Vitronectin receptor antagonists

This invention relates to pharmaceutically active compounds which inhibit the vitronectin receptor and are useful for the treatment of inflammation, cancer and cardiovascular disorders, such as atherosclerosis and restenosis, and diseases wherein bone résorption is a factor, such as osteoporosis.

BACKGROUND OF THE INVENTION

Integrins are a superfamily of cell adhesion receptors, which are transmembrane glycoproteins expressed on a variety of cells. These cell surface adhesion receptors include gpllb /Ilia (the fibrinogen receptor) and a_vB₃ (the vitronectin receptor). The fibrinogen receptor gpllb /Ilia is expressed on the platelet surface, and médiates platelet aggregation and the formation of a hemostatic clôt at the site of a bleeding wound. Philips, et al., Blood., 1988, 71, 831. The vitronectin receptor α_νβ₃ is expressed on a number of cells, including endothélial, smooth muscle, osteoclast, and tumor cells, and, thus, it has a variety of functions. The a_vB₃ receptor expressed on the membrane of osteoclast cells médiates the adhesion of osteoclasts to the bone matrix, a key step in the bone résorption process. Ross, et al., J. Biol. Chem., 1987, 262, 7703. A disease characterized by excessive bone résorption is osteoporosis. The a_vB₃ receptor expressed on human aortic smooth muscle cells médiates their migration into neointima, a process which can lead to restenosis after percutaneous coronary angioplasty. Brown, et al., Cardiovascular Res., 1994, 28,1815. Additionally, Brooks, et al., Cell, 1994, 79,1157 has shown that an α_νβ₃ antagonist is able to promote tumor régression by inducing apoptosis of angiogenic blood vessels. Thus, agents that block the vitronectin receptor would be useful in treating diseases, such as osteoporosis, restenosis and cancer.

The vitronectin receptor is now known to refer to three different integrins, designated α_νβμ α_νΒ₃^a°d ^αν^5· Horton, et al., Int. J. Exp. Pathol., 1990, 71, 741. a_vBj binds fîbronectin and vitronectin. α_νβ₃ binds a large variety of ligands, including fibrin, fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand's factor, osteopontin and bone sialoprotein I. a_vfi5 binds vitronectin. The vitronectin receptor a_vB5 has been shown to be involved in cell adhesion of a variety of cell types, including microvascular endothélial cells, (Davis, et al., J. Cell. Biol., 1993,51, 206), and its rôle in angiogenesis has been confirmed. Brooks, et al., Science, 1994, 264, 569. This integrin is expressed on blood vessels in human wound granulation tissue, but not in normal skin.

The vitronectin receptor is known to bind to bone matrix proteins which contain the tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al., Exp. Cell Res. 1991,195,

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368, disclose that RGD-containing peptides and an anti-vitronectin receptor antibody (23C6) inhibit dentine résorption and cell spreading by osteoclasts. In addition, Sato, et al.,

J. Cell Biol. 1990, lll, 1713 discloses that echistatin, a snake venom peptide which contains the RGD sequence, is a potent inhibitor of bone résorption in tissue culture, and inhibits attachment of osteoclasts to bone.

It has now been discovered that certain compounds are potent inhibitors of the a_vB₃ and a_vB₃ receptors. In particular, it has been discovered that such compounds are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor.

SUMMARY OF THE INVENTION

This invention comprises compounds as described hereinafter, which hâve pharmacological activity for the inhibition of the vitronection receptor and are useful in the treatment of inflammation, cancer and cardiovascular disorders, such as atherosclerosis and restenosis, and diseases wherein bone résorption is a factor, such as osteoporosis.

This invention is also a pharmaceutical composition comprising compounds as described hereinafter and a pharmaceutically carrier.

This invention is also a method of treating diseases which are mediated by the vitronectin receptor. In a particular aspect, the compounds of this invention are useful for treating atherosclerosis, restenosis, inflammation, cancer and diseases wherein bone résorption is a factor, such as osteoporosis.

DETAILED DESCRIPTION

This invention comprises novel compounds which are more potent inhibitors of the vitronectin receptor than the fibrinogen receptor. The novel compounds comprise a dibenzocycloheptene core in which a nitrogen-containing substituent is présent on one of the aromatic six-membered rings of the dibenzocycloheptene and an aliphatic substituent containing an acidic moiety is présent on the seven-membered ring of the dibenzocycloheptene. The dibenzocycloheptene ring System is believed to orient the substituent sidechains on the six and seven membered rings so that they may interact favorably with the vitronectin receptor. It is preferred that about twelve to fourteen intervening covalent bonds via the shortest intramolecular path will exist between the acidic group on the aliphatic substituent of the seven-membered ring of the dibenzocycloheptene and the nitrogen of the nitrogen-containing substituent on one of the aromatic six-membered ring of the dibenzocycloheptene.

Spécifie compounds of this invention are:

(±)-10,11 -Dihydro-3-[3-(4-amino-2-pyridylamino)-1 -propyloxy ]-5Hdibenzo[a,d]cycloheptene-10-acetic acid;

011076 (±)-10,ll-Dihydro-3-[2-[6-(ethylamino)-2-pyridyl]-l-ethoxy]-5Hdibenzo[a,d]cycloheptene-10-acetic acid; and (±)-10,ll-Dihydro-3-[3-(4-methyl-2-pyridylamino)-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetic acid;

or pharmaceutically acceptable salts thereof.

In cases wherein the compounds of this invention may hâve one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques.

Also included in this invention are prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug (compounds of the instant invention) in vivo.

The compounds of this invention inhibit the binding of vitronectin and other RGDcontaining peptides to the vitronectin receptor. Inhibition of the vitronectin receptor on osteoclasts inhibits osteoclastic bone résorption and is useful in the treatment of diseases wherein bone résorption is associated with pathology, such as osteoporosis and osteoarthritis.

In another aspect, this invention is a method for stimulating bone formation which comprises administering a compound which causes an increase in osteocalcin release. Increased bone production is a clear benefit in disease states wherein there is a deficiency of mineralized bone mass or remodeling of bone is desired, such as fracture healing and the prévention of bone fractures. Diseases and metabolic disorders which resuit in loss of bone structure would also benefit from such treatment. For instance, hyperparathyroidism, Paget’s disease, hypercalcemia of malignancy, osteolytic lésions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency, Behçet's disease, osteomalacia, hyperostosis and osteopetrosis, could benefit from administering a compound of this invention.

Additionally, since the compounds of the instant invention inhibit vitronectin receptors on a number of different types of cells, said compounds would be useful in the treatment of inflammatory disorders, such as rheumatoid arthritis and psoriasis, and cardiovascular diseases, such as atherosclerosis and restenosis. The compounds of the présent invention may be useful for the treatment or prévention of other diseases including, but not limited to, thromboembolie disorders, asthma, allergies, adult respiratory distress syndrome, graft versus host disease, organ transplant rejection, septic shock, eczema, contact dermatitis, inflammatory bowel disease, and other autoimmune diseases. The compounds of the présent invention may also be useful for wound healing.

The compounds of the présent invention are also useful for the treatment, including prévention, of angiogenic disorders. The term angiogenic disorders as used herein includes

011076 conditions involving abnormal neovascularization. Where the growth of new blood vessels is the cause of, or contributes to, the pathology associated with a disease, inhibition of angiogenisis will reduce the deleterious effects of the disease. An example of such a disease target is diabetic retinopathy. Where the growth of new blood vessels is required to support growth of a deleterious tissue, inhibition of angiogenisis will reduce the blood supply to the tissue and thereby contribute to réduction in tissue mass based on blood supply requirements. Examples include growth of tumors where neovascularization is a continuai requirement in order that the tumor grow and the establishment of solid tumor métastasés. Thus, the compounds of the présent invention inhibit tumor tissue angiogenesis, thereby preventing tumor metastasis and tumor growth.

Thus, according to the methods of the présent invention, the inhibition of angiogenesis using the compounds of the présent invention can ameliorate the symptoms of the disease, and, in some cases, can cure the disease.

Another therapeutic target for the compounds of the instant invention are eye diseases chacterized by neovascularization. Such eye diseases include comeal neovascular disorders, such as comeal transplantation, herpetic keratitis, luetic keratitis, pterygium and neovascular pannus associated with contact lens use. Additional eye diseases also include age-related macular degeneration, presumed ocular histoplasmosis, retinopathy of prematurity and neovascular glaucoma.

This invention further provides a method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of the présent invention and an antineoplastic agent, such as topotecan and cisplatin.

This invention also provides for a process for preparing a compound of formula (I):

<img file="OA11076A_D0001.tif" />

which process comprises réduction of a compound of formula (II):

<img file="OA11076A_D0002.tif" />

In another aspect of this invention is a process for preparing a compound of formula (II):

011076

<img file="OA11076A_D0003.tif" />

(Π) which process comprises cyclization of a compound of formula (III):

<img file="OA11076A_D0004.tif" />

(m).

This invention further provides for novel intermediates of the formulae (IV), (V), (VI) and (II):

<img file="OA11076A_D0005.tif" />

(IV);

<img file="OA11076A_D0006.tif" />

(VI); and

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<img file="OA11076A_D0007.tif" />

As used herein, Ci_6alkyl is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof.

Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to diisopropylethylamine, EDC refers to N-ethyl-N’(dimethylaminopropyl)-carbodiimide. HOBt refers to

1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DMF refers to dimethyl formamide, NBS refers to N-bromo-succinimide, Pd/C refers to a palladium on carbon catalyst, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, PPA refers to polyphosphoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate.

Compounds of the instant invention are prepared by the methods described in Bondinell et al., PCT Publication No. WO 97/01540 (International Application No. PCT/US96/11108), published January 16, 1997, the entire disdosure of which is incorporated herein by reference.

Additionally, compounds of this invention are prepared by methods analogous to those described in the schemes that are detailed hereinafter.

Scheme 1

<img file="OA11076A_D0008.tif" />

P50608

011076

<img file="OA11076A_D0009.tif" />

<img file="OA11076A_D0010.tif" />

<img file="OA11076A_D0011.tif" />

a) Tf2Û, 2,6-lutidine, CH2CI2; b) allyltributyltin, LiCl, (Ph3P)2PdC12, DMF; c) R11CI3, H5IO6, CCI4, CH3CN, H2O; d) PPA; e) EtOAc/LiHMDS, THF; f) H2, 10% Pd/C, conc. HCl, AcOH; g) EtSH, AICI3, CH2CI2; h) Tf20,2,6-lutidine, CH2CI2; i) CO, Pd(OAc)2, KOAc, dppf, DMSO.

2-Benzyl-4-methoxyphenol (J. Am. Chem. Soc. 1949, 71, 64) is converted to the corresponding trifluoromethanesulfonate ester, compound 2-Scheme l(e.g., 1-2) by reaction with trifluoromethanesulfonic anhydride (Tf2O) in the presence of a suitable base, for instance 2,6-lutidine, in an inert solvent, generally CH2CI2. Reaction of 1-2 with allyltributyltin in the presence of LiCl and a palladium catalyst, for example bis(triphenylphosphine)palladium (II) chloride ((Ph3P)2PdC12), in an inert solvent such as DMF, by the method described by Tilley (J. Org. Chem. 1990, 55, 906), affords 1-3. Oxidative cleavage of the olefin in 3-Scheme 1 to afford directly the carboxylic acid 1-4 can be accomplished by reaction with an appropriate oxidizing agent, classically KMnÛ4, in a suitable aqueous solvent, such as aqueous acetone or aqueous acetic acid. Preferably, however, oxidative cleavage of the olefin in 1-3 to afford directly the carboxylic acid 1-4 is conducted according to the general method of Sharpless (J. Org. Chem. 1981, 46, 3936; J. Org. Chem. 1985, 50, 1560, footnote 4), wherein RuO4 is generated in situ by the reaction of RuC13 or RuO2 with NaIO4 or H5IO6 in a solvent mixture of CCI4, CH3CN, and H2O. Alternatively, the oxidation might be conducted in two operations, involving in the first

