Hybrids of Benzenesulfonamide Oxadiazole Derivatives with Dual CA II and COX‑2 Inhibitory Activity Demonstrating Antiglaucoma and Anti-inflammatory Action: Synthesis, In Silico Insights, and In Vitro and In Vivo Bioevaluation

Abstract

In this study, new sulfonamide derivatives 5a–g and 10a–e were designed, synthesized, and biologically evaluated for their anti-inflammatory activity. In vitro COX inhibitory assays were performed, and among the synthesized compounds, 5b and 5d emerged as the most promising leads, combining COX-2 inhibition with remarkable selectivity (COX-2 IC₅₀ = 0.13 and 0.05 μM, SI = 9.25 and 12.02, respectively) and hCA II inhibition (K _i = 39.1 nM and 62.6 nM, respectively) with a high selectivity index over hCA I (SI = 933.8 and 704.2, respectively). In vivo evaluations confirmed that compounds 5b and 5d (50 mg/kg) possess promising analgesic and anti-inflammatory effects, with rapid onset, sustained duration of action, and a reduced ulcerogenic liability, indicating an improved gastrointestinal safety profile. Additionally, 5b showed significant and sustained IOP-lowering effects in antiglaucoma animal models. Computational studies and X-ray crystallography were performed as a proof of concept.

1. Introduction

Inflammation is the body’s normal defense mechanism for harmful assaults, including noxious stimuli, tissue injury, or infections. The most significant class of widely prescribed medications for the management of pain and inflammation are nonsteroidal anti-inflammatory drugs (NSAIDs) through blocking cyclooxygenases (COXs), the primary regulators of arachidonic acid conversion into prostaglandins. Long-term use of traditional NSAIDs is linked to gastrointestinal (GI) irritation, bleeding, and ulceration due to their high selectivity for COX-1 over COX-2. Therefore, developing anti-inflammatory medications that discriminate between therapeutic and adverse effects is a strategy for boosting COX-2 specificity and enhancing safety for long-term prophylaxis of chronic diseases. , Attempts to explore selective COX-2 inhibitors have yielded numerous diarylheterocycles and core ring pharmacophore models. Celecoxib I and SC-558 II (Figure ) are regarded as typical templates of a pyrazole central scaffold bearing 1,3-diphenyl moieties, , followed subsequently by valdecoxib. However, the global withdrawal of rofecoxib (Vioxx) after evidence linking its use to an increased risk of cardiovascular events raised significant concerns regarding the overall cardiovascular safety profile of COX-2 selective inhibitors.

1.

Design of pyrazole-based derivatives 5a–g and 10a–e targeting both hCA II and COX-2 isoforms.

The current understanding of inflammation extends beyond prostaglandin-mediated pathways to include additional contributing mechanisms, notably tissue acidosis, which plays a significant role in the sensitization of pain and the progression of the inflammatory responses. Therefore, targeting pH-regulating enzymes such as carbonic anhydrases (CAs) represents a complementary strategy that may enhance the anti-inflammatory efficacy when combined with COX-2 inhibition.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that exert a dual therapeutic effect: (i) as anti-inflammatory agents, they regulate pH by catalyzing the reversible hydration of carbon dioxide to yield protons and bicarbonate ions; , (ii) as antiglaucoma agents, remarkably, the inhibition of human carbonic anhydrase (hCA) I, II, and IV isoforms with a minor contribution from CA XII reduces intraocular pressure (IOP) by inhibiting aqueous humor production within the ciliary body of the eye.

The free sulfonamide functionality of CAs does not only act as a catalytic Zn²⁺ binding candidate for the most potent CA inhibitors (CAIs) but also as an important pharmacophoric feature for selective COX-2 inhibitor development (Figure ). Celecoxib I, a well-known sulfonamide-tethered anti-inflammatory drug, effectively inhibits hCA II, exhibiting IC₅₀ values in the submicromolar range. Compounds III and IV (Figure ) are aminoaryl pyrazole derivatives that have been reported to exhibit dual inhibitory activity against both COX-2 and carbonic anhydrase (CA) enzymes. − Sulfonamide-based hCAIs’ main drawback, however, is their lack of selectivity. Hence, Supuran’s group established the “tail approach” to overcome the selectivity hurdle and appropriately target their activity toward a particular CA isoform. The elongated molecule with its new tail (usually on a benzenesulfonamide scaffold) can precisely interact with the nonconserved amino acid residues in the middle/outer rims of the CA’s active region (Figure ). ,

2.

