Synthesis of New Triarylpyrazole Derivatives Possessing Terminal Sulfonamide Moiety and Their Inhibitory Effects on PGE2 and Nitric Oxide Productions in Lipopolysaccharide-Induced RAW 264.7 Macrophages

This article describes the design, synthesis, and in vitro anti-inflammatory screening of new triarylpyrazole derivatives. A total of 34 new compounds were synthesized containing a terminal arylsulfonamide moiety and a different linker between the sulfonamide and pyridine ring at position 4 of the pyrazole ring. All the target compounds were tested for both cytotoxicity and nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Compounds 1b, 1d, 1g, 2a, and 2c showed the highest NO inhibition percentages and the lowest cytotoxic effect. The most potent derivatives were tested for their ability to inhibit prostaglandin E2 (PGE2) in LPS-induced RAW 264.7 macrophages. The IC50 for nitric oxide inhibition, PGE2 inhibition, and cell viability were determined. In addition, 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX-2) protein expression as well as iNOS enzymatic activity.


Introduction
Inflammation is one of the most important and complicated defense mechanisms. Inflammation participates in vital pathological and physiological processes like infection and wound healing [1]. As a result of tissue damage, many chemical intermediates are released in the damaged area. The chemical intermediates (such as E, L, and P-selectin and chemokines) initiate activation and migration of white blood cells to the damaged area. Eosinophils and neutrophils are the first leucocytes that migrate to the affected area followed by macrophages that release a number of cytokines and growth factors that affect the surrounding tissues [2][3][4][5]. Inflammation can be acute, occurring as part of a healing process, or chronic inflammation, which arises from the over response of the immune system and can lead to tissue damage. Chronic inflammation contributes to several physiological disorders such as neurodegenerative diseases [6], cancer [7], inflammatory bowel disease [8], and arteriosclerosis [9].
At the inflammation site, monocytes are converted to macrophages that release a large amount of nitric oxide (NO). Nitric oxide is produced as a result of oxidation of L-arginine by one of the nitric oxide synthase family members: endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), which is a calcium-dependent enzyme, and inducible NOS (iNOS), which is calcium-independent enzyme) [10,11]. The presence of pro-inflammatory and chemical stimuli, such as lipopolysaccharide (LPS), leads to over-expression of iNOS [12]. Successful NO production inhibitory agents act through inhibition of iNOS protein expression and/or inhibition of iNOS enzymatic activity.
In addition to nitric oxide, prostaglandins are another important inflammation phospholipid by-product [13]. Prostaglandin E 2 (PGE 2 ) plays an important role in most inflammation conditions [14], such as glomerulonephritis, which may lead to renal failure [15]. The production of PGE 2 is initiated by membrane phospholipids that are converted to arachidonic acid under the effect of the phospholipases enzyme. Arachidonic acid is transformed first to prostaglandin H 2 , which finally produces PGE 2 [16]. The increase in both prostaglandin E 2 and nitric oxide in chronic inflammation cases can lead to severe complicated physiological disorders [17,18]. So, the inhibition of both PGE 2 and nitric oxide could result in the discovery of new anti-inflammatory drug candidates.
Several scaffolds have been investigated for their antiinflammatory activity, such as thiadiazole [19][20][21], chromones [22,23], triazoles [24], imidazole [25], and pyrazole. Many compounds with a pyrazole backbone have been proven to exhibit both anticancer [26][27][28][29][30][31] and antiinflammatory effects [32][33][34][35]. Celecoxib is an anti-inflammatory drug that contains diarylpyrazole as a back bone and works through inhibition of the COX-2 enzyme [36,37]. Previously, we reported the synthesis of a series of triarylpyrazoles [38][39][40][41], from which compound I (Figure 1) showed the highest activity for both nitric oxide and PGE 2 production inhibition [40]. In the current work and based on our previous work, we synthesized a new series of triarylpyrazole derivatives. The new series contains 2-substituted pyridine at position 4 of the pyrazole ring. The substitutions contain a terminal sulfonamide moiety and a different linker between the sulfonamide and pyridine ring. The linker we used to investigate the effect of linker length on the activity was either ethylene or propylene. The new series was screened for its ability to inhibit nitric oxide; their cytotoxicity on RAW 264.7 macrophages was also investigated.
The most potent compounds were tested for their inhibitory effect on PGE 2 and iNOS expression. Molecules 2018, 23,

Chemistry
The synthesis of the final target compounds 1a-i, 2a-i, 3a-h, and 4a-h was achieved by adopting the synthetic strategy illustrated in Scheme 1. We first synthesized the side chains 8a-i and 9a-i. The main intermediate 5 was synthesized according to previously reported procedures [42,43]. Eventually, the target compounds 1a-i and 2a-i were obtained by coupling compound 5 with 8a-i and 9a-i using pyridine as a solvent and refluxing for 12 h. Another pathway to obtain 1a-i and 2a-i was refluxing 5 with 1,2-ethylenediamine or 1,3-propylenediamine to produce 6 and 7, which, upon reaction with the appropriate arylsulfonyl chloride in the presence of triethylamine, produced the desired final compounds 1a-i and 2a-i. Demethylation of compounds 1 and 2 using boron tribromide produced the hydroxyl final analogues 3a-h and 4a-h (Scheme 1). The structures of the final target compounds and their yields are represented in Table 1.

