Pyrimidine thioethers: a novel class of HIV-1 reverse transcriptase inhibitors with activity against BHAP-resistant HIV.

A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.

Targeting delavirdine/atevirdine resistant HIV-1: identification of (alkylamino)piperidine-containing bis(heteroaryl)piperazines as broad spectrum HIV-1 reverse transcriptase inhibitors.

A novel class of bis(heteroaryl)piperazine (BHAP) analogs which possesses the ability to inhibit NNRTI (non-nucleoside reverse transcriptase inhibitor) resistant recombinant HIV-1 reverse transcriptase (RT) and NNRTI resistant variants of HIV-1 has been identified via targeted screening. Further investigation of the structure-activity relationships of close congeners of these novel (alkylamino)piperidine BHAPs (AAP-BHAPs) led to the synthesis of several compounds possessing the desired phenotype (e.g., activity against recombinant RTs carrying the Y181C and P236L substitutions). Further structural modifications were required to inhibit metabolism and modulate solubility in order to obtain compounds with the desired biological profile as well as appropriate pharmaceutical properties. The AAP-BHAPs with the most suitable characteristics were compounds 7, 15, and 36.

In vitro and in vivo inhibition of rat vascular smooth muscle cell migration and proliferation by a 2-aminochromone U-86983.

Vascular smooth muscle cell migration and proliferation are the primary events that govern neointimal thickening and thus they determine the extent to which delayed restenosis occurs after percutaneous transluminal coronary angioplasty. In this study, the in vitro and in vivo smooth muscle cell antichemotactic and antiproliferative properties of a 2-aminochromone, 2-(4-morpholinyl)-8-(3-pyridinylmethoxy)-4H-1-benzopyran-4-one (U-86983), were examined. Migration and proliferation of early-passage rat vascular smooth muscle cells were inhibited by U-86983 in a concentration-dependent manner (IC50S, approximately 10 microM and 3.5 microM, respectively). Longer-term studies showed that the proliferation of smooth muscle cells was inhibited by U-86983 for at least 7 days and was fully reversible on removal of the drug. In addition, the effect of U-86983 on neointimal formation was examined in rats subjected to left common carotid artery balloon dilatation injury. Continual (2-week) i.v. administration of U-86983 (216 mg kg-1 day-1) resulted in a mean plasma drug concentration of 2.39 micrograms/ml (blood level, approximately 3.5 microM) and a 42% (P = .003) reduction in the neointima/media ratio of the injured artery. In agreement with the in vitro reversibility results, administration of U-86983 for only 2, 4 or 7 days did not affect significantly the neointimal thickness measured at 14 days, which indicated that the stimuli for smooth muscle cell migration and/or proliferation were still present 1 week after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological evaluation of antiplatelet 2-aminochromones.

The synthesis and biological evaluation of a series of antiplatelet 2-morpholinylchromones has been described. Modification of the C-7 phenylmethoxy group of 8-methyl-7-(phenylmethoxy)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (2) has led to the discovery of a series of 7-[(amino-ethyl)oxy]-8-methyl derivatives which are potent inhibitors of ADP-induced platelet aggregation. Several members of this class proved active in preventing platelet-dependent thrombus formation in the dog, including 8-methyl-7-[2-(4-methyl-1-piperazinyl)ethoxy]-2-(4- morpholinyl)-4H-1-benzopyran-4-one (39) which was devoid of hemodynamic effects at the effective antithrombotic dose.

2-Aminochromones block human platelet aggregation by inhibiting cyclic AMP-dependent phosphodiesterase leading to reduced platelet phospholipase C activity.

We examined a series of 2-aminochromone analogs typified by U-84569 [8-methyl-2-(4-morpholinyl)-7-(1-naphthylenylmethoxy)-4H-1- benzopyran-4-one] as potential antithrombotic agents. U-84569 proved to be a potent inhibitor of human platelet aggregation regardless of the agonist used. Subsequent experiments showed that U-84569 increased platelet cyclic AMP (cAMP) levels in intact cells, but U-84569 did not directly stimulate adenylate cyclase. Our experiments showed that U-84569 was a potent inhibitor of the low Km cAMP-dependent phosphodiesterase with an IC50 of 300 nM in platelet cytosol. Isobutylmethylxanthine had an IC50 of 10 microM in the same system. Although U-84569 elevated cAMP by inhibiting cAMP metabolism, we were interested in the mechanism by which cAMP blocked aggregation. Our first experiments showed that U-84569 concentration-dependently blocked agonist-stimulated, but not phorbol myristate acetate-dependent, phosphorylation of the 47 kDa protein kinase C substrate in platelets. These data suggested that U-84569 could interrupt receptor-mediated signal transduction. In support of this hypothesis, U-84569 proved to be a potent inhibitor of thrombin-stimulated inositol phosphate synthesis, diacylglycerol formation and Ca++ mobilization in intact cells. These data indicate that agonist-stimulated phospholipase C activity was reduced in U-84569-treated cells. There was no direct influence of U-84569 on either basal or thrombin-stimulated phospholipase C activity in broken cells, suggesting that U-84569 (by inhibiting phosphodiesterase and elevating cAMP), indirectly blocked receptor-mediated phospholipase C activation and aggregation in platelets. The 2-aminochromones represent a new class of potent antithrombotic agents.

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.

Contribution of leukotriene B4 to airway inflammation and the effect of antagonists.

Inhalation of aerosols of ovalbumin in sensitized guinea pigs produced a marked, bronchoalveolar eosinophilia 24 hr after challenge. The lung eosinophilia was not prevented by the cyclooxygenase inhibitors, indomethacin or PAF antagonists (WEB-2086 and L-652731) but was inhibited by methylprednisolone, the 5-LO inhibitor, U-66858 and a series of structural analogs of LTB4, U-75302, U-77692, U-75485 and U-78489. The effectiveness of LTB4 antagonists but not PAF antagonists in vivo was consistent with in vitro studies in which LTB4 was shown to be far more chemotactic than PAF for guinea pig eosinophils. LTB4 elicited maximal directional migration of guinea pig eosinophils at concentrations from 10(-7) M to 10(-9) M while PAF showed no effect over the same concentration range. The structural analogs of LTB4 were shown to inhibit LTB4 induced chemotaxis of guinea pig eosinophils and produced a dose-related inhibition of binding of LTB4 to guinea pig eosinophil membranes. To add further proof to the hypothesis that LTB4 contributed to the antigen-induced lung eosinophilia we attempted to measure LTB4 release into BAL fluid immediately after and at various time points up to 24 hr after antigen inhalation. However, using a sensitive radioimmunoassay (detection limit 10 pg/ml) very low levels of LTB4 (24.9-67.9 pg/ml) or its metabolite, 20-OH LTB4 (24.9-98.2 pg/ml) were detected in BAL fluid and these levels did not increase significantly following antigen provocation. Inhalation of LTB4 aerosols in unsensitized Brown-Norway rats or inhalation of aerosols of ovalbumin in sensitized Brown-Norway rats also produced a marked "late-phase" eosinophil-rich influx of inflammatory cells into the lungs. The lung eosinophilia in the rat was prevented by two structurally unrelated leukotriene B4 (LTB4) antagonists, U-75302 and Ly255283. These data implicate LTB4 as a mediator of allergen-induced bronchopulmonary eosinophilia. Leukotriene B4 antagonists may provide leads for the development of compounds which inhibit the chronic airway inflammation associated with asthma in man.

Effect of the selective leukotriene B4 antagonist U-75302 on antigen-induced bronchopulmonary eosinophilia in sensitized guinea pigs.

The selective leukotriene B4 (LTB4) antagonist, U-75302, 6-(6-(3-hydroxy-1E,5Z-undecadien-1-yl)-2-pyridinyl)-1,5-hexa nediol) was examined for its ability to inhibit the "late-phase" bronchopulmonary eosinophilia that occurs 6 to 24 h after inhalation of specific antigen in sensitized guinea pigs. Groups of 6 male guinea pigs, sensitized with ovalbumin, were pretreated with U-75302, 1.0, 10.0, or 30.0 mg/kg, or vehicle 1 h before and 7 h after antigen inhalation. Twenty-four hours after antigen provocation, the lungs were lavaged for the enumeration of inflammatory cell populations. Doses of U-75302 (1.0, 10.0 and 30.0 mg/kg) administered orally produced 12.2%, (p greater than 0.05), 43.2% (p less than 0.05), and 61.1% (p less than 0.05) inhibition, respectively, of the antigen-induced influx of eosinophils into the bronchial lumen. Neutrophil populations were not significantly affected by treatment with U-75302. In a separate study, we compared the histopathological changes that occurred following antigen challenge in U-75302-treated or vehicle-treated guinea pigs. Vehicle-treated, sensitized animals exhibited marked changes in the airway at 8 min, 6 h, and 24 h after antigen challenge. U-75302 treatment produced a significant reduction in eosinophil adherence to peribronchial/peribronchiolar capillaries followed by a dramatic and specific reduction of peribronchial eosinophil infiltration (81% reduction at 6 h and 79% reduction at 24 h). Neutrophil migration appeared unaffected. These data implicate LTB4 as a mediator of antigen-induced bronchopulmonary eosinophilia in the guinea pig.

