Microwave-assisted synthesis of 2(1H)-pyridones and their glucosides as cell proliferation inhibitors

A new series of substituted 2(1H)-pyridones (4a-i) and their glucosides (5, 6a-e) were prepared as potential agents against leukemia (HL-60) cells. Glucosides (5,6a-e) were synthesized using three independent methods. Microwave protocol as an ecologically new method was used to synthesize the target compounds. Structures of the new products were confirmed using one- and two-dimensional NMR spectroscopy. In vitro exposure of pyridones substituted at position 4 with a 2-thienyl or 2-(trifluoromethyl)phenyl were found to exhibit high antiproliferation activities; in particular, 3-cyano-4-(thien-2'-yl)-6-(4'-chlorophenyl)-2(1H)-pyridone (4c) and its glucoside analogue (6c) had the highest activity.     

Synthesis and antimicrobial activity of some new substituted pyrido[3′,2′:4,5]thieno[3,2-d]-pyrimidinone derivatives

A series of new 3-substituted-7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one derivatives were synthesized as antimicrobial agents using 7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3′,2′:4,5]thieno[3,2-d]-[1,3]oxazin-4-one as a starting compound. Its condensation with substituted aniline derivatives or phenyl hydrazine gave the corresponding N-substituted derivatives. Treatment of the starting compound with hydrazine hydrate afforded the corresponding N-amino derivative, which was reacted with substituted phenylisocyanate and phenylisothiocyanate derivatives to give the corresponding semicarbazides and thiosemicarbazide derivatives. All the newly synthesized compounds were evaluated for their antimicrobial activities in comparison to streptomycin and fusidic acid as positive controls. The structure assignments of the new compounds are based on chemical and spectroscopic evidence.     

Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease

A series of new 7-substituted-4-chloro-3-alkoxy isocoumarin derivatives were synthesized and evaluated as inhibitors of representative classes of proteases: serine protease (alpha-chymotrypsin, trypsin), cysteine protease (Caspase-3), and aspartyl protease (HIV-protease), 20S proteasome and also as inhibitors of amyloid peptide gamma-secretase-mediated production. Protease inhibition selectivity is directly related to the structure of the substituent at the 7-position of the isocoumarin nucleus. 7-Nitro-isocoumarin derivatives (4c, 4d, 4f) are potent alpha-chymotrypsin inhibitors but slightly active or inactive on HIV-protease, as well as on cysteine protease. In contrast, only derivatives bearing a free amino (5d, 5f) or a substituted amino group (6f) at the 7-position of the isocoumarin nucleus, were found weakly active or inactive on alpha-chymotrypsin, trypsin, Caspase-3 and HIV-protease, but prevent gamma-secretase-mediated production of Abeta 40/42 amyloid peptides, which is known to be involved in Alzheimer's disease. Moreover, the most active compounds on beta-amyloid peptide production [JLK6 (5d), JLK2 (5f) and JLK7 (6f)] show only weak or moderate inhibitory activity on the 20S proteasome. The obtained results suggest that the described new isocoumarin analogues could be of interest, since compounds like JLK6 (5d), JLK2 (5f) and JLK7 (6f) can be considered as possible hits for the development of new agents directed towards Alzheimer's disease.     

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).     

Syntheses of phenylpyrazolodiazepin-7-ones as conformationally rigid analogs of aminopyrazole amide scaffold and their antiproliferative effects on cancer cells

Recently, we have reported the syntheses and antiproliferative activities of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives on melanoma cells. As a continuous work for antiproliferative agents in melanoma, here we report the synthesis of conformationally rigid analogs, phenyl-6,8-dihydropyrazolo[3,4-b][1,4]diazepin-7(1H)-one derivatives 7a-g, 8a-o and their antiproliferative activities against A375P melanoma cell line and U937 hematopoietic cell line. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, N-(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-4-chloro-3-(trifluoro methyl)benzamide-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl) phenyl) ureido)-2-methylbenzamide (7b) exhibited potent activities (GI(50)=0.43 μM and 0.06 μM) on both cell lines. It has been further confirmed to be a potent and selective Raf kinases inhibitor and also mild inhibitor of PI3Kα.     

Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological evaluation of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives as potential anticancer agents

A series of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives 5, 6 and 7 were prepared starting from 6-phenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 3 via selective N-alkylation, followed by reaction with different primary aliphatic amines, cyclic secondary amines or l-amino acids under different set of conditions. All the synthesized compounds 5, 6 and 7 were screened for anticancer activity against four cancer cell lines such as A549—Lung cancer (CCL-185), MCF7—Breast cancer (HTB-22), DU145—Prostate cancer (HTB-81) and HeLa—Cervical cancer (CCL-2). The compounds 5i and 6e are found to have promising bioactivity at micro molar concentration.     

Photosynthesis involvement in the mechanism of action of diphenyl ether herbicides

Photosynthesis is not required for the toxicity of diphenyl ether herbicides, nor are chloroplast thylakoids the primary site of diphenyl ether herbicide activity. Isolated spinach (Spinacia oleracea L.) chloroplast fragments produced malonyl dialdehyde, indicating lipid peroxidation, when paraquat (1,1′-dimethyl-4,4′-bipyridinium ion) or diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] were added to the medium, but no malonyl dialdehyde was produced when chloroplast fragments were treated with the methyl ester of acifluorfen (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid), oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene], or MC15608 (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chlorobenzoate). In most cases the toxicity of acifluorfen-methyl, oxyfluorfen, or MC15608 to the unicellular green alga Chlamydomonas eugametos (Moewus) did not decrease after simultaneous treatment with diuron. However, diuron significantly reduced cell death after paraquat treatment at all but the highest paraquat concentration tested (0.1 millimolar). These data indicate electron transport of photosynthesis is not serving the same function for diphenyl ether herbicides as for paraquat. Additional evidence for differential action of paraquat was obtained from the superoxide scavenger copper penicillamine (copper complex of 2-amino-3-mercapto-3-methylbutanoic acid). Copper penicillamine eliminated paraquat toxicity in cucumber (Cucumis sativus L.) cotyledons but did not reduce diphenyl ether herbicide toxicity.     

Competitive interaction of three peroxidizing herbicides with the binding of [3H]acifluorfen to corn etioplast membranes

The specific binding of the herbicide acifluorfen 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid to corn etioplast membranes is competitively inhibited by protoporphyrinogen IX, the substrate of protoporphyrinogen oxidase. Three other peroxidizing molecules, oxadiazon [5-terbutyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol -2-one], LS 82556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine], and M&B 39279 [5-amino-4-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazol], also compete with acifluorfen for its binding site. The four herbicides thus bind to the same site, or to closely located sites, on the enzyme protoporphyrinogen oxidase.     

A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2′-amino-4′-chlorophenyl) pyrrole.     

Tryptophan 7-Halogenase from Pseudomonas aureofaciens ACN Strain: Gene Cloning and Sequencing and the Enzyme Expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

Peroxidase catalyzed nitration of tryptophan derivatives. Mechanism, products and comparison with chemical nitrating agents.

The enzymatic nitration of tryptophan derivatives by oxidation of nitrite has been studied using lactoperoxidase and horseradish peroxidase, and compared with the chemical nitration produced by nitrogen dioxide and peroxynitrite. HPLC, mass spectra and NMR analysis of the mixture of products clearly show that nitration occurs at position 4-, 6-, 7-, and N1 of the indole ring, and nitrosation at position N1. Kinetic studies performed on peroxidase/NO2-/H2O2 systems showed substrate saturation behavior with all the tryptophan derivatives employed. The rate dependence on nitrite concentration was found to be linear with horseradish peroxidase while it exhibited saturation behavior with lactoperoxidase. The composition of the product mixture depends on the nitrating agent. While the production of 4-nitro, 6-nitro, 7-nitro and N1-nitro derivatives follows a similar trend, indicating that they are formed according to a similar mechanism, the ratio between the N1-nitroso derivative and other derivatives depends markedly on the nitrite concentration when tryptophan modification is performed by the peroxidase/H2O2/nitrite systems. Analysis of the data indicates that at low nitrite concentration the enzymatic reaction occurs through the classical peroxidase cycle. At high nitrite concentration the reaction proceeds through a different intermediate that we assume to be a protein bound peroxynitrite species.     

