5864 J. Med. Chem. 2009, 52, 5864–5871 DOI: 10.1021/jm900702b
Pyridine Analogues of Nimesulide: Design, Synthesis, and in Vitro and in Vivo Pharmacological Evaluation as Promising Cyclooxygenase 1 and 2 Inhibitors Jean-Franc-ois Renard,*,† Deniz Arslan,† Nancy Garbacki,‡ Bernard Pirotte,†,§ and Xavier de Leval†,§ †
Drug Research Center (CIRM), Laboratory of Medicinal Chemistry, University of Li ege, Avenue de l’H^ opital 1, B-4000 Li ege, Belgium, and Laboratory of Connective Tissues Biology, GIGA-R, University of Li ege, Avenue de l’H^ opital 3, B-4000 Li ege, Belgium. §The two last authors assumed equal supervision.
‡
Received May 25, 2009
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most prescribed medications, although the chronic use of such pharmacological agents is commonly associated with numerous side effects. The demonstration that the use of COX-2 selective or preferential inhibitors is associated with a better tolerability opened new horizons in the search of safer drugs for the management of inflammation. In the present study, we report the synthesis and the pharmacological evaluation of pyridine analogues of nimesulide, a COX-2 preferential inhibitor. The cyclooxygenases (COXs) inhibitory activities were evaluated in vitro using a human whole blood model. According to the in vitro results, a selection of compounds exhibiting moderate to high COX-2/COX-1 selectivity ratio (from weak COX-2 preferential inhibitors to compounds displaying a celecoxib-like selectivity profile) were further evaluated in vivo in a model of λ carrageenan-induced pleurisy in rats. Some of the selected compounds displayed similar or improved anti-inflammatory properties when compared to nimesulide and celecoxib.
Introduction Inflammation can be summarized as a complex process involving the release of several biochemical factors called inflammation mediators. These mediators can act both directly or synergistically in order to promote or maintain the inflammatory process. Among these mediators, prostanoids, lipidic mediators deriving from fatty acid metabolism, have been identified as key influencing mediators. Indeed, single inhibition of cyclooxygenases, the enzymes responsible for prostanoids biosynthesis, has been demonstrated for a long time to be a sufficient and an efficient strategy to reduce inflammation.1 Marketed COXsa inhibitors, whatever their chemical structures, are grouped together under the denomination of NSAIDs. They are widely used in the management of pain and acute inflammation but also in chronic inflammation states such as, for example, osteoarthritis and rheumatoid arthritis. Besides these therapeutic indications, the use of NSAIDs could be of interest in the prevention of several cancer types such as colon, lung, breast, prostate, and esophagus cancers.2-7 Cyclooxygenases also seem to be involved in the development of neurodegenerative diseases, especially Alzheimer’s and Parkinson’s diseases. However, although *To whom correspondence should be addressed. Phone: þ32-43664381. Fax: þ32-4-3664362. E-mail:
[email protected]. University of Liege Laboratory of Medicinal Chemistry 1, Avenue de l’H^ opital, tour 4(þ5), B-4000 Liege, Belgium. a Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; PGI2, prostacyclin; TXA2, thromboxane A2; CAI, carbonic anhydrase inhibitor; hCA, human carbonic anhydrase; DMSO, dimethylsulfoxide; RPMI, Roswell Park Memorial Institute culture medium; LPS, lipopolysaccharide; TXB2, thromboxane B2; PGE2, prostaglandin E2.
pubs.acs.org/jmc
Published on Web 09/03/2009
some COXs inhibitors displayed interesting preclinical activity,8 they failed to demonstrate convincing therapeutic effect in clinical practice.9,10 Cyclooxygenases exist under two isoforms called COX-1 and COX-2. COX-1, the constitutive form of the enzyme, is present in the stomach, intestines, kidneys, and platelets. This form is mainly responsible for the physiological production of prostanoids. COX-2, although it is also constitutively expressed in brain, spinal cord,11 and kidneys, is an inducible form and its expression is triggered under pathological conditions such as inflammation. Most of the first-generation anti-inflammatory drugs inhibit both cyclooxygenase isoforms. This broad inhibition profile induces a decrease of inflammation but also downregulates the basal or physiological prostaglandin production in all tissues. This lack of selectivity represents the theoretical basis of their well-known gastric12,13 and renal toxicity.14 In this context, the use of COX-2 specific inhibitors appears as an attractive approach to manage inflammation in a safer way. This promising concept of COX-2 selective inhibition led to the development of a large number of new molecules. Among these, the well-known ones are rofecoxib (1),15 celecoxib (2),16 etoricoxib (3),17 and valdecoxib (4)18 (Figure 1). Clinical trials and clinical use of these agents confirmed that COX-2 specific inhibitors display anti-inflammatory, antipyretic, and analgesic properties with fewer gastrointestinal side effects.19 Nevertheless, an increased risk of cardiovascular events associated with the use of rofecoxib was demonstrated in two independent clinical trials: the Vioxx Gastrointestinal Outcomes Research (VIGOR)20 and the Adenomatous Polyps Prevention on Vioxx (APPROVe)21 trials. This finding led to the withdrawal of this compound from the market and reached r 2009 American Chemical Society
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Figure 1. Chemical structures of COX-2 inhibitors.
