CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-part application 5 of U.S. Patent Application Serial No. 07/869,230 filed April 14, 1992, pending.

FIELD OF THE INVENTION

The present invention relates to the field of dibenz[c,e]azepine compounds. More particularly, the present 10 invention relates to novel dibenz[c,e]azepines that exhibit protein kinase C inhibitory activity and that are useful for treating inflammatory, cardiovascular, neoplastic and other diseases.

BACKGROUND OF THE INVENTION

15 Protein kinase C (PKC) is a family of calcium- and phospholipid-dependent serine/threonine-specific protein kinases which play an important role in cellular growth control, regulation, and differentiation. Protein kinase C is also fundamental to the processes involved in tumorigenicity,

20 since it is the major high-affinity receptor for several classes of tumor promoters as well as for endogenous cellular diacylglycerols. These tumor promoters also stimulate protein kinase C catalysis. Castagna, et al . , J. Biol . Chem . , 1982, 257, 7847, reported direct activation of protein kinase C by

25 tumor promoting phorbol esters. The mechanisms of protein kinase C action have been described in U.S. Patent 4,816,450

issued March 28, 1989 to Bell, et al . , the disclosures of which are specifically incorporated as if fully set forth herein. Protein kinase C is activated by diacylglycerol (DAG) , a neutral lipid, and when activated will transfer the 7-phosphate of MgATP to a serine or threonine residue on a substrate protein.

Since the activation of protein kinase C has been implicated in several human disease processes, including cancer tumors, inflammation, and reperfusion injury, inhibition of protein kinase C should be of great therapeutic value in treating these conditions.

Cancer is a disease characterized in part by uncontrolled cell growth. Protein kinase C is directly involved in cellular growth control and is believed to be involved in tumor formation. Protein kinase C is the major, if not exclusive, intracellular receptor of phorbol esters which are very potent tumor promoters. Phorbol esters and other tumor promoters bind to and activate protein kinase C. Since diacylglycerol (DAG) and phorbol esters interact at the same site, DAG's have been suggested to be the "endogenous phorbol esters" by analogy with the opiate receptor, where the conservation of a high affinity receptor implied the existence of an endogenous analogue. DAG has been shown to increase the affinity of protein kinase C for Ca ⁺² and phospholipid and, thus, to activate protein kinase C at cellular levels of these essential cofactors. Extracellular signals including hormones, growth factors, and neurotransmitters are known to stimulate phosphatidylinositol turnover, resulting in the generation of IP ₃ and DAG. Structures of 40 distinct oncogenes of viral and cellular origin have revealed that oncogenes encode altered forms of normal cellular proteins. Several of the gene products appear related to growth factors or other elements involved in transmembrane signalling. These oncogene products appear to function by altering the level of critical second messengers. Cells transformed with the oncogenes ras, sis, erbB , abl , and src have been shown to contain elevated levels of DAG, which is believed to activate protein kinase C. Indeed

studies on ras transformed cells have shown protein kinase C activation concomitant with elevation of DAG.

Phorbol esters such as phorbol myristate acetate (PMA) have complex effects on cells, including effects on membrane function, mitogenesis, differentiation, and gene expression. Synthetic diacylglycerols mimic many of the effects of PMA in vitro and inhibitors of protein kinase C have been shown to block PMA-induced effects on cells. Thus, protein kinase C may mediate the actions of certain oncogenes, such as ras , which cause intracellular increases in DAG and concomitant increases in protein kinase C. In addition, activation of protein kinase C leads to the expression of c-myc, c-fos , c-cis , and c-fms , nuclear protooncogenes important in cell transformation. Overexpression of protein kinase C in NIH 3T3 cells causes altered growth regulation and enhanced tumorigenicity. In rat fibroblasts such overexpression leads to anchorage-independent growth in soft agar. Overexpression of protein kinase C in these cells resulted in tumor formation in animals receiving transplanted cells. Several studies have shown increased expression of protein kinase C in certain tumor types such as breast and lung carcinomas. Activated protein kinase C has also been detected in human colon carcinomas, although increased expression at the gene level was not seen. Topoisomerases are directly modulated by protein kinase C as substrates for the enzyme. Protein kinase C inhibitors have been shown to potentiate the action of chemotherapy drugs such as cis-platin.

Protein kinase C inhibitors have been reported to potentiate the antitumor activity of cis-platin both in vitro and in vivo (Grunicke, et al . , Adv. Enzyme Regul . , 1989, 28 , 201 and German Offenlegungsschrift DE 3827974) . In addition, it has been suggested that protein kinase C would be a potential target for therapeutic design because of its central role in cell growth (Tritton, et al . , Cancer Cells, 1990, 2 , 95-102). Further, inflammation and reperfusion injury, particularly pertaining to cardiac injury, are common conditions for which there exists no definitive treatment

despite extensive research. Appropriate treatments for these conditions are needed.

Protein kinase C inhibitors have been demonstrated to block platelet aggregation and release of neutrophil activating agents such as platelet activating factor (PAF) (Schachtele, et al. , Biochem. Biophy. Res. Comm n. , 1988, 151, 542; Hannun, et al., J. Biol. Chen., 1987, 262, 13620; Yamada, etal., Biochem.

Pharmacol., 1988, 37, 1161). Protein kinase C inhibitors have also been shown to inhibit neutrophil activation, and che otactic migration (Mclntyre, et al., J. Biol Chem. , 1987,

262, 15730; Lambreth, et al., J. Biol. Chem., 1988, 263, 3818;

Pittet, et al., J. Biol. Chem., 1987, 262, 10072; and Gaudry, et al., Immunology, 1988, 63, 715), as well as neutrophil degranulation and release of proteolytic enzymes and reactive oxygen intermediates (Wilson, et al., J. Biol. Chem., 1986,

261, 12616; Fujita, et al. , Biochem. Pharmacol., 1986, 35,

4555; Berkow, et al., J. Leukoc, Biol., 1987, 41, 441;

Salzer, et al., Biochem. Biophys. Res. Commun., 1987, 148, 747;

Kramer, et al., J. Biol. Chem., 1989, 262, 5876; and Dewald, et al., Biochem. J., 1989, 264, 879). Thus inhibitors of protein kinase C have the capability of blocking all three of the most significant mechanisms of pathogenesis associated with myocardial reperfusion injury, and should thus have a decided therapeutic advantage. Additionally, the inhibitory effect of protein kinase C inhibitors on keratinocytes and on the oxidative burst in neutrophils should lead to an anti-inflammatory effect.

German Offenlegungsschrift DE 3827974 Al discloses therapeutic preparations comprising a protein kinase C inhibitor in combination with a lipid, a lipid analogue, a cytostatic agent or phospholipase inhibitor useful for cancer therapy.

Substituted dibenz[c,e]azepines havebeenpatented for pesticide uses (EP 368,176 issued May 16, 1990 to Zurfleh; EP 192,034 issued August 8, 1986 to Zurfleh) , anxiolytic uses

(U.S. Patent No. 4,315,926 issued February 16, 1982 to

Gschwend) , and hypolipidemic uses (U.S. Patent No. 4,689,326 issued August 25, 1987 to Hall). Hall, Acta Pharm . Nord. , 1990, 2 (6) , 387-400 and Pecherer, J. Med. Chem . , 1969, 12 (1) , 149-51 reported that certain substituted dibenzazepines may have anti-inflammatory activity. However, no dibenz[c,e]azepines have been disclosed as protein kinase C inhibitors.

U.S. Patent Number 2,619,484 issued November 25, 1952 to Wenner discloses 6,7-dihydro-5h-dibenz[c,e]azepine and derivatives thereof. The compounds disclosed are not substituted at positions equivalent to the R, - 1^ positions of the present invention.

U.S. Patent Number 3,075,966 issued January 29, 1963 to Hawthorne et al . disclose 5-hydroxy-5h-dibenz[c,e]azepine, derivatives thereof and a method for their manufacture. A process of reducing 5-hydroxy-5h-dibenz[c,e]azepine to 6,7- dihydro-5h-dibenz[c,e]azepine is disclosed. None of the compounds disclosed contain substitutions at positions equivalent to the R, - R ₆ positions of the present invention. U.S. Patent Number 3,116,283 issued December 31, 1963 to Boiler et al . disclose a method of preparing nitrogen heteroσyclic compounds including derivatives of 6,7-dihydro-5h- dibenz[c,e]azepine. None of the compounds disclosed have substitutions at positions equivalent to the R, - R ₆ positions of the present invention.

U.S. Patent Number 3,751,487 issued August 7, 1973 to Brossi et al . disclose substituted dibenz[c,e]azepines. Specifically, compounds are disclosed which are substitute with lower alkyl, lower alkoxy or benzyl alkoxy. No compounds are disclosed which are substituted with hydroxyl at position equivalent to the R ₁ - R ₆ positions of the present invention.

U.S. Patent Number 4,177,122 issued September 26, 1978 to Freed an discloses 11-aminoalykylmorphanthridin-ll-ols.

Freedman does not disclose compounds relevant to the presen invention but does indicate the level of the state of the art.

