Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2.

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.

Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and L.

Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC(50) values of 0. 37 and 0.45 M, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K(i) value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k(on) values and that the association of the compound with the enzyme fits an apparent one-step mechanism. (13)C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by (13)C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC(50) value of 0.7 M. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.

Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.

The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs.

Quinolines as potent 5-lipoxygenase inhibitors: synthesis and biological profile of L-746,530.

Leukotriene biosynthesis inhibitors have potential as new therapeutic agents for asthma and inflammatory diseases. A series of novel substituted 2-cyanoquinolines have been synthesized and the structure activity relationships were evaluated with respect to their ability to inhibit the formation of leukotrienes via the 5-lipoxygenase enzyme. [1S,5R]-2-Cyano-4-(3-furyl)-7-¿3-fluoro-5-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]-octanyl)]phenoxymethyl ¿quinoline (L-746,530) 3 represents a distinct class of inhibitors and possesses in vitro and in vivo potency comparable or superior to naphthalenic analog (L-739,010) 2.

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs.

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.

Substituted (pyridylmethoxy)naphthalenes as potent and orally active 5-lipoxygenase inhibitors; synthesis, biological profile, and pharmacokinetics of L-739,010.

Dioxabicyclooctanyl naphthalenenitriles have been reported as a class of potent and nonredox 5-lipoxygenase (5-LO) inhibitors. These bicyclo derivatives were shown to be metabolically more stable than their tetrahydropyranyl counterparts but were not well orally absorbed. Replacement of the phenyl ring in the naphthalenenitrile 1 by a pyridine ring leads to the potent and orally absorbed inhibitor 3g (L-739,010, 2-cyano-4-(3-furyl)-7-[[6-[3-(3-hydroxy-6,8-dioxabicyclo[3.2.1] octanyl)]-2-pyridyl]methoxy]naphthalene). Compound 3g inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50S of 20, 1.6, and 42 nM, respectively). Derivative 3g is orally active in the rat pleurisy model (inhibition of LTB4, ED50 = 0.3 mg/kg) and in the anesthetized dog model (inhibition of ex vivo whole blood LTB4 and urinary LTE4, ED50 = 0.45 and 0.23 microgram/kg/min, respectively, i.v. infusion). In addition, 3g shows excellent functional activity against ovalbumin-induced dyspnea in rats (60% inhibition at 0.5 mg/kg, 4 h pretreatment) and Ascaris-induced bronchoconstriction in conscious sheep (50% and > 85% inhibition in early and late phases, respectively at 2.5 micrograms/kg/min, i.v. infusion) and, more particularly in the conscious antigen sensitive squirrel monkey model (53% inhibition of the increase in RL and 76% in the decrease of Cdyn, at 0.1 mg/kg, po). In rats and dogs, 3g presents excellent pharmacokinetics (estimated half-lives of 5 and 16 h, respectively) and bioavailabilities (26% and 73% when dosed as its hydrochloride salt at doses of 20 and 10 mg/kg, respectively, in methocel suspension). Based on its overall biological profile, compound 3g has been selected for preclinical animal toxicity studies.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Altered sensitivity of aspirin-acetylated prostaglandin G/H synthase-2 to inhibition by nonsteroidal anti-inflammatory drugs.

Aspirin (ASA) acetylates Ser516 of prostaglandin G/H synthase-2 (PGHS-2) resulting in a modified enzyme that converts arachidonic acid to 15(R)-hydroxy-eicosatetraeroic acid [15(R)-HETE]. ASA has pharmacological benefits that may not all be limited to inhibition of prostaglandin synthesis, and this study was initiated to further investigate the properties of ASA-acetylated PGHS-2 and of the mutation of Ser516 to methionine, which mimics ASA acetylation. Both the S516M mutant and ASA-acetylated form of PGHS-2 (ASA-PGHS-2) synthesize 15(R)-HETE and have apparent K(m) values for arachidonic acid within 10-fold of the apparent K(m) value for untreated PGHS-2. The time courses of turnover-dependent inactivation were similar for reactions catalyzed by PGHS-2 and ASA-PGHS-2, whereas the PGHS-2(S516M) showed a decrease in both the initial rate of 15-HETE production and rate of enzyme inactivation. The production of 15-HETE by modified PGHS-2 was sensitive to inhibition by most nonsteroidal anti-inflammatory drugs (NSAIDs), including selective PGHS-2 inhibitors. As observed for the cyclooxygenase activity of PGHS-2, the inhibition of 15-HETE production by indomethacin was time-dependent for both ASA-PGHS-2 and PGHS-2(S516M). However, two potent, structurally related NSAIDs, diclofenac and meclofenamic acid, do not inhibit either ASA-PGHS-2 or the PGHS-2(S516M) mutant. These results demonstrate that the sensitivity to inhibition by NSAIDs of the 15-HETE production by ASA-treated PGHS-2 is different than that of prostaglandin production by PGHS-2 and that Ser516 plays an important role in the interaction with fenamate inhibitors. The results also indicate that the conversion of arachidonic acid to 15-HETE by ASA-PGHS-2 is an efficient process providing a unique mechanism among NSAIDs that will not lead to arachidonic acid accumulation or shunting to other biosynthetic pathways.

