Synthesis and anti-inflammatory activity of alkyl/arylidene-2-aminobenzothiazoles and 1-benzothiazol-2-yl-3-chloro-4-substituted-azetidin-2-ones

Ten new derivatives of 1-benzothiazol-2-yl-3-chloro-4-substituted-azetidin-2-ones (3a-j) were synthesized using various Schiff bases (alkyl/arylidene-2-aminobenzothiazoles; 2a-j), which in turn were prepared starting from 2-aminobenzothiazole (1). All the synthesised compounds were characterised by elemental analyses and spectral (IR, 1H-NMR, 13C-NMR and EI-MS) data. The title compounds 2a-j and 3a-j were screened in vivo using carrageenan-induced rat paw edema model. All the test compounds showed anti-inflammatory activity when tested in vivo. In general, compounds 3a-j were found to be more potent compared to compounds 2a-j. Among the compounds tested, compound 2g in the alkyl/arylidene-2-aminobenzothiazoles series and compound 3 g in the 1-benzothiazol-2-yl-3-chloro-4-substituted-azetidin-2-ones series were found to be the most potent. All the test compounds were also evaluated to check the gastric ulcer incidence. In gastric ulceration studies, all the test compounds were generally found to be safe at the 100 mg/kg dose level. Furthermore the most potent compounds 2 g and 3 g from each series were screened in vitro for inhibition of both COX-2 and COX-1 catalysed prostaglandin biosynthesis (radiochemical assay). Like most of the commercially available non-steroidal anti-inflammatory drugs (NSAIDs), in the in vitro study, compounds 2 g and 3 g showed anti-inflammatory activity by blocking the metabolism of arachidonic acid to prostaglandin via the cyclooxygenase pathways. In general, in the vitro assay, test compounds 2 g and 3 g were found to be more active after 15 min pre-incubation with the enzyme. Compound 3 g was found to be more COX-2 selective, while compound 2 g was found to be equally COX-2 and COX-1 selective.     

Enlarging the NSAIDs family: ether, ester and acid derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents

The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.     

Synthesis and biological evaluation of 3,4-diaryloxazolones: A new class of orally active cyclooxygenase-2 inhibitors

A series of 3,4-diaryloxazolones were prepared and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2). Extensive structure-activity relationship work was carried out within this series, and a number of potent and selective COX-2 inhibitors were identified. The replacement of the methyl sulfone group on the 4-phenyl ring by a sulfonamide moiety resulted in compounds with superior in vivo antiinflammatory properties. In the sulfonamide series, the introduction of a methyl group at the 5-position of the oxazolone ring gave rise to very COX-2-selective compounds but with decreased in vivo activity. Selected 3,4-diaryloxazolones exhibited excellent activities in experimental models of arthritis and hyperalgesia. The in vivo activity of these compounds was confirmed with the evaluation of their antipyretic effectiveness and their ability to inhibit migration of proinflammatory cells. As expected from their COX-2 selectivity, most of the active compounds lacked gastrointestinal toxicity in vivo in rats after a 4-day treatment of 100 mg/kg/day. Within this novel series, sulfonamides 9-11 have been selected for further preclinical evaluation.     

3-[5-(4,5-dihydro-1H-imidazol-2-yl)-furan-2-yl]phenylamine (Amifuraline), a promising reversible and selective peripheral MAO-A inhibitor

On the basis of the observation that the central side effects of MAO inhibitors may represent a major limit for their use in pathological processes involving peripheral MAOs, we investigated the possibility of generating novel inhibitors able to target specifically peripheral MAOs. To address this issue, we designed compounds 7-28. From biological results, the 2-(5-phenyl-furan-2-yl)-4,5-dihydro-1H-imidazole (Furaline, 17) proved to be a suitable lead. In fact, in enzyme assays on homogenate preparation from rat liver and HEK cells expressing MAO-A or MAO-B, compounds possessing the frame of 17 behaved as selective and reversible MAO-A inhibitors. Interestingly, in in vivo studies the amino derivative 21 (Amifuraline), endowed with good hydrophilic character, was able to significantly inhibit liver but not brain MAO-A.     

