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A series of 138 nonchiral 3-amidinobenzyl-1H-indole-2-carboxamides and analogues as inhibitors of the blood coagulation enzyme factor Xa (fXa) were designed, synthesized, and investigated by X-ray structure analysis and 3D quantitative structure-activity relationship (QSAR) studies (CoMFA, CoMSIA) in order to identify important protein-ligand interactions responsible for biological affinity and selectivity. Several compounds from this series are highly potent and selective inhibitors of this important enzyme linking extrinsic and intrinsic coagulation pathways. To rationalize biological affinity and to provide guidelines for further design, all compounds were docked into the factor Xa binding site. Those docking studies were based on X-ray structures of factor Xa in complex with literature-known inhibitors. It was possible to validate those binding modes by four X-ray crystal structures of representative ligands in factor Xa, while one ligand was additionally crystallized in trypsin to rationalize requirements for selective factor Xa inhibition. The 3D-QSAR models based on a superposition rule derived from these docking studies were validated using conventional and cross-validated r(2) values using the leave-one-out method and repeated analyses using two randomly chosen cross-validation groups plus randomization of biological activities. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which were found to correspond to experimentally determined factor Xa binding site topology in terms of steric, electrostatic, and hydrophobic complementarity. Subsets selected as smaller training sets using 2D fingerprints and maximum dissimilarity methods resulted in 3D-QSAR models with remarkable correlation coefficients and a high predictive power. The final quantitative SAR information agrees with all experimental data for the binding topology and thus provides reasonable activity predictions for novel factor Xa inhibitors.

The invention provides compounds which specifically inhibit factor Xa activity. The compounds consist of the structure X1 -YIR-X2, wherein X1 is H, acyl, alkyl, acylalkyl, arylalkyl or one or more amino acids, and X2 is a modified C-terminal group, one or more carboxy-protecting groups or one or more amino acids or other substituent, and Y, I and R are tyrosine, isoleucine and arginine, respectively, or peptidomimetic or organic structures that possess the same functional activity as Y, I and R, respectively. In addition, the present invention provides a compound having the structure A1-A2-(A3)m --B, where m is 0 or 1. A compound of the invention can be linear or cyclic and can be about 2 and 43 residues in length. A compound of the invention is characterized, in part, in that it exhibits a specific inhibition of factor Xa activity with a Ki of ≦100 μM, preferably ≦2 nM, and does not substantially inhibit the activity of other proteases involved in the coagulation cascade. The invention further provides methods of specifically inhibiting the activity of factor Xa and of inhibiting blood clotting in vitro and in an individual and methods of detecting factor Xa levels or activity.

A series of [1,2,4]triazolo[4,3-alpha][1,4]benzodiazepines bearing an ethynyl functionality at the 8-position and the isosteric thieno[3,2-f][1,2,4]triazolo[4,3-alpha][1,4]diazepines were prepared and evaluated as antagonists of platelet activating factor. The effects of substitution were explored in in vitro and in vivo test systems designed to measured PAF-antagonistic activity. Results are discussed and compared with previously published data. Many of the compounds had activity superior to WEB 2086, compound 1. In general, the thieno analogues exhibited better oral activity than the corresponding benzodiazepines. The duration of activity upon oral administration was modulated by the substitution on the acetylenic side chain. Compounds 71 and 81 were selected for further pharmacological evaluation as a result of their good oral potency and exceptionally long duration of action.

The syntheses of two novel pentacyclic ring systems, the thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepino-[4,5-a]benzimidazole and the indazolo[2,3-d][1,2,4]triazolo[4,3-a][1,4]benzodiazepine are described. Attachment of a propargyl linked quinolinone resulted in compounds 6 and 16 which showed PAF-antagonist activity by the intravenous route of administration in guinea pigs. The more potent compound 16 also exhibited good oral activity with an ID50 of 0.2 mg/kg in the bronchoconstriction assay.

The syntheses of two enantiomeric tetracyclic compounds, the thieno[3,2-f][pyrrolo[2,1-c][1,2,4]triazolo[4,3-a][1,4]diazepines 10a and 10b are described. Testing of these enantiomers as inhibitors of platelet activating factor (PAF) revealed good enantiospecificity, meaning that one enantiomer is responsible for the observed biological activity.

The syntheses of two enantiomeric tetracyclic compounds, the thieno[3,2-f][pyrrolo[2,1-c][1,2,4]triazolo[4,3-a][1,4]diazepines 10a and 10b are described. Testing of these enantiomers as inhibitors of platelet activating factor (PAF) revealed good enantiospecificity, meaning that one enantiomer is responsible for the observed biological activity.

