Synthesis, characterization and pharmacological evaluation of pyrazolyl urea derivatives as potential anti-inflammatory agents.

p38α mitogen activated protein kinase (MAPK) inhibitors provide a novel approach for the treatment of inflammatory disorders. A series of fifteen pyrazolyl urea derivatives (3a-o) were synthesized and evaluated for their p38α MAPK inhibition and antioxidant potential. Compounds 3a-e, 3g and 3h showed low micromolar range potency (IC50 values ranging from 0.037 ± 1.56 to 0.069 ± 0.07 µmol/L) compared to the standard inhibitor SB 203580 (IC50 = 0.043 ± 3.62 µmol/L) when evaluated for p38α MAPK inhibition by an immunosorbent-based assay. Antioxidant activity was measured by a 2,2'-diphenyl-1-picryl hydrazyl radical (DPPH) free radical scavenging method and one of the compounds, 3c, showed better percentage antioxidant activity (75.06%) compared to butylated hydroxy anisole (71.53%) at 1 mmol/L concentration. Compounds 3a-e, 3g and 3h showed promising in vivo anti-inflammatory activity (ranging from 62.25% to 80.93%) in comparison to diclofenac sodium (81.62%). The ulcerogenic liability and lipid peroxidation activity of these compounds were observed to be less in comparison to diclofenac sodium. These compounds also potently inhibited the lipopolysaccharide (LPS)-induced TNF-α release in mice (ID50 of 3a-c = 19.98, 11.32 and 9.67 mg/kg, respectively). Among the screened compounds, derivative 3c was found to be the most potent and its binding mode within the p38α MAPK is also reported.

Introduction

Inflammation is a multifactorial, protective attempt of the non-specific immune system. In response to infection stimulus, monocytes/macrophages lineage cells are activated, thereby generating an inflammatory environment by secreting proinflammatory cytokines. It is an important aspect in rheumatoid arthritis, osteoarthritis, Alzheimer׳s disease and obesity related diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the most widely prescribed agents for the management of various inflammatory diseases2, 3. NSAIDs act by counteracting the cyclooxygenase (COX) that converts arachidonic acid into prostaglandins in inflammatory processes. Commonly used NSAIDs, such as aspirin, indomethacin and diclofenac, are non-selective inhibitors and are responsible for adverse side effects, such as gastric ulceration, bleeding and renal function suppression. There are at least two mammalian COX isoforms5, 6. COX-1 is constitutive and provides cytoprotection in the gastrointestinal (GI) tract, while COX-2 is induced and responsible for pro-inflammatory conditions. Various selective COX-2 inhibitors, such as celecoxib, rofecoxib and valdecoxib, showed anti-inflammatory activity with minimum gastric side effects. Unfortunately, selective COX-2 inhibitors were found to cause cardiovascular side effects. Therefore, in view of the GI toxicity of conventional NSAIDs and the adverse cardiovascular side effects of selective COX-2 inhibitors, there is a need to develop anti-inflammatory agents with an improved safety profile.

p38α mitogen activated protein kinase (MAPK) has attracted considerable attention as a major target in developing anti-inflammatory drugs. Activated p38α phosphorylates a range of intracellular protein substrates that transcriptionally regulate the biosynthesis of inflammatory cytokines like tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The identification of p38 MAPK as the target of some pyridinyl-imidazole compounds, e.g. SB203580 (Fig. 1A), confirmed the role of this intracellular enzyme in the regulation of many physiological and pathological states and reinforced the importance of its modulation in the therapy of many inflammatory diseases. p38α MAPK belongs to the serine/threonine family of kinases and is a key enzyme of a cascade leading to the production of pro-inflammatory cytokines, such as IL-1β and TNF-α. Excessive levels of these cytokines are known to be involved in the progression of many inflammatory disorders, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis12, 13, 14. TNF-α is a major pleiotropic pro-inflammatory cytokine and is known to activate platelets and also participates in the genesis of fever and anemia. Increased production of TNF-α also modulates processes, such as immune cell activation, proliferation, apoptosis and leukocyte migration and is thereby associated with many inflammatory diseases, like Crohn׳s disease, psoriasis, multiple sclerosis and rheumatoid arthritis. p38α MAPK inhibition is therefore a promising therapeutic strategy to block the biosynthesis of TNF-α.

Figure 1

Compound structures of (A) SB 203580 and (B) BIRB 796 (Doramapimod).

Pyrazole derivatives are an important class of heterocycles because of their diverse pharmacological properties, such as antioxidant, anti-inflammatory, antimicrobial and anti-viral/anti-tumor effects. Rofecoxib and celecoxib (selective COX-2 inhibitors) having a pyrazole moiety have exhibited significant anti-inflammatory activity with reduced GI toxicity. Furthermore, pyrazoles and pyrazolyl urea derivatives have also been reported to be potential anti-inflammatory agents18, 19, 20. Compound BIRB-796 (Fig. 1B) having pyrazolyl urea moiety has shown significant anti-inflammatory activity through p38α MAPK and TNF-α inhibition and had advanced into clinical trials.

Encouraged by these observations and in the course of our research program on the synthesis of five membered heterocyclic compounds as anti-inflammatory agents10, 21, 22, 23, 24, we report herein the synthesis and evaluation of some new pyrazolyl urea derivatives as potential anti-inflammatory agents.

Results and discussion

Chemistry

The titled compounds 3a—o were synthesized as illustrated in Scheme 1. The pyrazoles 1a—o were synthesized as reported earlier. Compounds 1a—o were then treated with 4-nitrophenylchloroformate in acetonitrile in the presence of pyridine to afford phenylcarbamate derivatives 2a—o. The pyrazolyl urea derivatives 3a—o were synthesized by treating compounds 2a—o with ammonium acetate in THF in the presence of triethylamine. Many literature revealed the use of benzylisocyanate for converting amino groups into ureas. In the present study, benzylisocyanate was used for the preparation of urea derivatives but the reaction failed to yield the desired products. Further literature surveys revealed that amines when treated with 4-nitrophenylchloroformate, followed by the treatment with ammonium acetate, provided the corresponding ureas in high yield and purity even in aqueous environment. Following this method the titled compounds were obtained in good yields and were found to be pure.

Scheme 1

Reaction protocol for the synthesis of 3a—o. Reagent and conditions: (i) 4-nitrophenylchloroformate, pyridine, CH3CN; (ii) ammonium acetate, Et3N, THF.

