Chemotherapeutic agents, XXI: Synthesis of Pi-deficient pyrimidines as leishmanicides.

Synthesis of 5-cyano-6-(3-pyridyl)-2-thiouracil (1) has been described from 3-pyridinecarboxaldehyde, thiourea, and ethyl cyanoacetate. Alkylation of 1 with mono- and dihaloalkanes under different conditions, provided 2a,b, 3a,b, and 4a-i. Halogenation of 4g with POCl3 yielded 5 which underwent nucleophilic substitution with amines to give 6a-g. Fusion of 4a with aromatic and heterocyclic amines at 160 degrees C gave the substitution products 7a-c. Some of the compounds were screened for antileishmanial activity but only one of them (6d) demonstrated very significant activity.     

Synthesis and cardiotonic activity of 2-substituted 5-cyano-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids and their methyl or ethyl esters.

The synthesis of ethyl or methyl esters of 5-cyano-1,6-dihydro-6-oxo-3- pyridinecarboxylic acids carrying as 2-substituent a polar group such as CO2C2H5, (CH2)2CO2CH3, (CH2)3CO2C2H5, CH2OCH3, or CF3 group is described. Also 2-[5-cyano-1,6-dihydro-2-(1,1-dimethylethyl)-6-oxo-3-pyridyl]-2- oxoacetic acid and 2,5,6,8-tetrahydro-2,5-dioxo-1H-thiopyrano[3,4-b]pyridine-3-carbon itrile were prepared. Nearly all the above esters gave routinely the corresponding carboxylic acids by alkaline hydrolysis followed by acidification. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea-pigs. 5-Cyano-2-trifluoromethyl-1,6- dihydro-6-oxo-3-pyridinecarboxylic acid and, in a lesser degree, the relative ethyl ester showed an appreciable positive inotropic activity, although inferior to that of milrinone.     

Isoxazoles substituted with 4-pyridyl and o-chlorophenyl radicals]

By reaction of suitable hydroxamic acids chlorides with beta-ketoesters, we have prepared the ethyl esters of isoxazol-4-carboxylic acids, substituted in the 3- and 5-positions with 4-pyridyl and o-chlorophenyl groups, and some of their esters and amides of pharmaceutical interest. 3-(4-Pyridyl)-5-(o-chlorophenyl)isoxazole was obtained either by decarboxylation of the acid or by oxidation of the syn and anti oximes of 3-(o-chlorophenyl)-1-(4-pyridyl)-2-propen-1-one; from the syn oxime the corresponding isoxazoline was also obtained. Pharmacological screening shows that some of the new compounds have a myolytic activity.     

Synthesis and biological evaluation of some new 3 ,4-dihydropyrimidin-4-ones.

Condensation of 5-cyano-2-hydrazino-3-N-methyl-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (3a and 3b) with 2,4-bisalkyl/arylamino-6-chloro-s-triazine (4) gave the corresponding 2,4-bisalkyl/arylamino-6-[5'-cyano-3'-N-methyl]-6'-phenyl/pchlorophenyl-3',4'-dihydropyrimidin-4'-one-2'-yl-hydrazino-s-triazines (5a-n and 6a-n). The compounds 4 have been prepared by the condensation of cyanuric chloride and different alkyl/aryl amines. The reaction between 5-cyano-3-N-methyl-2-methylthio-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (2a and 2b) with hydrazine hydrate furnished 3a and 3b, respectively. The condensation of 6-phenyl/p-chlorophenyl/5-cyano-2-mercapto-3,4-dihydropyrimidin-4-one (1a and 1b) with methyl iodide yielded 2a and 2b, respectively. All the products have been evaluated in vitro for their antimicrobial activity against several microbes and antitubercular activity against Mycobacterium tuberculosis H37 Rv.     

