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J Balzarini

Publications and source records attributed to J Balzarini.

At least 163 records · Page 9Linked to original sources
PubMed1998

Mutational analysis of Tyr-318 within the non-nucleoside reverse transcriptase inhibitor binding pocket of human immunodeficiency virus type I reverse transcriptase.

The highly conserved Tyr-318 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type I reverse transcriptase (RT) and makes contact within 4 A with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318F mutant RTs retained substantial RT activity, whereas the catalytic activities of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity. The Y318F mutant RT retained substantial sensitivity to the majority of NNRTIs tested, whereas the Y318W mutant RT showed varying degrees of resistance to NNRTIs. Subunit-specific site-directed mutagenesis revealed that there was no difference in the catalytic activity or resistance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-318 mutation was introduced in both subunits or only in the p66 subunit of RT. Recombinant viruses harboring the Y318F or Y318W mutation in the RT showed a similar resistance/sensitivity pattern to NNRTIs as their corresponding 318 mutant recombinant RTs. Our findings stress a functional or structural role for Tyr-318 in wild-type RT and argue for the design of novel NNRTIs that interact more closely with this amino acid in the NNRTI-specific pocket of human immunodeficiency virus type I RT.

Amino Acid Substitution↗
PubMed1998

Synthesis of imidazo[1,2-a]pyridines as antiviral agents.

The synthesis of original imidazo[1,2-a]pyridines bearing a thioether side chain at the 3 position and their antiviral activity are reported. From the synthesized compounds, 4, 15, and 21 were highly active against human cytomegalovirus with a therapeutic index superior to 150. These compounds also showed pronounced activity against varicella-zoster virus. Their structure-activity relationship is discussed.

Animals↗
PubMed1998

Abasic analogues of TSAO-T as the first sugar derivatives that specifically inhibit HIV-1 reverse transcriptase.

With the aim of assessing the role that the thymine base of TSAO-T may play in the interaction of TSAO compounds with HIV-1 reverse transcriptase (RT), we have designed, synthesized, and evaluated for their anti-HIV-1 activity a series of 3-spiro sugar derivatives substituted at the anomeric position with nonaromatic rings or with amine, amide, urea, or thiourea moieties that mimic parts or the whole thymine base of TSAO-T. Also, a dihydrouracil TSAO analogue and O-glycosyl 3-spiro sugar derivatives substituted at the anomeric position with methyloxy or benzyloxy groups have been prepared. Compounds substituted at the anomeric position with an azido, amino, or methoxy group, respectively, were devoid of marked antiviral activity (EC50: 10-200 microM). However, the substituted urea sugar derivatives led to an increase in antiviral potency (EC50: 0.35-4 microM), among them those urea derivatives that mimic most closely the intact TSAO-T molecule retained the highest antiviral activity. Also, the dihydrouracil TSAO derivative retained pronounced anti-HIV-1 activity. None of the compounds showed any anti-HIV-2 activity. The results described herein represent the first examples of sugar derivatives that interact in a specific manner with HIV-1 RT. Molecular modeling studies carried out with a prototype urea derivative indicate that a heteroaromatic ring is not an absolute requirement for a favorable interaction between TSAO-T and HIV-1 RT. Urea derivatives, which can mimic to a large extent both the shape and the electrostatic potential of a thymine ring, can effectively replace this nucleic acid base when incorporated into a TSAO molecular framework with only moderate loss of activity.

Animals↗
PubMed1998

Lactate cannot substitute for alanine in d4T-based anti-HIV nucleotide prodrugs--despite efficient esterase-mediated hydrolysis.

As part of our on-going effort to deliver masked phosphates of antiviral nucleosides inside living cells we have previously discovered that amino acid-derived phosphoramidates are particularly effective. Here we report that lactate analogues, with a simple change of bridging nitrogen for oxygen, are virtually inactive as antiviral agents and apparently do not achieve intracellular nucleoside phosphate delivery.

Alanine↗
PubMed1998

7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase.

