Rational design and synthesis of novel thiazolidin-4-ones as non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel thiazolidin-4-one analogues, characterized by different substitution patterns at positions C-2 and N-3 of the thiazolidin-4-one scaffold for anti-HIV-1 activity has been investigated. Most of the compounds showed anti-HIV-1 activity at micromolar concentrations when tested in TZM-bl cells in vitro. Among the thirty-three compounds tested, compound 16 was the most potent inhibitor of HIV-1 replication against HIV-1_IIIB, HIV-1_ADA5, HIV-1_UG070 and HIV-1_VB59 (EC₅₀ = 0.02, 0.08, 0.08 and 0.08 μM, respectively) with selectivity index (SI = 6940, 1735, 1692 and 1692) against tested viral strains, respectively. The results of the present study suggested that the substitution of the nitro group at 6′ position of the C-2 phenyl ring and 4,6-dimethylpyridin-2-yl at the N-3 position of thiazolidin-4-one had a major impact on the anti-HIV-1 activity and was found to lower cytotoxicity. The substitution of the heteroaryl ring with bromo group and bicyclic heteroaryl ring at N-3 thiazolidin-4-one was found to lower anti-HIV-1 activity and increase cytotoxicity. The undertaken docking studies thus facilitated the identification of crucial interactions between the HIV-1 RT enzyme and thiazolidin-4-one inhibitors, which can be used to design new potential inhibitors.     

Mutagenicity of nitro-azabenzo[a]pyrene and its related compounds.

The mutagenicity of nitrated benzo[a]pyrene (BP) and the related compounds, 1- and 3-nitrobenzo[a]pyrene (NBP), 1- and 3-nitro-6-cyanobenzo[a]pyrene (N-6-CBP), 1- and 3-nitro-6-azabenzo[a]-pyrene (N-6-ABP), 1- and 3-nitro-6-azabenzo[a]-pyrene-N-oxide (N-6-ABPO) and 1,6- and 3,6-dinitrobenzo[a]-pyrene (DNBP), was investigated. The mutagenic activities of 3-N-6-CBP and 3-N-6-ABP were 117 and 76 times, respectively, that of 3-NBP. In addition, 3,6-DNBP was more mutagenic than 1,6-DNBP. It is suggested that the mutagenic activation differs with the position of NO2 substitution in the chemical structure. A nitro derivative with NO2 substitution at the 3 position of the aromatic ring of BP was more mutagenic than that with the substitution at the 1 or 6 position. The reducibility of DNBPs was then determined by detecting 1- or 3-amino-6-nitrobenzo[a]pyrene (A-6-NBP), a metabolite of DNBP; 3,6- and 1,6-DNBP were reduced to 3- and 1-A-6-NBP at frequencies of 958 +/- 26 and 79 +/- 8, respectively, pmole per mg of protein, when the compound was incubated anaerobically with rat liver S9 mix at 37 degrees C for 15 min. NO2 substituted at the 3 position of the aromatic ring of BP was readily reduced by a microsome enzyme to form an amino derivative. The result suggests that these compounds have a structure-activity relationship between mutagenicity and NO2 substitution of BP.     

Methylated 7-deazahypoxanthines as regiochemical probes of xanthine oxidase

7-Deazahypoxanthine was found to be oxidised by cow's milk xanthine oxidase exclusively at carbon 2. The resulting 7-deazaxanthine is a strong inhibitor of the enzymatic reaction. This offers a possibility for determining the structural requirements of ligand binding separately for the first step. All the monomethyl isomers of 7-deazahypoxanthine were tested as probes by measuring their Km, Ki and V values. While the N-3-methyl and C-7-methyl isomers are still processed, the N-9-methyl and 6-O-methyl isomers are bound as inhibitors to the active site. The N-1-methyl compound is neither an inhibitor nor a substrate. This demonstrates that HN(1) and O = C(6) are essential for the binding. Replacement of O = C(6) by S = C(6) changes the substrate into a strong inhibitor (Ki = 9 microM), implying that the electron transfer to the enzyme is hindered. Methylation of the thioxo group (S =) reduces the inhibition significantly. In contrast to 7-deazahypoxanthine, 2-thioxo-7-deazaxanthine is an activator at concentrations below 87 microM and a partial competitive inhibitor above this concentration, which implies the presence of a second binding site.     