the question of a rofecoxib-related toxicity or a pharmacological class associated cardiovascular toxicity. The reasons for this toxicity are currently not fully understood, but different hypothesis were investigated.22 Among these, some only concern compound 1 and are rather related to its particular chemical structure while others concern the COX-2 selective inhibition. First, a drastic and selective inhibition of COX-2 is associated with a decrease of prostacyclin (PGI2) levels, an antiaggregant and vasodilator agent mainly synthesized through the COX-2 catalytic activity without suppressing the thromboxane A2 (TXA2) production, a strong pro-aggregant and vasoconstrictor mediator mainly produced through the COX-1 pathway. The modification of the physiological balance between TXA2/PGI2 may generate an alteration of the vascular homeostasis.23,24 Regarding the compound-related toxicity hypothesis, 1 does not bear a sulfonamide moiety as do compounds 2 and 4, which possess an unsubstituted arenesulfonamide group and for which the clinical use is not associated with an increased incidence of cardiovascular events. Moreover, this sulfonamide moiety is a common pattern to many carbonic anhydrase inhibitors (CAIs). Compounds 2 and 4 have been shown to present a high carbonic anhydrase inhibitory activity25,26 that was similar to that of acetazolamide, a human carbonic anhydrase (hCA) inhibitor marketed for its diuretic and antihypertensive properties. Crystallographic studies have also confirmed the ability of 2 to bind hCA-II. Finally, sulfone COX-2 inhibitors such as 1 and 3 increase the susceptibility of biological lipids to oxidative modification through a nonenzymatic process. This pro-oxidant activity could contribute to the progression of atherogenesis and could, at least in part, explain the increase of cardiovascular risk observed with 1.27-29 As a consequence, COX-2 selective inhibitors, despite their good tolerability and therapeutic activities, may contribute or promote a cardiovascular toxicity. Our group has been involved for several years in the development of nimesulide30 (5) derivatives, a NSAID exhibiting a COX-2 preferential profile.31 Of particular interest is the excellent tolerability profile of this drug with a lack of demonstrated cardiovascular toxicity, therefore suggesting that a “sufficient” COX-2/ COX-1 selectivity ratio may lead to safe anti-inflammatory agents. In a previous work, we synthesized a nimesulide analogue for which the nitrobenzenic ring was replaced by a pyridinic one (6; Table 1).32 This compound displayed a better COX-1 inhibitory activity than that of the parent compound but was almost inactive against COX-2. Several
Table 1. IC50 COX-1 and IC50 COX-2 of Previous Pyridinic Analogues of Nimesulide and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
IC50 (μM) compd
R
X
COX-1
COX-2
ratio
6 7 8 9
CH3 CF3 CH3 CF3
O O S NH
0.41 ( 0.13 0.14 ( 0.01 3.62 ( 0.90 0.18 ( 0.06
>100 0.62 ( 0.18 1.19 ( 0.58 0.09 ( 0.03
0.22 3.05 2.00
pharmacomodulations were investigated in order to increase the COX-2 inhibitory properties and to recover a COX-2 preferential profile. The acidic character of the alkanesulfonamide moiety was increased by introducing a trifluoromethanesulfonamide group, the phenyl ring was substituted with various halogen atoms or groups, and finally the replacement of the ether linkage by a thioether, a sulfone, and a secondary or tertiary amine bridge was evaluated (compounds 7-9; Table 1). From all of these modifications, N-(3-phenylamino-4-pyridinyl)trifluoromethanesulfonamide (FJ29),33 bearing a trifluoromethanesulfonamide moiety, a nonsubstituted phenyl ring, and a secondary amine linkage, appeared as a promising approach. Indeed, 9 (FJ29) presented a strong inhibitory activity on both cyclooxygenases with a COX-2/ COX-1 selectivity ratio equal to 2 (COX-1 and COX-2 IC50 values for compound 9: 0.18 and 0.09 μM, respectively).33 In the present study, we aimed at increasing the COX-2 preferential profile of compound 9 while retaining its COXs inhibitory activity. For this purpose, we have substituted the phenyl ring with various halogen atoms and some alkyl or alkoxy chains. The benzenic ring was also replaced with a variety of cycloalkyl rings. Finally, we have kept the possibility to modulate the acidic character of the sulfonamide moiety by introducing methane- and trifluoromethanesulfonamide moieties. The influence of the length of this group was also evaluated by increasing the size of the alkyl chain. Results and Discussion Chemistry. The synthetic pathway (Scheme 1) used to prepare the new compounds consists of a five-step process
5866 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Renard et al.
Scheme 1a
(i) H2O2, CH3COOH; (ii) HNO3, H2SO4; (iii a) NH2-C6H5-R1; (iii b) NH2-CH(CH2)n (n = 4, 5, 6); (iv) Fe, CH3COOH/H2O; (v) R2-SO2Cl, K2CO3, CH3CN. a
starting from 3-bromopyridine (10). The synthesis of the common intermediate, 3-bromo-4-nitropyridine N-oxide (12), was achieved in two steps.34 First, 10 was oxidized by a mixture of acetic acid and hydrogen peroxide in order to generate in situ peracetic acid. This oxidation was followed by a nitration of 3-bromopyridine N-oxide (11) at the 4-position. This step was conducted in a nitric and sulfuric acid medium at 90 °C for 5 h. Compound 12 was obtained as a yellow solid. The next step consisted in the formation of the amine linkage by substitution of the bromine atom of 12 with the appropriate amine (13a-o, 18-20). The fourth step was the reduction of the nitro and the N-oxide moieties, which were simultaneously reduced using iron in an acetic acid and water medium. The corresponding aminopyridines (14a-o, 21-23) were obtained as oily compounds and were used without further purification. The final compounds were obtained by reaction between the aminopyridine derivatives and the suitable sulfonyl chloride. The inhibitory activity of the synthesized compounds against COX-1 and COX-2 was evaluated in vitro using a human whole blood model.35 During these experiments, the selectivity ratio toward COX-2 was calculated using the formula [IC50 COX-1 (μM)/IC50 COX-2 (μM)]. These in vitro studies enabled us to select compounds for further determination of their anti-inflammatory profile in a λ carrageenan-induced pleurisy model in rats.36 In Vitro COXs Inhibition Studies. The influence of the nature and the position of substitution on the phenyl ring on
the cyclooxygenases inhibitory activity was first evaluated. The results obtained with the newly synthesized compound bearing a trifluoromethanesulfonamide moiety are presented in Table 2. Compared to the unsubstituted compound with a trifluoromethanesulfonamide moiety (9), the substitution of the phenyl ring with a chlorine atom (compound 15a-c) led to compounds displaying drastic COXs inhibitory properties, although slightly weaker when compared to compound 9. Of particular interest was the finding that this weaker inhibitory profile mainly concerns the COX-1 activity when the chlorination is located at the 3- and the 4-position. As a consequence, compounds 15b and 15c exhibited an enhanced selectivity ratio when compared to compound 9 (COX-2 selectivity ratio of 4.10 and 2.81, respectively). The increase of steric hindrance and lipophilicity was further evaluated by replacing the chlorine atom by a bromine one (15d and 15e). Substitution of the phenyl ring with a bromine atom at the 3- and 4-position was investigated while 2-position substitution was, due to excessive steric hindrance, not achieved using the described synthesis pathway. Among these two compounds, compound 15d displayed strong COX-2 inhibitory properties (COX-2 IC50 value for 15d: 0.12 μM) and a COX-2 selectivity similar to that observed with compound 2 (COX-2 selectivity ratio: 7.48 and 7.46 for compound 15d and 2, respectively). The substitution of the phenyl ring with short alkyl chains was also explored. In this context, the hydrogen atom at the
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Table 2. IC50 COX-1 and IC50 COX-2 of Compounds 2, 5, 9, and 15a-n, and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
Table 3. IC50 COX-1 and IC50 COX-2 of Compounds 2, 5, and 16a-o and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
IC50 (μM) compd 5 2 9 15a 15b 15c 15d 15e 15f 15g 15h 15i 15j 15k 15l 15m 15n
5867
IC50 (μM)
R1
COX-1
COX-2
ratio
compd
H 2-Cl 3-Cl 4-Cl 3-Br 4-Br 2-CH3 3-CH3 4-CH3 2-C2H5 3-C2H5 4-C2H5 2-OCH3 3-OCH3 4-OCH3
3.76 ( 1.02 2.60 ( 0.20 0.18 ( 0.06 0.19 ( 0.09 1.09 ( 0.16 1.15 ( 0.48 0.91 ( 0.05 1.60 ( 0.15 0.48 ( 0.36 0.99 ( 0.10 0.17 ( 0.05 3.25 ( 0.71 0.69 ( 0.12 1.22 ( 0.29 1.11 ( 0.51 1.00 ( 0.27 0.10 ( 0.01
0.70 ( 0.23 0.35 ( 0.09 0.09 ( 0.03 2.11 ( 1.22 0.27 ( 0.11 0.41 ( 0.27 0.12 ( 0.14 0.91 ( 0.14 2.69 ( 1.16 0.45 ( 0.20 0.46 ( 0.27 7.24 ( 4.53 0.24 ( 0.13 0.91 ( 0.02 2.53 ( 2.05 1.58 ( 0.85 0.16 ( 0.04
5.37 7.46 2.00 0.09 4.10 2.81 7.48 1.77 0.18 2.19 0.38 0.44 2.84 1.35 0.44 0.63 0.60
5 2 16a 16b 16c 16d 16e 16f 16g 16h 16i 16j 16k 16l 16m 16n 16o