U.S. Patent Number 4,551,451 issued November 5, 198 to Pestellini et al . disclose tricyclic derivatives of 5,6

U.S. Patent Number 4,551,451 issued November 5, 1985 to Pestellini et al . disclose tricyclic derivatives of 5,6- dihydro-llh-dibenzo[b,e]azepin-6-one having pharmaceutical activity. These compounds are dissimilar from those claimed in the present invention, but reflect the level of the state of the art.

U.S. Patent Number 4,689,326 issued August 25, 1987 to Hall et al . discloses 6,7-dihydro-5h-dibenz[c,e]azepine derivatives. Hall et al. , which relates to a method of treating hyperlipidemia, does not disclose compounds substituted at positions equivalent to the R, - R ₆ positions of the present invention.

U.S. Patent Number 4,769,368 issued September 6, 1988 to Kaiser et al . discloses 2,3,4,8,9,9a-hexahydro-4-aryl-lh- indeno[l,7-cd]azepines. These compounds have a tricyclic core structure including a single benzene ring which can be substituted with hydroxyl groups.

SUMMARY OF THE INVENTION

The present invention provides substituted dibenz[c,e]azepines of the formula

wherein:

R,, R ₂ , R ₃ , R ₄ , Rg and R ₆ , independently, are H, halo, methoxy or hydroxyl, provided that at least one of R ₂ , R ₃ , R ₄ , R _j and R ₆ is hydroxyl; R ₇ and R ₈ are H or taken together form a carbonyl;

R ₁₂ and R ₁₃ are H or taken together form a carbonyl;

A is H or CR _j R^R^;

R, and R ₁₀ are H or taken together form a carbonyl;

R _n is H, C, through C ₁₇ alkyl, C, through C ₁₇ alkenyl C, through C ₁₇ alkynyl, or aryl; and,

R ₁₄ and R ₁₅ , independently are H, halo, methoxy o hydroxyl when at least 5 of R ₁ - R ₆ are hydroxyl; and, R _u an R ₁₅ are H when less than 5 of E, - R ₆ are hydroxyl.

The compounds of the invention inhibit protein kinas C and exert anti-inflammatory, anti-cancer, and reperfusio injury protection effects through their anti-proliferative an anti-inflammatory activities in human neutrophils and tumo cells. Also within the scope of the invention are th pharmaceutically acceptable salts and the optically activ stereoisomers of the compounds of the invention.

The present invention also provides novel methods fo treating conditions related to or affected by inhibition o protein kinase C activity, particularly cancer tumors inflammatory disease, reperfusion injury, and cardia dysfunctions related to reperfusion injury.

The present invention further provides pharmaceutica compositions comprising a compound of the invention and pharmaceutically acceptable carrier or diluent.

This invention is more particularly pointed out in th appended claims and is described in its preferred embodiment in the following description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of th formula

wherein:

R _1r R ₂ , R ₃ , R ₄ , Rg and Rg, independently, are H, halo, methoxy or hydroxyl, provided that at least one of R _l R ₂ , R ₃ , R ₄ , R _g and R _g is hydroxyl; R ₇ and R _g are H or taken together form a carbonyl;

R ₁₂ and R ₁₃ are H or taken together form a carbonyl;

A is H or CRR ₁₀ R ₁₁ ;

R, and R ₁₀ are H or taken together form a carbonyl; _1t is H, C ₁ through C ₁₇ alkyl, C ₁ through C ₁₇ alkenyl, C, through C ₁₇ alkynyl _r or aryl; and,

R ₁₄ and R ₁₅ , independently are H, halo, methoxy or hydroxyl when at least 5 of R, - Rg are hydroxyl; and, R ₁₄ and R ₁₅ are H when less than 5 of R _n - R _g are hydroxyl.

Preferably R ₁ , R ₂ , R ₃ , R ₄ , Rg and Rg are, independently, H or hydroxyl, provided that at least one of _1f R ₂ , R ₃ , R ₄ , _g and R _g is hydroxyl. More preferably, a plurality of R R ₂ , R ₃ , R ₄ , _g and R _g are hydroxyl. More preferably, at least five of R ₁ R ₂ , R ₃ , R ₄ , R _g and Rg is hydroxyl. Most preferably, all six of R _1f R ₂ , R ₃ , R, Rg and Rg are hydroxyl. When four or less of R,, R ₂ , R ₃ , R ₄ , R _g and R ₆ are hydroxyl, R ₁₄ and R ₁₅ are H. When five or all six of R _1f R ₂ , R ₃ , R ₄ , Rg and R _g are hydroxyl, R ₁₄ and R ₁₅ , independently are H, halo, methoxy or hydroxyl. If R ₁₄ and/or R ₁₅ are halo, it is preferably Cl. R ₇ and R _g are H or taken together form a carbonyl.

Preferably, R ₇ and R _g are H.

R ₁₂ and R ₁₃ are H or taken together form a carbonyl. Preferably, R ₁₂ and R ₁₃ are H.

A preferably is H or CR^^R^ wherein R, and R ₁₀ are H and ^ is H, G, through C ₁₇ alkyl or aryl. Preferably, R^ is C, through C ₁₇ alkyl or substituted phenyl. Substituted rings including aromatic rings may be substituted with alkyl, aryl, arylalkyl, alkylaryl, halogen, nitro, amino, acylamino, hydroxy, carboxyl, alkoxy, aryloxy, thioalkoxyl, alkylthio, arylthio or a fused aromatic ring. It is preferred that, if substituted at all, rings are substituted with lower aliphatic or aromatic groups, i.e. up to C ₂₀ or with hydroxyl. It is most

preferred that rings are substituted with hydroxyl or not substituted. Rings substituted with hydroxyl include those substituted with 1-5, preferably 1-3 hydroxyl.

Some preferred compounds are those in which: a plurality of R _1f R ₂ , R ₃ , R ₄ , R _g and R ₆ are hydroxyl;

R ₇ , R _g , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are each H; and, A is H or CRςR^^ wherein R and R ₁₀ are H and R is H, C ₁ through C ₁₇ alkyl, phenyl or substituted phenyl. Some preferred compounds are those in which: five of R _1f R ₂ , R ₃ , R ₄ , Rg and R ₆ are hydroxyl;

R ₇ , R _g , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are each H; and, A is H or CR^^R^ wherein R, and R ₁₀ are H and R,, is H, C, through C ₁₅ alkyl or substituted phenyl. In such embodiments, the one of R,, R ₂ , R ₃ , R ₄ , R _g and R ₆ that is not hydroxyl is preferrably H or halo.

Some preferred compounds are those in which: R _1# R ₂ , R ₃ , R ₄ , Rg and Rg are each hydroxyl; R ₇ , R _g , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are each H; and, A is H or CR-I^oR^ wherein R, and R ₁₀ are H and „ is H, C, through C ₁₅ alkyl or substituted phenyl.

The compounds of the invention have protein kinase C inhibiting activity, and exert anti-inflammatory, anti-cancer, and reperfusion injury protection effects through their anti- proliferative and anti-inflammatory activities in human neutrophils and tumor cells. The compounds and pharmaceutical compositions of the invention are useful for treating conditions related to or affected by inhibitions of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury. The compounds of the invention are selective for protein kinase C and have no effect on cyclic AMP

(cAMP) dependent protein kinase activity. The compounds should thus have no effect on the metabolic pathways associated with stimulation of protein kinase by cAMP. Accordingly, the present invention provides methods of inhibiting protein kinase C comprising contacting protein kinase C with an inhibitory concentration of a compound of the

invention. As used herein, the terms "protein kinase C inhibitory concentration", "protein kinase C inhibitory amount", "inhibitory concentration" and similar terms refer to the amount of a compound that will inhibit protein kinase C activity, reduce the amount of histone HI that is phosphorylated by protein kinase C, or inhibit the activity of protein kinase C by any other measure of protein kinase C activity.

A further aspect of the invention provides methods of inhibiting an oxidative burst in neutrophils which comprise contacting a neutrophil with a compound of the invention in an amount effective to inhibit the oxidative burst, or contacting the neutrophil with a protein kinase C inhibitory concentration of such compound. An additional aspect of the present invention provides methods for treating inflammation which comprise either administering to a mammal suffering from inflammation a compound of the invention in an amount effective to inhibit or lessen inflammation, or administering to the mammal such compound in an amount effective to inhibit protein kinase C. Inhibition of inflammation refers to the amelioration of symptoms of inflammation such as, but not limited to, reduction of swelling of tissue or reduction of pain.

Still another aspect of the invention provides methods for inhibiting growth of mammalian tumor cells and/or tumors which comprise either contacting a mammalian tumor cell or a mammalian tumor with a compound of the invention in an amount effective to inhibit growth of the tumor cell or tumor, or contacting the tumor cell or tumor with an amount of such compound effective to inhibit protein kinase C.

Still another aspect of the invention provides methods of inhibiting keratinocyte proliferation comprising either administering to a keratinocyte a compound of the invention in an amount effective to inhibit proliferation of the keratinocyte, or administering to the keratinocyte a protein kinase C inhibitory amount of such compound.