4-Heterocyclyloxy-2H-1-benzopyran potassium channel activators

The reaction of 2,4-dihydroxypyridine (2) with 3,4-epoxy-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-carbonitrile (1) yielded the 4-[(1,2-dihydro-2-oxo-4-pyridyl)oxy] compound 3a, accompanied by small amounts of the isomeric 4-(1,2-dihydro-4-hydroxy-2-oxo-1-pyridyl) compound 4. This could also be prepared by hydrogenation of the benzyloxy derivative 5. Reaction of 3,6-pyridazinediol (10) with 1 (R = CN) gave the 4-[(1,6-dihydro-6-oxo-3-pyridazinyl)oxy] compound 11a, which in turn rearranged on heating with NaH in DMSO into the 4-(1,6-dihydro-3-hydroxy-6-oxo-1-pyridazinyl) compound 12. A series of 6-substituted analogues (R = CO2Me, CSNH2, NO2, Br) of 3a and 11a were synthesized. N-Alkylation led to compounds 14a-c (R = Me, Et, CHMe2). The 4-heterocyclyloxychromenes 9 and 16a were obtained by alkaline hydrolysis of their 3-camphorsulfonates. The racemic pyridazinyloxy compounds 11a and 14a could be resolved via their diastereomeric camphorsulfonates or camphanates. The differences between the 4-heterocyclyloxychromanols and the isomeric N-substituted compounds 4 and 12 were elucidated in the course of extensive NMR investigations. While in DMSO the former appeared to be conformationally flexible molecules the latter were rigid. All compounds were tested for oral antihypertensive activity in spontaneously hypertensive rats, using doses of 1 mg/kg. High and long lasting activities were found for the pyridyloxy compounds 3a and 3d, the pyridazinyloxy compound 11a, and its N-alkylation products, as well as for the 3S,4R-enantiomers 20a and 22a. (-)-(3S,4R)-3,4-Dihydro-4-[(1,6-dihydro-1-methyl-6-oxo-3- pyridazinyl)oxy]-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-carbonitrile (22a) was selected for further development.     

Identification of adducts produced by the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol with deoxyguanosine and DNA

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL is present in the blood and urine of people exposed to tobacco products and has carcinogenic activity in rodents similar to that of NNK. DNA adducts specific to NNAL have not been previously identified. Metabolic activation of NNAL by alpha-methyl hydroxylation, a pathway known to occur in rodent and human microsomes, would produce pyridylhydroxybutylating agents that could react with DNA. We investigated this possibility in the present study by allowing 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNALCH(2)OAc) to react with dGuo and DNA. Products were identified by HPLC with UV detection, liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) and LC/ESI-tandem mass spectrometry (LC/ESI-MS/MS). In the dGuo reactions, selected ion monitoring for m/z 417, corresponding to pyridylhydroxybutylated dGuo, showed several peaks. One adduct was identified as 7-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo (21) by neutral thermal hydrolysis, which converted it to 7-[1-hydroxy-1-(3-pyridyl)but-4-yl]Gua (22) and 4-hydroxy-1-(3-pyridyl)-1-butanol (16). Adduct 22 was identified by comparison of its LC/ESI-MS and LC/ESI-MS/MS properties to those of standard 22. Two other adducts, O(6)-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo (17) and N(2)-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo (19), were identified by comparison of their LC/ESI-MS and LC/ESI-MS/MS properties to those of standard 17 and 19. Further evidence for the identity of 17 and 19 was obtained by mild acid hydrolysis, which converted them to the corresponding Gua bases 18 and 20, identified by comparison to synthetic standards. Neutral thermal hydrolysis of DNA that had been reacted with NNALCH(2)OAc produced 22, identified by comparison to a standard. Adducts 17 and 19 were identified in enzyme hydrolysates of this DNA by comparison to standards. Thus, DNA that had been allowed to react with NNALCH(2)OAc contained adducts 17, 19, and 21. The results of this study provide markers for investigating the role of specific NNAL-DNA adducts in carcinogenesis by NNAL and NNK.     