2-, 3-, or 4-position of the benzene ring was substituted with a methyl or an ethyl group. Regarding the methyl-substituted derivatives (15f-h), we noticed important COXs inhibitory properties, whatever the methyl substitution position, although inhibitory properties appeared slightly weaker when compared to compound 9 profile. Among these three derivatives, the best selectivity ratio was obtained, as it was already observed with the chlorinated and brominated derivatives when the methyl substituent is located at the 3position (compound 15g, COX-2 selectivity ratio of 2.19). Regarding now the ethyl derivatives, we found that the replacement of the methyl group with an ethyl one at the 2- and 4-position (compounds 15i and 15k, respectively) led to a significant decrease in COXs inhibitory properties without drastic modification of the COX-2 selectivity ratio. On the contrary, an ethyl group at the 3-position (compound 15j) showed slightly improved COXs inhibitory properties when compared to those of its methyl analogue (COX-1 and COX-2 IC50 values for 15g: 0.99 and 0.45 μM; for compound 15j: 0.69 and 0.24 μM, respectively). Moreover, the selectivity ratio of 15j was improved when compared to 15g (COX-2 selectivity ratio for compounds 15j and 15g: 2.84 and 2.19, respectively). Taken together, these data demonstrate that the substitution of the benzene ring with a halogen atom or an alkyl chain lead to compounds displaying strong inhibitory properties against COXs enzymes. Moreover, the best selectivity ratios are observed with compounds bearing a substituent at the 3-position. Regarding the steric hindrance, the replacement of the methyl substituent of 15g with an ethyl one (15j) was accompanied with an improved activity, subsequently suggesting that an ethyl group did not display sufficient steric hindrance to limit the COXs inhibitory activities. The last modulation investigated in the trifluoromethanesulfonamide series was the substitution of the benzene ring of 9 with a methoxy moiety (compounds 15l-n). Globally, all the methoxy derivatives, whatever the substitution position,
R1
COX-1
COX-2
ratio
H 2-Cl 3-Cl 4-Cl 3-Br 4-Br 2-CH3 3-CH3 4-CH3 2-C2H5 3-C2H5 4-C2H5 2-OCH3 3-OCH3 4-OCH3
3.76 ( 1.02 2.60 ( 0.20 2.26 ( 0.91 0.48 ( 0.13 7.15 ( 3.34 1.31 ( 0.27 20.02 ( 2.22 2.12 ( 0.59 0.56 ( 0.10 2.57 ( 0.55 1.31 ( 0.84 3.79 ( 0.28 60.39 ( 5.17 2.73 ( 0.92 9.76 ( 0.22 25.24 ( 2.47 0.56 ( 0.08
0.70 ( 0.23 0.35 ( 0.09 2.69 ( 0.62 9.38 ( 6.48 0.73 ( 0.04 5.93 ( 2.23 2.56 ( 0.69 21.9 ( 12.7 18.6 ( 12.43 8.81 ( 0.48 12.03 ( 3.36 >100 >100 8.15 ( 1.90 >100 >100 21.61 ( 3.22
5.37 7.46 0.85 0.05 9.83 0.22 7.81 0.10 0.08 0.29 0.11
0.33
0.03
displayed COX-2 selectivity ratios below 1 and therefore appeared more active against COX-1. Among these, it seems interesting to point out that compound 15n, although it has COX-1 preferential inhibitory activity, displayed particularly high COXs inhibitory properties (COX-1 and COX-2 IC50 values for 15n: 0.10 and 0.16 μM, respectively). In a second step, we aimed to evaluate the influence of the same substitution pattern in the methanesulfonamide series. Indeed, during our previous work,33 we demonstrated that the modulation of the sulfonamide acidic character impacted on both the cyclooxygenases inhibitory activity and the COX-2 selectivity. As a brief summary of these previous works on pyridine derivatives of nimesulide bearing an ether or thioether linkage, we showed that compounds with a methanesulfonamide moiety, when compared to their trifluoromethanesulfonamide analogues, were characterized by a weaker COXs inhibitory profile but often an increased selectivity ratio against COX-2. All the results of the in vitro pharmacological evaluations performed with these newly synthesized compounds bearing a methanesulfonamide moiety are presented in Table 3. In a first step, the analogue of compound 9, compound 16a, was synthesized and pharmacological results demonstrated that the replacement of the trifluoromethanesulfonamide with a methanesulfonamide moiety led to a drastic loss of COXs inhibitory properties (COX-1 and COX-2 IC50 values for compound 16a: 2.26 and 2.69 μM, respectively). Moreover, the COX-2 selectivity ratio was also strongly decreased when compared to that of compound 9 (COX-2 selectivity ratio for 9 and 16a: 2.00 and 0.85, respectively). The substitution of the benzene ring with a chlorine atom (16b-d) did not significantly modify the COX-2 inhibitory activity. Of particular interest are the results obtained with 16c for which the well-preserved COX-2 inhibitory activity was accompanied with a decreased COX-1 inhibitory activity (COX-1 and COX-2 IC50 values for compound 16c: 7.15 and 0.73 μM, respectively), subsequently leading to a
5868 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 Table 4. IC50 COX-1 and IC50 COX-2 of Compounds 9, the Cycloalkanes Derivatives, and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
Renard et al. Table 5. In Vivo Anti-inflammatory Activity of Compounds 2, 5, and Selected Compounds at 5, 10, and 20 mg/kg ip in the Rat CarrageenanInduced Pleurisy (mean ( SE, n = 3-6 Rats/Group) % inhibition of exudate compd
5 mg/kg
10 mg/kg
20 mg/kg
5 2 9 15b 15c 15d 15g 16c
31.05 ( 7.85* 15.67 ( 5.99 25.41 ( 3.30 16.14 ( 11.42 39.39 ( 3.65** 42.07 ( 3.74** 23.50 ( 13.75 11.48 ( 6.04
48.44 ( 3.09** 27.32 ( 5.60 55.68 ( 9.50** 40.03 ( 4.76* 51.41 ( 5.28** 52.16 ( 2.63** 50.29 ( 15.04* 18.31 ( 7.74
61.53 ( 6.75** 42.09 ( 9.70** 72.20 ( 6.28** 52.60 ( 4.17** 68.83 ( 9.95** 64.52 ( 3.21** 69.53 ( 4.17** 25.46 ( 2.50
* p < 0.05. ** p < 0.01 vs control, one way ANOVA, and Bonferroni’s multiple comparison test.
