Synthesis and antifungal evaluation of 7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates

7-Arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates were synthesized and tested for in vitro antifungal activity against two pathogenic strains of fungi. Most of tested compounds showed good antifungal activity. The results suggest that those 5,8-dioxo-5,8-dihydroisoquinolines would be potent antifungal agents.     

Synthesis and cytotoxic activities of 6-chloro-7-arylamino-5,8-isoquinolinediones.

6-Chloro-7-arylamino-5,8-isoquinolinediones were newly synthesized and evaluated for in vitro cytotoxic activities against five human solid tumor cell lines. Among them, 5b, 5c and 5d exhibited potent activities against the cell lines HCT-15 and SK-MEL-2.     

Biological evaluation of newly synthesized quinoline-5,8-quinones as Cdc25B inhibitors

Cdc25B protein phosphatase represents an attractive potential therapeutic target for small molecule intervention because of its central role in positively regulating cyclin dependent kinases and thus cell proliferation, as well as its elevated levels observed in many human tumors. Among the most potent previously identified Cdc25 inhibitors have been quinoline quinones, which have a rich legacy as therapeutic agents but have also been associated with nonspecific interactions. In this study, we have interrogated the structure-activity relationship of a focused series of C2-, C3-, or C4-modified quinoline-5,8-quinones on Cdc25B inhibition in vitro. Substitution at the C3-position in this small chemical series were slightly superior to substitutions at the C3-position. For all compounds, recombinant human Cdc25B was approximately 5-fold more sensitive compared to recombinant human PTP1B. Two compounds inhibited HeLa cell growth with IC50 values of approximately 2 microM. Consistent with other para-quinones, some members of this series generated intracellular reactive oxygen species and the in vitro enzyme inhibition was mitigated by addition of reductants or catalase. These results indicate that chemical modifications on the pyridine core are tolerated, providing additional sites for future structural modification of this biologically active pharmacophore.     

Structure-activity relationships of a series of antitumoral 5,8-quinazolinediones in mutagenicity studies.

Four antitumoral 5,8-quinazolinediones were examined for their ability to induce mutation in Salmonella typhimurium. Each compound was tested at several concentrations in 4 strains. Relationships were established between the structure of the quinones and their mutagenic activities. The mutagenicity was influenced by (i) the nature of the substituent(s) of the quinonic moiety: the methoxyquinone had no mutagenic properties and the aziridinylquinones were mutagenic in the 4 strains with or without activation by S9 mix; (ii) the presence or the absence of a diaminopolymethylenic chain in the 4 position; (iii) the monomeric or the dimeric structure of the tested compound. Interestingly, the data indicated that the aziridinylquinazolinedione bearing the dimethylaminopropylamino chain in the 4 position was less mutagenic and had greater antitumor activity than the dimeric quinone.     

Effects of 4,4-dimethyl-5,8-dihydroxynaphtalene-1-one and 4,4-dimethyl-5,8-dihydroxytetralone derivatives on tumor cell respiration

A set of structurally related compounds incorporating a carbonyl group in the ortho position with regard to a phenol function were tested against the TA3 mouse carcinoma cell line and its multidrug-resistant variant TA3-MTX-R. The series consists of 2'-hydroxyacetophenone, 4'-hydroxyacetophenone 2',5'-dihydroxyacetophenone, 4-acetyl-3,3-dimethyl-5-hydroxy-2-morpholino-2,3-dihydrobenzobfuran, five 4,4-dimethyl-5,8-dioxygenated naphtalene-1-ones and three 4,4-dimethyl-5,8-dioxygenated tetralones. A tentative structure-activity relationship was found for this family of substances, suggesting that a coplanar ortho-carbonyl-1,4-hydroquinone motif is able to cause inhibition of cellular respiration.     