Compounds according to the present invention inhibit protein kinase C activity. In assays reported below, 6,7- dihydro-5h-dibenz[c,e]azepine derivatives that contain either methoxy or -0(CO)CH ₃ at positions R _u R ₂ , R ₃ , R ₄ , R _g and R ₆ were found to be inactive as protein kinase C inhibitors while compounds hydroxylated at those positions were found to inhibit PKC. The prior art does not teach or suggest dibenz[c,e]azepines with substitutions of positions R., to R ₆ with hydroxyl groups. The prior art does not teach or suggest dibenz[c,e]azepines that inhibit protein kinase C and nothing in the prior art teaches or suggests that dibenzfc,e]azepineε substituted at positions R, to R _g with hydroxyl groups would inhibit protein kinase C.

The compounds and pharmaceutical compositions of the invention can be administered by any method that produces contact of the active ingredient with the agent's site of action in the body of a mammal or in the body fluid or tissue. These methods include but are not limited to oral, topical, hypodermal, intramuscular, intravenous, and intraparenteral. The compounds can be administered singly or in combination wit other compounds of the invention or other pharmaceutica compounds such as chemotherapeutic compounds. The compound also can be administered in conjunction with therapies, such a radiation treatment. Dibenz[c,e]azepine derivatives ar preferably administered with a pharmaceutically acceptabl carrier selected on the basis of the selected route o administration and standard pharmaceutical practice.

The compounds are administered to mammals, preferabl humans, in therapeutically effective amounts to inhibit protei kinase C, to inhibit tumor cell growth or tumor growth, t inhibit inflammation of tissue, to inhibit keratinocyt proliferation, to inhibit oxidative burst from neutrophils o to inhibit platelet aggregation. The dosage administered i any particular instance will depend upon factors such as th pharmacodynamic characteristics of the particular compound, it mode and route of administration, the age, health, and weigh of the recipient, the nature and extent of symptoms, kind o

concurrent treatment, frequency of treatment, and the effect desired.

Pharmaceutically acceptable salts of the compounds of the present invention can also be employed, as can pharmaceutical compositions containing the compounds or salts. Pharmaceutically acceptable salts useful in the invention include hydrochloride, hydrobromide, succinate, fumarate, oxalate, methanesulfonate, sulfate, maleate, malonate, acetate or lactate. It is contemplated that the daily dosage of the compounds will be in the range of from about 0.1 to about 40 mg per kg of body weight, preferably from about l to about 20 mg per kg body weight. The pharmaceutical compositions of the invention can be administered in any dosage form, including a single dosage, divided dosages, or in sustained release form. Persons of ordinary skill will ^* be able to determine dosage forms and amounts with only routine experimentation based upon the considerations of the invention. Isomers of the compounds and pharmaceutical compositions, particularly optically active stereoisomers, are also within the scope of the present invention.

The pharmaceutical compositions of the invention can also be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. They also can be administered parenterally in sterile liquid dosage forms or topically in a carrier. The pharmaceutical compositions _, of the invention can be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Remington ^f s Pharmaceutical Sciences, A. Osol, Mack Publishing Company, Easton, Pennsylvania.

For example, the compounds of the invention can be mixed with powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets. Both tablets and capsules can be manufactured as sustained release products for continuous release of medication over a period of hours. Compressed

tablets can be sugar coated or film coated to mask any unpleasant taste and to protect the tablet from the atmosphere or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance, in addition to a pharmaceutically acceptable diluent such as water, buffer or saline solution.

For parenteral administration, compounds useful in the invention can be mixed with a suitable carrier or diluent such as water, oil, saline solution, aqueous dextrose (glucose) and related sugar solutions, and glycols such as propylene glycol or polyethylene glycols. Solutions for parenteral administration preferably contain a water soluble salt of a compound of the invention. Stabilizing agents, antioxidizing agents and preservatives can also be added. Suitable antioxidizing agents include sodium bisulfite, sodium sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.

Animal studies have shown that perhaps 50% or more of ischemic-related myocardial damage can be attributed to polymorphonuclear leukocytes (neutrophils) which accumulate at the site of occlusion. Damage from the accumulated neutrophils may be due to the release of proteolytic enzymes from the activated neutrophils or the release of reactive oxygen intermediates (ROI) . Much of the "no reflow" phenomenon associated with myocardial ischemia is attributed to myocardial capillary plugging. The plugging of capillaries has been attributed to both aggregated platelets and aggregated neutrophils. Although both cell types are aggregated during the ischemic event, the relative contribution of each to capillary plugging has not yet been established. It is accepted that the damage by neutrophils to myocardial tissue proceeds through a cascade of events, one of the earliest bein the bonding of activated neutrophils to damaged vascula endothelium. However, the binding of the neutrophils is

significantly enhanced by their activation. Thus, an even earlier event is the generation of molecules (such as cytokines, and chemotactic factors) which can function as activation stimuli. These molecules probably originate from damaged and aggregated platelets, from damaged vascular endothelium, or from the oxidation of plasma proteins or lipids by endothelial-derived oxidants.

Strategies for overcoming the deleterious effects of reactive oxygen intermediates have centered on the development of scavengers for the molecules. Superoxide dismutase (SOD) has been shown to be a particularly effective scavenger of superoxide, but suffers from a very short half-life in the blood. Several companies have tackled this problem by creating versions of this enzyme with increased half-lives by techniques such as liposome encapsulation or polyethylene glycol conjugation. Reports on the effectiveness of these new version are mixed. Catalase, a scavenger of hydrogen peroxide, and hydroxyl radical scavengers have also been tested and found to be effective to varying degrees. However, none of the strategies designed to scavenge reactive oxygen intermediates will prevent the aggregation of platelets, the release of chemotactic molecules, the activation and adherence of neutrophils to vascular endothelium, or the release of proteolytic enzymes from activated neutrophils. One advantage of protein kinase C inhibitors as therapeutics for reperfusion injury is that they have been demonstrated to: 1) block platelet aggregation and release of neutrophil activating agents such as FAF; 2) block neutrophil activation, chemotactic migration, and adherence to activated or damaged endothelium; and 3) block neutrophil release of proteolytic enzymes and reactive oxygen intermediates. Thus, these agents have the capability of blocking all three of the most significant mechanisms of pathogenesis associated with reperfusion injury and should thus have a decided therapeutic advantage.

The compounds of the invention can be prepared by methods known in the part for preparing dibenz[c,e]azepines.

such as the methods disclosed herein, with the substitution an addition of appropriate substituents. For example, Scheme shows a method for synthesizing compounds of the invention tha begins with the coupling of 3 ,4,5-trimethoxybenzoyl chlorid and 3,4,5-trimethoxybenzlyamine through an amide linkage (se Example 1) .

Scheme I

X

The corresponding amine is formed with borane treatment and the acetamide is then formed (see Example 2) . The acetamide is dibrominated with bromine and potassium carbonate and coupled with copper powder to give the azepine (see Example 3) . The azepine is then deprotected to remove methyl groups (see Example 4) . Prior to the deprotection step, the acyl group can be substituted by hydrolysis with acid followed by reacylation as shown in Scheme II, wherein hydrolysis with acid followed by reaction with an acylchloride provides compound of the invention with different acyl groups.

Scheme II

Alternatively, as shown in Scheme I , N-3,4,5- trimethoxybenzyl-3,4,5-trimethoxybenzamidecanbeN-substituted prior to azepine ring formation by reaction with an appropriate iodide or bromide such as methyl iodide. Azepine rin formation can be accomplished by treating the N-substituted amide with ferric chloride (see Example 9) . Differing substitution patterns on the benzene rings produce analogous dibenzazepines.

The present invention is illustrated by the followin non-limiting examples.

EXAMPLE 1

N-3, ,5-Trimethoxybenzyl-3,4,5-trimethoxybenzamide.

3 ,4 ,5-Trimethoxybenzoyl chloride (5.0 g, 21.6 mmol) and 4-dimethylamino pyridine (DMAP) (26 mg, 0.21 mmol) were dissolved in methylene chloride (30 mL) . 3,4,5-Trimethoxy- benzyl amine (4.27 g, 21.6 mmol) and triethyl amine (2.19 g, 21.6 mmol) dissolved in methylene chloride (30 mL) were added dropwise at room temperature under N ₂ . After addition the mixture was stirred for an additional hour, then concentrated. The residue was partitioned between water (70 mL) and ethyl acetate (170 mL) . The solid at the solvent interface was filtered off to give 7 g of the product as a white powder. The organic layer was separated, washed with brine (40 mL) , dried (over MgS0 ₄ ) and concentrated to give 1.2 g of the product as a white powder. (Total yield: 8.2 g, 97%). The 1.2 g sample was recrystallized from ethyl acetate/methylene chloride to give 752 mg of a purified sample of the title compound, m.p. 164-166°C; ^T H-NMR (CDC1 ₃ , 300 MHz) : S 3.81 (3H, s) , 3.83 (6H, 2S) , 3.86 (3H, s) , 3.87 (6H, 2 S) , 4.54 (2H, d) , 6.55 (1H, br t, NH) , 7.02 (2H, s) , 7.26 (2H, s) ; IR (KBr) : 3318, 2941, 2837, 1637, 1583, 1535, 1501, 1465, 1419, 1336, 1235, 998 cm ^*1 . Anal. Calcd. for C ₂₀ H ₂₅ NO ₇ : C, 61.37; H, 6.44; N, 3.58. Found: C, 61.37; H, 6.45; N, 3.53.