Diagrammatic illustration of the tail approach for designing selective zinc-binding CAIs by the tail approach.

Designing single-entity ligands has emerged as a robust approach to address complex pathologies such as glaucoma, where the concurrent carbonic anhydrase and cyclooxygenase pathway modulation can provide synergistic therapeutic benefits. ,

Motivated by the aforementioned findings, we embarked on the design and synthesis of a series of oxadiazole-bearing (pyrazol-1-yl)­benzenesulfonamide derivatives 5a–g and 10a–e that maintain celecoxib’s 4-(pyrazol-1-yl)­benzenesulfonamide pharmacophoric moiety. The integrated free sulfonamide functional group is anticipated not only to bind the catalytic Zn²⁺ of hCA but also to induce COX-2 selectivity through the formation of a hydrogen bond with the ligand binding pocket’s hydrophilic amino acid residues. Krasavin et al. developed a series of oxadiazol-5-yl benzenesulfonamides V and VI as selective inhibitors of hCA II and IX isoforms at subnanomolar levels, surpassing acetazolamide (Figure ). Consequently, the inhibitory efficacy was expected to be augmented via incorporation of various extended alkyl tails through an oxadiazole linker, affording the targeted compounds 5a–g and 10a–e (Figures and ).

All of the synthesized compounds were assessed for COX-1 and COX-2 inhibition, in addition to their hCA I, II, and XII inhibitory activities. Moreover, the analgesic, anti-inflammatory, and ulcerogenic properties and antiglaucoma potential were estimated for pyrazoles 5b and 5d, exhibiting favorable COX-2 potency and selectivity while maintaining acceptable carbonic anhydrase II activity.

2. Results and Discussion

2.1. Chemistry

Two series of 4-(4-(5-(mercapto/thiosubstituted)-1,3,4-oxadiazol-2-yl)-5-phenyl-1H-pyrazol-1-yl)­benzenesulfonamide 5a–g and 4-(5-amino-4-(5-(thio)-1,3,4-oxadiazol-2-yl)-1H-pyrazol-1-yl)­benzenesulfonamide 10a–e derivatives have been synthesized as illustrated in Schemes and , respectively. Scheme illustrates the pathway for the synthesis of derivatives 5a–g, which started with the reflux of ethyl benzoyl acetate (1) with DMF–DMA (formylating/enaminating agent), affording the aminoacrylate enaminone derivative (2) (yield = 78%). Then, derivative (2) was cyclized with 4-aminosulfonylphenylhydrazine hydrochloride in refluxing absolute ethanol to give the pyrazole-4-carboxylate derivative (3) in 69% yield, which was employed in the next hydrazinolysis step through fusion with hydrazine hydrate to produce the hydrazide derivative (4) (yield = 56%). Regarding the second series, 10a–e (Scheme ), it started with the reflux of ethylcyanoacetate (6) with triethyl orthoformate in acetic anhydride, yielding 76% ethyl (ethoxyethylene)­cyanoacetate (7). Following this, derivative (7) cyclized with 4-aminosulfonylphenylhydrazine in a mixture of acetic acid/water (5:1) under reflux to give the pyrazole-4-carboxylate derivative (8) in 68% yield, which was subsequently subjected to hydrazinolysis to form the corresponding hydrazide derivative (9) (yield = 57%). Finally, the target 1,3,4-oxadiazole derivatives 5a–g (Scheme ) and 10a–e (Scheme ) were afforded by the reaction of the acid hydrazide derivatives 4 and 9, respectively, with carbon disulfide in DMF at 70 °C for 4 h via a multistep process involving the initial formation of a dithiocarbazate intermediate, followed by intramolecular cyclization and ring closure. Following cyclization, the thiol intermediate is deprotonated by triethylamine (TEA) to form a reactive thiolate, which then undergoes S-alkylation by stirring overnight with alkyl halides to yield the corresponding thioether products.