Synthesis, cytotoxicity, and antiviral activity of certain 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine nucleosides related to toyocamycin and sangivamycin

A number of 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their biological activity. Treatment of the sodium salt of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (1) with (2-acetoxyethoxy)methyl bromide (2) afforded a mixture of 4-amino-6-bromo-5-cyano-7-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d] pyrimidine (3) and the corresponding N1 isomer. Debromination of this mixture gave the corresponding 4-amino-5-cyano-7-[(2-acetoxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidi ne (4) and 4-amino-5-cyano-1-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d]pyrimidin e (5). Deacetylation of 4 and 5 furnished 4-amino-5-cyano-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (6) and the corresponding N1 isomer (7), respectively. The sites of attachment for the acyclic moiety for 6 and 7 were assigned on the basis of UV spectral studies as well as 13C NMR spectroscopy. Conventional functional group transformation of 6 provided a number of novel 5-substituted derivatives (8-10), including the sangivamycin derivative 8. The methyl formimidate derivative 10 was converted to the thioamide derivative 11 and the carbohydrazide derivative 12. Compounds 6 and 8-12 were tested for cytotoxicity to L1210 murine leukemic cells in vitro. None of these compounds caused significant inhibition of cell growth. Evaluation of compounds 4 and 6-12 for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) revealed that only the thioamide (11) was active. It inhibited HCMV but not HSV-1 at concentrations producing only slight cytotoxicity in human foreskin fibroblasts (HFF cells) and KB cells.     

Synthesis and reaction of cyanopyridone derivatives and their potential biological activities

4-Carboxy-3-cyano-6-biphenyl-pyrid-2-one (1) was prepared and then allowed to react with methyliodide, benzenesulphonyl chloride, phenylisothiocyanate, acetic anhydride, o-phenylenediamine, phenylmagnesium bromide and phosphorus pentasulphide affording the corresponding N-substituted pyrid-2-one 2-5, 4-(benzimidazol-2-yl)-pyrid-2-one 7, 2-hydroxy-2-phenyl-1,2-dihydro-pyridine 8 and 2-thiopyridone 9 derivatives, respectively. Treatment of 1 with dimethyl sulphate and phosphorus oxychloride to give 2-methoxy pyridine 12 and 2-chloro pyridine 13 derivatives. Reaction of 13 with amines and hydrazine hydrate afforded 14 and 15, respectively. The structural assignments of the new compounds were based on analytical, spectroscopic measurements and chemical reactions. Some of the obtained compounds showed interesting antibacterial and antifungal activities in vitro.     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure-activity relationships of the 4-aryl group

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.     

Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)- 1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitros- amino)-1-(3-pyridyl)-1-butanol with DNA

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.     

Synthesis of condensed heterocycles from 3-aryl-2,4-dicarbethoxy-5-methylcyclohexanones and their testing for antimicrobial activity.

Condensation of the title compounds (1) with hydroxylamine hydrochloride, hydrazines and/or aromatic amines resulted in the formation of the benzisoxazoles 2, oximes 3, indazolines 4 and beta-keto anilides 6. The oxime derivatives and anilides underwent cyclization to compounds 2. The interaction between 1 and thiourea gave the benzothiazines 7 and thiouracils 8. Compounds 8 on treatment with monochloroacetic acid gave the dioxo compounds 9, while their reaction with hydrazine hydrate afforded the hydrazino derivatives 10, which upon treatment with nitrous acid gave the azido or tetrazolo derivatives 11 and 12. Treatment of 1 with 2,3-diaminopyridine and/or 2-amino-3-hydroxy-pyridine gave the pyrimidoquinazolines 13 or 14. Some of the synthesized compounds were screened to test their antimicrobial properties.     

Synthesis of 4-alkyl (aryl)-6-aryl-3-cyano-2(1H)-pyridinones and their 2-imino isosteres as nonsteroidal cardiotonic agents.