EXAMPLE 2 N,N-Di(3,4,5-trimethoxybenzyl)acetamide.

N-3,4,5-trimethoxybenzyl-3,4,5-trimethoxybenzamide (7.06 g, 18.0 mmol) was slurried in tetrahydrofuran (THF) (50 mL) and treated with borane (IM in THF, 56 mL) . The mixture was heated to reflux for 16 h. The excess borane was then carefully quenched with methanol (20 mL) and heated at reflux for 8 h to destroy the boron complex. The reaction mixture was then concentrated to an oil and partitioned between ethyl acetate and water (250 mL ea.) The aqueous layer was washed with ethyl acetate (50 mL) and the organic layers were combined, washed with brine (50 mL) , dried (over MgS0 ₄ ) and concentrated to give 7.9 g (>100%) of

N,N-di(3,4,5-trimethoxybenzyl)amine as an oil. ¹ H-NMR (CDC1 ₃ , 300 MHZ) δ 3.73 (4H, s) , 3.81 (6H, 2s) , 3.84 (12H, s) , 6.56 (4H, S).

Crude di(3,4,5-trimethoxybenzyl)amine (6.43 g, 16.7 mmol) was dissolved in methylene chloride (100 mL) and treated with triethyl amine (1.87 g, 2.54 mL, 18.44 mmol) and DMAP (20 mg) . Acetyl chloride (1.38 g, 1.25 mL, 17.6 mmol) in methylene chloride (50 mL) was added dropwise. After addition and stirring for 1 h the reaction mixture was concentrated and partitioned between 1 N HCl (50 mL) and ethyl acetate (100 mL) . The organic layer was washed with brine (50 ml) , dried (over MgS0 ₄ ) and concentrated to an oil which crystallized on standing. The solid was recrystallized from methylene chloride and hexanes to give 4.6 g (75% from starting amide) of the product as a white crystalline solid, m.p. 100-101°C. ¹ H-NMR(CDC1 ₃ , 300 MHz) δ 2.20 (3H, s), 3.80 (18H, s) , 4.40 (2H, S) , 4.54 (2H, S) , 6.30 (2H, s) , 6.44 (2H, s) . IR (KBr) 2991, 2969, 2940, 2840, 1649, 1589, 1505, 1461, 1447, 1432, 1414, 1331, 1232 cm ^"1 . Anal. Calcd. for C ₂₂ H ₂₉ N0 ₇ : C, 62.99; H, 6.97; N, 3.34. Found: C, 62.77; H, 6.96; N, 3.29.

EXAMPLE 3

N-Acetyl-1,2,3-trimethoxybenzo-[5,4-c]-1,2,3-trimethoxybe nzo-

[ ,5-e]-azepine.

N,N-Di(3,4,5-trimethoxybenzyl)acetamide (4 g, 9.37 mmol) was dissolved in methylene chloride (100 mL) and treated with potassium carbonate (1.55 g, 11.2 mmol). The mixture was then treated dropwise with bromine (3 g, 18.7 mmol; 2 equiv. =

60 mL of 5 g/100 mL solution) and allowed to stir overnight.

The reaction mixture was poured onto a flash column an chromatographed (7 x 12 cm silica gel, 4/3/3 : hexanes/ethyl acetate/ ethylene chloride) to give 4.85 g (88%) of

(N,N-Di(2-Bromo-3,4,5-trimethoxybenzyl)acetamide as a clea oil. H-NMR (CDC1 ₃ , 300 MHz) : δ 2.15 (3H, s) , 3.73 (3H, s) ,

3.78 (3H, S), 3.79 (3H, s) , 3.80 (3H, s) , 3.81 (3H, s) , 3.83 (3H, S), 4.48 (2H, s) , 4.69 (2H, s) , 6.33 (1H, s) , 6.7

(1H, s).

N, -Di(2-Bromo-3,4,5-trimethoxybenzyl)acetamide (4.6 g, 7.97 mmol) and copper (7.5 g, 118 mmol) were slurried in nitrobenzene (15 mL) and heated to reflux for approximately 3 h. The reaction mixture was allowed to cool then poured onto a flash column and chromatographed (7 x 12 cm silica gel, ethyl acetate) to give 1.47 g of impure product. The product was dissolved in methylene chloride, heated with decolorizing carbon and rechromatographed (3.9 x 15 cm, ethyl acetate) to give product. The product was triturated with hexanes/ethyl acetate/methylene chloride and filtered to give 380 mg (11%) of the title compound as a white solid, m.p. 160-161°C. H-NMR (CDC1 ₃ , 300 MHz) δ 2.19 (3H, s) , 3.33 (1H, d, J = 13.4 Hz) , 3.68 (3H, S) , 3.72 (3H, s) , 3.82 (1H, d) , 3.86 (3H, s) , 3.88 (3H, S) , 3.89 (6H, 2s), 4.34 (1H, d, J = 12.88 Hz) , 5.19 (1H, d, J = 13.4 Hz), 6.61 (1H, s) , 6.70 (1H, s) ; IR (KBr) : 2981, 2941, 2843, 1639, 1597, 1488, 1463, 1424, 1406, 1322, 1244, 1127, 1106, 926, 859, 534 cm ^"1 . Anal. Calcd. for C ₂₂ H ₂₇ N0 ₇ : C, 63.30; H, 6.52; N, 3.36. Found: C, 63.33; H, 6.56; N, 3.35.

EXAMPLE 4 N-Acetyl-1,2,3-trihydroxybenzo-[5, -c]-1,2,3-trihydroxy- benzo-[4,5-e]-azepine*H ₂ 0 (Compound 1)

N-Acetyl-1,2,3-trimethoxybenzo-[5,4-c]-1,2,3- trimethoxybenzo-[4,5-e]-azepine (0.7 g, 1.67 mmol) was dissolved in methylene chloride (20 mL) and cooled to -78°C. Boron tribromide (15 mL of a l.OM solution in methylene chloride, 15 mmol) was added and the solution was allowed to warm to room temperature and stirred vigorously for 20 h. The resulting slurry was cooled to -78 ^β C and quenched with methanol (20 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 1 h and concentrated to a solid. Water (40 mL) was added to dissolve the solid and the mixture was concentrated. The solid was again dissolved in water and on swirling and heating, the product crystallized out. The product was filtered to give the title compound (495 mg, 84%) as a white solid, m.p. 235-240 ^β C dec. ^-N R (CDC1 ₃ , 300 MHz) δ 2.08 (3H, s) , 3.20- (1H, d, J = 13.1 Hz) , 3.56 (1H,

d, J « 12.76 HZ), 4.32 (1H, d, J = 12.76 Hz) , 4.82 (1H, d, J = 13.1 HZ), 6.37 (1H, s) , 6.53 (1H, s) , 8.49 (2H, br s) , 8.50 (2H, br s) , 9.08 (1H, br s) , 9.16 (1H, br s) ; IR (KBr) : 3467, 3326, 1589, 1458, 1340, 1287, 1055, 1016 cm ^"1 . Anal. Calcd. for C ₁₆ H ₁₅ N0 ₇ *H ₂ 0: C, 54.70; H, 4.88; N, 3.99. Found: C, 54.75; H, 4.91; N, 3.92.

EXAMPLE 5

N-Palmitoyl-1,2,3-trihydroxybenzo-[5,4-c]-1,2,3-trihydrox y- benzo- [ , 5 -e ] - zep ine • l/ 2H ₂ 0 (Compound 2) N-Acetyl-1,2, 3-trimethoxybenzo-[5,4-c]-l,2,3- trimethoxybenzo-[4,5-e]-azepine (Example 3) (425 mg, 1.02 mmol) was slurried in IN HCl (25 mL) and heated to reflux for 24 h. The reaction mixture was then concentrated to a solid. The solid was dissolved in methylene chloride (10 mL) and treated with triethyl amine (303 mg, 412 μl, 3 mmol) followed by palmitoyl chloride (302 mg, 1.1 mmol) and stirred for 24 h. The reaction mixture was then concentrated and chromatographed (silica gel, 4.1 x 15 cm, 30% ethyl acetate in hexanes) to give N-pa lmi toyl - 1 , 2 , 3 - tr imethoxyb en z o - [5,4-c]-l,2,3-trimethoxybenzo-[4,5-e]-azepine (420 mg, 67%) as a waxy solid. ¹ H-NMR (CDC1 ₃ , 300 MHz) δ 0.85 (3H, t, J = 6.9 Hz), 1.2-1.4 (10H, m) , 1.67 (2H, m) , 2.42 (2H, m) , 3.31 (1H, d, J = 13.5 HZ) , 3.67 (3H, s) , 3.72 (3H, s) , 3.78 (1H, d, J = 13) , 3.86-3.89 (4 X 3H, 4 s) , 4.39 (1H, d, J = 13 Hz) , 5.20 (1H, d, J = 13.5 Hz), 6.60 (1H, s) , 6.71 (1H, s) .