Chemistry
The synthesis of the final target compounds 1a-i, 2a-i, 3a-h, and 4a-h was achieved by adopting the synthetic strategy illustrated in Scheme 1. We first synthesized the side chains 8a-i and 9a-i. The main intermediate 5 was synthesized according to previously reported procedures [42,43]. Eventually, the target compounds 1a-i and 2a-i were obtained by coupling compound 5 with 8a-i and 9a-i using pyridine as a solvent and refluxing for 12 h. Another pathway to obtain 1a-i and 2a-i was refluxing 5 with 1,2-ethylenediamine or 1,3-propylenediamine to produce 6 and 7, which, upon reaction with the appropriate arylsulfonyl chloride in the presence of triethylamine, produced the desired final compounds 1a-i and 2a-i. Demethylation of compounds 1 and 2 using boron tribromide produced the hydroxyl final analogues 3a-h and 4a-h (Scheme 1). The structures of the final target compounds and their yields are represented in Table 1.  Figure 1. General structures of the target compounds, Celecoxib, and previously-reported pyrazole compound [40].

Chemistry
The synthesis of the final target compounds 1a-i, 2a-i, 3a-h, and 4a-h was achieved by adopting the synthetic strategy illustrated in Scheme 1. We first synthesized the side chains 8a-i and 9a-i. The main intermediate 5 was synthesized according to previously reported procedures [42,43]. Eventually, the target compounds 1a-i and 2a-i were obtained by coupling compound 5 with 8a-i and 9a-i using pyridine as a solvent and refluxing for 12 h. Another pathway to obtain 1a-i and 2a-i was refluxing 5 with 1,2-ethylenediamine or 1,3-propylenediamine to produce 6 and 7, which, upon reaction with the appropriate arylsulfonyl chloride in the presence of triethylamine, produced the desired final compounds 1a-i and 2a-i. Demethylation of compounds 1 and 2 using boron tribromide produced the hydroxyl final analogues 3a-h and 4a-h (Scheme 1). The structures of the final target compounds and their yields are represented in Table 1.  The tested derivatives exhibited diverse activity for NO production inhibition. All compounds inhibited NO release in a dose-dependent manner. For series 1a-i, most of the compounds inhibited the production of NO by 50% or more at 10 µM. Compound 1b (p-bromo) showed the highest inhibition at 68.66% followed by 1d (p-flouro) with inhibition of 61.28%, then 1g (p-CF 3 ) with inhibition 60.80%. Compound 1a, 1f, and 1i had moderate activity with 52.93%, 53.09%, and 51.51% inhibition, respectively. Regarding compounds 2a-i, the highest inhibition was obtained from compounds 2a (62.76%), 2c (59.09%), 2g (56.03%), 2d (55.00%), and 2b (52.44%). Generally for methoxy series, derivatives with ethylene bridges were more active compared to compounds with propylene bridges. In addition, compounds with electron-withdrawing groups were more potent compared to compounds with electron-donating groups, and para substitutions were slightly more active than meta substitutions. The electronic nature and the position of the substituents were other important factors that confer optimum affinity to the receptor site.
Derivatives containing hydroxyl group, 3a-h and 4a-h, were less active compared to methoxy derivatives. The highest percent inhibition for 3a-h was exhibited by 3a (48.97%). Series 4a-h showed good inhibition with the highest demonstrated by compound 4b (63.85%) followed by 4e (59.69%), 4a (59.25%), 4f (53.82%), and 4g (51.15%), as illustrated in Table 2. The methoxy group is more hydrophobic and bulkier than hydroxyl, and this might affect the activity by enhancing the molecule's ability to cross the cell membrane and/or increasing the affinity with the target receptor site. Furthermore, the most active compounds (e.g., 1b) were more active than the lead compound possessing no tether on the pyridyl ring [33]. So, this side chain is an important contributor to the inhibition activity, which could improve the molecular affinity to its receptor site.
In addition to NO inhibition, the cytotoxic activity of compounds 1a-i, 2a-i, 3a-h, and 4a-h in RAW 264.7 macrophages were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to check whether the effects on the production of NO was caused by nonspecific cytotoxicity. The IC 50 values for both nitric oxide inhibition and cell viability are presented in Table 3. Compounds 1a-i and 2a-i had high IC 50 values in the cell viability test and all compounds had an IC 50 of more than 169 µM. The IC 50 for nitric oxide production inhibition was less than 14 µM. The most potent compound among the methoxy derivatives was 1b with an IC 50 of 7.90 µM followed by 2a, 1d, and 2c with IC 50 values of 8.04, 8.2, and 8.68 µM, respectively. These most potent molecules showed extreme safety expressed by very high IC 50 values as cytotoxic agents. This means that their inhibitory effect against NO production is not due to the cytotoxic effect. Compounds 3a-h and 4a-h showed cytotoxic effects at low doses and the IC 50 s for nitric oxide inhibition were close to the IC 50 s of the cell viability test. From Table 3, it can be predicted that the inhibitory effect of hydroxyl-containing compounds is due to the cytotoxic effect.