Thirty new 1,2-dihydropyridine derivatives of the general formula 4-alkyl (aryl)-6-aryl-3-cyano-2(1H)-pyridinones (1-15) and 4-alkyl (aryl)-6-aryl-3-cyano-2(1H)-iminopyridines (16-30) were synthesized using one-pot multicomponent reactions of the properly substituted acetophenone, appropriate aldehyde, ammonium acetate and ethyl cyanoacetate (1-15) or malononitrile (16-30) in ethanol. These target compounds (1-30) were evaluated for their cardiotonic activity using the spontaneously beating atria model, from reserpine-treated guinea pigs. The best pharmacological profile was obtained with 3-cyano-6-(3,4-dimethoxyphenyl)-4-(4-hydroxyphenyl)-2(1H)-pyridinone (9) which displaced selectivity for increasing the force of contraction (108.7 +/- 6.7,% change over control) rather than the frequency rate (40.8 +/- 5.3,% change over control) at a 5 x 10(-4) M concentration. The effects of structural changes upon activity are reported.     

Synthesis and antimicrobial testing of some new S-substituted-thiopyridines,thienopyridines, pyridothienopyrimidines and pyridothienotriazines

The reaction of 5-acetyl-4-aryl-3-cyano-6-methylpyridine-2(1H)-thiones (1a, b) with 4-methylphenacyl bromide, chloro-N-arylacetamides or chloroacetonitrile gave the corresponding S-substituted thiopyridines 2a-c, 4a-f and 6a-c, respectively. The latter compounds underwent intramolecular Thorpe-Ziegler cyclization to give 2-substituted 5-acetyl-3-amino-4-aryl-6-methylthieno[2,3-b]pyridines 3a-c, 5a-f and 7a-c. Compounds 5a-f and 7b, c are key intermediates in the synthesis of the target compounds. Some compounds showed remarkable antimicrobial activity.     

3-Methyl-2H-1-benzopyran potassium channel activators

By aldol condensation of 4-chromanones with paraformaldehyde, 3-alkylenechromanones 10 were obtained which gave 3-alkylchromenes following reduction and dehydration. Subsequent 3-chloroperbenzoic acid oxidation produced the versatile epoxide intermediates 15, from which 3,4-epoxy-3,4-dihydro-2,2,3-trimethyl-2H-1-benzopyran-6-carbonitrile (15a) was resolved into its enantiomers by entrainment. In addition to the methyl group, the benzyl, alkyloxymethyl, and 2-nitroethyl residues could be introduced in the 3-position. Treatment of 15a with 2-pyridone simultaneously gave N- and O-substituted products 19a and 20. 19a easily gave 4-(1,2-dihydro-2-oxo-1-pyridyl)chromene 21 by treatment with base. The corresponding pyrrolidinone compounds 26 and 27 were obtained by a slightly modified procedure. Reaction with 2,4-dihydroxypyridine or 3,6-pyridazinediol resulted in the exclusive formation of 4-(heterocyclyloxy)chromanols (31 and 32). Treatment of 15a with 3-amino-6-pyridazinol gave 4-(3-amino-1,6-dihydro-6-oxo-1-pyridazinyl)chromanol derivative 34 lacking an NH bridge. This could be established after methylation of the ring-nitrogen atom (----35). Trans-configurated 3-methyl-4-pyridone compound 36 was obtained by addition of methyllithium to chromene 3. Hyperpolarizing and antispasmodic or relaxing effects of the compounds were determined in organ bath studies using pig coronary arteries precontracted with acetylcholine or rabbit main pulmonary arteries precontracted with noradrenaline. In the 3-methyl series the classical pyridone and pyrrolidinone structures (9, 21, 26, 27) were only weakly active or inactive, but the corresponding 4-(heterocyclyloxy) and 4-(heterocyclylamino) derivatives (31, 32, 35) were even more potent than the demethyl analogues. In conformation/activity investigations it was found that the activity of the 4-substituted benzopyran derivatives seems to be dependent on the relative orientation of their ring systems.     