1. Synthesis of Compounds 5a–g .

^a Reagents and conditions: (i) DMF–DMA, reflux 2 h; (ii) p-SO₂NH₂C₆H₄NHNH₂·HC1/abs. Ethanol/reflux 4 h; (iii) N₂H₄·H₂O/reflux 3 h; (iv) CS₂/DMF/stir overnight/rt; (v) RBr/TEA/stir/rt.

2. Synthesis of Compounds 10a–e .

^a Reagents and conditions; (i) triethylorthoformate/acetic anhydride/reflux 10 h; (ii) p-SO₂NH₂C₆H₄NHNH₂·HCl/acetic acid/H₂O/reflux 4 h; (iii) N₂H₄·H₂O/reflux 4 h; (iv) CS₂/DMF/stir overnight/rt; (v) RBr/TEA/stir/rt.

¹H NMR spectra confirmed the structures of the target derivatives 5a–g and 10a–e. The benzyl derivatives 5a and 10a demonstrated additional singlet signals at δ 4.36 and 4.5 ppm, respectively, of the benzylic CH₂ protons. Also, the spectrum of derivative 5g showed a singlet D₂O exchangeable signal of the mercapto group at δ 14.51 ppm. Moreover, after stirring overnight with the appropriate alkyl halide, the corresponding S-alkylated derivatives 5c–f and 10c–e demonstrated the aliphatic signals with a range of δ 0.85–3.28 ppm assigned to these groups. Also, the allylic protons of 5d and 10d were confirmed as they showed signals around δ 5.07–5.99 ppm. Furthermore, for acetic acid derivatives 5b and 10b, the spectra showed singlet signals of carboxylic CH₂ at δ 3.79 and 4.18 ppm, respectively. Regarding the ¹³C NMR spectra, they revealed the S-alkyl substitution with aliphatic signals between δ 13.18 and 40.58 ppm for derivatives 5c, 5e, 5f, 10c, and 10e. Additionally, for the S-benzyl substituted derivatives 5a and 10a, the spectra showed a signal for the benzylic carbon at δ 36.29 and 36.52 ppm, respectively. Referring to the carboxylic acid derivatives 5b and 10b, a signal at 168.93 and 169.50, respectively, is assigned to the carboxylic carbonyl carbon. Also, the S-allyl substituted derivatives 5d and 10d exhibited three signals in the range of δ 35.06–133.31 ppm attributed to the three allylic carbons.

2.2. Biological Evaluation

2.2.1. COX-1 and COX-2 Inhibitory Assay

All the new target sulfonamide derivatives 5a–g and 10a–e were in vitro assessed for both their COX-1 and COX-2 inhibitory activities using celecoxib as a reference drug, and the results were displayed as IC₅₀ values. In addition, the compounds’ safety profile was investigated and the selectivity indices (SIs) toward COX-2 were determined. As depicted in Table , six compounds 5b, 5c, 5d, 5g, 10c, and 10e (IC₅₀ ranges between 0.05 and 0.17 μM) had comparable COX-2 inhibitory activity compared to the reference celecoxib (IC₅₀ = 0.05 μM), while derivatives 5a and 10d revealed moderate potency with IC₅₀ values of 0.46 and 0.42 μM, respectively.

1. IC₅₀ Values of the New Sulfonamide Derivatives 5a–g and 10a–e as COX Inhibitors in Comparison with Celecoxib as a Reference Drug.

Compound	R1	R2	COX-1 inhibition [IC50 μM ± SD]	COX-2 inhibition [IC50 μM ± SD]	SI (COX-1/COX-2)
5a	Ph	–CH2–C6H5	0.45 ± 0.20	0.46 ± 0.18	0.97
5b	Ph	–CH2–COOH	1.19 ± 0.50	0.13 ± 0.05	9.25
5c	Ph	–CH2CH2CH3	1.19 ± 0.48	0.17 ± 0.06	7.07
5d	Ph	–CH2CHCH2	0.64 ± 0.26	0.05 ± 0.02	12.02
5e	Ph	CH2CH2CH2CH3	5.55 ± 1.97	0.62 ± 0.23	8.98
5f	Ph	–CH2CH(CH3)2	2.35 ± 0.86	1.21 ± 0.46	1.94
5g	Ph	H	1.34 ± 0.62	0.17 ± 0.07	7.95
10a	NH2	–CH2–C6H5	6.59 ± 2.21	1.54 ± 0.50	4.27
10b	NH2	–CH2–COOH	9.51 ± 3.32	2.65 ± 0.84	3.59
10c	NH2	–CH2CH2CH3	0.96 ± 0.40	0.15 ± 0.06	6.35
10d	NH2	–CH2CHCH2	2.40 ± 0.88	0.42 ± 0.17	5.71
10e	NH2	–CH2CH3	0.29 ± 0.13	0.07 ± 0.03	4.18
Celecoxib		-	1.51 ± 0.66	0.05 ± 0.11	29.22

^aThe data are presented as the average IC₅₀ values (mean ± SD) of three independent replicates.

Comparing the COX-1 and COX-2 isozymes’ inhibitory effects, all new sulfonamide derivatives 5a–g and 10a–e revealed enhanced COX-2 inhibitory efficacy (IC₅₀ ranges between 0.05 and 2.65 μM) in comparison to COX-1 (IC₅₀ ranges between 0.29 and 9.51 μM) with good selectivity indices. In particular, 1,5-diphenylpyrazoles 5b, 5c, and 5d demonstrated outstanding selectivity indices (SI ranges between 7.07 and 12.02) rather than their corresponding (5-amino-1H-pyrazol-1-yl)­benzenesulfonamide analogues 10b, 10c, and 10d (SI ranges between 3.59 and 6.35) against the COX-2 isoenzyme, except for the 5-(benzylthio)-1,3,4-oxadiazol-2-yl derivative 5a, which showed a lower selectivity index (SI = 0.97) than its corresponding analogue 10a (SI = 4.27).

Considering the series 5a–g, the SAR investigation revealed that COX-2 inhibitory activity was improved in the derivatives 5c and 5d (IC₅₀ range = 0.17 and 0.05 μM, respectively) with alkylated side chain substitution or with freely ionizable centers in 5b and 5g with IC₅₀ in the range 0.13–0.17 μM, except for butyl 5e and isopropyl 5f derivatives that exhibited lower IC₅₀ values of 0.62 μM and 1.21 μM, respectively. Moreover, the introduction of a benzylic side chain led to moderate COX-2 inhibitory activity, as demonstrated by compound 5a (IC₅₀ = 0.46 μM). However, in series 10a–e, the derivatives with alkylated side chain substitution in 10c–10e demonstrated greater potency (IC₅₀ range = 0.07–0.42 μM) rather than the ionizable carboxylic acid derivative 10b (IC₅₀ = 2.65 μM) or the derivative inserted with a benzylic side chain as 10a (IC₅₀ = 1.54 μM). It is worth mentioning that compounds 5b and 5d have emerged as the most promising compounds combining notable COX-2 inhibition with excellent selectivity indices of 9.25 and 12.02, respectively.

Moreover, COX-2 selectivity was strongly affected by both the pyrazole C₅ substituent (R₁) and the oxadiazole thio substituent (R₂). The 1,5-diphenylpyrazole derivatives 5a–g (R₁ = Ph) were generally more selective than the corresponding 5-amino analogues 10a–e (R₁ = NH₂), indicating that the bulky phenyl group better fits the larger COX-2 secondary pocket. Compounds 5b, 5c, and 5d showed selectivity indices higher than those of 10b, 10c, and 10d. Among R₂ substituents, the allyl derivative 5d showed the highest selectivity (SI = 12.02), followed by the carboxymethyl derivative 5b (SI = 9.25), suggesting that small flexible alkyl chains and ionizable groups enhance selective COX-2 binding rather than bulky benzylic, long-chain, or branched alkyl substituents. Thus, compounds 5b and 5d were the most promising selective COX-2 inhibitors.

2.2.2. Carbonic Anhydrase (CA) Inhibitory Assay

A carbonic anhydrase inhibition assay for all derivatives has been performed against three human carbonic anhydrase (hCA) isoforms: hCA I, hCA II, and hCA XII. The method used in the assay was the stopped-flow CO₂ hydrase assay (Table ).

2. Inhibition Assay for the Synthesized 5a–g and 10a–e against hCA I, hCA II, and hCA XII Using Acetazolamide (AAZ) as a Reference Drug.