Biosynthesis and cytokinin activity of 8-hydroxy and 2,8-dihydroxy derivatives of zeatin and N-6(increment-2-isopentenyl)adenine.

8-Hydroxy and 2,8-dihydroxy derivatives of the cytokinins, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and N-6-(increment -2-isopentenyl)adenine, have been biosynthesized by xanthine oxidase oxidation. 8-Hydroxy derivatives have been shown to be the major intermdeiates. These compounds were tested for cytokinin activity in the tobacco bioassay. The results suggest that substitution of the 8 position with a hydroxyl group causes less decrease of cytokinin activity than substitution of both the 2 and 8 positions with hydroxyl groups.     

Calcium-independent activation of calcium ion dependent cyclic nucleotide phosphodiesterase by synthetic compounds: quinazolinesulfonamide derivatives

Quinazolinesulfonamides are synthetic compounds which calcium-independently stimulate Ca2+-dependent cyclic nucleotide phosphodiesterase. As this activation was observed with 2,4-dipiperidino-6-quinazolinesulfonamides but not with 4-piperidino-6-quinazolinesulfonamides, the activation seems to be dependent on the piperidine residue at the 2 and 4 position of the quinazoline ring, and the extent of hydrophobicity of each compound was thus enhanced. 2,4-Dipiperidino-6-quinazolinesulfonamide activates Ca2+-dependent phosphodiesterase in the absence of Ca2+-calmodulin (CaM). These quinazolinesulfonamides did not further enhance the activity of Ca2+-dependent phosphodiesterase activated by the Ca2+-CaM complex. These compounds are also potent inhibitors of cyclic AMP and GMP phosphodiesterases. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), its derivatives, and chlorpromazine and prenylamine inhibited selectively the quinazolinesulfonamide-induced activations of the phosphodiesterase. These quinazolinesulfonamides, in a high concentration, had only a slight stimulatory effect on myosin light chain kinase activity. All these findings suggest that the quinazolinesulfonamides are calcium-independent activators of Ca2+-dependent phosphodiesterase and they are proving to be useful tools for the study of CaM and phosphodiesterase, in vitro.     

Anti-cancer activities of 5-acyl-6-[2-hydroxy/benzyloxy-3-(amino)-propylamino]-1,3-dialkyl-1H-pyrimidin-2,4-diones

All the nine 1,3-dialkylated-pyrimidin-2,4-diones investigated are active against all the 59 human tumor cell lines. Compounds 2, 3, 4, and 6 show significant anti-cancer activities at some specific cell lines while compounds 7 and 9 exhibit anti-cancer activities against more number of cell lines. The structure–activity relationship studies indicate that the presence of piperidine/pyrrolidine at the end of C-6 chain, benzoyl group at C-5, and benzyl groups at N-1, N-3 of the pyrimidine ring increases the anti-cancer activities of these molecules.     

Pteridines as inhibitors of xanthine oxidase: structural requirements

Different pteridine derivatives were investigated for their inhibitory action on xanthine oxidase. From 27 investigated compounds, 13 showed concentration-dependent inhibition of the enzyme. Concentrations necessary for 50% inhibition ranged from <0.1 up to >100 microM. Different types of inhibition were found concerning xanthine and pterin as substrates: competitive, noncompetitive and mixed type. Out of 18 aromatic compounds tested, 12 were inhibitors. Only one out of nine reduced derivatives served as inhibitor. A simple regression model was used to specify the structural requirements for a pteridine to be an inhibitor. The most characteristic features of an inhibitor are aromaticity and no substitution at position 7 of the pteridine ring.     

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.     

