Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC(50) = 0.19 microM) but is much less potent with PGHS-1 (IC(50) = 20 microM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC(50) = 14 microM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various darbufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one-step slow-binding inhibition. A two-step slow-binding inhibition model is proposed in which the E.I complex (K(i) = 6.2 +/- 1.9 to 14 +/- 1 microM) slowly transforms (k(5) = 0.015-0.030 s(-)(1)) to a tightly bound E.I form with K(i) = 0.63 +/- 0.07 microM and k(6) = 0.0034 s(-)(1). In steady-state kinetics inhibition experiments performed with no preincubation, we find that darbufelone is a noncompetitive inhibitor of PGHS-2 (K(i) = 10 +/- 5 microM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda(ex) = 280 nm) with K(d) = 0.98 +/- 0.03 microM. The PGHS substrate, arachidonate, and various cyclooxygenase inhibitors do not alter this binding affinity of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhibit PGHS-2 by exploiting a previously unrecognized binding site on the enzyme.

Structure-activity relationships and pharmacokinetic analysis for a series of potent, systemically available biphenylsulfonamide matrix metalloproteinase inhibitors.

A series of biphenylsulfonamide derivatives of (S)-2-(biphenyl-4-sulfonylamino)-3-methylbutyric acid (5) were prepared and evaluated for their ability to inhibit matrix metalloproteinases (MMPs). For this series of compounds, our objective was to systematically replace substituents appended to the biphenyl and alpha-position of 5 with structurally diverse functionalities to assess the effects these changes have on biological and pharmacokinetic activity. The ensuing structure-activity relationship (SAR) studies showed that biphenylsulfonamides substituted with bromine in the 4'-position (11c) significantly improved in vitro activity and exhibited superior pharmacokinetics (C(max), t(1/2), AUCs), relative to compound 5. Varying the lipophilicity of the alpha-position by replacing the isopropyl group of 11c with a variety of substituents, in general, maintained potency versus MMP-2, -3, and -13 but decreased the oral systemic availability. Subsequent evaluation of its enantiomer, 11c', showed that both compounds were equally effective MMP inhibitors. In contrast, the corresponding hydroxamic acid enantiomeric pair, 16a (S-isomer) and 16a' (R-isomer), stereoselectivity inhibited MMPs. For the first time in this series, 16a' provided nanomolar potency against MMP-1, -7, and -9 (IC(50)'s = 110, 140, and 18 nM, respectively), whereas 16a was less potent against these MMPs (IC(50)'s = 24, 78, and 84 microM, respectively). However, unlike 11c, compound 16a' afforded very low plasma concentrations following a single 5 mg/kg oral dose in rat. Subsequent X-ray crystal structures of the catalytic domain of stromelysin (MMP-3CD) complexed with inhibitors from closely related series established the differences in the binding mode of carboxylic acid-based inhibitors (11c,c') relative to the corresponding hydroxamic acids (16a,a').

Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain.

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.

Synthesis, structure-activity relationships, and in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and orally active cyclooxygenase-2 inhibitors. 1. Thiazolone and oxazolone series.

Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). Initial mass screening and subsequent structure-activity relationship (SAR) studies have identified 4b (PD138387) as the most potent and selective COX-2 inhibitor within the thiazolone and oxazolone series of di-tert-butylphenols. Compound 4b has an IC50 of 1.7 microM against recombinant human COX-2 and inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.17 microM. It was inactive against purified ovine COX-1 at 100 microM and did not inhibit COX-1 activity in platelets at 20 microM. Compound 4b was also orally active in vivo with an ED40 of 16 mg/kg in the carrageenan footpad edema (CFE) assay and caused no gastrointestinal (GI) damage in rats at the dose of 100 mg/kg but inhibited gastric prostaglandin E2 (PGE2) production in rats' gastric mucosa by 33% following a dose of 100 mg/kg. The SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes. A simple isosteric replacement led to the reversal of selectivity.

Synthesis, structure-activity relationships, and in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and orally active cyclooxygenase-2 inhibitors. 2. 1,3,4- and 1,2,4-thiadiazole series.

Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had IC50 values of 0.14 and 100 microM against recombinant human COX-2 and purified ovine COX-1, respectively. It inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.18 microM and inhibited COX-1 activity in platelets with an IC50 of 3.1 microM. The choline salt of compound 6b was also orally active in vivo with an ED40 of 7. 1 mg/kg in the carrageenan footpad edema (CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI damage. SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes.

Effects of novel anti-inflammatory compounds on healing of acetic acid-induced gastric ulcer in rats.

Nonsteroidal anti-inflammatory drugs often cause development of significant GI lesions. Selective inhibitors of prostaglandin G/H synthase/cyclooxygenase-2 (PGHS-2) enzyme and some dual inhibitors of PGHS/5-lipoxygenase (5-LO) enzymes have been reported to be potent anti-inflammatory compounds that carry a much lower risk of having GI irritating effects. We have evaluated the anti-inflammatory effect and the GI safety profile of three new anti-inflammatory compounds: the selective PGHS-2 inhibitors NS-398 and PD 138387 and the PGHS/5-LO dual inhibitor PD 137968. All the compounds tested showed an anti-inflammatory activity in the carragenan footpad edema test in rats. None of these compounds caused either gastric damage 4 h after p.o. administration of 100 mg/kg in rats or inhibition of PGE2 synthesis in the stomach. However, when administered p.o. at an effective anti-inflammatory dose to rats with pre-existing acetic acid-induced gastric ulcer, NS-398 caused a statistically significant delay of ulcer healing. No impairment of the ulcer healing was observed with the other compounds evaluated. Derivatives of 2,6-di-tert-butylphenol, whose members may act as PGHS-1/PGHS-2 inhibitors, selective PGHS-2 inhibitors or PGHS/5-LO dual inhibitors, are novel anti-inflammatory compounds that are devoid of GI irritating effects and do not affect the rate of pre-existing gastric ulcer healing.

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

1. 15-Lipoxygenase (15-LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15-LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol-fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. 2. PD 146176 inhibited rabbit reticulocyte 15-LO through a mixed noncompetitive mode with a Ki of 197 nM. The drug had minimal effects on either copper or 2,2'-azobis(2-amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. 3. Control New Zealand rabbits were fed a high-fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg-1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nM). During the 12 week study, there were no significant differences in weight gain haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. 4. The drug plasma concentrations achieved in vivo did not inhibit low-density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176-treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. 5. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15 +/- 4 to 0% (P < 0.02); esterified cholesterol content was reduced from 2.1 +/- 0.7 to 0 micrograms mg-1 (P < 0.02) in this region. Immunostainable lipid-laden macrophages present in aortic intima of control animals were totally absent in the drug-treated group. 6. Results of these studies are consistent with a role for 15-LO in atherogenesis.

The role of arachidonic acid in the secretion of the amyloid precursor protein (APP).

We have studied the activation of human ml-muscarinic receptors in a genetically engineered Chinese hamster ovary cell line (CHO-ml) to determine which second messenger systems affect the secretion of APP via the non-amyloidogenic route. Carbachol activation of the signaling pathways in CHO-ml cells promotes APP secretion by activation of both protein kinase C (PKC)-dependent or Ca(++)-dependent second messenger pathways. Both pathways converge to increase the enzyme activity of phospholipase A2 (PLA2), the enzyme that releases arachidonic acid from cellular stores. Directly activating PLA2 with melittin, a peptide from bee venom, or by adding arachidonic acid directly to cultured cells increases the secretion of APP. Thus, our results indicate that arachidonic acid is yet another cellular second messenger involved in regulating the metabolism of APP in addition to PKC and cytoplasmic Ca++. Moreover, activation of PLA2 appears to be an obligatory event in increasing the secretion of APP from CHO-ml cells by the various methods of activation that we have tried thus far.

Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons.