Substrate specificity of mammalian folylpoly-gamma-glutamate synthetase for 5,8-dideazafolates and 5,8-dideaza analogues of aminopterin

The substrate specificity of pig liver folylpolyglutamate synthetase (tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming), EC 6.3.2.17) for classical 5,8-dideaza analogues of folic acid, isofolic acid aminopterin and isoaminopterin has been investigated. 5,8-Dideazafolate and 5,8-dideazaaminopterin are very effective substrates with activities approaching those of the best reduced folate substrates. The analogous isofolate analogues are less effective substrates, but still better than folic acid. The 5-chloro substituent is the only modification that consistently increases the on rate, with 5-chloro-5,8-dideazaaminopterin being the most effective substrate found, thus far, for the enzyme. Methylation at positions 9 or 10 generally decreases binding, while 5-methylation increases the binding of 4-oxoquinazolines, but decreases the binding of their 4-amino counterparts. The presence of a formyl group at N9 or N10 has the opposite effect, decreasing the binding of 4-oxo analogues while increasing the rate for 4-amino derivatives. Increases in on rate with methyl, formyl or 4-amino substitutions are only significant when the parent compound is a poor substrate, suggesting that these groups do not interact directly with the enzyme but cause conformational changes in the structure of the substrate that influence binding to the enzyme.     

Exogenous quinones inhibit photosynthetic electron transfer in Chloroflexus aurantiacus by specific quenching of the excited bacteriochlorophyll c antenna.

In the photosynthetic green filamentous bacterium Chloroflexus aurantiacus, excitation energy is transferred from a large bacteriochlorophyll (BChl) c antenna via smaller BChl a antennas to the reaction center. The effects of substituted 1,4-naphthoquinones on BChl c and BChl a fluorescence and on flash-induced cytochrome c oxidation were studied in whole cells under aerobic conditions. BChl c fluorescence in a cell suspension with 5.4 microM BChl c was quenched to 50% by addition of 0.6 microM shikonin ((R)-2-(1-hydroxy-4-methyl-3-pentenyl)-5,8-dihydroxy-1, 4-naphthoquinone), 0.9 microM 5-hydroxy-1,4-naphthoquinone, or 4 microM 2-acetyl-3-methyl-1,4-naphthoquinone. Between 25 and 100 times higher quinone concentrations were needed to quench BChl a fluorescence to a similar extent. These quinones also efficiently inhibited flash-induced cytochrome c oxidation when BChl c was excited, but not when BChl a was excited. The quenching of BChl c fluorescence induced by these quinones correlated with the inhibition of flash-induced cytochrome c oxidation. We concluded that the quinones inhibited electron transfer in the reaction center by specifically quenching the excitation energy in the BChl c antenna. Our results provide a model system for studying the redox-dependent antenna quenching in green sulfur bacteria because the antennas in these bacteria inherently exhibit a sensitivity to O(2) similar to the quinone-supplemented cells of Cfx. aurantiacus.     

Synthesis and antifungal activity of 6-arylamino-phthalazine-5,8-diones and 6,7-bis(arylthio)-phthalazine-5,8-diones

6-Arylamino-phthalazine-5,8-diones and 6,7-bis(arylthio)-phthalazine-5,8-diones were synthesized and tested for in vitro antifungal activity against two pathogenic strains of fungi. Among those tested, many compounds showed good antifungal activity. The results suggest that phthalazine-5,8-diones would be potent antifungal agents.     

Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes.

The effect of hydroxy substitution on 1,4-naphthoquinone toxicity to cultured rat hepatocytes was studied. Toxicity of the quinones decreased in the series 5,8-dihydroxy-1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 1,4-naphthoquinone greater than 2-hydroxy-1,4-naphthoquinone, and intracellular GSSG formation decreased in the order 5,8-dihydroxy-1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone much greater than 1,4-naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. The electrophilicity of the quinones decreased in the order 1,4-naphthoquinone much greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4-naphthoquinone much greater than 2-hydroxy-1,4-naphthoquinone. Treatment of the hepatocytes with BSO (buthionine sulfoximine) or BCNU (1,3-bis-2-chloroethyl-1-nitrosourea) increased 5-hydroxy-1, 4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone toxicity, whereas neither BSO nor BCNU largely affected 1,4-naphthoquinone and 2-hydroxy-1, 4-naphthoquinone toxicity. Dicumarol increased the toxicity of 1,4-naphthoquinone dramatically and somewhat the toxicity of 2-hydroxy-1,4- naphthoquinone, whereas 5-hydroxy-1,4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone toxicity increased only slightly. The toxicity of 5,8-dihydroxy-1,4-naphthoquinone decreased dramatically in reduced O2 concentration, whereas 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 2-hydroxy-1,4-naphthoquinone toxicity was not largely affected. It was concluded that 5,8-dihydroxy-1,4-naphthoquinone toxicity is due to free radical formation, whereas the toxicity of 1,4-naphthoquinone and of 5-hydroxy-1,4-naphthoquinone also has an electrophilic addition component. The toxicity of 2-hydroxy-1,4-naphthoquinone could not be fully explained by either of these phenomena.     