The syntheses of two novel pentacyclic ring systems, the thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepino-[4,5-a]benzimidazole and the indazolo[2,3-d][1,2,4]triazolo[4,3-a][1,4]benzodiazepine are described. Attachment of a propargyl linked quinolinone resulted in compounds 6 and 16 which showed PAF-antagonist activity by the intravenous route of administration in guinea pigs. The more potent compound 16 also exhibited good oral activity with an ID50 of 0.2 mg/kg in the bronchoconstriction assay.

A series of [1,2,4]triazolo[4,3-alpha][1,4]benzodiazepines bearing an ethynyl functionality at the 8-position and the isosteric thieno[3,2-f][1,2,4]triazolo[4,3-alpha][1,4]diazepines were prepared and evaluated as antagonists of platelet activating factor. The effects of substitution were explored in in vitro and in vivo test systems designed to measured PAF-antagonistic activity. Results are discussed and compared with previously published data. Many of the compounds had activity superior to WEB 2086, compound 1. In general, the thieno analogues exhibited better oral activity than the corresponding benzodiazepines. The duration of activity upon oral administration was modulated by the substitution on the acetylenic side chain. Compounds 71 and 81 were selected for further pharmacological evaluation as a result of their good oral potency and exceptionally long duration of action.

The quinazoline 3-oxides 2 were found to convert to the benzisoxazoloquinazolines 3 by heating in acetic anhydride. The dihydroquinazoline 3-oxides 1 underwent aromatization to 4 under the same conditions. Hydrolysis of 3 or 2 led to the 3-(2-aminophenyl)benzisoxazoles 8 which could be rearranged to the 3-(2-hydroxyphenyl)indazoles 11 by treatment with hydride. Possible mechanisms are discussed.

The preparation and oxidation of 2,3-dihydroindazoles to 1H, 2H or 3H-indazoles is described. A method for the synthesis of indazole 2-oxides has been found. Oxidation of 2-acetyl-2,3-dihydro-3,3-disubstituted indazoles 5a and 5c gave quinoid compounds 20a, 20b, 24a and 24b, which could be isomerized to 3H-indazoles upon removal of the acetyl group. A quinoid compound 21 was also obtained on treatment of 5a with tetracyanoethylene.

1,4-Benzodiazepine-2-acetic acid derivatives were prepared and converted to compounds with a heterocyclic ring fused to the a-face of the benzodiazepine system. Representatives of pyrido[1,2-a][1,4]benzodiaze-pines, pyrimido[1,6-a][1,4]benzodiazepines and [1,3]oxazino[3,4-a][1,4]benzodiazepines are described. Some of the compounds showed marked CNS-activity as measured by the antimetrazole test which is a well established primary screening method for assessment of benzodiazepine type activity.

The synthesis of the 1-phenylimidazobenzodiazepine 5 from 1 and the anion of the nitrone 2 is described. The 3-phenyl-derivative 14 was prepared via the ammo alcohol 11 which was obtained by condensation of the nitrosamine 9 with benzaldehyde followed by catalytic hydro-genolysis of the nitroso group.

1,4-Benzodiazepines bearing a carbon substituent at the 2-position were obtained by reaction of 2-chloromethylquinazoline 3-oxides with stabilized carbanions. The carbanions of alkyl acetates, N,N-disubstituted acetamides, acetonitrile, dimethylsulfone, N,N-dimethyl methanesulfonamide and 2-methylpyridine were successfully applied. The conversion of some of the 2-carbon substituted 1,4-benzodiazepines to imidazo[1,5-a][1,4]benzodiazepines and [1,2,5]oxadiazino[5,4-a][1,4]benzodiazepines is described.

The facile synthesis of [1,2,3]triazolo((4,5-d][2]benzazepines and dibenzo[c,f][1,2,3]triazolo[3,4-a]azepines by the addition of sodium azide to acetylenic benzophenones is described. Examination of the pharmacological data indicates that selected triazolobenzazepines are as potent as diazepam in the anti-pentylenetetrazole test and are weaker in the inclined screen and rotarod tests, suggesting that these compounds have antianxiety properties similar to diazepam with fewer deficits in motor coordination. In addition, a possible diazepam antagonist was found in the triazolo-benzazepine series. The dibenzotriazoloazepines were found to be inactive in four standard CNS screening procedures.

Pyrano[3,2-d][2]benzazepines were synthesized by reaction of the beta-dimethylamino unsaturated ketone 1 with malonic ester or the anion of t-butyl acetate. Conversion of the pyrano derivative to the pyrido[3,2-d]-[2]benzazepine was achieved in low yield. A pyrrolo[3,2-d][2]benzazepine was prepared via an oxazepino-[6,7-d][2]benzazepine which was obtained by reaction of 1 with the anion of ethyl isocyanoacetate.