011076 stage an oxidative cleavage of the olefin to the corresponding aldéhyde, which can be accomplished by procedures well known to those of skill in the art, followed by oxidation of the aldéhyde to the carboxylic acid using, for example, NaClC&gt;2 as described by Pinnick (Tetrahedron 1981, 37, 2091) or by Dalcanale and Montanari (J. Org. Chem. 1986, 51, 567). Cyclization of 1-4 to 1-5 can be accomplished using polyphosphoric acid, according to the method described by Proctor, Renfrew, and Savage (J. Chem. Soc. (C) 1969, 1000). Altematively, 1-4 can be converted to 1-5 via the corresponding acid chloride of 1-4, which can be prepared by methods well-known to those of skill in the art. Treatment of this acid chloride with an appropriate Friedel-Crafts catalyst, such as AICI3 or SnC14, in an inert solvent, such as CH2CI2 or CS2, provides the cyclic ketone 1-5. Reaction of 1-5 in an aldol-type reaction with the enolate of ethyl acetate, which can be generated from ethyl acetate on exposure to an appropriate amide base, for instance lithium diisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), gives 1-6. Frequently, THF is the solvent of choice for an aldol reaction, although THF in the presence of various additives, for instance HMPA or TMEDA, is often used. Réduction of 1-6 to give 1-7 can be accomplished by hydrogenolysis over an appropriate catalyst, for example palladium métal on activated carbon (Pd/C), in an appropriate solvent, such as acetic acid, in the presence of a minerai acid such as HCl. Altematively, this réduction can be accomplished by treatment of 1-6 with triethylsilane in the presence of boron trifluoride etherate by the general method of Orphanopoulos and Smonu (Synth. Commun. 1988, 833). Removal of the methyl ether of 1-7 to give 1-8 can be accomplished with BBrç in an inert solvent, for example CH2CI2, of by reaction with ethanethiol and AICI3 in an inert solvent, preferably CH2CI2. Other useful methods for removal of a methyl ether are described in Greene, Protective Groups in Organic Synthesis (published by John Wiley and Sons). 1-9, the trifluoromethanesulfonate ester of 1-8, prepared by the method described earlier for the conversion of 1-1 to 1-2, reacts with carbon monoxide in the presence of potassium acetate, l,l’-bis(diphenylphosphino)ferrocene (dppf), and a palladium catalyst, for instance palladium acetate (Pd(OAc)2), in a suitable solvent, preferably DMSO, according to the general method described by Cacchi and Lupi (Tet. Lett. 1992, 33, 3939), to give 1-10.

Scheme 2 is illustrative of a carbon-oxygen bond forming coupling method that may be used to form an ether linkage.

011076

<img file="OA11076A_D0012.tif" />

<img file="OA11076A_D0013.tif" />

a) 2-[(3-hydroxy-l-propyl)amino]pyridine-N-oxide, DEAD, (Ph^P, DMF; b) cyclohexene, 10% Pd/C, 2-propanol; c) 1.0 N NaOH, EtOH, then acidification.

Compound 1 of Scheme 2 (2-1) is reacted with 2-[(3-hydroxy-lpropyl)amino]pyridine-N-oxide in a Mitsunobu-type coupling reaction (Organic Reactions 1992, 42, 335-656; Synthesis 1981,1-28) to afford 2-2. The reaction is mediated by the complex formed between diethyl azodicarboxylate and triphenylphosphine, and is conducted in an aprotic solvent, for instance THF, CH2CI2, or DMF. The pyridine-Noxide moiety of 2-2 is reduced to the corresponding pyridine 2-3 under transfer hydrogénation conditions using a palladium catalyst, preferabiy palladium métal on activated carbon, in an inert solvent, for instance methanol, éthanol, or 2-propanol. Cyclohexene, 1,4-cyclohexadiene, formic acid, and salts of formic acid, such as potassium formate or ammonium formate, are commonly used as the hydrogen transfer reagent in this type of reaction. The ethyl ester of 2-3 is saponified as described in Scheme 1 to afford 2-

4.

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Altemate methods for preparing certain intermediate compounds described hereinbefore was accomplished as shown below in Schemes 3 and 4.

Scheme 3 o

<img file="OA11076A_D0014.tif" />

o co₂h co₂h

a) 10% Pd/C, HOAc; b) SOC1₂, toluene; c) À1C1_3&gt; CH₂C1₂

Scheme 3 summarizes an altemate method for preparing Scheme 1, formula 5 (1-5) compounds. According to this scheme, 3-1 (J. Med. Chem., 1981, 24, 998) is hydrogenated over an atmosphère of hydrogen gas at a suitable pressure, for example, 50 psi, in the presence of a palladium catalyst, for example, 10% Pd/C, in a suitable solvent, for example, glacial acetic acid, at a suitable température, for example, 25°C, to give 3-2. Cyclization of

3-2 to 3-3, is accomplished by first converting 3-2 to the corresponding acid chloride in the presence of a suitable chlorinating agent, for example, thionyl chloride, in a suitable solvent, for example, benzene, at a suitable température, for example, 85°C. Treatment of this acid chloride with a suitable Friedel-Crafts catalyst, for example, AICI3, in a suitable solvent, for example, CH2CI2, at a suitable température, for example, 25°C, gives 6-3.

011076

Scheme 4

<img file="OA11076A_D0015.tif" />

<img file="OA11076A_D0016.tif" />

<img file="OA11076A_D0017.tif" />

<img file="OA11076A_D0018.tif" />

<img file="OA11076A_D0019.tif" />

a) LiN(TMS)₂, ethyl bromoacetate; b) Jones reagent, OsO₄; c) H₂, 10% Pd/C, HOAC;

d) C₂O₂C1₂, DMF; e) A1C1₃, CH₂C1₂, RT; f) H₂, 10% Pd/C, HOAC

Scheme 4 summarizes an altemate method for preparing Scheme 1, formula 7 (1-7) compounds. According to this scheme, 4-1 (J. Med. Chem., 1981, 24, 998) is reacted with ethyl bromoacetate in the presence of a suitable base, for example, LiN(TMS)2, in a suitable solvent, for example, THF, at a suitable température, for example, -78°C, to give

4-2. 4-2 is oxidatively cleaved in the presence of a suitable mixture of oxidants, for example, OsO^Jones reagent, in a suitable solvent, for example, acetone, at a suitable température, for example, 25°C, to give 4-3 (J. Org. Chem., 1993, 58, 4745). 4-3 is further hydrogenated over an atmosphère of hydrogen gas at a suitable pressure, for example, 50 psi, in the presence of a palladium catalyst, for example, 10% Pd/C, in a suitable solvent, for example, glacial acetic acid, at a suitable température, for example, 25°C, to give 4-4.

Cyclization of 4-4 to 4-5, is accomplished by first converting 4-4 to the corresponding acid chloride in the presence of a suitable chlorinating agent, for example, oxalyl chloride, in the %

011076 presence of catalytic amount of an additive, for example, DMF, in a suitable solvent, for example, CH2CI2, at a suitable température, for example, 25°C. Treatment of this acid chloride with a suitable Friedel-Crafts catalyst, for example, AICI3, in a suitable solvent, for example, CH2CI2, at a suitable température, for example, 25°C, gives 4-5. 4-5 is further hydrogenated over an atmosphère of hydrogen gas at a suitable pressure, for example, 50 psi, in the presence of a palladium catalyst, for example, 10% Pd/C, in a suitable solvent, for example, glacial acetic acid, at a suitable température, for example, 25°C, to give 4-6.

Acid addition salts of the compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺and NH4⁴· are spécifie examples of cations présent in pharmaceutically acceptable salts.

This invention also provides a pharmaceutical composition which comprises a compound according as described herein and a pharmaceutically acceptable carrier. Accordingly, the compounds of this invention may be used in the manufacture of a médicament. Pharmaceutical compositions of the compounds of this invention prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parentéral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonie, aqueous solution. Examples of suitable diluents are normal isotonie saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parentéral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be désirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Altemately, these compounds may be encapsulated, tableted or prepared in a émulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate préparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnésium stéarate or stearic acid, talc, pectin, acacia, 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

011076 with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical préparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the préparation will be in the form of a syrup, élixir, émulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.

The compounds described herein are antagonists of the vitronectin receptor, and are useful for treating diseases wherein the underlying pathology is attributable to ligand or cell which interacts with the vitronectin receptor. For instance, these compounds are useful for the treatment of diseases wherein loss of the bone matrix créâtes pathology. Thus, the instant compounds are useful for the treatment of ostoeporosis, hyperparathyroidism, Paget’s disease, hypercalcemia of malignancy, osteolytic lésions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency. The compounds of this invention are also believed to hâve utilîty as antitumor, anti-angiogenic, antiinflammatory and anti-metastatic agents, and be useful in the treatment of atherosderosis and restenosis.

The compound is administered either orally or parenterally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone résorption, or other such indication. The pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg. For acute therapy, parentéral administration is preferred. An intravenous infusion of the peptide in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parentéral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The précisé level and method by which the compounds are administered is readily determined by one routinely skilled in the art by comparing the blood level of the agent to the concentration required to hâve a therapeutic effect.

This invention further provides a method for treating osteoporosis or inhibiting bone loss which comprises administering stepwise or in physical combination a compound of this invention and other inhibitors of bone résorption, such as bisphosphonates (i.e.,

011076 allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In addition, this invention provides a method of treatment using a compound of this invention and an anabolic agent, such as the bone morphogenic protein, iproflavone, useful in the prévention of bone loss and/or to increase bone mass.

Additionally, this invention provides a method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of this invention and an antineoplastic agent. Compounds of the camptothecin analog class, such as topotecan, irinotecan and 9-aminocamptothecin, and platinum coordination complexes, such as cisplatin, ormaplatin and tetraplatin, are well known groups of antineoplastic 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 4,342,776, 4,513,138, 4,399,276, EP Patent Application Publication Nos . 0 418 099 and 0 088 642, Wani, et al., J. Med. Chem., 1986, 29, 2358, Wani, et al., J. Med. Chem., 1980, 23, 554, Wani, et al., J. Med. Chem., 1987, 30, 1774, and Nitta, et al., Proc. 14th International Congr. Chemotherapy., 1985, Anticancer Section 1, 28, the entire disclosure of each which is hereby incorporated by reference. The platinum coordination complex, cisplatin, is available under the name Platinol® from Bristol Myers-Squibb Corporation. Useful formulations for cisplatin are described in U.S. Patent Nos. 5,562,925 and 4,310,515, the entire disclosure of each which is hereby incorporated by reference.

In the method of inhibiting tumor growth which comprises administering stepwise or in physical combination a compound of this invention and an antineoplastic agent, the platinum coordination compound, for example cisplatin, can be administered using slow intravenous infusion. The preferred carrier is a dextrose/saline solution containing mannitol. The dose schedule of the platinum coordination compound may be on the basis of from about 1 to about 500 mg per square meter (mg/nT) of body surface area per course of treatment. Infusions of the platinum coordiation compound may be given one to two times weekly, and the weekly treatments may be repeated several times. Using a compound of the camptothecin analog class in a parentéral administration, the course of therapy generally employed is from about 0.1 to about 300.0 mg/m^ of body surface area per day for about five consecutive days. Most preferably, the course of therapy employed for topotecan is from about 1.0 to about 2.0 mg/m^ of body surface area per day for about five consecutive days. Preferably, the course of therapy is repeated at least once at about a seven day to about a twenty-eight day interval.

The pharmaceutical composition may be formulated with both the compound of this invention and the antineoplastic agent in the same container, but formualtion in different containers is preferred. When both agents are provided in solution form, they can

011076 be contained in an infusion/injection System for simultaneous administration or in a tandem arrangement.

For convenient administration of the compound of this invention and the antineoplastic agent at the same or different times, a kit is prepared, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the compound of this invention for parentéral administration, as described above, and an effective amount of the antineoplastic agent for parentéral administration, as described above. Such kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for reconstitution. A variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use. With such an arrangement, the antineoplastic agent and the compound of this invention may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.

When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement. For example, the compound of this invention may be in an i.v. injectable form, or infusion bag linked in sériés, via tubing, to the antineoplastic agent in a second infusion bag. Using such a system, a patient can receive an initial bolus-type injection or infusion of the compound of this invention foiiowed by an infusion of the antineoplastic agent.

The compounds may be tested in one of several biological assays to détermine the concentration of compound which is required to hâve a given pharmacological effect.

Inhibition of vitronectin binding

Solid-Phase [3Hj-SK&amp;F-107260 Binding to α_νβ$: Human placenta or human piatelet α_νβ3 (0.1-0.3 mg/mL) in buffer T (containing 2 mM CaC12 and 1% octylglucoside) was diluted with buffer T containing 1 mM CaC12,1 mM MnC12, 1 mM MgC12 (buffer A) and 0.05% NaN₃, and then immediately added to 96-well ELISA plates (Coming, New York, NY) at 0.1 mL per well. 0.1 - 0.2 pg of α_νβ3 was added per well. The plates were incubated overnight at 4°C. At the time of the experiment, the wells were washed once with buffer A and were incubated with 0.1 mL of 3.5% bovine sérum albumin in the same buffer for 1 hr at room température. Following incubation the wells were aspirated completely and washed twice with 0.2 mL buffer A.

