Synthesis of amino acid derived seven-membered lactams by RCM and their evaluation against HIV protease

A versatile synthesis of hydroxylated and epoxy 1-azepin 2-ones substituted at N1, C-3 and C-4 or C-7 has been developed. The sequence involves ring-closing metathesis of an amino acid derived diene amide, followed by either epoxidation or dihydroxylation, of the resulting alkene. Assay of the product epoxides (10, 18, 25) and diols (9a, 17, 24) against HIV protease reveals micromolar inhibition.     

Naphthalene-dioxygenase-catalysed cis-dihydroxylation of azaarene derivatives: Part 1: 2-Pyridones and 2-quinolones

The scope of the biotransformation of 2-pyridone- and 2-quinolone-derived compounds by recombinant whole-cells of E. coli JM109(DE3)(pDTG141) expressing the naphthalene-dioxygenase system from Pseudomonas sp. NCIB 9816-4 was explored, using a series of N- and C-substituted derivatives. Among them, only the N-methyl substituted compounds were good substrates for a regio- and stereoselective dihydroxylation reaction leading to cis-dihydroxydihydro pyridone derivatives, corresponding to the general pattern expected for this enzyme. In the absence of dihydroxylation reactions, N-dealkylations and monohydroxylations on external methyl groups were observed.     

Synthesis of 11 alpha-hydroxyprogesterone haptens

C-4 and C-6 bridged haptens of 11 alpha-hydroxyprogesterone, an anti-androgen, were respectively synthesized via 4, 5 and 5 alpha, 6 alpha epoxide ring openings using 3-mercaptopropanoic acid. Substitution of 6-bromo derivative of progesterone using the same reagent unexpectedly afforded a C-4 substituted product instead of the normal C-6 substituted product previously reported in the literature.     

Synthesis of 4-azasteroids by an intramolecular Ugi reaction

In this paper we report the use of an intramolecular Ugi reaction to synthesize new 4-azacholestanes diversely substituted both at N-4 and C-5. Both the scope and the stereochemical outcome of this approach were studied by varying the nature of the components necessary for this multicomponent reaction. In sight of our results we concluded that this methodology can be applied to obtain 4-azasteroids targeted to find new biologically active compounds.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Structural elucidation of the major Hex4 lipopolysaccharide glycoform from the lgtC mutant of Haemophilus influenzae strain Eagan

Lipopolysaccharide (LPS) oligosaccharide epitopes are major virulence factors of Haemophilus influenzae. The structure of LPS glycoforms of H. influenzae type b strain Eagan containing a mutation in the gene lgtC is investigated. LgtC is involved in the biosynthesis of globoside trisaccharide [alpha-D-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-D-Glcp-(1-->], an LPS epitope implicated in the virulence of this organism. Glycose and methylation analyses provided information on the composition while electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS (LPS-OH) indicated the major glycoform to contain 4 hexoses attached to the common H. influenzae triheptosyl inner-core unit. The structure of the Hex4 glycoform in LPS-OH and core oligosaccharide samples was determined by NMR. It consists of an l-alpha-D-HepIIIp-(1-->2)-[PEtn-->6]-l-alpha-D-HepIIp-(1-->3)-l-alpha-D-HepIp-(1-->5)-[P-->4]-alpha-D-Kdop-(2--> to which a beta-D-Glcp-(1-->4)-alpha-D-Glcp disaccharide unit is extended from HepII at the C-3 position, while HepI and HepIII are substituted at the C-4 and C-2 positions with beta-D-Glcp and beta-D-Galp, respectively. This structure corresponds to that expressed as a subpopulation in the parent strain. 31P NMR studies permitted the identification of subpopulations of LPS containing Kdo substituted at the C-4 position with monophosphate or pyrophosphoethanolamine (PPEtn). HepIII was found to be substituted with either phosphate at the C-4 position or acetate at the C-3 position, but not both of them together in the same subpopulation. The subpopulations containing phosphate and acetate at HepIII and their location have not previously been reported.     

Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 37b4

Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4' of the nonreducing glucosamine. 1,4'-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3' of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3') is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6' of the glucosamine disaccharide are unsubstituted. C-6' being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.     

Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Exchange of oxime functions: a useful reaction in GC-ms analysis of steroids

Simple and various O-substituted oxime derivatives of steroids may be interconverted by an acid-catalyzed reaction of the oxime with the appropriate substituted hydroxylamine hydrochloride in pyridine. For example, unsubstituted oximes of common hormonal steroids with keto groups in positions 3, 17, and 20 are converted into O-methyloximes by reaction of the oximes with 5 mg of methoxyamine hydrochloride in 50 μl of pyridine for 1 hr at 100°C. The reaction involves cleavage of the carbon-nitrogen bond and follows pseudo-first-order kinetics in the presence of excess reagent. The conversion of an oxime with a small O-substituent into one with a larger O-substituent was faster than the reverse reaction at the C-3 position. The rate of conversion depends on the position of the oxime function and decreases in the order: C-3 > C-20 > C-17 > C-11. The proximity and the configuration of other functional groups also influence the rates. Thus, determination of reaction rates with the aid of gas chromatography-mass spectrometry may be used to determine the positions of functional groups in steroids. The reactions are likely to be applieable to other groups of compounds possessing keto or aldehyde functions.     

New acyclic quinoxaline nucleosides. Synthesis and anti-hiv activity

A series of acyclonucleosides substituted 1-(4,5-dihydroxypentyl) (13-8) and 2-(4,5-dihydroxypentyloxy)quinoxalines (19-24) were synthesized by the sharpless asymmetric dihydroxylation of the derivatives 1-6 and 7-12, respectively. Treatment of the quinoxaline base 26 with (R)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl-p-toluenesulfonate (27) in the presence of NaH/DMF furnished 28. Acid hydrolysis of 28 gave 1-(2,3-dihydroxypropyl)-6,7-dimethyl-quinoxaline-2-one (29). Alternatively, 29 was prepared by sharpless dihydroxylation of 30. All the compounds were evaluated for their in vitro anti-HIV-1 and HIV-2 in MT-4 cell and found inactive, except 29, which showed inhibition of HIV-1 with EC50 value of 0.15 +/- 0.1 microg/ml and a therapeutic index (SI) of 73.     

Synthesis of 9-[1-(substituted)-3-(phosphonomethoxy)propyl]adenine derivatives as possible antiviral agents

Acyclic N9 adenine nucleosides substituted at C-1' position were prepared by the Mitsunobu reaction of 1-tert-butyldimethylsilyl-4-pivaloylbutan-1,2,4-triol (5) with adenine. Pivaloyl hydroxyl was modified to the phosphonomethoxy derivatives, and the tert-butyldimethylsilyl hydroxyl was converted to methoxy, azido, amino, fluoro, and c-hydroxyethyl and was eliminated to give vinyl. The resulting phosphonic acids were converted to prodrugs also.     

Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.     

1,4-dihydropyridines: reactivity of nitrosoaryl and nitroaryl derivatives with alkylperoxyl radicals and ABTS radical cation

In the present paper, a direct quenching of radical species by a number of synthesized nitrosoaryl 1,4-dihydropyridines and their parent nitroaryl 1,4-dihydropyridines was determined in aqueous media at pH 7.4. These two series of compounds were compared with the C-4 unsubstituted 1,4-dihydropyridines derivatives and the corresponding C-4 aryl substituted 1,4-dihydropyridines derivatives. Kinetic rate constants were assessed by UV-Vis spectroscopy. Nitrosoaryl derivatives were more reactive than the parent nitroaryl 1,4-dihydropyridines.

Our results strongly support the assumption that the reactivity between the synthesized 1,4-dihydropyridines derivatives with alkylperoxyl radicals involves electron transfer reactions, which is documented by the presence of pyridine as final product of reaction and the complete oxidation of the nitroso group to give rise the nitro group in the case of the nitrosoaryl 1,4-dihydropyridines derivatives.     

