Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Studies on 5-lipoxygenase inhibitors. II. Discovery, optical resolution and enantioselective synthesis of FR110302, a highly potent non-redox type 5-lipoxygenase inhibitor

A novel series of 2,2-dialkyl-5-(2-quinolylmethoxy)-1,2,3, 4-tetrahydro-1-naphthols was synthesized and evaluated as 5-lipoxygenase (5-LO) inhibitors. Systematic optimization led to identification of several highly potent non-redox type 5-LO inhibitors with nanomolar IC50s as racemic mixtures. Optical resolution of racemate 50 indicated that its 5-LO inhibitory activity was enantiospecific and due to the (+)-enantiomer. An efficient synthetic route to the (+)-enantiomers via asymmetric reduction of tetralone intermediates was established. The best compound, (+)-2,2-dibutyl-5-(2-quinolylmethoxy)-1,2,3,4-tetrahydro-1-naphtho l (FR110302, (+)-50), showed potent inhibitory activity against leukotriene (LT) biosynthesis by intact neutrophiles in rats (IC50 4.9 nM) and in humans (IC50 40 nM). Furthermore oral administration of FR110302 significantly inhibited neutrophil migration in the rat air pouch model at 1 mg/kg.     

Antiherpesvirus activities of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine (A-5021) in cell culture

Antiherpetic activity of (1'S,2'R)-9-([1',2'-bis(hydroxymethyl)cycloprop-1'yl]methyl)guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures. In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells. Against clinical isolates of herpes simplex virus type 1 (HSV-1, n = 5) and type 2 (HSV-2, n = 6), mean 50% inhibitory concentrations (IC50s) for A-5021 were 0.013 and 0.15 microgram/ml, respectively, in MRC-5 cells. Corresponding IC50s for ACV were 0.22 and 0.30 microgram/ml, and those for PCV were 0.84 and 1.5 micrograms/ml, respectively. Against clinical isolates of varicella-zoster virus (VZV, n = 5), mean IC50s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 micrograms/ml, respectively, in human embryonic lung (HEL) cells. A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time. The selectivity index, the ratio of 50% cytotoxic concentration to IC50, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV. In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds. These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.     

5-Lipoxygenase: a target for antiinflammatory drugs revisited

Arachidonate 5-lipoxygenase is the key enzyme in leukotriene biosynthesis and catalyzes the initial steps in the conversion of arachidonic acid to biologically active leukotrienes. Leukotrienes are considered as potent potent mediators of inflammatory and allergic reactions which are locally released by leukocytes and other 5-LO expressing cells and exert their effects via binding to specific membrane receptors and, as suggested recently, the nuclear receptor PPARa. Because of the proinflammatory profile of leukotrienes it was assumed that leukotriene biosynthesis inhibitors and leukotriene receptor antagonists have a therapeutical potential in a variety of inflammatory diseases. Clinical studies confirmed the therapeutic value of the antileukotriene therapy in asthma but the results with leukotriene biosynthesis inhibitors in psoriasis, arthritis and inflammatory bowel disease were more or less disappointing. This review summarizes the biochemistry of the 5-lipoxygenase pathway, the pharmacology of FLAP and 5 lipoxygenase inhibitors and discusses possible criteria for the development of these drugs.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.     

Six primary squamous cell carcinomas of the head and neck

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.     

Characterization of the inhibitory prostanoid receptors on human neutrophils

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Leukotrienes in allergic inflammatory reactions

Production of leukotrienes, lipooxygenase products of arachidonic acid metabolism, plays an important role in inflammatory reactions, particularly well studied in bronchial asthma. Lipooxigenase-5 and lipooxygenase-activating protein-5 are crucial in the production of leukotrienes with potent biological activities. Leukotriene B4 is a leukocytic chemoattractant and it induces aggregation and adherence of leukocytes to endothelial vasculature. Sulfidopeptid leukotrienes (C4, D4 and E4) are potent bronchoconstrictors, producing mucous secretion in the airways and increasing vascular permeability. Leukotrienes participate in the process of inflammation, as well as in early and late asthmatic responses. They are found in the blood, liquid obtained upon bronchoalveolar lavage as well as in the urine, irrespectively whether bronchospasm developed spontaneously or it was induced by an allergen. Administration of the specific leukotriene receptor antagonists or leukotriene synthesis inhibitors ameliorates the symptoms and signs of bronchial asthma.     

Antitumor agents. 178. Synthesis and biological evaluation of substituted 2-aryl-1,8-naphthyridin-4(1H)-ones as antitumor agents that inhibit tubulin polymerization

As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-naphthyridin-4(1H)-one series, we have synthesized two series of 3'-substituted 2-phenyl-1,8-naphthyridin-4(1H)-ones and 2-naphthyl-1,8-naphthyridin-4(1H)-ones. All compounds showed significant cytotoxic effects (log GI50 < -4.0; log molar drug concentration required to cause 50% growth inhibition) against a variety of human tumor cell lines of the National Cancer Institute's in vitro screen, including cells derived from solid tumors such as non-small cell lung, colon, central nervous system, melanoma, ovarian, prostate, and breast cancers. All 3'-substituted compounds demonstrated strong cytotoxic effects in almost all tumor cell lines. Introduction of an aromatic ring at the 2'- and 3'-positions also generated compounds with potent antitumor activity. Incorporation of an aromatic ring at the 3'- and 4'-positions produced compounds with reduced activity. Interestingly, introduction of a halogen at the 3'-position yielded compounds with different selectivity for the tumor cell lines tested. All 3'-halogenated compounds (29-36) and compounds 38 and 42-44 were potent inhibitors of tubulin polymerization with activities nearly comparable to those of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4. Active agents also inhibited the binding of [3H]colchicine to tubulin.     

Myocardial protection by the leukotriene synthesis inhibitor BAY X1005: importance of transcellular biosynthesis of cysteinyl-leukotrienes

Perfusion of the isolated rabbit heart with 5 x 10(6) human polymorphonuclear leukocytes, under recirculating conditions (50 ml), and challenge with A-23187 (0.5 microM) caused an increase in coronary perfusion pressure (from a prechallenge value of 46 +/- 1.1 to 176.2 +/- 29.7 mm Hg, 30 min after challenge, n = 6-4), which was linearly correlated (P < .006) with formation of cysteinyl leukotrienes (29.7 +/- 7.3 pmol/ml, 30 min after challenge). Pretreatment with the leukotriene synthesis inhibitor BAY X1005 (1 microM) (n = 6) resulted in significant protection against the increase in coronary perfusion pressure (76.7 +/- 12.8 mm Hg, 30 min after challenge) and in almost complete inhibition of sulfidopeptide leukotriene synthesis (3.2 +/- 1.7 pmol/ml, 30 min after challenge). In in vivo experiments, ligation of the left anterior descending coronary artery in the rabbit (n = 10) resulted in acute myocardial infarction marked by a mortality rate of 60% compared with sham-operated animals (n = 10). Intravenous treatment of the rabbits with BAY X1005 (10 mg/kg/h, for 2 h) (n = 10) markedly reduced the mortality rate (20%), protected the rabbits against the marked electrocardiogram derangement and abolished the significant increase in plasma creatine phosphokinase activity and cardiac tissue myeloperoxidase activity induced by coronary artery ligation. BAY X1005 exerts a significant cardioprotection and suggests that specific leukotriene synthesis inhibitors may lead to innovative therapy in myocardial ischemia.     

Synthesis and antiallergic activity of pyridothienopyrimidines

The synthesis of a series of pyridothienopyrimidines and their evaluation as inhibitors or inducers of the release of histamine from rat mast cells is reported. The activity was measured after immunological stimulation with ovoalbumin and chemical stimulation with polymer 48/80 and the drugs adryamicin and vinorelbine. The experiments were carried out with and without preincubation of the stimulus with the cells before addition of the drug. Several pyridothienopyrimidines show inhibitory IC50 values in the range 2-25 microM, indicating they are up to 100 times more potent than cromoglycate (DSCG) and 10 times greater than Ketotifen. Compound 9l is a potent inhibitor in all the conditions tested and shows IC50 = 9-25 microM. Pyridothienopyrimidines 4l and 9e are very strong inducers of histamine release in the immunological (4l, 170-230%) and chemical (9e, 100-150%) assays, respectively. Compounds 4l and 9i are cytotoxic in vitro (IC50 = 0.1-0.2 microgram/mL) against P-388, A-549, HT-29, and MEL-28 tumor cell lines.     

Novel hepatitis C virus protease inhibitors: thiazolidine derivatives

This study evaluated the inhibitory effects of thiazolidine derivatives on hepatitis C virus (HCV) protease and other human serine proteases. The inhibition efficacy was tested with a reversed-phase high-performance liquid chromatography (HPLC) assay system using a NS3-NS4A fusion protein as the HCV protease and a synthetic peptide substrate that mimics the NS5A-5B junction. Nine thiazolidine derivatives showed more than 50% inhibition at 50 microg/ml. The most potent derivative was RD4-6250, with 50% inhibition at a concentration of 2.3 microg/ml; this concentration was lower than those of other protease inhibitors reported previously. The most selective derivative was RD4-6205, with 50% inhibition at a concentration of 6.4 microg/ml, a lower concentration than those on other serine proteases (chymotrypsin, trypsin, plasmin, and elastase). These results suggest that the RD4-6205 skeleton is an important structure for inhibitory activity on the HCV protease NS3-NS4A.     