Recent identification of a second, inducible form of prostaglandin H synthase (PGHS-2) led to the hypothesis that constitutively expressed PGHS (PGHS-1) is involved in the homeostatic role of eicosanoids, whereas the inducible enzyme is responsible for their inflammatory actions. We report here the purification of PGHS-2 from near-term sheep placental cotyledons. The PGHS-2 from this tissue was purified in multimilligram quantities by a combination of anion-exchange, size-exclusion, and affinity chromatography. This enzyme is different from ovine seminal vesicle PGHS-1 and was characterized as PGHS-2 based on (a) chromatographic properties, (b) immunochemical reactivities with isoenzyme-specific antibodies, (c) amino acid microsequencing, (d) kinetics of reaction with arachidonic acid (Km = 2.1 +/- 0.2 microM vs 8.3 +/- 0.2 microM for ovine PGHS-1), and (e) different sensitivities for several non-steroidal antiinflammatory drugs. Since the first identification of PGHS, ram seminal vesicles served as a rich source of the enzyme (PGHS-1). Our studies establish the sheep placental cotyledons as a rich natural source of PGHS-2.

Hydroxylamine analogs of 2,6-di-t-butylphenols: dual inhibitors of cyclooxygenase and 5-lipoxygenase or selective 5-lipoxygenase inhibitors.

The preparation of hydroxylamine analogs of 2,6-di-tert-butylphenols (DTBP) and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is discussed.

Quantitative structure-activity relationships of 5-lipoxygenase inhibitors. Inhibitory potency of pyridazinone analogues.

Quantitative structure-activity relationships (QSAR) of 72 1-phenyltetrahydropyridazin-3(2H)-one (I) analogues are examined for the inhibitory potency (IC50) of 5-lipoxygenase in a broken cell. The potency is increased by lipophilic substituents at the 3'- and 4'-positions. Substituents with positive F value at the 4'-position also increase the potency, while substituents at the 3'-position with a positive R value decrease it. The potency also decreases as the size of the 2'- and/or 4'-substituents increases. Thioketone analogues are about 5 times more potent than the corresponding carbonyl analogues.

The pharmacologic effects of 5-[3,5-bis(1,1-dimethylethyl)-4- hydroxyphenyl]-1,3,4-thiadiazole-2(3H)-thione, choline salt (CI-986), a novel inhibitor of arachidonic acid metabolism in models of inflammation, analgesia and gastric irritation.

CI-986 is a potent inhibitor of 5-lipoxygenase and cyclooxygenase pathway product biosynthesis from rat basophilic leukemia (RBL) cells. Because metabolites from these pathways have proinflammatory properties, CI-986 was evaluated in several acute and chronic models of inflammation and hyperalgesia. The compound inhibited swelling in the carrageenan footpad edema, Mycobacterium foot-pad edema and adjuvant arthritis models of inflammation with ID40 values of 1.0, 7.7., and 7.2 mg/kg, respectively. It was roughly equivalent in potency to the standard selective cyclooxygenase inhibitor, naproxen (ID40 = 0.7, 6.3, and 3.8 mg/kg, respectively). CI-986 was also evaluated in the acetic acid induced writhing hyperalgesia assay (ID50 = 0.23 mg/kg) and was approximately equipotent with indomethacin (ID50 = 0.87 mg/kg). Although the effects of CI-986 were similar to those of standard nonsteroidal antiinflammatory drugs (NSAIDs) in the inflammation models, its gastrointestinal profile was unique. CI-986 caused no gastrointestinal irritation at doses up to 200 mg/kg in acute and chronic studies. In contrast, standard NSAIDs caused ulcers at doses of 3.7-37 mg/kg after a single dose. Moreover, CI-986 inhibited the release of LTC4 and PGE2 by gastric mucosa and reduced mucosal and vascular damage induced by oral administration of absolute ethanol to rats. These results indicate that CI-986 is a potent nonulcerogenic antiinflammatory agent with novel pharmacologic properties.

Synthesis and biological evaluation of 5-[[3,5-bis(1,1-dimethylethyl)- 4-hydroxyphenyl]methylene]oxazoles, -thiazoles, and -imidazoles: novel dual 5-lipoxygenase and cyclooxygenase inhibitors with antiinflammatory activity.