Zinc dependent activation of cAMP-specific phosphodiesterase (PDE4A).

Cyclic nucleotide phosphodiesterases (PDE), including PDE4A, contain two consensus sequences (HX3HX24-26E) which have been associated with Zn2+ binding and activation with other proteins. This study shows that Zn2+ activates purified recombinant human PDE4A with an EC50 of <1 microM. The EC50 for Mg2+, the generally accepted activating metal ion, is approximately 100 microM. Zn2+ concentrations higher than 5 microM are inhibitory. Mn2+, Co2+ and Ni2+ also activate PDE4A with EC50 values of approximately 2, 3 and 10 microM, respectively. PDE4A binds 65Zn2+ with a Kd of 0.4 microM and approximately 1:1 stoichiometry. Titrations of PDE4A inhibition with Mg2+ and Zn2+ as activating metal ions showed that the competitive inhibitors R-Rolipram, CDP-840, RS-14203 and KF18280 are shifted at least 10-fold to lower potency in the presence of Zn2+. The effect is likely at the site of inhibitor binding as the Km for cAMP in the presence of Mg2+ and Zn2+ is similar (1-3 microM). The Kd of [3H]-R-Rolipram for PDE4A was increased at least 30-fold from 3 nM (with Mg2+) by the presence of Zn2+. The high affinity of Zn2+ for PDE4A indicates that this metal may play a role in the regulation of PDE4A activity.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

Dioxabicyclooctanyl naphthalenenitriles as nonredox 5-lipoxygenase inhibitors: structure-activity relationship study directed toward the improvement of metabolic stability.

Naphthalenic lignan lactone 3a (L-702,539), a potent and selective 5-lipoxygenase (5-LO) inhibitor, is extensively metabolized at two different sites: the tetrahydropyran and the lactone rings. Early knowledge of the metabolic pathways triggered and directed a structure-activity relationship study aimed toward the improvement of metabolic stability in this series. The best modifications discovered, i.e., replacement of the lactone ring by a nitrile group, replacement of the tetrahydropyran ring by a 6,8-dioxabicyclo[3.2.1]octanyl moiety, and replacement of the pendant phenyl ring by a 3-furyl ring, were incorporated in a single molecule to produce inhibitor 9ac (L-708,780). Compound 9ac inhibits the oxidation of arachidonic acid to 5-hydroperoxy-eicosatetraenoic acid by 5-LO (IC50 = 190 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 3 nM) as well as in human whole blood (IC50 = 150 nM). The good inhibitory profile shown by naphthalenenitrile 9ac is accompanied by an improved resistance to oxidative metabolism. In addition, 9ac is orally active in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.1 mg/kg).

Inhibitor-induced changes in the intrinsic fluorescence of human cyclooxygenase-2.

The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.

Arginine 120 of prostaglandin G/H synthase-1 is required for the inhibition by nonsteroidal anti-inflammatory drugs containing a carboxylic acid moiety.

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.

Characterization of the arachidonate and ATP binding sites of human 5-lipoxygenase using photoaffinity labeling and enzyme immobilization.

The arachidonic acid and the ATP binding sites of human 5-lipoxygenase were characterized using photoaffinity labeling and immobilization of the enzyme on ATP-agarose. Photoaffinity labeling of the active site of 5-lipoxygenase was achieved with a novel thiopyranoindole inhibitor containing a 4-azido-3-iodobenzenesulfonyl moiety (L-708,714). This probe was found to inhibit the activity of 5-lipoxygenase (IC50 = 0.3 microM) and to covalently label the enzyme after UV light irradiation. The labeling was inhibited by arachidonic acid, N-hydroxyurea, and dihydrobenzofuranol inhibitors which have been shown to reduce the non-heme iron center of 5-lipoxygenase. Photoaffinity labeling of 5-lipoxygenase by L-708,714 was dependent on the presence of both Ca2+ ions and phospholipids and was independent of ATP. It occurred at similar levels using native (Fe2+), oxidized (Fe3+), or H2O2-inactivated enzyme, but was abolished by heat inactivation of the enzyme. Competition of the labeling by various thiopyranoindoles and other inhibitors such as L-697,198,ZD-2138, and zileuton was found to be related to their inhibitory potency. Immobilized 5-lipoxygenase on ATP-agarose was found to be selectively eluted by adenine nucleotides (ATP > ADP > AMP) but not by solutions containing high salt concentrations, mild detergents, arachidonic acid, or inhibitors. 5-Lipoxygenase inhibitors were selectively retained on the immobilized enzyme and eluted by buffer containing arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiopyranol[2,3,4-c,d]indoles as inhibitors of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase.