compound displaying a selectivity ratio of 9.83. Results obtained with the brominated derivatives (16e and 16f) confirmed that the best selectivity profile was obtained when the substituent is located at the 3-position (COX-2 selectivity ratio for compound 16e: 7.81), although the COXs inhibitory properties appeared weaker when compared to the chlorinated analogues 16c and 16d. The substitution of the phenyl ring of 16a with a short chain was also investigated. Among the methylated derivatives (16g-i), we observed a decrease in the COX-2 inhibitory property when compared to their chlorinated analogues, whatever the substitution position. Moreover, the selectivity ratios measured were below the unit, identifying these three compounds as COX-1 preferential inhibitors. The loss of activity against COXs enzymes was even more pronounced when evaluating the ethylated derivatives (16j-l). Indeed, derivatives 16j and k, bearing an ethyl chain at the 2- and 3-position, were characterized as weak COX-2 inhibitors with IC50 greater than 100 μM. Compound 16l displayed a slightly better inhibitory profile with an IC50 against COX-2 of 8.15 μM, although its selectivity ratio remained below the unit. Finally, derivatives bearing a methoxy moiety (16m-o) behaved in the same manner as the ethyl derivatives with 2- and 3-methoxy derivatives showing a COX-2 IC50 greater than 100 μM. As observed with the ethyl derivatives, the best results in this substitution pattern were achieved with the 4-methoxy derivative (COX-1 and COX-2 IC50 values for compound 16o: 0.56 and 21.61 μM, respectively). The third pharmacomodulation investigated was the replacement of the benzene ring of 9 with several cycloalkane rings (Table 4). Indeed, the replacement of the benzene ring of 5 with a cyclohexane one led to the [N-(2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398).37 This compound exhibited an important cyclooxygenase inhibitory potency and an enhanced COX-2 preferential profile in different tests38,39 when compared to compound 5.
Cycloalkanes used for these investigations bore 5, 6, or 7 carbon atoms. All of these compounds showed a COX-1 preferential profile. Compounds bearing a trifluoromethanesulfonamide moiety demonstrated a higher COXs inhibitory potency than their methanesulfonamide analogues. Moreover, the size of the cycloalkane ring seemed to be important for the COXs inhibitory activity. Cyclohexane (compounds 25a and 25b) provided drugs with a strong COXs inhibitory activity. The increase of the size of the cycloalkane (26a and 26b) slightly decreased this activity, while a cyclopentane (compounds 24a and 24b) led to compounds exhibiting a poor inhibitory activity against the two cyclooxygenases. Compound 24b bearing a methanesulfonamide moiety was even inactive on COX-2. Finally, the influence of the length of the sulfonamide moiety was evaluated by using the 3,3,3-trifluoropropanesulfonamide group. The replacement of the trifluoromethanesulfonamide moiety of 9 by a 3,3,3-trifluoropropanesulfonamide (17) drastically decreased the COXs inhibitory potency. Furthermore, this decrease was more important regarding the COXs inhibitory profile and COX-2 selectivity ratio when compared to compound 9. In Vivo Anti-inflammatory Activity. The in vitro results allowed us to select compounds exhibiting a high COXs inhibitory activity and/or an interesting COX-2/COX-1 selectivity ratio for further investigation. Compounds 15b, 15c, 15d, 15g, and 16c were therefore selected and evaluated for their in vivo anti-inflammatory properties in an animal model of carrageenan-induced pleurisy (Table 5). Compounds 5, 2, and our lead compound 9 were evaluated in the same test as reference drugs. In this test, the inhibition by 5 of the exudate production, which is the reflection of the anti-inflammatory activity, was statistically significant whatever the dose administered (5, 10, and 20 mg/kg) while the anti-inflammatory activity of 2 was found to be only significant at the dose of 20 mg/kg. Compound 9, our lead compound, displayed a strong antiinflammatory activity, reaching 72% inhibition at the dose of 20 mg/kg. Nevertheless, the decrease of exudate volume was only statistically significant at the two higher doses (10 and 20 mg/kg) investigated (p < 0.001). Compound 15c exhibited an inhibition profile similar to that of 9, but its activity was already statistically significant at the dose of 5 mg/kg (p < 0.01). The anti-inflammatory properties of 15b were similar to that of 2 at the dose of 5 mg/kg (% of inhibition: 16.1% and 15.7%, respectively). At higher doses, 15b displayed a stronger anti-inflammatory activity when compared to 2 but failed to reach the efficiency of compound