A variety of benzylideneoxazoles, -thiazoles, and -imidazoles derived from 2,6-di-tert-butylphenol were prepared and evaluated as dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia (RBL-1) cells. The target compounds exhibit varying degrees of selectivity toward the two enzymes. Several compounds are orally active in the rat carageenan footpad edema (CFE) and mycobacterium footpad edema (MFE) antiinflammatory models. Structure-activity relationships are discussed. From this work, (Z)-5-[[3,5-bis(1,1-dimethylethyl)-4- hydroxyphenyl]-methylene]-2-imino-4-thiazolidinone methanesulfonate salt (CI-1004) was identified as a potent dual inhibitor of 5-lipoxygenase (IC50 = 0.77 microM) and cyclooxygenase (IC50 = 0.39 microM), with oral activity (ID40 = 0.6 mg/kg) in the rat MFE model of inflammation.

1,3,4-Oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole analogs of the fenamates: in vitro inhibition of cyclooxygenase and 5-lipoxygenase activities.

N-Arylanthranilic acids, known generically as the fenamates, are nonsteroidal antiinflammatory drugs (NSAIDs) that block the metabolism of arachidonic acid by the enzyme cyclooxygenase (CO). Substitution of the carboxylic acid functionality of several fenamates with acidic heterocycles provided dual inhibitors of CO and 5-lipoxygenase (5-LO) activities when tested in an intact rat basophilic leukemia (RBL-1) cell line. Compound 5b (IC50 = 0.77 microM (5-LO), 0.27 microM (CO)) which contains an 1,3,4-oxadiazole-2-thione replacement and 10b (IC50 = 0.87 microM (5-LO), 0.85 microM (CO)) which contains a 1,3,4-thiadiazole-2-thione are the most potent inhibitors of 5-LO and CO activities from these series. Both of these heterocyclic analogs of flufenamic acid are also active in carageenin-induced rat footpad edema (CFE), a model of acute inflammation. When dosed orally the ID50s for 5b and 10b in CFE are 8.5 and 4.7 mg/kg, respectively.

Design of 5-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,3,4-thiadiazoles, -1,3,4-oxadiazoles, and -1,2,4-triazoles as orally-active, nonulcerogenic antiinflammatory agents.

To discover dual inhibitors of 5-lipoxygenase (LO) and cyclooxygenase (CO) with improved pharmacokinetic properties, we have designed and synthesized series of 1,2,4-triazole, 1,3,4-oxadiazole, and 1,3,4-thiadiazole di-tert-butylphenol derivatives which exhibit a wide range of log P (2.3 to > 4) and pKa (5.5-12) values. From this work 5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)- thione, choline salt (12a, CI-986) was found to be a potent inhibitor of 5-LO (IC50 = 2.8 microM) and CO (IC50 = 0.8 microM), orally active in rat models of inflammation and nonulcerogenic.

Novel 1,2,4-oxadiazoles and 1,2,4-thiadiazoles as dual 5-lipoxygenase and cyclooxygenase inhibitors.

A series of 1,2,4-oxadiazoles and 1,2,4-thiadiazoles containing a 2,6-di-tert-butylphenol substituent were prepared and evaluated as dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia (RBL-1) cells. Several of these compounds show oral efficacy in the rat carrageenan footpad edema (CFE) and mycobacterium footpad edema (MFE) antiinflammatory models, without concomitant gastric ulceration. Structure-activity relationships are discussed. The best compounds (ID40 values in MFE of 3-8 mg/kg po) contain guanidine-derived substituents on the heterocyclic ring.

4-hydroxythiazole inhibitors of 5-lipoxygenase.

4-Hydroxythiazoles have been identified as potent inhibitors of 5-lipoxygenase in vitro exhibiting IC50's of less than 1 microM. An investigation of structure-activity relationships showed that the most potent inhibitors of this series are the 5-phenyl derivatives. The corresponding thiazolidin-4-one analogues were found to be relatively inactive. The 4-hydroxythiazoles were active inhibitors against 5-lipoxygenase in both intact rat polymorphonuclear leukocytes and human whole blood. The compounds were also selective inhibitors of 5-lipoxygenase, displaying only weak activity against other related enzymes, cyclooxygenase and 12- and 15-lipoxygenase.