Synthesis and biological evaluation of novel hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines as potent and selective 5-HT(2C) receptor agonists

Further lead optimization efforts on previously described 1,2,3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indoles led to the new class of 5,5a,6,7,8,9-hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines culminating in the discovery of (5aR,9R)-2-[(cyclopropylmethoxy)methyl]-5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[3', 2':4,5]pyrrolo[1,2-a]pyrazine 18 as a potent, full 5-HT(2C) receptor agonist with an outstanding selectivity profile and excellent hERG and phospholipidosis properties.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

A new assay for N-hydroxyarylamine O-acetyltransferase: reduction of N-hydroxyarylamines through N-acetoxyarylamines

A simple fluorometric assay for N-hydroxyarylamine O-acetyltransferase is described and compared with a nucleic acid-binding assay. The assay method is based on the finding that the highly mutagenic 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) was reduced to the corresponding amine, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), through N-acetoxy-Glu-P-1 as the reactive intermediate in the presence of N-hydroxyarylamine O-acetyltransferase, acetyl-CoA, and a sulfhydryl compound. The formation of Glu-P-1 was determined by its characteristic fluorescence intensity at 445 nm with excitation wavelength at 376 nm. The reductive reaction was inhibited by the addition of tRNA, DNA, and poly(G), to which the enzymatic product, N-acetoxy Glu-P-1, bound effectively due to its electrophilic nature. Since the fluorometric assay for the O-acetyltransferase is rapid, simple, and sensitive as compared with the nucleic acid-binding method using radioisotope-labeled N-hydroxyarylamine, this method is applicable to the general assay for the formation of reactive N-acetoxy-Glu-P-1.     

Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a:3',2-d)-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.     

A pyrimidopyrimidoindole nucleoside (dC PPI): photophysical properties and thermal stability of the modified DNA duplexes

A new fluorescent deoxycytidine analog, 10-(2-deoxy-beta -D-ribofuranosyl)-pyrimido[4',5' :4,5]-pyrimido[1,6-a]indole-6,9(7H)-dione (dC(PPI)) was synthesized. Its fluorescent properties were studied in detail. It was found that this fluorescent nucleoside dC(PPI) could be used as a fluorescent label for DNA probes with minimal disturbance of their overall structure.     

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.     

DNA mediated resonance energy transfer from 4',6-diamidino-2-phenylindole to [Ru(1,10-phenanthroline)2L]2+

The binding site of Delta- and Lambda-[Ru(phenanthroline)2L]2+ (L being phenanthroline (phen), dipyrido[3,2-a:2'3'-c]phenazine (DPPZ), and benzodipyrido[3,2-a:2'3'-c]phenazine (benzoDPPZ)), bound to poly[d(A-T)2] in the presence and absence of 4',6-diamidino-2-phenylindole (DAPI) was investigated by circular dichroism and fluorescence techniques. DAPI binds at the minor groove of poly[d(A-T)2] and blocks the groove. The circular dichroism spectrum of all Ru(II) complexes are essentially unaffected whether the minor groove of poly[d(A-T)2] is blocked by DAPI or not, indicating that the Ru(II) complexes are intercalated from the major groove. When DAPI and Ru(II) complexes simultaneously bound to poly[d(A-T)2], the fluorescence intensity of DAPI decreases upon increasing Ru(II) complex concentrations. The energy of DAPI at excited state transfers to Ru(II) complexes across the DNA via the F?rster type resonance energy transfer. The efficiency of the energy transfer is similar for both [Ru(phen)2DPPZ]2+ and [Ru(phen)2benzoDPPZ]2+ complexes, whereas that of [Ru(phen)3]2+ is significantly lower. The distance between DAPI and [Ru(phen)3]2+ is estimated as 0.38 and 0.64 F?rster distance, respectively, for the Delta- and Lambda-isomer.     

Non-enzymatic glutathione conjugation of 2-nitroso-6-methyldipyrido [1,2-a: 3',2'-d] imidazole (NO-Glu-P-1) in vitro: N-hydroxy-sulfonamide, a new binding form of arylnitroso compounds and thiols

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.     