1,4-Dihydropyridines: Reactivity of Nitrosoaryl and Nitroaryl Derivatives with Alkylperoxyl Radicals and ABTS Radical Cation

In the present paper, a direct quenching of radical species by a number of synthesized nitrosoaryl 1,4-dihydropyridines and their parent nitroaryl 1,4-dihydropyridines was determined in aqueous media at pH 7.4. These two series of compounds were compared with the C-4 unsubstituted 1,4-dihydropyridines derivatives and the corresponding C-4 aryl substituted 1,4-dihydropyridines derivatives. Kinetic rate constants were assessed by UV-Vis spectroscopy. Nitrosoaryl derivatives were more reactive than the parent nitroaryl 1,4-dihydropyridines.

Our results strongly support the assumption that the reactivity between the synthesized 1,4-dihydropyridines derivatives with alkylperoxyl radicals involves electron transfer reactions, which is documented by the presence of pyridine as final product of reaction and the complete oxidation of the nitroso group to give rise the nitro group in the case of the nitrosoaryl 1,4-dihydropyridines derivatives.     

Structural effects on the reactivity 1,4-dihydropyridines with alkylperoxyl radicals and ABTS radical cation

A series of eight commercial C-4 substituted 1,4-dihydropyridines and other synthesized related compounds were tested for direct potential scavenger effect towards alkylperoxyl radicals and ABTS radical cation in aqueous Britton-Robinson buffer pH7.4. A direct quenching radical species was established. The tested 1,4-dihydropyridines were 8.3-fold more reactive towards alkylperoxyl radicals than ABTS cation radical, expressed by their corresponding kinetic rate constants. Furthermore, NPD a photolyte of nifedipine and the C-4 unsubstituted 1,4-DHP were the most reactive derivatives towards alkylperoxyl radicals. The pyridine derivative was confirmed by GC/MS technique as the final product of reaction. In consequence, the reduction of alkylperoxyl and ABTS radicals by 1,4-dihydropyridines involved an electron transfer process. Also, the participation of the hydrogen of the 1-position appears as relevant on the reactivity. Results of reactivity were compared with Trolox.     

Superoxide organic chemistry within the liposomal bilayer, part II: a correlation between location and chemistry

Coumarin ester derivatives 1, substituted at C-4 and/or C-12 with alkyl chains, were synthesized and intercalated within DMPC liposomal bilayers. By correlating the 13C chemical shift with medium polarity [E(T)(30)], the relative location of these substrates within the liposomal bilayer was determined. The length of the alkyl chain substituents clearly influences the lipophilicity of the substrates and their location and orientation within the liposome: Superoxide readily saponifies the C-12 esteric linkage of 1, when this reaction site lies in a polar region of the liposome (E(T)(30) > 45 kcal/mol), to give the corresponding 7-hydroxy coumarin derivatives 2. However, when C-12 lies deeper and is hence less available to O(2)(*-), the lactonic carbon C-2, which lies in a shallower region (E(T)(30) = 43-49), is the preferred site for superoxide-mediated cleavage. When coumarin 1 is disubstituted with long chains at both C-12 and C-4, these derivatives lie deep within the bilayer and react only slowly with O(2)(*-). These results indicate there is indeed a correlation between location within the bilayer and substrate reactivity. Contrary to the suggestion of Dix and Aikens (Chem. Res. Toxicol.6:2-18; 1993) superoxide can penetrate deep within the liposomal bilayer. Nevertheless, its concentration drops precipitously (to approximately 16% of what it is near the interface) below E(T) values of 38, thereby precluding substantial reaction with many highly lipophilic substrates. This work also confirms the findings of others that reactions of small oxy-radicals occur within cellular membranes and appear to be of significant biological importance.     

Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.     

The effect of vindoline C-16 substituents on the biomimetic coupling reaction: synthesis and cytotoxicity evaluation of the corresponding vinorelbine analogues

A new series of vinorelbine analogues are designed and synthesized to explore the vindoline C-16 substituent effects on the biomimetic coupling with catharanthine, and the structure-activity relationships of these vinorelbine analogues as cytotoxic agents are also studied. The results show that introduction of severe steric hindrance and/or electron-withdrawing group at C-16 site are not propitious to improving the yields of the coupling reaction, and the SAR information collected so far suggests that small alkyl groups substituted at C-16 of vindoline are conductive to maintaining the cytotoxicity.