Selective cyclooxygenase-2 inhibitors: heteroaryl modified 1,2-diarylimidazoles are potent, orally active antiinflammatory agents.

A series of heteroaryl modified 1,2-diarylimidazoles has been synthesized and found to be potent and highly selective (1000-9000-fold) inhibitors of the human COX-2. 3-Pyridyl derived COX-2 selective inhibitor (25) exhibited excellent activity in acute (carrageenan induced paw edema, ED(50) = 5.4 mg/kg) and chronic (adjuvant induced arthritis, ED(50) = 0.25 mg/kg) models of inflammation. The relatively long half-life of 25 in rat and dog prompted investigation of the pyridyl and other heteroaromatic systems containing potential metabolic functionalities. A number of substituted pyridyl and thiazole containing compounds (e.g., 44, 46, 54, 76, and 78) demonstrated excellent oral activity in every efficacy model evaluated. Several orally active diarylimidazoles exhibited desirable pharmacokinetics profiles and showed no GI toxicity in the rat up to 100 mg/kg in both acute and chronic models. The paper describes facile and practical syntheses of the targeted diarylimidazoles. The structure-activity relationships and antiinflammatory properties of a series of diarylimidazoles are discussed.

Pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib in rats.

The pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide, a cyclooxygenase-2 inhibitor, were investigated in rats. Celecoxib was metabolized extensively after i.v. administration of [(14)C]celecoxib, and elimination of unchanged compound was minor (less than 2%) in male and female rats. The only metabolism of celecoxib observed in rats was via a single oxidative pathway. The methyl group of celecoxib is first oxidized to a hydroxymethyl metabolite, followed by additional oxidation of the hydroxymethyl group to a carboxylic acid metabolite. Glucuronide conjugates of both the hydroxymethyl and carboxylic acid metabolites are formed. Total mean percent recovery of the radioactive dose was about 100% for both the male rat (9.6% in urine; 91.7% in feces) and the female rat (10.6% in urine; 91.3% in feces). After oral administration of [(14)C]celecoxib at doses of 20, 80, and 400 mg/kg, the majority of the radioactivity was excreted in the feces (88-94%) with the remainder of the dose excreted in the urine (7-10%). Both unchanged drug and the carboxylic acid metabolite of celecoxib were the major radioactive components excreted with the amount of celecoxib excreted in the feces increasing with dose. When administered orally, celecoxib was well distributed to the tissues examined with the highest concentrations of radioactivity found in the gastrointestinal tract. Maximal concentration of radioactivity was reached in most all tissues between 1 and 3 h postdose with the half-life paralleling that of plasma, with the exception of the gastrointestinal tract tissues.

Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benze nesulfonamide (SC-58635, celecoxib).

A series of sulfonamide-containing 1,5-diarylpyrazole derivatives were prepared and evaluated for their ability to block cyclooxygenase-2 (COX-2) in vitro and in vivo. Extensive structure-activity relationship (SAR) work was carried out within this series, and a number of potent and selective inhibitors of COX-2 were identified. Since an early structural lead (1f, SC-236) exhibited an unacceptably long plasma half-life, a number of pyrazole analogs containing potential metabolic sites were evaluated further in vivo in an effort to identify compounds with acceptable pharmacokinetic profiles. This work led to the identification of 1i (4-[5-(4-methylphenyl)-3-(trifluoromethyl)- H-pyrazol-1-yl]benzenesulfonamide, SC-58635, celecoxib), which is currently in phase III clinical trials for the treatment of rheumatoid arthritis and osteoarthritis.

Diarylspiro[2.4]heptenes as orally active, highly selective cyclooxygenase-2 inhibitors: synthesis and structure-activity relationships.

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure-activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

1,2-Diarylcyclopentenes as selective cyclooxygenase-2 inhibitors and orally active anti-inflammatory agents.