7-Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration-dependent manner. At 1 mM, it almost completely prevented the TPase-catalysed hydrolysis of dThd to thymine. The 50% inhibitory concentration (IC50 of 7DX was 40 microM in the presence of 100 microM of the natural substrate dThd. 7DX is also endowed with a marked inhibitory effect on angiogenesis. It significantly prevents neovascularisation in the chicken chorioallantoic membrane during development. 7DX is the first purine derivative shown to be a potent inhibitor of purified TPase and angiogenesis.

Allantois↗
PubMed1998

Crystal structures of HIV-1 reverse transcriptase in complex with carboxanilide derivatives.

The carboxanilides are nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT), of potential clinical importance. The compounds differ in potency and in their retention of potency in the face of drug resistance mutations. Whereas UC-84, the prototype compound, only weakly inhibits many RTs bearing single point resistance mutations, inhibition by UC-781 is little affected. It has been proposed that UC-38 and UC-781 may form quaternary complexes with RT at a site other than the known binding pocket of other NNIs. X-ray crystal structures of four HIV-1 RT-carboxanilide complexes (UC-10, UC-38, UC-84, and UC-781) reported here reveal that all four inhibitors bind in the usual NNI site, forming binary 1:1 complexes with RT in the absence of substrates with the amide/thioamide bond in cis conformations. For all four complexes the anilide rings of the inhibitors overlap aromatic rings of many other NNIs bound to RT. In contrast, the second rings of UC-10, UC-84, and UC-781 do not bind in equivalent positions to those of other "two-ring" NNIs such as alpha-APA or HEPT derivatives. The binding modes most closely resemble that of the structurally dissimilar NNI, Cl-TIBO, with a common hydrogen bond between each carboxanilide NH- group and the main-chain carbonyl oxygen of Lys101. The binding modes differ slightly between the UC-10/UC-781 and UC-38/UC-84 pairs of compounds, apparently related to the shorter isopropylmethanoyl substituents of the anilide rings of UC-38/UC-84, which draws these rings closer to residues Tyr181 and Tyr188. This in turn explains the differences in the effect of mutated residues on the binding of these compounds.

Anilides↗
PubMed1998

4-(beta-Arylvinyl)-3-(beta-arylvinylketo)-1-ethyl-4-piperidinols and related compounds: a novel class of cytotoxic and anticancer agents.

The syntheses of a series of 1-aryl-5-diethylamino-1-penten-3-one hydrochlorides 1 and 1-aryl-3-diethylamino-1-propanone hydrochlorides 4 were accomplished. Attempts to prepare the corresponding bis(5-aryl-3-oxo-4-pentenyl)ethylamine hydrochlorides 2 and bis(3-aryl-3-oxopropyl)ethylamine hydrochlorides 5 led to the formation of a series of 4-(beta-arylvinyl)-3-(beta-arylvinylketo)-1-ethyl-4-piperidi nol hydrochlorides 9 and 4-aryl-3-arylketo-1-ethyl-4-piperidinol hydrochlorides 11, most of which were converted subsequently into the corresponding quaternary ammonium salts 10 and 12, respectively. The structures of these compounds were determined by 1H NMR spectroscopy and confirmed by X-ray crystallography of representative molecules. Most compounds displayed significant cytotoxicity toward murine P388 and L1210 cells as well as human tumors. In general, Mannich bases containing olefinic bonds were more cytotoxic than the analogues without this functional group, while the piperidines 9 and 11 were more potent than the acyclic analogues 1 and 4, respectively. Correlations were noted between various physicochemical constants in the aryl rings and cytotoxicity. Compound 9d displayed promising in vivo activity against colon cancers. This study has revealed that the piperidines 9 and 11 constitute new classses of cytotoxic agents.

Animals↗
PubMed1998

Novel 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazine derivatives as non-nucleoside reverse transcriptase inhibitors that inhibit human immunodeficiency virus type 1 replication.

The 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs) represent a recently discovered chemical class of non-nucleoside reverse transcriptase inhibitors that selectively block human immunodeficiency virus type 1 replication. In a search for a better understanding of their mode of binding and with the aim of obtaining novel lead compounds, a second series of TTD derivatives was synthesized and evaluated for antiviral activity. The design of the new compounds was based on a variety of chemical modifications which were carried out in the original prototype 20a (QM 96521). Substitution of a halogen at the meta position of the N-2 benzyl group resulted in an improvement of the antiviral activity by 1 order of magnitude. Compounds bearing at the N-4 position a cyanomethyl, propargyl, or benzyl substituent were found to be the most potent of the series. Modifying the thieno[3,4-e] ring fused to the 1,2,4-thiadiazine moiety to other heterocyclic ring systems decreased the potency. The results obtained in this investigation have provided new indications for the design of even more effective TTDs.

