

Compounds of formula (I) are described in Bondinell et al., PCT Application Publication WO 93/000, published January 7, 1993, the entire contents of which are hereby incorporated by reference.
95 and Bondinell et al., PCT Application Publication WO 94/14776.

In addition, compounds of formula (I) are prepared by the methods described in detail in the scheme below.

[0024]

Embedded image

Compound I-1 prepared by the general methods described in Bondinell et al., PCT Application Publication WO 93/00095 and Bondinell et al., PCT Application Publication WO 94/14776, published Jan. 7, 1993. To Mitsunobu-type coupling reactions (Organic Reactions 19)
92, 42, 335-656; Synthesis 1981, 1-28) to react with 2- [N- (3-hydroxy-1-propyl) -N- (tert-butoxycarbonyl) amino] pyridine-N-oxide I-2 is obtained. The reaction is mediated by the complex formed between diethyl azodicarboxylate and triphenylphosphine in an aprotic solvent such, THF, carried out in CH ₂ Cl ₂ or in DMF. The resulting product I-2 can be alkylated at the 2-position (benzazepine numbering) under standard alkylation conditions well known to those skilled in the art. For example, I-2 can be treated with sodium hydride, L in a suitable solvent, usually THF, DMF, DME or a mixture thereof.
Treatment with a base such as DA or lithium hexamethyldisilazide gives the amide NH
Can be deprotonated. The resulting anionic species to a suitable electrophile,
For example, treatment with 2,3,4-trifluorobenzyl bromide causes N-alkylation to give the product 1-3. The tert-butoxycarbamate (Boc) group can be removed from I-3 under standard acidic conditions to give I-4. Such conditions are well known to those skilled in the art and can be found in appropriate reference books, eg, Greene, “Protect
ive Groups in Organic Synthesis "(issued by Wiley-Interscience). Transfer hydrogenation using a palladium catalyst, preferably palladium metal on activated carbon, in an inert solvent such as methanol, ethanol, or 2-propanol. Under conditions, the pyridine-N-oxide moiety of I-4 is reduced to the corresponding pyridine I-5.Cyclohexane, 1,4-cyclohexadiene, formic acid, and salts of formic acid, such as potassium formate or ammonium formate, Commonly used as a hydrogen transfer reagent in reaction types, the methyl ester of I-5 can be converted to an aqueous base such as LiOH in aqueous THF or NaO in aqueous methanol or ethanol.
Hydrolysis with H to convert the intermediate carboxylate to a suitable acid such as TFA or H
Acidification with Cl gives carboxylic acid I-6. Alternatively, if desired, the intermediate carboxylate can be isolated, or the carboxylate of the free carboxylic acid can be prepared by methods well known to those skilled in the art.

The acid addition salt of a compound is prepared in a standard manner, in a suitable solvent, in a suitable solvent and an excess of an acid such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoro, and the like. Prepared from acetic, maleic, succinic or methanesulfonic acid. The cation salt is prepared by treating the parent compound with an excess of an alkaline reagent containing a suitable cation, such as a hydroxide, carbonate or alkoxide; or with a suitable organic amine. Cations such as Li ⁺ , Na ⁺ , K ⁺ , Ca ⁺⁺ , Mg ⁺⁺ and NH ₄ ⁺ are special examples of cations present in pharmaceutically acceptable salts.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. Thus, the compounds of formula (I) may be used in the manufacture of a medicament. The pharmaceutical composition of the compound of formula (I) prepared as described above may be formulated as a solution for parenteral administration or a lyophilized powder. The powder may be reconstituted before use with the addition of a suitable diluent or other pharmaceutically acceptable carrier. The liquid formulation may be a buffered isotonic aqueous solution. Examples of suitable diluents are usually isotonic saline solution, 5% dextrose in standard water or buffered sodium or ammonium acetate solution. Such formulations are particularly suitable for parenteral administration, but may be used for oral administration or may be contained in a metered dose inhaler or nebulizer for inhalation. Polyvinylpyrrolidone, gelatin, hydroxycellulose, gum arabic, polyethylene glycol, mannitol,
It may be desirable to add excipients such as sodium chloride or sodium citrate.

[0028] Alternatively, these compounds may be encapsulated, tableted, or prepared in an emulsion or syrup for oral administration. A pharmaceutically acceptable solid or liquid carrier may be added for bulking or stabilizing the composition or for facilitating the preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium or stearic acid, talc, pectin, gum arabic, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate alone or with a wax. The amount of solid carrier varies but, preferably, will be from about 20 mg to about 1 g per dosage unit. Pharmaceutical preparations are prepared by conventional techniques of pharmacy, including for tablets, grinding, mixing, granulating and, if necessary, compression; or for hard gelatin capsules, grinding, mixing and filling. If a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid preparations may be administered directly orally or may be filled into soft gelatin capsules.

For rectal administration, the compounds of the present invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycol to form suppositories.

The compounds described herein are antagonists of the vitronectin receptor and are useful for treating diseases in which the underlying condition is caused by ligands or cells that interact with the vitronectin receptor. For example, these compounds are useful for treating diseases in which loss of bone matrix produces a medical condition. Thus, the compounds of the present invention
It is useful in the treatment of osteoporosis, hyperparathyroidism, Paget's disease, hypercalcemia of malignancy, osteolytic lesions caused by bone metastases, bone loss due to fixation or sex hormone deficiency. The compounds of the present invention also have utility as anti-tumor, anti-angiogenic, anti-inflammatory and anti-metastatic agents and are believed to be useful in the treatment of atherosclerosis and restenosis.

The compound is administered to the patient either orally or parenterally in such a way that the concentration of the drug is sufficient to inhibit bone resorption or other such signs. Pharmaceutical compositions containing the compound are administered at an oral dosage of from about 0.1 to about 50 mg / kg in a manner appropriate to the condition of the patient. Preferably, the oral dose is about 0
It will be between about 0.5 and about 20 mg / kg. For the treatment of acute diseases, parenteral administration is preferred. Intravenous infusion of the peptide in water or 5% dextrose in normal saline, or a similar formulation with suitable excipients, is most effective, but intramuscular bolus injections are also useful. Typically, parenteral dosages will be from about 0.01 to about 100 mg / kg; preferably, from 0.1 to 20 mg / kg. The compounds are administered one to four times daily at a level such that the total daily dose reaches about 0.4 to about 400 mg / kg / day. Precise dosages and modes of administration for the compounds are readily determined by those skilled in the art by comparing blood levels of the drug to the concentrations required to achieve a therapeutic effect.

The present invention further relates to a method of treating osteoporosis or inhibiting bone loss, comprising a compound of formula (I) and another inhibitor of bone resorption, such as a bisphosphonate (ie alendronate) G), hormone replacement therapy, stepwise or physically combined administration of antiestrogen or calcitonin. Further, the present invention provides a therapeutic method using the compound of the present invention and an anabolic agent, for example, iproflavone, a bone morphogenetic protein useful for preventing bone loss and / or increasing bone mass.