Compounds were dissolved in 100% DMSO to give a 2 mM stock solution, which was diluted with binding buffer (15 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaC12, 1 mM MnC12, 1 mM MgC12) to a final compound concentration of 100 μΜ. This solution is

011076 then diluted to the required final compound concentration. Various concentrations of unlabeled antagonists (0.001 - 100 μΜ) were added to the wells in triplicates, followed by the addition of 5.0 nM of [³H]-SK&amp;F-107260 (65 - 86 Ci/mmol).

The plates were incubated for 1 hr at room température. Following incubation the wells were aspirated completely and washed once with 0.2 mL of ice cold buffer A in a well-to-well fashion. The receptors were solubilized with 0.1 mL of 1% SDS and the bound [³H]-SK&amp;F-107260 was determined by liquid scintillation counting with the addition of 3 mL Ready Safe in a Beckman LS Liquid Scintillation Counter, with 40% efficiency. Nonspecific binding of [³H]-SK&amp;F-107260 was determined in the presence of 2 μΜ SK&amp;F-107260 and was consistently less than 1% of total radioligand input. The IC50 (concentration of the antagonist to inhibit 50% binding of [³H]-SK&amp;F-107260) was determined by a nonlinear, least squares curve-fitting routine, which was modified from the LUNDON-2 program. The Kj (dissociation constant of the antagonist) was calculated according to the équation: Kj = IC5()/(1 + L/Κμ), where L and K&lt;j were the concentration and the dissociation constant of [³H]-SK&amp;F-107260, respectively.

Compounds of this invention are also tested for in vitro and in vivo bone résorption in assays standard in the art for evaluating inhibition of bone formation, such as the pit formation assay disclosed in EP 528 587, which may also be performed using human osteoclasts in place of rat osteoclasts, and the ovarectomized rat model, described by Wronski et al., Cells and Materials 1991, Sup. 1, 69-74.

Vascular smooth muscle cell migration assay

Rat or human aortic smooth muscle cells were used. The cell migration was monitored in a Transwell cell culture chamber by using a polycarbonate membrane with pores of 8 um (Costar). The lower surface of the filter was coated with vitronectin. Cells were suspended in DMEM supplemented with 0.2% bovine sérum albumin at a concentration of 2.5 - 5.0 x 10^ cells/mL, and were pretreated with test compound at various concentrations for 20 min at 20°C. The solvent alone was used as 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 sérum albumin. Incubation was carried out at 37°C in an atmosphère of 95% air/5% CO2 for 24 hr. After incubation, the non-migrated cells on the upper surface of the filter were removed by gentle scraping. The filter was then fixed in methanol and stained with 10% Giemsa stain. Migration was measured either by a) counting the number of cells that had migrated to the lower surface of the filter or by b) extracting the stained cells with 10% acetic acid followed by determining the absorbance at 600 nM.

011076

Thyroparathyroîdectomized rat model

Each experimental group consists of 5-6 adult male Sprague-Dawley rats (250-400g body weight). The rats are thyroparathyroîdectomized (by the vendor, Taconic Farms) 7 days prior to use. Ail rats receive a replacement dose of thyroxine every 3 days. On receipt of the rats, circulating ionized calcium levels are measured in whole blood immediately after it has been withdrawn by tail venipuncture into heparinized tubes. Rats are included if the ionized Ca level (measured with a Ciba-Coming model 634 calcium pH analyzer) is &lt;1.2 mM/L. Each rat is fitted with an indwelling venous and arterial cathéter for the delivery of test material and for blood sampling respectively. The rats are then put on a diet of calcium-free chow and deionized water. Baseline Ca levels are measured and each rat is administered either control vehicle or human parathyroid hormone 1-34 peptide (hPTHl-34, dose 1.25 ug/kg/h in saline/0.1% bovine sérum albumin, Bachem, Ca) or a mixture of hPTHl-34 and test material, by continuous intravenous infusion via the venous cathéter using an extemal syringe pump. The calcémie response of each rat is measured at two-hourly intervals during the infusion period of 6-8 hours.

Human osteoclast résorption and adhesion assays

Pit résorption and adhesion assays hâve been developed and standardized using normal human osteoclasts derived from osteoclastoma tissue. Assay 1 was developed for the measurement of osteoclast pit volumes by laser confocal microscopy. Assay 2 was developed as a higher throughput screen in which collagen fragments (released during résorption) are measured by competitve ELISA.

Assay 1 (using laser confocal microscopy) • Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37°C and washed xl in RPMI-1640 medium by centrifugation (lOOOrpm, 5 mins at 4°C).

• The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.

• The cells are washed x2 with cold RPMI-1640 foliowed by centrifugation (1000 rpm, 5 mins at 4°C) and the cells are then transferred to a stérile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.

• Sufficient magnetic beads (5 t mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, NY) are removed from their stock bottle and placed into 5 ml of

011076 fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.

• The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.

• The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a stérile 50 ml centrifuge tube.

• Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xlO. The bead-coated cells are discarded.

• The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.

• The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteoclasts is variable from tumor to tumor), supplemented with 10% fêtai calf sérum and 1.7g/liter of sodium bicarbonate.

• 3ml aliquots of the cell suspension (per compound treatment) are decanted into 15ml centrifuge tubes. The cells are pelleted by centrifugation.

• To each tube, 3ml of the appropriate compound treatment are added (diluted to 50 uM in the EMEM medium). Also included are appropriate vehicle controls, a positive control (anti-vitronectin receptor murine monoclonal antibody [87MEM1] diluted to 100 ug/ml) and an isotype control (IgG_2a diluted to 100 ug/ml). The samples are incubated at 37°C for 30 mins.

• 0.5ml aliquots of the cells are seeded onto stérile dentine slices in a 48-well plate and incubated at 37°C for 2 hours. Each treatment is screened in quadruplicate.

• The slices are washed in six changes of warm PBS (10 ml / well in a 6-well plate) and then placed into fresh medium containing the compound treatment or control samples. The samples are incubated at 37°C for 48 hours.

Tartrate résistant acid phosphatase (TRAP) procedure (sélective stainfor cells ofthe osteoclast lineage) • The bone slices containing the attached osteoclasts are washed in phosphate buffered saline and fixed in 2% gluteraldehyde (in 0.2M sodium cacodylate) for 5 mins.

• They are then washed in water and are incubated for 4 minutes in TRAP buffer at 37°C (0.5 mg/ml naphthol AS-BI phosphate dissolved in Ν,Ν-dimethylformamide and mixed with 0.25 M citrate buffer (pH 4.5), containing 10 mM sodium tartrate.

• Following a wash in cold water the slices are immersed in cold acetate buffer (0.1 M, pH 6.2) containing 1 mg/ml fast red gamet and incubated at 4°C for 4 minutes.

011076 • Excess buffer is aspirated, and the slices are air dried following a wash in water.

• The TRAP positive osteoclasts (brick red/ purple precipitate) are enumerated by bright-field microscopy and are then removed from the surface of the dentine by sonication.

• Pit volumes are determined using the Nikon/Lasertec ILM21W confocal microscope.

Assay 2 (using an ELISA readout)

The human osteoclasts are enriched and prepared for compound screening as described in the initial 9 steps of Assay 1. For clarity, these steps are repeated hereinbelow.

• Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37°C and washed xl in RPMI-1640 medium by centrifugation (lOOOrpm, 5 mins at 4°C).

• The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.

• The cells are washed x2 with cold RPMI-1640 followed by centrifugation (1000 rpm, 5 mins at 4°C) and the cells are then transferred to a stérile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.

• Sufficient magnetic beads (5 / mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, NY) are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.

• The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.

• The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a stérile 50 ml centrifuge tube.

• Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xlO. The bead-coated cells are discarded.

• The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.

• The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteoclasts is variable from tumor to tumor), supplemented with 10% fêtai calf sérum and 1.7g/liter of sodium bicarbonate.

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In contrast to the method desribed above in Assay 1, the compounds are screened at doses to obtain an IC₅₀, as outlined below:

• The osteoclast préparations are preincubated for 30 minutes at 37°C with test compound (4 doses) or controls.

• They are then seeded onto bovine cortical bone slices in wells of a 48-well tissue culture plate and are incubated for a further 2 hours at 37°C.

• The bone slices are washed in six changes of warm phosphate buffered saline (PBS), to remove non-adherent cells, and are then retumed to wells of a 48 well plate containing fresh compound or controls.

• The tissue culture plate is then incubated for 48 hours at 37°C.

• The supematants from each well are aspirated into individual tubes and are screened in a compétitive ELISA that detects the c-telopeptide of type I collagen which is released during the résorption process. This is a commercially available ELISA (Osteometer, Denmark) that contains a rabbit antibody that specifically reacts with an 8amino acid sequence (Glu-Lys-Ala-His- Asp-Gly-Gly-Arg) that is présent in the carboxy-terminal telopeptide of the al-chain of type I collagen. The results are expressed as % inhibition of résorption compared to a vehicle control.

Human osteoclast adhesion assay

The human osteoclasts are enriched and prepared for compound screening as described above in the inital 9 steps of Assay 1. For clarity, these steps are repeated hereinbelow.

• Aliquots of human osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37°C and washed xl in RPMI-1640 medium by centrifugation (lOOOrpm, 5 mins at 4°C).

• The medium is aspirated and replaced with murine anti-HLA-DR antibody then diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on ice and mixed frequently.

• The cells are washed x2 with cold RPMI-1640 followed by centrifugation (1000 rpm, 5 mins at 4°C) and the cells are then transferred to a stérile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.

• Sufficient magnetic beads (5 / mononuclear cell), coated with goat anti-mouse IgG (Dynal, Great Neck, NY) are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.

011076 • The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.

• The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a stérile 50 ml centrifuge tube.

• Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xlO. The bead-coated cells are discarded.

• The viable osteoclasts are enumerated in a counting chamber, using fluorescein diacetate to label live cells. A large-bore disposable plastic pasteur pipet is used to add the sample to the chamber.

• The osteoclasts are pelleted by centrifugation and the density adjusted to the appropriate number in EMEM medium (the number of osteoclasts is variable from tumor to tumor), supplemented with 10% fêtai calf sérum and 1.7g/liter of sodium bicarbonate.

• Osteoclastoma-derived osteoclasts are preincubated with compound (4 doses) or controls at 37°C for 30 minutes.

• The cells are then seeded onto osteopontin-coated slides (human or rat osteopontin, 2.5ug/ml) and incubated for 2 hours at 37°C.

• Non adhèrent cells are removed by washing the slides vigorously in phosphate buffered saline and the cells remaining on the slides are fixed in acetone.

• The osteoclasts are stained for tartrate-resistant acid phosphatase (TRAP), a sélective marker for cells of this phenotype (see steps 15 -17), and are enumerated by light microscopy. The results are expressed as % inhibition of adhesion compared to a vehicle control.

Cell Adhesion Assay

Cells and Cell Culture

Human embryonic kidney cells ( HEK293 cells) were obtained from ATCC (Catalog No. CRL 1573). Cells were grown in Earl’s minimal essential medium (EMEM) medium containing Earl’s salts, 10% fêtai bovine sérum, 1% glutamine and 1% PenicillinSteptomycin.

Constructs and Transfections

A 3.2 kb EcoRI-Kpnl fragment of the a_v subunit and a 2.4 kb Xbal- Xhol fragment of the ββ subunit were inserted into the EcoRI - EcoRV cloning sites of the pCDN vector (Aiyar et al., 1994 ) which contains a CMV promoter and a G418 selectable marker by blunt end ligation. For stable expression, 80 x 10 6 HEK 293 cells were electrotransformed with α_ν+β3 constructs (20 gg DNA of each subunit) using a Gene Puiser (Hensley et al., 1994 ) and plated in 100 mm plates (5x10⁵ cells/plate). After 48 hr, the growth medium

011076 was supplemented with 450 pg/mL Geneticin (G418 Sulfate, GIBCO-BRL, Bethesda, MD). The cells were maintained in sélection medium until the colonies were large enough to be assayed.

Immunocytochemical analysis of transfected cells

To détermine whether the HEK 293 transfectants expressed the vitronectin receptor, the cells were immobilized on glass microscope slides by centrifugation, fixed in acetone for 2 min at room température and air dried. Spécifie reactivity with 23C6, a monoclonal antibody spécifie for the α_νβ3 complex was demonstrated using a standard indirect immunofluorescence method.

