Design,Synthesis and Biological Evaluation of New N-Acyl Hydrazones with a Methyl Sulfonyl Moiety as Selective COX-2 Inhibitors

The mechanism of action of nonsteroidal anti-inflammatory drugs (NSAIDs) is inhibition of specific prostaglandin (PG) synthesis by inhibition of cyclooxygenase (COX) enzymes. The two COX isoenzymes show 60 % similarity. It is known that the nonspecific side effects of conventional NSAIDs are physiologically caused by inhibition of the COX-1 enzyme. Therefore, the use of COX-2 selective inhibitors is seen to be a more beneficial approach in reducing these negative effects. However, some of the existing COX-2 selective inhibitors show cardiovascular side effects. Therefore, studies on the development of new selective COX-2 inhibitors remain necessary. It is important to develop new COX-2 inhibitors in the field of medicinal chemistry. Accordingly, novel N-acyl hydrazone derivatives were synthesized as new COX-2 inhibitors in this study. The hydrazone structure, also known for its COX activity, is important in terms of many biological activities and was preferred as the main structure in the design of these compounds. A methyl sulfonyl pharmacophore was added to the structure in order to increase the affinity for the polar side pocket present in the COX-2 enzyme. It is known that methyl sulfonyl groups are suitable for polar side pockets. The synthesis of the compounds ( 3a – 3j ) was characterized by spectroscopic methods. Evaluation of in vitro COX-1/COX-2 enzyme inhibition was performed by fluorometric method. According to the enzyme inhibition results, the obtained compounds displayed the predicted selectivity for COX-2 enzyme inhibition. Compound 3j showed important COX-2 inhibition with a value of IC₅₀=0.143 uM. Interaction modes between the COX-2 enzyme and compound 3j were investigated by docking studies.     

Cyclooxygenase-2 inhibitors: promise or peril?

The discovery of two isoforms of the cyclooxygenase enzyme, COX-1 and COX-2, and the development of COX-2-specific inhibitors as anti-inflammatories and analgesics have offered great promise that the therapeutic benefits of NSAIDs could be optimized through inhibition of COX-2, while minimizing their adverse side effect profile associated with inhibition of COX-1. While COX-2 specific inhibitors have proven to be efficacious in a variety of inflammatory conditions, exposure of large numbers of patients to these drugs in postmarketing studies have uncovered potential safety concerns that raise questions about the benefit/risk ratio of COX-2-specific NSAIDs compared to conventional NSAIDs. This article reviews the efficacy and safety profiles of COX-2-specific inhibitors, comparing them with conventional NSDAIDs.     

In vitro antiproliferative activity against human colon cancer cell lines of representative 4-thiazolidinones. Part I

The characterization of two cyclooxygenase isoforms (COX), the rate-limiting enzyme for the synthesis of prostaglandins (PGs) from arachidonic acid, has allowed the development of COX-2 selective inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs) with significant gastric tolerability. However, PGs are also important in cancer pathogenesis. Thus, there is an increasing interest in studying COX-2 inhibitors as potential drugs aimed at the prevention and treatment of cancer, especially colorectal cancer. The purpose of this study was to determine the inhibitory effects of some representative 4-thiazolidinones, already widely investigated as potential NSAIDs, on the growth of five human colon carcinoma cell lines with a different COX-2 expression, and to correlate them with COX-2 inhibitory properties. Our results preliminarily revealed that 2-phenylimino derivative 3 and 2,4-thiazolidindione 4 were the most active compounds. In particular, 3 mainly inhibited the HT29 cell line characterized by a high COX-2 expression, whereas 4 showed antiproliferative properties on all tested cell lines, suggesting molecular targets other than COX-2 inhibition.     