Further, the present invention provides a method for inhibiting tumor growth, which comprises administering a compound of formula (I) and an antitumor agent in a stepwise or physical combination. I do. Camptothecin analog class compounds, such as topotecan,
Irinotecan and 9-aminocamptothecin and platinum coordination complexes such as cisplatin, ormaplatin and tetraplatin are a well-known group of antitumor agents. Compounds of the camptothecin analog class are described in U.S. Patent Nos. 5,004,758, 4,604,463, 4,473,692, 4,545,880, 4,545,880, the entire disclosure of which is hereby incorporated by reference.
4,342,776, 4,513,138, 4,399,276, European Patent Application Publication Nos. 0 418 099 and 0 088 642, Wani et al., J. Med.Chem. 1986, 29, 2358, Wani et al., J. Me.
d. Chem. 1980, 23, 554; Wani et al., J. Med. Chem. 1987, 30, 1774, and Nitt.
a et al., Proc. 14th International Congr. Chemotherapy, 1985, Anticancer Sect.
ion 1, 28. Cisplatin, a platinum coordination complex, is available from Bristol Myers-Squibb Corporation under the name Platinol®. Useful formulations for cisplatin are described in US Pat. Nos. 5,562,925 and 4,310,515, the entire disclosures of each being incorporated herein by reference.

In a method of inhibiting tumor growth comprising administering a compound of formula (I) and an anti-tumor agent in a stepwise or physical combination, a platinum coordinating compound such as cisplatin is slowly Intravenous infusion can be administered. A preferred carrier is a dextrose / saline solution containing mannitol. Dosage regimens for platinum coordination compounds are based on about 1 to about 500 mg / m ^{2 of} body surface area per unit of treatment (mg / m ² ). Infusion of platinum coordination compound
It is given once or twice a week, and this weekly treatment may be repeated several times. When a compound of the camptothecin analog class is used for parenteral administration, the commonly used therapeutic units are from about 0.1 mg to about 300.0 mg / m ² of body surface area per day for about 5 consecutive days. Most preferably, the treatment unit used for topotecan is from about about 1.0 per day 5 consecutive days about 2.0 mg / body surface area m ^2. Preferably, the treatment unit is repeated at least once at an interval of about 7 days to about 28 days.

The pharmaceutical compositions may be formulated in the same container with both the compound of formula (I) and the antitumor agent, but are preferably formulated in separate containers. If both agents are provided in solution form, they can be included in an infusion / injection system or tandem device for simultaneous administration.

To conveniently administer the compound of formula (I) and the anti-tumor agent simultaneously or separately, in a single container, such as a box, carton or other container, each as described above. An individual bottle, bag, vial or other container containing an effective amount of a compound of formula (I) for parenteral administration and an effective amount of an anti-tumor agent for parenteral administration as described above. A kit is prepared containing Such a kit comprises, for example, both agents, which may be in separate containers or as lyophilized plugs in the same container,
And a container for the reconstitution solution. A variation on this is to include a reconstitution solution and a lyophilization plug in two chambers of a single container that can be mixed before use. Using such a device, the anti-tumor agent and the compound of the present invention may be packaged separately in two containers, or may be lyophilized together as a powder and provided in a single container.

When both agents are provided in solution form, they can be included in an infusion / injection system or tandem device for simultaneous administration. For example, the compound of formula (I) may be in intravenous form or in an infusion bag connected in series via a tubing to an antitumor agent in a second infusion bag. Using such a system, patients can receive a bolus injection or infusion of a compound of formula (I) first, followed by an infusion of an antitumor agent.

The compounds may be tested in one of several biological assays to determine the concentration of the compound required to have a given pharmacological effect.

Inhibition of vitronectin binding Solid phase [ ³ H] -SK & F-107260 binding to α _v β ₃ : human placenta or human platelets α _{v in} buffer T (containing 2 mM CaCl ₂ and 1% octyl glucoside) β ₃ (0.1-0.3 mg / mL) was diluted in buffer T containing 1 mM CaCl ₂ , 1 mM MnCl ₂ , 1 mM MgCl ₂ (buffer A) and 0.05% NaN ₃ , and then immediately diluted with 96 ml. Well ELISA plates (Corning, New York, NY) were added at 0.1 mL per well.
Α _v β ₃ 0.1-0.2 μg was added per well. Plates were incubated overnight at 4 ° C. At the time of the experiment, wells were washed once with buffer A and incubated for 1 hour at room temperature with 0.1 mL of 3.5% bovine serum albumin in the same buffer. After incubation, the wells were aspirated completely and washed twice with 0.2 mL of Buffer A.

Compounds were dissolved in 100% DMSO to give a 2 mM stock solution, which was combined with binding buffer (15 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl ₂ , 1 mM to a final compound concentration of 100 μM. (MgCl ₂ ). The solution was then diluted to the desired final compound concentration. Various concentrations of unlabeled antagonist (0.001-100 μM) were added to the wells in triplicate, followed by the addition of 5.0 nM [ ³ H] -SK & F-107260 (65-86 Ci / mmol).

The plate was incubated at room temperature for 1 hour. After the incubation, the wells were completely aspirated, and washed once in a well-to-well fashion using 0.2 mL of ice-cold buffer A to transfer from well to well. Receptor is 1% SDS 0.1
solubilized mL, Beckman (Beckman) LS liquid scintillation counters, by liquid scintillation counting with the addition of Ready Safe (Ready Safe) 3 mL at 40% efficiency, Bound ^{[3 H] -SK & F-} 107260 It was measured. Non-specific binding of [ ³ H] -SK & F-107260 was determined by 2 μM SK & F
Measured in the presence of -107260, consistently less than 1% of total radioligand input. The IC ₅₀ (concentration of antagonist to inhibit the binding of [ ³ H] -SK & F-107260 by 50%) was determined by a non-linear least-squares curve-fit method modified with the LUNDON-2 program. K _i (antagonist dissociation constant) the Hohodo _{formula: K i = IC 50 / (} 1 + L / K d) ( here, L and _the K _d, respectively, ^[3 H] concentration and the dissociation constant of -S K & F-107260 ). The compounds of the present invention are available in SK & F 107260 at a concentration of about 0.003 micromolar.
Inhibits vitronectin binding to

The compounds of the invention may also be used in vitro in assays standard in the art for assessing inhibition of bone formation, such as the bone resorption pit formation assay disclosed in EP 528 587. And in vivo bone resorption, which was also described in Wronski et al., Cells and Materials 1991, Sup. 1, 69-74, using human osteoclasts instead of rat osteoclasts, and ovary. This may be performed using a resected rat model.

Vascular Smooth Muscle Cell Migration Assay Rat or human aortic smooth muscle cells were used. Cell migration was monitored in a Transwell cell culture chamber by using a polycarbonate membrane with 8 um pores (Costar). The lower surface of the filter was coated with vitronectin. The cells are suspended in DMEM supplemented with 0.2% bovine serum albumin at a concentration of 2.5-5.0 × 10 ⁶ cells / mL and various concentrations of the test compound are used for 20 minutes at 20 ° C. Pre-processed. Solvent alone was used as a control. 0.2 mL of the cell suspension was placed in the upper compartment of the chamber. The lower compartment contained 0.6 mL of DMEM supplemented with 0.2% bovine serum albumin. Incubations were performed for 24 hours at 37 ° C. in an atmosphere of 95% air / 5% CO ₂ . After incubation, non-migrating cells on the top surface of the filter were removed by gentle scraping. The filters were then fixed in methanol and stained with 10% Giemsa stain. Migration was measured either by: a) counting the number of cells that migrated to the lower surface of the filter, or b) extracting the stained cells with 10% acetic acid and then measuring the absorbance at 600 nm.

Thyroid Parathyroidectomy Rat Model Each experimental group consisted of adult male Sprague-Dawley rats (weight 250-400 g) 5-6
Consists of animals. Rats should be purchased 7 days before use (distributor, Taconic Farms).
thyroid parathyroidectomy). All rats receive a replacement dose of thyroxine every three days. After administration to rats, circulating ionized calcium levels in whole blood are measured immediately after whole blood is collected by tail vein puncture into test tubes containing heparin. Rats are included in the test group if the ionized Ca level (measured on a Ciba-Corning model 634 calcium pH analyzer) is <1.2 mM / L. Each rat is equipped with an indwelling venous and arterial catheter for delivery of the test substance and blood collection, respectively. The rats are then fed a calcium-free diet and deionized water. Baseline Ca levels were measured and each rat was given a control vehicle or human parathyroid hormone 1-34 peptide (hPTH1-34).
A dose of 1.25 μg / kg / hr in saline / 0.1% bovine serum albumin, Bachem, Ca) or a mixture of hPTH1-34 and the test substance,
It is administered by continuous intravenous infusion via an intravenous catheter using an external syringe pump. The calcemia response of each rat is measured at 2 hour intervals during the 6-8 hour infusion period.

Human Osteoclast Resorption and Adhesion Assay A bone resorption fossa resorption and adhesion assay was developed and standardized using normal human osteoclasts from osteoclastoma tissue. Assay 1 was developed to measure osteoclast bone resorption volume by laser confocal microscopy. Assay 2 was developed as a high-throughput screen that measures collagen fragments (released during absorption) by competitive ELISA.

Assay 1 (using laser confocal microscopy) Aliquots of cell suspension from human osteoclastoma were removed from liquid nitrogen storage, quickly warmed to 37 ° C, and centrifuged (1000 rpm, 4 rpm). 5 min at ℃
Wash once with MI-1640 medium. -Aspirate medium and replace with murine anti-HLA-DR antibody, then RPMI-
Dilute 1: 3 with 1640 medium. Incubate the suspension on ice for 30 minutes,
Mix frequently. Wash cells twice with cold RPMI-1640, then centrifuge (1000 r
pm, 4 ° C for 5 minutes) and then transfer the cells to a 15 ml sterile centrifuge tube. The number of mononuclear cells is counted in the modified Neubauer counting chamber.

Sufficient magnetic beads (5 per mononuclear cell) coated with goat anti-mouse IgG (Dynal, Great Neck, NY) are removed from their storage bottles and placed in 5 ml of fresh medium. (This will wash away toxic azide preservatives). The media is removed by fixing the beads on a magnet and replaced with fresh media. -Mix beads with cells and incubate suspension on ice for 30 minutes. Mix the suspension frequently. • Fix the bead-coated cells on a magnet and decant the remaining cells (osteoblast-rich fraction) into a 50 ml sterile centrifuge tube. • Add fresh media to the bead-coated cells to migrate any captured osteoclasts. This washing process is repeated 10 times. Discard the bead-coated cells.

Label viable cells with fluorescein diacetate and count viable osteoclasts in the counting chamber. Add the sample to the chamber using a large bore disposable plastic Pasteur pipette. Pellet the osteoclasts by centrifugation and adjust the density to the appropriate number in EMEM medium supplemented with 10% fetal calf serum and 1.7 g / l sodium bicarbonate (the number of osteoclasts varies by tumor ). • Decant 3 ml aliquots of cell suspension (per compound treatment) into 15 ml centrifuge tubes. The cells are pelleted by centrifugation.

Add 3 ml of the appropriate compound treatment to each centrifuge tube (50 μM in EMEM medium)
Diluted). Further, a suitable vehicle controls (anti diluted to 100 [mu] g / ml - vitronectin receptor murine monoclonal antibody [87MEM1]) positive control and isotype control (IgG was diluted to 100 [mu] g / ml _2a) are also encompassed. Incubate the sample at 37 ° C. for 30 minutes. • Seed a 0.5 ml aliquot of cells on sterile dentin slices in a 48 well plate and incubate at 37 ° C for 2 hours. Each treatment is screened in a quadruplicate test. Wash the slices with 6 changes of warm PBS (10 ml / well in a 6-well plate), then place in fresh medium containing compound treatments or control samples. Incubate the sample at 37 ° C. for 48 hours.

Tartrate-resistant acid phosphatase (TRAP) method (selective staining for cells of the osteoclast lineage) Bone slices containing adherent osteoclasts were washed with phosphate buffered saline and 2%
Fix in glutaraldehyde (in 0.2 M sodium cacodylate) for 5 minutes. • They are then washed with water, 0.5 mg / ml naphthol in TRAP buffer (dissolved in N, N-dimethylformamide and mixed with 0.25 M citrate buffer containing 10 mM sodium tartrate, pH 4.5). (AS-BI phosphate) at 37 ° C. for 4 minutes. After washing with cold water, the slices are immersed in cold acetate buffer (0.1 M, pH 6.2) containing 1 mg / ml Fast Red Garnet and incubated at 4 ° C. for 4 minutes.・ Aspirate excess buffer, wash the slices with water, and air dry. TRAP-positive osteoclasts (red brick / purple sediment) are counted by brightfield microscopy and then removed from the dentin surface by sonication. • Measure the bone resorption fossa volume using a Nikon / Lasertec ILM21W confocal microscope.

Assay 2 (using an ELISA readout) Human osteoclasts are expanded and prepared for compound screening as described in the first 9 steps of Assay 1. For clarity, these steps are repeated below. An aliquot of the cell suspension from human osteoclastoma was removed from liquid nitrogen storage, quickly warmed to 37 ° C, and centrifuged (1000 rpm, 4 ° C for 5 minutes) to RP
Wash once with MI-1640 medium. -Aspirate medium and replace with murine anti-HLA-DR antibody, then RPMI-
Dilute 1: 3 with 1640 medium. Incubate the suspension on ice for 30 minutes,
Mix frequently. Wash cells twice with cold RPMI-1640, then centrifuge (1000 r
pm, 4 ° C for 5 minutes) and then transfer the cells to a 15 ml sterile centrifuge tube. The number of mononuclear cells is counted in a modified Neubauer counting chamber.

Remove enough magnetic beads (5 per mononuclear cell) coated with goat anti-mouse IgG (Dinal, Great Neck, NY) from their storage bottles and place in 5 ml of fresh medium (this is , Wash off toxic azide preservatives). The media is removed by fixing the beads on a magnet and replaced with fresh media.
-Mix beads with cells and incubate suspension on ice for 30 minutes. Mix the suspension frequently. • Fix the bead-coated cells on a magnet and decant the remaining cells (osteoblast-rich fraction) into a 50 ml sterile centrifuge tube. • Add fresh media to the bead-coated cells to migrate any captured osteoclasts. This washing process is repeated 10 times. Discard the bead-coated cells.

Label viable cells with fluorescein diacetate and count viable osteoclasts in the counting chamber. Add the sample to the chamber using a large bore disposable plastic Pasteur pipette. Pellet the osteoclasts by centrifugation and adjust the density to the appropriate number in EMEM medium supplemented with 10% fetal calf serum and 1.7 g / l sodium bicarbonate (the number of osteoclasts varies by tumor ).