Compounds 1b, 1d, 1g, 2a, and 2c, which exhibited the highest activities regarding nitric oxide inhibition and the highest IC 50 values in the cell viability test, were investigated for their ability to inhibit PGE 2 production in LPS-induced RAW 264.7 macrophages at 1, 5, and 10 µM. The investigation results are shown in Table 4. Compounds 1b, 1g, 2a, and 2c were able to inhibit more than 50% of the prostaglandin production at a dose 5 µM. The five compounds were able to reduce PGE 2 production by over 75% at a dose of 10 µM. Compound 1g was the most potent compound with an IC 50 of 4.55 µM followed by 2c, 1b, 2a, and 1d with IC 50 values of 4.68, 4.72, 4.87, and 5.06 µM, respectively. The cumulative activities of compounds 1b, 1d, 1g, 2a, and 2c are illustrated in Figure 2 using N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) and N 6 -(1-Iminoethyl)-L-lysine (L-NIL) as standard compounds for PGE 2 production inhibition and NO production inhibition, respectively. The tested compounds showed low cytotoxic activity in the viability test. A significant reduction in both nitric oxide and PGE 2 production was observed starting from 5 µM. At 20 µM, the production of both inflammatory mediators was restored to normal levels.  The cumulative activities of compounds 1b, 1d, 1g, 2a, and 2c are illustrated in Figure 2 using N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) and N 6 -(1-Iminoethyl)-L-lysine (L-NIL) as standard compounds for PGE2 production inhibition and NO production inhibition, respectively. The tested compounds showed low cytotoxic activity in the viability test. A significant reduction in both nitric oxide and PGE2 production was observed starting from 5 μM. At 20 μM, the production of both inflammatory mediators was restored to normal levels.  As a result of their activity against both NO and PGE2 production and low cellular toxicity, compounds 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on the expression of both iNOS and COX-2. The cellular lysates were prepared from the with-and without-pretreatment tested compounds (5, 10, 20 μM) for one hour and then with LPS (1 μg/mL) for 24 h, using β-actin as a reference. The results are shown in Figure 3. Compound 1g, possessing an ethylene spacer, 3-methoxyphenyl at position 3 of the pyrazole ring, and a p-(trifluoromethyl)phenyl terminal ring, showed complete inhibition of iNOS expression at 20 µ M. Compounds 1b and 1d exhibited a partial inhibitory effect against iNOS at the same concentration (Figures 3 and 4). Compound 1g might express its inhibitory effect on NO production mainly through inhibition of iNOS protein expression and partially through inhibition of iNOS enzyme activity.  1b, 1d, 1g, 2a, and 2c. Data are presented as the means ± SD of three independent experiments. # p < 0.05 versus the control cells; *** p < 0.001 versus lipopolysaccharide-stimulated cells; ** p < 0.05 versus lipopolysaccharide-stimulated cells; * statistical significances were compared using ANOVA and Dunnett's post hoc test.
As a result of their activity against both NO and PGE 2 production and low cellular toxicity, compounds 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on the expression of both iNOS and COX-2. The cellular lysates were prepared from the with-and without-pretreatment tested compounds (5, 10, 20 µM) for one hour and then with LPS (1 µg/mL) for 24 h, using β-actin as a reference. The results are shown in Figure 3. Compound 1g, possessing an ethylene spacer, 3-methoxyphenyl at position 3 of the pyrazole ring, and a p-(trifluoromethyl)phenyl terminal ring, showed complete inhibition of iNOS expression at 20 µM. Compounds 1b and 1d exhibited a partial inhibitory effect against iNOS at the same concentration (Figures 3 and 4). Compound 1g might express its inhibitory effect on NO production mainly through inhibition of iNOS protein expression and partially through inhibition of iNOS enzyme activity.   of compounds 1b, 1d, 1g, 2a, and 2c on iNOS activity. Following pretreatment with lipopolysaccharide (LPS, 1 µ g/mL) for 12 h and wash with phosphate buffer solution (PBS), cells were treated with 1g (5, 10, or 20 μM) for 12 h N 6 -(1-Iminoethyl)-L-lysine. (L-NIL) (40 μM) was used as the positive control in the assay. Levels of NO in culture media were quantified using the Griess reaction assay. Data are presented as the means ± SD of three independent experiments. # p < 0.05 versus the control cells; *** p < 0.001 versus LPS-stimulated cells; * statistical significances were compared using ANOVA and Dunnett's post hoc test.