Novel alkaloids from the roots of Stemona sessilifolia

Four new Stemona alkaloids, sessilistemonamines A-C (1-3, resp.) and dihydrostemoninine (4), were isolated from the roots of Stemona sessilifolia. Their structures and relative configurations were elucidated by means of in-depth 1D- and 2D-NMR-spectroscopic as well as mass-spectrometric experiments; and the structure of 4 was solved by X-ray single-crystal diffraction. The stereoisomeric compounds 1-3 share an unprecedented tetracyclic decahydro-1H-furo[2',3':4,5]cyclopenta[1,2-b]pyrrolo[1,2-a]azepine nucleus. Compounds 1 and 2 were found to be moderately active in terms of acetylcholinesterase (AchE) inhibition, with IC50 values of 68.8+/-9.5 and 17.1+/-2.5 microM, resp.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

Synthesis of new azino fused benzimidazolium salts. a new family of DNA intercalating agents. I

A series of new pyrido[1,2-a]- and pyridazino[1,6-a]benzimidazolium salts have been synthesized from readily available 1,3-disubstituted 2-alkylbenzimidazolium salts. Their affinity to DNA and in vitro cytotoxicity versus HT-29 have been tested. The initial results show that the title compounds are a new family of intercalating agents.     

Synthesis and antitumoral evaluation of indole alkaloid analogues containing an hexahydropyrrolo[1',2',3':1,9a,9]imidazo[1,2-a]indole skeleton

The scope of acid-mediated cyclative additions of electrophiles to tryptophan-derived alpha-amino nitriles for the synthesis of 10b-substituted-1,2,4,5,10b,10c-hexahydropyrrolo[1',2',3':1,9a,9]imidazo[1,2-a]indoles analogues of indole alkaloids has been studied. The results demonstrate the high potential of the methodology for the synthesis of 10b-bromo-derivatives, by bromination with NBS, 10b-allyl-derivatives, by bromo-allyl exchange, and 10b-prenyl-derivatives, by reaction with prenyl bromide in the presence of Mg(NO(3))(2).6H(2)0. Some of the new pyrroloimidazoindole derivatives displayed moderate microM cytotoxicities in human cancer cell lines and at 10 microg/mL inhibited more than 50% EGFR or HIF-1alpha.     

Efficient synthesis of a new L-shaped dimer of naphthalene, and its analogs

Efficient syntheses of 14H-dinaphtho[1,8-bc:1',8'-fg]oxocin-14-one (2), 14H-dinaphtho[1,8-bc:1',2'-f]oxepin-14-one (3), and 2,2'(2H,2'H)-spirobi[naphtho[1,8-bc]furan] (9) are described. The putative structure of 2 has been reported previously, but the synthetic route was not reproducible. 7H-Dibenzo[c,h]xanthen-7-one (4), a known compound, was obtained by a different method. Possible reaction mechanism are proposed.     

Oxidation of the 2-hydroxyamino derivative of 2-amino-6-methyl-dipyrido[1,2-a: 3',2'-d] imidazole (Glu-P-1) to its 2-nitroso form, an ultimate form reacting with hemoglobin thiol groups

The binding to hemoglobin of synthetic 2-hydroxyamino-6-methyldipyrido[1,2-a: 3',2'-d] imidazole from the carcinogenic product of L-glutamic acid pyrolysis 2-amino-6-methyldipyrido[1,2-a: 3',2'-d] imidazole were investigated in vitro. The hydroxylamine required oxidation to its nitroso derivative to bind to rat hemoglobin through thiol groups. Oxidation of the hydroxylamine to the nitroso form was found to be enhanced by oxyhemoglobin and superoxide dismutase at pH 7.4 under aerobic conditions. Since these conditions might also enhance this oxidation in vivo, the conversion of the DNA-reactive arylhydroxylamines to the DNA-non-reactive nitroso compounds and their subsequent binding to highly abundant thiol groups of proteins could be considered as a process for detoxification of toxic arylhydroxylamines.     