Synthesis and HPLC chiral recognition of regioselectively carbamoylated cellulose derivatives

Four regioselective-carbamoylated cellulose derivatives having two different substituents at 2-, 3-, and 6-position were prepared and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. Investigations showed that the nature and arrangement of the substituents significantly influenced the chiral recognition abilities of the heterosubstituted cellulose derivatives and each derivative exhibited characteristic enantioseparation. Some racemates were better resolved on these derivatives than the corresponding homogeneously substituted cellulose derivatives including a commercial CSP, Chiralcel OD. Racemic compounds shown in this study were most effectively discriminated on cellulose 2,3-(3-chloro-4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) and 2,3-(3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate).     

Electron spin resonance investigations of mitochondrial electron transport in Neurospora crassa. Characterization of paramagnetic intermediates in a standard strain.

1. Submitochondrial particles from Neurospora strain inl-89601 have been analyzed by electron spin resonance spectroscopy (ESR). Numerous signals due to iron-sulfur proteins are observed at low temperatures. Analysis of these ESR signals at various temperatures allows the assignment of resonances to iron-sulfur centers 1-5 that have been described in other organisms. There are no discrepancies between the signals seen in Neurospora and those described in other organisms and it is likely that Neurospora mitochondria contain the same iron-sulfur centers that are observed elsewhere. 2. NADPH and NADH act to reduce the iron-sulfur centers of respiratory complex I. 3. The drug pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorphenyl)pyrrole] is an effective inhibitor of both NADH-supported and succinate-supported electron transport in Neurospora. 4. Analysis of pyrrolnitrin inhibition curves, respiration studies, ESR spectra, and the steady-state level of reduction of cytochrome b in the presence and absence of the drug shows that pyrrolnitrin acts to inhibit electron transport in Neurospora mitochondria at multiple sites in the region between ubiquinone and cytochrome b.     

Mefloquine-oxazolidine derivatives, derived from mefloquine and arenecarbaldehydes: In vitro activity including against the multidrug-resistant tuberculosis strain T113

Ten new mefloquine-oxazolidine derivatives, 4-[(1S,8aR)-3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline (1: aryl=substituted phenyl) and 4-[(1S,8aR)-3-(heteroaryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline [2: heteroaryl=5-nitrothien-2-yl (2a); 5-nitrofuran-2-yl (2b) and 4H-imidazol-2-yl) (2c)], have been synthesized and evaluated against Mycobacterium tuberculosis. Compounds 1f (aryl=3-ethoxyphenyl), 1g (Ar=3,4,5-(MeO)(3)-C(6)H(2)) and 2c were slightly more active than mefloquine (MIC=33μM) with MICs=24.5, 22.5 and 27.4, respectively, whereas compounds 1e (aryl=3,4-(MeO)(2)-C(6)H(3)) and 2a (MICs=11.9 and 12.1μM, respectively) were ca. 2.7 times more active than mefloquine, with a better tuberculostatic activity than the first line tuberculostatic agent ethambutol (MIC=15.9). The compounds were also assayed against the MDR strain T113 and the same MICs were observed. Thus the new derivatives have advantages over such anti-TB drugs as isoniazid, rifampicin, ethambutol and ofloxacin, for which this strain is resistant. The most active compounds were not cytotoxic to Murine Macrophages Cells in a concentration near their MIC values.     

1,2,4-Triazino-[5,6b]indole derivatives: effects of the trifluoromethyl group on in vitro antimalarial activity

In an attempt to search for new and alternative antimalarial agents, a series of unsubstituted and 6-trifluoromethyl-1,2,4-triazino[5,6b]indole and 5H-1,2,4-triazolo[1',5',2,3]-1,2,4-triazino[5,6b]indole derivatives were synthesized and their chemical structures confirmed by 1H NMR and 13C NMR, elemental, IR and mass spectrophotometric analyses. The in vitro antimalarial activities of these compounds were evaluated against the chloroquine-sensitive (D10) and the chloroquine-resistant (RSA11) strains of Plasmodium falciparum. The 1,2,4-triazino[5,6b]indole derivatives (4, 6 and 8) with a trifluoromethyl group at position 6 exhibit increased in vitro activity when compared to the unsubstituted analogues, which are all devoid of activity. The presence of the trifluoromethyl group in the 5H-1,2,4-triazolo[1',5',2,3]-1,2,4-triazino[5,6b]indole ring system leads to compounds with diminished antimalarial activity when compared to the corresponding unsubstituted analogues. The compounds associate with ferriprotoporphyrin IX and interact with DNA to more or less the same extent.     