Cell Line↗
PubMed1998

Potent differentiation-inducing properties of the antiretroviral agent 9-(2-phosphonylmethoxyethyl) adenine (PMEA) in the rat choriocarcinoma (RCHO) tumor cell model.

9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its closely related structural analogue (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) are potent inhibitors of retroviruses and hepatitis B virus. In its oral prodrug form (adefovir dipivoxil), PMEA is currently the subject of advanced phase II/III clinical trials for the treatment of HIV infections. PMEA has also been shown to be a potent differentiation-inducing agent. In the present study, PMEA was found to have a strong differentiation-inducing effect on rat choriocarcinoma (RCHO) cells, comparable to that of methotrexate, which is the drug of choice for the chemotherapy of choriocarcinoma in humans. PMEA induced differentiation of choriocarcinoma trophoblast cells in a concentration-dependent manner within the 2- to 50-microM concentration range, as ascertained by giant cell formation, alkaline phosphatase induction, progesterone secretion, and the disappearance of a cytotrophoblast-specific surface antigen. PMEA had to be exposed to the rat choriocarcinoma cell cultures for at least 2-3 days to achieve optimal growth inhibition and differentiation of the tumor cells. Unlike PMEA, (R)-9-(2-phosphonylmethoxypropyl)adenine failed to induce differentiation of proliferating cytotrophoblasts into nonproliferating, hormonally active giant cells. This points to the specificity of PMEA as an inducer of choriocarcinoma cell differentiation.

Adenine↗
PubMed1998

Inactivation of S-adenosyl-L-homocysteine hydrolase and antiviral activity with 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosine analogues (4'-haloacetylene analogues derived from adenosine).

Treatment of a protected 9-(5, 6-dideoxy-beta-D-ribo-hex-5-ynofuranosyl)adenine derivative with silver nitrate and N-iodosuccinimide (NIS) and deprotection gave the 6'-iodo acetylenic nucleoside analogue 3c. Halogenation of 3-O-benzoyl-5,6-dideoxy-1, 2-O-isopropylidene-alpha-D-ribo-hex-5-enofuranose gave 6-halo acetylenic sugars that were converted to anomeric 1,2-di-O-acetyl derivatives and coupled with 6-N-benzoyladenine. These intermediates were deprotected to give the 6'-chloro 3a, 6'-bromo 3b, and 6'-iodo 3c acetylenic nucleoside analogues. Iodo compound 3c appears to inactivate S-adenosyl-L-homocysteine hydrolase by a type I ("cofactor depletion") mechanism since complete reduction of enzyme-bound NAD+ to NADH was observed and no release of adenine or iodide ion was detected. In contrast, incubation of the enzyme with the chloro 3a or bromo 3b analogues resulted in release of Cl- or Br- and Ade, as well as partial reduction of E-NAD+ to E-NADH. Compounds 3a, 3b, and 3c were inhibitory to replication of vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus, and reovirus-1 (3a < 3b < 3c, in order of increasing activity). The antiviral effects appear to correlate with type I mechanism-based inhibition of S-adenosyl-L-homocysteine hydrolase. Mechanistic considerations are discussed.

Adenosine↗
PubMed1998

Discovery of type II (covalent) inactivation of S-adenosyl-L-homocysteine hydrolase involving its "hydrolytic activity": synthesis and evaluation of dihalohomovinyl nucleoside analogues derived from adenosine.