The NH2 protons of the pyrazoles 1a—o were observed at δ 5.17–5.24, which disappeared in the phenyl carbamate derivatives 2a—o. Appearance of a singlet for CONH protons from δ 10.21–10.47 confirmed the formation of phenyl carbamate derivatives. The formation of 3a—o was confirmed by the appearance of a singlet for NH2 protons from δ 6.12–6.23 showing the presence of a urea group in the compounds. Compounds 3a—o also showed a singlet for the CONH protons from δ 10.13 to 10.55. The 13C NMR spectral data of 3a—o showed characteristic peaks for C=O carbons from δ 170.11 to 171.96. The pyrazole carbons were detected at δ 159.21 to 161.48 (pyrazole C3), δ 102.48 to 105.68 (pyrazole C4) and δ 150.97 to 159.66 (pyrazole C5). The OCH2 carbon showed distinct peaks at δ 70.19 to 72.54. Mass spectra of compounds 3a—o showed molecular ion peaks M+ at an m/z corresponding to their molecular formula.

p38α MAPK assay

The modulation of p38α MAPK activity by all the synthesized compounds 3a–o was determined in an enzyme assay measuring the inhibition of the p38α MAPK mediated ATF-2 phosphorylation. Compound 3c with a 4-chlorophenyl group at position 1 of the pyrazole ring exhibited the strongest p38α MAPK inhibition (IC50 = 0.037 ± 1.56 µmol/L) in comparison to the reference standard, SB 203580 (IC50 = 0.043 ± 3.62 µmol/L). Substitution of the 4-chlorophenyl group by 2-chloro (3a), 3-chloro (3b), 4-fluoro (3e) and 4-bromo (3g) groups also led to similar p38α MAPK activity (IC50 = 0.039 ± 0.04, 0.039 ± 1.50, 0.048 ± 0.01, 0.042 ± 1.22 µmol/L, respectively). The IC50 value was decreased slightly for compound 3h having a 4-nitro group (IC50 = 0.067 ± 0.95 µmol/L). Replacement with a disubstituted electron withdrawing groups, such as 3,4-dichloro (3d), 2,4-dinitro (3i) and 4-fluoro-3-chloro (3f), resulted in further decrease of p38α MAPK inhibitory activity (IC50 = 0.079 ± 0.65, 0.112 ± 0.04 and 0.110 ± 0.17 µmol/L, respectively). When electron withdrawing groups were replaced with electron donating groups like 2-methyl (3j), 3-methyl (3k), 4-methyl (3l), 2-methoxy (3n) and 4-methoxy (3o), led to a further decrease of activity (IC50 ranging from 0.123 ± 1.74 to 0.210 ± 0.11 µmol/L). It was observed that when these electron donating groups were replaced by disubstituted methyl group (2,6-dimethyl, 3m), the activity was found to be minimum (IC50 = 0.270 ± 4.22 µmol/L) (Table 1). Thus the initial activity profile suggests that electron withdrawing groups attached to the position 1 of pyrazole ring were more active than electron donating groups. Furthermore, monosubstituted compounds were found to be more active than disubstituted compounds. On the basis of these results, seven compounds (3a—e, 3g and 3h) showing good inhibitory activity were selected for the in vivo anti-inflammatory activity screening.

Table 1

IC50 values against p38α MAPK and XP docking glide score of 3a—o and the standard, SB 203580.

Compd.	R	IC50 value (µmol/L)a	Glide scoreb
3a	2-Cl	0.039±0.04	−8.197
3b	3-Cl	0.039±1.50	−8.782
3c	4-Cl	0.037±1.56	−8.872
3d	3,4-diCl	0.079±0.65	−8.083
3e	4-F	0.048±0.01	−8.705
3f	4-F-3-Cl	0.110±0.17	−9.175
3g	4-Br	0.042±1.22	−8.818
3h	4-NO2	0.067±0.95	−7.610
3i	2,4-diNO2	0.112 ±0.04	−7.159
3j	2-CH3	0.123±1.74	−8.227
3k	3-CH3	0.210±0.11	−8.520
3l	4-CH3	0.169±0.07	−8.945
3m	2,6-diCH3	0.270±4.22	−7.567
3n	2-OCH3	0.206±0.37	−8.012
3o	4-OCH3	0.170±0.87	−7.753
SB 203580	–	0.043±3.62	−8.795

Mean ± SEM of three experiments.

Glide score denotes g score obtained for docking with p38α MAPK (PDB ID: 3D83).

Antioxidant activity

In this study all the synthesized compounds were evaluated for their free radical scavenging property by measuring the decrease in the absorption of the stable 2,2′-diphenyl-1-picryl hydrazyl radical (DPPH) at 517 nm. This bleaching occurs when the odd electron of the radical is paired and is independent of any enzymatic activity. All the tested compounds showed antioxidant activity ranging from 75.06% to 31.75% when compared to a standard butylated hydroxy anisole (BHA) (71.53%). The most active compound of the series was found to be 3c showing 75.06% antioxidant activity. Unfortunately compounds 3j, 3n and 3o did not exhibit any significant antioxidant activity (Fig. 2).

Figure 2

Antioxidant activity of derivatives 3a—o. Compounds 3j, 3n and 3o did not exhibit significant antioxidant activity. Data are expressed as mean ± SD, n=3. *P<0.05 compared to BHA. BHA: butylated hydroxy anisole.

Anti-inflammatory activity

Compounds 3a—e, 3g and 3h showing good p38α MAPK inhibitory activity (IC50 ranging from 0.037 ± 1.56 to 0.079 ± 0.65 µmol/L) were further evaluated for their anti-inflammatory activity (in vivo) by the carrageenan-induced rat paw edema method. The compounds and standard drug, diclofenac sodium, were tested at an equimolar oral dose (10 mg/kg body weight). The tested compounds showed anti-inflammatory activity ranging from 62.25% to 80.93%, whereas the standard drug showed 81.72% inhibition after 4h. It was observed that compound 3c having a 4-chloro group showing high p38α MAPK activity also showed the highest anti-inflammatory activity (80.93% inhibition). Replacement of 4-chloro group by 2-chloro (3a) and 3-chloro (3b) groups resulted in a slight decrease of activity (76.19% and 78.06% inhibition, respectively). Substitution with a 4-fluoro (3e), 4-bromo (3g) and 4-nitro (3h) groups resulted in further decrease of anti-inflammatory activity (72.33%, 74.01% and 68.38% inhibition, respectively). Compound 3d having a disubstituted group (3,4-dichloro) showed minimum anti-inflammatory activity (62.25% inhibition, Table 2). It was observed that monosubstituted electron withdrawing groups in the phenyl ring attached to the pyrazole nucleus showed good anti-inflammatory activity in both the in vitro and in vivo models.