Pyrrolnitrin Production by an Enterobacter agglomerans Strain with a Broad Spectrum of Antagonistic Activity Towards Fungal and Bacterial Phytopathogens

Strain IC1270 of Enterobacter agglomerans has been previously described as a producer of a complex of chitinolytic enzymes and as an antagonist of many fungal phytopathogens [Chernin et al. (1995) Appl. Env. Microbiol. 61:1720–1726]. Here we show that this strain also produces an antibiotic that was purified by TLC and HPLC and identified by UV, IR, MS, and NMR analyses as pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole]. The purified antibiotic is efficient against many phytopathogenic bacteria and fungi in vitro. This is the first piece of evidence showing that pyrrolnitrin can be produced by bacteria other than Pseudomonas and that one bacterial strain can simultaneously produce chitinolytic enzymes and pyrrolnitrin. The possible role of a combination of chitinases and pyrrolnitrin in antagonism is discussed.     

Structural activity relationship between Salmonella-mutagenicity and nitro-orientation of nitroazaphenanthrenes

Nitroazaphenanthrenes (NAphs) and their N-oxides (NAphOs) were synthesized as derivatives with nitrogen atoms in the 1, 4, and 9 positions of phenanthrene rings, and as nitrated derivatives substituted at the 1, 2, 3, 4, 5, 6, 7, and 8 positions of phenanthrene rings. To determine the structure activity relationship of these derivatives, all 19 isomers were bioassayed with Salmonella tester strains. NAphs substituted at the 4, 6, 7 and 8 positions were mutagenic for TA98, and 1-, 2-, and 3-N-9-AphOs, 6-N-1-AphO and 6-N-4-AphO were mutagenic for TA98 and TA100 without the S9 mix, while 5-N-1-AphO and 5-N-9-AphO were non- or weakly mutagenic. Nitrated derivatives, 6-N-4-Aph, 6-N-9-Aph, 6-N-1-AphO, and 6-N-4-AphO, were powerful mutagens for TA98 and TA100. Mutagenicity was enhanced by mutant strains producing nitroreductase, such as YG1021 and 1026, and by those producing O-acetyltransferase, such as YG1024 and 1029. Nitro derivatives substituted at positions 4 and 5 in the phenanthrene rings were perpendicular, while those at positions 2, 3, 6 and 7 were coplanar to the phenanthrene rings. NAphs substituted at the 1 and 8 positions were noncoplanar due to steric hindrance of the aromatic proton at the peri position. On the other hand, 1,5- and 1,8-dinitro-4-azaphenanthrenes showed high mutagenicity for strains TA98 and TA100 in the absence of the S9 mix, and were strongly enhanced by nitroreductase and O-acetyltransferase, over-producing mutants. Therefore, it was found that the mutagenic potency of NAphs and NAphOs was closely associated with the chemical properties and orientation of nitro substitution of aromatic rings.     

Design and synthesis of novel 7-heterocycle-6-trifluoromethyl-3-oxoquinoxaline-2-carboxylic acids bearing a substituted phenyl group as superior AMPA receptor antagonists with good physicochemical properties

We describe the design, synthesis, and physicochemical and biological properties of a novel series of 7-heterocycle-6-trifluoromethyl-3-oxoquinoxaline-2-carboxylic acids bearing a substituted phenyl group joined through a urethane or urea linkage to the heterocycle at the 7 position. Introduction of the trifluoromethyl group at the 6 position conferred good biological activity, including neuroprotective effects, as well as good physicochemical properties. In terms of alpha-amino-3-hydroxy-5-methylisoxazole propionate receptor (AMPA-R) affinity, a urea linkage was equivalent to a urethane linkage and a pyrrole ring at the 7 position reduced affinity in comparison with an imidazole ring. Among this series, compound 14h (KRP-199), which has a 4-carboxyphenyl group joined through a urethane linkage to a 7-imidazolyl heterocycle, was found to possess high potency and selectivity for the AMPA-R in vitro and to exhibit good neuroprotective effects in vivo. Furthermore, the compound showed good physicochemical properties, including stability to light and good solubility in aqueous solutions.