Cell Adhesion Studies

Coming 96-well ELIS A plates were precoated overnight at 4°C with 0.1 mL of human vitronectin (0.2 pg/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 hr at room température. Transfected 293 cells were resuspended in RPMI medium, supplemented with 20 mM Hepes, pH 7.4 and 0.1% BSA at a density of 0.5 x 10^ cells/mL. 0.1 mL of cell suspension was added to each well and incubated for 1 hr at 37°C, in the presence or absence of various α_νβ3 antagonists. Following incubation, 0.025 mL of a 10% formaldéhyde solution, pH 7.4, was added and the cells were fixed at room température for 10 min. The plates were washed 3 times with 0.2 mL of RPMI medium and the adhèrent cells were stained with 0.1 mL of 0.5% toluidine blue for 20 min at room température. Excess stain was removed by extensive washing with deionized water. The toluidine blue incorporated into cells was eluted by the addition of 0.1 mL of 50% éthanol containing 50 mM HCl. Cell adhesion was quantitated at an optical density of 600 nm on a microtiter plate reader (Titertek Multiskan MC, Sterling, VA).

Solid-Phase α_νβ5 Binding Assay:

The vitronectin receptor α_νβ5 was purified from human placenta. Receptor préparation was diluted with 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM CaC12, 1 mM MnC12, 1 mM MgC12 (buffer A) and was immediately added to 96-well ELISA plates at 0.1 ml per well. 0.1-0.2 pg of α_νβ3 was added per well. The plates were incubated overnight at 4°C. At the time of the experiment, the wells were washed once with buffer A and were incubated with 0.1 ml of 3.5% bovine sérum albumin in the same buffer for 1 hr at room température. Following incubation the wells were aspirated completely and washed twice with 0.2 ml buffer A.

011076

In a [^HJ-SK&amp;F-107260 compétition assay, various concentrations of unlabeled antagonists (0.001-100 μΜ) were added to the wells, followed by the addition of 5.0 nM of [3HJ-SK&amp;F-107260. The plates were incubated for 1 hr at room température. Following incubation the wells were aspirated completely and washed once with 0.2 ml of ice cold buffer A in a well-to-well fashion. The receptors were solubilized with 0.1 ml of 1% SDS and the bound pHj-SK&amp;F-107260 was determined by liquid scintillation counting with the addition of 3 ml Ready Safe in a Beckman LS 6800 Liquid Scintillation Counter, with 40% efficiency. Nonspecific binding of [^HJ-SK&amp;F-107260 was determined in the presence of 2 μΜ SK&amp;F-107260 and was consistently less than 1% of total radioligand input. The IC50 (concentration of the antagonist to inhibit 50% binding of [^H]-SK&amp;F-107260) was determined by a nonlinear, least squares curve-fitting routine, which was modified from the LUNDON-2 program. The Kj (dissociation constant of the antagonist) was calculated according to Cheng and Prusoff équation: Kj = IC50/ (1 + L/Kj), where L and K4 were the concentration and the dissociation constant of pH]-SK&amp;F-107260, respectively.

Inhibition of RGD-mediated GPIIb-IIIa binding

Purification of GPIIb-IIIa

Ten units of outdated, washed human platelets (obtained from Red Cross) were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4,140 mM NaCl, 2 mM CaC12 at 4°C for 2 h. The lysate was centrifuged at 100,000g for 1 h. The supematant obtained was applied to a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibrated with 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1% octylglucoside (buffer A). After 2 h incubation, the column was washed with 50 mL cold buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A containing 10% dextrose. Ail procedures were performed at 4°C. The GPIIb-IIIa obtained was &gt;95% pure as shown by SDS polyacrylamide gel electrophoresis.

Incorporation of GPIIb-IIIa in Liposomes.

A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti Polar Lipids) were dried to the walls of a glass tube under a stream of nitrogen. Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg/mL and mixed with the phospholipids in a protein:phospholipid ratio of 1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator for 5 min. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cutoff dialysis tubing against a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaC12 (with 2 changes). The GPIIb-IIIacontaining liposomes wee centrifuged at 12,000g for 15 min and resuspended in the dialysis

011076 buffer at a final protein concentration of approximately 1 mg/mL. The liposomes were stored at -70C until needed.

Compétitive Binding to GPIIb-IIIa

The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect compétitive binding method using PHJ-SK&amp;F-107260 as an RGD-type ligand. The binding assay was performed in a 96-well filtration plate assembly (Millipore Corporation, Bedford, MA) using 0.22 um hydrophilic durapore membranes. The wells were precoated with 0.2 mL of 10 pg/mL polylysine (Sigma Chemical Co., St. Louis, MO.) at room température for 1 h to block nonspecific binding. Various concentrations of unlabeled benzazepines were added to the wells in quadruplicate. [^H]-SK&amp;F-107260 was applied to each well at a final concentration of 4.5 nM, followed by the addition of 1 pg of the purified platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at room température. The GPIIb-IIIa-bound [3HJ-SK&amp;F-107260 was seperated from the unbound by filtration using a Millipore filtration manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL). Bound radioactivity remaining on the filters was counted in 1.5 mL Ready Solve (Beckman Instruments, Fullerton, CA) in a Beckman Liquid Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific binding was determined in the presence of 2 pM unlabeled SK&amp;F-107260 and was consistently less than 0.14% of the total radioactivity added to the samples. Ail data points are the mean of quadruplicate déterminations.

Compétition binding data were analyzed by a nonlinear least-squares curve fitting procedure. This method provides the IC50 of the antagonists (concentration of the antagonist which inhibits spécifie binding of pHJ-SK&amp;F-107260 by 50% at equilibrium). The IC50 is related to the equilibrium dissociation constant (Ki) of the antagonist based on the Cheng and Prusoff équation: Ki = IC50/(l+L/Kd), where L is the concentration of [3H]SK&amp;F-107260 used in the compétitive binding assay (4.5 nM), and Kd is the dissociation constant of [3HJ-SK&amp;F-107260 which is 4.5 nM as determined by Scatchard analysis.

Preferred compounds of this invention hâve an affinity for the vitronectin receptor relative to the fibrinogen receptor of greater than 10:1. Most preferred compounds hâve a ratio of activity of greater than 100:1.

The efficacy of the compounds of this invention alone or in combination with an antineoplastic agent may be determined using several transplantable mouse tumor models. See U. S. Patent Nos. 5,004,758 and 5,633,016 for details of these models

011076

The examples which follow are intended in no way to limit the scope of this invention, but are provided to illustrate how to make and use the compounds of this invention. Many other embodiments will be readily apparent to those skilled in the art.

EXAMPLES

General

Nuclear magnetic résonance spectra were recorded at either 250 or 400 MHz using, respectively, a Bruker AM 250 or Bruker AC 400 spectrometer. CDCI3 is deuteriochloroform, DMSO-dè is hexadeuteriodimethylsulfoxide, and CD3OD is tetradeuteriomethanol. Chemical shifts are reported in parts per million (δ) downfield from the internai standard tetramethylsilane. Abbreviations for NMR data are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, app = apparent, br = broad. J indicates the NMR coupling constant measured in Hertz. Continuous wave infrared (IR) spectra were recorded on a PerkinElmer 683 infrared spectrometer, and Fourier transform infrared (Fl'lR) spectra were recorded on a Nicolet Impact 400 D infrared spectrometer. IR and FTIR spectra were recorded in transmission mode, and band positions are reported in inverse wavenumbers (cm'l). Mass spectra were taken on either VG 70 FE, PE Syx API III, or VG ZAB HF instruments, using fast atom bombardment (FAB) or electrospray (ES) ionization techniques. Elemental analyses were obtained using a Perkin-Elmer 240C elemental analyzer. Melting points were taken on a Thomas-Hoover melting point apparatus and are uncorrected. Ail températures are reported in degrees Celsius.

Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Both flash and gravity chromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel. Analytical and préparative HPLC were carried out on Rainin or Beckman chromatographs. ODS refers to an octadecylsilyl derivatized silica gel chromatographie support. 5 μ Apex-ODS indicates an octadecylsilyl derivatized silica gel chromatographie support having a nominal particle size of 5 μ, made by Jones Chromatography, Littleton, Colorado. YMC ODS-AQ® is an ODS chromatographie support and is a registered trademark of YMC Co. Ltd., Kyoto, Japan. PRP-1® is a polymeric (styrene-divinylbenzene) chromatographie support, and is a registered trademark of Hamilton Co., Reno, Nevada. Celïte® is a filter aid composed of acid-washed diatomaceous silica, and is a registered trademark of Manville Corp., Denver, Colorado.

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Préparation 1

Préparation of ethyl (±1-10,1 l-dihydro-3-carboxy-5H-dibenzoia.dlcycloheptene-10-acetate

a) 3-Benzyl-4-(trifluoromethanesulfonyloxy)anisole

Trifluoromethanesulfonic anhydride (10.0 mL, 60 mmol) was added over 3 min to a solution of 2-benzyl-4-methoxyphenol (10.71 g, 50 mmol; prepared according toJ. Am. Chem. Soc. 1949, 71, 64) and anhydrous 2,6-lutidine (12.0 mL, 100 mmol) in anhydrous CH2CI2 (250 mL) at -78°C under argon. The reaction was stirred at -78°C for 0.5 h, then was warmed to RT. After 1 h, the reaction was diluted with hexanes (250 mL) and washed sequentially with 1.0 N HCl (2 x 100 mL), 1.0 N NaOH (2 x 50 mL), H2O (100 mL) and brine (50 mL). Drying (Na2SO4), concentration, and silica gel chromatography (10% EtOAc/hexanes) gave the title compound as a light yellow solid (16.65 g, 96%): TLC Rf 0.51 (10% EtOAc/hexanes); NMR (250 MHz, CDCI3) δ 7.10-7.40 (m, 6H), 6.77 (dd, J = 9.0, 3.1 Hz, 1H), 6.66 (d, J = 3.1 Hz, 1H), 4.03 (s, 2H), 3.73 (s, 3H); FTIR (CCI4) 1492, 1423, 1405, 1249, 1216, 1161, 1144, 1039, 869 cm’¹; MS (ES) m/e 369 (M+Na)+, 364.0 (M+NH4)⁺, 347.0 (M +H)+.

b) 4-AllyI-3-benzylanisole

LiCl (3.08 g, 72.8 mmol) in a roundbottom flask was flame-dried in high vacuum, and the System was allowed to cool to RT under argon. 3-Benzyl-4(trifluoromethanesulfonyloxy)anisole (21.0 g, 60.6 mmol), bis(triphenylphosphine)palladium(II) chloride (2.13 g, 3.0 mmol), anhydrous DMF (150 mL), and allyItributyltin (22.6 mL, 72.8 mmol) were added, and the mixture was purged with argon through three evacuation/argon flush cycles. The mixture was heated in an oil bath preset at 95°C, affording a yellow, homogeneous solution. After 1.5 h, the dark mixture was concentrated on the rotavap (high vacuum), and the residue was reconcentrated from xylenes. The resulting residue was taken up in Et20 (120 mL) and stirred briskly with 10% KF (120 mL) for 0.5 h. The layers were separated, and the aqueous layer was extracted with Et20 (2 x 120 mL). The combined organics were fîltered through celite® to remove the insoluble solids, and the filtrate was washed sequentially with H2O (60 mL) and brine (60 mL). Drying (MgSO4) and concentration left a cloudy, yellow oil. Chromatography (silica gel, 5% EtOAc/hexanes) gave the title compound as a light yellow oil (14.21 g, 98%); TLC Rf (5% EtOAc/hexanes) 0.51; NMR (250 MHz, CDCI3) δ 7.03-7.31 (m, 6H), 6.74 (dd, J = 8.3, 2.7 Hz, 1H), 6.66 (d, J = 2.7 Hz, 1H), 5.79-5.98 (m, 1H), 4.89-5.07 (m, 2H), 3.97 (s, 2H), 3.75 (s, 3H), 3.21-3.33 (m, 2H); FTIR (CCI4) 1610, 1496, 1256, 1046, 914 cm'¹; MS (ES) m/e 239.2 (M +H)⁺.