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5 even at the highest investigated dose (% inflammation inhibition at the dose of 20 mg/kg for 15b and 5: 52.6% and 61.5%, respectively). Its methanesulfonamide analogue, compound 16c, showed a weak and non-statistically significant anti-inflammatory activity in vivo (% inhibition at the dose of 20 mg/kg: 25.5%), leading to the conclusion that the weaker COXs inhibitory profile of the methanesulfonamide compound 16c when compared to 15b is associated with a drastically decrease in anti-inflammatory activity although this compound displayed a high COX-2 selectivity ratio. Substitution of the benzene ring of 9 with a methyl group at the 3-position (15g) did not significantly modify the antiinflammatory activity. Indeed, the anti-inflammatory properties of 15g and 9 were similar at all the doses investigated with a decrease of the pleural exudate volume statistically significant at the doses of 10 and 20 mg/kg while no statistically significant effect was observed at 5 mg/kg. Finally, compound 15d, which presented a COX-2 inhibitory profile similar to 9 (COX-2 IC50 value for 15d and 9: 0.12 and 0.09 μM, respectively), but a COX-2 selectivity ratio comparable to compound 2 (COX-2 selectivity ratio for 15d and 2: 7.48 and 7.46, respectively) was evaluated. 15d displayed statistically significant anti-inflammatory properties at the doses of 5, 10, and 20 mg/kg (p < 0.001). The strongest inhibition of inflammation response at the dose of 5 mg/kg was obtained with this drug while it exhibited a decrease of exudate development similar to that of compounds 9, 15c, and 15g at the doses of 10 and 20 mg/kg, therefore identifying this compound as the most active drug synthesized during this study. Conclusion In the present study, we synthesized 36 original pyridine derivatives of nimesulide bearing an amino linkage by using a five-step synthesis pathway. These 36 original compounds can be classified into two series: the methanesulfonamide and the trifluoromethanesulfonamide derivatives. All these original derivatives were evaluated for their ability to inhibit human COX-1 and COX-2 enzymes in a human whole blood model. Results obtained demonstrated that, when comparing methane and trifluoromethanesulfonamide analogues, the highest inhibitory properties were observed with trifluoromethanesulfonamide derivatives while the highest COX-2 selectivity ratios were found in the methanesulfonamide series. For example, compounds 15d and 15n exhibited the most important inhibitory properties against COXs enzymes (COX-1 and COX-2 IC50 values for compound 15d: 0.91 and 0.12 μM; for compound 15n: 0.10 and 0.16 μM, respectively), while compounds 16c and 16e exhibited the highest COX-2 selectivity ratios (COX-2 selectivity ratio for compounds 16c and 16e: 9.83 and 7.81, respectively). In a second step, we selected several synthesized compounds on the basis of their COXs inhibitory properties and COX-2 selectivity and we investigated their ability to reduce inflammation in an acute inflammation model in rats. In this in vivo model, all the evaluated compounds, except 16c, displayed a strong anti-inflammatory activity at the dose of 20 mg/kg. Of particular interest were the results obtained with compounds 15c and 15d that demonstrated an enhanced antiinflammatory activity at the dose of 5 mg/kg when compared to nimesulide and celecoxib, therefore identifying these two compounds as promising anti-inflammatory agents. Moreover, results obtained also evidenced that compounds with weaker COXs inhibitory properties (compound 16c)
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displayed weak anti-inflammatory profile in vivo, whatever their COX-2 selectivity ratio. Regarding now the safety profile, comparison of nonselective and COX-2 selective agents evidenced that the use of COX-2 selective inhibitors such as compound 1 or 2 was associated with a significantly decreased risk of gastrointestinal toxicity when compared to nonselective NSAIDs. Nevertheless, the occurrence of cardiovascular side effects observed with 1 led to its withdrawal from the market. Although not yet fully elucidated, this toxicity seems to be related to either its high COX-2 selectivity ratio or its chemical structure, more precisely to its methylsulfone moiety. Having obtained compounds of which the best representatives showed a COX2 selectivity ratio in the range of that displayed by 2 (compounds 15d, 16c, and 16e) and characterized by the presence of a methane- or trifluoromethanesulfonamide moiety instead of a methylsulfone moiety, we can theoretically expect that the use of our compounds should be associated with a good safety profile. Finally, some cases of nimesulide-induced hepatotoxicity were recently reported and led to the withdrawal of this compound first in Finland and soon after in Spain. More recently, 5 was also withdrawn in Belgium for similar hepatic injuries. This hepatotoxicity