Synthesis, biological evaluation, and enzyme docking simulations of 1,5-diarylpyrrole-3-alkoxyethyl ethers as selective cyclooxygenase-2 inhibitors endowed with anti-inflammatory and antinociceptive activity

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic ester moiety with an alkoxyethyl group still led to new, highly selective and potent COX-2 inhibitors. In the in vitro cell culture assay, all the compounds proved to be potent and selective COX-2 inhibitors. In the human whole blood (HWB) assay, compound 8a had a comparable COX-2 selectivity to valdecoxib, while it was more selective than celecoxib but less selective than rofecoxib. The potential anti-inflammatory and antinociceptive activities of compounds 7a, 8a, and 8d were evaluated in vivo, where they showed a very good activity against both carrageenan-induced hyperalgesia and edema in the rat paw test. In the abdominal constriction test compound 7a, 8a, and 8d were able to reduce the number of writhes in a statistically significant manner. Furthermore, the affinity data of these compounds have been rationalized through enzyme docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site by means of the software package Autodock 3.0.5, GRID 21, and MacroModel 8.5 using the complex between COX-2 and SC-558 (1b), refined at a 3 A resolution (Brookhaven Protein Data Bank entry: 6cox ).     

Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.     

Pharmacophore elucidation and molecular docking studies on 5-phenyl-1-(3-pyridyl)-1h-1,2,4-triazole-3-carboxylic acid derivatives as COX-2 inhibitors

A set of 5-phenyl-1-(3-pyridyl)-1H-1,2,4-triazole-3-carboxylic acid derivatives (16a32) showing anti-inflammatory activity was analyzed using a three-dimensional qualitative structure-selectivity relationship (3D QSSR) method. The CatalystHipHop approach was used to generate a pharmacophore model for cyclooxygenase-2 (COX-2) inhibitors based on a training set of 15 active inhibitors (1a15). The degree of fitting of the test set compounds (16a32) to the generated hypothetical model revealed a qualitative measure of the more or less selective COX-2 inhibition of these compounds. The results indicate that most derivatives (16, 18, 20a25, and 30a32) are able to effectively satisfy the proposed pharmacophore geometry using energy accessible conformers (E(conf) < 20 kcal/mol). In addition, the triazole derivatives (16a32) were docked into COX-1 and COX-2 X-ray structures, using the program GOLD. Based on the docking results it is suggested that several of these novel triazole derivatives are active COX inhibitors with a significant preference for COX-2. In principle, this work presents an interesting, comprehensive approach to theoretically predict the mode of action of compounds that showed anti-inflammatory activity in an in vivo model.     

Alkylamino derivatives of 4-aminomethylpyridine as inhibitors of copper-containing amine oxidases

The first substratelike, reversible inhibitors of different copper amine oxidases (CAOs) with IC50 (M) as low as 2.0 x 10(-8) corresponding to derivatives of 4-aminomethylpyridine with alkoxy (1a-d), alkylthio (2a,b), and alkylamino (3a-e, 4a-j) groups in the positions 3 and 5 have been prepared and studied. The inhibitors 1a-d are active on benzylamine oxidase and semicarbazide-sensitive amine oxidase and are very selective with respect to diamine oxidase, lysyl oxidase, and monoamine oxidases. The inhibitors 2a,b are selective for benzylamine oxidase whereas 2a is also a new type of good substrate of diamine oxidase. The inhibitors 3a-e and 4a-j are substratelike, reversible, nonselective inhibitors of various CAOs including pea seedling amine oxidase and Hansenula polymorpha amine oxidase, whose enzymatic sites are known from X-ray structure determinations. The inhibitors 3b,c and 4b,c are excellent substratelike tools for studies correlating CAOs that afford crystals suitable for X-ray structure determinations with CAOs from mammals.     