011076

c) 2-Benzyl-4-methoxyphenylacetic acid

A solution of H5IO6 (23.83 g, 104.5 mmol) in H₂O (56 mL) was added to a solution of 4-allyl-3-benzylanisole (5.30 g, 22.24 mmol) in CCI4 (28 mL) and CH3CN (28 mL), and the well-stirred mixture was cooled thoroughly to 0°C. RuCl₃ (231 mg, 1.11 mmol) was added, and the reaction was stirred briskly at 0°C for 4 h, then at RT for 45 min. The mixture was filtered through celite®, and the filter pad was washed with CH₂C12 (120 mL) then with H₂O (120 mL). The layers were separated and the aqueous layer was extracted with CH2CI2 (3 x 120 mL). Drying (Na2SO4) and concentration left a brown oil. This was partitioned between Et20 (90 mL) and 0.25 N NaOH (90 mL), and the layers were separated. The Et₂O layer was extracted with 0.25 N NaOH (2 x 10 mL), and the combined aqueous layers were acidified (pH 2) with conc. HCl. CH2CI2 extraction, drying (Na2SO4), and concentration gave the title compound as a yellow oil which solidified to a yellow solid (4.19 g, 74%): *H NMR (250 MHz, CDC1₃) δ 7.05-7.35 (m, 6H), 6.77 (dd, J = 8.3, 2.7 Hz, 1H), 6.71 (d, J = 2.7 Hz, 1H), 4.00 (s, 2H), 3.76 (s, 3H), 3.54 (s, 2H); FTIR (CCI4) 2300-3500 (broad), 1710, 1611, 1502, 1496, 1285, 1257, 1045 cm'¹; MS (ES) m/e 279.0 (M+Na)⁺, 274.0 (M+NH4)+, 257.0 (M +H)+.

d) 3-Methoxy-5H-dibenzo[a,d]cycloheptene-10(l lH)-one

Finely powdered 2-benzyl-4-methoxyphenylacetic acid (3.26 g, 12.72 mmol) was added to well-stirred polyphosphoric acid (165 g) at 100-110°C. After 15 min, the reaction was poured onto ice (330 g). Et20 (330 mL) was added, and the mixture was stirred briskly for 15 min. The layers were separated, and the aqueous layer was extracted with Et20 (330 mL). The combined organic layers were washed with 5% NaHCO₃ (2 x 80 mL) then with brine (80 mL), dried (MgSO4), and concentrated. The residue was reconcentrated from toluene, then was chromatographed (silica gel, 20% EtOAc/hexanes). The title compound was obtained as a yellow solid (1.44 g, 48%): TLC Rf (20% EtOAc/hexanes) 0.46; ^]H NMR (250 MHz, CDC1₃) δ 8.07-8.15 (m, 1H), 7.39-7.49 (m, 1H), 7.25-7.48 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 2.6 Hz, 1H), 6.71 (dd, J = 8.3,

2.6 Hz, 1H), 4.21 (s, 2H), 4.11 (s, 2H), 3.77 (s, 3H); FTIR (CCI4) 1680, 1501, 1282,1270 cm'¹; MS (ES) m/e 261 (M+Na)+, 256.0 (M+NH4)+, 239.0 (M +H)+.

e) Ethyl (±)-10,l l-dihydro-10-hydroxy-3-methoxy-5H-dibenzo[a,d]cycloheptene-10acetate

Anhydrous EtOAc (0.58 mL, 6.6 mmol) was added dropwise to a solution of lithium bis(trimethylsilyl)amide (1.0 M in THF, 6 mL, 6 mmol) in dry THF (24 mL) in a flame-dried flask at -78°C under argon. The yellow solution was stirred at -78°C for 0.5 h,

011076 then a solution of 3-methoxy-5H-dibenzo[a,d]cycloheptene-10(l lH)-one (715 mg, 3 mmol) in dry THF (3 mL) was added dropwise over 3 min. Additional dry THF (0.4 mL) was used in transfer. After 0.5 h at -78°C, the reaction was quenched with saturated NH4CI (15 mL), warmed to RT, and extracted with EtOAc (2 x 30 mL). Drying (MgSO4), concentration, and chromatography (silica gel, 10% EtOAc/hexanes (400 mL), then 20% EtOAc/hexanes) gave recovered 3-methoxy-5H-dibenzo[a,d]cycloheptene-10(l lH)-one (305.4 mg, 43%) as a yellow solid, followed by the title compound as a light yellow oil (531.9 mg, 54%); TLC Rf 0.37 (20% EtOAc/hexanes); NMR (250 MHz, CDCI3) δ

7.63 (d, J = 7.7 Hz, 1H), 7.00-7.30 (m, 4H), 6.80 (d, J = 2.6 Hz, 1H), 6.69 (dd, J = 8.2, 2.6 Hz, 1H), 3.95-4.35 (m, 2H), 4.07 (s, 2H), 3.76 (s, 3H), 3.68 (s, 1H), 3.64 (d, J = 14.2 Hz, 1H), 3.35 (d, J = 14.2 Hz, 1H), 2.79 (d, J = 16.0 Hz, 1H), 2.66 (d, J = 16.0 Hz, 1H), 1.22 (t, J = 7.2 Hz, 3H); FTIR (CCI4) 3580 (sharp), 3509 (broad), 1735, 1715, 1503, 1261, 1198, 1156, 1044 cm'¹; MS (ES) m/e 675.2 (2M+Na)+, 653.2 (2M +H)+.

f) Ethyl (±)-10,l l-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

10% Pd/C (242 mg, 0.23 mmol) was added to a solution of ethyl (±)-10,lldihydro-10-hydroxy-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate (741.1 mg, 2.27 mmol) and conc. HCl (0.19 mL, 2.27 mmol) in glacial AcOH (23 mL), and the mixture was shaken on a Parr apparatus at RT under H2 (50 psi). After 6 h, the reaction was filtered through celite®, and the filter pad was washed with EtOAc. The filtrate was concentrated, and the residue was reconcentrated from toluene. The resulting faintly yellow, oily residue was chromatographed (silica gel, 20% EtOAc/hexanes) to afford the title compound as a colorless oil (643.6 mg, 91%): TLC Rf 0.57 (20% EtOAc/hexanes); !h NMR (250 MHz, CDCI3) δ 7.05-7.22 (m, 4H), 7.01 (d, J = 8.2 Hz, 1H), 6.76 (d, J = 2.7 Hz, 1H), 6.67 (dd, J = 8.2, 2.7 Hz, 1H), 4.30 (d, J = 15.0 Hz, 1H), 4.11-4.25 (m, 2H), 3.85 (d, J = 15.0 Hz, 1H), 3.70-3.90 (m, 1H), 3.77 (s, 3H), 3.31 (dd, J = 15.0, 4.1 Hz, 1H), 2.93 (dd, J = 15.0, 9.2 Hz, 1H), 2.64 (dd, J = 15.6, 5.0 Hz, 1H), 2.52 (dd, J = 15.6, 9.3 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H); FTIR (CCI4) 1734, 1611, 1504, 1285, 1263,1155, 1044 cm’¹; MS (ES) m/e 333.0 (M+Na)+, 328.0 (M+NH4)+, 311.0 (M +H)+, 265.0 (M+H-EtOH)+.

g) Ethyl (±)-10,11 -dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate

Anhydrous AICI3 (1.38 g, 10.35 mmol) was added ail at once to a solution of ethyl (±)-10,1 l-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate (643.6 mg, 2.07 mmol) in anhydrous CH2CI2 (21 mL) at 0°C under argon. The yellow solution was warmed to RT and stirred for 3 h, then was cooled to 0°C and quenched with cold 3 N HCl (10 mL). The layers were separated, and the aqueous layer was extracted with CH2CI2. The combined organic layers were dried (MgSO4) and concentrated. Silica gel

011076 chromatography (25% EtOAc/hexanes) gave the title compound as a nearly colorless oil (611.7 mg, 100%): TLC Rf 0.26 (20% EtOAc/hexanes); NMR (400 MHz, CDCI3) δ 7.03-7.22 (m, 4H), 6.93 (d, J = 8.1 Hz, 1H), 6.69 (d, J = 2.6 Hz, 1H), 6.58 (dd, J = 8.1, 2.6 Hz, 1H), 5.00 (s, 1H), 4.25 (d, J = 14.9 Hz, 1H), 4.11-4.25 (m, 2H), 3.73-3.88 (m, 1H), 3.79 (d, J = 14.9 Hz, 1H), 3.28 (dd, J = 15.0, 4.1 Hz, 1H), 2.91 (dd, J = 15.0, 9.3 Hz, 1H), 2.65 (dd, J = 15.6, 4.9 Hz, 1H), 2.53 (dd, J = 15.6, 9.5 Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H); FTIR (CCI4) 3611 (Sharp), 3447 (broad), 1734, 1504, 1291, 1272, 1176, 1152 cm'¹; MS (ES) m/e 314.2 (M+NH4)^{i) * * 4}·, 297.2 (M +H)⁺.

h) Ethyl (±)-10,l l-dihydro-3-(trifluoromethanesulfonyloxy)-5Hdibenzo[a,d]cycloheptene-10-acetate

Trifluoromethanesulfonic anhydride (0.45 mL, 2.68 mmol) was added dropwise to a solution of ethyl (±)-10,ll-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate (611.7 mg, 2.06 mmol) and 2,6-lutidine (0.48 mL, 4.12 mmol) in anhydrous CH2CI2 (10.3 mL) at -78°C under argon. After 0.5 h, the reaction was warmed to RT and stirred for 1 h. The yellow solution was diluted with Et2Û (50 mL) and washed sequentially with 1.0 N HCl (5 mL), 5% NaHCC&gt;3 (5 mL), and brine (5 mL). Drying (MgSO4), concentration, and silica gel chromatography (20% EtOAc/hexanes) gave the title compound as a colorless oil (808.9 mg, 92%); TLC Rf (20% EtOAc/hexanes) 0.58; iH NMR (250 MHz, CDCI3) δ 6.98-7.30 (m, 7H), 4.35 (d, J = 15.2 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.91 (d, J = 15.2 Hz, 1H), 3.78-3.95 (m, 1H), 3.37 (dd, J = 15.2, 4.1 Hz, 1H), 3.02 (dd, J = 15.2, 9.6 Hz, 1H),

2.70 (dd, J = 15.8, 4.8 Hz, 1H), 2.53 (dd, J = 15.8, 9.6 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H); FTIR (CCI4) 1735, 1493, 1427, 1250, 1215, 1144, 961, 856 cm’¹; MS (ES) m/e 451.1 (M+Na)+, 446.2 (M+NH4)+, 429.2 (M +H)+.

i) Ethyl (±)~ 10,1 l-dihydro-3-carboxy-5H-dibenzo[a,d]cycloheptene-10-acetate

A mixture of ethyl (+)-10,1 l-dihydro-3-(trifluoromethanesulfonyloxy)-5Hdibenzo[a,d]cycloheptene-10-acetate (808.9 mg, 1.89 mmol), KOAc (742 mg, 7.56 mmol),

Pd(OAc)2 (21.2 mg, 0.095 mmol), l,l'-bis(diphenylphosphino)ferrocene (210 mg, 0.38 mmol), and anhydrous DMSO ( 11 mL) was purged with carbon monoxide (three evacuation/CO flush cycles, followed by bubbling CO through the mixture for 5 min), then was stirred under a balloon of CO in an oil bath set at 70°C. After 3.5 h, the reaction was diluted with H2O (11 mL), cooled to 0°C, and acidified with 1.0 N HCl (ca. 8 mL). CH2CI2 extraction (3 x 30 mL), drying (Na2SO4), concentration, and reconcentration from toluene left a reddish-orange liquid (2-3 mL). Chromatography (silica gel, 3:2:0.1 EtOAc/toluene/AcOH; mixed fractions again with 1:1:0.1 EtOAc/toluene/AcOH) gave the title compound (581.9 mg, 95%) as a viscous, yellow oil which partially crystallized in high

011076 vacuum at 40°C: TLC Rf (3:2:0.1 EtOAc/toluene/AcOH) 0.60; NMR (250 MHz, CDCI3) δ 7.95 (d, J = 1.5 Hz, IH), 7.87 (dd, J = 7.8, 1.5 Hz, IH), 7.00-7.35 (m, 5H), 4.40 (d, J = 15.2 Hz, IH), 4.19 (q, J = 7.1 Hz, 2H), 3.97 (d, J = 15.2 Hz, IH), 3.82-4.00 (m, IH), 3.43 (dd, J = 15.3, 4.0 Hz, IH), 3.07 (dd, J = 15.3, 9.5 Hz, IH), 2.69 (dd, J = 15.8, 4.8 Hz, IH), 2.53 (dd, J = 15.8, 9.5 Hz, IH), 1.28 (t, J = 7.1 Hz, 3H); FTIR (CCI4) 2357-3378 (broad), 1735,1692,1280 cm'¹; MS (ES) m/e 342.2 (M+NH4)⁴·, 325.2 (M +H)+, 307.2 (M+H-H2O)⁴-.