N-Palmitoy1-1,2,3-trimethoxybenzo-[5,4-c]-1,2,3- trimethoxy-benzo-[4,5-e]-azepine (406 mg, 0.66 mmol) was dissolved in methylene chloride (10 mL) and cooled to -78°C. Boron tribromide (5.95 mL of a l.OM solution in methylene chloride, 5.95 mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously for 20 h. The clear mixture was cooled to -78 ^β C and quenched with methanol (18 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 3 h and concentrated to a solid. Water and methanol (25 mL ea.) were added to dissolve the solid and the mixture was slightl

concentrated to effect precipitation of the product. The slurry was stirred for 48 h. The product was filtered, air dried, then dried in the vacuum oven (65°C at 0.5 mm Hg) to give the title compound (311 mg, 88%) as an off-white solid. m.p. 140-145°C dec. ¹ H-NMR (DMS0-d ₆ , 300 MHz) δ 0.85 (3H, t, J - 6.22 HZ), 1.2-1.35 (10H, m) , 1.51 (2H, m) , 2.34 (1H, m) , 2.41 (1H, m) , 3.03 (1H, d, J = 13.1 Hz) , 3.50 (1H, d, J = 12.9 HZ) , 4.36 (1H, d, J = 12.9 Hz) , 4.83 (1H, d, J = 13.1 Hz) , 6.36 (1H, S) , 6.50 (1H, S) ; IR (KBr) : 3329, 2919, 2849, 2361, 1573, 1468, 1369, 1205, 1051 cm ^"1 . Anal. Calcd. for C ₃₀ H ₄₃ NO ₇ »0.5 H ₂ 0: C, 66.89; H, 8.23; N, 2.60. Found: C, 66.69; H, 8.23; N, 2.60.

EXAMPLE 6

N-Myristoyl-l,2,3-trihydroxybenzo-[5, -c]-1,2,3-trihydroxy- benzo-[4,5-e]-azepine«l/2H ₂ 0 (Compound 3) N-Acetyl-1,2, 3-trimethoxybenzo-[5, 4-c]-l,2,3- trimethoxybenzo-[4,5-e]-azepine (425 mg, 1.02 mmol) was slurried in IN HCl (25 mL) and heated to reflux for 24 h. The reaction mixture was then concentrated to a solid. The solid was dissolved in methylene chloride (10 mL) and treated with triethyl amine (303 mg, 412 μl, 3 mmol) followed by myristoyl chloride (271 mg, 1.1 mmol) and stirred for 24 h. The reaction mixture was then concentrated and chromatographed (silica gel, 4.1 x 15 cm, 35% ethyl acetate in hexanes) to give N-myristoyl- 1 , 2 , 3 - tr imethoxybenz o- [5,4-c]-l,2,3-trimethoxybenzo-[4,5-e]-azepine (410 mg, 69%) as a waxy solid. -NMR (CDC1 ₃ , 300 MHz) δ 0.85 (3H, t, J = 6.5 Hz), 1.2-1.4 (8H, m), 1.68 (2H, m) , 2.41 (2H, m) , 3.32 (1H, d, J = 13.4 HZ), 3.68 (3H, S) , 3.72 (3H, s) , 3.78 (1H, d, J = 12.9), 3.87-3.89 (4 X 3H, 4 s) , 4.39 (1H, d, J = 13 Hz) , 5.20 (1H, d, J = 13.4 HZ), 6.60 (1H, s) , 6.71 (1H, s) .

N-Myristoyl-1,2,3-trimethoxybenzo-[5,4-c]-1,2,3- trimethoxy-benzo-[4,5-e]-azepine (437 mg, 0.75 mmol) was dissolved in methylene chloride (10 mL) and cooled to -78°C. Boron tribromide (6.71 mL of a l.OM solution in methylene chloride, 6.71 mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously for

20 h. The clear mixture was cooled to -78°C and quenched with methanol (18 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 3 h and concentrated to a solid. Water and methanol (25 mL ea.) were added to dissolve the solid and the mixture was slightly concentrated to effect precipitation of the product. The slurry was stirred for 48 h. The product was filtered, air dried, then dried in the vacuum oven (65°C at 0.5 mm Hg) to give the title compound (326 mg, 87%) as an off-white solid. m.p. 145-150 ^β C dec. ¹ H-NMR (DMSO-d ₆ , 300 MHz) δ 0.86 (3H, t, J = 6.22 Hz), 1.2-1.35 (8H, m) , 1.52 (2H, m) , 2.34 (1H, m) , 2.41 (1H, m) , 3.03 (1H, d, J = 13.1 Hz) , 3.50 (1H, d, J = 12.9 HZ), 4.36 (1H, d, J = 12.9 Hz) , 4.83 (1H, d, J = 13.1 Hz) , 6.36 (1H, s) , 6.50 (1H, s) ; IR (KBr) : 3230, 2919, 2851, 1596, 1204, 1050 cm ^"1 . Anal. Calcd. for C ₂₈ H ₃₉ NO ₇ «0.5 H ₂ 0: C, 65.86; H, 7.90; N, 2.74. Found: C, 65.78; H, 7.94; N, 2.73.

EXAMPLE 7

1,2,3-Trihydroxybenzo-[5,4-c]-1,2,3-trihydroxybβnzo-[4,5 -e]-a zepine«HCl (compound 4) N-Acetyl-1,2,3-trihydroxybenzo-[5,4-c]-l,2,3- trihydroxy-benzo-[4,5-e]-azepine»H ₂ 0 (Example 4) (300mg, 0.85 mmol) was slurried in IN HCl (50 mL) , heated to reflux for 18 h and allowed to cool to room temperature. The reaction mixture was diluted with isopropanol then concentrated until solid fell out of solution. The solid was filtered off under nitrogen and vacuum and dried under nitrogen for 96 h. The solid was then vacuum dried (0.5 mm at 60°C) for 16 h to give the product (155 mg, 55%) as a white solid, m.p. 220°C dec. ¹ H-NMR (DMSO-dg, 300 MHz) δ 3.20 (2H, d, J = 12.6 Hz), 3.84 (2H, d, J = 12.6 Hz), 6.52 (2H, s) , 8.8 (2H, br s) , 9.29 (2H, br s) ; IR (KBr): 3283, 1623, 1449, 1303, 1042 cm ^*1 . Anal. Calcd. for C ₁₄ H ₁₃ NO ₆ »HCL*0.33 H ₂ 0: C, 50.39; H, 4.43; N, 4.20; Cl, 10.62. Found: C, 50.38,50.34; H, 4.42, 4.43; N, 4.2, 4.11; Cl, 10.57, 10.51.

EXAMPLE 8

N-Methyl-N-3,4,5-trimethoxybenzyl-3, ,5-trimethoxybenzamide

N-3,4,5-Trimethoxybenzyl-3,4,5-trimethoxy- benzamide (2 g, 5.1 mmol) and potassium tert-butoxide (0.63 g, 5.6 mmol) were dissolved in DMF (5 mL) and treated with methyl iodode (4.34 g, 31 mmol). The mixture was heated to reflux («45°C) for 5 h. The mixture was then poured into ethyl acetate and water (50 mL ea.). The organic layer was washed with IN HCl solution (2 x 50 mL) , brine (50 mL) , dried (MgS0 ₄ ) and concentrated. The resulting yellow oil was chromatographed (silica gel, 4.1 x 15 cm, 80% ethyl acetate in hexanes) to give the product (1.75 g, 84%) as a clear oil. H-NMR indicated an equilibrating (on the nmr time scale) mixture of rotamers ¹ H-NMR (CDC1 ₃ , 300 MHz) δ 2.90 (minor 3H, br s) , 3.06 (major 3H, s) , 3.6-3.9 (18H, br s's) , 4.47 (major 2H, br s) , 4.62 (minor 2H, br s) , 6.37 (major 2H, br s) , 6.55 (minor 2H, br s) , 6.65 (both 2H, br s) .

