Roger Guedj

Synthesis and anti-HIV activity of glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir

With the aim at improving the transport of the current HIV protease inhibitors across the intestinal and blood brain barriers and their penetration into the central nervous system, the synthesis of various acyl and carbamatoyl glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis, and their anti-HIV activity have been investigated. D-Glucose, which is actively transported across these barriers, was connected through its 3-hydroxyl to these antiproteases via a linker. The liberation of the active free drug during the incubation time of the prodrugs with the cells was found to be crucial for HIV inhibition. The labile ester linking of the glucose-containing moiety to the peptidomimetic hydroxyl of saquinavir or to the indinavir C-8 hydroxyl, which is not part of the transition state isostere, is not an obstacle for anti-HIV activity. This is not the case for its stable carbamate linking to the peptidomimetic hydroxyl of saquinavir, indinavir and nelfinavir. The chemical stability with respect to hydrolysis of some of the saquinavir and indinavir prodrugs reported here, the liberation rate of the active free drug and the HIV inhibitory potency are acceptable for an in vivo use of these prodrugs. These glucose-linked ester and carbamate prodrugs display a promising therapeutic potential provided that their bioavailability, penetration into the HIV sanctuaries, and/or the liberation of the active free drug from the carbamate prodrugs are improved. Furthermore, no cytotoxicity was detected for the prodrugs for concentrations as high as 10 or even 100 microM, thus indicating an encouraging therapeutic index.

Liquid phase synthesis of a peptidic nucleic acid dimer

Abstract The first liquid phase synthesis of a peptidic nucleic acid (PNA_) dimer containing guinine and adenine has been achieved in good yields. A new strategy was elaborated in order to circumvent difficult coupling of the protected PNA.

Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients

ABSTRACT We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml−1 limit of detection and thus ∼100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml−1 limit of detection was observed) on a minimum of 30 μl of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.

Quantification of plasma and intracellular levels of the HIV protease inhibitor ritonavir by competitive ELISA

The HIV protease inhibitor ritonavir (Norvir; ABT-578), currently used in combination with nucleoside analogs and other protease inhibitors in anti-HIV therapy, has previously been quantified by an HPLC procedure. Here, we report the first convenient one-step competitive ELISA for measuring plasma and intracellular ritonavir in HIV patients. Anti-ritonavir antibody was raised in rabbits using ritonavir-KLH conjugate as immunogen, and the enzymatic tracer was prepared by coupling the drug to acetylcholine esterase. Samples for analysis were first extracted with methanol. Bound/free separation was achieved in a microtiter plate previously coated with anti rabbit IgG monoclonal antibody. Fifty percent inhibition was observed at 1 ng/ml ritonavir and the method accurately and specifically detected as little as 3-4 ng/ml of plasma ritonavir as well as intracellular drug in the peripheral blood mononuclear cells of patients undergoing ritonavir therapy. Within-run and day to day coefficients of variation were below 10% and the drugs currently used in HIV therapy did not interfere with the test. The ELISA was applied to the measurement of plasma ritonavir and to the determination of the extracellular/intracellular drug level ratios in HIV patients receiving long-term multidrug therapy.

New synthetic routes to β-fluoro β-phenyllactic acid derivatives and β-fluorocyanohydrins

Abstract Alkyl phenyl 2,3-epoxycarboxylates from the well-known Darzens glycidic esters synthesis react under very mild conditions with pyridinium-poly-hydrogen fluoride to give corresponding 3-fluoro 3-phenyllactates in almost quantitative yields with a high regio and stereoselectivity. This method can be applied successfully to other glycidic derivatives : glycidoamides, glycidonitriles, glycidoiminoesters… The spectrometric propertities (IR, NMR) are presented.

Nouvelle voie de synthese d'acides amines monofluores

Resume Ring opening of the glycidonitriles (IIa-e) by HF/pyridine leads to the fluorocyanohydrins (IIIa-e). Treatment of the nitriles (IIIa-e) with ammonia in methanol the α-amino-β-fluoro nitriles (IVa-e) which upon acidic hydrolysis afford theβp-fluoro-α-amino acids (Va-e) in good overall yield.

LIQUID-PHASE SYNTHESIS OF POLYAMIDE NUCLEIC ACIDS (PNA)

Abstract Three liquid-phase processes for the elaboration of short orthogonally protected PNA have been devised. Two of these methods are similar to the convergent and divergent approaches in peptide synthesis. The third process consists in building a fully protected polyamide backbone, by using as many different and orthogonal protecting groups as there are different types of nucleic bases in the targeted polyPNA. Simultaneous and selective cleavage of one kind of protecting group allows the simultaneous attachment of several identical nucleobase units.

Synthesis and anti-HIV activity of some novel arylphosphate and H-phosphonate derivatives of 3′-azido-2′,3′-dideoxythymidine and 2′,3′-didehydro-2′,3′-dideoxythymidine

Phosphate and H-phosphonate derivatives of anti-HIV nucleoside analogues (AZT and d4T) were prepared as potential prodrugs of the bio-active free nucleotide and they were evaluated for their inhibitory effects on the replication of HIV-1 in several cell culture systems. One compound exhibited an important anti-HIV-1 activity and proved to be significantly more efficient than the parent nucleoside.

Formation d'aziridines dans KF/acetonitrile - obtention de 3-fluoro amino-acides par ouverture avec HF/Py

Abstract Synthesis of 1-trityl-2-phenylaziridine, 1-trityl-2-methylaziridine and 2-methylcarboxylates of 1-tritylaziridine,1-trityl-3-methylaziridine, 1-trityl-3-phenylaziridine by reacting N-triphenylmethyl-α-chloroamines and methyl esters of N-triphenylmethyl-O-mesylsulfonyl-α-aminoacids with KF in acetonitrile under reflux is described. Functionalized aziridines opened with hydrogen fluoride in pyridine (70 %) give isobutyl 3-fluoroalanine, methyl 3-fluorophenylalanine and methyl 2-amino-3-fluorobutyrate.

Fluorinated peptides incorporating a 4-fluoroproline residue as potential inhibitors of HIV protease

Abstract N-Fmoc-4-fluoro-L-proline methyl ester was prepared as an attractive synthon for both solid and solution phase peptide synthesis. Its use for the synthesis of Fmoc-Phe-Pro(F)-OMe and Fmoc-Pro(F)-Val-Val-OMe is presented. Direct fluorination with DASTof a 4-hydroxy proline residue incorporated into a peptide and elongation from the terminal amino group allowed preparation of the hexapeptide Boc-AlaAla-Phe-Pro (F) -Val-Val-OMe, analogous to the p 17-p24 gag junction of structural HIV proteins. None of the fluoropeptides in the paper displayed anti-protease or anti-HIV activity.