5,8-Dihydroxy-3,6,7-trimethoxyflavone from Gnaphalium gaudichaudianum

5,8-Dihydroxy-3,6,7-trimethoxyflavone and 5,8-dihydroxy-6,7-dimethoxyflavone were isolated from aerial parts of Gnaphalium gaudichaudianum and identified by spectral data.     

Synthesis and antifungal activity of 6,7-bis(arylthio)-quinazoline-5,8-diones and furo[2,3-f]quinazolin-5-ols

6,7-Bis(arylthio)-quinazoline-5,8-dione and furo[2,3-f]quinazolin-5-ol derivatives were synthesized and tested for in vitro antifungal activity against Candida, Aspergillus species, and Cryptococcus neoformans. Among them tested, many of furo[2,3-f]quinazolin-5-ols and 6,7-bis(arylthio)-quinazoline-5,8-diones showed good antifungal activity. The compounds completely inhibited the growth of all against Candida and Aspergillus species tested at the MIC level of 12.5μg/mL. The results suggest that furo[2,3-f]quinazolin-5-ols and 6,7-bis(arylthio)-quinazoline-5,8-diones would be promising antifungal agents.     

Studies on quinones. Part 41: synthesis and cytotoxicity of isoquinoline-containing polycyclic quinones

In the search for new potentially anticancer drugs, isoquinolinequinone-containing polycyclic compounds have been designed and synthesized through highly regiocontrolled cycloaddition reactions of methyl 1,3-dimethyl-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate with polarized 1,3-dienes and a thiazole-o-quinodimethane. The new N-heterocyclic quinones were tested on normal human fibroblasts and four distinct human cancer cell lines. Two of the evaluated compounds displayed significant in vitro activity (IC50: 0.44-5.9 microM) comparable to that of the reference drug etoposide.     

Synthesis and antiplatelet, antiinflammatory, and antiallergic activities of substituted 3-chloro-5,8-dimethoxy-1,4-naphthoquinone and related compounds

2-Amino (6), 2-alkylamino (7–8), 2-methoxy (9), 2-acetamido (10), and 5,8-diacetoxy (11) derivatives of the lead compound 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (4) were synthesized, together with 6,7-dichloro-5,8-dimethoxy-1,4-naphthoquinone (5), a positional isomer of 4. Antiplatelet, antiinflammatory, and antiallergic activities were evaluated, and most compounds were quite potent in all assays. Compounds 5 and 9–11 were especially active; however, 5 was ineffective against neutrophil superoxide formation, and 10 was ineffective against mast cell degranulation.     

Redox properties of novel antioxidant 5,8-Dihydroxycoumarin: implications for its prooxidant cytotoxicity

The aim of this work was to characterize the redox properties of the new antioxidant 5,8-dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochlo? odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8-DHC at pH 7.0 was characterized by the midpoint potential (E(p/2)) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H202 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 +/- 5.5) microM. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.     

The molecular basis of leucine auxotrophy of quinone-treated Escherichia coli. Active site-directed modification of leucyl-tRNA synthetase by 6-amino-7-chloro-5,8-dioxoquinoline.

