Symmetric building blocks and combinatorial functional group transformation as versatile strategies in combinatorial chemistry.

A combination of symmetric building blocks and combinatorial functional group transformation for synthesis of pyrimidines was investigated. The purpose of the study was to maximize the return on invested synthetic efforts of reaction development for libraries. A representative set of symmetric diacids was coupled onto deprotected TentaGel Rink Amide resin. The amino function served as a model of a chemical process providing a functional group for additional synthetic steps, while the symmetric building blocks served as a model to connect synthesis protocols and to switch to a different synthesis paradigm consecutively. The reaction sequence was continued in a noncombinatorial step by coupling a bifunctional reagent (3-aminoacetophenone) to the remaining carboxy function of the symmetric diacid. The ketone served as a model of a reagent prepared for combinatorial functional group transformation. The arylmethylketone was reacted with a set of aryl- and heteroarylaldehydes to give alpha,beta-unsaturated ketones. Subsequently, guanidine, alkyl-, and arylcarboxamidines were introduced in combinatorial synthesis of substituted pyrimidines by reaction with the alpha, beta-unsaturated ketone functionality. The combination of symmetric building blocks and combinatorial functional group transformation created a versatile reaction sequence ideally suited for production of libraries from libraries with added diversity.

Time-resolved fluorescence anisotropy of HIV-1 protease inhibitor complexes correlates with inhibitory activity.

The tryptophan time-resolved fluorescence intensity and anisotropy of the HIV-1 protease dimer is shown to be a quick and efficient method for the conformational characterization of protease inhibitor complexes. Four fluorescence lifetimes were needed to adequately describe the fluorescence decay of the two tryptophan residues, W6 and W42, per protease monomer. As a result of the wavelength dependence of the respective amplitudes, the 2.06 ns and the 4.46 ns decay constants were suggested to be the intrinsic fluorescence lifetimes of the more solvent-exposed W6 and the less exposed W42 residues, respectively. Analysis of the fluorescence anisotropy decay yielded a short correlation time of 250 ps corresponding to local chromophore motions, and a long correlation time of 12.96 ns resulting from overall rotation of the protease enzyme. Fluorescence lifetimes and rotational correlation times changed when inhibitors of the HIV-1 protease were added. The effects of 11 different inhibitors including statine-derived, hydroxyethylamine-derived, and 2 symmetrical inhibitors on the protease fluorescence dynamics were investigated. Inhibitor binding is shown to induce an increase of the mean fluorescence lifetime taumean, an increase of the short rotational correlation time phi1, as well as a decrease of the long rotational correlation time phi2. The mean rotational correlation time phimean was identified as the global dynamic parameter for a given molecular complex, which correlates with the inhibitor dissociation constant Ki, and therefore with the activity of the inhibitor.

Paracyclophanes: a novel class of water-soluble inhibitors of HIV proteinase.

A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes constitute a novel building block for potent human immunodeficiency virus (HIV) protease inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with appropriate alpha, omega-diamines and consecutive ring closure under high dilution conditions. The resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracyclophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with Ki values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained. The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral bioavailability in mice are given for selected compounds.

Inhibitors of human immunodeficiency virus type 1 protease containing 2-aminobenzyl-substituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, activity, and oral bioavailability.

Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-valyl]-amino]-3-hydroxy-2-[(4- methoxybenzyl)amino]-5-phenylpentanoyl)-L-valine 2-(aminomethyl)- benzimidazole amide led to a novel series of inhibitors with shortened, modified carboxy terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported. Of particular interest are derivatives featuring the (1S,2R)-1-amino-2-hydroxyindan moiety at the P2'-position since some of them exhibit substantial oral bioavailability in mice. The influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors is discussed. Optimum enhancement of oral bioavailability was observed with L-tert-leucine in P2-position, resulting in the discovery of (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-tert-leucyl]- amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hydroxyindan amide which combines high antiviral activity (IC50 = 250 nM) with a good pharmacokinetic profile (AUC = 82.5 microM.h at a dose of 125 mg/kg po in mice).

SDZ PRI 053, an orally bioavailable human immunodeficiency virus type 1 proteinase inhibitor containing the 2-aminobenzylstatine moiety.

A series of inhibitors of human immunodeficiency virus type 1 (HIV-1) proteinase containing the 2-aralkyl-amino-substituted statine moiety as a novel transition-state analog was synthesized, with the aim to obtain compounds which combine anti-HIV potency with oral bioavailability. The reduced-size 2-aminobenzylstatine derivative SDZ PRI 053, which contains 2-(S)-amino-3-(R)-hydroxyindane in place of an amino acid amide, is a potent and orally bioavailable inhibitor of HIV-1 replication. The antiviral activity of SDZ PRI 053 was demonstrated in various cell lines, in primary lymphocytes, and in primary monocytes, against laboratory strains as well as clinical HIV-1 isolates (50% effective dose = 0.028 to 0.15 microM). Cell proliferation was impaired only at 100- to 300-fold-higher concentrations. The mechanism of antiviral action of the proteinase inhibitor SDZ PRI 0.53 was demonstrated to be inhibition of gag precursor protein processing. The finding that the inhibitory potency of SDZ PRI 053 in chronic virus infection, determined by p24 release, was considerably lower than that in de novo infection may be explained by the fact that the virus particles produced in the presence of SDZ PRI 053 are about 50-fold less infectious than those from untreated cultures. Upon intravenous administration, half-lives in blood of 100 and 32 min in mice and rats, respectively, were measured. Oral bioavailability of SDZ PRI 053 in rodents was 20 to 60%, depending on the dose. In mice, rats, and dogs, the inhibitor levels after oral administration remained far above the concentrations needed to efficiently block HIV replication in vitro for a prolonged period. This compound is thus a promising candidate for clinical use in HIV disease.