Table 2

Anti-inflammatory activity of 3a—e, 3g, 3h and diclofenac sodium.

Compd.	Increase in paw edema (mL)a	Inhibition (%)	Activity relative to diclofenac sodium
3a	0.40±0.08	76.19b	93.23
3b	0.37±0.20	78.06b	95.52
3c	0.32±0.06	80.93b	99.03
3d	0.64±0.05	62.25	76.17
3e	0.47±0.16	72.33	88.50
3g	0.44±0.06	74.01b	90.56
3h	0.53±0.12	68.38	83.67
Control	1.69±0.04	—	—
Diclofenac sodium	0.31±0.13	81.72	100

— not applicable.

Data are expressed as mean±SEM, and analyzed by Student׳s t-test for n=6.

Values are statistically significant compared to control group (P<0.05).

Ulcerogenic potential and lipid peroxidation

Compounds 3a—e, 3g and n class="Chemical">3h were also tested for their ulcerogenic activity at an oral dose of 30 mg/kg. Compound 3c showed minimum ulcerogenicity (severity index 0.58 ± 0.37). Compounds 3a, 3b, 3d, 3e, 3g and 3h also showed reduction in severity index (ranging from 0.67 ± 0.25 to 1.08 ± 0.37) superior to the standard drug, diclofenac (severity index 1.83 ± 0.27) (Table 3). Thus it was observed that the tested compounds have better GI safety profile than the standard drug.

Table 3

Ulcerogenic, lipid peroxidation and TNF-α inhibition activities of 3a—e, 3g, 3h and the standards.

Compd.	Severity indexa	Lipid peroxidation (nmol MDA/100 mg tissue)a	TNF-α inhibition (%)a	ID50 (mg/kg)
Control	0.00±0.00	3.26±0.12	—	—
3a	0.67±0.25b	5.47±0.44b	60.87±2.65b	19.98
3b	0.67±0.40b	5.19±0.48b	62.45±0.07b	11.32b
3c	0.58±0.37b	5.12±0.55b	62.56±0.24	9.67b
3d	1.08±0.37b	6.83±0.48b	52.60±2.16	—
3e	0.83±0.22	6.08±0.15	57.34±3.05	—
3g	0.75±0.27	6.02±0.17a	58.13±0.28	—
3h	0.91±0.20	6.84±0.18	52.76±2.08	—
Diclofenac sodium	1.83±0.27	6.52±0.42	—	—
SB 203580	—	—	52.11±1.08b	28.40b

— not applicable.

Data were expressed as mean±SEM and analyzed by Student׳s t-test for n=6.

Values are statistically significant compared to respective standard (P<0.05).

The compounds showing high anti-inflammatory activity and reduced ulcerogenic potential were also tested for their lipid peroxidation, which was measured as nmol of malondialdehyde (MDA) per 100 mg of gastric mucosal tissue. The control group showed 3.26 ± 0.12 nmol/100 mg of lipid peroxidation, whereas diclofenac sodium (standard drug) showed 6.52 ± 0.42 nmol/100 mg MDA. It was found that all the tested compounds showed reduced lipid peroxidation superior to the standard drug except compounds 3d and 3h, which showed slightly higher lipid peroxidation (Table 3). Thus it may be concluded that the protection of gastric mucosa might be related to the inhibition of lipid peroxidation.

TNF-α production inhibition evaluation

Compounds 3a—e, 3g and n class="Chemical">3h were also evaluated for their inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production in mice. Compound 3c was found to be most effective with an ID50 value of 9.67 mg/kg, which was more active than that of SB 203580 (ID50 = 28.40 mg/kg). Analogs 3a and 3b also demonstrated good TNF-α inhibitory efficacy with ID50 values of 19.98 and 11.32 mg/kg, respectively (Table 3).

Docking study

The crystal structures of p38α MAPK complexed with inhibitors were selected as the protein target used for the docking study. Among the compounds studied for p38α MAPK inhibition, compound 3c showing high p38α MAPK activity (IC50= 0.037 ± 1.56 µmol/L) and in vivo anti-inflammatory activity (80.93% inhibition) was found to be potent. The most stable docking pose was selected according to the best docking scored conformation predicted by the Glide scoring function. The orientation and conformation of the docked compound are similar to those of SB 203580 (p38α prototype inhibitor). The binding modes of 3c and the prototype inhibitor (SB 203580) to a rearranged (DFG-out) form of p38α are presented in Fig. 3. Compound 3c and SB 203580 showed a common interaction with amino acid residue ASP 168 and LYS 53. The nitrogen atom of pyrazole scaffold of 3c formed hydrogen bond with the backbone of ASP 168 (N….HN, 2.65 Å). The oxygen atom in the carbonyl functionality in 3c forms a hydrogen bond with the side chain of LYS 53 (C=O….HN, 3.84 Å) residue in the binding pocket, shown in Fig. 3A. Moreover the urea moiety forms additional hydrogen bonds with THR 106 (H….OH, 2.14 Å) and Ala 51 (O….HN, 2.25 Å; O….HN, 3.73 Å). The receptor surface view of compound 3c in the protein is shown in Fig. 3B and superimposed docked pose of compound 3c with SB 203580 is also represented in Fig. 3D, which confirms their identical orientation and alignment. Furthermore, the docked pose of SB 203580 revealed that the pyrazole NH and N atoms formed two hydrogen bonds with the side chain of GLU 71 (1.89 Å, H…O) and backbone of ASP 168 (2.70 Å, N…H) in the binding pocket. Finally, SB 203580 adopts a favorable conformation in which one of the phenyl ring formed a π-cation interaction with LYS 53 (4.21 Å, Phenyl…+HN). The glide scores of 3a—o were in the range of −9.175 to −7.159. Compound 3c showed a glide score of −8.872 and SB 203580 had a glide score of −8.795 (Table 1).

Figure 3

(A) Docked pose of compound 3c (green color) represented as tube in the binding site of p38α MAPK showing hydrogen bond interaction (yellow dashed lines) with ASP 168, LYS 53, ALA 51 and THR 106; (B) Receptor surface view of compound 3c (green color) (C) Docked pose of SB 203580 (pink color) represented as tube in the binding site of p38α MAPK showing hydrogen bond interaction (yellow dashed lines) with ASP 168, GLU 71 and π-cation interaction with LYS 53; (D) Superimposed docked pose of compound 3c (green color) with SB 203580 (pink color) in the binding site.