			K i (nM)			Selectivity Indices (SI)
Compound	R1	R2	hCA I	hCA II	hCA XII	I/II	XII/II
5a	Ph	–CH2–C6H5	42,140	43.7	60.9	964.3	1.39
5b	Ph	–CH2–COOH	36,510	39.1	34.2	933.8	0.87
5c	Ph	–CH2CH2CH3	46,630	67.8	61.1	687.8	0.90
5d	Ph	–CH2CHCH2	44,080	62.6	69.7	704.2	1.11
5e	Ph	–CH2CH2CH2CH3	50,270	48.2	54.6	1042.9	1.13
5f	Ph	–CH2CH(CH3)2	47,390	54.1	75.3	875.9	1.39
5g	Ph	H	57,860	50.5	39.2	1145.7	0.78
10a	NH2	–CH2–C6H5	938.6	85.4	53.4	10.9	0.63
10b	NH2	–CH2–COOH	425.8	61.6	52.9	6.9	0.86
10c	NH2	–CH2CH2CH3	1387	73.9	74.3	18.8	1.01
10d	NH2	–CH2CHCH2	2546	102.3	66.4	24.9	0.65
10e	NH2	–CH2CH3	1798	93.1	61.8	19.3	0.66
AAZ	-	-	250.0	12.0	5.7	20.8	0.48

^aThe mean of 3 different assays, by a stopped-flow technique (errors were in the range of ±5–10% of the reported values).

Compared to the reference compound AAZ, all derivatives exhibited a less favorable inhibitory profile against the off-target isoform hCA I as well as against the target isoforms hCA II and hCA XII while still retaining potency in the low double-digit nanomolar range toward the latter two isoforms. Nevertheless, this modulation resulted in compounds 5a–g with a markedly improved selectivity relative to AAZ (SI I/II = 687.8–1145.7), together with a slightly enhanced selectivity profile favoring hCA XII over hCA II (SI XII/II = 0.63–1.39) for all compounds. Such selectivity is particularly important for minimizing the off-target side effects commonly associated with CA inhibitors.

Based on the results of CA inhibition assays, it is observed that replacing the small 5-amino group of 10a–e derivatives (hCA I K _i range: 425.8 nM (10b) to 2546 nM (10d)) with a bulky phenyl moiety in 5a–g series (hCA I K _i values above 36,500 nM) drastically decreases the affinity toward hCA I, which improves the derivatives’ selectivity.

Interestingly, the acetic acid derivative 10b with the 5-amino group showed superior potency against hCA I (K _i = 425.8 nM), suggesting a favorable interaction binding mode with hCA I and helping design highly selective inhibitors. Moreover, 10b is unique among the derivatives in its selectivity toward hCA XII when compared to hCA II.

Conspicuously, the most suited derivatives for hCA XII inhibition are those with a 5-phenyl moiety and with polar tails such as the carboxylic acid derivative 5b (K _i = 34.2 nM) and mercapto derivative 5g (K _i = 39.2 nM).

2.2.3. In Vivo Biological Evaluations

2.2.3.1. Analgesic Activity

The analgesic activities of compounds 5b and 5d were assessed over a 90 min period to evaluate both their efficacy and duration of action. As illustrated in Figure , both compounds produced a pronounced and statistically significant increase in latency time relative to the vehicle-treated control group (P < 0.001).

3.

Time-dependent analgesic effects of compounds 5b and 5d were evaluated using the hot plate assay. Ibuprofen served as the standard reference drug. Results are presented as mean ± SEM (n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA followed by Tukey’s post hoc test to assess significance. The exact P values, mean differences, and 95% confidence intervals for all treatment groups across key time points are provided in Table S2 of the Supporting Information.

Compound 5d demonstrated a rapid onset of action, with significant analgesic effects observed as early as 10 min postadministration (P < 0.01). At this early point, its efficacy was statistically compared to that of the reference drug, ibuprofen (P > 0.05, Tukey’s test), indicating a similar pharmacodynamic onset. In contrast, compound 5b exhibited a relatively slower onset of action, with activity that appeared lower than that of Ibuprofen at the 10 min time point (P < 0.01). Nevertheless, a highly significant pharmacological effect (P < 0.001) was observed at subsequent time intervals under the present experimental conditions.