Synthesis of nitro esters of prednisolone, new compounds combining pharmacological properties of both glucocorticoids and nitric oxide

Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development.     

Cyclooxygenase-2 inhibitors. 1,5-diarylpyrrol-3-acetic esters with enhanced inhibitory activity toward cyclooxygenase-2 and improved cyclooxygenase-2/cyclooxygenase-1 selectivity

The important role of cyclooxygenase-2 (COX-2) in the pathogenesis of inflammation and side effect limitations of current COX-2 inhibitor drugs illustrates a need for the design of new compounds based on alternative structural templates. We previously reported a set of substituted 1,5-diarylpyrrole derivatives, along with their inhibitory activity toward COX enzymes. Several compounds proved to be highly selective COX-2 inhibitors and their affinity data were rationalized through docking simulations. In this paper, we describe the synthesis of new 1,5-diarylpyrrole derivatives that were assayed for their in vitro inhibitory effects toward COX isozymes. Among them, the ethyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-[3-fluorophenyl]-1H-pyrrol-3-acetate (1d), which was the most potent and COX-2 selective compound, also showed a very interesting in vivo anti-inflammatory and analgesic activity, laying the foundations for developing new lead compounds that could be effective agents in the armamentarium for the management of inflammation and pain.     

Synthesis and structure-activity relationship of novel, highly potent metharyl and methcycloalkyl cyclooxygenase-2 (COX-2) selective inhibitors

A novel series of benzo-1,3-dioxolane metharyl derivatives was synthesized and evaluated for cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) inhibition in human whole blood (HWB). In the present study, structure-activity relationships (SAR) in the metharyl analogues were investigated. The spacer group and substitutions in the spacer group were found to be quite important for potent COX-2 inhibition. Compounds in which a methylene group (8a-c), carbonyl group (12a-c), or methylidene group (7a-c) connected cycloalkyl groups to the central benzo-1,3-dioxolane template were found to be potent and selective COX-2 inhibitors. Aryl-substituted compounds linked to the central ring by either a methylene or a carbonyl spacer resulted in potent, highly selective COX-2 inhibitors. In this series of substituted-(2H-benzo[3,4-d]1,3-dioxolan-5-yl))-1-(methylsulfonyl)benzene compounds, SAR studies demonstrated that substitution at the 3-position of the aryl group optimized COX-2 selectivity and potency, whereas substitution at the 4-position attenuated COX-2 inhibition. Mono- or difluoro substitution at meta position(s), as in 22c and 22h, was advantageous for both in vitro COX-2 potency and selectivity (e.g., COX-2 IC(50) for 22c = 1 microM and COX-1 IC(50) for 22c = 20 microM in HWB assay). Several novel compounds in the (2H-benzo[3,4-d]1,3-dioxolan-5-yl))-1-(methylsulfonyl)benzene series, as shown in structures 7c, 8a, 12a, 21c, 22c, 22e, and 22h, selectively inhibited COX-2 activity by 40-50% at a test concentration of 1 microM in an in vitro HWB assay.     

1,5-Diarylpyrrole-3-acetic acids and esters as novel classes of potent and highly selective cyclooxygenase-2 inhibitors

A small set of substituted 1,5-diarylpyrrole-3-acetic and -glyoxylic acid derivatives have been synthesized, and their cyclooxygenase (COX-1 and COX-2) inhibiting properties have been evaluated. Some compounds proved to be highly selective COX-2 inhibitors, and their affinity data have been rationalized through docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site.     