Conclusions

Fifteen new pyrazolyl urea derivatives have been synthesized and subjected to in vitro screening for p38α MAPK inhibition and antioxidant activities. Moreover, the in vivo anti-inflammatory, ulcerogenic, lipid peroxidation and TNF-α inhibition activities of the compounds have also been tested. The results of the in vitro activities revealed that compounds 3a—e, 3g and 3h bearing 2-chloro, 3-chloro, 4-chloro, 3,4-dichloro, 4-fluoro, 4-bromo and 4-nitro phenyl groups, respectively, at the position 1 of pyrazole ring showed better p38α MAPK inhibition as compared to the standard inhibitor (SB 203580). Compound 3c was found to have superior antioxidant activity in comparison to the reference compound, BHA. It was noted that compound 3c was a potent anti-inflammatory agent comparable to the standard reference drug diclofenac sodium. This compound also showed reduced ulcerogenic potential and the weakest activity in inducing oxidative stress in tissues compared to the standard drug. Compound 3c also demonstrated good inhibition of LPS-induced TNF-α production in mice. It was observed that compounds showing significant in vitro p38α MAPK activity also showed good in vivo anti-inflammatory and TNF-α inhibitory activities. In summary, among all the tested compounds, 1-[4-[5-(4-(benzyloxy)phenyl)-1-(4-chlorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3c) was the most potent anti-inflammatory agent having reduced ulcerogenic liability and lipid peroxidation.

Our earlier studies on pyrazole derivatives having a sulfonamide moiety also showed good anti-inflammatory and TNF-α inhibitory activities. Compound 4a (Fig. 4) having 2-chloro group at the position 1 of pyrazole ring has shown high anti-inflammatory and TNF-α inhibitory activities. Whereas in the present studies, pyrazole derivatives having a urea pharmacophore also showed comparable anti-inflammatory activity along with improved TNF-α inhibitory properties. Furthermore, pyrazolyl urea derivatives were also studied for their p38α MAPK inhibition and showed significant activity.

Figure 4

Structure of compound 4a

Experimental

Melting points (mp, °C) were recorded using a Labtronics digital melting point apparatus (Haryana, India) and were uncorrected. IR spectra were recorded on a Perkin-Elmer 1720 FTIR spectrometer (New York, USA). 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz) were obtained on a Bruker Avance NMR spectrometer (Zurich, Switzerland) using tetramethysilane (TMS) as the internal reference with complete proton decoupling. MS analyses were performed on a Jeol SX-102 spectrometer (Tokyo, Japan). Thin layer chromatography (TLC) was performed on silica gel G (Merck) and spots were visualized under the ultraviolet light (UV 254 nm). Elemental analyses (C, H and N) were conducted using a CHNS Vario EL III machine (Elementar Analysen systeme GmbH, Germany).

4-[5-[4-(Benzyloxy)phenyl]-1-substituted phenyl-1H-pyrazol-3-yl]anilines (1a—o)

The compounds were synthesized using our earlier reported method.

General method for the synthesis of 4-nitrophenyl 4-[5-(4-(benzyloxy)phenyl)-1-substituted-1H-pyrazol-3-yl]phenylcarbamates (2a—o)

A mixture of one equivalent of the 4-[5-(4-(benzyloxy)phenyl)-1-substituted phenyl-1H-pyrazol-3-yl]anilines 1a—o (5 mmol), five equivalents of 4-nitrophenylchloroformate (25 mmol) and 5 equivalents of pyridine (5 mL) in the presence of acetonitrile (20 mL) was stirred at room temperature for 3–5 h. The reaction mixture was quenched with water (100 mL). An oily material thus obtained was kept aside overnight at room temperature. Solid thus obtained was filtered, washed with water, dried and recrystallized from ethanol.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2a)

Yield 82%; mp 157–158 °C; IR (cm−1, KBr): 3031 (C-H), 1670 (CONH), 1598 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.27 (s, 1H, CONH, D2O exchangeable), 7.42–7.89 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.12 (s, 2H, OCH2); Anal. Calcd. for C35H25ClN4O5: C, 68.13; H, 4.08; N, 9.08; Found: C, 68.20; H, 4.02; N, 9.11.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(3-chlorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2b)

Yield 84%; mp 154–156 °C; IR (cm−1, KBr): 3031 (C–H), 1666 (C=O), 1592 (C=N); n class="Chemical">1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.47–7.88 (m, 21H, Ar–H), 7.02 (s, 1H, pyrazole-H-4), 5.16 (s, 2H, OCH2); Anal. Calcd. for C35H25ClN4O5: C, 68.13; H, 4.08; N, 9.08; Found: C, 68.23; H, 4.14; N, 9.07.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-chlorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2c)

Yield 79%; mp 163–164 °C; IR (cm−1, KBr): 3035 (C-H), 1668 (CONH), 1595 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.36 (s, 1H, CONH, D2O exchangeable), 7.43–7.89 (m, 21H, Ar-H), 7.09 (s, 1H, pyrazole-H-4), 5.21 (s, 2H, OCH2); Anal. Calcd. for C35H25ClN4O5: C, 68.13; H, 4.08; N, 9.08; Found: C, 68.06; H, 4.03; N, 9.05.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(3,4-dichlorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2d)

Yield 77%; mp 158–159 °C; IR (cm−1, KBr): 3031 (C-H), 1672 (CONH), 1596 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.47 (s, 1H, CONH, D2O exchangeable), 7.42–7.91 (m, 20H, Ar-H), 7.11 (s, 1H, pyrazole-H-4), 5.13 (s, 2H, OCH2); Anal. Calcd. for C35H24Cl2N4O5: C, 64.52; H, 3.71; N, 8.60; Found: C, 64.56; H, 3.64; N, 8.65.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2e)

Yield 82%; mp 186–187 °C; IR (cm−1, KBr): 3032 (C-H), 1663 (CONH), 1585 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.44 (s, 1H, CONH, D2O exchangeable), 7.39–7.88 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.18 (s, 2H, OCH2); Anal. Calcd. for C35H25FN4O5: C, 69.99; H, 4.20; N, 9.33; Found: C, 69.96; H, 4.13; N, 9.27.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-fluoro-3-chlorophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2f)

Yield 75%; mp 202–203 °C; IR (cm−1, KBr): 3033 (C-H), 1672 (CONH), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.21 (s, 1H, CONH, D2O exchangeable), 7.42–7.85 (m, 20H, Ar-H), 7.08 (s, 1H, pyrazole-H-4), 5.16 (s, 2H, OCH2); Anal. Calcd. for C35H24ClFN4O5: C, 66.20; H, 3.81; N, 8.82; Found: C, 66.16; H, 3.83; N, 8.75.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-bromophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2g)