Notably, both compounds maintained their analgesic effects throughout the entire observation period. At later time points (60–90 min), both 5b and 5d continued to display significantly elevated latency times compared to the control group (P < 0.001 at 60 min). Collectively, these findings suggest that compounds 5b and 5d may possess promising analgesic activity accompanied by a sustained duration of action compared with the reference nonsteroidal anti-inflammatory drug, ibuprofen. Nevertheless, these observations remain preliminary and should be further validated through expanded pharmacological and mechanistic investigations.

2.2.3.2. Anti-inflammatory Activity

The in vivo anti-inflammatory activities of compounds 5b and 5d were investigated using the carrageenan-induced rat paw edema model over a 6 h period, with ibuprofen employed as the reference standard. Subplantar administration of carrageenan produced a progressive localized edema in the negative control group, reaching maximal swelling between 3 and 6 h postinjection.

As depicted in Table and Figure , both compounds (50 mg/kg) demonstrated promising anti-inflammatory activity, with an apparent early onset of action under the current experimental conditions. At 2 h post carrageenan injection, compounds 5b and 5d significantly reduced paw edema relative to the carrageenan-treated group, whereas the reference drug ibuprofen (25 mg/kg) did not exhibit a statistically significant effect at this time point. The most pronounced anti-inflammatory responses were observed at 3 and 4 h, during which both derivatives markedly attenuated paw edema compared with the carrageenan group and displayed considerable anti-inflammatory activity relative to the standard reference treatment. However, comparisons between the tested compounds and ibuprofen should be interpreted cautiously due to differences in the administered dose levels.

3. Percentage Inhibition of Carrageenan-Induced Paw Edema in Rats Following Treatment with Compounds 5b and 5d .

Treatment	Dose (mg/kg)	1 h	2 h	3 h	4 h	5 h	6 h
Carrageenan	1%	-	-	-	-	-	-
Ibuprofen	25	0%	0%	23.3%***	30.8%***	68.1%***	77.1%***
5b	50	23.3%**	22.8%***	49.2%***	56.6%***	73.0%***	72.3%***
5d	50	23.6%**	9.8%***	49.2%***	49.2%***	67.9%***	72.3%***

^aData are presented as percentage inhibition (n = 3). Statistical significance was determined based on raw paw volume measurements relative to the carrageenan-induced control group using two-way repeated-measures ANOVA followed by Tukey’s post hoc test. Significance levels are indicated as **P < 0.01 and ***P < 0.001.

4.

In vivo anti-inflammatory effects of compounds 5b and 5d were assessed using the carrageenan-induced rat paw edema model. Paw volume (mL) was recorded at 1, 2, 3, 4, 5, and 6 h following carrageenan administration. Ibuprofen served as the positive reference control. Data are presented as mean ± SEM (n = 3). Statistical analysis was conducted using two-way repeated-measures ANOVA followed by Tukey’s multiple comparisons test. The exact P values, mean differences (effect size), and 95% confidence intervals for all treatment groups across key time points are provided in Table S3 of the Supporting Information.

This potential anti-inflammatory effect was sustained for the duration of the experiment. At 5 and 6 h, both compounds maintained paw volumes approaching those of the baseline normal control group. Comparative analysis suggested that compound 5b produced a somewhat greater inhibitory effect on edema at 5 h than compound 5d; nevertheless, both derivatives demonstrated sustained anti-inflammatory activity throughout the experimental period. Under the present experimental conditions, the tested compounds (50 mg/kg) exhibited anti-inflammatory effects relative to the reference treatment, ibuprofen (25 mg/kg). However, such comparisons should be interpreted cautiously owing to the differences in the administered dose levels.

2.2.3.3. Ulcerogenic Activity

The gastric safety of compounds 5b and 5d was systematically assessed over a 24 h period and benchmarked against the reference drug, ibuprofen. As illustrated in Table and Figures and S75, ibuprofen induced a progressive, time-dependent increase in gastric mucosal injury, with the Ulcer Index (UI) rising from 7.33 at 1 h to a maximum of 19.66 at 24 h.

4. Assessment of the Ulcerogenic Potential and Gastroprotective Efficacy of Ibuprofen in Comparison with Compounds 5b and 5d in Rats across Multiple Time Intervals.