Xanthine phosphoribosyltransferase from Streptococcus faecalis. Properties and specificity

Streptococcus faecalis (ATCC 8043) was shown to have a purine phosphoribosyltransferase specific for xanthine. This enzyme was separated from interfering activities by heat treatment, ammonium sulfate fractionation, hydroxylapatite chromatography, and affinity chromatography. The xanthine phosphoribosyltransfer activity of this preparation was stable between pH 5.6 and 10, had a pH optimum between pH 7.4 and 8.8, and had a particle weight of 42,000 as determined by G-100 Sephadex chromatography. An initial velocity analysis when plotted in double-reciprocal form resulted in a family of parallel lines which when extrapolated to infinite concentration gave K_m values for xanthine and PP-ribose-P of 20 and 53 μm, respectively. Inhibition studies with 42 purine and purine analogs indicated that oxo groups at positions 2 and 6 of the purine ring were required for optimal binding. The substitution of thio for oxo reduced binding to the enzyme ca. 20-fold. In contrast to its rigid specificity with respect to the 2,6-dioxo substituents, the enzyme bound a variety of 4,5-condensed pyrimidine systems containing a nitrogen at the position corresponding to the N-7 of xanthine. At concentrations of 1 mm, hypoxanthine, adenine, and 4,6-dihydroxypyrazolo[3,4-d]pyrimidine were converted to their corresponding ribonucleotides at rates approximately 0.1% of the rate for xanthine. Guanine was not detected as a substrate (rate <0.007% that of xanthine). The enzyme was inhibited by the ribonucleoside mono-, di-, and triphosphates of xanthine and guanine but not by those of adenine.     

Substituent--directed aralkylation and alkylation reactions of the tricyclic analogues of acyclovir and guanosine

Aryl or tert-butyl substituent in the 6 position of 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-6-R-5H-imidazo[1,2-a]purine (6-R-TACV) 1 partly directs aralkylation reactions into unusual positions: N-4 to give 3 and C-7 to give N-5,7-disubstituted or N-4,7-disubstituted derivatives. In the case of alkylation the effect is limited to aryl substituent and position N-4. Replacement of acyclic moiety of 1 with a ribosyl one like in 7 prevents N-4 substitution. Cleavage of the third ring of 3b to give 3-benzylacyclovir 10 is an example of a new short route to 3-aralkyl-9-substituted guanines.     

Novel N-3 substituted TSAO-T derivatives: synthesis and anti-HIV-evaluation

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.     

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase.

1. Pteridin-4-ones, methylated at nitrogen or carbon, N-methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine : oxygen oxidoreductase, EC 1.2.3.2). 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N-Methylation may increase or reduce rates of oxidation. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2) = N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6) = C(7). 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.     

Quaternary salts of 4,3' and 4,4' bis-pyridinium monooximes. Part 2: synthesis and biological activity

In continuation of our investigations of unsymmetrical bisquaternary monooximes, we synthesized four new series of compounds bridged by hexyl, heptyl, octyl and nonyl groups. All eight monooximes viz., dibromides of 1-(4-hydroxyiminomethylpyridinium)6-(3/4-carbamoylpyridinium)hexane, 1-(4-hydroxyiminomethylpyridinium)-7-(3/4-carbamoylpyridinium)heptane, 1-(4-hydroxyiminomethylpyridinium)-8-(3/4-carbamoylpyridinium)octane, 1-(4-hydroxyiminomethylpyridinium)-9-(3/4-carbamoylpyridinium)nonane as well as the corresponding bis-oximes were synthesized and characterized by spectral data. Their ability to reactivate tetraethylpyrophosphate (TEPP) inhibited mouse total brain cholinesterase was investigated and compared with the conventional oxime 2-pyridinealdoxime chloride (2-PAM). Mouse brain homogenate was used as the source of acetylcholinesterase. Among all the compounds, tested the compound with the hexylene bridge (6b) and a 3-carbamoyl group on the second pyridine ring was found to be the most active acetylcholinesterase reactivator (72%) which is greater than that of 2-PAM (56%). However, the activity was reversed; as the chain length increased from a heptylene to a nonylene bridge, they potentiated the inhibitory effect of TEPP rather than reactivation. It is interesting to note that compound 6b with a carbamoyl group at the 3rd position of the pyridine ring showed dose dependent reactivation whereas the corresponding compound 6a with the carbamoyl group present at the 4th position of the pyridine ring showed reactivation at lower concentration (30 microM) and potentiation of TEPP inhibition at higher concentrations (100 and 300 microM).     