Treatment of the 5'-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2',3'-O-isopropylideneadenosine (1) with the "(bromofluoromethylene)triphenylphosphorane" reagent and deprotection gave 9-(6-bromo-5, 6-dideoxy-6-fluoro-beta-d-ribo-hex-5-enofuranosyl)adenine (4). Parallel treatment with a "dibromomethylene Wittig reagent" and deprotection gave 9-(6,6-dibromo-5, 6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by successive bromination and dehydrobromination of the 6'-bromohomovinyl nucleoside 8. Bromination-dehydrobromination of the 5'-bromohomovinyl analogue 11 and deprotection gave (E)-9-(5, 6-dibromo-5,6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and 15 were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-l-homocysteine (AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could generate electrophilic acyl halide or alpha-halo ketone species that could undergo nucleophilic attack by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.

Adenosylhomocysteinase↗
PubMed1998

Preclinical studies on thiocarboxanilide UC-781 as a virucidal agent.

BACKGROUND: Thiocarboxanilide UC-781 is a highly potent and selective non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1, which also has virucidal properties. Recent studies have shown that UC-781 would seem an ideal candidate for application as a vaginal virucide. OBJECTIVE: To investigate the antiviral potency and stability of UC-781 in a lipophilic gel formulation. METHODS: UC-781 was formulated in replens gel at different concentrations and administered intravaginally to rabbits at 5% in replens gel for 10 days. UC-781 was also exposed to temperatures of 4, 37 and 50 degrees C, and to low pH (6.0, 4.3, 2.0 and 1.2). A number of microorganisms were exposed in culture to serial dilutions of UC-781. RESULTS: The drug was stable under low pH conditions and did not lose its antiviral potency upon 4 h exposure to pH 3.5 (the estimated vaginal pH). UC-781 can be easily formulated into a lipophilic gel (replens; up to 5%) and proved fully stable at 50 degrees C for 30 days. There was no effect on the growth of microorganisms (i.e., Candida and Lactobacillus strains) that are present in the vaginal flora. Neither systemic side-effects, nor local inflammation or damage of the vaginal mucosa or epithelium were observed in rabbits to which 5% UC-781 in replens gel had been administered. UC-781, formulated as 0.5, 0.2 and 0.05% replens gel, and UC-38, alpha-APA and zidovudine, formulated as 0.5 or 0.2% replens gel, were effective in protecting CEM cells in the very beginning against productive HIV-1 replication. This points to an efficient diffusion of the drugs from the lipophilic gel to the hydrophilic culture medium. However, subsequent subcultivations at a dilution rate of 1:10 every 3-4 days resulted in a rapid breakthrough of virus with all drugs except UC-781 in its 0.5 and 0.2% gel formulation. These cultures were fully protected against HIV-1 and remained completely cleared from virus for at least 10 subcultivations. CONCLUSIONS: The virus that emerged under 0.05% UC-781 remained highly sensitive to the NNRTI, including UC-781, in cell culture, suggesting a lack of resistance development under our experimental conditions.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Design and synthesis of bio-isosteres of thymidine triphosphate.

This paper describes the design and synthesis of potential isosteres of triphosphates which should show enhanced metabolic stability and lipophilicity compared to triphosphates. The triphosphate isosteres were then linked to nucleosides and evaluated for their inhibitory activity against HIV infection.

Computer Simulation↗
PubMed1998

Potent antitumor activity of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine in choriocarcinoma-bearing rats.

The acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective antiretroviral agent which is currently evaluated in its oral prodrug form, bis(POM)PMEA (adefovir dipivoxil), in phase II and III clinical trials in human hepatitis B virus (HBV)- and human immunodeficiency virus (HIV)-infected individuals, respectively. We have now found that PMEA is also a potent inhibitor of growth of the highly aggressive choriocarcinoma tumor arising from rat choriocarcinoma RCHO cells grafted under the kidney capsule of syngeneic WKA/H rats. In untreated rats, massive invasive RCHO tumors, covering the whole surface of the kidney and resulting in a marked enlargement of the kidney, were observed at day 10 after tumor cell grafting. Daily treatment with PMEA at 25 mg/kg/day afforded a marked reduction in tumor size (i.e., smaller tumors and slight, if any, enlargement of the kidney). Increasing the PMEA dose to 50, 100 or 250 mg/kglday resulted in a gradual increase of the antitumor effect of the compound. At the highest dose tested, i.e., 250 mg/kg/day, PMEA completely suppressed tumor growth. The antitumor activity of PMEA persisted for at least 10 days after termination of drug treatment. In addition, delayed treatment with PMEA at a dose of 200 mg/kg/day, started at a time point where choriocarcinoma tumors had already developed, stopped further growth and even induced regression of the tumors. PMPA, a closely related structural analogue of PMEA, failed to inhibit choriocarcinoma tumor growth. This observation points to the specificity of PMEA as an antitumor agent. In view of our findings, the therapeutic potential of PMEA for the treatment of neoplastic diseases appears to merit further investigation.