A novel genetic model of selective COX-2 inhibition: comparison with COX-2 null mice

Prostaglandin H Synthase (PGHS) is a bi-functional enzyme with a cyclooxygenase (COX) activity and a functionally linked peroxidase (POX) activity that exists in two isoforms (COX-1, COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs), including the selective COX-2 inhibitors, block COX activity while leaving POX activity unscathed. Recently, some selective COX-2 inhibitors were withdrawn from the market due to elevated cardiovascular risk in placebo-controlled trials. Mice deficient for PGHS2 were developed in 1995 and through numerous subsequent studies have revealed significant roles in renal development, ductus arteriosus patency/closure, skin carcinogenesis and cardiovascular function. In this short review, we compare a novel genetic COX-2 selective inhibition mouse model with the originally described COX-2 null mice in these different physiological functions.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

The role of the cylooxygenase pathway in nociception and pain

Cycloxygenase (COX) pathways have long been targeted for the treatment of inflammatory pain, initially through the use of NSAIDs. With the demonstration of two major COX isoforms, COX-1 and COX-2, involved in the production of prostanoids, but with different distribution and regulation, selective COX-2 inhibitors have been developed. This review covers factors influencing COX enzyme activity, the role of their products in the development and maintenance of pain and discusses recent safety concerns of COX-2 inhibitors.     

Cyclooxygenase isoforms and atherosclerosis

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of arthritis and pain. However, their long-term use is limited by gastrointestinal (GI) side effects such as gastric ulcers. NSAIDs act by inhibiting an enzyme called cyclooxygenase. Cyclooxygenase (COX) catalyses the generation of prostaglandins from arachidonic acid. Two isoforms of the enzyme exist--COX-1 and COX-2--both of which are targets for NSAIDs. Although they are associated with GI toxicity, NSAIDs have important antithrombotic and anti-inflammatory effects. The GI injury has been attributed to COX-1 inhibition and the anti-inflammatory effects to COX-2 inhibition. As COX-2 is traditionally viewed as an inducible enzyme, selective inhibition of COX-2 by 'coxibs' (selective COX-2 inhibitors) has been employed to achieve anti-inflammatory and analgesic effects without GI side effects. However, recently there have been suggestions that chronic administration of coxibs might increase the risk of cardiovascular events, such as atherosclerosis, compared with traditional NSAIDs. In vascular disease, there is increased expression of both COX-1 and COX-2, resulting in enhanced prostaglandin generation. The specific role of COX-1 and COX-2 in vascular regulation is still unknown but such knowledge is essential for the effective use of coxibs. Although more evidence is pointing to selective COX-1 inhibition as a therapeutic measure in inflammatory atherosclerosis, there are some studies that suggest that inhibition of COX-2 might have a potential benefit on atherosclerosis.     

Cardiovascular and gastrointestinal effects of COX-2 inhibitors and NSAIDs: achieving a balance

Conventional 'nonselective' nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation; however, the potential gastrointestinal risks associated with their use can be a cause for concern. In response to the adverse effects that can accompany nonselective NSAID use, selective cyclo-oxygenase (COX)-2 inhibitors were developed to target the COX-2 isoenzyme, thus providing anti-inflammatory and analgesic benefits while theoretically sparing the gastroprotective activity of the COX-1 isoenzyme. Data from large-scale clinical trials have confirmed that the COX-2 inhibitors are associated with substantial reductions in gastrointestinal risk in the majority of patients who do not receive aspirin. However, some or all of the gastrointestinal benefit of COX-2 inhibitors may be lost in patients who receive low, cardioprotective doses of aspirin, and recent evidence suggests that some of these agents, at some doses, may be associated with an increased risk for cardiovascular adverse events compared with no therapy. The risks and benefits of conventional NSAIDs and of COX-2 inhibitors must be weighed carefully; in clinical practice many patients who might benefit from NSAID or COX-2 therapy are likely to be elderly and at relatively high risk for gastrointestinal and cardiovascular adverse events. These patients are also more likely to be taking low-dose aspirin for cardiovascular prophylaxis and over-the-counter NSAIDs for pain. Identifying therapies that provide relief from arthritis related symptoms, confer optimum cardioprotection, and preserve the gastrointestinal mucosa is complex. Factors to consider include the interference of certain NSAIDs with the antiplatelet effects of aspirin, differences in the adverse gastrointestinal event rates among nonselective NSAIDs and selective COX-2 inhibitors, emerging data regarding the relative risks for cardiovascular events associated with these drugs, and the feasibility and cost of co-therapy with proton pump inhibitors.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Targeting PGE2 receptor subtypes rather than cyclooxygenases: a bridge over troubled water?