EXAMPLE 9

N-Methyl-2-oxo-l,2,3-trimethoxybenzo-[5,4-c]-l-chloro-2,3 ,4- trimethoxybenzo-[5,6-e]-azepine and N-methyl-2-oxo-l,2,3- trimethoxybenzo-[5,4-c]-1,2,3-trimethoxybenzo-[ ,5-e]-azepine

N-Methyl-N-3 , 4 , 5-trimethoxybenzyl-3 ,4,5- trimethoxybenzamide (1.75 g, 4.3 mmol) was dissolved in methylene chloride (25 mL) and added to a solution of ferric chloride (7.0 g, 43 mmol) and acetic acid (2.59 g, 43 mmol) in methylene chloride (60 ml) . After stirring for about 15 m, thin layer chromatography (TLC) (100% ethyl acetate silica gel) indicated no starting material (R _f = 0.57) and two new products

(R _f = 0.78 and 0.48). The reaction mixture was then washed with water (2 x 60 mL) , IN HCl (50 ml) , and IN NaOH (2 x 60 mL) , dried (over MgS0 ₄ ) and concentrated to an oil. The resulting yellow oil was chromatographed (silica gel, 4.1 x 15 cm, 100% ethyl acetate) to give N-methyl-2-oxo-1,2,3- trimethoxybenzo-[5,4-c]-l-chloro-2,3,4-trimethoxybenzo- [5,6-e]-azepine (R _f = 0.78, 0.33 g, 19%; MS (FAB) m/z 438 (M ⁺¹ ) ; ^-NMR (DMSO-dg, 300 MHz) δ 3.11 (6H, s) , 3.51 (6H, s) , 3.70

(6H, S) , 3.85 (6H, s) , 3.89 (12H, s) , 3.90 (6H, s) , 4.08 (2H, d, J = 14.7 HZ), 4.50 (2H, d, J = 14.7 Hz) , 7.15 (2H, s) ) and N-methyl-2-oxo-l , 2 , 3-trimethoxybenzo- [ 5 , 4-c] - 1,2, 3-trimeth¬ oxybenzo- [4, 5-e] -azepine (R _f = 0.48, 0.47 g, 27%; H-NMR (DMSO-d _g , 300 MHz) δ 2.97 (3H, s) , 3.48 (3H, s) , 3.57 (3H, s) , 3.75 (3H, s) , 3.77 (3H, s) , 3.78 (6H, s) , 3.6-3.8 (1H, buried), 4.18 (1H, d, J = 14.3 Hz) , 6.52 (1H, s) , 7.06 (1H, s) ) , both as clear glasses. EXAMPLE 10 N-Methyl-2-oxo-l, 2 , 3-trihydroxybenzo- [5, 4-c] -l-chloro-2 , 3 , 4-t rihydroxybenzo- [ 5 , 6-e ] -azepine (Compound 5)

N-Methyl-2-oxo-l, 2 , 3-trimethoxybenzo- [5 , 4-c] - 4-chloro-l, 2, 3-trimethoxybenzo- [4, 5-e] -azepine (Example 9) (0.3g, 0.37 mmol) was dissolved in methylene chloride (15 mL) and cooled to -78 °C. Boron tribromide (6.7 mL of a l.OM solution in methylene chloride, 6.7 mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously for 19 h. The clear mixture was cooled to -78°C and quenched with methanol (20 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 3 h and concentrated to an oil. The oil was dissolved in water (100 mL) and concentrate to give a white solid. The solid was resuspended in water (50 mL) and filtered, air dried, then dried in the vacuum oven (50°C at 0.5 mm Hg) for 16 h to give the title compound (164 mg, 66%) as a white solid. m.p. 240 ^β C dec. ¹ H-NMR (DMS0-d ₆ , 300 MHz) δ 2.95 (3H, s) , 3.97 (1H, d, J = 14.6 Hz), 4.39 (1H, d, J = 14.6 Hz), 6.74 (1H, s) , 8.3-9.6 (6H, 4 br S) ; IR (KBr): 3474, 3217, 1573, 1430, 1343, 1301,1083, 1030 cm ^"1 . Anal. Calcd. for C ₁₅ H ₁₂ C1N0 ₇ »0.5 H ₂ 0: C, 49.67; H, 3.61; N, 3.86. Found: C, 49.81; H, 3.54; N, 3.88.

EXAMPLE 11

N-Methyl-2-oxo-l, 2 , 3-trihydroxybenzo- [5, -c] -1, 2 , 3-trihydr- oxybenzo- [ 4 , 5 -e ] -azepine ( Compound 6 )

N-Methyl-2-oxo-l, 2 , 3-trimethoxybenzo- [ 5 , 4-c] l,2,3-trimeth-oxybenzo-[4,5-e]-azepine (0.47 g, 1.2 mmol) was dissolved in methylene chloride (20 mL) and cooled to -78'C.

Boron tribromide (10.4 mL of a l.OM solution in methylene chloride, 10.4 mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously for 19 h. The clear mixture was cooled to -78 ^β C and quenched with methanol (20 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 3 h and concentrated to an oil. The oil was dissolved in water (100 mL) and concentrated to give a white solid. The solid was resuspended in water (50 mL) and filtered, air dried, then dried in the vacuum oven (50 ^β C at 0.5 mm Hg) for 16 h to give the title compound (88 mg, 22%) as a white solid, m.p. 260°C dec. ¹ H-NMR (DMSO-dg, 300 MHz) δ 2.89 (3H, s) , 3.70 (1H, d, J = 14.2 HZ), 3.99 (1H, d, J = 14.2 Hz), 6.46 (1H, s) , 6.72 (1H, s), 8.3-9.4 (6H, 4 br s) ; IR (KBr): 3283, 1568, 1454, 1357, 11004455 ccmm ^""11 .. AAnnaall.. CCaallccdd.. ffoorr CC ₁₁₅₅ HH _ll33 NN00 ₇₇ **00..55 HH ₂₂ 00:: CC, 54.14; H, 4.39; N, 4.21. Found: C, 54.03; H, 4.43; N, 4.16.

EXAMPLE 12

1,2,3-trimethoxybenzo-[5,4-c]-1,2,3-trimethoxybenzo-

[ 4 , 5 - e ] - a z e p i n e . N-Acetyl-1,2,3-trimethoxybenzo-[5,4-c]-l,2,3- trimethoxybenzo-[4,5-e]-azepine (Example 3) (300 mg, 0.72 mmol) was slurried in IN HCl (15 mL) and heated to reflux for 16 h.The reaction mixture was concentrated to give the product hydrochloride as a crystalline solid (m.p. 230-232 ^β C) . The solid was partitioned between ethyl acetate and cold IN NaOH (25 mL ea.) . The organic layer was washed with brine (25 mL) , dried (over MgS0 ₄ ) and concentrated to a solid. The solid was dissolved in methanol and diluted with water. On partial concentration, a solid crystalized from the solution. The crystals were filtered off to give the title compound (90 mg, 33%) as a white crystalline solid. m.p. 164-165 ^β C. ¹ H-NMR (DMSO-d ₆ /D ₂ 0, 300 MHz) d 3.03 (2H, d, J = 12.1 Hz), 3.47 (6H, S) , 3.05 (2H, d, J = 12.1 HZ) , 3.71 (6H, S) , 3.78 (6H, s) , 6.76 (2H, S) ; IR (KBr): 3314, 2949, 1596, 1461, 1320, 1239, 1105, 699, 536 cm ^"1 . Anal. Calcd. for C ₂₀ H ₂₅ NO ₆ : C, 63.99; H, 6.71; N, 3.73. Found: C, 63.91; H, 6.66; N, 3.74.

EXAMPLE 13

N-Octanoy1-1,2,3-trimethoxybenzo-[5,4-c]-l,2,3-trimethoxy ¬ benzo-[ ,5-e]-azepine.

1, 2 , 3-Trimethoxybenzo- [ 5 , 4-c] -1 , 2 , 3 - trimethoxybenzo-[4,5-e]-azepinehydrochloride (Example 12) (500 mg, 1.21 mmol), octanoylchloride (0.22g, 1.33 mmole) , and dimethylaminopyridine (0.015 g, 126 μmol) were dissolved in methylene chloride (15 mL) and treated with triethylamine (0.381 g, 518 μl , 3.77 mmol). After stirring for 2 h, the reaction mixture was poured onto a column and chromatographed (silica gel, 4.1 x 15 cm, 40% ethyl acetate, 60% hexane) to give the product as an oil (570 mg, 94%) . ¹ H-NMR (CDC1 ₃ , 300 MHZ) δ 0.86 (3H, t) , 1.27 (4H, m) , 1.33(4H, m, ), 1.67 (2H, m) , 2.41 (2H, m) , 3.33 (IH, br d, J=12.8HZ), 3.70 (6H, d) , 3.79 (IH, br d, J=12.8HZ), 3.89 (12H, s) , 4.40 (IH, br d, J=13.2Hz), 5.21 (IH, br d, J=13.2HZ), 6.61 (1H,S), 6.72 (lH,s).