3-(2-Arninophenyl)indazoles 2 were prepared by the reaction of the o-fluorobenzophenones 1 with hydrazines. These indazoles were converted to the tetracyclic title compounds by conventional methods.

Variously substituted indolo[2,3-c]quinolines, benzofurano[2,3-c]quinolines and their positional isomers indolo[3,2-c]quinolines and benzofurano[3,2-c]quinolines respectively, were prepared from both indole and quinoline derivatives by intramolecular nucleophilic displacement of aromatic fluorine. The formation of an indolo[2,3-d]benzazepine was also observed.

A facile synthesis of the 4H-imidazo[5,1-c][1,4]benzothiazine ring system is described. The synthesis utilized the annulation of an imidazole ring onto a 2H-1,4-benzothiazin-3(4H)-one via the addition of the anion of ethyl isocyanoacetate. Methods for the functionalization of the 1-, 3-, 4-, and 5-positions of the ring system are presented.

Some new triazolylbenzophenones 3 were prepared by reaction of the corresponding quinazolines 2 with formic acid. Conversions of compounds 3 to known triazolobenzodiazepines are described. One of the processes proceeds via the double bond isomer 13. The reaction of the aldehyde 9 with hydrazine interestingly yielded the triazoloquinolines 11 and 12. The cyclization of the benzophenones 3 to the triazoloindoles 15 and the alkylation of the latter to derivatives with basic and acidic side chains are also reported. Quaternization of compound 16 with ethyl bromoacetate followed by treatment with hydroxide resulted in the formation of the triazinoindole 12.

The thieno[3,2-e][1,4]diazepin-2-one (1a), the thieno[2,3-e] [ 1,4] diazepin-2-one (1b), the pyrazolo[3,4-e][1,4]diazepin-2-one (1c) and a chloro analog of 1b, compound 1d, were each converted to derivatives of the novel tricyclic ring systems 4H-imidazo[1,5-a]thieno[2,3-f] [1,4]-diazepine, 4Himidazo[1,5a]thieno[2,3f][1,4]diazepine and 4H-imidazo[ 1,5-a]pyrazolo[4,3-f]-[1,4]diazepine. Depending on the substituents desired on the imidazo ring, two different synthetic pathways were employed.

s-Triazolo[4,3-a][1,4]benzodiazepines bearing various substituents in the 1 position were prepared by oxidative cyclization of the appropriate aldehyde hydrazones of 2-hydrazinobenzodiazepines. Diethyl azodicarboxylate and activated manganese dioxide were used as oxidizing agents. The new triazolo compounds were active in the CNS tests but none of them reached the potency of the known triazolobenzodiazepines.

The preparation of the title compounds 3 and 4 using two different methods of synthesis is described. These compounds are readily reduced to 2,3-dihydro-1H-derivatives 5. Oxidation of 2-alkylthio-1H-1,4-benzodiazepines leads to the corresponding sulfoxides and sulfones. The oxidative rearrangement of sulfones 9 to a 2H-1,4-benzodiazepin-2-one derivative 10 is also given. The “normal” addition of azodicarboxylate together with an unusual addition of two moles of acetylenedicarboxylate to the enamine double bond of 1H compounds is discussed.

The 2-hydrazinobenzodiazepines 1 were reacted with acelylacelone to form the hydroxy-pyrazolines 2 which were converted to the triazolobenzodiazcpines 6 and the 2-pyrazolyl-benzodiazepine 4. Ring cleavage of the hydrazone 8 obtained from cyclohexane-1,3-dione and 1a led similarly to the 1-(4-oxopentyl)triazolol)enzodiazepine 12.

The addition of diethyl azodicarboxylate to an electron rich double bond was employed as the key step for the preparation of the title compounds. Novel tetracyclic [1,2,4]triazino[1′,6′:3,4]imidazo[1,5-a][1,4]benzodiazepines were accessible from these 2-aminoimidazo[1,5-a][1,4]benzodiazepin-1-ones.

A series of pyrazino[1,2-a][1,4]benzodiazepines were prepared by acylating the primary amino group of an α-amino-1,4-benzodiazepine-2-ylideneacetic acid ester (1) with α-chloroacyl chlorides followed by cyclo-dehydrohalogenation with triethylamine in dimethylformamide. Some pharmacological data for CNS-activity are discussed.

The displacement of the nitro group in nitrooximes by other nucleophiles was used to prepare various 2-(hydroxyimino)methyl- and 2-(methoxyimino)methyl-1,4-benzodiazepines. These compounds were converted to imidazo[1,5-a][1,4]benzodiazepines bearing a tertiary amine, methoxy or thiomethyl group in the 3-position.