Studies on synthesis and evaluation of quantitative structure-activity relationship of 5-[(3'-chloro-4',4'-disubstituted-2-oxoazetidinyl)(N-nitro)amino]-6-hydroxy-3-alkyl/aryl[1,3]azaphospholo[1,5-a]pyridin-1-yl-phosphorus dichlorides

A new series of 5-[(3'-chloro-4',4'-disubstituted-2-oxoazetidinyl)(N-nitro)amino]-6-hydroxy-3-alkyl/aryl[1,3]azaphospholo[1,5-a]pyridin-1-yl-phosphorus dichlorides has been synthesized and subjected to acute antibacterial and antifungal screening studies. All the derivatives belonging to this series delineated remarkable activity as compared to standard drugs (ampicillin and clotrimazole). Compounds are quantitatively analyzed in relation to their different physicochemical parameters. Significant correlations were obtained between biological activity and polarizability parameter (MR).     

Generation and exploration of new classes of antitubercular agents: The optimization of oxazolines, oxazoles, thiazolines, thiazoles to imidazo[1,2-a]pyridines and isomeric 5,6-fused scaffolds

Tuberculosis (TB) is a devastating disease resulting in a death every 20s. Thus, new drugs are urgently needed. Herein we report ten classes of compounds-oxazoline, oxazole, thiazoline, thiazole, pyrazole, pyridine, isoxazole, imidazo[1,2-a]pyridine, imidazo[1,2-a]pyrimidine and imidazo[1,2-c]pyrimidine-which have good (micromolar) to excellent (sub-micromolar) antitubercular potency. The 5,6-fused heteroaromatic compounds were the most potent with MIC's as low as <0.195 μM (9 and 11). Overall, the imidazo[1,2-a]pyridine class was determined to be most promising, with potency similar to isoniazid and PA-824 against replicating Mtb H(37)Rv, clinically relevant drug sensitive, multi- and extensively resistant Mtb strains as well as having good in vitro metabolic stability.     

Synthesis and analgesic/anti-inflammatory evaluation of fused heterocyclic ring systems incorporating phenylsulfonyl moiety

A series of pyrazolo[1,5-a]pyrimidine, triazolo[1,5-a]pyrimidine, and pyrimido[1,2-a]benzimidazole ring systems incorporating phenylsulfonyl moiety were synthesized via the reaction of 3-(N,N-dimethylamino)-1-aryl-2-(phenylsulfonyl)prop-2-en-1-one derivatives 2a,b with appropriate nitrogen nucleophiles. The analgesic and anti-inflammatory activities of the newly synthesized compound were investigated in vivo. 3-Bromo-2-phenyl-6-(phenylsulfonyl)-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5e) was found to have an excellent analgesic activity in comparison with indomethacin as a reference drug, while the highest anti-inflammatory effect was observed in the case of 2-(4-bromophenyl)-6-(phenylsulfonyl)-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5d). From the structure-activity relationship (SAR) point of view, the analgesic/anti-inflammatory activity of pyrazolo[1,5-a]pyrimidine derivatives was found to be much higher than triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]benzimidazole derivatives.     

The imidazo[2,1-a]isoindole system. A new skeletal basis for antiplasmodial compounds

The in vitro antiplasmodial activity of some dihydrostilbenamides, phtalazinones, imidazo[2,1-a]isoindole and pyrimido[2,1-a]isoindole derivatives related to the natural dihydrostilbenoid isonotholaenic acid is reported. The evaluation was performed on cultures of F32 strain of Plasmodium falciparum and potent representative compounds were also evaluated in the ferriprotoporphyrin IX biomineralization inhibition test (FBIT). Compounds having the imidazo[2,1-a]isoindole skeleton were the most active and one compound of this group resulted to be as potent as chloroquine, but acting through a mechanism different that of the inhibition of heme biomineralization.     

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase

The glutathione (GSH) conjugation reaction of the active metabolite of a potent protein-pyrolysate carcinogen, 2-nitroso-6-methyldipyrido[1,2-a:3',2'-d]imidazole (NO-Glu-P-1), occurred in glycerol matrix inside a fast atom bombardment (FAB) mass spectrometer. The short lived GSH-conjugates were detected by in situ FAB analysis. The precursor-product relationship between the conjugates was indicated by following the reaction by measuring [M + H]+ ions of the conjugates.