EXAMPLE 14

N-Octanoyl-1,2,3-trihydroxybenzo-[5, -c]-1,2,3-trihydroxy- benzo- [ 4 , 5 -e ] -azep ine ( Compound 7 ) . N-Octanoyl-1, 2, 3-trimethoxybenzo- [5, 4-c] - l,2,3-trimethoxy-benzo-[4,5-e]-azepine (0.57g, 1.13 mmol) was dissolved in methylene chloride (10 mL) and cooled to -78°C. Boron tribromide (10.21 mL of a l.OM solution in methylen chloride, 10.21mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously fo 20 h. The clear mixture was cooled to -78 ^β C and quenched wit methanol (30 mL) added dropwise. The mixture was allowed t warm to room temperature, stirred for about 3 h an concentrated to an oil. Upon addition of water (25 ml) an methanol (25 ml) , the product crystallized out. The produc was washed with water and filtered to give the title compoun (358 mg, 75%) as a white solid. m.p. 155-158°C, ^-N (DMSO-d _g , 300 MHZ) d 0.86 (3H, t) , 1.27 (8H,m) , 1.51 (2H, b m) , 2.34 (IH, br m) , 2.41 {IH, br m) , 3.03 (IH, d, J=12.9), 3.50, (IH, d, J=12.7), 4.36 (IH, d, J=12.7), 4.83 (IH, d, J=12.9), 6.35 (1H,S), 6.50(lH,s), 8.48 (2H, br s) , 8.68 (2H, b

s) , 9.07 (IH, br S), 9.16 (IH, br s) ; IR (KBr): 3456, 3323, 2921, 2852, 1573, 1467, 1370, 1310 cm ^'1 . Anal. Calcd. for C ₂₂ H ₂₇ NO ₇ *0.75 H ₂ 0: C, 61.31; H, 6.67; N, 3.25. Found: C, 61.47; H, 6.70; N, 3.27. EXAMPLE 15

N-3, ,5-Trimethoxybenzoyl-l,2,3-trimethoxybenzo-[5,4-c]- 1, 2 , 3-trimethoxybenzo- [ 4 , 5-e] -azepine . 1, 2 , 3-Trimethoxybenzo- [5, 4-c] -1, 2 , 3 - trimethoxybenzo-[4,5-e]-azepinehydrochloride (Example 12) (500 mg, 1.21 mmol) , 3,4,5-trimethoxybenzoyl chloride (0.29 g, 1.26 mmol), and dimethylaminopyridine (0.015 g, 126 μmol) were dissolved in methylene chloride (10 mL) and treated with triethylamine (0.381 g, 518 μl, 3.77 mmol). After stirring for 16 h, the reaction mixture was poured onto a column and chromatographed (silica gel, 4.1 x 15 cm, 100% ethyl acetate) to give the product as a solid (668 mg, 97%) . An aliquot of the product was recrystallized to give the title compound as a white crystalline solid, m.p. 175-177 ^β c. ¹ H-NMR (CDC1 ₃ , 300 MHz) δ 3.61 (IH, d, J = 13.2 Hz) , 3.7-3.91 (28H) , 4.38 (H, d, J = 13.2 HZ), 5.078 (H, d, J = 13.3 Hz) , 6.31 (IH, br s) , 6.67 (2H, S) , 6.81 (IH, br S) ; IR (KBr): 2942, 2838, 1627, 1583, 1461, 1404, 1324, 1238, 1126, 1004 cm ^"1 . Anal. Calcd. for C ₃₀ H ₃₅ NO ₁₀ : C 63.26; H, 6.19; N, 2.46. Found: C, 63.06; H, 6.25; N, 2.42. EXAMPLE 16

N-3, ,5-Trihydroxybenzoyl-l,2,3-trihydroxybenzo-[5,4-c]- 1,2,3-trihydroxybenzo-[4,5-e]-azepine (Compound 8) N-3 , 4 , 5-Trimethoxybenzoyl-l , 2 , 3 - trimethoxybenzo-[5,4-c]-1,2,3-trimethoxybenzo-[4,5-e]-azepin e (0.42 g, 0.74 mmol) was dissolved in methylene chloride (20 mL) and cooled to -78'C. Boron tribromide (9.95 mL of a l.OM solution in methylene chloride, 9.95 mmol) was added slowly and the solution was allowed to warm to room temperature and stirred vigorously for 20 h. The clear mixture was cooled to -78°C and quenched with methanol (20 mL) added dropwise. The mixture was allowed to warm to room temperature, stirred for about 3 h and concentrated to an oil. Water (40 mL) was added

to dissolve the solid and the mixture was concentrated. The solid was again dissolved in water and on swirling, the product crystallized out. The product was filtered and air dried for 5 days to give the title compound (340 mg, 99%) as an off-white solid, m.p. 250°C dec. ¹ H-NMR (DMSO-d ₆ , 300 MHz) d 3.25 (IH, br d) , 3.53 (IH, br d) , 4.23 (IH, br d) , 4.57 (IH, br d) , 6.07 (IH, br s) , 6.34 (2H, s) , 6.46, (IH, br s) ; IR (KBr): 3507, 3299, 1625, 1547, 1460, 1329, 1030 cm ^"1 . Anal. Calcd. for C ₂₁ H ₁₇ NO ₁₀ *l.25 H ₂ 0: C, 54.14; H, 4.22; N, 3.01. Found: C, 54.14; H, 4.17; N, 3.04.

EXAMPLE 17

Table 1 shows examples of the compounds of the invention.

10

<_o o

15

Compound 12 is a hydroxylated dibenz(c,ez)azepine that has less than five hydorxyl g at positions R., - R ₆ and Cl at positions R14 and R15,

12 R ¹ R ³ R ⁵ &R ⁶ =OH H- CH, 286-287 R &R=H +1, 1' dichloro

Table 2 shows compounds that are not substituted with hydroxyl at the R - R posi

10

15

EXAMPLE 18

Protein Kinase C Inhibition

The protein kinase C (PKC) assay is designed to duplicate the in vivo conditions required for protein kinase C function. Therefore, pH, salt and cofactor concentrations are similar to physiologic levels. Histone HI (lysine rich) is used in the assay as the phosphorylation acceptor protein because it is readily available and serves as a good substrate for protein kinase C. The enzyme is prepared from rat brain and is purified to apparent homogeneity as determined by a single band on silver stained SDS-polyacrylamide. Studies on the mechanism of regulation of protein kinase C by phospholipids, DAG and Ca ⁺ have been hampered by the physical properties of the lipid cofactors. In the screening assay, phosphatidylserine (PS) and DAG are co-sonicated to form unilamellar and multilamellar vesicles. The concentration of lipids in the assay are suboptimal to maximize the detection potential of the assay for inhibitors. Potential inhibitor compounds are added to the assay in dimethylsulfoxide at three concentrations to give final inhibitor concentrations of 4.3, 43 and 218 mM, respectively. The assay is started with the addition of enzyme and stopped after 10 min by the addition of 25% trichloroacetic acid (TCA) and 1.0 mg/mL bovine serum albumin (BSA) . The radioactive histone product is retained and washed on glass fiber filters that allow the unreacted ³² P-ATP to pass through. The amount of phosphorylation is determined by the radioactivity measured in a scintillation counter. Controls are included in every assay to measure background activity in the absence of enzyme, activity in the absence of lipids and the maximum enzyme activity with saturating levels of the activator lipids. Assay components and concentrations are given in Table 3.

Table 3

Assay Component Concentration

Hepes pH 7.5 20 mM

MgCl ₂ 20 mM"

CaCl ₂ 100 μM

EGTA 95 μM

Histone HI 200 mg/mL

Phosphatidylserine 40 mg/mL

Diacylglycerol 1.8 mg/mL

Protein Kinase C 0.6 mg/mL γ- P-ATP 20 μM

Results of the protein kinase C assay are shown in Table 4 in the column labeled PKC. Results are shown as IC ₅₀ , which is the concentration of test compound needed to inhibit 50% of the protein kinase C activity as compared with levels of protein kinase C activity in controls. Compounds of the invention were able to effectively inhibit protein kinase activity. IC ₅₀ values for compounds 1-8 and 11, shown in Table 4, ranged from 14mM - 65mM. Compounds 13-19, which are substituted at positions R, - Rg with methoxy or -0(CO)CH ₃ , did not inhibit protein kinase C. Compound 12, which had four hydroxyl groups but which had Cl at positions R ₁₄ and R ₁₅ did not inhibit protein kinase C. The comparative data indicates that the presence of the hydroxyl groups confers PKC inhibitory activity while dibenz(c,ez)azepines substituted with -OCH ₃ or 0(CO)CH ₃ do not share this activity. Hydroxylated dibenz(c,e)azepines according to the present invention (Compounds 1-11) demonstrated an ability to inhibit PKC.

Table 4

EXAMPLE 19 cAMP Dependent Protein Kinase (PKA) Assay

Compounds found to be inhibitors of protein kinase C are tested for inhibitory activity against protein kinase

(PKA) . This enzyme, like protein kinase C, plays an important role in cell-cell communication and is activated by a second messenger, cAMP. Secondary screening against PKA is useful for ascertaining the selectivity of the compounds of the invention.

The standard assay conditions are given in Table 5. The catalytic subunit of PKA (Sigma Chemical Company, St. Louis,

Missouri) is mixed with buffer before addition of the inhibitor in dimethylsulfoxide (DMSO) . The assay is started by the addition of P-ATP and the reaction is allowed to proceed for

10 min before stopping with 25% trichloroacetic acid (TCA) and

1.0 ταg/mL bovine serum albumin (BSA) . The phosphorylated protein is isolated by filtration and the radioactivity is counted in a beta scintillation counter.

Table 5

Assay Components Concentration

Hepes pH7.520 mM Histone H1200 mg/mL Dithiothreitol 32 mg/mL Pratein Kinase A2.6 mg/mL 7- -ATP 20 μM

Results of the PKA assay are shown in Table 4 (in Example 17) in the column labeled PKA. The results are shown as IC ₅₀ which is the concentration of that compound needed to inhibit 50% of the protein kinase (PKA) activity as compared with levels of PKA activity as compared with levels of PKA activity in controls. As shown in Table 4, compounds 1-8 each had an IC ₅₀ greater than 150 mM indicating that the compounds had no effect on PKA. The tested compounds of the invention are selective for protein kinase C, and have no effect on cAMP

dependent protein kinase. The compounds of the inventio should thus have no effect on the metabolic pathways associate with stimulation of protein kinase by cAMP.