Adenine↗
PubMed1998

cycloSal-2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (cycloSal-d4TMP): synthesis and antiviral evaluation of a new d4TMP delivery system.

The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-d4TMP derivatives 3a-h of the anti-HIV dideoxynucleoside 2',3'-dideoxy-2',3'-didehydrothymidine (d4T, 1) are reported. This pro-nucleotide concept has been designed to deliver d4TMP (2) by selective chemical hydrolysis. All compounds 3a-h were synthesized using phosphorus(III) chemistry in good yields and in somewhat lower yields using phosphorus(V) chemistry starting from substituted salicyl alcohols 6a-h. The phosphotriesters 3 were obtained without stereochemical preference with respect to the configuration at the phosphorus center as 1:1 diastereomeric mixtures. However, a few of the triesters 3 could be separated into the diastereomers by means of semipreparative HPLC. In a 1-octanol/phosphate buffer mixture, all compounds 3 exhibited 9-100-fold higher lipophilicity as judged from their Pa values as compared to d4T (1). Furthermore, in hydrolysis studies 3 decomposed under mild aqueous basic conditions releasing solely d4TMP (2) and the diols 6 following the designed tandem reaction sequence. A correlation of the electronic properties introduced by the substituents and the half-lives of triesters 3 was observed. Thus, by varying the substituent, the half-lives of 3 could be adjusted over a wide range of compounds still delivering d4TMP (2) selectively. Phosphotriesters 3 exhibited considerable activity against HIV-1 and HIV-2 in wild-type human T-lymphocyte (CEM/O) cells as well as mutant thymidine kinase-deficient (CEM/TK-) cells. Surprisingly, we observed a 3-80-fold difference in antiviral activity between the two diastereomers. Our data clearly prove that the cycloSal-d4TMPs deliver exclusively the nucleotide d4TMP not only under simulated hydrolysis conditions but also under cellular conditions and thus fulfill the thymidine kinase-bypass premise. Therefore, the cycloSal-nucleotide concept is the first reported pro-nucleotide system that delivers the dideoxynucleotide by a pH-driven, chemically activated, tandem reaction without the requirement of an enzymatic contribution.

Animals↗
PubMed1998

Marked inhibitory activity of masked aryloxy aminoacyl phosphoramidate derivatives of dideoxynucleoside analogues against visna virus infection.

Lipophilic masked aryloxyaminoacylphosphoramidate derivatives of 2',3'-dideoxynucleoside (ddN) analogues with potent anti-HIV activity (i.e., stavudine [d4T], azidothymidine [AZT], dideoxycytidine [ddC], 3'thio-2',3'-dideoxy cytidine [3TC], dideoxyadenosine [ddA], and 2',3'-didehydro-2',3'-dideoxyadenosine [d4A]) activity were evaluated for their activity against visna virus (VV) in sheep choroid plexus (SCP) cells. The activity of several prodrug derivatives against VV proved markedly superior to that of the corresponding free ddN analogues. In particular, the d4A and ddA prodrug derivatives were exquisitely inhibitory in this model system (50% effective concentration [EC50], < or = 0.003 microM), and their anti-VV potency exceeded by at least 200-fold the antiviral potency of the corresponding free nucleosides. Marked differences were noted in the anti-VV potencies of several of the test compounds depending on the nature of the amino acid linked to the 5'-phosphate moiety, the nature of the nucleoside, or both. In view of the stability of the prodrugs in lamb serum, the VV infection model in lambs may be considered highly useful for investigating the in vivo antiretroviral efficacy of these type of drugs, particularly the d4T, ddA, and d4A prodrug derivatives.

Animals↗
PubMed1998

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

Animals↗