HIV proteinase inhibitors containing 2-aminobenzylstatine as a novel scissile bond replacement: biochemical and pharmacological characterization.

Derivation of the 2-aminobenzylstatine containing HIV-1 proteinase (PR) inhibitor I led to a series of compounds with considerably improved antiviral activity, the most potent derivatives inhibiting HIV-1 with IC50 values below 25 nM. This was achieved by the combination of several structural modifications, most prominently by introduction of a benzimidazole heterocycle into the inhibitor. The mode of action of the 2-aminobenzylstatine PR inhibitors was demonstrated to be inhibition of gag precursor processing. The antiviral efficacy of the PR inhibitors was demonstrated in various cell lines, in primary T4 lymphocytes and in monocytes. The most potent compound (XI) inhibited replication of several HIV-1 clinical isolates in primary cells with IC50 values of 8 to 23 nM. The analysis of the pharmacokinetic behaviour of compounds I and VII revealed blood half-lives in rodents in the range of about 1.5 h. Compound I also showed appreciable oral uptake in mice (18%), but yielded no detectable blood levels in rats after oral administration. Benzimidazole containing compounds like VII were not orally bioavailable to a significant extent, neither in mice nor in rats. Thus, while introduction of a benzimidazole group into the PR inhibitors was a successful structural modification with regard to antiviral activity in cell culture, it completely abolished oral bioavailability.

Inhibitors of HIV-1 proteinase containing 2-heterosubstituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, enzyme inhibition, and antiviral activity.

A convenient procedure for the synthesis of 2-heterosubstituted statine derivatives as novel building blocks in HIV-protease inhibitors has been developed. The synthesis starts with protected L-phenylalaninols, which were converted to gamma-amino alpha, beta-unsaturated esters in a one-pot procedure. A highly diastereoselective epoxidation of the N-protected (E)-enoates, followed by regioselective ring opening of the corresponding 2,3-epoxy esters with a variety of heteronucleophiles, resulted in 2-heterosubstituted statine derivatives. The overall stereo-chemical outcome of the transformations meets the required configuration of HIV-protease inhibitors. The short, synthetically flexible, and highly diastereoselective synthesis of 2-heterosubstituted statines has enabled a broad derivation, covering the S3, S2, and S1'-S3' sites of the enzyme. In a series of 46 derivatives, several potent inhibitors were obtained with Ki values as low as 3.4 nM and antiviral activity in the lower nanomolar-range. The structural parameters of the compounds which determine the potency of inhibition and selectivity for the viral enzyme are discussed.

Helix-loop-helix motif in HIV-1 Rev.

Circular dichroism (CD) spectra of C-terminal deletion mutants of the HIV-1 Rev protein, Rev M9 delta 14 (missing aa 68-112) and Rev M11 delta 14 (lacking aa 92-112), indicated that Rev contains 46-49 residues in alpha-helical conformation within the N-terminal 71 or 95 amino acids of the 116 residue protein. Complexation with a 40-nucleotide fragment of the Rev responsive element, RRE, (G39 to C78), containing the minimal element for Rev binding, induced an A to B form structural transition in the RRE fragment, whereas the percentage of alpha-helical conformation in the protein stays constant on substrate binding. When complexed to the RNA, neither mutant protein showed structural changes upon raising the temperature to 40 degrees C, as determined by the lack of decrease of the signal intensity at 222 nm, indicative for alpha-helical conformation. In contrast, Rev M9 delta 14, which is shorter than Rev M11 delta 14 by 24 amino acids, in the absence of RNA, lost about 60% of the spectral minima at 222 nm at the same temperature. The Rev M11 delta 14 mutant, in the absence of RNA, showed a decrease of 20% in spectral intensity upon heating to 40 degrees C. Free and RNA-bound mutant proteins showed reversible transitions upon heating to 80 degrees C and subsequent cooling down to 10 degrees C overnight. The Rev peptide Cys 75-93, spanning the Rev transactivation domain, showed secondary structure in 40% and 60% hexafluoropropanol (HFP) solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

[Splenic complications of chronic pancreatitis. Clinical and experimental study]. / Complications spléniques de la pancréatite chronique. Etude clinique et expérimentale.

Splenic complications of chronic pancreatitis appear to be less exceptional than is usually accepted, particularly since preoperative diagnosis has been facilitated by ultrasound and abdominal scan imaging. Complications noted in 37 cases were: infarcts (2 cases), hematoma or false blood cysts (26 cases) and rupture (9 cases). The splenic infarcts were revealed by digestive hemorrhage, the false blood cysts of spleen by a painful mass in left hypochondrium associated with pleural effusion and rupture of spleen by an acute hemoperitoneum. Treatment included splenectomy in 19 cases, splenectomy caudal pancreatectomy in 17 cases and drainage of a splenic hematoma in one patient. Operative mortality was 16.2% and the long-term prognosis was poor and related to underlying condition. Data from an experimental study suggest that the effect of an episode of acute pancreatitis on the splenic pedicle is the most important physiopathologic factor. A hemorrhagic infarct or infarction of splenic parenchyma are common starting points for all clinicopathologic forms described.