Préparation 2

Préparation of Ethyl 2-Carboxy-10,ll-dihydro-5H-dibenzo[a,dl cycloheptenyl-10-acetate

a) Methyl 2-Benzoyl-5-methoxyphenylacetate

Methyl 3-methoxy-phenylacetate was treated with benzoyl chloride and aluminum chloride as described in J. Chem. Soc., Perkin Trans 1,1991, 171 to give the title compound.

b) Methyl 2-Benzyl-5-methoxyphenylacetate

The compound of Préparation 2(a) is treated with sodium borohydride and trifluoroacetic acid in dichloromethane according to the general procedure of Synthesis 1978, 763, to give the title compound.

c) 2-Benzyl-5-methoxyphenylacetic Acid

The compound of Préparation 2(b) is treated with aqueous sodium hydroxide and methanol and stirred. The mixture is concentrated and treated with dilute hydrochloric acid to give the title compound.

d) 5,11 -Dihydro-2-methoxy-10H-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(c) is added to a mixture of phosphoric acid and phosphorous pentoxide stirred and heated to 80°C according to the general procedure of United States Patent 3,567,730 to give the title compound.

e) 5,1 l-Dihydro-2-hydroxy-10H-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(d) is treated with ethanethiol and aluminum chloride according to the general procedure of Tetrahedron. Letters 1978, 5211 to give the title compound.

011076

f) 5,11 -Dihydro-2-(trifluoromethanesulfony l)oxy-1 OH-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(e) is treated with triflic anhydride according to the general procedure of J. Chem. Soc., Chem. Commun. 1987, 904 to give the title compound.

g) 5,1 l-Dihydro-2-methoxycarbonyl-10H-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(f) is treated with carbon monoxide, methanol, palladium acetate and l,3-bis(diphenylphosphino)propare in dimethyl sulfoxide according to the general procedure of J. Chem. Soc., Chem. Commun. 1987, 904 to give the title compound.

h) 5,1 l-Dihydro-2-carboxy-10H-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(g) is stirred with dilute aqueous sodium hydroxide. The mixture is treated with dilute hydrochloric acid to give the title compound.

i) 5,ll-Dihydro-2-tert-butoxycarbonyl-10H-dibenzo[a,d]cyclohepten-10-one

The compound of Préparation 2(h) is treated with Ν,Ν-dimethylformamide di-tertbutyl acetal accoding to the general procedure of Synthesis 1983, 2,135 to give the title compound.

j) Ethyl 2-tert-Butoxycarbonyl-5H-dibenzo[a,d]cycloheptene-10-acetate

The compound of Préparation 2(i) is treated with zinc powder and ethyl bromoacetate according to the general procedure of Or g. Reactions 1947,1, 1 and J. Am. Chem. Soc. 1938, 60, 2947 to give the title compound.

k) Ethyl 2-Carboxy-5H-dibenzo[a,d]cycloheptene-10-acetate

The compound of Préparation 2(j) was treated with trifluoroacetic acid in dichloromethane and stirred. The mixture is concentrated to give the title compound.

Préparation 3

Préparation of 2-[(2-aminoethyI)aminolpyridine dihydrochloride

a) Mono-Boc-l,2-ethylenediamine

A solution of di-ierr-butyl dicarbonate (10.91 g, 50 mmole) in CH2CI2 (50 mL) was added dropwise over 30 min to a briskly stirred solution of 1,2-ethylenediamine (33 mL, 500 mmole) in CH2CI2 (250 mL) at 0°C under argon. A precipitate separated during the addition. When the addition was complété, the reaction was warmed to RT, stirred for 1

011076 hr, and concentrated on the rotavap. The residue was taken up in H₂O (100 mL) and filtered to remove a small amount of insoluble material. The filtrate was extracted with CH₂C1₂ (3 x 100 mL), and the combined organics were dried (MgSOq.) and concentrated to afford the title compound (6.00 g, 75 %) as a cloudy liquid: NMR (250, CDCI3) δ 4.75

- 5.00 (m, 1 H), 3.05 - 3.25 (m, 2 H), 2.65 - 2.85 (m, 2 H), 1.46 (s, 9 H), 1 12 (br s, 2 H).

b) 2-[[2-(Boc-amino)ethyl]amino]pyridine-N-oxide

A mixture of mono-Boc-l,2-ethyIenediamine (5.83 g, 36.39 mmole), 2chloropyridine-N-oxide hydrochloride (7.25 g, 43.67 mmole), NaHCOj (15.29 g, 182 mmole), and tert-amyl alcohol (36 mL) was heated at reflux. After 47 hr, the dark brown mixture was cooled, diluted with CH₂C1₂ (100 mL), and suction filtered to remove insoluble materials. The filtrate was concentrated and reconcentrated from toluene. Silica gel chromatography (10% MeOH/CHC^) gave impure title compound (8.23 g, 89%) as a yellow solid which was used without further purification: TLC (10% MeOH/CHClg) Rf 0.42; !h NMR (250, CDCI3) δ 8.16 (dd, J = 6.5, 1.3 Hz, 1 H), 7.05 - 7.30 (m, 2 H), 6.68 (br d, J = 8.6 Hz, 1 H), 6.50 - 6.65 (m, 1 H), 5.70 - 5.95 (m, 1 H), 3.25 - 3.60 (m, 4 H), 1.44 (s, 9 H); MS (ES) m/e 254 (M + H)+.

c) 2-[[2-(Boc-amino)ethyl]amino]pyridine

10% Pd/C (106.4 mg, 0.10 mmole) was added to a solution of 2-[[2-(Bocamino)ethyl]amino]pyridine-N-oxide (126.7 mg, 0.5 mmole) and cyclohexene (0.25 mL, 0.25 mmole) in absolute EtOH (5 mL), and the mixture was heated to reflux. After 16 hr, the reaction was filtered through celite® and the filtrate was concentrated. The residue was combined with the residue obtained from a separate préparation (0.5 mmole scale), and the combined materials were purified by silica gel chromatography (5% MeOH/CHC^). The title compound (148.4 mg, 63% based on 1 mmole of 2-[[2-(Bocamino)ethyl]amino]pyridine-N-oxide) was obtained as a yellow oil: TLC (5% MeOH/CHCl₃) R_f 0.43; NMR (400, CDCI3) δ 8.05 - 8.12 (m, 1 H), 7.37 - 7.46 (m, 1 H), 6.53 - 6.61 (m, 1 H), 6.41 (d, J = 8.3 Hz, 1 H), 5.12 (br s, 1 H), 4.86 (br s, 1 H), 3.26 -

3.51 (m, 4 H), 1.44 (s, 9 H); MS (ES) m/e 238 (M + H)⁺.

d) 2-[(2-Aminoethyl)amino]pyridine dihydrochloride

N HCl in dioxane (3.2 mL) was added in a stream to a solution of 2-[[2-(Bocamino)ethyl]amino]pyridine (148.4 mg, 0.63 mmole) in anhydrous CH₂C1₂ (3.2 mL) at 0°C, then the reaction was warmed to RT. After 2 hr, the mixture was suction filtered to collect the precipitated solid, which was washed with anhydrous Et₂O and dried to afford the title compound (132.8 mg, quantitative) as a yellow solid: NMR (400, CD3OD) Ô

011076

7.99 - 8.07 (m, 1 H), 7.92 - 7.98 (m, 1 H), 7.19 (d, J = 9.1 Hz, 1 H), 6.98 - 7.04 (m, 1 H), 3.76 (t, J = 6.2 Hz, 2 H), 3.27 (t, J - 6.2 Hz, 2 H, partially obscured by residual solvent signal); MS (ES) m/e 138 (M + H)+.

Préparation 4

Préparation of 2-[(3-hydroxy-l-propyl)amino1pyridine-N-oxide

a) 2-[(3-Hydroxy-l-propyl)amino]pyridine-N-oxide

A mixture of 2-chloropyridine-N-oxide (16.6 g, 0.1 mole), 3-amino-l-propanol (15.3 mL, 0.2 mole), NaHCO₃ (42 g, 0.5 mole), and ieri-amyl alcohol (100 mL) was heated to reflux. After 21 hr, the reaction was cooled, diluted with CH2CI2 (300 mL), and suction filtered to remove insoluble materials. The filtrate was concentrated and reconcentrated from toluene to leave a yellow oil. Silica gel chromatography ( 20% MeOH/CHCl₃) gave the title compound (15.62 g, 93%) as a yellow solid: TLC (20% MeOH/CHCl₃) Rf 0.48; !h NMR (250, CDC1₃) δ 8.07 (dd, J = 6.6, 1.2 Hz, 1 H), 7.34 (br t, 1 H), 7.10 - 7.30 (m, 1 H), 6.64 (dd, J = 8.5, 1.4 Hz, 1 H), 6.40 - 6.60 (m, 1 H), 4.49 (br s, 1 H), 3.65 - 3.90 (m, 2 H), 3.35 - 3.60 (m, 2 H), 1.75 - 2.00 (m, 2 H); MS (ES) m/e 169 (M+ H)+.

Préparation 5

Préparation of 2-lf3-hydroxy-l-propyl)amino]-4-nitropyridine-N-oxide

a) 2-[(3-Hydroxy-l-propyl)amino]-4-nitropyridine-N-oxide

According to the procedure of Préparation 12, except substituting 2-chloro-4nitropyridine-N-oxide (see Jain, P. C.; Chatterjee, S. K.; Anand, N. Indian Journal of Chemistry 1966, 403) for the 2-chloropyridine-N-oxide hydrochloride, the title compound was prepared as an orange solid: MS (ES) m/e 214.2 (M + H)⁺.

Préparation 6

Préparation of 2-[(3-hydroxy-l-propyl)amino1-4-methylpyridine-N-oxide

a) 2-[(3-Hydroxy-l-propyl)amino]-4-methylpyridine-N-oxide

According to the procedure of Préparation 12, except substituting 2-chloro-4methylpyridine-N-oxide (see Brown, E. V. J. Amer. Chem. Soc. 1957, 79, 3565) for the 2

011076 chloropyridine-N-oxide hydrochloride, the title compound was prepared as an off-white solid: MS (ES) m/e 183 (M + H)+.

Préparation 7

Préparation of 2-(ethylamino)-6-pyridylethanol

a) 2-(N-Boc-amino)-6-picoline

To a stirred solution of 2-amino-6-picoline (4.33 g, 40 mmol), Et3N (6.2 mL, 40 mmol) and CH2CI2 (50 mL) at 0°C was added di-ierr-butyl dicarbonate (9.6 g, 44 mmol). After stirring at RT overnight, the reaction mixture was concentrated in vacuum, diluted with H2O, and extracted with CH2CI2 (2 x 50 mL). Drying (MgSC&gt;4) and concentration gave the title compound as colorless oil: MS (ES) m/e 209 (M+H)+.

b) 2-(N-Boc-N-ethylamino)-6-picoline

To a suspension of NaH (60% dispersion in minerai oil, 1.15 g, 29.5 mmol) in DMF (50 mL) at 0°C was added a solution of 2-(N-Boc-amino)-6-picoline in DMF (30 mL). The reaction was stirred at 0°C for 15 min; then ethyl iodide (4.6 g, 30 mmol) was added. The reaction mixture was stirred at RT overnight; then was concentrated in vacuum. The residue was diluted with H2O and extracted with CH2CI2 (3 x 50 mL). Drying (MgSOfo, concentration, and silica gel chromatography (20% EtOAc/hexane) gave the title compound as colorless oil: MS (ES) m/e 237 (M+H)⁺.

c) Ethyl 2-(N-Boc-N-ethylamino)-6-pyridylacetate

LDA (0.018 mol) was prepared in THF (30 mL), cooled to -78°C, and 2-(N-Boc-Nethylamino)-6-picoline (3.5 g, 15 mmol) was added, forming a deep red solution. After 15 min, diethyl carbonate (2.2 mL, 17.9 mmol) was added, the burgundy-colored solution was stirred at 78°C for an additional 15 min, then the reaction was quenched with saturated NH4CI. The mixture was warmed to RT and extracted with EtOAc(3 x 30 mL). The combined organic layers were washed with brine, dried (MgSO4), ^an^ concentrated. Silica gel chromatography (10% EtOAc/hexane) gave the title compound as colorless oil: MS (ES) m/e 309 (M+H)⁺.

d) Ethyl 2-(ethylamino)-6-pyridylacetate

A solution of ethyl 2-(N-Boc-N-ethylamino)-6-pyridylacetate (1.5 g, 4.87 mmol) and 4M HCl/dioxane (5 mL, 20 mol) was stirred at RT overnight, then was concentrated. Reconcentration from toluene gave the title compound as white solid: MS (ES) m/e 209 (M+H)⁺.

011076

e) 2-(Ethylamino)-6-pyridylethanol

To a mechanically stirred solution of L1AIH4 in THF(1.0 M, 20 mL, 20.4 mmol) was added dropwise a solution of ethyl 2-(ethylamino)-6-pyridylacetate (1 g, 4.1 mmol) in THF (30 mL). After the addition was completed, the reaction mixture was heated to reflux. After 5 hr, the reaction was cooled to 0°C and quenched with 10% NaOH solution. The solids were removed by filtration, and the filtrate was concentrated in vacuum. The residue was dissolved in CH2CI2, and the solution was dried (MgSC&gt;4) ^an&lt;^ concentrated. Reconcentration from toluene (3 x) gave the title compound as a colorless oil: MS (ES) 167 (M+H)+.

Préparation 8

Préparation of 10,1 l-Dihydro-3-methoxy-5H-dibenzo[a,dlcyclohepten-10-one

a) 2-Benzyl-4-methoxyphenylacetic acid

A solution of 2-benzoyl-4-methoxyphenylacetic acid (13.0 g, 0.048 mol), prepared by the method of J. Med. Chem. 1981,24, 998, in glacial acetic acid (600 mL) was treated under argon with 4.3 g. of 10% Pd/C and hydrogenated at 50 psi for 17 hours. The mixture was filtered using celite® and the filtrate was concentrated and reconcentrated from toluene and methylene chloride to give 14.2 of the title compound: NMR (400 MHz, CDCI3) δ

3.52 (s, 2H), 3.75 (s, 3H), 4.0 (s, 3H), 6.7 (m, 2H), 7.15 (m, 6H).

b) 10,11 -Dihydro-3-methoxy-5H-dibenzo[a,d]cyclohepten-10-one

A solution of 2-benzyl-4-methoxyphenylacetic acid (14.2 g, 0.055m) in benzene (120 mL) and thionyl chloride (28 mL) was refluxed for 1 hour and concentrated. The acid chloride was dissolved in dry methylene chloride (40 mL), and the solution was added dropwise under argon to a solution of AICI3 (14.7 g, 0.11 mol) in methylene chloride (600 mL). The reaction was stirred under an argon atmosphère for 2.5 hours at room température, then was quenched with ice-water (200 mL). The layers were separated, and the organic phase was washed sequentially with 10% NaOH solution, water, and dil. HCl. The resulting solution was diluted with ether (200 mL), dried over MgSO4, and concentrated. The solid residue was triturated with ether/hexane (1:1) and 9.35 g of the title compound was collected by filtration: Mp 105 - 106°C; NMR (400 MHz, CDCI3) δ 3.72 (s, 3H), 4.1 (s, 2H), 4.2 (s, 2H), 6.7 (d, 1H), 6.82 (s, 1H), 7.30 (m, 4H), 8.1 (d, 1H).

Préparation 9

Préparation of ethyl (tMO.ll-dihydro-S-methoxy-SH-dibenzola.dlcycloheptene-lO-acetate

a) Ethyl (±) 3-(3-methoxyphenyl)indeneacetate

To a cold solution of 3-(3-methoxyphenyl)indene (4 g, 18 mmol), prepared by the method of J. Med. Chem. 1981, 24, 998, in THF (15 mL) at 0°Cwas added dropwise a solution of LiN(TMS)2 (20 mL, IM in THF) over 5 min. The resulting solution was added dropwise to a solution of ethyl bromoacetate (3.34 g, 20 mmol) in THF (15 mL) at -78°C over 30 min. After 2.5 h, the mixture was quenched with saturated ammonium chloride solution and the layers were separated. The organic layer was dried over MgSO„ and concentrated to give the crude product which was purified by column chromatography (SiO/2-4% EtOAc/hexane) to give title compound (1.1g): ^H NMR (400 MHz, CDCI3) δ

1.30 (t, 3H), 2.50 (m, 1H), 2.85 (m, 1H), 3.85 (s, 3H), 4.0 (m, 1H), 4.20 (q, 2H), 6.6 (s, 1H), 6.9 (m, 1H), 7.2 (s, 1H), 7.35 (m, 6H).

b) Ethyl (±) 3-[(3-methoxybenzoyl)]phenylsuccinate

A solution of ethyl (±) 3-(3-methoxyphenyl)indeneacetate (1.1 g, 3.6 mmol) in acetone (30 mL) was treated with 4% aqueous solution of osmium tetroxide (0.5 mL) followed by a dropwise addition of 1.2 M Jones reagent (5 mL, 6 mmol) according to the literature procedure (J. Or g. Chem. 1993, 58, 4745). After stirring ovemight at room température, the dark reaction mixture was quenched with isopropanol (2.5 mL), followed by sodium bisulfite (0.9 g) and water (30 mL). The product was extracted with ethyl acetate, washed with brine, dried over MgSOzj., and concentrated to give a solid residue. Trituration with 1:1 ether / hexane gave 0.76 g of the title compound: ^H NMR (400 MHz, CDCI3) δ 1.18 (t, 3H), 2.90 (m, 1H), 3.3 (m, 1H), 3.92 (s, 3H), 4.1 (q, 2H), 4.4 (m, 1H), 4.4 (d, 1H), 7.25 (m, 2H), 7.5 (m, 6H).

c) Ethyl (±) 3-[(3-methoxybenzyl)]phenyIsuccinate

A mixture of ethyl (±) 3-[(3-methoxybenzoyl)]phenylsuccinate (0.76 g., 2.1 mmol) and 10% Pd/C (0.6 g) in glacial acetic acid (35 mL) was hydrogenated at 50 psi for 17 hours. The mixture was filtered using celite® and the filter pad was washed with acetic acid. The filtrate was concentrated and reconcentrated from toluene and methylene chloride to give 0.65 g of the title compound: ÏH NMR (400 MHz, CDCI3) δ 1.20 (t, 3H), 2.20 (m, 1H), 3.0 (m, 1H), 3.74 (s, 3H), 4.1 (q, 2H), 4.18 (q, 2H), 4.4 (d, 1H), 6.2 (m, 2H),

7.22 (m, 6H).

011076

d) Ethyl (±)-10,l l-dihydro-3-methoxy-l l-oxo-5H-dibenzo[a,d]cycloheptene-10-acetate

To a magnetically stirred solution of ethyl (±) 3-[(3methoxybenzyl)]phenylsuccinate (0.65 g, 1.9 mmol) in dry methylene chloride (10 mL) were added DMF (0.2 mL) and oxalyl chloride (0.2 mL, 2.28 mmol). After 1.5 h, the solution was added dropwise to a suspension of aluminum chloride (0.6 g, 4.5 mmol) in dry methylene chloride (15 mL). The mixture was quenched after 2 h with ice water, the layers were separated, and the aqueous layer was extracted with methylene chloride. The combined organic layers were dried over MgSC&gt;4 and concentrated. The residue was purified by column chromatography (SiO/2-4% EtOAc/hexane) to give title compound (0.3 g): !h NMR (400 MHz, CDCI3) δ 1.28 (t, 3H), 2.88 (m, 1H), 3.55 (m, 1H), 3.84 (s, 3H), 3.88 (d, 1H), 4.18 (q, 2H), 4.85 (d, 1H), 4.95 (m, 1H), 5.8 (m, 2H), 7.22 (m, 4H), 8.1 (s, 1H).

e) Ethyl (±)-10,l l-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

A mixture of ethyl (±)-10,ll-dihydro-3-methoxy-ll-oxo-5H-dibenzo[a,d]cycloheptene-10acetate (0.3 g., 0.93 mmol) and 10% Pd/C (0.3 g) in glacial acetic acid (25 mL) was hydrogenated at 50 psi for 18 hours. The mixture was filtered using celite® and washed with acetic acid. The filtrate was concentrated and reconcentrated from toluene and methylene chloride to give 0.25 g of the title compound: ^H NMR (400 MHz, CDCI3) δ

1.28 (t, 3H), 2.60 (m, 2H), 2.90 (m, 1H), 3.30 (m, 1H), 3.80 (s, 3H), 3.85 (d, 1H), 4.18 (q, 2H), 4.30 (d, 1H), 6.70 (m, 2H), 7.0 (d, 1H), 7.22 (m, 4H).

The following compounds illustrate methods for preparing the biologically active compounds of this invention from intermediate compounds such as described in the foregoing Préparations.

Example 1

Préparation of (+)-10,1 l-dihydro-3-i3-(2-pyridylamino)-l-propyloxyl-5Hdibenzo|a,d]cycloheptene-10-acetic acid

a) Ethyl (±)-10,ll-dihydro-3-[3-[2-(N-oxopyridyl)amino]-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

A solution of 2-[(3-hydroxy-l-propyl)amino]pyridine-N-oxide (1.94 g, 11.55 mmole) and diethyl azodicarboxylate (1.8 mL, 11.55 mmole) in anhydrous DMF (58 mL) was added dropwise over 10 min to a solution of ethyl (+)-10,1 l-dihydro-3-hydroxy-5Hdibenzo[a,d]cycloheptene-10-acetate (1.37 g, 4.62 mmole) and triphenylphosphine (3.27 g,

011076

12.47 mmole) in anhydrous DMF (23 mL) at RT under argon. After 23.5 hr, the reaction was concentrated on the rotavap, and the residue was reconcentrated from xylenes to remove residual DMF. Silica gel chromatography (30% EtOAc/hexanes (0.5 L), then EtOAc (1 L), then 5% MeOH/CHC^) gave recovered ethyl (±)-10,ll-dihydro-3-hydroxy5H-dibenzo[a,d]cycloheptene-10-acetate (298.1 mg, 22%), then the title compound (1.54 g, 75 %) as a yellow oil: ^!H NMR (250 MHz, CDCI3) δ 8.10 (dd, J = 6.6, 1.3 Hz, 1 H), 6.85 -

7.30 (m, 7 H), 6.78 (d, J = 2.6 Hz, 1 H), 6.68 (dd, J = 8.2, 2.6 Hz, 1 H), 6.61 (dd, J = 8.5,

1.6 Hz, 1 H), 6.45 - 6.57 (m, 1 H), 4.29 (d, J = 15.1 Hz, 1 H), 4.10 - 4.25 (m, 2 H), 4.06 (t, J = 5.7 Hz, 2 H), 3.84 (d, J = 15.1 Hz, 1 H), 3.70 - 3.92 (m, 1 H), 3.49 (q, J = 6.5 Hz, 2 H),

3.30 (dd, J = 15.0, 4.2 Hz, 1 H), 2.93 (dd, J = 15.0, 9.3 Hz, 1 H), 2.65 (dd, J = 15.6, 5.0 Hz,

H), 2.51 (dd, J = 15.6, 9.4 Hz, 1 H), 2.05 - 2.25 (m, 2 H), 1.27 (t, J = 7.1 Hz, 3 H); MS (ES) m/e 447 (M + H)+.

b) Ethyl (±)-10,11 -dihydro-3-[3-(2-pyridylamino)-1 -propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

A mixture of ethyl (±)-10,ll-dihydro-3-[3-[2-(N-oxopyridyl)amino]-l-propyloxyJ5H-dibenzo[a,d]cycloheptene-10-acetate (1.54 g, 3.45 mmole), 10% Pd/C (0.73 g, 0.69 mmole), cyclohexene (7 mL, 6.9 mmole), and isopropanol (35 mL) was heated at reflux for hr, then the catalyst was removed by filtration through celite®. Concentration left a yellow oil, which was resubmitted to the reaction conditions. After 17 hr, the reaction was worked up as before, and yellow residue was again submitted to the reaction conditions, using 1:1 EtOAc/isopropanoI (35 mL) instead of isopropanol as solvent. The mixture was heated at reflux for 5 hr, Pd black (73 mg, 0.69 mmole) was added, and reflux was continued for another 18.5 hr. Work up as before followed by silica gel chromatography ( 1:1 EtOAc/hexanes) gave the title compound (0.94 g, 63%) as a light yellow oil: TLC Rf (1:1 EtOAc/hexanes) 0.38; ÎH NMR (400 MHz, CDCI3) δ 8.02 - 8.11 (m, 1 H), 7.33 - 7.42 (m, 1 H), 7.02 - 7.20 (m, 4 H), 7.00 (d, J = 8.2 Hz, 1 H), 6.77 (d, J = 2.6 Hz, 1 H), 6.67 (dd, J = 8.2, 2.6 Hz, 1 H), 6.50 - 6.60 (m, 1 H), 6.39 (d, J = 8.5 Hz, 1 H), 4.69 (br t, 1 H), 4.29 (d, J = 15.0 Hz, 1 H), 4.11 - 4.25 (m, 2 H), 4.05 (t, J = 5.8 Hz, 2 H), 3.84 (d, J = 15.0 Hz, 1 H), 3.75 - 3.90 (m, 1 H), 3.48 (app. q, 2 H), 3.30 (dd, J = 15.0, 4.1 Hz, 1 H), 2.93 (dd, J = 15.0, 9.2 Hz, 1 H), 2.64 (dd, J = 15.6, 4.8 Hz, 1 H), 2.52 (dd, J = 15.6, 9.5 Hz, 1 H), 2.02 -