The attachment of an arylacetic or benzoic acid moiety to the thiopyrano[2,3,4-c,d]indole nucleus results in compounds which are highly potent and selective 5-lipoxygenase (5-LO) inhibitors. These compounds are structurally simpler than previous compounds of similar potency in that they contain a single chiral center. From the data presented, 2-[[1-(3-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]- 4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]-phenylacetic acid, 14b, was shown to inhibit 5-hydroperoxyeicosatetraenoic acid (5-HPETE) production by human 5-LO (IC50 of 18 nM). The acid 14b is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or human leukocyte leukotriene A4 (LTA4) hydrolase (IC50 > 20 microM). In addition, 14b was inactive in a 5-lipoxygenase-activating protein (FLAP) binding assay at 10 microM. In vivo studies showed that 14b is bioavailable in rat and functionally active in the hyperreactive rat model of antigen-induced dyspnea (74% inhibition at 0.5 mk/kg po; 2 h pretreatment). In the conscious squirrel monkey model of asthma, 14b showed excellent functional activity at 0.1 mg/kg against antigen-induced bronchoconstriction (94% inhibition of the increase in RL and 100% inhibition in the decrease in Cdyn; n = 4). Resolution of this compound gave (-)-14b, the most potent enantiomer (IC50 = 10 nM in the human 5-LO assay), which was shown to possess the S configuration at the chiral center by X-ray crystallographic analysis of an intermediate. Subsequent studies on the aryl thiopyrano[2,3,4-c,d]indole series of inhibitors led to the discovery of potent dual inhibitors of both FLAP and 5-LO, the most potent of which is 2-[[1-(4-chlorobenzyl)-4-methyl-6-(quinolin-2-ylmethoxy)-4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]phenylacetic acid, 19. Acid 19 has an IC50 of 100 nM for the inhibition of 5-HPETE production by human 5-LO and is active in a FLAP binding assay with an IC50 of 32 nM. Furthermore, thiopyrano[2,3,4-c,d]indoles such as 1 and 14b are capable of inhibiting the LTC4 synthase reaction in a dose dependent manner (IC50s of 11 and 16 microM, respectively, compared to that of LTC2 at 1.2 microM) in contrast to other, structurally distinct 5-LO inhibitors. It has also been observed that the thiopyrano[2,3,4-c,d]indole class of compounds strongly promotes the translocation of 5-LO from the cytosol to a membrane fraction in the presence or absence of the ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective inhibitors of COX-2.

The main target of non-steroidal anti-inflammatory drugs (NSAIDs) is prostaglandin G/H synthase (PGHS), also known as cyclooxygenase (COX), which exists as two isoforms. In order to evaluate the contributions of PGHS isoforms to physiological and pathological conditions and their sensitivity to inhibition by non-steroidal anti-inflammatory drugs, we have established high level expression systems of recombinant human PGHS isoforms. The inducible form of PGHS, termed PGHS-2, has been purified and characterized with respect to substrate specificity, product formation, enzymatic activity, glycosylation, heme content, quaternary structure, and modification by aspirin. Pharmacological profiles of the recombinant PGHS isoforms indicate that conventional NSAIDs show little selectivity for either enzyme, however, the recently described NSAID, NS-398, exhibits a high degree of specificity for PGHS-2 through a time dependent mechanism.

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid.

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.

Naphthalenic lignan lactones as selective, nonredox 5-lipoxygenase inhibitors. Synthesis and biological activity of (methoxyalkyl)thiazole and methoxytetrahydropyran hybrids.

Combinations of structural elements found in (methoxyalkyl)thiazole 1a and methoxytetrahydropyran 2a with a naphthalenic lignan lactone produce the potent 5-lipoxygenase (5-LO) inhibitors 3 and 4. While the nature of link Y-Z has a major effect on the in vitro activity of compounds 1 and 2, inhibitors 3 and 4 retain their potencies with either an oxymethylene (Y = O, Z = CH2) or a methyleneoxy (Y = CH2, Z = O) link. Compound 4b inhibits the oxidation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid by 5-LO (IC50 = 14 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 1.5 nM) as well as in human whole blood (IC50 = 50 nM). Compound 4b is a selective 5-LO inhibitor showing no significant inhibition of human 15-lipoxygenase or porcine 12-lipoxygenase or binding to human 5-lipoxygenase-activating protein up to 10 microM and inhibits leukotriene biosynthesis by a direct, nonredox interaction with 5-LO. Compound 15, the open form of lactone 4b, is well absorbed in the rat and is transformed into the active species 4b. In addition, 15 is orally active in the rat pleurisy model (ED50 = 0.6 mg/kg) and in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.3 mg/kg).