Design, synthesis and pharmacological evaluation of 4-[2-alkylthio-5（4）-（4- substitutedphenyl）imidazole-4（5）yl]benzenesulfonamides as selective COX-2 inhibitors

Aim： To design and synthesize a series of benzenesulfonamide derivatives, 4-[2- alkylthio-5 （4）-（4-substitutedphenyl）imidazole-4（5）-yl]benzenesul fonamides （4a-4j）, which are intended to act as cyclooxygenase-2 （COX-2） inhibitors with good COX-2 inhibitor activity, and which will exert anti-inflammatory activities in vivo. Methods： Benzenesulfonamide derivatives were designed and synthesized through multi-step chemical reactions. All the synthesized compounds were evaluated in an in vitro assay. The active compound 4a-4f was selected for further evaluation in a carrageenan-induced rat paw edema model. Results： Docking studies showed that compound 4 bind into the primary binding site of COX-2 with the sulfonamide SO2NH2 moiety interacting with the secondary pocket amino acid residues. In the in vitro assay, compound 4 inhibited COX-2 with an inhibition concentration IC50 value of 1.23-8 nmol/L, compared to celecoxib with IC50 value of 1.5 nmol/L. Compound 4b and 4c had good potency and selectivity in comparison to the celecoxib. In the in vivo model, compound 4a-4f exhibited a moderate potency to inhibit 50% carrageenan-induced paw edema with value of 1.58-4.3 mg/kg. In the latter experiment, compound 4c was the most active compound. Conclusion： The antiinflammatory effects obtained for compound 4a-4j could be due to the presence of fluorine or hydrogen substituents in the para position of the phenyl ring of these compounds.     

Synthesis and effects on the COX-1 and COX-2 activity in human whole blood ex vivo of derivatives containing the [1]benzothienol-[3, 2-d]pyrimidin-4-one heterocyclic system

Methyl and phenyl derivatives containing the [1]Benzothieno [3, 2-d]pyrimidin-4-one system have been synthesized and tested as inhibitors of COX-1 and COX-2 activities in human whole blood (HWB) ex vivo; all compounds turned out to be weak inhibitors of COX-1 activity, as deduced from the TXB(2) (thromboxane B) generation; the acid phenyl derivative 11 b was an interesting inhibitor of COX-2 activity, as deduced from the PGE(2) (prostaglandine E) generation.     

Synthesis of NO-donor bisphosphonates and their in-vitro action on bone resorption

A new class of bisphosphonates containing nitrooxy NO-donor functions has been developed. The products proved to display affinity for hydroxyapatite. Injection of (99m)Tc-labeled derivatives 11 and 18 into male rats showed a preferential accumulation of the compounds in bone as compared to blood and muscles. The products were found to inhibit the differentiation of pre-osteoclasts to functional osteoclasts induced by receptor activator of NF-kappaB ligand (RANKL), through a prevalent NO-dependent mechanism.     

Characterization of eltenac and novel COX-2 selective thiopheneacetic acid analogues in vitro and in vivo

We assessed the effect of novel selective thiopheneacetic acids on cyclooxygenase isoenzymes in vitro and in vivo. Thiopheneacetic acid Eltenac and derivatives were investigated in this study. In human whole blood experiments these derivatives were potent inhibitors of COX-2 (IC(50)=0.02-0.4 microM) with less pronounced effect on COX-1 (IC(50)=0.15-5.6 microM). With COX-1/COX-2 ratios between 7.5- and 16-fold they are in the range of Celecoxib (13-fold). The parent drug Eltenac demonstrated no selectivity for COX-2. In a rat paw edema model, these compounds showed reduction of edema volume in the range of 36-45% at 10 mg/kg (Eltenac 52%, Diclofenac 51%). However, the compounds were superior to Diclofenac and Eltenac with respect to their ulcerogenic and gastrointestinal properties. Introduction of a nitrate-ester moiety to either Eltenac or a derivative did neither improve selectivity or potency in vitro, nor ulcerogenicity in vivo. Molecular modeling of selective thiopheneacetic acid derivatives to the active site of human COX-2 suggested similar binding properties as Lumiracoxib and Diclofenac. In summary, modification of Eltenac generates moderately selective COX-2 drugs in the range of Celecoxib with respect to potency and selectivity. The drugs showed potent anti-inflammatory properties and significant improvement of animal survival in a sub-chronical experimental set up. Thiopheneacetic derivatives are characterized by low pK(a) values, short microsomal half-lives and binding mode to COX-2 similar to Diclofenac and Lumiracoxib. These properties may also have an impact on the transient inhibition of COX-2-dependent prostacyclin, thereby being less associated with vascular complications.     