Yield 77%; mp 171–172 °C; IR (cm−1, KBr): 3031 (C-H), 1658 (CONH), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.26 (s, 1H, CONH, D2O exchangeable), 7.40–7.84 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.16 (s, 2H, OCH2); Anal. Calcd. for C35H25BrN4O5: C, 63.55; H, 3.81; N, 8.47; Found: C, 63.61; H, 3.89; N, 8.45.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-nitrophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2h)

Yield 81%; mp 188–189 °C; IR (cm−1, KBr): 3030 (C-H), 1671 (CONH), 1594 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.33 (s, 1H, CONH, D2O exchangeable), 7.36–7.82 (m, 21H, Ar-H), 7.07 (s, 1H, pyrazole-H-4), 5.09 (s, 2H, OCH2); Anal. Calcd. for C35H25N5O7: C, 66.98; H, 4.02; N, 11.16; Found: C, 66.96; H, 4.09; N, 11.09.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-3-yl]phenyl carbamate (2i)

Yield 89%; mp 178–179 °C; IR (cm−1, KBr): 3031 (C-H), 1673 (C=O), 1597 (C=N); n class="Chemical">1H NMR (DMSO-d6): δ 10.39 (s, 1H, CONH, D2O exchangeable), 7.42–7.86 (m, 20H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.16 (s, 2H, OCH2); Anal. Calcd. for C35H24N6O9: C, 62.50; H, 3.60; N, 12.49; Found: C, 62.56; H, 3.68; N, 12.54.

4-Nitrophenyl 4-[5-(4-(benzyloxy)phenyl)-1-(2-methylphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2j)

Yield 81%; mp 181–183 °C; IR (cm−1, KBr): 3029 (C-H), 1656 (CONH), 1588 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.29 (s, 1H, CONH, D2O exchangeable), 7.42–7.84 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.10 (s, 2H, OCH2), 2.27 (s, 3H, CH3); Anal. Calcd. for C36H28N4O5: C, 72.47; H, 4.73; N, 9.39; Found: C, 72.52; H, 4.79; N, 9.31.

4-Nitrophenyl 4-[5-(4-(benzyloxy)phenyl)-1-(3-methylphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2k)

Yield 84%; mp 184–185 °C; IR (cm−1, KBr): 3037 (C-H), 1674 (CONH), 1592 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.34 (s, 1H, CONH, D2O exchangeable), 7.41–7.87 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.11 (s, 2H, OCH2), 2.29 (s, 3H, CH3); Anal. Calcd. for C36H28N4O5: C, 72.47; H, 4.73; N, 9.39; Found: C, 72.48; H, 4.76; N, 9.35.

4-Nitrophenyl 4-[5-(4-(benzyloxy)phenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2l)

Yield 81%; mp 185–186 °C; IR (cm−1, KBr): 3031 (C-H), 1663 (CONH), 1585 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.38 (s, 1H, CONH, D2O exchangeable), 7.36–7.93 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.22 (s, 2H, OCH2), 2.30 (s, 3H, CH3); Anal. Calcd. for C36H28N4O5: C, 72.47; H, 4.73; N, 9.39; Found: C, 72.53; H, 4.68; N, 9.34.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(2,6-dimethylphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2m)

Yield 74%; mp 193–195 °C; IR (cm−1, KBr): 3037 (C-H), 1668 (CONH), 1592 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.23 (s, 1H, CONH, D2O exchangeable), 7.42–7.82 (m, 20H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.12 (s, 2H, OCH2), 2.17 (s, 6H, CH3); Anal. Calcd. for C37H30N4O5: C, 72.77; H, 4.95; N, 9.17; Found: C, 72.73; H, 4.98; N, 9.24.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(2-methoxyphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2n)

Yield 72%; mp 165–166 °C; IR (cm−1, KBr): 3029 (C–H), 1661 (CONH), 1595 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.35 (s, 1H, CONH, D2O exchangeable), 7.42–7.88 (m, 21H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 5.18 (s, 2H, OCH2), 3.69 (s, 3H, OCH3); Anal. Calcd. for C36H28N4O6: C, 70.58; H, 4.61; N, 9.15; Found: C, 70.53; H, 4.68; N, 9.09.

4-Nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-(4-methoxyphenyl)-1H-pyrazol-3-yl]phenyl carbamate (2o)

Yield 77%; mp 169–170 °C; IR (cm−1, KBr): 3032 (C-H), 1674 (CONH), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.41 (s, 1H, CONH, D2O exchangeable), 7.38–7.92 (m, 21H, Ar-H), 7.03 (s, 1H, pyrazole-H-4), 5.21 (s, 2H, OCH2), 3.72 (s, 3H, OCH3); Anal. Calcd. for C36H28N4O6: C, 70.58; H, 4.61; N, 9.15; Found: C, 70.51; H, 4.57; N, 9.11.

General method for the synthesis of 1-[4-[5-(4-(benzyloxy)phenyl)-1-(substituted phenyl)-1H-pyrazol-3-yl]phenyl]ureas (3a—o)

A mixture of 4-nitrophenyl-4-[5-(4-(benzyloxy)phenyl)-1-substituted-1H-pyrazol-3-yl]phenylcarbamates2a—o(5 mmol)n class="Chemical">and ammonium acetate (20 mmol) in the presence of triethylamine (20 mmol) and tetrahydrofuran (20 mL) was stirred at room temperature for 4–6 h. The reaction mixture was then poured into ice water (30 mL) while stirring. The compound obtained was filtered, washed with water and dried. The residue thus obtained was triturated in hot DCM, filtered and recrystallized from ethanol.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3a)

Yield 64%; mp 210–211 °C; IR (cm−1, KBr): 3352 (N-H), 3031 (C-H), 1652 (n class="Chemical">CONH), 1598 (C=N); 1H NMR (DMSO-d6): δ 10.32 (s, 1H, CONH, D2O exchangeable), 7.49–8.16 (m, 17H, Ar-H), 7.12 (s, 1H, pyrazole-H-4), 6.18 (s, 2H, NH2, D2O exchangeable), 5.23 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 170.17 (C=O), 161.25 (pyrazole C3), 150.97 (pyrazole C5), 144.74, 138.25, 135.11, 135.07, 134.18, 133.82, 129.78, 129.15, 128.43, 128.38, 128.05, 127.83, 125.62, 122.09, 121.84, 121.09, 117.98, 112.99, 103.97 (pyrazole C4), 72.09 (OCH2); ESI-MS (m/z): 510 [M+H]+, 511 [M+2]+; Anal. Calcd. for C29H23ClN4O2: C, 70.37; H, 4.68; N, 11.32; Found: C, 70.41; H, 4.61; N, 11.28.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(3-chlorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3b)