In contrast to the method described above for Assay 1, compounds are screened at four doses to determine the IC ₅₀ as outlined below. • Pre-incubate the osteoclast preparation with the test compound (4 doses) or control at 37 ° C for 30 minutes. -Then they are seeded on bovine cortical bone slices in wells of a 48-well tissue culture plate and incubated at 37 ° C for a further 2 hours. -The bone slices are washed with 6 changes of warm phosphate buffered saline (PBS) to remove non-adherent cells and then returned to the wells of the 48-well plate containing fresh compound or control. -The tissue culture plate is then incubated at 37 ° C for 48 hours. -Aspirate the supernatant from each well into a separate tube and release I during the absorption process
Screen in a competitive ELISA to detect c-telopeptide of type collagen. This corresponds to the 8 amino acid sequence present in the carboxy-terminal telopeptide of the a1-chain of type I collagen (Glu-Lys-Ala-His-Asp-Gly-GI
Commercially available ELISA (Osteometer, Denmark) containing a rabbit antibody that specifically reacts with y-Arg). Results are expressed as% inhibition of absorption compared to vehicle control.

Human Osteoclast Adhesion Assay Human osteoclasts are grown and prepared for compound screening as described above in the first nine steps of Assay 1. For clarity, these steps are repeated below. An aliquot of the cell suspension from human osteoclastoma was removed from liquid nitrogen storage, quickly warmed to 37 ° C, and centrifuged (1000 rpm, 5 minutes, 4 ° C).
Wash once with MI-1640 medium. -Aspirate medium and replace with murine anti-HLA-DR antibody, then RPMI-
Dilute 1: 3 with 1640 medium. Incubate the suspension on ice for 30 minutes,
Mix frequently. Wash cells twice with cold RPMI-1640, then centrifuge (1000 r
pm, 4 ° C for 5 minutes) and then transfer the cells to a 15 ml sterile centrifuge tube. The number of mononuclear cells is counted in a modified Neubauer counting chamber.

Remove enough magnetic beads (5 per mononuclear cell) coated with goat anti-mouse IgG (Dinal, Great Neck, NY) from their storage bottles and place in 5 ml of fresh medium (this is , Wash off toxic azide preservatives). The media is removed by fixing the beads on a magnet and replaced with fresh media. -Mix beads with cells and incubate suspension on ice for 30 minutes. Mix the suspension frequently. • Fix the bead-coated cells on a magnet and decant the remaining cells (osteoblast-rich fraction) into a 50 ml sterile centrifuge tube. • Add fresh media to the bead-coated cells to migrate any captured osteoclasts. This washing process is repeated 10 times. Discard the bead-coated cells.

Label viable cells with fluorescein diacetate and count viable osteoclasts in the counting chamber. Add the sample to the chamber using a large bore disposable plastic Pasteur pipette. Pellet the osteoclasts by centrifugation and adjust the density to the appropriate number in EMEM medium supplemented with 10% fetal calf serum and 1.7 g / l sodium bicarbonate (the number of osteoclasts varies by tumor ).

Pre-incubate osteoclasts from osteoclastoma with compound (four doses) or control at 37 ° C. for 30 minutes. -The cells are then seeded on osteopontin-coated slides (human or rat osteopontin 2.5 μg / ml) and incubated at 37 ° C for 2 hours. -Remove non-adherent cells by vigorously washing the slide with phosphate buffered saline and fix the cells remaining on the slide in acetone. O Osteoclasts are stained for tartrate-resistant acid phosphatase (TRAP), a selectable marker for cells of the phenotype (see steps 15-17) and counted by light microscopy. Results are expressed as% inhibition of adhesion compared to vehicle control.

Cell Adhesion Assay Cells and Cell Culture Human embryonic kidney cells (HEK293 cells) were obtained from the ATCC (catalog number CRL1573). Earle's salt, 10% fetal bovine serum, 1% glutamine and 1
Eagle's minimum essential medium containing 5% penicillin-streptomycin (EME
Cells were grown in M).

[0060] The 2.4 kb XbaI-XhoI fragment of 3.2 kb EcoRI-KpnI fragment and beta ₃ subunits of the construction and transfection alpha _v subunit, by blunt end ligation, containing CMV promoter and G418 selection markers pC
It was inserted into the EcoRI-EcoRV cloning site of the DN vector (Aiyar et al., 1994). For stable expression, Gene Pulser (Hensley et al.)
80 × 10 ⁶ HEK293 cells were electrotransformed with the α _v + β ₃ construct (20 μg of DNA for each subunit) and seeded in 100 mm plates (5 × 10 ⁵ cells / plate). Forty-eight hours later, 450 μg / mL geneticin (Geneticin, G418 sulfate, GIBCO-BRL, Bethesda, MD) was added to the growth medium. Cells were maintained in selective media until colonies were large enough to be assayed.

Immunocytochemical Analysis of Transfected Cells To determine whether HEK293 transfected cells express the vitronectin receptor, cells were fixed on microscope slides by centrifugation,
Fix in acetone for 2 minutes at room temperature and air dry. The specific reactivity to 23C6, a monoclonal antibody specific for the α _v β ₃ complex, was demonstrated using standard indirect immunofluorescence.

Cell Adhesion Studies Corning 96-well ELISA plates were precoated overnight at 4 ° C. with 0.1 mL of human vitronectin (0.2 μg / mL in RPMI medium). At the time of the experiment, the plates were washed once with RPMI medium and blocked with 3.5% BSA in RPMI medium for 1 hour at room temperature. Transfected 293 cells were
Cells were resuspended in RPMI medium supplemented with mM Hepes, pH 7.4 and 0.1% BSA at a density of 0.5 × 10 ⁶ cells / mL. 0.1 mL of cell suspension was added to each well and incubated at 37 ° C for 1 hour in the presence or absence of various α _v β ₃ antagonists. After incubation, a 10% formaldehyde solution (pH
7.4) 0.025 mL was added and the cells were fixed at room temperature for 10 minutes. RP plate
After washing three times with 0.2 mL of MI medium, the adherent cells were stained with 0.1 mL of 0.5% toluidine blue for 20 minutes at room temperature. Excess dye was removed by extensive washing with deionized water. Toluidine blue incorporated into the cells was eluted by the addition of 0.1 mL of 50% ethanol containing 50 mM HCl. 6 cell adhesion
Quantification was performed on a microtiter plate reader (Titertek Multiskan MC, Sterling, VA) at an optical density of 00 nm.

Solid phase α _v β ₅ binding assay: The vitronectin receptor α _v β ₅ was purified from human placenta. 50 m of receptor preparation
M Tris-HCl, pH 7.5, diluted with 100 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl ₂ , 1 mM MgCl ₂ (buffer A) and immediately added to a 96-well ELISA plate at 0.1 ml per well. α _v β ₃ 0.1-0.2 μg
Was added per well. Plates were incubated overnight at 4 ° C. At the time of the experiment, wells were washed once with buffer A and incubated for 1 hour at room temperature with 0.1 ml of 3.5% bovine serum albumin in the same buffer. After incubation, the wells were aspirated completely and washed twice with 0.2 ml of buffer A.

