Synthesis and in vitro evaluation of novel anti-varicella-zoster virus (VZV) nucleosides.

A series of alkyl-aryl, -phenoxy, and -thiophenoxy bicyclic furo pyrimidine nucleosides have been successfully synthesised by Pd-coupling of 5-iodo-2'-deoxyuridine (IDU) with terminal alkynes, followed by in situ copper-cyclisation. Synthesised compounds (4a-i) showed an anti-VZV activity at low microM concentration, comparable to that of current treatment acyclovir.

Design, synthesis, and evaluation of a novel class of enantioselective electrophilic fluorinating agents: N-fluoro ammonium salts of cinchona alkaloids (F-CA-BF(4)).

The first enantiopure N-fluoro quaternary ammonium salts of cinchona alkaloids as enantioselective fluorinating agents are reported. A one-step transfer-fluorination on the naturally occurring cinchona alkaloids gave the fluorinating agents F-CA-BF(4). This new generation of fluorinating agents exhibited asymmetric induction up to 61% on fluorination of enolates and silyl enol ethers of 2-methyl-1-tetralone.

A QSAR study investigating the effect of L-alanine ester variation on the anti-HIV activity of some phosphoramidate derivatives of d4T.

A QSAR study, involving the use of calculated physical properties (TSAR), and the use of a neural network approach (TSAR), has been performed concerning the anti-HIV activity and cytotoxic effects of a series of d4T phosphoramidate derivatives with varying L-alanine esters. Models were obtained which allow reliable predictions for the anti-HIV activity, and cytotoxicity, of these derivatives.

Activities of masked 2',3'-dideoxynucleoside monophosphate derivatives against human immunodeficiency virus in resting macrophages.

The anti-human immunodeficiency virus (HIV) activity of aryloxyphosphoramidate protides of a number of anti-HIV nucleoside analogues was assessed in resting primary monocyte-macrophages (M/M). While 2',3'-dideoxythymidine (d4T), 2',3'-dideoxyadenosine (ddA), and 2',3'-dideoxy-2',3'-didehydroadenosine (d4A) protides showed an anti-HIV activity that was 25- to 625-fold greater than the parent nucleotides d4T, ddA, and d4A, respectively, other aryloxyphosphoramidate protides showed similar or even lower anti-HIV activities than their parent compounds. This variable anti-HIV effect is most likely related to the different dynamics of intracellular nucleoside monophosphate release from the protides. Our results indicate the potential advantage of therapeutic use of this approach for some nucleotide analogues to affect HIV replication in M/M, one of the major reservoirs of HIV in vivo.

Interactive computerized microscopy as a tool for quantifying vascular remodelling effects of diabetes and V1a receptor antagonist SR 49059 on rat mesenteric arterial bed.

A methodology using interactive computerized microscopy (ICM) was developed to quantify in the mesenteric arterial bed the morphometric changes associated with diabetes and the influence of treatment with SR 49059, an antagonist of vasopressin V1a receptors. Four groups of rats were studied: untreated normal (N) or streptozotocin- (60 mg/kg i.v.) induced diabetic (D), and treated (0.4 mg/g SR 49059 included in food) normal (NT) or diabetic (DT) animals. Treatment was initiated 4 days after diabetes induction and continued for 3 weeks. Nested (hierarchical) analysis of variance of ICM data was performed on raw diameter or after logarithmic normalization of area and nuclei values. Diabetes was associated with an increase in arterial diameters, and in total vessel, wall, media, adventitia, and lumen areas. The same parameters, with the exception of the lumen, were also increased in DT as compared to D. The number of nuclei in the media or adventitia was increased in D as compared to N, and in DT as compared to D. In summary, ICM is allowed to further characterize the vascular mesenteric changes and describe for the first time the enlargement of adventitia associated with diabetes. Our study also suggested that the blockade of Via receptors is unable to prevent diabetes-related vascular changes, although the slight increase in food intake associated with SR 49059 treatment may have had an indirect influence on angiopathy development.

Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.

Synthesis, anti-human immunodeficiency virus activity and esterase lability of some novel carboxylic ester-modified phosphoramidate derivatives of stavudine (d4T).

We report the design, synthesis and antiviral evaluation of a series of lipophilic, masked phosphoramidate derivatives of the anti-human immunodeficiency virus (HIV) nucleoside analogue d4T, designed to act as membrane-soluble prodrug forms for the free nucleotide. In particular, we report a series of 12 novel compounds with systematic variation in the structure of the carboxylate ester function. In order to rationalize the changes in antiviral action with variation of this moiety we applied our recently developed 31P NMR-based assay for carboxyesterase lability to this series. However, no clear positive correlation emerged, indicating that, at least within this series, factors other than simple esterase lability may be the major determinants of antiviral potency.

Metabolism and anti-HIV activity of phosphoramidate derivatives of D4T-MP with variations in the amino acid moiety.

The metabolism of different phosphoramidate prodrugs of d4T-MP, in which the phosphate group is linked to a phenyl group and the alkyl ester of an amino acid was studied in crude CEM cell extracts. Significant (80-100%) conversion to the amino acyl d4T-MP metabolite was obtained with derivatives containing L-alanine or methyl-L-aspartic acid. A lower degree of conversion was seen with derivatives containing L-phenylalanine, L-methionine, methyl-L-glutamic acid or L-leucine. Derivatives containing D-alanine, beta-alanine, glycine, L-valine or L-lactate showed no conversion to the amino acyl d4T-MP metabolite. Overall, there was a close correlation between the anti-HIV activity of these prodrugs and their conversion rate to the amino acyl d4T-MP metabolite. Our data suggest that the enzymes involved in the formation of the amino acyl d4T-MP metabolite have a rather stringent specificity for L-alanine as the amino acid moiety. In addition, these enzymes were found to be markedly species-dependent, their activities being highest in mouse serum, followed by guinea pig serum, but only minimal in human serum. Mouse serum therefore appears to be the medium of choice to isolate and identify the enzymes that are involved in the metabolism of these phosphoramidate prodrugs.