Synthesis and antiviral activity of some 7-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine analogues of sangivamycin and toyocamycin

The sodium salt of 4-amino-3-cyanopyrazolo[3,4-d]pyrimidine (1) was condensed with (2-acetoxyethoxy)methyl bromide (2) to provide the corresponding protected acyclic nucleoside, 4-amino-3-cyano-1-[(2-acetoxyethoxy)methyl]-pyrazolo[3,4-d]pyrimid ine (3). Treatment of 3 with sodium methoxide in methanol provided a good yield of methyl 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- formimidate (4). Treatment of the imidate (4) with sodium hydrogen sulfide gave the thiocarboxamide derivative 5. Aqueous base transformed 4 into 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- carboxamide (6) in good yield. Treatment of 5 with mercuric chloride furnished the toyocamycin analogue 7. Evaluation of compounds 1, 3-7 revealed that only the heterocycle (1) and the thiocarboxamide acyclic nucleoside (5) were active. Compound 5 was the more potent with activity against human cytomegalovirus and herpes simplex virus type 1.     

Xanthone aus Chromon-Derivaten

Xanthones from Chromone Derivatives The 3-acyl-2-(methylthio)chromones 2a - 2c and the 6-acetyl-7-(methylthio)furochromone 10 react with the CH-acidic compounds 5a-5f, 11 and potassium tert.-butylate to yield the 3-acylchromones 4a - 4d and 9 , the xanthones 1 und 6a - 6d and the furoxanthones 13a-13c and 20 . With ammonium acetate, 4b and 4c gave the benzopyranopyridinones (azaxanthones) 8a - 8b .     

Puryl-6-ammonium- and -imonium-derivatives as substrates for xanthine oxidase

Puryl-6-ammonium- and -imonium-Derivatives as Substrates for Xanthine Oxidase N-Dimethyl-β-amino-ethanol, chinuclidine-3-ol and N-methylpiperidine react with 6-chloropurine (1) to yield the ammonium salts 2, 3 and 4 . With pyridine, 3-hydroxy-pyridine, 3-amino-pyridine, nicotinic acid amide and 1-(2-or 3-pyridyl)-2-(3- or 4-pyridyl)-ethylene as reactants one obtains the imonium salts 5, 6, 7, 8, 14 and 15 . 6-Chloro-purine reacts with 4-hydroxy- and 4-amino-pyridine to form the tertiary amines 9 and 10 and with 3- and 4-picolylamine and 2-pyridyl-β-ethylamine to the secondary amines 11, 12 and 13 . In contrast to the imonium salts 5, 6, 7, 8, 14 and 15 the quarternary ammonium salts cannot serve as substrates for xanthine oxidase. The secondary amines 11,12 and 13 serve as substrates as expected.     

Guanidine derivatives as combined thromboxane A2 receptor antagonists and synthase inhibitors

A new series of omega-disubstituted alkenoic acid derivatives derived from samixogrel 5 were designed and synthesized as combined thromboxane A2 receptor antagonists/thromboxane A2 synthase inhibitors with improved solubility and reduced protein binding compared to 5. Hexenoic acid derivatives with a 3-pyridyl group and 3-(2-cyano-3-alkyl-guanidino)phenyl substituent were found to be optimal with regard to this dual mode of action. The most potent compound, E-6-(3-(2-cyano-3-tert-butyl-guanidino)phenyl)-6-(3-pyridyl)hex-5-eno ic acid, "terbogrel" 32 inhibits the thromboxane A2 synthase in human gel-filtered platelets with an IC50 value of 4.0 +/- 0.5 nM (n = 4). Radioligand binding studies with 3H-SQ 29,548 revealed that 32 blocks the thromboxane A2/endoperoxide receptor on washed human platelets with an IC50 of 11 +/- 6 nM (n = 2) and with an IC50 of 38 +/- 1 nM (n = 15) in platelet-rich plasma. Terbogrel inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an IC50 of 310 +/- 18 nM (n = 8) and 52 +/- 20 nM (n = 6), respectively. This was shown to translate into a potent antithrombotic effect in vivo as demonstrated in studies using a model of arterial thrombosis in rabbits (ED50 = 0.19 +/- 0.07 mg/kg; n = 20). Thus, terbogrel is the first compound with a guanidino moiety demonstrating both a potent TXA2 synthase inhibition and a potent TXA2 receptor antagonism and has been selected for further clinical development.     

Synthesis and cytotoxic evaluation of some 6-arylidene-2-(alpha-hydroxyamino-alpha-arylmethyl)cyclohexanone oximes and related compounds.

Reaction of 2,6-bis-(phenylmethylene)cyclohexanone (1) with a 4-molar excess of hydroxylamine hydrochloride and sodium acetate to produce the corresponding oxime 2 gave rise to 2-(alpha-hydroxyamino-alpha-phenylmethyl)-6-phenylmethylenecyclohexan one oxime (5a), whose structure was deduced from high-resolution proton nuclear magnetic resonance spectroscopy and confirmed by X-ray analysis. Compound 2 was eventually prepared from 1 with hydroxylamine per se and not with a mixture of hydroxylamine hydrochloride and sodium acetate. Ten analogues of 5a, namely 5b-5k, were prepared and evaluated for cytotoxicity. Six of the 11 compounds in series 5, as well as 1, showed activity in the 240-950 microM range against murine mammary EMT6 cells. Series 5 was also examined for cytotoxicity in an in vitro screen conducted by the National Cancer Institute with approximately 54 cell lines, and four compounds demonstrated selective toxicity toward various groups of tumors.     

Examination of newly synthesized 2-chloroethylnitrosoureas in preterminal leukemia L 5222 and in two transplanted neurogenic tumors

The chemotherapeutic activities of 11 chloroethylnitrosoureas, among them 10 newly synthesized compounds, were investigated in rat leukemia L 5222 and in two neurogenic rat tumors. 1-(4-Amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU, compound 1) and cyclophosphamide (12) served as reference substances. The newly synthesized compounds were 1-(2-chloroethyl)-1-nitroso-3-(2-carboxyethyl)-urea (2-carboxyethyl-CNU, 2), 1-(2-chloroethyl)-1-nitroso-4-methyl-4-formyl-semicarbazide (methyl-formyl-amino-CNU, 3), 1-(2-chloroethyl)-1-nitroso-3-(methylene-2-pyridyl)-urea(methylene-2-pyridyl- CNU, 4), 1-(2-chloroethyl)-1-nitroso-3-(methylene-2-pyridyl)-urea hydrochloride (methylene-2-pyridyl-CNU . HCl, 5) 1-(2-chloroethyl)-1-nitroso-3-(methylene-4-pyridyl)-urea hydrochloride (methylene-4-pyridyl-CNU . HCl, 6), 1-(2-chloroethyl)-1-nitroso-3-(3-pyridino)-urea (pyridyl-3-CNU, 7), 1-(2-chloroethyl)-1-nitroso-3-(3-pyridyl)-urea hydrochloride (pyridyl-3-CNU . HCl, 8), 1-(2-chloroethyl)-1-nitroso-3-[4(2,6-dimethyl-morpholino)]-urea (dimethyl-morpholino-CNU, 9), 1-(2-chloroethyl)-1-nitrosocarbamoyl-morpholine (chloroethyl-nitroso-carbamoyl-morpholine, 10), 1-(2-chloroethyl)-1-nitroso-carbamoyl-2,6-dimethyl-morpholine (chloroethyl-nitroso-carbamoyl-dimethylmorpholine, 11). Against both neurogenic tumors cyclophosphamide was distinctly superior to all nitrosoureas. In leukemia L 5222 all nitrosoureas except compounds 7, 8, 11 effected cures. Remarkable differences in toxicity could be observed between the nitroso compounds investigated.