Isatins inhibit cyclooxygenase-2 and inducible nitric oxide synthase in a mouse macrophage cell line

Isatin is a versatile compound with a diversity of effects. We designed to investigate the inhibitory effect of isatin derivatives on lipopolysaccharide/interferon-gamma-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, production of prostaglandin E(2) (PGE(2)), nitric oxide (NO), tumor necrosis factor (TNF-alpha), and their capacity to scavenge NO. Isatins inhibit TNF-alpha production and iNOS and COX-2 protein expression resulting on reduced levels of NO and PGE(2). Our results indicate isatin and it derivatives as inhibitors of iNOS and COX-2 enzymes, which might be used as anti-inflammatory and antitumoral agents.     

Synthesis and three-dimensional qualitative structure selectivity relationship of 3,5-disubstituted-2,4-thiazolidinedione derivatives as COX2 inhibitors

In our effort for synthesis of selective COX2 inhibitors, certain new 2,4-thiazolidinedione derivatives were synthesized. It necessitates preparation of potassium salt of 2,4-thiazolidinedione 2, which condensed with intermediate 4a. The resulting 3-[2-(4-methylphenyl)-2-oxo-l-phenylethyl]-2,4-thiazolidinedione 8 was condensed with appropriate aldehyde to afford compounds 10a, 10i-l, 10o and 10p. Compounds (9a-l, 10a-n, 10p, 11 and 12) were obtained through the preparation of 5-arylmethylidene-2,4-thiazolidinediones 6a-p and reaction of its potassium salt 7a-p with compounds 4a, 4b, and 5. Some compounds displayed significant analgesic activity as compared to reference standards. The anti-inflammatory activity of the synthesized compounds revealed that intermediate 8 and compounds 9c, 10c and 10d showed good results. Compound 10c produced no significant mucosal injury. HipHop methodology of Catalyst program was used to build up hypothetical model of selective COX2 inhibitors followed by fitting the synthesized compounds to this model. Compounds 10c and 10d were suspected to be promising selective COX2 inhibitors. Also, compounds (6c, 8, 9a,c,d,k, 10a,c,d,k, 11 and 12) were docked into COX1 and COX2 X-ray structures, using DOCK6 program. Docking results suggested that several of these derivatives are active COX inhibitors with a significant preference for COX2.     

Influence of 2-aryl-3-halogen/3-hydroxy-1,4-naphthoquinones with salicylic and cinnamic acid partial structures on the arachidonic acid cascade

Searching for more potent 5-lipoxygenase (LO) inhibitors one tert-butyl group of the selective 5-LO-inhibitor 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-naphthoquinone (1) was substituted by polar functions (-CHO, -COOH, -CH=CH-COOR, -CH2-CH2-COOR). At the same time the 5-LO selectivity of the new compounds within the arachidonic acid cascade was investigated. For this 12-LO- and COX-1-assays with activated human platelets were used. Screening the test compounds new selective 5-LO-inhibitors (4, 9 and 16) and a COX-1-inhibitor (10) as well as dual 5-LO/COX-1- (23) and 12-LO/COX-1- (12) inhibiting compounds were found. Obviously in this class of compounds 5-LO and 12-LO inhibition are mutually excluded for a structural reason. In addition to the well known 3-chloro- (19) and 3-bromo- (20) analogues of 1 the 3-fluoro- (22), 3-iodo- (23) and the 3-carbonitrile- (24) derivatives were synthesized. All 3-halogen compounds, except 23 and the nitrile, are potent non selective inhibitors of all three enzymes. Causative for this unselectivity is the inhibition of the arachidonic acid release by inhibition of the cytosolic phospholipase A2 (cPLA2) with the exception of 24.