was found to be idiosyncratic (predisposition of polymorphism in either of P450 1A2 and P450 2C19).40 More precisely, it seems that several nimesulide metabolites are involved in this hepatotoxicity. Indeed, several metabolites of this compound have been identified including 4-hydroxynimesulide and amino des-nitro nimesulide in which the nitrobenzene group has been reduced. Of particular interest is the ability of amino des-nitro nimesulide to be further oxidized by P450 enzyme to diiminoquinone, highly reactive species able to alkylate proteins. In a second step, modified peptides generated from such nucleophilic addition can act as antigens and trigger an immune response.40 This metabolism pathway may represent the mechanism of the acute liver failure described in some patients under nimesulide therapy. Therefore, this occasionally observed toxicity seems to be associated with the in vivo metabolism of the nitrobenzene ring of nimesulide (reduction followed by the formation of a reactive diiminoquinone). Since we performed the replacement of the nitrobenzene ring of nimesulide with a pyridinic one, we believe that the use of our newly synthesized compounds should not be associated with such toxicity. To conclude, we have achieved the synthesis of pyridinic derivatives of nimesulide displaying high COXs inhibitory properties associated, for some of them, with a COX-2 selectivity ratio similar to that of 2. Among these derivatives, compound 15d, displaying a drastic inhibition of COX-2 (COX-2 IC50 value for compound 15d: 0.12 μM) with a COX2 selectivity ratio of 7.48 and a strong anti-inflammatory profile in vivo appeared as the most promising agent. Experimental Section Chemistry. All commercial chemicals (Sigma-Aldrich, Belgium and Fluorochem, United Kingdom) and solvents are reagent grade and were used without further purification. Melting points were determined on a Stuart SMP3 apparatus in open capillary tubes and are uncorrected. IR spectra were recorded as KBr pellets on a Perkin-Elmer 1000 FTIR spectrophotometer. NMR spectra were recorded on a Bruker Avance (500 MHz) spectrometer using DMSO-d6 as solvent and tetramethylsilane as internal standard; chemical shifts are reported in
5870 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
δ values (ppm) relative to internal tetramethylsilane. The abbreviation s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and bs = broad signal are used throughout. Elemental analyses (C, H, N, S) were determined either on a Carlo-Erba EA 1108 or a Thermo Flash EA 1112 series elemental analyzer and were within (0.4% of the theoretical values. All reactions were followed by TLC (silica gel 60F254 Merck) and visualization was accomplished with UV light (254 nm). Experimental procedures for the preparation of 4-nitropyridine N-oxide (13a-o, 18-20) and 4-aminopyridine derivatives (14a-o, 21-23) are presented in the Supporting Information. General Procedure for the Preparation of Alkanesulfonamides with Alkanesulfonyl Chlorides. The appropriate aminopyridine (14a-o, 21-23, 5 mmol) was dissolved in dry acetonitrile (40 mL). Anhydrous potassium carbonate (30 mmol) was added, and the suspension was stirred for 5 min and the appropriate alkanesulfonyl chloride (6-7.5 mmol) was added to the mixture. When the reaction was finished (1-4 h), the suspension was filtered and the filtrate was evaporated under reduced pressure. The residue was taken up with NaOH (10% aqueous sol. w/v) and then was filtered. The filtrate was neutralized with 1 N HCl, and the crude product was collected by filtration. The resulting product was crystallized in ethanol/water or purified by column chromatography on silica gel using a chloroform/methanol mixture (18:2) as eluent to give the product as a white solid. N-(3-Phenylamino-4-pyridinyl)trifluoromethanesulfonamide (9). Yield: 52%; mp: 188-189 °C. 1H NMR (DMSO-d6) δ 7.05 (t, 1H, H-40 ), 7.30 (d, 2H, H-20 þ H-60 ), 7.35 (t, 2H, H-30 þ H-50 ), 7.40 (s, 1H, NH), 7.61 (d, 1H, H-5), 7.96 (d, 1H, H-6), 8.13 (s, 1H, H-2), 13.52 (s, 1H, NHþ). Anal. (C12H10F3N3O2S) C, H, N, S. N-[3-(3-Chlorophenylamino)-4-pyridinyl]trifluoromethanesulfonamide (15b). Yield: 65%; mp: 211-213 °C. 1H NMR (DMSO-d6) δ 7.00 (d, 1H, H-60 ), 7.21 (d, 1H, H-40 ), 7.31 (bm, 2H, H-20 þ H-50 ), 7.63 (m, 2H, H-5 þ NH), 8.01 (d, 1H, H-6), 8.23 (s, 1H, H-2), 13.55 (s, 1H, NHþ). Anal. (C12H9ClF3N3O2S) C, H, N, S. N-[3-(4-Chlorophenylamino)-4-pyridinyl]trifluoromethanesulfonamide (15c). Yield: 27%; mp: 203-205 °C. 1H NMR (DMSO-d6) δ 7.30 (d, 2H, H-20 þ H-60 ), 7.35 (d, 2H, H-30 þ H-50 ), 7.52 (s, 1H, NH), 7.62 (d, 1H, H-5), 7.98 (d, 1H, H-6), 8.13 (s, 1H, H-2), 13.54 (s, 1H, NHþ). Anal. (C12H9ClF3N3O2S) C, H, N, S. N-[3-(3-Bromophenylamino)-4-pyridinyl]trifluoromethanesulfonamide (15d). Yield: 69%; mp: 241-242 °C. 1H NMR (DMSOd6) δ 7.26 (m, 