EXAMPLE 20 Human Tumor Cell Growth Inhibition

MCF-7 a human breast tumor cell line and MCF-7/ADR a adriamycin resistant line of MCF-7 cells were obtained from th National Cancer Institute, Frederick, Maryland. CEM cell (ATCC accession number CCL 119) were obtained from the America Type Culture Collection, Rockville, Maryland.

Human tumor cells are trypsinized (0.05% trypsin GIBCO) , counted with a hemacytometer and seeded at concentration of 10,000 cells/well in a 96 well microtite plate. After allowing cells to attach to the surfac overnight, the culture medium is aspirated and replaced wit 100 mL of fresh medium. Test agents are diluted to determin dose response at 2X final concentration and added i quadruplicate at 100 mL/well to bring the total volume of eac well to 200 mL. The microtiter plate is then incubated at 37° 5% C0 ₂ overnight (18-24 hrs) before H-thymidine is added at concentration of 0.5 mCi/well in 50 mL culture medium. Th plate is incubated again for 4 hrs under the same conditions a above. Supernatant is then aspirated and 50ml trypsin (0.05% GIBCO) is added to each well. Cells are checke microscopically to determine detachment from surfaces, an plates are then harvested with a cell harvester (PHD, Cambridg Technology, Inc.) Filter papers corresponding to wells ar placed in scintillation vials and counted to determine th amount of H-thymidine incorporated by the cells. Test agen response is compared to a positive control of cell wells wit culture media only to determine the IC ₅₀ . IC ₅₀ is th concentration of test compound required to inhibit fifty pe cent of the incorporation of H-thymidine into proliferatin cells not exposed to test agent. Uptake of H-thymidine is standard test for measuring the metabolism of cells. Cell which are actively proliferating take up H-thymidine, wherea

cells that are not proliferating take up H-thymidine at much slower rates or not at all. Test agents that inhibit the uptake of H-thymidine thus slow the growth of cells.

As shown in Table 6, compounds 1-3 of the invention were able to inhibit ³ H-thymidine uptake with an IC ₅₀ of 17.4 mM to

25 mM and thus inhibit the proliferation of the tested cell lines. Compound 4 inhibited the proliferation of the tested cell line with an IC ₅₀ greater than 25 mM.

Table 6 ic ₅₀ (mM)

Compound MCF-7

1 21

2 25

3 17.4 4 >25

EXAMPLE 21

Human Keratinocyte Inhibition

Proliferating keratinocytes (NHEK cells purchased from Clonetics, Inc., San Diego, California) in second passage were grown in Keratinocyte Growth Medium (KGM) (Clonetics, Inc.) Cells are trypsinized (0.025% trypsin, Clonetics) , counted with a hemacytometer (Scientific Products) , and seeded at a concentration of 2,500 cells/well in a 96 well microtiter plate. After allowing cells to attach to the surface overnight, the culture medium is aspirated and replaced with 100 ml of fresh KGM. Test agents are evaluated and IC ₅₀ 's are determined according to the H-thymidine incorporation procedures described as in Example 20. IC ₅₀ is the concentration of test compound required to inhibit fifty per cent of the incorporation of H-thymidine into proliferating cells not exposed to test agent.

As shown in Table 7, compounds of the invention are active against human keratinocytes, and will be useful in treating topical inflammatory conditions such as psoriasis and other conditions where hyperproliferation of keratinocytes is a symptom.

Table 7

EXAMPLE 22 Neutrophil Superoxide Anion (0 ₂ -) Release Assay

Neutrophils are isolated form whole blood collected from human volunteers. All reagent materials are obtained from Sigma Chemical Company with the exception of isotonic saline (Travenol Laboratories, Inc. , Deerfield, Illinois) and lymphocyte separation medium (Organon Teknika, Durham, North Carolina) . Neutrophil Isolation

Whole blood is drawn and mixed with sodium heparin (final cone. 10 units/mL) to prevent clotting. An equal volume of dextran (3.0%) in isotonic saline is added, mixed, and allowed to settle for 30 min to bind red blood cells (RBC) . Supernatant is removed, underlayered with lymphocyte separation medium and centrifuged for 40 min at 400 xg in a centrifuge (Beckman GPR, Norcross, Georgia) . The pellet is alternately resuspended in 0.2% and 1.6% NaCl to lyse RBCs before washing with Hank's Balanced Salt Solution (HBSS) . The washed pellet is resuspended in 10 mL HBSS and placed on ice before counting on a hemacytometer. Assay Procedure The neutrophil cell concentration is adjusted to 2xl0 cells/mL with HBSS before adding 0.8 mL cells to 12 X 75 m polypropylene test tubes (Fisher Scientific) . Test agents are diluted to determine dose response and added at 10X final concentration at a volume of 0.1 mL/tube in duplicate. The 10X concentrations of cytochrome C (15 mg/mL) with catalas (3000 units/mL) either alone or containing 25 ng/mL phorbo 12-myristate 13-acetate (PMA) are added at a volume o O.lmL/tube and incubated at 37°C for 30 min before stopping th

reaction by placing tubes on ice. Tubes are then centrifuged at 900 xg for 10 min, 0.5 mL supernatant is removed and added to 0.5 mL H ₂ 0 in a microcuvette. Optical density (OD) of cytochrome c is read in a spectrophotometer (Shimadzu) at 550 nm. The OD of cytochrome c is obtained between PMA-stimulated and non-stimulated tubes, and the dose responses of the test agents are compared to the positive controls (which contain HBSS in place of test agents) . PMA stimulates 0 ₂ ^" production which reduces cytochrome c. Reducing cytochrome c increases its absorbance, and the change in OD of cytochrome c is proportional to the amount of 0 ₂ ^" produced by PMA stimulation. Inhibition of the 0 ₂ ^" burst by test compounds of the invention is seen as a reduction in the change in optical density. Inhibition is expressed as IC ₅₀ mM and is the amount of test compound that will inhibit fifty per cent of the PMA-stimulated respiratory outburst, i.e. 0 ₂ ^" production.

As shown in Table 8, compounds of the invention were able to inhibit 0 ₂ ^" production by PMA-stimulated neutrophils.

Table 8 Neutrophil Superoxide Release

(Int. = intefers w/assay)

EXAMPLE 23

Inhibition of Protein Kinase C Isoenzymes

As a means of studying the PKC inhibitory activity of the compounds of the present invention in greater detail, a series of assays were performed which use the various isoenzymes of PKC. In particular, α PKC isoenzyme from rat and βu _r 7, S, e and ξ PKC isoenzymes from humans were used in assays similar to those described in Example 18.

The protein kinase C (PKC) isoenzyme assays were designed to duplicate the in vivo conditions required for

protein kinase C function. Therefore, pH, salt and cofactor concentrations were similar to physiologic levels. Histone HI (lysine rich) was used in the assay as the phosphorylation acceptor protein because it was readily available and serves as a good substrate for protein kinase C. The isoenzymes, which are well known, were prepared from readily available starting materials using well known techniques.

Studies on the mechanism of regulation of protein kinase

C by phospholipids, DAG and Ca +2 have been hampered by the physical properties of the lipid cofactors. In the screening assay, phosphatidylserine (PS) and DAG were co-sonicated to form unilamellar and multilamellar vesicles. The concentration of lipids in the assay were suboptimal to maximize the detection potential of the assay for inhibitors. Potential inhibitor compounds were added to the assay in dimethylsulfoxide at three concentrations to give final inhibitor concentrations of 4.3, 43 and 218 mM, respectively. The assay was started with the addition of enzyme and stopped after 10 min by the addition of 25% trichloroacetic acid (TCA) and 1.0 mg/mL bovine serum albumin (BSA) . The radioactive histone product was retained and washed on glass fiber filters that allow the unreacted ² P-ATP to pass through. The amount of phosphorylation was determined by the radioactivity measured in a scintillation counter. Controls were included in every assay to measure background activity in the absence of enzyme, activity in the absence of lipids and the maximum enzyme activity with saturating levels of the activator lipids. Assay components and concentrations are given in Table 9.

Table 9

Assay Component Concentration

Hepes pH 7.5 20 mM

MgCl ₂ 20 mM

CaCl ₂ 100 μM

EGTA 95 μM

Histone HI 200 mg/mL

Phosphatidylserine 40 mg/mL

Diacylglycerol 1.8 mg/mL Pr&tein Kinase C isoenzyme 0.6 mg/mL 7- P-ATP 20 μM

Results of the protein kinase C assay are shown in Table

10 in the column labeled PKC. Results are shown as IC ₅₀ , which is the concentration of test compound needed to inhibit 50% of the protein kinase C activity as compared with levels of protein kinase C activity in controls. Compounds of the invention were able to effectively inhibit protein kinase activity.

The data demonstrate that the hydroxylate dibenz(c,ez)azepine are actively inhibit some or all of the PK isoenzymes except the dichloro compound, compound 12 which has less than 5 hydroxyl groups. The non-hydroxylate dibenz(c,ez)azepines were not active, i.e they did not inhibi any isoenzymes of PKC.