General
All chemicals were commercially available and used with no further purification. The final compounds and intermediates were purified by column chromatography using silica gel (0.040-0.063 mm, 230-400 mesh) and technical grade solvents. Analytical thin layer chromatography (TLC) was adopted on silica gel 60 F 254 plates from Merck (Merck, Massachusetts, MA, USA). Purity percentages of the target compounds were confirmed to be more than 96% by liquid chromatography-mass spectrometry (LC-MS). Proton nuclear magnetic resonance ( 1 H-NMR) and carbon NMR ( 13 C-NMR) spectra were recorded on a Bruker Avance 400 or 300 spectrometer (Massachusetts, MA, USA) using tetramethylsilane as an internal standard and signals are described as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet), brs (broad singlet), or dd (doublet of doublets). LC-MS analysis was carried out using the following system: Waters 2998 photodiode array detector, Waters 3100 mass detector, Waters SFO system fluidics organizer, Waters 2545 binary gradient module, Waters reagent manager, Waters 2767 sample manager, Waters 2998 photodiode and Sunfire™ C18 column (4.6 × 50 mm, 5 µm particle size) (Waters, Massachusetts, MA, USA). The solvent gradient = 95% A at 0 min, 1% A at 5 min. Solvent A was 0.035% trifluoroacetic acid (TFA) in water, solvent B was 0.035% TFA in CH 3 OH, and the flow rate was 3.0 mL/min. The area under the curve (AUC) was calculated using Waters MassLynx 4.1 Waters, Massachusetts, MA, USA) software. Solvents and liquid reagents were transferred using hypodermic syringes. Melting points were obtained on a Walden Precision Apparatus Electro thermal 9300 apparatus (Stone, Staffordshire, England) and were uncorrected.

Method A
To a solution of compound 6 or 7 (0.2 mmol) in anhydrous dichloromethane (5 mL), triethylamine (50.5 mg, 0.5 mmol) was added at 0 • C. A solution appropriate arylsulfonyl chloride (0.21 mmol) in anhydrous dichloromethane (1 mL) was added dropwise. The reaction mixture was stirred at room temperature for 24 h. When the reaction was finished, the solvent was removed in vacuo, and the residue was partitioned between ethyl acetate (5 mL) and water (5 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The combined organic layer was washed with saturated saline (2 × 5 mL) and the organic solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane-ethyl acetate 4:1 v/v) to produce the required product.

Method B
A mixture of compound 5 (81 mg, 0.2 mmol) and compound 8 or 9 (0.2 mmol) in pyridine was heated at 100 • C for 24 h. After complete reaction, monitored by thin-layer chromatograph (TLC), pyridine was removed under reduced pressure. The residue was partitioned between water (50 mL) and ethylacetate (50 mL). The organic layer was separated and washed three times with distilled water (3 × 50 mL) and dried over Na 2 SO 4 . The organic layer was evaporated and the residue was purified using column chromatography.  159.7, 158.5, 147.4, 143.1, 142.1, 140.1,  139.6, 139.3, 137.1, 131.1, 129.9, 129.5, 128.8, 127.6, 127.0, 125.1, 122.7, 120.0, 115.7, 114.7, 112   To a mixture of compound (1a-i) or (2a-i) (0.1 mmol) in methylene chloride (5 mL), BBr 3 (0.13 g, 1.0 mmol) was added dropwise at −78 • C under nitrogen, and the reaction mixture was stirred at 0 • C for 24 h. The mixture was quenched with saturated aqueous NaHCO 3 . Ethyl acetate (10 mL) was added and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (3 × 10 mL). The combined organic layer extracts were washed with brine and dried over anhydrous Na 2 SO 4 . The organic solvent was evaporated under reduced pressure and the residue was purified by column chromatography.