Leucyl-tRNA synthetase from Escherichia coli is rapidly inactivated by 6-amino-7-chloro-5,8-dioxoquinoline (quinone), a model substance for cytostatic quinones. Loss of activity follows pseudo-first order kinetics. The quinone masks essential--SH groups that are reactive with N-ethylmaleimide. Specific protection of the enzyme by leucine provides evidence for active site-directed modification. Half-maximal protection is found at a concentration of 150 micron which is identical with the dissociation constant of the enzyme.substrate complex. The competitive inhibitor leucinol also protects the enzyme from inactivation by the quinone. MgATP enhances the protective effect of leucinol about 250-fold, thus substantiating recently published findings on synergistic coupling of ligands to aminoacyl-tRNA synthetases. The results support the assumption that the bacteriostatic quinone directly interferes with leucyl-tRNA synthetase in growing cells. Active-site-directed inhibition of the enzyme could adequately explain the phenotypically observed auxotrophy for leucine of quinone-treated E. coli.     

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.     

Synthesis of [3alpha-3H]cholesta-5,8-dien-3beta-ol and tritium-labeled forms of other sterols of potential importance in the Smith-Lemli-Optiz syndrome

Five unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome have been prepared in high radiochemical purity with a tritium label at the 3alpha position. Swern oxidation of cholesta-5,8-dien-3beta-ol and other unlabeled C27 sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH4 gave the desired 3alpha-3H sterols, with double bonds at the delta(5,8), delta(5,8(14)), delta(6,8), delta(6,8(14)), and delta8 positions. High radiochemical purity of the tritiated sterols was demonstrated by normal phase, reversed phase, and silver-ion (Ag+) high-performance liquid chromatography (HPLC). In the course of this work, we developed a medium-pressure variant of Ag+-HPLC for purifying radiolabeled samples, documented significant isotopic fractionation of the 3alpha-tritiated sterols and their acetates on Ag+-HPLC, and discovered unexpected effects of a delta(8(14)) bond on the conformation of 3-keto-delta5-steroids. The synthetic and analytical methodologies described herein should provide a sound basis for investigating the origin and metabolism of sterols involved in the Smith-Lemli-Opitz syndrome and in late stages of cholesterol biosynthesis.     

The anti-cancer,anti-inflammatory and tuberculostatic activities of a series of 6,7-substituted-5,8-quinolinequinones

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone (10a) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone (12), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone (18a) exhibiting the highest AI activity (0.11 μM), while 6,7-dichloro-5,8-quinolinequinone (7a), 6,7-dichloro-2-methyl-5,8-quinolinequinone (7b), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol (19) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone (11) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone (14), exhibited low MICs (1.56–3.13 μg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.     

Rational design of quinazoline-based irreversible inhibitors of human erythrocyte purine nucleoside phosphorylase

Described herein is the rational design of irreversible inhibitors of human erythrocyte purine nucleoside phosphorylase (PNPase). Inhibitor design started with the observation that the amino group of 8-aminoquinazolin-4(3H)-one interacts with enzyme-bound phosphate. This observation correctly predicted that the 5,8-dione (quinone) and 5,8-dihydroxy (hydroquinone) derivatives of quinazolin-4(3H)-ones would enter the active site. The amine-phosphate interaction also served to confirm that a quinazolin-4(3H)-one binds in the PNPase active sites like a purine substrate. From models of the PNPase active site it was possible to design quinazoline-based quinones that undergo a reductive-addition reaction with an active-site glutamate residue. The best inhibitor studied, 2-(chloromethyl)quinazoline-4,5,8(3H)-trione, rapidly inactivates PNPase by a first-order process with an inhibitor to enzyme stoichiometry of 150. The active-site hydroquinone adduct of this inhibitor eliminates a leaving group to afford a quinone methide species positioned to alkylate another active-site glutamate residue. Thus, this inhibitor is designed to cross-link the PNPase active site by reductive addition followed by the generation of an alkylating quinone methide species.     

Isolation of 5,8-oxyretinoic acid from rat intestinal mucosa.

Polar metabolites of retinoic acid accumulate in the intestine of vitamin A-deficient rats 3 h after administration of 450 μg of [11,12-³H]retinoic acid. Using new Chromatographic procedures developed for the purification of vitamin A metabolites, a major polar derivative of retinoic acid was isolated from intestine in pure form as its methyl ester and positively identified as 5,8-oxyretinoic acid.