Prostaglandins (PG) are synthesized by the sequential action of phosholipases, cyclooxygenases (COX)-1 and COX-2, and specific terminal synthases, and exert their diverse biological effects through several membrane receptors. In particular, PGE2 is involved in many normal and pathological pathways that are mediated by four different E prostanoid receptors (EP1-4). Selective COX-2 inhibitors (Coxibs) have analgesic and antipyretic effects that are indistinguishable from those of nonsteroidal anti-inflammatory drugs (NSAIDs), but some possess hazardous cardiovascular side effects. Recent results indicate that EP1 and EP4 antagonists might prove useful for inhibiting the unwanted actions of COX-2. Has the time come for research to examine earnestly the selective antagonism of EP subtypes rather than further the development of direct COX-2 inhibitors?     

Fluorescence quenching analysis of the association and dissociation of a diarylheterocycle to cyclooxygenase-1 and cyclooxygenase-2: dynamic basis of cyclooxygenase-2 selectivity

Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.     

Mediators of PGE2 synthesis and signalling downstream of COX-2 represent potential targets for the prevention/treatment of colorectal cancer

Colorectal cancer is a major cause of mortality and whilst up to 80% of sporadic colorectal tumours are considered preventable, trends toward increasing obesity suggest the potential for a further increase in its worldwide incidence. Novel methods of colorectal cancer prevention and therapy are therefore of considerable importance. Non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive against colorectal cancer, mainly through their inhibitory effects on the cyclooxygenase isoform COX-2. COX enzymes represent the committed step in prostaglandin biosynthesis and it is predominantly increased COX-2-mediated prostaglandin-E2 (PGE2) production that has a strong association with colorectal neoplasia, by promoting cell survival, cell growth, migration, invasion and angiogenesis. COX-1 and COX-2 inhibition by traditional NSAIDs (for example, aspirin) although chemopreventive have some side effects due to the role of COX-1 in maintaining the integrity of the gastric mucosa. Interestingly, the use of COX-2 selective NSAIDs has also shown promise in the prevention/treatment of colorectal cancer while having a reduced impact on the gastric mucosa. However, the prolonged use of high dose COX-2 selective inhibitors is associated with a risk of cardiovascular side effects. Whilst COX-2 inhibitors may still represent viable adjuvants to current colorectal cancer therapy, there is an urgent need to further our understanding of the downstream mechanisms by which PGE2 promotes tumorigenesis and hence identify safer, more effective strategies for the prevention of colorectal cancer. In particular, PGE2 synthases and E-prostanoid receptors (EP1-4) have recently attracted considerable interest in this area. It is hoped that at the appropriate stage, selective (and possibly combinatorial) inhibition of the synthesis and signalling of those prostaglandins most highly associated with colorectal tumorigenesis, such as PGE2, may have advantages over COX-2 selective inhibition and therefore represent more suitable targets for long-term chemoprevention. Furthermore, as COX-2 is found to be overexpressed in cancers such as breast, gastric, lung and pancreatic, these investigations may also have broad implications for the prevention/treatment of a number of other malignancies.     

Mediators of PGE2 synthesis and signalling downstream of COX-2 represent potential targets for the prevention/treatment of colorectal cancer

Colorectal cancer is a major cause of mortality and whilst up to 80% of sporadic colorectal tumours are considered preventable, trends toward increasing obesity suggest the potential for a further increase in its worldwide incidence. Novel methods of colorectal cancer prevention and therapy are therefore of considerable importance. Non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive against colorectal cancer, mainly through their inhibitory effects on the cyclooxygenase isoform COX-2. COX enzymes represent the committed step in prostaglandin biosynthesis and it is predominantly increased COX-2-mediated prostaglandin-E₂ (PGE₂) production that has a strong association with colorectal neoplasia, by promoting cell survival, cell growth, migration, invasion and angiogenesis. COX-1 and COX-2 inhibition by traditional NSAIDs (for example, aspirin) although chemopreventive have some side effects due to the role of COX-1 in maintaining the integrity of the gastric mucosa. Interestingly, the use of COX-2 selective NSAIDs has also shown promise in the prevention/treatment of colorectal cancer while having a reduced impact on the gastric mucosa. However, the prolonged use of high dose COX-2 selective inhibitors is associated with a risk of cardiovascular side effects. Whilst COX-2 inhibitors may still represent viable adjuvants to current colorectal cancer therapy, there is an urgent need to further our understanding of the downstream mechanisms by which PGE₂ promotes tumorigenesis and hence identify safer, more effective strategies for the prevention of colorectal cancer. In particular, PGE₂ synthases and E-prostanoid receptors (EP1–4) have recently attracted considerable interest in this area. It is hoped that at the appropriate stage, selective (and possibly combinatorial) inhibition of the synthesis and signalling of those prostaglandins most highly associated with colorectal tumorigenesis, such as PGE₂, may have advantages over COX-2 selective inhibition and therefore represent more suitable targets for long-term chemoprevention. Furthermore, as COX-2 is found to be overexpressed in cancers such as breast, gastric, lung and pancreatic, these investigations may also have broad implications for the prevention/treatment of a number of other malignancies.     