Yield 58%; mp 192–193 °C; IR (cm−1, KBr): 3344 (N-H), 3038 (C-H), 1656 (n class="Chemical">CONH), 1592 (C=N); 1H NMR (DMSO-d6): δ 10.13 (s, 1H, CONH, D2O exchangeable), 7.38–8.07 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.16 (s, 2H, NH2, D2O exchangeable), 5.15 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 171.83 (C=O), 160.83 (pyrazole C3), 159.07 (pyrazole C5), 151.52, 144.65, 138.27, 136.93, 136.48, 134.72, 130.45, 130.18, 128.90, 128.73, 128.67, 128.42, 125.13, 123.58, 122.38, 121.27, 119.83, 113.79, 105.38 (pyrazole C4), 71.49 (OCH2); ESI-MS (m/z): 510 [M+H]+, 511 [M+2]+; Anal. Calcd. for C29H23ClN4O2: C, 70.37; H, 4.68; N, 11.32; Found: C, 70.32; H, 4.63; N, 11.36.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(4-chlorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3c)

Yield 62%; mp 210–211 °C; IR (cm−1, KBr): 3345 (N-H), 3035 (C-H), 1631 (n class="Chemical">CONH), 1595 (C=N); 1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.42–8.09 (m, 17H, Ar-H), 7.07 (s, 1H, pyrazole-H-4), 6.19 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 170.21 (C=O), 159.67 (pyrazole C3), 158.12 (pyrazole C5), 151.36, 143.25, 138.31, 136.72, 136.16, 135.94, 130.19, 129.87, 128.97, 128.83, 128.58, 128.14, 125.66, 122.93, 122.62, 120.93, 117.61, 113.18, 104.75 (pyrazole C4), 70.56 (OCH2); ESI-MS (m/z): 510 [M+H]+, 511 [M+2]+; Anal. Calcd. for C29H23ClN4O2: C, 70.37; H, 4.68; N, 11.32; Found: C, 70.39; H, 4.72; N, 11.39.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(3,4-dichlorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3d)

Yield 62%; mp 253–254 °C; IR (cm−1, KBr): 3348 (N-H), 3031 (C-H), 1629 (C=O), 1596 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.42–8.09 (m, 16H, Ar-H), 7.07 (s, 1H, pyrazole-H-4), 6.15 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 171.65 (C=O), 160.29 (pyrazole C3), 159.05 (pyrazole C5), 152.07, 144.37, 138.16, 136.54, 136.37, 135.12, 130.86, 130.65, 128.34, 128.14, 128.11, 128.06, 124.95, 123.61, 122.98, 122.65, 119.57, 114.06, 104.32 (pyrazole C4), 71.32 (OCH2); ESI-MS (m/z): 544 [M+H]+, 545 [M+2]+; Anal. Calcd. for C29H22Cl2N4O2: C, 65.79; H, 4.19; N, 10.58; Found: C, 65.84; H, 4.22; N, 10.64.

1-[4-[5-(4-(benzyloxy)phenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-yl]phenyl]urea (3e)

Yield 66%; mp 261–262 °C; IR (cm−1, KBr): 3356 (N-H), 3032 (C-H), 1631 (C=O), 1585 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.44 (s, 1H, CONH, D2O exchangeable), 7.49–8.16 (m, 17H, Ar-H), 7.14 (s, 1H, pyrazole-H-4), 6.19 (s, 2H, NH2, D2O exchangeable), 5.23 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 170.34 (C=O), 159.21 (pyrazole C3), 158.36 (pyrazole C5), 152.65, 144.16, 138.51, 135.43, 135.23, 134.86, 129.65, 129.12, 128.95, 128.63, 128.44, 127.09, 124.07, 123.54, 121.58, 120.87, 118.43, 114.17, 104.69 (pyrazole C4), 71.62 (OCH2); ESI-MS (m/z): 494 [M+H]+; Anal. Calcd. for C29H23FN4O2: C, 72.79; H, 4.84; N, 11.71; Found: C, 72.76; H, 4.90; N, 11.77.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(3-chloro-4-fluorophenyl)-1H-pyrazol-3-yl]phenyl] urea (3f)

Yield 63%; mp 220–221 °C; IR (cm−1, KBr): 3351 (N-H), 3033 (C-H), 1632 (C=O), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.21 (s, 1H, CONH, D2O exchangeable), 7.42–7.82 (m, 16H, Ar-H), 7.10 (s, 1H, pyrazole-H-4), 6.13 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 170.69 (C=O), 159.54 (pyrazole C3), 158.79 (pyrazole C5), 151.94, 144.72, 138.09, 135.17, 135.10, 134.94, 129.89, 129.13, 128.82, 128.78, 128.52, 128.17, 124.37, 123.16, 122.43, 120.96, 117.65, 112.65, 105.66 (pyrazole C4), 72.11 (OCH2); ESI-MS (m/z): 528 [M+H]+, 529 [M+2]+; Anal. Calcd. for C29H22ClFN4O2: C, 67.90; H, 4.32; N, 10.92; Found: C, 67.84; H, 4.39; N, 10.95.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(4-bromophenyl)-1H-pyrazol-3-yl]phenyl]urea (3g)

Yield 68%; mp 230–231 °C; IR (cm−1, KBr): 3357 (N-H), 3031 (C-H), 1626 (C=O), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.15 (s, 1H, CONH, D2O exchangeable), 7.42–7.87 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.23 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 171.17 (C=O), 160.62 (pyrazole C3), 158.12 (pyrazole C5), 151.39, 144.51, 137.62, 136.15, 135.82, 134.15, 130.65, 130.09, 128.73, 128.65, 128.32, 127.85, 125.62, 123.65, 121.54, 120.32, 118.58, 112.29, 103.81 (pyrazole C4), 72.18 (OCH2); ESI-MS (m/z): 554 [M+H]+, 555 [M+2]+; Anal. Calcd. for C29H23BrN4O2: C, 64.57; H, 4.30; N, 10.39; Found: C, 64.53; H, 4.37; N, 10.44.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(4-nitrophenyl)-1H-pyrazol-3-yl]phenyl]urea (3H)

Yield 61%; mp 215–216 °C; IR (cm−1, KBr): 3341 (N-H), 3030 (C-H), 1631 (C=O), 1590 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.42–8.09 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.12 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 171.39 (C=O), 161.15 (pyrazole C3), 157.37 (pyrazole C5), 150.62, 143.09, 137.37, 136.12, 136.09, 135.26, 130.73, 129.08, 128.57, 128.42, 128.13, 128.08, 125.37, 123.19, 122.64, 120.43, 118.43, 112.89, 103.07 (pyrazole C4), 70.34 (OCH2); ESI-MS (m/z): 521 [M+H]+; Anal. Calcd. for C29H23N5O4: C, 68.90; H, 4.59; N, 13.85; Found: C, 68.98; H, 4.62; N, 13.91.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-3-yl]phenyl]urea (3i)

Yield 64%; mp 222–223 °C; IR (cm−1, KBr): 3624, 3346 (N-H), 3031 (C-H), 1626 (C=O), 1585 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.39 (s, 1H, CONH, D2O exchangeable), 7.42–7.88 (m, 16H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.20 (s, 2H, NH2, D2O exchangeable), 5.16 (s, 2H, OCH2); 13C NMR (DMSO-d6): δ 170.74 (C=O), 160.37 (pyrazole C3), 157.95 (pyrazole C5), 150.38, 142.96, 138.65, 136.09, 135.26, 134.09, 130.56, 129.13, 128.97, 128.32, 128.05, 127.57, 124.92, 123.42, 121.90, 120.56, 119.37, 112.48, 102.48 (pyrazole C4), 70.19 (OCH2); ESI-MS (m/z): 566 [M+H]+; Anal. Calcd. for C29H22N6O6: C, 63.27; H, 4.03; N, 15.27; Found: C, 63.33; H, 4.09; N, 15.20.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(2-methylphenyl)-1H-pyrazol-3-yl]phenyl]urea (3j)

Yield 57%; mp 200–201 °C; IR (cm−1, KBr): 3632, 3347 (N-H), 3029 (C-H), 1639 (C=O), 1588 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.42–7.83 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.22 (s, 2H, NH2, D2O exchangeable), 5.10 (s, 2H, OCH2), 2.06 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 170.11 (C=O), 160.96 (pyrazole C3), 158.07 (pyrazole C5), 152.77, 142.08, 138.14, 136.02, 135.87, 135.13, 130.75, 129.62, 128.59, 128.32, 128.17, 127.27, 124.85, 122.96, 121.87, 120.61, 119.01, 114.83, 103.67 (pyrazole C4), 72.54 (OCH2), 17.83 (CH3); ESI-MS (m/z): 490 [M+H]+; Anal. Calcd. for C30H26N4O2: C, 75.93; H, 5.52; N, 11.81; Found: C, 75.85; H, 5.59; N, 11.76.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(3-methylphenyl)-1H-pyrazol-3-yl]phenyl]urea (3k)

Yield 55%; mp 202–203 °C; IR (cm−1, KBr): 3635, 3350 (N-H), 3037 (C-H), 1635 (C=O), 1592 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.25 (s, 1H, CONH, D2O exchangeable), 7.42–7.81 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.14 (s, 2H, NH2, D2O exchangeable), 5.11 (s, 2H, OCH2), 2.06 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 170.35 (C=O), 161.48 (pyrazole C3), 159.53 (pyrazole C5), 152.09, 143.73, 138.95, 135.47, 135.05, 134.85, 129.86, 129.15, 128.83, 128.48, 128.39, 128.11, 125.16, 122.58, 121.74, 120.13, 117.46, 114.61, 104.17 (pyrazole C4), 71.56 (OCH2), 17.18 (CH3); ESI-MS (m/z): 490 [M+H]+; Anal. Calcd. for C30H26N4O2: C, 75.93; H, 5.52; N, 11.81; Found: C, 75.96; H, 5.55; N, 11.87.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl]phenyl]urea (3l)

Yield 60%; mp 215–216 °C; IR (cm−1, KBr): 3613, 3358 (N-H), 3031 (C-H), 1635 (C=O), 1585 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.39 (s, 1H, CONH, D2O exchangeable), 7.36–7.91 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.12 (s, 2H, NH2, D2O exchangeable), 5.22 (s, 2H, OCH2), 2.12 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 171.49 (C=O), 160.55 (pyrazole C3), 159.66 (pyrazole C5), 151.08, 143.02, 138.54, 135.39, 135.17, 135.06, 130.52, 129.34, 128.72, 128.65, 128.43, 128.31, 125.08, 123.81, 121.31, 120.07, 117.32, 113.35, 105.04 (pyrazole C4), 71.97 (OCH2), 17.66 (CH3); ESI-MS (m/z): 490 [M+H]+; Anal. Calcd. for C30H26N4O2: C, 75.93; H, 5.52; N, 11.81; Found: C, 75.97; H, 5.48; N, 11.83.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(2,6-dimethylphenyl)-1H-pyrazol-3-yl]phenyl]urea (3m)

Yield 68%; mp 213–214 °C; IR (cm−1, KBr): 3628, 3349 (N-H), 3037 (C-H), 1635 (C=O), 1592 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.21 (s, 1H, CONH, D2O exchangeable), 7.42–7.80 (m, 16H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.18 (s, 2H, NH2, D2O exchangeable), 5.12 (s, 2H, OCH2), 2.11 (s, 6H, CH3); 13C NMR (DMSO-d6): δ 171.96 (C=O), 160.91 (pyrazole C3), 159.27 (pyrazole C5), 152.56, 143.95, 137.53, 135.91, 135.08, 134.73, 129.31, 129.06, 128.56, 128.52, 128.37, 128.08, 125.37, 123.49, 122.06, 120.95, 118.78, 113.47, 105.25 (pyrazole C4), 71.82 (OCH2), 17.53 (2 CH3); ESI-MS (m/z): 489 [M+H]+; Anal. Calcd. for C31H28N4O2: C, 76.21; H, 5.78; N, 11.47; Found: C, 76.18; H, 5.71; N, 11.42.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(2-methoxyphenyl)-1H-pyrazol-3-yl]phenyl]urea (3n)

Yield 55%; mp 240–241 °C; IR (cm−1, KBr): 3633, 3353 (N-H), 3029 (C-H), 1621 (C=O), 1595 (C=n class="Chemical">N); 1H NMR (DMSO-d6): δ 10.23 (s, 1H, CONH, D2O exchangeable), 7.42–7.86 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.23 (s, 2H, NH2, D2O exchangeable), 5.18 (s, 2HH, OCH2), 3.72 (s, 3H, OCH3); 13C NMR (DMSO-d6): δ 170.27 (C=O), 159.76 (pyrazole C3), 151.32 (pyrazole C5), 143.01, 137.08, 134.96, 134.01, 133.12, 133.01, 130.63, 129.27, 128.87, 128.75, 128.69, 128.57, 125.36, 123.16, 122.53, 121.28, 118.03, 113.26, 105.68 (pyrazole C4), 70.65 (OCH2), 56.81 (OCH3); ESI-MS (m/z): 491 [M+H]+; Anal. Calcd. for C30H26N4O3: C, 73.45; H, 5.34; N, 11.42; Found: C, 73.41; H, 5.28; N, 11.47.

1-[4-[5-(4-(Benzyloxy)phenyl)-1-(4-methoxyphenyl)-1H-pyrazol-3-yl]phenyl]urea (3o)

Yield 61%; mp 243–244 °C; IR (cm−1, KBr): 3345 (N-H), 3032 (C-H), 1673 (n class="Chemical">CONH), 1590 (C=N); 1H NMR (DMSO-d6): δ 10.55(s, 1H, CONH, D2O exchangeable), 7.42–7.91 (m, 17H, Ar-H), 7.05 (s, 1H, pyrazole-H-4), 6.21 (s, 2H, NH2, D2O exchangeable), 5.21 (s, 2H, OCH2), 3.74 (s, 3H, OCH3); 13C NMR (DMSO-d6): δ 170.68 (C=O), 159.63 (pyrazole C3), 152.61 (pyrazole C5), 143.68, 138.82, 134.62, 134.13, 134.06, 133.65, 130.54, 129.09, 128.75, 128.63, 128.51, 127.28, 124.19, 123.47, 122.01, 121.79, 119.12, 113.54, 103.21 (pyrazole C4), 72.05 (OCH2), 56.39 (OCH3); ESI-MS (m/z): 491 [M+H]+; Anal. Calcd. for C30H26N4O3: C, 73.45; H, 5.34; N, 11.42; Found: C, 74.38; H, 5.37; N, 11.46.

Pharmacology

p38α MAPK assay was performed using the CycLex p38 Kinase assay kit (Cat# CY-1177) procured from MBL, USA. CycLex p38α positive control (Cat# CY-E1177) was also purchased from MBL, USA. The commercially available ELISA kit (Cat# KB2052, Krishgen Biosystems, Mumbai, India) was used for TNF-α estimation in the mice plasma samples. LPS from Escherichia coli 0111:B4 (Cat# 9028) was obtained from Chondrex, USA. All the pharmacological experiments were conducted in compliance with ethical principles after Institutional Animal Ethics Committee (IAEC, No. 173/CPCSEA, 2000) approval. Animals were obtained from central animal house facility, Hamdard University, New Delhi. The experiments were performed in albino rats of Wistar strain of either sex, weighing 180–200 g. The animals were maintained at 25 ± 2 °C, 50 ± 5% relative humidity and 12 h light/dark cycle. Food and water were freely available up to the time of experiments.

p38α MAPK assay

Inhibition of p38α MAPK activity was determined according to the method of Forrer et al.. All the samples were diluted with a kinase buffer as needed. In the test sample wells kinase reaction buffer (80 µL) and 10× inhibitor compounds or standard (SB 203580) (10 µL) were added. In the solvent control experiment, 80 µL of kinase reaction buffer and 10 µL of solvent for inhibitor were added. In the inhibition control wells, 80 µL of kinase reaction buffer and 10 µL of 10× SB 203580 (20 µmol/L) were added. The reaction in all wells was initiated by adding 10 µL of p38α positive control to each well and mixing thoroughly at room temperature. The plate was covered and incubated at 30 °C for 30 min. The wells were washed five times with a wash buffer. 100 µL of anti-phospho-ATF-2 Thr71 polyclonal antibody PPT-09 was pipetted into each well, covered and incubated at room temperature for 30 min. The wells were washed five times with a wash buffer. HRP-conjugated anti-rabbit IgG (100 µL) was pipetted into each well, covered and incubated at room temperature for 30 min. The wells were washed five times with a wash buffer. 100 µL of substrate reagent (tetramethylbenzidine, TMB) was added to each well and incubated at room temperature for 5–15 min. Finally 100 µL of a stop solution (1 mol/L H2SO4) was added to each well and absorbance was measured in each well using a spectrophotometric plate reader at a wavelength of 450 nm. IC50 values were calculated. All samples were assayed in triplicates.

Antioxidant assay

All the compounds (3a—o) were evaluated for their in vitro free radical scavenging activity by the 2,2′-diphenyl-1-picrylhydrazyl (n class="Chemical">DPPH) radical scavenging method described by Blois et al..

Anti-inflammatory activity

Compounds 3a—e, 3g and n class="Chemical">3h were evaluated for their anti-inflammatory activity by the carrageenan-induced rat paw edema method of Winter et al. using Digital Plethysmometer-PLM-01 Plus (Orchid Scientifics and Innovatives India Pvt., Ltd., Mumbai, India).

Ulcerogenic test and lipid peroxidation

The most active compound of the series 3a—e, 3g and n class="Chemical">3h were evaluated for their ulcerogenic potential in rats by already reported procedure,. Lipid peroxidation in the gastric mucosa was determined according to the already reported method of Ohkawa et al..

TNF-α production inhibition evaluation

TNF-α production inhibition in n class="Species">mice (BALB/c) weighing 20–30 g was carried out using the already reported procedure31, 32.

Molecular docking study

To analyze the biological activity on the basis of structure, molecular docking studies of the compounds were carried out by taking X-ray crystal structure data to establish the interactions of the compounds with the p38α MAPK (PDB code: 3D83 having resolution of 1.90 Å). The molecular docking study was used to understand the possible best binding pose of compounds (3a—o) by which it could be sorted out for identifying promising p38α MAPK inhibitor using Glide extra precision (XP) Maestro 10.1 Schrodinger, (2015-Release-4) running on the Linux 64 operating system. Molecular docking studies mainly involve selection and preparation of appropriate protein, grid generation, ligand preparation followed by docking and analysis. The ligands and the receptors were prepared using the LigPrep and Protein preparation wizard, respectively. Missing hydrogens were added using prime and unwanted water molecules were also removed.

Statistical analysis

Results are expressed as the mean ± SEM (standard error of the mean) of six animals per group. The data obtained from pharmacological experiments were analyzed using the Student׳s t- test. A P value of less than 0.05 was considered statistically significant.