Synthesis and cytotoxic evaluation of new (4,5,6,7-tetrahydro-indol-1-yl)-3-R-propionic acids and propionic acid ethyl esters generated by molecular mimicry

Indolones 4 and 5, and indolyl-aminoacids 6a-e, 7a-e, and 8a and 8b were designed by structural modification of lead compound 3. These compounds were tested on six tumor cell lines to determine the role of the azepinone ring and the N-phenyl substituent in the cytotoxicity of 3. Our results show that 4 and 5 have dramatically reduced cytotoxicity, due to the loss of the azepinone moiety of lead compound 3. In contrast, indolyl-aminoacids 6a, 7a, and 8a (N-(L)-cysteine ethyl ester derivatives) inhibited the proliferation of almost all cancer cell lines tested, even though they lack the azepinone ring. In addition, derivative 6c (N-(D)-alanine methyl ester group) was selectively cytotoxic to HCT-15 cells. Preliminary structure-activity relationship (SAR) studies with these compounds revealed the importance of the ethyl ester moiety on the amino acid moiety. Compounds 6a-e, 7a-e, and 8a and 8b were obtained in good yields by a catalytic Paal-Knorr reaction carried out under microwave irradiation using commercially available chiral amino esters or amino acids and 1,4-dicarbonyl compounds.     

Synthesis and in vitro binding studies of substituted piperidine naphthamides. Part I: Influence of the substitution on the basic nitrogen and the position of the amide on the affinity for D2L, D4.2, and 5-HT2A receptors

A series of 1- and 2-naphthamides has been prepared and tested for in vitro binding to D(2L), D(4.2), and 5-HT(2A) receptors. Different compounds display selectivity for D(4.2) and 5-HT(2A) receptors versus D(2L) receptors. N-(1-Arylalkyl-piperidin-4-yl) carboxamides have higher affinities than the corresponding N-(4-arylalkylamino-piperidin-1-yl) carboxamide analogues. A benzyl moiety in position 1 of the piperidine in the 2-naphthamide series (2) appears to be the best choice for a favorable interaction with D(4.2) and 5-HT(2A) receptors. Increasing the linker length between the phenyl ring and the basic nitrogen led to a decreased affinity for these receptors. In the 1-naphthamide series, the most potent D(4.2) ligand (7) possesses a phenylpropyl moiety while its affinity for 5-HT(2A) receptors is strongly reduced. All compounds with significant affinity for D(4.2) and 5-HT(2A) receptors were antagonists.     

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.     

2-Phenyl-1-[4-(2-piperidine-1-yl-ethoxy)benzyl]-1H-benzimidazoles as ligands for the estrogen receptor: Synthesis and pharmacological evaluation

2-Phenyl-1H-benzimidazoles 7a–e were synthesized and tested for gene activation on ERα-positive MCF-7 breast cancer cells, stably transfected with the reporter plasmid ERE_wtcluc (MCF-7-2a cells). None of the compounds showed agonistic properties, but they antagonized dependent on hydroxyl groups at the benzimidazole core (5- or 6-OH) and at the aromatic ring in the 2-position (4-OH) in high concentrations the gene activation induced by estradiol (E2, 1 nM). All compounds exhibited significant antiproliferative properties on MCF-7 cells but they were inactive against hormone independent, ER negative MDA-MB-231 cells.     

Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase.

1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3) NH, (9) N or (3) N, (9) NH. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.