Role of cyclooxygenase isoforms in gastric mucosal defence.

A complex system of interacting mediators exists in the gastric mucosa to strengthen its resistance against injury. In this system prostaglandins play an important role. Prostaglandin biosynthesis is catalysed by the enzyme cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. Initially the concept was developed that COX-1 functions as housekeeping enzyme, whereas COX-2 yields prostaglandins involved in pathophysiological reactions such as inflammation. In the gastrointestinal tract, the maintenance of mucosal integrity was attributed exclusively to COX-1 without a contribution of COX-2 and ulcerogenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) were believed to be the consequence of inhibition of COX-1. Recent findings, however, indicate that both COX-1 and COX-2 either alone or in concert contribute to gastric mucosal defence. Thus, in normal rat gastric mucosa specific inhibition of COX-1 does not elicit mucosal lesions despite near-maximal suppression of gastric prostaglandin formation. When a selective COX-2 inhibitor which is not ulcerogenic when given alone is added to the COX-1 inhibitor, severe gastric damage develops. In contrast to normal gastric mucosa which requires simultaneous inhibition of COX-1 and COX-2 for breakdown of mucosal resistance, in the acid-challenged rat stomach inhibition of COX-1 alone results in dose-dependent injury which is further increased by additional inhibition of COX-2 enzyme activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. COX-2 inhibitors do not damage the normal or acid-challenged gastric mucosa when given alone. However, when nitric oxide formation is suppressed or afferent nerves are defunctionalized, specific inhibition of COX-2 induces severe gastric damage. Ischemia-reperfusion of the gastric artery is associated with up-regulation of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone augment ischemia-reperfusion-induced gastric damage up to four-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE(2). Selective inhibition of COX-1 is less effective. Furthermore, COX-2 inhibitors antagonize the protective effect of a mild irritant or intragastric peptone perfusion in the rat stomach, whereas the protection induced by chronic administration of endotoxin is mediated by COX-1. Finally, an important function of COX-2 is the acceleration of ulcer healing. COX-2 is up-regulated in chronic gastric ulcers and inhibitors of COX-2 impair the healing of ulcers to the same extent as non-selective NSAIDs. Taken together, these observations show that both COX isoenzymes are essential factors in mucosal defence with specific contributions in various physiological and pathophysiological situations.     

Synthesis and evaluation of carbaborane derivatives of indomethacin as cyclooxygenase inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological activities by inhibiting cyclooxygenase (COX)-1 and COX-2. Previous studies have shown that esters and amides of non-selective inhibitors such as indomethacin are selective against COX-2, which is the therapeutically relevant isoform. Structure-activity analysis indicates that substituted phenyl rings are tolerated as ester components. In the present study, the introduction of inorganic ortho- and meta-carbaborane moieties was explored with the aim to create COX-2 inhibitors and more importantly to investigate the validity of using these boron clusters as drug entities. Interestingly, only the ortho-carbaborane ester was active whereas the meta isomer was not. A similar lack of inhibitory potency was observed when an adamantyl substituent or alkylene spacers at the carbaborane were introduced in the ester functionality.     

Computer-aided, rational design of a potent and selective small peptide inhibitor of cyclooxygenase 2 (COX2)

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors. Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX1 and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Inhibition of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) by cyclooxygenase inhibitors and chemopreventive agents

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. This enzyme is markedly induced by androgens in hormone-sensitive human prostate cancer cells (Tong M., Tai H. H. Biochem Biophys Res Commun 2000; 276: 77-81) and may be involved in tumorigenesis. Inhibition of this enzyme may be of value in anticancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenases (COXs) have been shown to be chemopreventive in epidemiological and animal-model studies. However, chemoprevention by these drugs may not be directly related to their inhibition of COXs. Other targets may be also involved in their chemopreventive activity. We have examined a variety of NSAIDs including COX-2 selective inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists and phytophenolic compounds which have been shown to be chemopreventive for their effect on 15-PGDH. It was found that most of these compounds were potent inhibitors of 15-PGDH. Among these compounds, ciglitazone appeared to be the most powerful inhibitor (IC(50)=2.7 microM). Inhibition by ciglitazone was non-competitive with respect to NAD(+) and uncompetitive with respect to PGE(2).     