3H, H-40 þ H-50 , H-60 ), 7.47 (s, 1H, H-20 ), 7.63 (d, 1H, H-5), 7.64 (s, 1H, NH), 8.01 (d, 1H, H-6), 8.23 (s, 1H, H-2), 13.55 (s, 1H, NHþ). Anal. (C12H9BrF3N3O2S) C, H, N, S. N-[3-(3-Methylphenylamino)-4-pyridinyl]trifluoromethanesulfonamide (15g). Yield: 42%; mp: 167-169 °C. 1H NMR (DMSO-d6) δ 2.29 (s, 3H, CH3), 6.87 (d, 1H, H-60 ), 7.09 (d, 1H, H-40 ), 7.10 (s, 1H, H-20 ), 7.23 (t, 1H, H-50 ), 7.31 (s, 1H, NH), 7.60 (d, 1H, H-5), 7.96 (d, 1H, H-6), 8.13 (s, 1H, H-2), 13.50 (s, 1H, NHþ). Anal. (C13H12F3N3O2S) C, H, N, S. N-[3-(3-Chlorophenylamino)-4-pyridinyl]methanesulfonamide (16c). Yield: 23%; mp: 185-187 °C. 1H NMR (DMSO-d6) δ 2.89 (s, 3H, SO2CH3), 6.93 (d, 1H, H-60 ), 7.16 (d, 1H, H-40 ), 7.28 (bm, 3H, H-5 þ H-20 þ H-50 ), 7.43 (s, 1H, NH), 7.78 (d, 1H, H-6), 8.00 (s, 1H, H-2), 12.52 (bs, 1H, NHþ). Anal. (C12H9ClF3N3O2S) C, H, N, S. N-(3-Cyclohexylamino-4-pyridinyl)trifluoromethanesulfonamide hydrochloride (25a). Yield: 71%; mp: 217-218 °C. 1H NMR (DMSO-d6): δ 1.15-1.95 (bm, 10H, Hcyclohexyl), 3.33 (m, 1H, H-10 ), 7.44 (bs, 1H, H-6), 7.70 (d, 1H, H-2), 7.80 (d, 1H, H-5), 13.81 (s, 1H, NHþ). Anal. (C12H17ClF3N3O2S) C, H, N, S. Pharmacology. In Vitro COX-1 and COX-2 Inhibitory Activity Determination Using Human Whole Blood Assay. COX-1 assay. Fresh human blood was collected into heparinized tubes by venipuncture and directly diluted with RPMI (Roswell Park Memorial Institute culture medium) (1:4). Then 0.25 mL diluted
Renard et al.
blood aliquots were transferred into siliconized tubes preloaded with 1 μL of vehicle (DMSO) or test compounds at final concentration ranging from 0.01 to 100 μmol/L. The tubes were vortexed and incubated at 37 °C under constant agitation for 5 min, and then 10 μL of calcium ionophore A-23,187 (0.2 mg/mL) were added and the aliquots were incubated at 37 °C under constant agitation for 15 min. At the end of the incubation, the serum was obtained by centrifugation (3200g for 5 min) and was assayed for thromboxane B2 (TXB2) production using an enzyme immunoassay kit (Cayman Chemical TXB2 EIA Kit, Ann Arbor, MI, and Assay design TXB2 Correlate-EIA Kit, Ann Arbor, MI) according to the manufacturer’s instructions. COX-2 assay. Fresh human blood was collected in heparinized tubes by venipuncture. One mL aliquots of blood were transferred in heparinized tubes preloaded with either 4 μL of vehicle (DMSO) or 4 μL of test compound at a final concentration ranging from 0.01 to 100 μmol/L. The tubes were vortexed and incubated at 37 °C under constant agitation for 5 min. This first treatment was followed by incubation of the blood with 20 μL of lipopolysaccharide (LPS from E. coli serotype B8) at final concentration of 100 μg/mL under constant agitation for 24 h at 37 °C in order to induce COX-2 expression. Appropriate controls (LPS replaced by 20 μL of PBS) were used as blanks. After incubation, the tubes were centrifugated at 3200g for 5 min. Plasma was assayed for PGE2 production by a specific enzyme immunoassay (Cayman Chemical PGE2 EIA Kit, Ann Arbor, MI) according to the manufacturer’s instructions. Carrageenan-Induced Pleurisy. Animals. Male Wistar rats, weighing 230-260 g, were housed in the University of Liege animal facilities in accordance with local guidelines. The animals were maintained on a standard laboratory diet with free access to water. The experiments were conducted as approved by the Animal Ethics Committee of the University of Liege, Belgium. Carrageenan-Induced Pleurisy. Rats were pretreated with an intraperitoneal (ip) injection of saline or drugs (5, 10, or 20 mg/kg) 30 min before the intrapleural injection of carrageenan. They were then anaesthetized with sodium pentobarbital (40 mg/kg, ip) and λ carrageenan (0.2 mL, 10 mg/mL) was administered into the right pleural cavity. Each experimental group contained at least three animals. Four hours later, the animals were anaesthetized with sodium pentobarbital (80 mg/kg, ip). The chest was carefully opened and the pleural cavity rinsed with 2.0 mL of saline solution containing heparin (5 U/mL). Exudates and washing solutions were collected by aspiration. Exudates with blood were rejected. The volume of the exudates was calculated by subtracting the volume of the washing solution (2.0 mL) from the total volume harvested.
Acknowledgment. This study was supported by Belgian grants of the “Fonds pour la Recherche dans l’Industrie et l’Agriculture” (FRIA) and the “Leon Fredericq Foundation”. Supporting Information Available: Experimental procedure for 4-nitropyridine N-oxide derivatives (13a-o, 18-20), 4-aminopyridine derivatives (14a-o, 21-23), 4-alkylsulfonylaminopyridine derivatives (15a, 15e-f, 15h-n, 16a-b, 16d-o, 24ab, 25b, 26a-b, 17), and elemental analysis of the compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.
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