In the [ ³ H] -SK & F-107260 competition assay, various concentrations of unlabeled antagonist (0.001-100 μM) were added to the wells, followed by 5.0 nM.
[ ³ H] -SK & F-107260 was added. Plates were incubated for 1 hour at room temperature. After the incubation, the wells were completely aspirated and washed once using 0.2 ml of ice-cold buffer A, transferring from well to well. One receptor
% SDS solubilized and bound in 0.1 ml, bound [ ³ H] -SK & F-107260 was added to a Beckman LS6800 liquid scintillation counter by adding 3 ml of Ready Safe and liquid scintillation counting to 40% efficiency. It was measured. Non-specific binding of [ ³ H] -SK & F-107260 was measured in the presence of 2 μM SK & F-107260 and was consistently less than 1% of total radioligand loading. The IC ₅₀ (the concentration of antagonist that inhibits the binding of [ ³ H] -SK & F-107260 by ₅₀ %) was determined by a non-linear least-squares curve-fitting method modified on the LUNDON-2 program. K _i (the dissociation constants of the antagonist) Chen and equations Prusoff _{(Cheng and Prusoff): K i} = IC 50 / (1 + L / K d) ( here, L and _the K _d, respectively, ^[3 H] - SK & F-107260
Concentration and dissociation constant).

Inhibition of RGD-Mediated GPIIb-IIIa Binding Purification of GPIIb-IIIa 10 units of old washed human platelets (obtained from the Red Cross) were replaced with 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCl. , 2 mM C
Among NaCl _2, and dissolved by stirring gently for 2 hours at 4 ° C.. The lysate was centrifuged at 100,000 g for 1 hour. The resulting supernatant was washed with 20 mM Tris-HCl, p
H7.4, 100 mM NaCl, 2 mM CaCl ₂ , applied to a 5 mL Lentil Lectin Sepharose 4B column (EY Labs) pre-equilibrated with 1% octylglucoside (buffer A). After a 2 hour incubation, the column was washed with 50 mL of cold buffer A. GPIIb-IIIa retained on the lectin was eluted with buffer A containing 10% dextrose. 4 ° C for all procedures
I went in. The resulting GPIIb-IIIa was> 95% pure as shown by SDS polyacrylamide gel electrophoresis.

Incorporation of GPIIb-IIIa into liposomes A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti Polar Lipids) was dried on the walls of glass tubes under a stream of nitrogen. . Purified GPIIb-IIIa was brought to a final concentration of 0
It was diluted to 0.5 mg / mL and mixed with phospholipids at a protein: phospholipid ratio of 1: 3 (w: w). The mixture was resuspended and sonicated for 5 minutes in a sonication bath. Then, the mixture was subjected to 50 to 12,000-14,000 molecular weight separation dialysis tubing.
mM Tris -HCl, pH7.4,100mM NaCl, 100 0-fold excess liquid 2 mM CaCl ₂ (with two changes) was dialyzed overnight against. GPIIb-IIIa containing liposomes were centrifuged at 12,000 g for 15 minutes and resuspended in dialysis buffer at a final protein concentration of about 1 mg / mL. Liposomes at -70 ° C until needed
Stored at

Competitive Binding to GPIIb-IIIa Binding to fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect competitive binding method using [ ³ H] -SK & F-107260 as an RGD-type ligand. Binding assays were performed using a 0.22 μm hydrophilic durapore membrane in a 96-well filtration plate assembly (Millipore Corporation, Bedford, Mass.). Wells were pre-coated with 0.2 mL of 10 μg / mL polylysine (Sigma Chemical Co., St. Louis, Mo.) for 1 hour at room temperature to block non-specific binding. Various concentrations of unlabeled benzazepine were added to the wells in quadruplicate. [ ³ H] -SK & F-107260 was added to each well at a final concentration of 4.
5 nM, then purified platelet GPIIb-IIIa containing liposome 1
μg was added. The mixture was incubated at room temperature for 1 hour. GPIIb-II
Ia-bound [ ³ H] -SK & F-107260 was separated from unbound by filtration using a Millipore filtration manifold, then washed with ice-cold buffer (2 times, 0.2 mL each). The combined radioactivity remaining in the filter is analyzed in a Beckman liquid scintillation counter (Model LS6800) in 1.5 mL of Ready Solve (Beckman Instruments, Fullerton, CA) in 1.5 mL. Counted at 40%. Measuring non-specific binding in the presence of 2 μM unlabeled SK & F-107260,
Consistently less than 0.14% of the total activity added to the sample. All data points are the average of four measurements.

[0068] Competitive binding data was analyzed by non-linear least squares curve fitting. The method comprises:
Provides the IC _{50 of the} antagonist (the concentration of antagonist that inhibits the specific binding of [ ³ H] -SK & F-107260 by 50% at equilibrium). The IC _50, Cheng and Prusoff _{equation: K i = IC 50 / (} 1 + L / K d) ( here, L is used to competitive binding assays ^[3 H] -SK & concentration of F-107260 (4.5nM) Where K _d is the dissociation constant of [ ³ H] -SK & F-107260, which is 4.5 nM as determined by Scatchard analysis, based on the equilibrium dissociation constant of the antagonist ( K _i ).

The compounds of the present invention have an affinity for the vitronectin receptor relative to the fibrinogen receptor of greater than 10: 1. This compound has 100
It has an activity ratio greater than 1: 1.

The potency of the compounds of formula (I), alone or in combination with an anti-tumor agent, may be determined using several implantable mouse tumor models. See U.S. Patent Nos. 5,004,758 and 5,633,016 for details of these models.

The following examples are not intended to limit the scope of the invention in any way, but are provided to illustrate methods of making and using the compounds of the invention. Many other embodiments will be readily apparent to those skilled in the art.

EXAMPLES General Description ¹ H nuclear magnetic resonance (NMR) spectra were recorded at either 250 or 400 MHz. Chemical shifts are given in parts per million (ppm) downfield (δ) below the internal standard tetramethylsilane (TMS). The abbreviations for the NMR data are as follows: s = singlet, d = doublet, t = triplet, q = quadruple, m = multiplet, dd
= Double double line, dt = Double triple line, app = apparent, br = broad. J is
Shown are NMR binding constants measured in Hertz. CDCl ₃ is deuteriochloroform, DMSO-d ₆ is hexadeuteriodimethylsulfoxide, and CD ₃ OD is tetradeuteriomethanol. The infrared (IR) spectrum is recorded by a transmission method, and the band position is indicated by the reciprocal of the wavelength (cm ^-1 ). Mass spectra were obtained using electrospray (ES) ionization techniques. Elemental analysis was performed by Quantitative Technologies Inc., White House, NJ. Melting points are obtained on a Thomas-Hoover melting point apparatus and are uncorrected. All temperatures are given in degrees Celsius. Analtech Silica gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Flash and gravity chromatography are both used for E. Merck Kieselgel 60 (230
-400 mesh) on silica gel. Analytical and preparative HPLC were performed on a Rainin or Beckman chromatograph. ODS indicates an octadecylsilyl-derivatized silica gel chromatography support. 5μApex
-ODS is available from Jones Chromatography in Littleton, Colorado.
1 shows an octadecylsilyl-derivatized silica gel chromatography support with a nominal particle size of 5μ manufactured by Chromatography. YMC ODS-AQ (registered trademark) is an ODS chromatography support and is a registered trademark of YMC Co. Ltd., Kyoto, Japan. PRP-1
® is a polymer (styrene-divinylbenzene) chromatography support, available from Hamilton Co. of Reno, Nevada.
Is a registered trademark of Microsoft Corporation. Celite® is a filter aid composed of acid-washed diatomaceous earth silica and is a registered trademark of Manville Corp. of Denver, Colorado.

Example 1 (S) -3-oxo-8- [3- (pyridin-2-ylamino) -1-propyloxy] -2- [2,3,4- (trifluorobenzyl) -2, Preparation of 3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid

Preparation Example 1 Preparation of (±) -8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-methyl acetate a) 4-bromo-3-bromo Methylanisole 2-bromo-5-methoxytoluene (20 g, 0.10 mol), N-bromosuccinimide (19.6 g, 0.11 mol), benzoyl peroxide (1 g, 4 g)
Mmol), and a mixture of methylene chloride (200 mL) with a flood lamp.
Irradiated for 8 hours and gently refluxed. The mixture was then cooled to -10 C for several hours and the solution was decanted to remove the precipitated succinimide. The solution was concentrated and the residue was crystallized from chloroform / hexane to give the title compound as pale yellow prisms (19.7 g, 70%): ¹ H NMR (CDCl ₃ ) δ 7.45 (d, J). = 8.9Hz, 1H), 6.99 (d, J = 3Hz, 1H), 6.74 (dd, J = 8.9, 3Hz, 1H), 4.55 (s, 2H), 3. 80 (s, 3H).

B) 3-bis (tert-butoxycarbonyl) aminomethyl-4-bromoanisole 4-bromo-3-bromomethylanisole (24 g, 86 mmol) and potassium di-tert-butyliminodicarboxylate (24 g, 94 (Mmol) in dimethylformamide (200 mL) was stirred at room temperature under argon for 18 hours. The reaction was then concentrated in vacuo and the residue was partitioned between ethyl acetate and water. The organic phase was washed with water and brine, dried (MgSO _4), and concentrated. The residue was recrystallized from hexane to give the title compound as a white solid (15 g, 42%
): ¹ H NMR (CDCl ₃ ) δ 7.40 (d, J = 8.6 Hz, 1H), 6.68 (m
, 2H), 4.81 (s, 2H), 3.74 (s, 3H), 1.44 (s, 18H).

C) (±) -3-carbomethoxy-4- [2-bis (tert-butoxycarbonyl)
) Aminomethyl-4-methoxyphenyl] -3-butenoate 3-bis (tert-butoxycarbonyl) aminomethyl-
4-bromoanisole (15 g, 36 mmol), dimethyl itaconate (7.5 g, 47 mmol), tri-o-tolylphosphine (1 g, 3 mol), palladium acetate (0.4 g, 2 mmol), diisopropylethylamine (12.8 mL,
72 mmol), and propionitrile (150 mL). The mixture was purged with argon (several evacuation / argon flush cycles) and then heated to reflux for 1 hour under argon. The reaction was cooled to room temperature and then poured into ice-cold ethyl ether (500 mL). The obtained precipitate was removed by filtration, and the filtrate was concentrated. Silica gel chromatography of the residue (10% -20% ethyl acetate in hexane)
The title compound was obtained as a pale yellow oil (11.8 g, 66%): ¹ H NMR (CDCl ₃ ) δ 7.94 (s, 1 H), 7.15 (d, J). = 8
.1Hz, 1H), 6.77 (d, J = 8.1Hz, 1H), 6.76 (s, 1H), 4.7
3 (s, 2H), 3.81 (s, 3H), 3.79 (s, 3H), 3.71 (s, 3H), 3.38 (s, 2H), 1.45 (s, 18H) ).

D) (±) -3-carbomethoxy-4- [2-bis (tert-butoxycarbonyl)
) Methyl aminomethyl-4-methoxyphenyl] butanoate (±) -3-carbomethoxy-4- [2-bis (tert-butoxycarbonyl) aminomethyl-4-methoxyphenyl] -3-butenoate (11. A pressure vessel containing 8 g), ethyl acetate (120 mL), and 10% palladium on charcoal (1 g) was shaken under 45 psi of hydrogen for 18 hours. The mixture was then filtered and the filtrate was concentrated to give the title compound as a colorless oil (12 g, 100%): ¹ H NMR (C
DCl ₃ ) δ 7.00 (d, J = 8.2 Hz, 1H), 6.71 (m, 2H), 4.81 (s, 2H), 3.75 (s, 3H), 3.66 ( s, 3H), 3.63 (s, 3H), 3.05 (m, 2H), 2.73 (m, 2H), 2.42 (dd, J = 16.0, 4.8 Hz,
1H), 1.44 (s, 18H).

E) Methyl (±) -3-carbomethoxy-4- [2- (aminomethyl) -4-methoxyphenyl] butanoate (±) -3-carbomethoxy-4- [2-bis (tert- A solution of methyl butoxycarbonyl) aminomethyl-4-methoxyphenyl] butanoate (12 g) in chloroform (100 mL) and trifluoroacetic acid (50 mL) was stirred at room temperature under argon for 4 hours. The solution was then concentrated in vacuo to give the title compound as a viscous oil (10 g, 100%): MS (ES) m / e 296.2 (M + H) ⁺ .

F) Methyl (±) -8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (±) -3-carbomethoxy-4- [ A solution of methyl 2- (aminomethyl) -4-methoxyphenyl] butanoate (10 g, 24 mmol) and triethylamine (17 mL, 120 mmol) in toluene (100 mL) was heated at reflux for 18 hours. Then the reaction was concentrated and the residue was partitioned between ethyl acetate and water. The aqueous layer was extracted twice with ethyl acetate, and the combined organic extracts were washed with brine, dried (M
gSO ₄ ) and concentrated to give the title compound as a tan solid (4.8 g, 76%): MS (ES) m / e 264.2 (M + H) ⁺ .

G) (±) -8-Hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzoazepine-4-acetate Methyl (±) -8 at 0 ° C. under argon. Salification of methyl methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (3.0 g, 11 mmol) and ethanethiol (4.2 mL, 57 mmol) Anhydrous aluminum chloride (7.6 g, 57 mmol) was added dropwise to the stirred solution in methylene (100 mL). The resulting mixture was warmed to room temperature, stirred overnight, and then concentrated. The residue was triturated with ice-water and the resulting solid was collected by filtration and dried to give the title compound as an off-white solid (2.64 g, 91%): MS (ES) m / e. 250.2 (M + H) ⁺ .

Preparation Example 2 HPLC separation of the enantiomer of methyl (±) -8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzoazepine-4-acetate a) (R)- Methyl (+)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate and (S)-(-)-8-hydroxy-3-oxo -2,3,4,5-tetrahydro-1H-2-benzazepine-4-methyl acetate (±) -8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzoazepine -4-Methyl acetate was resolved into its enantiomers by chiral HPLC using the following conditions: Diacel Chiralpak AS
ralpak AS® column (21.2 × 250 mm), EtOH mobile phase, flow rate 7 mL / min, uv detection at 254 nm, injection 70 mg; (R)-(+)-8-hydroxy-3-oxo- T _{R of} 2,3,4,5-tetrahydro-1H-2-benzazepine-4-methyl acetate = 21.5 min; (S)-(−)-8-hydroxy-3-oxo-
2,3,4,5-tetrahydro t _R = 39.1 min-1H-2-benzazepine-4-acetic acid methyl.

Preparation Example 3 HPLC separation of enantiomers of methyl (±) -8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzoazepine-4-acetate a) (R)- (+)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-
Methyl 1H-2-benzazepine-4-acetate and (S)-(-)-8-methoxy-
Methyl 3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (±) -8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H- Methyl 2-benzoazepine-4-acetate was separated into its enantiomers by chiral HPLC using the following conditions: Diacel Chiralpak AS® column (21.2 × 250 mm), CH ₃ CN mobile phase, flow rate. 15mL / min, 254n
uv detection at m, infusion 500mg; (R) - (+ ) - 8- methoxy-3-oxo-2, 3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid methyl t _R = 10 .2 min; (S) - (-) - 8- methoxy-3-oxo-2,3,4,5-tetrahydro-t _R = 19.0 min-1H-2-benzazepine-4-acetic acid methyl.