A series of 1,2-diarylcyclopentene methyl sulfones and sulfonamides have been shown to be remarkably potent and selective cyclooxygenase-2 (COX-2) inhibitors. The methyl sulfone analogs 7 showed excellent COX-2 activity, with IC50s ranging from 0.003 (7f,n) to 0.87 (7o) microM. In addition, most analogs of 7 showed no activity (IC50 > 100 microM) against the COX-1 enzyme. Replacement of the methyl sulfone moiety with a sulfonamide group gave a slightly more potent (typically 2-5-fold) but less selective COX-2 inhibitor, mainly due to an increase (20- > 100-fold) in COX-1 activity. However, in vitro COX-1/COX-2 selectivity for the sulfonamides 8 could be increased in many cases by simply incorporating a substituent at the 3-position of the phenyl group. Furthermore, in vitro selectivity increased with the size and number of substituents, as demonstrated in the selectivity trend of 8k (8000) > 8j (1900) > 8i (500) > 8h (100). More importantly, the sulfonamide COX-2 inhibitors showed greatly enhanced oral activity in the rat model of established adjuvant-induced arthritis, with inhibition values of 79.0% (8a), 81.5% (8c), and 83.0% (8g) at 1 mg/kg. On the basis of its overall biological profile, sulfonamide 8c was evaluated as a potential clinical candidate, displaying an ED50 of 22 mpk in the rat carrageenan-induced paw edema model and an ED50 of 0.16 mpk in the rat established adjuvant-induced arthritis model with no indication of gastrointestinal toxicity in rats and mice at 200 mpk. In addition, a preparative-scale synthetic route to sulfonamide 8c has been developed.

Sulphation of proteochondroitin and 4-methylumbelliferyl beta-D-xyloside-chondroitin formed by mouse mastocytoma cells cultured in sulphate-deficient medium.

Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.

A model of human small intestinal absorptive cells. 1. Transport barrier.

The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37 degrees C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, or alanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.

Physiological and cytotoxic effects of Ca(2+) ionophores on Caco-2 paracellular permeability: relationship of 45Ca(2+) efflux to 51 Cr release.

The human intestinal cell line, Caco-2, and the Ca2+ ionophores, A23187 and ionomycin, were used to determine the interrelationships of 45Ca(2+) efflux, transepithelial electrical resistance (Rt), and [3H]-mannitol flux to changes in 51Cr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 mumol/l showed similar 45Ca(2+) efflux rate constants and coefficients. Analysis of the control and ionomycin-induced 45Ca(2+) efflux values showed the data to best fit a three Ca(2+) compartmental model. All changes in Caco-2 Rt and [3H]-mannitol flux were reversible with no significant increases in 51Cr release with ionomycin concentrations of less than or equal to 2.5 mumols/l. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 mumols/l showed rapid non-exponential 45Ca(2+) effluxes with irreversible changes in Rt, [3H]-mannitol flux, and significant increases in 51Cr release. There was no changes in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 mumols/l for 60 min. The results of our study show that: (1) disruption of Ca(2+) homeostasis in Caco-2 cells will occur with the addition of Ca(2+) ionophores at concentrations of greater than 2.50 mumols/l; (2) high concentrations (greater than 2.5 mumols/l) of ionomycin will cause non-exponential 45Ca(2+) efflux rates with irreversible changes in intracellular Rt and 14C-mannitol flux, and (3) early signs of Ca(2+) ionophore-induced damage can be detected by 51Cr release from Caco-2 cells into the media and not by changes in LDH media activity.

Increased proteoglycan synthesis following the differentiation of F9 embryonal carcinoma cells: formation of a differentiation-specific proteoheparan sulfate.