2.15 (m, 2 H), 1.27 (t, J = 7.2 Hz, 3 H); MS (ES) m/e 431 (M + H)+.

c) (+)-10,1 l-Dihydro-3-[3-(2-pyridylamino)-l-propyloxy]-5H-dibenzo[a,djcycloheptene10-acetic acid

A mixture of ethyl (±)-10,l l-dihydro-3-[3-(2-pyridylamino)-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-l0-acetate (0.94 g, 2.18 mmole) and 1.0 N NaOH (2.6 mL, 2.62

011076 mmole) in absolute EtOH (19.2 mL) is warmed in an oil bath set at 50°C. After 28.5 hr, the reaction was concentrated on the rotavap and the residue was purified by ODS chromatography (1:1 MeOHÆ^O). Concentration left a cloudy, aqueous solution, which was made homogeneous by addition of a little 1.0 N NaOH. The pH was adjusted to 7 with 1.0 N HCl, and the solid precipitate was collected and washed with H2O. The mother liquors were concentrated, and the residue treated similarly to afford a small second crop.

The combined materials were dried in high vacuum at 40°C to afford the title compound (0.70 g, 79%) as a nearly colorless solid: HPLC (Hamilton PRP-1®, 40% CH3CN/H2O containing 0.1% TFA) k’ = 1.2; !h NMR (400 MHz, DMSO-dg) δ 7.94 (d, J = 4.4 Hz, 1

H), 7.32 - 7.41 (m, 1 H), 7.03 - 7.22 (m, 4 H), 6.97 (d, J = 8.4 Hz, 1 H), 6.83 (d, J = 2.4 Hz,

H), 6.60 - 6.74 (m, 2 H), 6.41 - 6.50 (m, 2 H), 4.20 (d, J = 14.7 Hz, 1 H), 4.00 (t, J = 6.2 Hz, 2 H), 3.88 (d, J = 14.7 Hz, 1 H), 3.60 - 3.70 (m, 1 H), 3.26 - 3.40 (m, 2 H, partially obscured by residual solvent signal), 3.20 (dd, J = 15.1,4.3 Hz, 1 H), 2.83 (dd, J = 15.1, 10.3 Hz, 1 H), 2.60 (dd, J = 15.9, 5.3 Hz, 1 H), 2.50 (dd, 1 H, partially obscured by residual solvent signal), 1.88 - 2.00 (m, 2 H); MS (ES) m/e 403 (M + H)+. Anal. Calcd for ^C25^H26^N2°3 · °-25 H₂O: C, 73.78; H, 6.56; N, 6.88. Found: C, 73.85; H, 6.47; N, 6.75.

Example 2

Préparation of (±~)-10,l l-dihydro-3-r3-(4-amino-2-pyridylamino)-l-propyloxyl-5Hdibenzo[a,dlcycloheptene-10-acetic acid

a) Ethyl (±)-10,11 -dihydro-3- [3- [2-(4-nitro-N-oxopyridyl)amino]-1 -propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

According to the procedure of Example l(a), except substituting 2-[(3-hydroxy-lpropyl)amino]-4-nitropyridine-N-oxide for the 2-[(3-hydroxy-l-propyl)amino]pyridine-Noxide, the title compound was obtained following silica gel chromatography (gradient: 2:1 EtOAc/hexanes, then EtOAc, then 5% MeOH in 1:1 EtOAc/CHC13): MS (ES) m/e 492 (M + H)+.

b) Ethyl (±)-10,l l-dihydro-3-[3-(4-amino-2-pyridylamino)-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

According to the procedure of Example 1 (b), except substituting ethyl (±)-10,l 1dihydro-3-[3-[2-(4-nitro-N-oxopyridyl)amino]-l-propyloxy]-5H-dibenzo[a,d]cycloheptene10-acetate for the ethyl (±)-10,1 l-dihydro-3-[3-[2-(N-oxopyridyl)amino]-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-l 0-acetate, the title compound was obtained following silica gel chromatography (20% MeOH/CHC^): MS (ES) m/e 446 (M + H)⁺.

011076

c) (+)-10,11 -Dihy dro-3-[3-(4-amino-2-pyridy lamino)-1 -propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetic acid

1.0 N LiOH (0.74 mL, 0.74 mmole) was added ail at once to a solution of ethyl (±)10,11 -dihydro-3-[3-(4-amino-2-pyridylamino)-1 -propyloxy]-5H-dibenzo[a,d]cycloheptene10-acetate (216.3 mg, 0.49 mmole) in THF (2.5 mL) and H2O (1.8 mL) at RT. The twophase mixture was warmed for 1 hr in an oil bath set at 40°C, then absolute EtOH (ca. 1 mL) was added to combine the phases. The reaction was kept at 40°C for 19.5 hr, then was concentrated, and the residue was dissolved in 1:1 CH3CN/H2O (5 mL). The solution was acidified with TFA (0.11 mL, 1.47 mmole) and concentrated. ODS chromatography (40% CH3CN/H2O containing 0.1% TFA) followed by concentration to remove CH3CN left an oily, aqueous solution, which was made homogeneous by addition of a little CH3CN. The pH was adjusted to 7 with 1.0 N NaOH, and an oily semisolid precipitated. The mixture was concentrated to remove the CH3CN, and the resulting solid was collected. This was dissolved in aqueous NaOH, and the pH was adjusted to 7. The solid was collected, washed with plenty of H2O, and dried in high vacuum at 45°C to afford the title compound (133.3 mg, 61%) as an off-white solid: HPLC (Hamilton PRP-1®, 35% CH3CN/H2O containing 0.1% TFA) k’ = 3.0; *H NMR (400 MHz, DMSO-d₆) δ 7.48 (d, J = 6.3 Hz, 1 H), 7.04 -

7.22 (m, 4 H), 6.95 (d, J = 8.2 Hz, 1 H), 6.88 - 7.02 (m, 1 H), 6.82 (d, J = 2.4 Hz, 1 H), 6.67 (dd, J = 8.2, 2.4 Hz, 1 H), 6.27 (br s, 2 H), 5.95 (dd, 1 H), 5.65 (narrow d, 1 H), 4.18 (d, J =

14.7 Hz, 1 H), 4.00 (t, J = 6.1 Hz, 2 H), 3.88 (d, J = 14.7 Hz, 1 H), 3.59 - 3.70 (m, 1 H), 3.13 - 3.30 (m, 3 H), 2.82 (dd, J = 15.2, 10.0 Hz, 1 H), 2.46 (dd, J = 15.8, 8.8 Hz, 1 H, partially obscured by residual solvent signal), 2.58 (dd, J = 15.8, 5.2 Hz, 1 H), 1.86 - 2.01 (m, 2 H); MS (ES) m/e 418 (M + H)+. Anal. Calcd for C25H27N3O3 · 1.67 H₂O: C, 67.09; H, 6.83; N, 9.39. Found: C, 66.99; H, 6.59; N, 9.34.

Example 3

Préparation of (±)-10,l l-Dihydro-3-[3-(4-methyl-2-pyridylamino)-l-propyloxyl-5Hdibenzoia.dlcycloheptene-10-acetic acid

a) Ethyl (±)-10,l l-dihydro-3-[3-[2-(4-methyl-N-oxopyridyl)amino]-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

According to the procedure of Example l(a), except substituting 2-[(3-hydroxy-lpropyl)amino]-4-methylpyridine-N-oxide for the 2-[(3-hydroxy-l-propyl)amino]pyridineN-oxide, the title compound was obtained as a pale yellow oil following silica gel

011076 chromatography (gradient: 30 ,50,100% EtOAc in hexane, then 1-5% MeOH in CHCI3): MS (ES) m/e 461.3 (M + H)+.

b) Ethyl (±)-10,1 l-dihydro-3-[3-(4-methyl-2-pyridylamino)-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate

According to the procedure of Example l(b), except substituting ethyl (±)-10,11dihydro-3-[3-[2-(4-methyl-N-oxopyridyl)amino]-l-propyloxy]-5Hdibenzo[a,d]cycloheptene-10-acetate for the ethyl (±)-10,ll-dihydro-3-[3-[2-(Noxopyridyl)amino]-l-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetate, the title compound was obtained following silica gel chromatography (gradient: 30 - 50% EtOAc in hexane): MS (ES) m/e 445.3(M + H)⁺.

c) (±)-10,ll-Dihydro-3-[3-(4-methyl-2-pyridyIamino)-l-propyloxy]-5Hdibenzo[a,d]cycloheptene- 10-acetic acid

1.0 N NaOH (0.144 mL, 0.144 mmole) was added to a solution of ethyl (±)-10,l 1dihydro-3-[3-(4-methyl-2-pyridylamino)-l-propyloxy]-5H-dibenzo[a,d]cycloheptene-10acetate (35 mg, 0.08 mmole) in EtOH(5 mL) at RT. The mixture was warmed ovemight in an oil bath set at 32°C, then was concentrated. The residue was dissolved in H₂O (3 mL), and the solution was acidified with 20% TFA. ODS chromatography (5-10% CH3CN/H₂O containing 0.1% TFA) followed by concentration and lyophilization gave the title compound as a white powder: MS (ES) m/e 417.3(M + H)+. Anal. Calcd for C₂₆H₂₈N₂O₃ · ²·⁰ TFA ’ ¹⁰ H₂O: ^c· 54.38; H, 4.87; N, 4.23. Found: C, 54.66; H, 6.4.53; N, 4.32.

Example 4

Préparation of (+)-10,1 l-dihydro-3-[2-i6-(ethylamino)-2-pyridyll-l-ethoxyl-5Hdibenzoia.dlcycloheptene-10-acetic acid

a) Ethyl (±)-10,l l-dihydro-3-[2-[6-(ethylamino)-2-pyridyl]-l-ethoxy]-5Hdibenzo [a,d]cycloheptene-10-acetate

According to the procedure of example l(a), except substituting 2-(ethylamino)-6pyridylethanol for the 2-[(3-hydroxy-l-propyl)amino]pyridine-N-oxide, the title compound was obtained as colorless oil following silica gel chromatography (5% MeOH/CH₂Cl₂): MS (ES) 445 (M+H)+.

011 Ü 7 ό

b) (±)-10,ll-Dihydro-3-[2-[6-(ethylamino)-2-pyridyl]-l-ethoxy]-5H-dibenzo[a,d]cycloheptene-10acetic acid

Ethyl (±)-10,11 -dihydro-3-[2- [6-(ethy lamino)-2-pyridy 1]-1 -ethoxy]-5Hdibenzo[a,d]cycloheptene-10-acetic acid (80 mg, 0.18 mmol) was dissolved in MeOH (3 mL), and

1.0 N NaOH (0.22 mL, 0.22 mmol) was added. The solution was stirred at RT ovemight, then was concentrated in vacuum. The residue was dissolved in H2O (3 mL), and the solution was acidified with 20% TFA. Chromatography on C-18 Bond Elute (30% CH3CN/H2O containing 0.1% TFA) gave the title compound as a white solid: MS(ES) 417 (M+H)⁺. Anal. Calcd for C26H28N2O3 1.3 CF3CO2H: C, 60.83; H, 5.23; N, 4.96. Found: C, 60.64; H, 5.26; N, 4.26.

The above description fully discloses how to make and use the présent invention.

However, the présent invention is not limited to the particular embodiments described hereinabove, but includes ail modifications thereof within the scope of the following claims. The various references to joumals, patents and other publications which are cited 15 herein comprises the state of the art and are incorporated herein by reference as though fully set forth.