Preparation 4 Demethylation of methyl (S) -8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate a) (S) -8 -Hydroxy-3-oxo-2,3,4,5-tetrahydro-1 H-2-benzazepine-4-methyl acetate Boron tribromide (20.53 mL, 0.217 mol) at -8 ° C under argon. CH
Cl ₃ (160 mL) was added a solution (S)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-methyl acetate (15.0 g, 0.0
The CHCl ₃ (160 mL) a solution of 57 moles), while maintaining the temperature at -5 ° C. ~0 ° C., was added dropwise over 30 minutes. The reaction mixture was stirred at about -8 ° C for 30 minutes, then MeOH (200 mL) was added dropwise first, maintaining the temperature at about 0 ° C. The reaction mixture was concentrated to give a viscous oil, which was re-concentrated from MeOH (100 mL). The oil was dissolved in H ₂ O / MeOH and a small amount of dark solid was filtered off. The filtrate was neutralized (to pH 7) with 50% sodium hydroxide, during which time a white solid precipitated. The pH of the suspension was adjusted to 4.5 by the addition of a small amount of acetic acid, and the solid was collected and dried in vacuo to give the title compound (9.7 g, 68%). The products were assayed for chiral purity by HPLC: Chiralpak AS®
Column (4.6 × 50 mm), 100% EtOH mobile phase, flow rate 0.5 mL / min, uv detection at 215 nm; t _R = 7.5 min (S-enantiomer, 99%); t _R = 4. 4 min (R-enantiomer, 1%).

Preparation Example 5 2- [N- (3-hydroxy-1-propyl) -N- (tert-butoxycarbonyl
Preparation of) amino] pyridine-N-oxide

A) 2-[(3-Hydroxy-1-propyl) amino] pyridine-N-oxide 2-Chloropyridine-N-oxide (16.6 g, 0.1 mol), 3-amino-
1-propanol (15.3 mL, 0.2 mol), NaHCO ₃ (42 g, 0.5 molar), was heated to reflux tert- amyl alcohol (100 mL). After 21 hours, the reaction was cooled, diluted with CH ₂ Cl ₂ (300 mL) and filtered with suction to remove insoluble material. The filtrate was concentrated and re-concentrated from toluene to give a yellow oil. Silica gel chromatography (20% MeOH / CHCl ₃ ) afforded the title compound as a yellow solid (15.62 g, 93%); TLC (20% MeOH / CH3)
Cl ₃ ) R _f 0.48; ¹ H NMR (250, CDCl ₃ ) δ 8.07 (dd, J = 6.
6,1.2 Hz, 1H), 7.34 (brt, 1H), 7.10-7.30 (m, 1H), 6.64 (dd, J = 8.5, 1.4 Hz, 1H) , 6.40-6.60 (m, 1H),
4.49 (br s, 1H), 3.65-3.90 (m, 2H), 3.35-3.60 (m, 2H), 1.75-2.00 (m, 2H); MS (ES) m / e 169 (M + H) ^<+> .

B) 2- [N- (3-hydroxy-1-propyl) -N- (tert-butoxycarbonyl) amino] pyridine-N-oxide 2-[(3-hydroxy-1-propyl) amino] pyridine A solution of -N-oxide (8.0 g, 47.6 mmol) in tert-BuOH (80 mL) was treated with di-tert-butyl dicarbonate (11.4 g, 55.3 mmol). After 18 hours, the solution was concentrated and the residue was triturated with hexane. The resulting solid was dried in vacuo to give the title compound as an off-white solid (12.5 g, 98%); MS (ES) m / e 269.3 (M + H) ⁺ .

Preparation Example 6 (S) -3-oxo-8- [3- (pyridin-2-ylamino) -1-propyloxy] -2- (2,3,4-trifluorobenzyl) -2,3 , 4,5-tetrahydro-
Preparation of 1H-2-benzazepine-4-acetic acid

A) (S) -8- [3- [N- (1-oxopyridin-2-yl) -N- (tert-butoxycarbonyl) amino] -1-propyloxy] -3-oxo-2 , 3,4,5
Methyl tetrahydro-1H-2-benzazepine-4-acetate At room temperature, methyl (S) -8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (0.65 g, 2.75 mmol) and triphenylphosphine (1.94 g, 7.4 mmol) in anhydrous DMF (13
2- [N- (3-hydroxy-1-propyl) -N-tert-butoxycarbonyl) amino] pyridine-N-oxide (1.8 g, 6.84 mmol)
And a solution of diethyl azodicarboxylate (1.19 g, 6.84 mmol) in anhydrous DMF (22.5 mL) was added dropwise over 10 minutes. After 16 hours, the solution was evaporated to a brown oil which was combined with ethyl acetate (500 mL) and water (100 mL).
L). The organic layer was washed with water (2 × 100 mL) and brine (100
mL), dried (Na ₂ SO ₄ ) and concentrated to give a light brown oil. Purification by silica gel chromatography (5% MeOH / CH ₂ Cl ₂ ) afforded the title compound (0.85 g, 60%). MS (ES) m / e 50.3 (M
+ H) ⁺ .

B) (S) -8- [3- [N- (1-oxopyridin-2-yl) -N- (tert-butoxycarbonyl) amino] -1-propyloxy] -2- (2, (S) -8- [3,4-trifluorobenzyl) -3-oxo-2,3,4,5-tetrahydro-1H-2-benzoazepine-4-methyl acetate at -15 ° C under argon -[N- (1-oxopyridin-2-yl) -N- (tert-butoxycarbonyl) amino] -1-propyloxy] -3-oxo-2,3,4,5-tetrahydro-1H-2- To a solution of methyl benzazepine-4-acetate (0.470 g, 0.94 mmol) in anhydrous DMF (10.0 mL) was added 1.0 M of lithium bis (trimethylsilyl) amide (1.0 mL, 1.0 mmol). The solution was added dropwise. After 10 minutes, a solution of 2,3,4-trifluorobenzyl bromide (0.22 g, 0.96 mmol) in anhydrous THF (5.0 mL) was added dropwise. The solution was warmed to room temperature over 2 hours. After 18 hours, the solution was evaporated and the residue was partitioned between ethyl acetate (200mL) and water (30mL). The organic layer was washed with water (2
× 25 mL) and brine (30 mL), dried (Na ₂ SO ₄ ) and concentrated to give a pale yellow solid. Purification by silica gel chromatography (5% MeOH / CH ₂ Cl ₂ ) afforded the title compound (0.45 g, 75%).
MS (ES) m / e 644.1 (M + H) ^<+> .

C) (S) -8- [3- [N- (1-oxopyridin-2-yl) amino] -1-propyloxy] -2- (2,3,4-trifluorobenzyl)- 3-oxo-2,3
Methyl 4,4,5-tetrahydro-1H-2-benzazepine-4-acetate (S) -8- [3- [N- (1-oxopyridin-2-yl) -N- (tert-butoxycarbonyl) Amino] -1-propyloxy] -2- (2,3,4-trifluorobenzyl) -3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-methyl acetate (0 .39, 0.61 mmol) in 4.0M in 1,4-dioxane
Dissolved in a solution of hydrogen chloride (20 mL) and kept the reaction at room temperature for 16 hours. Removal of the solvent gave an oil which was solidified by trituration with ether. This material was dissolved in methylene chloride (80 mL) and washed with saturated NaHCO ₃ (10 mL). The organic layer was dried (Na ₂ SO ₄ ) and concentrated to give the title compound (0.33 g, 9
8%). MS (ES) m / e 544.1 (M + H) ^<+> .