Novel peptidyl phosphorus derivatives as inhibitors of human calpain I

Dipeptidyl phosphorus compounds were synthesized as potential bioisosteric mimics of peptide alpha-ketoesters and alpha-ketoacids. alpha-Ketophosphonate Cbz-Leu-Leu-P(O)(OCH3)2 (1b), containing an alpha-ketoester bioisostere, inhibits human calpain I with an IC50 = 0.43 microM. The potency of 1b compares very favorably with that of alpha-ketoester Cbz-Leu-Leu-CO2Et (IC50 = 0.60 microM). Monomethyl ketophosphonate Cbz-Leu-Leu-P(O)(OH)(OCH3) (1a, IC50 = 5.2 microM), an alpha-ketoacid mimic, is less potent. Dibutyl and dibenzyl alpha-ketophosphonates 1c,e,f are much less potent calpain inhibitors than dimethyl alpha-ketophosphonate 1b. alpha-Ketophosphinate 1g (IC50 = 0.37 microM) and alpha-ketophosphine oxide 1h (IC50 = 0.35 microM) are also potent calpain inhibitors.     

Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation

The anti-inflammatory activity of drugs more selective for cyclooxgenase isoform inhibition (cyclooxygenase 1, cyclooxygenase 2), were compared in rat carrageenin-induced pleurisy. Suppression of inflammation by cyclooxygenase 2-selective inhibitors, NS-398 (N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) and nimesulide (4-nitro-2-phenoxy-methanesulfonanilide), and by piroxicam and aspirin, more selective for cyclooxygenase 1, was measured. Piroxicam and aspirin significantly inhibited inflammatory cell influx, exudate and prostaglandin E2 formation, 6 h after carrageenin injection. Cyclooxygenase 2 inhibitors had little effect on these parameters with NS-398 alone reducing prostaglandin E2 levels, but increasing levels of leukotriene B4. In contrast, at 3 h after carrageenin injection, cyclooxygenase 2 inhibitors significantly inhibited all inflammatory parameters however suppression with piroxicam and aspirin was greater, and more pronounced than at 6 h. NS-398 and nimesulide dosing did not reduce thromboxane B2 production from platelets isolated from rats with carrageenin-induced pleurisy, demonstrating that at the doses used, cyclooxygenase 2 inhibitors did not inhibit cyclooxygenase 1, as platelets contain only this isoform. Therefore, in the rat carrageenin-induced pleurisy, drugs more selective for the inhibition of cyclooxygenase 1 attenuate inflammation over a wider time frame than cyclooxygenase 2-selective drugs, suggesting a significant role for cyclooxygenase 1 in this model. Inhibition of cyclooxygenase 2 by NS-398 however, resulted in an increase in the potent chemoattractant leukotriene B4.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Regulation of hsp expression during rodent spermatogenesis

As part of an effort to develop peptides with selective kappa-opioid antagonist activity, a series of N-alkylated [D-Pro10]dynorphin A-(1-11) derivatives were made through solid-phase peptide synthesis: R-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [3H]diprenorphine (approximately 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 microM) adenylyl cyclase activity. Displacement of [3H]diprenorphine specific binding by these peptides was observed with a rank order of affinity (Ki, nM) = [D-Pro10]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N-cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl- (12.6) approximately N-benzyl- (18.3) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl cyclase inhibition (IC50, nM): [D-Pro10]dynorphin A-(1-11) (0.12) approximately dynorphin A-(1-13)NH2 (0.19) > N-cyclopropylmethyl- (2.7) > N,N-dicyclopropylmethyl- (13.2) approximately N,N-diallyl- (18.0) approximately N-benzyl-[D-Pro10]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cyclase activity: dynorphin A-(1-13)NH2 (100%) approximately N-cyclopropylmethyl- (94.3%) approximately [D-Pro10]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) > N,N-dicyclopropylmethyl- (23.6%) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11)(18.9%). As the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) alone. This weak partial agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists.     

Inhibition of thrombin generation in plasma by inhibitors of factor Xa

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.     

Structure-activity relationship studies of chalcones as SRS-A receptor antagonists

A simple, reliable and highly sensitive bioassay with sensitized longitudinal strips of guinea pig ileum was used for screening the receptor antagonists of slow reacting substance of anaphylaxis (SRS-A). The SRS-A receptor antagonistic activities of 17 chalcones were studied. Most compounds in these chalcones were found to have SRS-A receptor antagonistic action at the concentration of 10(-4) mol.L-1. Among them, compounds 5, 13 and 17 were highly effective with IC50s of 7.5 x 10(-6), 7.5 x 10(-6) and 6.8 x 10(-5) mol.L-1, respectively. Under the same conditions, the IC50 of FPL 55712, a known leukotriene D4 receptor antagonist, was shown to be 3 x 10(-4) mol.L-1. It would appear that compounds 5, 13 and 17 were 40, 40 and 4.4 times more potent, respectively, than FPL 55712. From analysis of structure-activity relationship of chalcones, these results suggest that the following factors may be important for an active antagonist of SRS-A receptors: (a) There is a system of pi, pi conjugation in the molecule; (b) The ester group in the B ring of chalcones is more favorable than the carboxyl group; (c) Antagonism for meta- or para-substituted derivatives of carboxyl or ester group in the B ring are more potent than ortho-substituted compounds; (d) The length of carbon chain of alkyl group in the A ring of chalcones is more effective for 1, 4 or 6 carbon atoms than for 10 or 14 carbon atoms.     

Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.