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.

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.

Phosphoramidate derivatives of d4T as inhibitors of HIV: the effect of amino acid variation.

Phosphoramidate derivatives of the nucleoside analogue, 2',3'-dideoxy-2',3'-didehydro thymidine (d4T) have been prepared as potential membrane-soluble pro-drugs of the big-active free phosphate forms. In particular phenyl phosphates, linked via nitrogen to methyl-esterified amino acids, were studied. All compounds were fully characterised by a range of methods (high-field multinuclear NMR, mass spectrometry and high performance liquid chromatography (HPLC)) and were subjected to in vitro evaluation of their anti-HIV efficacy. The nature of the amino acid appeared to be extremely important for the eventual antiviral action. Of the amino acids studied, L-alanine was the most efficacious, whilst L-proline and glycine were particularly poor. However, an unnatural amino acid moiety, dimethylglycine, could substitute for alanine with little or no loss of activity.

Antiretrovirus specificity and intracellular metabolism of 2',3' -didehydro-2',3'-dideoxythymidine (stavudine) and its 5'-monophosphate triester prodrug So324.

2',3'-Didehydro-2',3'-dideoxythymidine (d4T) and its lipophilic 5'-monophosphate triester prodrug, So324, were evaluated for their antiretroviral and metabolic properties in four different animal species cell lines. The antiretrovirus activity of So324 was approximately 4-10-fold greater than that of d4T against human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus in human T lymphocyte CEM and MT-4 cells and against feline immunodeficiency virus in feline Crandell kidney cells, 50-fold greater against visna virus in sheep choroid plexus cells, but 5-fold inferior against murine (Moloney) sarcoma virus in murine embryo fibroblast (C3H) cells. Although the administration of both d4T and So324 resulted in the formation of the 5'-monophosphate (d4T-MP), 5'-diphosphate, and 5'-triphosphate in the different cell lines, a new d4T metabolite markedly accumulated in So324-treated cells and exceeded d4T-TP levels by 13-242-fold depending on the cell line used. This metabolite could be identified as alaninyl d4T-MP. Alanyl d4T-MP may be considered to be an intracellular depot form of d4T and/or d4T-MP, which may account for the superior antiretroviral activity of the lipophilic d4T-MP triester So324 compared with d4T.

Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives.

So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester of alanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at approximately 13- to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra- and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.

Aryl phosphoramidate derivatives of d4T have improved anti-HIV efficacy in tissue culture and may act by the generation of a novel intracellular metabolite.

New phosphate derivatives of the anti-HIV nucleoside analogue d4T were prepared as potential membrane-soluble prodrugs of the bioactive free nucleotide. The enhanced antiviral potency and/or reduced cytotoxicity of the derivatives leads to an increase in selectivity relative to the parent nucleoside analogue. Moreover, the derivatives appear to bypass the dependence of the nucleoside on thymidine kinase-mediated activation, retaining full activity in thymidine kinase-deficient cells. This strongly suggests the successful intracellular delivery of free nucleotides by the masked phosphate triester prodrugs. This is further confirmed by studies using radiolabeled compound which clearly demonstrate the generation of d4T mono-, di- and triphosphates from the prodrug, even in thymidine kinase-deficient cells. Moreover, we herein report the generation of a new metabolite, a partially hydrolyzed phosphate diester, alaninyl d4T monophosphate. We suggest that at least part of the antiviral action of the prodrugs derives from the intracellular generation of such novel diesters which may add considerable weight to the suggested further preclinical development of the phosphate prodrugs.

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Distribution profile and properties of peripheral-type benzodiazepine receptors on human hemopoietic cells.

The cellular localization of peripheral-type benzodiazepine receptors (PBRs) was characterized in several human blood cell subpopulations including erythrocytes, platelets, monocytes and polymorphonuclear neutrophils (PMN), B, NK, T8 and T4-cells. Pharmacological properties of the PBR were established by binding studies and PBR mRNA expression was measured by quantitative polymerase chain reaction based method. These data clearly indicate 1) the PBR is pharmacologically homogeneous in the various types of blood cells, 2) the rank order of PBR cell density is monocytes = PMN > lymphocytes >> platelets > erythrocytes, 3) the PBR appears to be transcriptionally regulated since mRNA levels are roughly correlated with PBR density.

Impairment of contractility associated with muscarinic supersensitivity in trachea isolated from diabetic rats: lack of correlation with ultrastructural changes or quinuclidinyl benzylate binding to lung membranes.

Evolution of cholinergic response of rat isolated trachea was determined after various durations of diabetes (17, 40, 90, 150 and 210 days). Long-term diabetes was associated with both impairment of contractility and supersensitivity to cholinergic stimulation. However, the mechanism of these alterations remains to be determined, as response to field stimulation was not specifically altered while electron microscopy studies could not detect any significant change in the aspect of nerves, smooth muscle or epithelium. As well, binding studies of lung cholinergic receptors using the antagonist ligand [3H]-quinuclidinyl benzylate and the agonist carbachol did not detect any change in diabetic animals.

Toxicological studies on a benzofuran derivative. III. Comparison of peroxisome proliferation in rat and human hepatocytes in primary culture.

Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid beta-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 microM in rat hepatocyte primary cultures, induced, after 96 hr, a dose-related increase of the peroxisomal beta-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist.