Novel pyrazole carboxylate derivatives as lonazolac bioisosteres with selective COX-2 inhibition: design, synthesis and anti-inflammatory activity

Abstract

Two novel series of di-aryl/tri-aryl substituted pyrazole ester derivatives 15a-h and 19a-d were designed, synthesized as novel non-acidic lonazolac analogs and tested for its COX-2, 5-LOX, 15-LOX, iNOS, pro-inflammatory cytokines TNF-α and PGE2 inhibitory activities. All the tested compounds showed excellent COX-2 inhibitory activity (IC₅₀ = 0.059–3.89 μM), compared to that of celecoxib (IC₅₀ = 0.22 μM), where derivatives 15c, 15d, 15 h and 19d were found to be the most potent showing COX-2 selectivity index in range of (S.I. = 28.56–98.71) compared to celecoxib (S.I. = 13.65). Moreover, the most potent four derivatives 15c, 15d, 15 h and 19d showed outstanding 5-LOX and 15-LOX inhibitory activities (IC₅₀ = 0.24–0.81, 0.20–2.2 respectively, compared to zileuton IC₅₀ = 1.52 and 0.54, respectively). Further investigation of the anti-inflammatory mechanistic study of derivatives 15c, 15d, 15 h and 19d revealed that these four compounds exhibited comparable TNF-α and PGE2 (LPS-induced pro-inflammatory cytokines) inhibitory activities (IC₅₀ = 0.77–1.20 μM and 0.28–0.52 μM respectively) when compared to celecoxib (IC₅₀ = 0.87 μM and 0.38 μM respectively) as reference drug using lipopolysaccharide-activated RAW 264.7 macrophages. Based on the advanced inhibitory activity of compounds 15c, 15d, 15 h and 19d against LPS-induced pro-inflammatory mediators (TNF-α and PGE2), inducible nitric oxide synthase (iNOS) inhibition assay was carried out. Remarkably, compounds 15c, 15d, 15 h and 19d showed higher potency with lower IC₅₀ (0.41–0.61 µM) when compared to the reference drug celecoxib (0.48 µM). Prior to in vivo anti-inflammatory activity screening, cytotoxicity testing was performed to ascertain safe and non-toxic concentrations of each compound. Safe doses of compounds were determined using lipopolysaccharide-activated RAW 264.7 macrophages, moreover results showed that compounds 15c, 15d, 15 h and 19d were more safer (less cytotoxic) with higher IC₅₀ (178.95–301.40 µM) when compared to the reference drug celecoxib (148.90 µM). In vivo anti-inflammatory activity of the target compounds 15c, 15d, 15 h and 19d reinforced the results of in vitro screening as the derivatives 15c, 15d, 15 h and 19d showed (ED₅₀ = 8.22–31.22 mg/kg, respectively) and were more potent than celecoxib (ED₅₀ = 40.39 mg/kg). All screened derivatives 15c, 15d, 15 h and 19d were less ulcerogenic (ulcer indexes = 1.22–3.93) than lonazolac (ulcer index = 20.30) and comparable to celecoxib (ulcer index = 3.02). In silico docking and ADME studies were carried out in order to clarify the interactions of the most active derivatives 15c, 15d, 15 h and 19d with the target enzymes and their pharmacokinetic parameters.

Introduction

Inflammation is the body tangled protective mechanism against any pathogens, injuries, or bacterial invasion [1]. This mechanism is characterized by swelling, redness and pain associated with vascular and cellular alterations [2]. It is a complex process in which many enzymes and mediators are involved such as inducible NO synthetase (iNOS), 5-LOX, 15-LOX and COX-2 that leads to development of inflammation-related diseases, such as cardiovascular disorders and cancer [3, 4].

The first stage in the inflammatory response is the release of pro-inflammatory mediators (e.g., prostaglandins, histamine, leukotrienes and inducible nitric oxide NO), this leads to vasodilation, as a consequence, a series of biochemical proceedings and passage of leukocytes occurs from blood to the affected tissue [5].

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs due to their anti-inflammatory, antipyretic and analgesic actions [6]. The anti-inflammatory activity of NSAIDs is due to their ability to inhibit cyclooxygenase (COX)-mediated production of pro-inflammatory mediators like; prostaglandins (PGs), cytokines tumor necrosis factor (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6) and thromboxanes (TXs) [7]. Additionally, lipopolysaccharide (LPS) is one of the essential elements promote macrophage activation, causing the release of transcription factors and pro-inflammatory mediators [8]. Controlling the NO and PGE2 generation in LPS-stimulated macrophages is therefore thought to be a great model for investigating of the effectiveness of the anti-inflammatory drugs [9, 10].

On the other hand, lipoxygenases (LOXs) are a non-heme iron-containing dioxygenases that catalyze the synthesis of leukotrienes from arachidonic acid and other unsaturated fatty acids by adding molecular oxygen (O₂) in the form of a hydro-peroxyl (HO₂) residue [11, 12]. From literature survey, 5-lipoxygenase (5-LOX) has been escorting to disorders like asthma, chronic bronchitis, rheumatoid arthritis and cancer through controlling LOX pathway which is mediated by inflammation and hyper-proliferation [13–15]. Furthermore, inducible-NOS (iNOS) are one of the nitric oxide synthase isoforms that can be stimulated by bacterial lipopolysaccharide (LPS) or cytokines such as TNF-α. Inducible-NOS produce large, cytotoxic amounts of NO that can mediate inflammation and innate immune response [16–18].

Many manuscripts revealed that, Coxibs containing the bioactive pharmacophores, either (SO₂NH₂) as in celecoxib (1) with pyrazole scaffold or (SO₂CH₃) as in rofecoxib (2) with furanone as central ring are characterized by the Y-shaped structural design and considered as selective COX-2 inhibitors with the most brilliant scaffolds displaying advanced anti-inflammatory activity [19–22]. Lonazolac (3) is 1,3-disubstituted-pyrazole acetic acid drug, with potent analgesic and anti-inflammatory activities, but is non-selective COX-2 inhibitor so it associated with gastrointestinal side effects like other traditional NSAIDs.

In 2018, it was reported by our team through some structural modifications of lonazolac (3), the synthesis of non-acidic 1,3,4-trisubstituted-pyrazole derivatives as lonazolac analogs (4a-l) [19]. Additionally, the pyrazolyl oxime derivative (5) was designed, synthesized and tested for anti-inflammatory activities; it was found to be more selective for COX-2 isozyme in comparison to celecoxib (1) [23]. Previously, synthesis of 1,5-diarylpyrazole carboxylic acid (6) was reported, which had higher COX-2 selectivity index (S.I. = 2.94) compared to that of celecoxib (S.I. = 7.70) [24].

Furthermore, a novel series of fluorinated triaryl-pyrazoles (7a-c) was reported to be with advanced anti-inflammatory activity at all-time intervals (% edema inhibition = 42.1–87.9) with better gastric profile (ulcer index U.I. = 1.25–2.5) than the traditional NSAID; indomethacin (ulcer index U.I. = 14) and were close to the selective COX-2 inhibitor; celecoxib (ulcer index U.I. = 1.75) [25]. Moreover, compounds (8a) (IC₅₀ = 0.67 μM) and (8b) (IC₅₀ = 0.58 μM) showed better COX-2 inhibitory activity than celecoxib (IC₅₀ = 0.87 μM) with selectivity index (S.I. = 8.41 and 10.55, respectively) relative to celecoxib (S.I. = 8.85) [26]. As well as, compounds (8a and 8b) exhibited advanced inhibitory activity against 5-LOX (IC₅₀ = 1.92, 2.31 μM) higher than zileuton as reference drug (IC₅₀ = 2.43 μM) [26].

In addition, compounds (9a and 9b) were found to be the most potent COX-2/5-LOX inhibitors compared to standard celecoxib (S.I. = 3.52) [27]. Finally, pyrazole containing acid derivative (10) exhibited promising inhibitory activity towards 5-LOX (IC₅₀ = 5.88 µM), the anti-inflammatory activity of this compound was confirmed by high iNOS and PGE2 inhibitory activities in LPS-stimulated RAW cells with IC₅₀ values of 4.93 µM and 10.98 µM, respectively [28] (Fig. 1).

Fig. 1.

Selective COX-2 inhibitors (rofecoxib (1), celecoxib (2), lonazolac (3), reported derivatives 4–10 and rationale design of the target compounds 15a-h and 19a-d

Synthesis of highly effective and enhanced safety-profiled anti-inflammatory drugs has turned out to be a stimulating chore in the drug innovation process. Though several improvements have been made, there is still essential need to invent more effective anti-inflammatory drugs with minimal side effects [29]. Based on the aforementioned data and in continuation of our research specialized with the design, synthesis and anti-inflammatory assessment of celecoxib and lonazolac analogs [30–43], we have proposed the synthesis of new hybrid mimic structures of triaryl and diaryl-pyrazole derivatives (15a-h) and (19a-d) with an ester substituent instead of carboxylic acid moiety of lonazolac to decrease the local ulceration side effect and investigated their anti-inflammatory properties and COX-2 selectivity inhibition through incorporation of bioactive pharmacophore, Y-shaped design with central pyrazole containing (SO₂CH₃) moiety.

Rationale and structure-based design

Our idea in the rationale of the target derivatives 15a-h and 19a-d comes from that, these derivatives exposed the essential pharmacophoric features of COX-2 inhibitors, through bioisosteric modification strategies of selective COX-2 inhibitors (celecoxib (1), rofecoxib (2)), lonazolac (3), non-acidic 1,3,4-trisubstituted-pyrazole derivatives 4a-l and Y-shaped pyrazole containing derivatives 5–10. As COX-2 inhibitors, i) pyrazole central ring in the target compounds was maintained, resembles that in celecoxib (1) and lonazolac (3). ii) The essential pharmacophore SO₂NH₂ in celecoxib (1) was replaced with the other active one, SO₂CH₃ as in rofecoxib (2) which is significant for enhancing COX-2 selectivity of our designed lonazolac analogues. iii) Moreover, the 4-chlorophenyl moiety at pyrazole C-3 of lonazolac (3) was maintained or replaced with another electronegative moiety as 4-nitrophenyl, 2-bromophenyl and 4-bromophenyl (as in our previously reported derivatives 4a-l) or electron-donating groups like 4-toloyl, 4-methoxyphenyl and 3,4,5-trimethoxyphenyl in the target compounds 15a-h and 19a-d to study the effect of these different groups on COX-2 selectivity. iv) Acetic acid moiety in position-4 of the pyrazole nucleus of lonazolac (3) or the more bulky moiety (methane/aminosulfonylphenylhydrazinomethene) in position-4 of its previously reported analogue, pyrazole derivatives (4a-l) was replaced with either steric arm (phenoxyacetic acid ester) as in target compounds 15a-h or (ethyl carboxylate moiety) as in target derivatives 19a-d, these replacements are supposed to reduce acidity of target compounds in order to have safer gastric profile. v) In addition, triaryl-substitution of pyrazoline nucleus was carried out as in target compounds 15a-h in order to resemble that of previously reported compounds (7a-c) (excellent anti-inflammatory potency). vi) Furthermore, the phenylhydrazone group in position-4 of the pyrazole nucleus of compounds 8a, 8b, 9a and 9b was replaced with the steric arm (phenoxyacetic acid ethyl ester) as in target compounds 15a-h. vii) Finally, the presence of the carboxylic group or its pro-ester function as in derivative (10), which is essential for 5-LOX and iNOs activities; was maintained in the target derivatives 19a-d in form of ester moiety. All these modifications prompted us to study the structure activity relationship of the target compounds 15a-h and 19a-d as anti-inflammatory compounds.

Results and discussion

Chemistry

Schemes for synthesis of the final target compounds 15a-h and 19a-d

In scheme 1, the general reactions used for the preparation of the final target ethyl pyrazolyl-ester derivatives 15a-h were outlined. The chalcone acetic acid derivatives 13a-h were obtained using 4-acetylphenoxyacetic acid 11 that prepared according to reported procedure [44] and various aromatic aldehydes 12a-h as starting materials, according to the literature through a base-catalyzed Claisen–Schmidt condensation at room temperature [44–46]. Furthermore, cyclo-condensation of the appropriate chalcone acetic acid derivatives 13a-h with 4-methanesulfonylphenylhydrazine hydrochloride 14 [47, 48] in aqueous ethanol afforded the respective 1,3,5-triaryl-4,5-dihydro-1H-pyrazole 15a-h in good yields (61–88%). The prepared compounds have been characterized by IR, ¹H NMR, DEPTQ-¹³C NMR spectra and elemental analyses, the IR spectra of compounds 15a-h showed a sharp peak at 1755–1732 cm⁻¹ corresponding to the ester C = O group, two sharp peaks at 1300–1400 cm⁻¹ and 1130–1141 cm⁻¹ corresponding to SO₂CH₃ group. ¹H NMR spectra of 15a-h revealed the presence of three signals as a doublet of doublet (dd) each of one proton intensity, one at δ 3.11–3.22 ppm, the second at δ 3.89–4.03 ppm and the third at δ 5.54–5.89 ppm with three different J values (17.6 Hz, 12.0 Hz, 4.8–5.2 Hz) corresponding to three protons of the 4,5-di-hydropyrazole ring. The highest J value was due to the geminal coupling of two protons at position 4, while the other two J values were due to the coupling of two geminal protons with the vicinal proton at position-5. Likewise, DEPTQ-¹³C NMR of the pyrazoline derivatives 15a-h revealed signals at δ 61.20–62.66 ppm and at δ 42.53–44.63 ppm indicating the CH, CH₂ of the pyrazoline ring respectively (Scheme 1).

Scheme 1.

Reagents and conditions: a NaOH, EtOH (95%), stirring at room temp., 24 h; b EtOH (95%), reflux, 36 h

An interesting formation of the ethyl ester derivatives 15a-h on the expense of acid derivatives was proved by the careful investigation of ¹H NMR and IR spectra for these compounds 15a-h as in IR spectra, the dragged carboxylic peak was unobserved in pyrazolyl ester derivatives 15a-h but only the appearance of sharp carbonyl group peak at 1755–1732 cm⁻¹. In addition, ¹H NMR spectra showed the characteristic signals of ethyl ester group in all derivatives 15a-h as triplet and quartet peaks at δ 1.20–1.23 ppm and at 4.15 ppm respectively. Moreover, DEPTQ-¹³C NMR of the pyrazoline derivatives 15a-h revealed signals at δ 14.49–14.52 ppm and at δ 65.15–65.19 ppm indicating the CH₃, CH₂ of the ester group respectively. Formation of ethyl ester derivatives on expense of acid derivatives was contributed to the Fisher esterification of acidic group of derivatives 13a-h by ethanol that used as solvent and with the aid of the hydrochloride of 4-methanesulfonylphenylhydrazine hydrochloride 14 during cyclization of these chalcone derivatives 13a-h into the pyrazole derivatives 15a-h.

In scheme 2, the reaction pathways for the synthesis of the diaryl-pyrazolyl ester derivatives 19a-d were outlined. A one-pot reaction using an ethanolic solution of oxone is used to synthesize the ester derivatives 19a-d from the corresponding aldehydes 18a-d that were previously prepared through Vilsiemier Haack reaction conditions of their preliminary hydrazone derivatives 17a-d [19]. The ester formation resulted from the oxidation of aldehyde to carboxylic acid followed by Fischer-type esterification of the acids in alcoholic solvent was illustrated by IR, ¹H NMR, DEPTQ-¹³C NMR spectra and elemental analyses, IR spectra showed a characteristic absorption band at 1701–1693 cm⁻¹ attributed to the carbonyl group of ethyl ester moiety. As well, ¹H NMR spectra demonstrated a singlet signal at δ 1.26 ppm and at δ 4.22–4.23 ppm corresponding to CH₃, CH₂ of the ester group respectively. Also, DEPTQ-¹³C NMR of the pyrazoline derivatives 19a-d revealed signals at δ 14.56–14.60 ppm and at δ 60.79–60.62 ppm indicating the CH₃, CH₂ of the ethyl ester functionality (Scheme 2).

Scheme 2.

Reagents and conditions: a ethanol (95%), reflux, 24 h; b DMF, POCl₃, reflux, 24 h; c Oxone, ethanol (95%), stirring at room temp., 24 h

Biological evaluation

In vitro anti-inflammatory activity against COX-1 and COX-2 enzymes

The in vitro COX-1/COX-2 inhibition was assessed using the COX-1 Inhibitor Screening Kit-K548 and the COX-2 Inhibitor Screening Kit-K547 from Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA [29]. The results showed that compounds 15c, 15d, 15 h and 19d have strong inhibitory activity against COX-2 isozyme (IC₅₀ = 0.059–3.89 μM), compared to that of celecoxib (IC₅₀ = 0.22 μM), where derivatives 15c, 15d, 15 h and 19d were found to be the most potent showing COX-2 selectivity index in range of (28.56–98.71) compared to celecoxib (S.I. = 13.65) (Table 1).

Table 1.

In vitro COX-1 and COX-2 inhibitory activities of derivatives 15a-h, 19a-d and the reference celecoxib

Compound	COX-1 IC50 μMa	COX-2 IC50 μMa	COX-2 S.I.b
15a	7.422 ± 0.45	3.897 ± 0.129	1.90
15b	11.42 ± 0.42	1.94 ± 0.064	5.89
15c	11.341 ± 0.45	0.397 ± 0.013	28.56
15d	15.519 ± 0.61	0.221 ± 0.007	70.22
15e	3.006 ± 0.12	0.742 ± 0.025	4.05
15f	6.826 ± 0.25	1.586 ± 0.052	4.30
15 g	8.589 ± 0.33	0.575 ± 0.019	15.07
15 h	6.884 ± 0.25	0.103 ± 0.003	66.83
19a	14.561 ± 0.53	2.33 ± 0.077	6.24
19b	9.417 ± 0.4	2.473 ± 0.100	3.80
19c	18.070 ± 0.66	2.005 ± 0.080	9.01
19d	5.824 ± 0.23	0.059 ± 0.002	98.71
Celecoxib	3.005 ± 0.12	0.22 ± 0.007	13.65

^aThe concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme, the result is the mean of six values ± standard deviation. ^bThe in vitro COX-2 selectivity index (COX-1 IC₅₀/COX-2 IC₅₀)

In vitro anti-inflammatory activity against 5-LOX/15-LOX enzymes

The in vitro 5-LOX/15-LOX inhibition was assessed using the 5-Lipoxygenase Inhibitor Screening Kit (Catalog # K980-100) from Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA and the Lipoxygenase Inhibitor Screening assay Kit (Item # 760,700) from Cayman Chemical, 1180, East Ellsworth Rd., Ann Arbor, MI 48108, USA [49, 50]. The test compounds potency was revealed by calculating IC₅₀. The results exhibited that compounds 15c, 15d, 15 h and 19d had excellent inhibitory action against 5-LOX isozyme (IC₅₀ = 0.24–0.81 μM range) and enhanced inhibitory activity against 15-LOX isozyme (IC₅₀ = 0.20–2.2 μM range) compared to zileuton IC₅₀ = 1.52 and 0.54, respectively) (Table 2).

Table 2.

In vitro 5-LOX/15-LOX, TNF-α, PGE2 and iNOS production and cytotoxicity in LPS-activated RAW 264.7 macrophages inhibitory activities of derivatives 15c, 15d, 15 h, 19d and reference compounds (Zileuton and celecoxib)

Compound	5-LOX IC50 μMa	15-LOX IC50 μMa	TNF-α IC50 μMa	PGE2 IC50 μMa	iNOS IC50 μMa	Cytotoxicity IC50 μM (LPS-induced RAW 264.7 cells)
15c	0.453 ± 0.018	0.412 ± 0.016	0.77 ± 0.025	0.52 ± 0.029	0.410 ± 0.06	301.40 ± 10.7
15d	0.241 ± 0.009	2.232 ± 0.087	1.20 ± 0.060	0.33 ± 0.021	0.531 ± 0.05	182.99 ± 6.52
15 h	0.813 ± 0.032	0.488 ± 0.019	1.04 ± 0.042	0.28 ± 0.032	0.610 ± 0.13	178.95 ± 6.37
19d	0.337 ± 0.013	0.208 ± 0.008	1.01 ± 0.041	0.45 ± 0.026	0.481 ± 0.12	209.45 ± 7.46
Celecoxib	––-	––-	0.87 ± 0.030	0.38 ± 0.027	0.480 ± 0.04	148.90 ± 5.30
Zileuton	1.524 ± 0.059	0.542 ± 0.021	––-	––-	––-	–––

^aThe concentration of test compound produce 50% inhibition, the result is the mean of six values ± standard deviation

In vitro anti-inflammatory mechanistic study of compounds 15c, 15d, 15 h and 19d against pro-inflammatory mediators TNF-α and PGE2 cytokines production in LPS-activated RAW 264.7 macrophages

Further investigation of the anti-inflammatory mechanistic study of compounds 15c, 15d, 15 h and 19d via assessing their inhibitory action against LPS-induced pro-inflammatory cytokines (TNF-α and PGE2) production. The results were summarized in Table 2. The results revealed that compounds 15c, 15d, 15 h and 19d exhibited excellent TNF-α and PGE2 inhibitory activities (IC₅₀ = 0.77–1.20 μM and 0.28–0.52 μM respectively) when compared to celecoxib (IC₅₀ = 0.87 μM and 0.38 μM respectively) as a reference drug (Table 2).

Inducible nitric oxide synthase (iNOS) inhibition

In LPS-induced RAW 264.7 cells, the inhibitory effects of compounds 15c, 15d, 15 h and 19d on iNOS activity production were investigated (Table 2). When tested on LPS-induced RAW 264.7 cells, the active four compounds showed a significant inhibitory effect on iNOS (0.41–0.61 µM) when compared to the reference drug celecoxib (0.48 µM).

Cytotoxicity determination of the target compounds

Prior to assessing the in vivo anti-inflammatory activity, the MTS assay was used to evaluate the cytotoxicity of the most potent synthesized compounds 15c, 15d, 15 h and 19d against RAW 264.7 macrophages [36–42]. None of the tested compounds demonstrated any noticeable cytotoxicity and showed high IC₅₀ concentrations (Table 2). Therefore, the in vivo anti-inflammatory activity of these derivatives was further assessed.

In vivo anti-inflammatory activity

Using a dose of 50 mg/kg body weight, the most selective COX-2 inhibitors, 15c, 15d, 15 h and 19d as well as celecoxib as a reference drug, were evaluated for their in vivo anti-inflammatory efficacy using the carrageenan-induced rat paw edema assay, in accordance with the reported procedure [35]. The anti-inflammatory activity was determined using paw-thickness changes at 1 h, 3 h, and 5 h following carrageenan injection, as shown in (Table 3). When compared to carrageenan, the most potent compounds 15c, 15d, 15 h and 19d were found to significantly reduce inflammation at all times. Comparable studies comparing the tested compounds’ anti-inflammatory activity to celecoxib's at different time intervals found that, after 1 h, the tested compounds had good anti-inflammatory activity (A.I. = 38.3–61.1%) compared to celecoxib's (A.I. = 30.1%). All compounds showed increased anti-inflammatory activity after 3 h (A.I. = 41.9–82.0%). In a similar way, all compounds showed increased anti-inflammatory effect after 5 h (A.I. = 40.0–92.2%). Furthermore, in comparison to celecoxib, the most effective anti-inflammatory derivatives 15c, 15d, 15 h and 19d had their dose inducing 50% edema inhibition (ED₅₀) evaluated. Derivatives 15c, 15d, 15 h and 19d (ED₅₀ values of 8.22–31.22 mg/kg) were more potent than celecoxib (ED₅₀ values of 40.39 mg/kg). (Table 4).

Table 3.

In vivo anti-inflammatory activity of derivatives 15c, 15d, 15 h, 19d and celecoxib

Compound No	Paw edema thickness (mm) ± SEM (% inhibition)
	1 h	3 h	5 h
15c	3.1 ± 0.002 (38.3%)	2.56 ± 0.0034 (55%)	2.1 ± 0.0084 (66.1%)
15d	2.23 ± 0.0045 (61.1%)	2.79 ± 0.011 (41.9%)	2.90 ± 0.002 (40%)
15 h	2.57 ± 0.0071 (54.85%)	2.61 ± 0.018 (55.9%)	2.44 ± 0.0099 (59.3%)
19d	2.85 ± 0.025 (40.7%)	1.3 ± 0.006 (82%)	0.21 ± 0.0094 (92.2%)
Celecoxib	3.43 ± 0.004 (30.1%)	3.1 ± 0.011 (38.3%)	2.52 ± 0.0095 (56.3%)

Values represent means ± SEM of four animals for each group

**Means significant difference with celecoxib at p < 0.05

***Means highly significant difference with celecoxib at p < 0.005

Table 4.

ED₅₀ for the most active derivatives 15c, 15d, 15 h, 19d and reference drug celecoxib

Compound No	% inhibition
	10 mg/kg	25 mg/kg	50 mg/kg	ED50 (mg/kg)a
15c	22.32%	31.51%	70.02%	24.1
15d	29.52%	36.61%	80.29%	31.22
15 h	19.49%	37.98%	82.28%	29.9
19d	35.11%	52.23%	74.99%	8.22
Celecoxib	55.48%	34.97%	97.48%	40.39

^aThe concentration of test compound produce 50% edema inhibition

Ulcerogenic liability

The most potent compounds (15c, 15d, 15 h and 19d) were subjected to further evaluation to determine their ulcerogenic effect (ulcer index) [51] in comparison with celecoxib and lonazolc using 50 mg/kg dose. The results revealed that all compounds were less ulcerogenic (ulcer indexes = 1.22–2.93) than lonazolac (ulcer index = 20.30) and comparable to celecoxib (ulcer index = 3.02) which greatly supported our main objective to avoid gastric ulceration caused by COX-1 inhibition (Table 8). The diaryl pyrazole ester derivative (19d) was the most safe derivative (ulcer index = 1.22) which is about 20-fold less ulcerogenic than lonazolac and showed in our assay an ulcerogenic potential less than that of celecoxib (Table 5).

Table 8.

Physicochemical, Pharmacokinetics parameters, lipophilicity, water solubility and drug likeness of compounds 15c, 15d, 15 h, 19d and celecxoib

Compound No	M.W	No of rotoable atoms	No of H-bond acceptors	No of H-bond donors	Molar refractivity	TPSA (Å2)	Log (p0/w)	Log S (ESOl)	GI absorption	BBB permeant	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	Lipinski	Bioavailability score	Lead likeness	Pains
15c	508.59	10	7	0	144.05	102.88	3.95	−5.53	High	No	No	Yes	Yes	1 violation	0.55	3 violations	0 alert
15d	568.64	12	9	0	157.03	121.34	3.78	−5.70	High	No	No	Yes	Yes	1 violation	0.55	3 violations	0 alert
14 h	557.46	9	6	0	145.25	93.65	4.03	−6.37	High	No	No	Yes	Yes	1 violation	0.55	3 violations	0 alert
19d	449.32	6	5	0	105.88	86.64	3.43	−5.03	High	No	Yes	Yes	Yes	0 violations	0.55	2 violations	0 alert
Celecoxib	381.34	4	7	1	89.96	86.36	2.56	−4.57	High	No	Yes	No	Yes	0 violations	0.55	1 violation	0 alert
Lonazolac	312.75	4	3	1	85.55	55.12	2.60	−4.49	High	Yes	No	Yes	Yes	0 violations	0.85	1 violation	0 alert

Table 5.

Ulcer index for the most potent derivatives 15c, 15d, 15 h, 19d, reference drugs celecoxib and lonazolac

Compound	Average severity	Average no of ulcera	% incidence /10	Ulcer index
15c	0.17 ± 0.004***b	0.55 ± 0.012***b	2	2.61
15d	0.51 ± 0.013b	0.47 ± 0.006b	2	2.93
15 h	0.29 ± 0.009***b	0.40 ± 0.006b	2	2.60
19d	0.15 ± 0.002***b	0.12 ± 0.003***b	1	1.22
Celecoxib	0.66 ± 0.026***b	0.48 ± 0.015***b	2	3.02
Lonazolac	2.29 ± 0.13	8.1	10	20.30

^aValues represent means ± SEM of ten animals for each group

***Means significant difference with celecoxib at p < 0.001

^bMeans significant difference with lonazolac at p < 0.001

Molecular modeling studies

In order to investigate the possible binding mode of the synthesized compounds with either COX-2 or 5-LOX receptors, molecular docking study was performed using Molecular Operating Environment (MOE) version 2015.10 modeling software. The X-ray, crystal structure data for the enzymes were obtained from the protein data bank with code (PDB ID: 3LN1) [52] and (PDB ID: 3V99) [53] respectively. Docking scores, amino acid residues forming hydrogen bonding interactions and their lengths were summarized in Tables 6 and 7.

Table 6.

Molecular data for compounds 15a-h, 19a-d and celecoxib during docking inside COX-2 receptor

Compound No	E-score Kcal/mol	No of hydrogen bonds	Hydrogen bonding residues	Hydrogen bonding type	Distance (Ao)
15a	−12.90	1	Ser339	Arene-H	4.00
15b	−16.43	2	Ser339 Ala513	H-donor Arene-H	3.25 3.67
15c	−17.64	2	Arg449 Ala513	H-accept Arene-H	2.74 3.86
15d	−17.64	2	Ser339 Ser339	H-donor Arene-H	3.18 3.93
15e	−16.74	2	Arg449 Glu510	H-accept H-donor	2.73 3.13
15f	−16.44	2	Ser339 Ser516	Arene-H H-accept	3.88 3.10
15 g	−17.30	2	Arg449 Ala513	H-accept Arene-H	2.92 4.33
15 h	−17.67	2	Ser339 Arg449	Arene-H H-accept	3.66 2.73
19a	−13.57	1	Ser339	Arene-H	3.82
19b	−13.53	2	Ser339	Arene-H	3.78
19c	−15.67	1	Arg449	H-accept	2.89
19d	−18.17	5	Leu338 Arg449 Ser339 Ser516 Gln178	Arene-H H-accept Arene-H H-accept H-donor	4.21 2.71 3.99 3.00 3.46
Celecoxib	−13.57	4	Arg449 Leu338 Ser339 Gln178	H-accept H-donor H-donor H-donor	3.53 3.04 2.93 3.06

Table 7.

Molecular data for compounds 15c, 15d, 15 h, 19d, zileuton and celecoxib during docking inside 5-LOX receptor

Compound No	E-score Kcal/mol	No of hydrogen bonds	Hydrogen bonding residues	Hydrogen bonding type	Distance (Ao)
15c	−16.25	1 + 7 with HOH	Gly50 HOH 393 HOH 356 HOH 338 HOH 459 HOH 343 HOH 459 HOH 595	H-accept H-accept Arene-H H-accept H-accept H-accept H-accept H-accept	3.42 3.26 3.52 3.23 3.10 2.91 3.13 3.00
15d	−11.78	3 + 7 with HOH	Gly50 Lys67 Glu121 HOH 393 HOH 357 HOH 475 HOH 352 HOH 577 HOH 338 HOH 459	H-accept H-accept H-donor H-accept Arene-H H-donor H-accept H-donor H-accept H-accept	3.54 2.99 2.74 3.50 3.74 3.64 2.81 3.68 3.32 3.26
15 h	−14.17	1 + 5 with HOH	Lys67 HOH 499 HOH 352 HOH 352 HOH 475 HOH 596	H-accept H-accept H-accept H-accept H-accept H-accept	2.83 3.47 2.83 3.52 3.57 3.16
19d	−18.17	2 + 10 with HOH	Gly50 Lys67 HOH 543 HOH 596 HOH 336 HOH 338 HOH 459 HOH 462 HOH 352 HOH 412 HOH 462 HOH 543	H-accept H-accept H-donor H-donor H-accept H-accept H-accept H-accept H-accept H-accept H-accept H-accept	3.05 3.09 3.23 3.13 3.57 2.83 2.81 3.49 2.58 2.83 2.72 3.33
Zileuton	−12.00	1 + 8 with HOH	Gly50 HOH 393 HOH 356 HOH 475 HOH 577 HOH 343 HOH 393 HOH 459 HOH 356	H-accept H-donor H-donor H-donor H-donor H-accept H-accept H-accept Arene-H	3.60 3.30 2.83 3.36 3.42 3.22 3.04 3.30 3.77
Celecoxib	−13.41	2 + 11 with HOH	Gly50 Lys67 HOH 596 HOH 336 HOH 352 HOH 412 HOH 462 HOH 543 HOH 338 HOH 459 HOH 462 HOH 459 HOH 543	H-accept H-accept H-donor H-accept H-accept H-accept H-accept H-accept H-accept H-accept H-accept H-accept H-accept	3.17 2.96 3.15 3.48 2.52 2.54 2.52 3.27 2.75 2.58 2.95 2.82 3.00

Regarding COX-2 enzyme, docking of celecoxib into the COX-2 isozyme afforded four hydrogen bonding interactions (HBs) and (distance Aº) (i) SO₂NH₂ with Leu338 (3.04 Aº), (ii) SO₂NH₂ with Ser339 (2.93 Aº), (iii) SO₂NH₂ with Gln178 (3.06 Aº), (iv) SO₂NH₂ with Arg499 (3.53 Aº) with energy score -13.57 kcal/mol (Fig. 2). The docking results of the synthesized compounds fitted well to COX-2 active site inside the pocket and showed variable binding mode of interactions with energy score ranged from −12.90 to −18.17 kcal/mol. The binding mode of interactions for the most active compounds 15c, 15d, 15 h and 19d with COX-2 active site were represented as in Fig. 2. They exhibited higher binding affinity toward COX-2 enzyme by forming 2–5 hydrogen-bond interactions in addition to arene-hydrogen bonds with different amino acids such as (Leu338, Ser339, Gln178, and Arg449) similar to the ligand compound in addition to Ser516 and Ala513 amino acids with good energy scores range (−17.64 to −18.17 kcal/mole) and these results elucidate their good selectivity toward COX-2.

Fig. 2.

Binding of ligand celecoxib, compound 15c, 15d, 15 h and 19d inside COX-2 active site. A 2D interactions of the proposed binding mode of celecoxib or selected compound inside the active site of COX-2. B 3D interactions of celecoxib or selected compond

Concerning the docking results of the most active compound 19d, it was observed that its binding mode in COX-2 active site was very similar to celecoxib mode, it showed three hydrogen bonding interaction (HBs) (i) benzene ring with Ser339 (3.99 Aº), (ii) SO₂CH₃ with Gln178 (3.46 Aº), (iii) COOC₂H₅ with Ser516 (3.00 Aº), in addition to two hydrophobic π-H interactions with the key amino (iv) SO₂CH₃ with Arg499 (2.71 Aº), and (v) pyrazole ring with Leu338 (4.21 Aº) with energy score -18.17 kcal/mol (Fig. 2). On the other hand, the binding mode of the least active selective COX-2 compound 15a showed only one hydrogen bond interaction with Ser339 (4.00Aº) with energy score -12.90 kcal/mol. The docking results including energy scores, hydrogen bonding interaction between amino acid residues and functional groups of docked compounds and their length were summarized in Table 6.

For 5-LOX enzyme, the binding pattern of zileuton revealed a H-bond interaction between linker (O) and the key amino acid Gly50 (3.60 Aº), in addition to eight hydrogen bonds and hydrophobic π−H interactions with water molecules with energy score −12.00 kcal/mol (Fig. 3). On the other hand celecoxib showed two H-bond interactions between (O) of (SO₂NH₂) and the key amino acid Gly50 and Lys67 (3.17 Aº) and (2.96 Aº) respectively, in addition to eleven hydrogen bonds with water molecules with energy score −13.49 kcal/mol (Fig. 3). The results of the present in silico docking simulation for the most active compounds 15c, 15d, 15 h and 19d with 5-LOX active site revealed that they showed good binding interaction by making at least one hydrogen bond interaction with the main amino acids, in addition to several hydrogen bonds and hydrophobic π−H interactions with water molecules in similar manner to the both ligands (zileuton and celecoxib) with energy score range (−1.78 to −18.92 kcal/mol) (Fig. 3).

Fig. 3.

Binding of zileuton, compound 15c, 15d, 15 h and 19d inside 5-LOX active site. A 2D interactions of the proposed binding mode of zileuton or selected compound inside the active site of COX-2. B 3D interactions of zileuton or selected compound

The docking results of the significant compound 19d showed two hydrogen bonding interactions (HBs) (i) SO₂CH₃with Gly50 (3.05 Aº), (ii) SO₂CH₃ with Lys67 (3.09 Aº), in addition to ten hydrogen bonds with water molecules with low energy score -18.92 kcal/mol (Fig. 3). While the docking results of compound 15 h which showed the lower 5-LOX inhibitory activity exhibited that, it form only one H-bond with Lys67 amino acid (2.83 Aº) with high energy score (−11.78 kcal/mol), in addition to formation of the least number of hydrogen bonds with water molecules (5 H-bonds) and that illustrate its lower 5-LOX inhibitory activity (Fig. 3).

The docking results including energy scores, hydrogen bonding interaction between amino acid residues and functional groups of docked compounds and their length were represented in Table 7.

The results confirmed that there is a parallel relationship between docking study and the in vivo anti-inflammatory activity.

SwissADME studies

Drug development involves the assessment of efficacy and toxicity of the new drug candidates. A critical piece in drug discovery and development is conducting DMPK (drug metabolism and pharmacokinetics) studies, often referred to as ADMET (absorption, distribution, metabolism, elimination and toxicity) studies. The initiation of early absorption, distribution, metabolism and excretion (ADME) screening has dramatically decreased the proportion of compounds failing in clinical trials [54].

ADME study was assessed to new most active synthesized compounds 15c, 15d, 15 h, 19d, reference drugs celecoxib and lonazolac using the SwissADME web tool. Many parameters were detected, which are presented in (Table 8).

Drug likeliness parameters were studied by using different rules such as Lipinski rule of five, Ghose rule, Veber, Egan rule, and Muegge rule [54]. Particularly, the compliance of compound to taraditional Lipiniskiʼs rule “rule of five” which indicates that orally active substance in humans is an important indication for drugs pharmacokinetics. This simple rule states that oral active drug has no more than one violation of the following criteria: molecular weight (M wt) less than 500 Dalton (Da); no more than five hydrogen bond donors (HBD); no more than 10 hydrogen bond acceptors (HBA); and calculated octanol–water partition coefficient (clogP) not greater than 5 or (MlogP) not greater than 4.15 [55]. Also, topological polar surface area (TPSA) and the number of routable bonds are other critical properties that have been linked to the drug bioavailability. The reports suggested that compounds with a TPSA of more than 140 Ų and more than ten routable bonds are thought to have low oral bioavailability (Fig. 4) [55].

Fig. 4.

Physicochemical diagram of the most active compounds 15c, 15d, 15 h, 19d and celecoxib

From the obtained results, compounds 15c, 15d, and 15 h have M wt higher than 500 gm/ mol ranged from 508.59 to 568.64. Also, they have number of routable bonds (9 to 12) in comparison with number of routable bonds of standard drugs celecoxib and lonazolac. Furthermore, compounds 15c, 15d, and 15 h have no H-bond donors in comparison with celecoxib. In addition, 15c, 15d, and 15 h compounds have number of H-bond acceptors (6 to 9) of 1 relative to celecoxib and lonazolac (Table 8). The TPSA of compounds 15c, 15d and 15 h are ranged from 93.65 to 121.34 Ų within standard range 140 Ų in comparison with that of celecoxib (86.36 Ų) and lonazolac (55.12 Ų). Sufficient lipophilicity is required for having a good biological activity; their parameters can be determined by using MLOGP [56]. Concerning lipophilicity all compounds displayed the least value of partition coefficient 3.78–4.03 (Log P ≤ 5) relative to that of 2.56 of celecoxib (Table 8).

Fortunately, the most active compound (19d) fulfilled all Lipinskisʼ guidelines similar to the clinically used celecoxib and lonazolac, including molecular weight 449.3 Da compared to the molecular weight of celecoxib (381.76 Da) and that of lonazolac (312.75 Da). The number of hydrogen bond donors is zero compared to one HBD in celecoxib. Hydrogen bond acceptors were 5 compared to seven HBA in celecoxib and lonazolac (Table 8). The TPSA of compound (19d) is within standard range 140 Ų equal to 86.64 Ų which was very similar to of celecoxib (86.36 Ų) and lonazolac (55.12 Ų). Additionally, the partition coefficient of it was 3.43 relative to that of 2.56 of celecoxib and 2.60 for lonazolac (Table 8).

As our synthesized compounds have TPSA larger than 60 Å, all compounds don’t pass blood brain barrier as observed by our calculations. The bioavailability of drugs is affected by gastrointestinal absorption. All tested compounds have high GIT absorption comparable to celecoxib and lonazolac.

Cytochromes P450 (CYPs) are a family of enzymes that are responsible for over 90% of oxidative metabolic processes and play a major role in breaking down a variety of endogenous and xenobiotic substances [54–56]. Inhibition of CYP enzymes causes failure in inhibitory drug metabolism. As a result, studying the inhibitory activity of proposed derivatives against a certain CYP is very important in medication development. The results of the inhibitory prediction for two CYP isoforms (CYP1A2, and CYP2C19) are showed in (Table 8). All our evaluated compounds were anticipated to inhibit both CYP2C9 enzymes, which means they are lower exposed to inhibitory drug metabolism.

Regarding to bioavailability score, all compounds had similar score to that of reference drugs celecoxib (0.55) and lonazolac (0.85) which means that all ligands can reach the systemic circulation (Table 8).

In conclusion, the ADME studies support the discovery of the prepared compounds 15c, 15d, 15 h and 19d as active oral bio-available anti-inflammatory drugs.

Structural activity relationship (SAR)

SAR was performed based on the biological activity results of di-aryl/tri-aryl substituted pyrazole ester derivatives 15a-h and 19a-d as anti-inflammatory agents, as shown in Fig. 5. It was evident that in phenoxyacetic acid ester series 15a-h when R = 4-OCH₃, 3,4,5-(OCH₃)₃, 4-Br, these derivatives (15c, 15d and 15 h) showed the highest COX-2 potency, selectivity and 5-LOX, 15-LOX inhibitory activity. In addition, substitution with 4-OCH₃ group as in compound 15c showed the best TNF-α, iNOS inhibition and in vivo anti-inflammatory potential than 3,4,5-(OCH₃)₃ and 4-Br analogues. Moreover, substitution with 4-Br group as in compound 15 h exhibited the best PGE2 inhibition than 4-OCH₃ and 3,4,5-(OCH₃)₃ analogues.

Fig. 5.

SAR study of the target pyrazole ester derivatives 15a-h and 19a-d as anti-inflammatory agents

On the other hand, in ethyl carboxylate ester series 19a-d when R = Br this derivative 19d recorded the highest COX-2 potency and selectivity than H, CH₃ and Cl. Furthermore, when R = Br the same derivative displayed the highest in vivo anti-inflammatory potential in both series. Substitution with Br afforded the safest COX-2 inhibitor (lower ulcer index) than H, CH₃ and Cl analogues.

Modifications in anti-inflammatory activity upon replacement of substituents or rings from reported compounds

• Incorporation of SO₂CH₃ pharmacophore in the target compounds results in increase of COX-2 selectivity comparable to lonazolac.

• Replacement of 4-chlorophenyl moiety at pyrazole C-3 of lonazolac with electron-donating groups like 4-methoxyphenyl and 3,4,5-trimethoxyphenyl in the target compounds 15a-h and 19a-d increased COX-2 selectivity (COX-2 S.I. 28.56 and 70.22, respectively).

• Replacement of acetic acid moiety in position-4 of the pyrazole nucleus of lonazolac (3) or the hydrazone moity of reported compounds 4a-c (COX-2 S.I. = 9.22–98.7) with steric arm (phenoxyacetic acid ester) as in target compounds 15a-h or (ethyl carboxylate moiety) as in target derivatives 19a-d, increased COX-2 selectivity (COX-2 S.I. 28.56 and 70.22, respectively).

• Keeping Y-shaped diaryl pyrazole scaffold in the target compounds resembling to the previously reported excellent COX-2 selective inhibitors (7a-c COX-2 S.I. 50.6–253.1) resulted in keeping the high COX-2 selectivity to some extent.

• Replacement of hydrazone moiety in position 4 of pyrazole ring of the reported compounds 8a, 8b, 9a and 9b increased COX-2 selectivity from (8.41–10.55) to (28.56–70.22). In addition of increasing 5-LOX potency from (1.92, 2.31 µM compounds 8a, 8b) to (0.241–0.81 µM of our target compounds).

• Replacement of acid moiety of compound 10 with ester scaffold increased 5-LOX potency from 5.88 µM to (0.241–0.81 µM of our target compounds).

Experimental

Chemistry

Melting points were determined on a Griffin apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Shimadzu 435 spectrometer using KBr discs. ¹H NMR and ¹³C NMR spectra were measured on a Bruker 400 MHz spectrometer (Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt) in D₂O, (DMSO-d₆) with TMS as the internal standard, where J (coupling constant) values were estimated in hertz (Hz). Mass spectra were run on Hewlett Packard 5988 spectrometer. Microanalysis was performed for C, H, N at the Micro Analytical Center, Cairo University, Egypt and was within ± 0.4% of theoretical values. All other reagents, purchased from the Acros Chemical Company (Milwaukee, WI). The intermediates 4-methanesulfonylphenylhydrazine hydrochloride 12 [47, 48], chalcone derivatives 13a-h [44–46], hydrazone derivatives 17a-d and the pyrazole aldehydes 18a-d [19] were prepared according to reported procedure.

General method for preparation of 1,3,5-triarylpyrazolines (15a-h)

A solution of the appropriate chalcone (13a-h, 0.1 mol) in ethanol (50 mL) was heated under reflux with 4-methanesulfonylphenylhydrazine hydrochloride (12, 0.1 mol) for 36 h, cooled and diluted with cold water. The precipitated crude product was filtered and recrystallized from ethanol to give 15a-h. Physical and spectral data are listed below:

Ethyl 2-(4-(1-(4-(methylsulfonyl)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy) acetate (15a)

79% yield; reddish brown solid; m.p. 232–234 °C; IR (KBr disk) 1732 (C = O), 1377, 1138 (SO₂); ¹H NMR (DMSO-d₆) δ 1.23 (t, J = 3.2 Hz, 2H, OCH₂CH₃), 3.07 (s, 3H, SO₂CH₃), 3.15 (dd, J = 5.2, 17.6 Hz, 1H, pyrazole H-4), 3.93 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 3.2 Hz, 3H, OCH₂CH₃), 4.85 (s, 2H, CH₂), 5.59 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 7.03 (d, J = 8.8 Hz, 2H, phenyl H-2, H-6), 7.09 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.26 (m, 3H, phenyl H-3, H-4, H-5), 7.34 (d, J = 7.2 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.64 (d, J = 8.4 Hz, 2H, phenoxy H-2, H-6), 7.74 (d, J = 8.4 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.52, 43.75, 44.66, 61.20, 62.66, 65.19, 112.43, 115.30, 125.31, 126.17, 128.30, 129.02, 129.19, 129.65, 142.09, 147.66, 150.84, 159.21, 168.97; MS (m/z, relative abundance %): 478.16 (M^+.); Anal. Calcd for C₂₆H₂₆N₂O₅S: C, 65.25; H, 5.48; N, 5.85; Found: C, 65.44; H, 5.32; N, 5.55.

Ethyl 2-(4-(1-(4-(methylsulfonyl)phenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15b)

75% yield; yellowish brown solid; m.p. 244–246 °C; IR (KBr disk) 1743 (C = O), 1334, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 7.2 Hz, 2H, OCH₂CH₃), 3.71 (s, 3H, CH₃), 3.06 (s, 3H, SO₂CH₃), 3.11 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.90 (dd, J = 12.0, 17.2 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 7.2 Hz, 3H, OCH₂CH₃), 4.84 (s, 2H, CH₂), 5.54 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 7.00 (d, J = 8.4 Hz, 2H, 4-methylphenyl H-3, H-5), 7.08 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.15 (m, 4H, 4-methylphenyl H-2, H-6, aminosulfonylphenyl H-3, H-5), 7.62 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 7.72 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.49, 21.07, 43.90, 44.64, 62.36, 62.43, 65.15, 112.44, 115.29, 125.34, 126.09, 128.27, 128.97, 130.18, 137.42, 139.05, 147.65, 150.84, 159.16, 169.01; MS (m/z, relative abundance %): 531.17 [M + K]⁺; Anal. Calcd for C₂₇H₂₈N₂O₅S: C, 65.83; H, 5.73; N, 5.69; Found: C, 65.74; H, 5.78; N, 5.45.

Ethyl 2-(4-(5-(4-methoxyphenyl)-1-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15c)

83% yield; yellowish brown solid; m.p. 222–224 °C; IR (KBr disk) 1743 (C = O), 1334, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.21 (t, J = 7.2 Hz, 2H, OCH₂CH₃), 3.07 (s, 3H, SO₂CH₃), 3.12 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.71 (s, 3H, OCH₃), 3.89 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 7.2 Hz, 3H, OCH₂CH₃), 4.85 (s, 2H, CH₂), 5.54 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 6.89 (d, J = 8.4 Hz, 2H, 4-methoxyphenyl H-3, H-5), 7.01 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.10 (d, J = 8.8 Hz, 2H, 4-methoxyphenyl H-2, H-6), 7.18 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.63 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 7.73 (d, J = 8.4 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.52, 43.75, 44.67, 55.53, 61.20, 62.17, 65.19, 112.45, 114.98, 115.30, 125.40, 127.45, 128.26, 128.97, 129.07, 133.96, 147.65, 150.84, 159.13, 159.18, 168.98; MS (m/z, relative abundance %): 508.17 (M^+.); Anal. Calcd for C₂₇H₂₈N₂O₆S: C, 63.76; H, 5.55; N, 5.51; Found: C, 63.64; H, 5.68; N, 5.25.

Ethyl 2-(4-(1-(4-(methylsulfonyl)phenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15d)

88% yield; white solid; m.p. 256–258 °C; IR (KBr disk) 1751 (C = O), 1323, 1130 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 7.2 Hz, 2H, OCH₂CH₃), 3.08 (s, 3H, SO₂CH₃), 3.12 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.63 (s, 3H, 4-methoxyphenyl), 3.70 (s, 6 H, 3,5-dimethoxyphenyl), 3.91 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 7.2 Hz, 3H, OCH₂CH₃), 4.85 (s, 2H, CH₂), 5.45 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 6.59 (s, 2H, 3,4,5-trimethoxyphenyl H-2, H-6), 7.01 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.13 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.67 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 7.74 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.51, 43.85, 44.66, 56.37, 56.42, 61.20, 63.30, 65.00, 65.17, 103.33, 112.58, 115.28, 125.12, 125.33, 128.32, 129.00, 129.35, 137.24, 137.96, 148.14, 151.03, 153.87, 159.20, 169.00, 170.42; MS (m/z, relative abundance %): 591.19 [M + Na]⁺; Anal. Calcd for C₂₉H₃₂N₂O₈S: C, 61.25; H, 5.67; N, 4.93; Found: C, 61.44; H, 5.88; N, 5.15.

Ethyl 2-(4-(5-(4-chlorophenyl)-1-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15e)

69% yield; yellowish white solid; m.p. 218–220 °C; IR (KBr disk) 1755 (C = O), 1334, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 3.07 (s, 3H, SO₂CH₃), 3.15 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.92 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 4.84 (s, 2H, CH₂), 5.62 (dd, J = 5.2, 12.4 Hz, 1H, pyrazole H-5), 7.00 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H-3, H-5), 7.08 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.27 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H-2, H-6), 7.40 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.67 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 7.73 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.51, 43.52, 44.65, 61.17, 61.95, 62.18, 65.19, 112.47, 115.30, 125.20, 128.19, 128.33, 129.08, 129.24, 129.63, 132.69, 140.99, 147.50, 150.88, 159.25, 168.97; MS (m/z, relative abundance %): 512.12 (M +); Anal. Calcd for C₂₆H₂₅ClN₂O₅S: C, 60.87; H, 4.91; N, 5.46; Found: C, 60.78; H, 4.82; N, 5.65.

Ethyl 2-(4-(1-(4-(methylsulfonyl) phenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15f)

61% yield; yellowish white solid; m.p. 238–240 °C; IR (KBr disk) 1751 (C = O), 1343, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 3.08 (s, 3H, SO₂CH₃), 3.22 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.99 (dd, J = 12.4, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 4.85 (s, 2H, CH₂), 5.81 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 7.01 (d, J = 8.8 Hz, 2H, 4-nitrophenyl H-3, H-5), 7.09 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.54 (d, J = 8.4 Hz, 2H, 4-nitrophenyl H-2, H-6), 7.66 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.74 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 8.22 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.52, 44.63, 44.69, 61.99, 65.19, 112.52, 115.32, 124.92, 125.04, 127.75, 128.42, 129.17, 129.70, 147.41, 147.51, 149.50, 150.97, 159.34, 168.95; MS (m/z, relative abundance %): 523.14 (M +); Anal. Calcd for C₂₆H₂₅N₃O₇S: C, 59.65; H, 4.81; N, 8.03; Found: C, 59.86; H, 4.98; N, 8.16.

Ethyl 2-(4-(5-(2-bromophenyl)-1-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15 g)

68% yield; yellowish white solid; m.p. 278–280 °C; IR (KBr disk) 1735 (C = O), 1300, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 7.2 Hz, 2H, OCH₂CH₃), 3.08 (s, 3H, SO₂CH₃), 3.16 (dd, J = 5.2, 17.6 Hz, 1H, pyrazole H-4), 4.03 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 7.2 Hz, 3H, OCH₂CH₃), 4.84 (s, 2H, CH₂), 5.72 (dd, J = 5.2, 12.0 Hz, 1H, pyrazole H-5), 6.96 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.00 (d, J = 2.4 Hz, 2H, 2-bromophenyl H-6), 7.02 (d, J = 8.8 Hz, 2H, phenoxy H-2, H-6), 7.25 (t, J = 6.0 Hz, 2H, 2-bromophenyl H-5), 7.31 (t, J = 7.6 Hz, 2H, 2-bromophenyl H-4), 7.68 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.75 (d, J = 2.0 Hz, 2H, 2-bromophenyl H-6), 7.76 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.51, 42.53, 44.64, 61.21, 62.63, 65.18, 112.27, 115.29, 121.80, 125.08, 128.28, 128.39, 129.14, 129.23, 129.57, 130.34, 133.90, 139.87, 147.32, 150.97, 159.28, 168.97; MS (m/z, relative abundance %): 595.07 [M + K]⁺; Anal. Calcd for C₂₆H₂₅BrN₂O₅S: C, 56.02; H, 4.52; N, 5.03; Found: C, 56.08; H, 4.58; N, 5.16.

Ethyl 2-(4-(5-(4-bromophenyl)-1-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)acetate (15 h)

65% yield; yellowish white solid; m.p. 232–234 °C; IR (KBr disk) 1739 (C = O), 1400, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.20 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 3.07 (s, 3H, SO₂CH₃), 3.17 (dd, J = 4.8, 17.6 Hz, 1H, pyrazole H-4), 3.92 (dd, J = 12.0, 17.6 Hz, 1H, pyrazole H'-4), 4.15 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 4.85 (s, 2H, CH₂), 5.62 (dd, J = 4.8, 12.0 Hz, 1H, pyrazole H-5), 7.01 (d, J = 8.8 Hz, 2H, phenoxy H-3, H-5), 7.09 (d, J = 8.8 Hz, 2H, 4-bromophenyl H-3, H-5), 7.22 (d, J = 8.4 Hz, 2H, phenoxy H-2, H-6), 7.54 (d, J = 8.8 Hz, 2H, 4-bromophenyl H-2, H-6), 7.65 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.73 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 14.52, 43.47, 44.66, 61.20, 65.19, 112.46, 115.31, 121.21, 125.20, 128.34, 128.55, 129.09, 129.40, 132.55, 141.43, 147.48, 150.90, 159.26, 168.97; MS (m/z, relative abundance %): 595.07 [M + K]⁺; Anal. Calcd for C₂₆H₂₅BrN₂O₅S: C, 56.02; H, 4.52; N, 5.03; Found: C, 56.25; H, 4.80; N, 5.40.

General procedure for synthesis of the pyrazolyl ester derivatives (19a-d)

A mixture of the appropriate pyrazole aldehyde (18a-d, 1.0 mmol) and oxone (0.92 g, 1.5 mmol) in ethanol (20 mL) was stirred at room temperature for 24 h. After completion of the reaction the solvent was evaporated, and the formed precipitate was washed with water (150 mL), the obtained residue was recrystallized from methanol to give the final pyrazolyl ester derivatives (19a-d): Physical and spectral data are listed below:

Ethyl 1-(4-(methylsulfonyl)phenyl)-3-phenyl-1H-pyrazole-5-carboxylate (19a)

Yield 75%; white solid; m.p. 256–258 °C; IR (KBr disk) 1701 (C = O), 1355, 1149 (SO₂); ¹H NMR (DMSO-d₆) δ 1.26 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 3.29 (s, 3H, SO₂CH₃), 4.23 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 7.54 (d, J = 8.4 Hz, 2H, phenyl H-2, H-6), 7.86 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 7.89 (m, 1H, phenyl H-4), 8.09 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 8.29 (d, J = 8.4 Hz, 2H, aminosulfonlphenyl H-2, H-6), 9.34 (s, 1H, pyrazole H-5); ¹³C NMR (DMSO-d₆) δ 14.58, 43.99, 60.75, 114.60, 119.96, 128.48, 129.29, 130.83, 131.42, 134.21, 134.99, 139.63, 142.48, 152.78, 162.45; MS (m/z, relative abundance %): 370.10 (M +) (100%). Anal. Calcd. For C₁₉H₁₈N₂O₄S: C, 61.61; H, 4.90; N, 7.56; Found; C, 61.98; H, 5.12; N, 7.88.

Ethyl 1-(4-(methylsulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-5-carboxylate (19b)

Yield 71%; white solid; m.p. 267–269 °C; IR (KBr disk) 1693 (C = O), 1355, 1149 (SO₂); ¹H NMR (DMSO-d₆) δ 1.26 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 2.37 (s, 3H, CH₃), 3.28 (s, 3H, SO₂CH₃), 4.22 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 7.26 (d, J = 8.0 Hz, 2H, 4-methylphenyl H-3, H-5), 7.70 (d, J = 8.4 Hz, 2H, 4-methylphenyl H-2, H-6), 8.07 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 8.26 (d, J = 8.4 Hz, 2H, aminosulfonlphenyl H-2, H-6), 9.28 (s, 1H, pyrazole H-5); ¹³C NMR (DMSO-d₆) δ 14.60, 21.40, 44.01, 60.62, 119.83, 128.94, 129.28, 129.45, 129.49, 134.72, 138.85, 139.40, 142.57, 142.66, 153.95, 162.59; MS (m/z, relative abundance %): 384.11 (M +) (100%). Anal. Calcd. For C₂₀H₂₀N₂O₄S: C, 62.48; H, 5.24; N, 7.29; Found; C, 62.64; H, 5.38; N, 7.58.

Ethyl 3-(4-chlorophenyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazole-5-carboxylate (19c)

Yield 76%; white solid; m.p. 285–287 °C; IR (KBr disk) 1701 (C = O), 1355, 1149 (SO₂); ¹H NMR (DMSO-d₆) δ 1.26 (t, J = 6.8 Hz, 2H, OCH₂CH₃), 3.29 (s, 3H, SO₂CH₃), 4.23 (q, J = 6.8 Hz, 3H, OCH₂CH₃), 7.53 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H-2, H-6), 7.85 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H-3, H-5), 8.08 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5), 8.28 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6), 9.33 (s, 1H, pyrazole H-5); ¹³C NMR (DMSO-d₆) δ 14.57, 43.99, 60.76, 114.60, 119.96, 128.47, 129.29, 130.82, 131.42, 134.20, 134.97, 139.61, 142.47, 152.77, 162.45; MS (m/z, relative abundance %): 406.06 (M +) (100%). Anal. Calcd. For C₁₉H₁₇ClN₂O₄S: C, 56.36; H, 4.23; N, 6.92; Found; C, 56.44; H, 4.48; N, 7.15.

Ethyl 3-(4-bromophenyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazole-5-carboxylate (19d)

Yield 66%; white solid; m.p. 293–295 °C; IR (KBr disk) 1693 (C = O), 1355, 1141 (SO₂); ¹H NMR (DMSO-d₆) δ 1.26 (t, J = 7.2 Hz, 2H, OCH₂CH₃), 3.28 (s, 3H, SO₂CH₃), 4.23 (q, J = 7.2 Hz, 3H, OCH₂CH₃), 7.66 (d, J = 8.4 Hz, 2H, 4-bromophenyl H-2, H-6), 7.78 (d, J = 8.4 Hz, 2H, 4-bromophenyl H-3, H-5), 8.08 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5), 8.27 (d, J = 8.8 Hz, 2H, aminosulfonlphenyl H-2, H-6), 9.32 (s, 1H, pyrazole H-5); ¹³C NMR (DMSO-d₆) δ 14.56, 44.00, 60.79, 114.58, 119.96, 122.91, 129.30, 131.15, 131.39, 131.67, 134.94, 139.57, 142.46, 152.84. 162.45; MS (m/z, relative abundance %): 448.01 (M +) (100%). Anal. Calcd. For C₁₉H₁₇BrN₂O₄S: C, 50.79; H, 3.81; N, 6.23; Found; C, 50.54; H, 3.52; N, 6.14.

High performance liquid chromatography (HPLC) purity analysis for the final most potent compounds 15b, 15e, 15f, 19b and 19c

The percent purity of the final compounds 15b, 15e, 15f, 19b and 19c was determined through high performance liquid chromatography (HPLC) analysis using (Agilent HPLC Model No. 1100, Serial No. DEO3010942 utilizing GL Sciences Inertsil ph-3–5 µm, 4.6 × 250 mm column) (Cat. No. 5020–01921, Serial No. TH5-5726, Japan) at Faculty of Graduate Studies for Advanced Sciences, Beni-Suef University, using buffer: 2.7 g/L of monobasic potassium phosphate adjusted with phosphoric acid to a pH of 3.0 and mobile phase: methanol, acetonitrile and buffer (3:1:6) at 1.5 mL/min flowrate, sample solution is 0.5 mg/mL of each compound 16d and 19b in diluent (methanol/water 3:1). HPLC chromatograms for compounds 15b, 15e, 15f, 19b and 19c were provided in supplementary section with all detailed reports which showed that retention time of solvent mobile phase was 1.738 min. and that for compounds 15b, 15e, 15f, 19b and 19c were 2.164, 2.419, 2.418, 2.417 and 2.413 min, respectively. Solvent mobile phase chromatogram was provided to establish a base line for comparison. It was found that the purity of these compounds was almost 97.9–98.9%.

Biological evaluation

In vitro anti-inflammatory activity against COX-1 and COX-2 enzymes

Determination of the in vitro COX-1/COX-2 inhibition was carried out using COX-1 Inhibitor Screening Kit-K548 (Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA) and COX-2 Inhibitor Screening Kit-K547 (Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA). The IC₅₀ value of the tested compounds was determined. Moreover, the COX-2 selectivity index (S.I values), which are calculated using the formula IC₅₀ (COX-1)/IC₅₀ (COX-2), were determined and compared to that of the used reference celecoxib (as a selective COX-2 inhibitor).

COX-1 inhibitor Screening Protocol: Dissolve the tested compounds in DMSO. Use COX assay buffer to dilute the relevant test concentration by 10X before using. Place 10 µl of diluted test inhibitor or assay buffer in the specified wells instead of the sample screen (S) or enzyme control (EC; no inhibitor). To one of the wells, add 2 µl of SC560 and 8 µl of COX Assay Buffer as an inhibitor control (IC). Before using, combine 2 µl of COX Cofactor with 398 µl of COX Assay Buffer to dilute COX cofactor 200 times. Blend thoroughly. Just before using, mix 5 µl of the provided arachidonic acid with 5 µl of NaOH to make the arachidonic acid solution. Combine quickly using a vortex. To dilute the arachidonic acid/NaOH solution 10 times, add 90 µl of water. There should be 80 µl of reaction mix in each well. Using a multi-channel pipette, add 10 µl of diluted arachidonic acid/NaOH solution to each well to initiate all the reactions at once. Fluorescence (Ex/Em = 535/587 nm) measured kinetically at 25 °C for 5–10 min.

COX-2 inhibitor Screening Protocol: Dissolve the tested compounds in DMSO. Use COX Assay Buffer to dilute the relevant test concentration by 10X before using. Place 10 µl of diluted test inhibitor or Assay Buffer in the specified wells instead of the sample screen (S) or enzyme control (EC; no inhibitor). To one of the wells, add 2 µl of celecoxib and 8 µl of COX Assay Buffer as an inhibitor control (IC). Before using, combine 2 µl of COX Cofactor with 398 µl of COX Assay Buffer to dilute COX Cofactor 200 times. Blend thoroughly. Just before using, mix 5 µl of the provided arachidonic acid with 5 µl of NaOH to make the arachidonic acid solution. Combine quickly using a vortex. To dilute the arachidonic acid/NaOH solution 10 times, add 90 µl of water. There should be 80 µl of reaction mix in each well. Using a multi-channel pipette, add 10 µl of diluted arachidonic acid/NaOH solution into each well to initiate the reactions concurrently. Fluorescence (Ex/Em = 535/587 nm) measured kinetically at 25 °C for 5–10 min.

In vitro anti-inflammatory activity against 5-LOX/15-LOX enzymes

The in vitro 5-LOX/15-LOX inhibition was assessed using the 5-lipoxygenase Inhibitor Screening Kit (Catalog # K980-100) from Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA and the Lipoxygenase Inhibitor Screening assay Kit (Item # 760,700) from Cayman Chemical, 1180, East Ellsworth Rd., Ann Arbor, MI 48108, USA [49, 50]. The test compounds potency was determined by calculating the concentration that inhibits an enzyme by 50% (IC₅₀).

5-LOX inhibitor Screening Protocol: dissolve the test compounds in DMSO. The solution was prepared at a concentration so that the final 100 µl reaction volume per well contains no more than 2 µl of the test chemical solution added to it. Each well was filled in the 96-well white plate with 2 µl of the test substance. In test wells, 2 µl of the solvent used was added to generate the test compound solution at its final concentration for the “Solvent Control” and 2 µl of the supplied LOX inhibitor, zileuton, for the “Inhibitor Control.” 38 µl of LOX Assay Buffer was added to each well to increase the volume to 40 µl. To the “Enzyme Control” well, was filled with 40 µl of LOX Assay Buffer. In order to run the required number of assays, enough reagents were mixed. 40 µl of the following mix was prepared for each well: Buffer for Reaction MIx LOX Assay 34 µl LOX Probe 2 µl 5. LOX Enzyme 4 µl After thoroughly mixing, the Reaction Mix was transferred to the wells holding the Test Compounds, Enzyme Control, Inhibitor Control, and “Solvent Control.” substrate was added after 10 min of RT incubation of the plate. The wells shouldn’t contain any bubbles. To create a 500 X solution, the supplied LOX substrate was diluted (12,500 X) in LOX Assay Buffer using a 1:25 dilution factor. To obtain the 5X solution, the 500 X solution with LOX Assay Buffer was diluted at a ratio of 1:100, depending on the number of reactions to be conducted. For each reaction, 20 µl of 5X solution will be required. How much substrate is needed based on the number of reactions. The final substrate functioning solution needs to be consumed that same day and stored on ice. The remaining stock solution should be immediately stored at -20ºC. 20 µl of 5X LOX Substrate should be added with a multichannel pipette to every well. The second minute after the substrate is added; begin recording fluorescence at Ex/Em 500/536 nm at 30-s intervals for 30 to 60 min. To get the RFU for each sample, the RFU was subtracted at time t1 from the RFU at time t2, making sure that both t2 and t1 fall within the assay's linear range. The slope for each sample (including the “enzyme control”)was multiplied by the time Δt (t2 – t1) to find the ΔRFU. In the equations below, the values was substituted for “Solvent Control” for “Enzyme Control” if the two slopes disagree.

$$\%Inhibition=[slopeof(enzymecontrol)-slopeof(testcompound)]/slopeof(enzymecontrol)\times 100$$

$$\%Relativeactivity=[slope(testcompound)]/slope(enzymecontrol)\times 100$$

15-LOX inhibitor screening protocol: Blank wells: 100 µl of assay buffer was added to at least two wells, Positive control wells: 90 µl of 15-LOX standard was added to 10 µl of assay buffer to at least two wells, 100% initial activity wells: 90 µl of Lipoxygenase enzyme was added to 10 µl of solvent to at least two wells, Inhibitor wells: 90 µl of Lipoxygenase enzyme was added to 10 µl of inhibitor wells to. It was incubated for five minutes at room temperature. The reaction was initiated by adding 10 µl of substrate to all the wells. 96well-plates were placed on a shaker for at least 10 min. 100 µl of chromogen was added to each well to stop the enzyme catalysis. The reaction was covered with plate cover. 96well-plates were placed on a shaker for at least 5 min. The cover was removed and absorbance was read using plate reader at absorbance 490–500 nm.

TNF-α and PEG2 productions in LPS-activated RAW 264.7 macrophages

The inhibition of LPS-induced inflammation via attenuating TNF-α and PGE2 cytokines production was investigated for compounds 15c, 15d, 15 h and 19d as detailed in previous procedure [9, 57]. Briefly, LPS (1 µg/mL) was added for an additional 20 h. after RAW 264.7 macrophages had been incubated with compounds 15c, 15d, 15 h and 19d in concentrations of (12.5, 25, and 50 µM) for two h. Following the manufacturer's instructions, TNF-α (ab181421 Human TNF alpha Simple Step ELISA® Kit, Abcam Inc. 152 Grove Street Waltham, MA 02453 USA) and PEG2 (Catalog Number KGE004B, Inc., MN, USA) IC₅₀ were measured in the cell culture supernatants using these commercially available ELISA kits [9, 29, 57, 58].

Inducible nitric oxide synthase (iNOS) inhibition

NO is produced from L-arginine by iNOS and is utilized in a variety of cell signaling processes. An inducible member of the NOS family, iNOS is increased during pro-inflammatory cytokine activity as a host-defense mechanism. A commercially available quantitative ELISA kit (ab253219 Mouse iNOS Simple Step ELISA® Kit) will be used to evaluate the iNOS enzyme activity of the substances in the cell lysate in accordance with the manufacturer’s instructions [59].

Cell culture and cytotoxicity assay

Using Dulbecco’s Modified Eagle Medium (DMEM, Biowest L0060) supplemented with 10% foetal bovine serum (FBS) (Biowest, S1810) and 1% anti-biotic and anti-mycotic (Biowest, L0010-100), the RAW264.7 macrophage cell line (ATCC ® TIB-71TM) was cultivated. The cells were then incubated at 37 °C with 5% CO₂ until they were confluent, which should be between 70 and 80%. After two to three days, the growth media was regularly changed. Trypsin–EDTA was then used to wash and collect the cells (Biowest, L0931-500).

The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) test (Promega, ab197010) was used to determine cell viability. In short, a 96-well plate was seeded with 5 × 103 cells per well, and the cells were then incubated for 24 h at 37 °C and 5% CO₂. The cells were then re-incubated for 24 h at 37 °C and 5% CO₂ after the medium was discarded and replaced with 180 µl of fresh growth media and 20 µl of sample (1000 ug, 250 ug, 63 ug, 16 ug, and 4 ug µg/mL). 20 µl of MTS was then added to each well, and the mixture was incubated for three hours at 37 °C with 5% CO₂. A spectrophotometer (Multiskan GO Thermo Scientific 51,119,300, Thermo Fisher Scientific, USA) was used to detect the absorbance at 490 nm. Control cells were those that received no treatment [60].

In vivo anti-inflammatory activity

Using an in vivo carrageenan-induced rat foot paw edema model, the anti-inflammatory properties of the most effective compounds 15c, 15d, 15 h and 19d were assessed. As previously described [35], paw thickness was evaluated at 1, 3, and 5 h following carrageenan injection at a dose of 50 mg/kg. Furthermore, using the procedures outlined in [35], the dose at which 50% edema inhibition (ED₅₀) was achieved for the strongest anti-inflammatory compounds 15c, 15d, 15 h and 19d was determined in relation to celecoxib.

Ulcerogenic liability

The ulcerogenic effect of 15c, 15d, 15 h, 19d, celecoxib and lonazolac was determined using the previously reported procedures [49]. Rats were divided into 7 groups and fasted for 18 h before drug administration. The control group received the vehicle (2.5% Tween 80). Other groups were received test compounds, celecoxib or lonazolac as reference drugs at a dose of 50 mg/kg. After 2 h, animals were fed. Rats were given the required dose orally for three successive days. After 2 h of the last dose, rats were sacrificed; the stomach of each rat was removed then opened along the greater curvature and rinsed with saline. In order to examine the stomach, it was stretched by pins on a corkboard. The gastric mucosa was carefully inspected for the occurrence of ulcers with the aid of an illuminated magnifying lens (l0x), then ulcer index was calculated according to the method described by Cho and Ogle [51]. Lesions were counted and measured along the greater diameter using transparent ruler. Every five hemorrhagic spots were considered equivalent to 1 mm of ulcer. The ulcer index (mm) was calculated from the sum of the total length of ulcers and hemorrhagic spots in each stomach.

Molecular modeling study

Docking was performed to secure a confirmation and binding energy ranking prediction between target enzymes and new designed compounds using Molecular Operating Environment (MOE) version 2015.10 modeling software. All docking studies were carried out using enzyme downloaded from Protein Data Bank (PDB). The crystal structure of the reference drug celecoxib and zileuton bound at the COX-2 and 5-LOX active site obtained from protein data bank at Research Collaboration for Structural Bioinformatics (RSCB) protein data bank COX-2 enzyme (PDB ID:3LN1) and 5-LOX enzyme (PDB ID: 3V99) The co-crystallized ligand (celecoxib) in COX-2 and zileuton in 5-LOX receptors were docked firstly to study its energy score, root mean standard deviation (RMSD) and interaction of different amino acids. The best crystal-like poses of them were analyzed and the least energetic conformer was detected. London DG force and force field energy were used for the refinement of results. Docking of the tested compound was executed after their 3D protonation using the 3D structure built by MOE, running conformational analysis then selecting the least energetic conformer. The same protocol used for ligands and designed compounds after minimizing energy was performed. Poses displaying the best superimposition mode on the ligand and binding energy score for each compound were analyzed to identify potential interactions with amino acids in the active site of each enzyme.

Results that obtained from docking amino acid interactions, hydrogen bond lengths and energy scores were summarized in Tables 6 and 7.

Estimation of in silico ADME properties

The ADME study was performed by using the SwissADME web tool (http://www.swissadme.ch) after drawing the compounds by Chem Sketch (v.12) and converting them to SMILE to examine the ability of the molecule to be used as a drug. It predicts the physiochemical properties, absorption, distribution, metabolism, elimination, and pharmacokinetic properties of molecules, so it is considered the key endeavor to further clinical trials. The determined parameters which were established to the most selective compounds 15c, 15d, 15 h and 19d in addition to celecoxib were listed in (Table 8).

Conclusion

As new non-acidic lonazolac analogues, two new series of pyrazole ester derivatives, 15a-h and 19a-d, were designed, synthesized and evaluated for their anti-inflammatory activity. The most potent derivatives 15c, 15d, 15 h, and 19d, showed COX-2 selectivity index in the range of (28.56–98.71) when compared to celecoxib (S.I. = 13.65). Additionally, the four most powerful derivatives shown exceptional 5-LOX and 15-LOX inhibitory actions (IC₅₀ = 0.24–0.81 µM, 0.20–2.2 µM, respectively), in comparison to zileuton. Upon using lipopolysaccharide-activated RAW 264.7 macrophages, derivatives 15c, 15d, 15 h, and 19d showed similar inhibitory activities against TNF-α and PGE2 (IC₅₀ = 0.77–1.20 μM and 0.28–0.52 μM, respectively) compared to celecoxib (IC₅₀ = 0.87 μM and 0.38 μM, respectively) as a reference compound. Compounds 15c, 15d, 15 h and 19d remarkably shown greater inhibition of inducible nitric oxide synthase (iNOS) with lower IC₅₀ (0.41–0.61 µM), when compared to the reference drug celecoxib (0.48 µM). Additionally, the results indicated that compounds 15c, 15d, 15 h, and 19d were less cytotoxic and safer, with greater IC₅₀ values (178.95–301.40 µM) than the reference drug, celecoxib (148.90 µM). Concerning in vivo anti-inflammatory activity, these four compounds (ED₅₀ = 8.22–31.22 mg/kg, respectively) were more effective than celecoxib (ED₅₀ = 40.39 mg/kg). Finally, in comparison to lonazolac (ulcer index = 20.30) and celecoxib (ulcer index = 3.02), all of the screened derivatives 15c, 15d, 15 h, and 19d were less ulcerogenic (ulcer indexes = 1.22–2.93).

Finally, it is clear that; substitution of the acidic moiety of lonazolac with the ethyl ester one in the target compounds 15a-h and 19a-d resulted in increasing the safety profiles of these analogues. Also, the incorporation of SO₂CH₃-COX-2 selective inhibition pharmacophore in the para position of the pyrazole-N-1-phenyl moiety achieved the COX-2 selectivity of these analogues over that of lonazolac.

Author contributions

Idea was created by WAAF; synthesis of the compounds was carried out by WAAF. Data analysis, interpretation and discussion part were done by MTMN and AHA. Introduction and experimental parts were reedited by WAAF, NAI. Docking, SwissAdme studies and (SAR part) were carried out by DMEA. In vitro, in vivo screening and IC₅₀ determinations were performed by HKA, HHH, AMM and AE. All the authors shared in writing, reviewing the manuscript in its final form.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors declare that they have no financial sources of support for this study.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors have declared no conflict of interest.