Receptor antagonism of leukotriene B4 myotropic activity by the 2,6 disubstituted pyridine analog U-75302: characterization on lung parenchyma strips.

Leukotriene B4 constricts guinea pig lung parenchyma strips in a concentration-dependent manner. The LTB4 structural analog U-75302, 6-(6-[3-hydroxy-1E,5 Z-undecadienyl]-2-pyridinyl)-1,5-hexanediol, was a partial agonist in this system with a potency 300-1000 times less than LTB4. U-75302 constricted lung parenchyma strips only at concentrations greater than 0.3 microM. At concentrations lacking agonist activity U-75302 was an effective antagonist, displacing the LTB4 dose-response curve. Half-maximal responses required 0.10 microM LTB4 in the presence of 0.3 microM U-75302 and 0.01-0.02 microM LTB4 in its absence. The maximal force of contraction was unaffected at this concentration. Concurrent with antagonism of the myotropic response, U-75302 inhibited the LTB4-dependent release of thromboxane B2 from lung parenchyma. This effect was attributable to receptor antagonism, not enzymatic inhibition of phospholipase, cyclooxygenase, or thromboxane synthase. For instance, 0.3 microM U-75302 did not inhibit thromboxane B2 formation by lung parenchyma stimulated with calcium ionophore A23187 and it did not inhibit thromboxane B2 formation by human platelets stimulated with arachidonic acid. U-75302 selectively antagonized the activity of LTB4 and not other myotropic agonists including the thromboxane A2 mimetic U-46619, LTC4, LTD4, AGEPC, PGF2 alpha, and histamine. Receptor antagonists of leukotriene B4 may have multiple beneficial effects on asthmatic or respiratory disorders. These include (i) direct antagonism of LTB4 myotropic actions; (ii) antagonism of LTB4-dependent mediator release; and (iii) antagonism of LTB4 chemotactic action associated with leukocyte infiltration during anaphylactic late phase reactions.

Novel molecules that antagonize leukotriene B4 binding to neutrophils.

A series of LTB4 analogues have been synthesized that replace carbons 7-9 of the cis-trans-trans triene unit of LTB4 with a stable ring structure. Meta-substituted pyridine analogues are more potent inhibitors than benzene or furan analogues. C-1 alcohols are often more potent inhibitors than free carboxylic acids, and 5,6-cis double bond compounds are more potent than 5,6-trans compounds. Compounds such as these may prove to be useful in the treatment of inflammatory diseases.

On the stereochemistry and biosynthesis of lipoxin B.

Lipoxin B (LXB) was prepared by incubation of (15S)-15-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with human leukocytes. Comparison with a number of trihydroxyicosatetraenes prepared by total synthesis showed that biologically derived LXB is (5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-icosatetraenoi c acid. Two isomers of LXB were identified by using an improved isolation procedure. These compounds were shown to be (5S,14R,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid (8-trans-LXB) and (5S,14S,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid [(14S)-8-trans-LXB]. Experiments with 18O2 showed that formation of LXB and its two isomers occurred with incorporation of molecular oxygen at C-5 but not at C-14. These results together with the finding that (15S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) is a precursor of LXB compounds in activated leukocytes suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-icosatetraenoic acid or its equivalent is a common intermediate in the biosynthesis of LXB and its two isomers.

The synthesis and biological evaluation of 7 beta-[2-(5-amino-[1,2,4] oxadiazol-3-yl)-2-Z-methoximinoacetamido]-cephalosporin derivatives.

A series of 7 beta-[2-(5-aminooxadiazol-3-yl)-2-Z-methoximinoacetamido] -3-cephem-4-carboxylic acids (7a-g) were synthesized and evaluated microbiologically Although somewhat less active than cefotaxime 7a-g showed good antimicrobial activity against a wide variety of Gram-positive and Gram-negative bacteria. The beta-lactamase stability of 7a and 7f was also discussed.