D) (S) -3-oxo-8- [3- (pyridin-2-ylamino) -1-propyloxy] -2- (2,3,4-trifluorobenzyl) -2,3, Methyl 4,5-tetrahydro-1H-2-benzazepine-4-acetate (S) -8- [3- [N- (1-oxopyridin-2-yl) amino] -1-propyloxy] -2- (2,3,4-trifluorobenzyl) -3-oxo-2,3,4,5
-Methyl tetrahydro-1H-2-benzazepine-4-acetate (0.28 g, 0.2
52 mmol) and cyclohexene (0.28 g, 3.35 mmol) in 10
A solution of% Pd / C (0.270 g) in methanol (15 mL) was heated at reflux for 16 hours, cooled and filtered through celite®. The filtrate was concentrated to give the title compound (0.27 g, 99%). MS (ES) m / e 528
.1 (M + H) ⁺ .

E) (S) -3-oxo-8- [3- (pyridin-2-ylamino) -1-propyloxy] -2- (2,3,4-trifluorobenzyl) -2,3, 4,5-tetrahi
Dro-1H-2-benzazepine-4-acetic acid (S) -3-oxo-8- [3- (pyridin-2-ylamino) -1-propyloxy] -2- (2,3,4-trifluoro (Benzyl) -2,3,4,5-tetrahydro-
A solution of methyl 1H-benzazepine-4-acetate (0.27 g, 0.52 mmol) was prepared.
A solution of a mixture of ethanol (15 mL) and 0.5 N NaOH (3.0 mL) was
Heated at 0 ° C. After 2 hours, the reaction was cooled to room temperature, treated with TFA (0.50 mL) and concentrated to give a pale yellow solid. Semi-preparative HPLC (YMC ODS-AQ, 10 μm, 120 A, 50 mm × 250 mm, 40% CH containing 0.1% TFA)_ThreeCN / H_TwoO) to give the title compound (0.210 g, 67%). MS (ES) m / e 514.4 (M + H)⁺. Elemental analysis (C₂₇H₂₆F_ThreeN _Three O_Four・ 1.3CF_ThreeCO_TwoH: C, 53.73; H, 4.16; N, 6.35. measured value
: C, 53.64; H, 4.11; N, 6.14.

Example 2 Parenteral Dosage Unit Composition A preparation containing 20 mg of the compound of Example 1 as a sterile dry powder is prepared as follows: 20 mg of the compound is dissolved in 15 mL of distilled water. The solution is filtered under sterile conditions into 25 mL multidose ampoules and lyophilized. The powder is reconstituted by the addition of 20 mL of 5% dextrose in water (D5W) for intravenous or intramuscular injection. Dosage is determined by the volume of the injection. The metered volume of this dosage unit may then be diluted by adding it to another volume of D5W for injection, or the metered dose may be added to an IV drip bottle or bag or other injection-infusion system. May be added to another device for dispensing the drug.

Example 3 Oral Dosage Unit Composition A capsule for oral administration is prepared by mixing 50 mg of the compound of Example 1 with 75 mg of lactose and 5 mg of magnesium stearate and pulverizing. The resulting powder is screened and filled into hard gelatin capsules.

Example 4 Oral Dosage Unit Composition Tablets for oral administration are prepared by mixing 20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 1 with a 10% gelatin solution and granulating. I do. The wet granules are screened and dried, 10 mg of starch,
Mix with 5 mg talc and 3 mg stearic acid; compress into tablets.

The above description fully discloses the methods of making and using the present invention. However, the invention is not intended to be limited to the specific embodiments described hereinabove, but to encompass all modifications thereof within the scope of the following claims.
Various references to periodicals, patents and other publications cited herein are
It constitutes the current state of the art and is incorporated herein by reference as if fully set forth by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 19/10 A61P 19/10 29/00 29/00 35/00 35/00 35/04 35/04 ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AU, BA, BB, BG, BR, CA, CN, CZ, EE, GE, HU, ID, IL, IS, JP, K P, K R, LC, LK, LR, LT, LV, MG, MK, MN, MX, NO, NZ, PL, RO, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU F term (reference) 4C086 BC32 GA07 GA08 MA03 MA05 NA14 ZA45 ZA97 ZB11 ZB21 ZB26 ZC42

Claims (29)

[Claims] (1) Formula (I): Or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier. 3. A pharmaceutical composition comprising the compound according to claim 1, an antitumor agent and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to claim 3, wherein the antitumor agent is topotecan. 5. The pharmaceutical composition according to claim 3, wherein the antitumor agent is cisplatin. 6. A method for treating a condition requiring antagonism of the α _v β ₃ receptor, comprising administering the compound according to claim 1 to a subject in need of such treatment. 7. A method for treating a condition requiring antagonism of the α _v β ₅ receptor, comprising administering the compound according to claim 1 to a subject in need of such treatment. 8. A method of treating osteoporosis, comprising administering a compound according to claim 1 to a subject in need of such treatment. 9. A method of inhibiting angiogenesis, comprising administering to a subject in need of such inhibition a compound according to claim 1. 10. A method of inhibiting tumor growth or metastasis, comprising administering a compound of claim 1 to a subject in need of such inhibition. 11. A method of treating atherosclerosis or restenosis, comprising administering to a subject in need of such treatment a compound according to claim 1. 12. A method of treating inflammation, comprising administering a compound of claim 1 to a subject in need of such treatment. 13. A method for inhibiting tumor growth, comprising administering the compound of claim 1 and an antitumor agent to a subject in need of such inhibition in a stepwise or physical manner. 14. The method according to claim 13, wherein the antitumor agent is topotecan. 15. The method according to claim 13, wherein the antitumor agent is cisplatin. 16. A compound of formula (II): Or a pharmaceutically acceptable salt thereof. 17. A compound of formula (III): Or a pharmaceutically acceptable salt thereof. 18. The compound according to claim 1, for use as a medicament. 19. Use of the compound according to claim 1 in the manufacture of a medicament for treating a disease requiring antagonism of the α _v β ₃ receptor. 20. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of a disease in which antagonism of the α _v β ₅ receptor is required. 21. Use of a compound according to claim 1 in the manufacture of a medicament for treating osteoporosis. 22. Use of a compound according to claim 1 in the manufacture of a medicament for inhibiting angiogenesis. 23. Use of a compound according to claim 1 in the manufacture of a medicament for inhibiting tumor growth or tumor metastasis. 24. Use of a compound according to claim 1 in the manufacture of a medicament for treating atherosclerosis or restenosis. 25. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of inflammation. 26. Use of a compound according to claim 1 and an antitumor agent in the manufacture of a medicament for inhibiting tumor growth, either physically formulated or for gradual administration. 27. The use according to claim 26, wherein the antitumor agent is topotecan. 28. The use according to claim 26, wherein the antitumor agent is cisplatin. 29. Use of a compound of claim 1 and an inhibitor of bone resorption in the manufacture of a medicament for the treatment of osteoporosis, either physically formulated or for gradual administration.