We have examined changes in proteoglycan synthesis by F9 embryonal carcinoma cells after the cells have been treated with retinoic acid or retinoic acid plus cholera toxin. Retinoic acid is known to stimulate the differentiation of this cell type to a primitive endoderm-like cell characterized by the production of basement membrane components such as type IV collagen, laminin and proteoglycans. We have now demonstrated that proteoglycan synthesis and secretion were further stimulated when cholera toxin was added in addition to retinoic acid. Moreover, media of these fully differentiated cells was found to contain a different species of proteoheparan sulfate not produced by stem cells or retinoic acid-treated cells. This proteoheparan sulfate had a high density upon CsCl gradient centrifugation. The protein core of this proteoheparan sulfate was estimated by SDS gel electrophoresis to be approximately 15,000 daltons.

Simultaneous sulfation of endogenous chondroitin sulfate and chondroitin-derived oligosaccharides. Studies with separate 4-sulfating and 6-sulfating microsomal systems.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage and from chondroitin 4-sulfate-producing mouse mastocytoma cells were incubated with varying concentrations of 3'-phosphoadenylylphospho[35S]sulfate and chondroitin hexasaccharide in the presence or absence of Triton X-100. [35S]Sulfate incorporation into hexasaccharide and into endogenous microsomal chondroitin 6-sulfate or endogenous microsomal chondroitin 4-sulfate was measured. With both microsomal systems, Triton X-100 increased the incorporation of [35S]sulfate into hexasaccharide but had much less effect on the incorporation into endogenous chondroitin sulfate. Higher concentrations of hexasaccharide inhibited the incorporation of [35S]sulfate into endogenous chondroitin sulfate. The apparent Km for 3'-phosphoadenylylphosphosulfate for both the 6-sulfotransferase and 4-sulfotransferase with hexasaccharide and with endogenous chondroitin sulfate in the presence or absence of Triton X-100 were all similar. However, the apparent Km for hexasaccharide was lower in the presence of Triton X-100 for both the microsomal sulfotransferases. This is consistent with solubilization of sulfotransferases, and indicates that hexasaccharide access to these enzymes had been limited in the particulate system. Examination of 35S-oligosaccharide products formed with each system demonstrated good 6-sulfation or 4-sulfation of penultimate GalNAc at the non-reducing end of the chondroitin hexasaccharide. However, no 6-sulfation of terminal GalNAc at the non-reducing end of a chondroitin pentasaccharide derived from hexasaccharide was observed, and there was only minimal 4-sulfation of this terminal GalNAc. Concurrent addition of GalNAc to hexasaccharide resulting in heptasaccharide did not appear to promote significant 6-sulfation or 4-sulfation of newly added non-reducing terminal GalNAc.

The effect of penultimate N-acetylgalactosamine 4-sulfate on chondroitin chain elongation.

Previous work has shown that odd-numbered oligosaccharides containing nonreducing terminal, non-sulfated N-acetylgalactosamine (GalNAc) or 6-sulfated GalNAc are excellent acceptors for enzymic addition of glucuronic acid (GlcA). However, the presence of a 4-sulfated GalNAc group blocks further addition. We have now used even-numbered oligosaccharides (a mixture of 4-sulfated, 6-sulfated, and non-sulfated) as acceptors of [3H]GalNAc to investigate the effect of sulfate residues on the GalNAc in the penultimate position. 3H-Labeled oligosaccharides were partially degraded with chondroitin AC lyase. The labeled trisaccharides, consisting of the added [3H]GalNAc and the nonreducing terminal disaccharides of the oligosaccharide acceptors, were then characterized. Both non-sulfated and mono-sulfated 3H-trisaccharides were observed. However, the 3H-trisaccharides were shown by chromatography with prepared standards to be non-sulfated or to be sulfated only at the 6-position. Thus the oligosaccharides containing a 4-sulfated, penultimate GalNAc at the nonreducing end did not serve as acceptors for [3H]GalNAc. Microsomes from mastocytoma cells, which make only chondroitin 4-sulfate, exhibited the same substrate specificity for exogenous oligosaccharide acceptors as did microsomes from chick cartilage which makes chondroitin 6-sulfate.