Current state of therapy for pain and inflammation

Nonsteroidal anti-inflammatory drugs (NSAIDs), including both traditional nonselective NSAIDs and the selective cyclo-oxygenase (COX)-2 inhibitors, are among the most widely used medications in the USA. Traditional NSAIDs, although effective at relieving pain and inflammation, are associated with a significant increase in the risk for gastrointestinal adverse events. Throughout the 1990s these events were estimated to result in approximately 100,000 hospitalizations and 16,500 deaths each year nationally. Recent studies have indicated that the risk for serious NSAID gastropathy has declined substantially during the past decade as a result of a number of factors, including lower doses of NSAIDs, the use of gastroprotective agents such as proton pump inhibitors and misoprostol, and the introduction of the selective COX-2 inhibitors. One therapeutic approach that may reduce the risk for gastrointestinal side effects associated with traditional NSAIDs while retaining their efficacy is the inclusion of co-therapy with a proton pump inhibitor; these agents inhibit acid secretion and have been demonstrated to promote ulcer healing in patients with NSAID-related gastric ulcers. Alternatively, COX-2 selective agents have been used to treat patients at high risk for such events. Both nonselective and selective COX-2 inhibitors have now been shown to be associated with an increased risk for cardiovascular events. These studies, together with the outcomes of the recent US Food and Drug Administration decision to require 'black box' warnings regarding potential cardiovascular risks associated with NSAIDs, suggest that the use of COX-2 inhibitors as the sole strategy for gastroprotection in patients with arthritis and other pain syndromes must be reconsidered, particularly among those at risk for cardiovascular events.     

Surface plasmon resonance studies and biochemical evaluation of a potent peptide inhibitor against cyclooxygenase-2 as an anti-inflammatory agent

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are mediators of inflammation [D.L. Dewitt, W.L. Smith, Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence, Proc. Natl. Acad. Sci. USA 85 (1988) 1412-1416, 1]. It exists mainly in two isoforms COX-1 and COX-2 [A. Raz, A. Wyche, N. Siegel, P. Needleman, Regulation of fibroblast cyclooxygenase synthesis by interleukin-1, J. Biol. Chem. 263 (1988) 3022-3028, 2]. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) have adverse gastrointestinal side-effects, because they inhibit both isoforms [T.D. Warner, F. Guiliano, I. Vojnovic, A. Bukasa, J.A. Mitchell, J.P. Vane, Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis, Proc. Natl. Acad. Sci. USA 96 (1999) 7563-7568, 3; L.J. Marnett, A.S. Kalgutkar, Cyclooxygenase 2 inhibitors: discovery, selectivity and the future, Trends Pharmacol. Sci. 20 (1999) 465-469, 4; J.R. Vane, NSAIDs, Cox-2 inhibitors, and the gut, Lancet 346 (1995) 1105-1106, 5]. Therefore drugs which selectively inhibit COX-2, known as coxibs were developed. Recent reports on the harmful cardiovascular and renovascular side-effects of the anti-inflammatory drugs have led to the quest for a novel class of COX-2 selective inhibitors. Keeping this in mind, we have used the X-ray crystal structures of the complexes of the COX-1 and COX-2 with the known inhibitors for a rational, structure based approach to design a small peptide, which is potent inhibitor for COX-2. The peptides have been checked experimentally by in-vitro kinetic studies using surface plasmon resonance (SPR) and other biochemical methods. We have identified a tripeptide inhibitor which is a potential lead for a new class of COX-2 inhibitor. The dissociation constant (K(D)) determined for COX-2 with peptide WCS is 1.90x10(-10)M, the kinetic constant (K(i)) determined by spectrophotometry is 4.85x10(-9)M and the IC(50) value is 1.5x10(-8)M by ELISA test.     

Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC₅₀ values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.