Compound	Time (h)	Number of animals with ulcers	% Incidence divided by 10	Average number of ulcers	Average severity	Ulcer Index	% Protection (vs Ibuprofen)
Control	1	0/6	0	0	0	0	-
Ibuprofen	1	3/6	5	0.67	1.66	7.33	-
5b	1	1/6	1.76	0.33	2	4.09	44.20
5d	1	0/6	0	0	0	0	100.00
Control	3	0/6	0	0	0	0	-
Ibuprofen	3	5/6	8.33	2.6	2.8	13.73	-
5b	3	2/6	3.33	1.5	2	6.83	50.25
5d	3	1/6	1.66	2	2	5.66	58.78
Control	6	0/6	0	0	0	0	-
Ibuprofen	6	6/6	10	5	3.16	18.16	-
5b	6	3/6	5	2.66	2.33	9.99	44.99
5d	6	3/6	5	2.33	2	9.33	48.62
Control	24	0/6	0	0	0	0	-
Ibuprofen	24	6/6	10	5.66	4	19.66	-
5b	24	4/6	6.67	3.25	2.75	12.67	35.55
5d	24	3/6	5	2.36	2.5	9.86	49.85

5.

Time-dependent profile of ulcerogenic activity following administration of ibuprofen, 5b, and 5d in rats. The Ulcer Index (UI) was determined by integrating the incidence, number, and severity of gastric lesions at 1, 3, 6, and 24 h post-treatment. Each data point represents the mean ulcer index for the respective experimental group (n = 6).

In contrast, both synthesized derivatives exhibited a markedly lower ulcerogenic potential. Compound 5d, in particular, demonstrated pronounced gastroprotective properties, with complete protection observed at 1 h (100% inhibition of ulcer formation). Notably, even after 24 h, compound 5d maintained a relatively low UI of 9.86, corresponding to a 49.8% reduction in gastric damage compared with ibuprofen.

Similarly, compound 5b attenuated gastric irritation, affording 50.2% protection at 3 h and 35.5% protection at the 24 h time point. Collectively, these findings indicate that both derivatives possess improved gastric safety profiles relative to the reference drug, with compound 5d exhibiting superior protective efficacy. Although the obtained results suggest notable biological activity, definitive conclusions regarding the superiority or equivalence to ibuprofen cannot be established at this stage.

2.2.3.4. Antiglaucoma Activity

Compounds 5b and 5d were selected for antiglaucoma evaluation based on a balanced dual-enzymatic target profile rather than optimization toward a single enzyme. Given that COX-2 inhibition represents a key determinant of anti-inflammatory activity in this study, priority was assigned to compounds exhibiting superior COX-2 potency and selectivity while maintaining an acceptable carbonic anhydrase II activity.

The intraocular pressure (IOP)-lowering effects of compounds 5b and 5d were evaluated by using a rabbit model of glaucoma. Control experiments were conducted by intravitreal injection of 0.05 mL of a hypertonic saline solution (5% NaCl in distilled water) into both eyes. Rabbits with induced elevated IOP received topical administration of 0.05 mL of compound 5b prepared as 1% solution and 0.1 mL of compound 5d prepared as 0.05% solution.

Compounds 5b and 5d demonstrated intraocular pressure (IOP)-lowering activity in the rabbit model of induced glaucoma, supporting their potential ocular hypotensive effects. Following a substantial increase in IOP after glaucoma induction, topical administration of compound 5b produced a rapid and significant reduction in IOP, with a noticeable effect observed at 60 min and sustained over the experimental period. The statistically significant difference compared with the sham-treated eye indicates a promising pharmacological activity of this derivative in the tested model.

In contrast, compound 5d exhibited a slower and less pronounced effect on the IOP. Although a gradual reduction was observed at later time points, the changes did not reach statistical significance, indicating a comparatively weaker efficacy under the tested conditions. Overall, these findings suggest that compound 5b possesses superior IOP-lowering activity and represents a more promising candidate for further investigation (Figure ).

6.

Time-course profile of intraocular pressure (IOP) in a rabbit model of induced glaucoma was evaluated following a single topical administration of derivatives 5b (black) and 5d (blue). Data are presented as mean ± SEM (n = 4). Solid lines with closed circles denote treated (Rt) eyes, whereas dashed lines with open circles indicate contralateral sham-treated (Lt) eyes. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. Derivative 5b induced a significant reduction in IOP relative to the sham-treated eye at 60 min (**P = 0.0061). The exact P values, mean differences (effect size), and 95% confidence intervals for all treatment groups across key time points are provided in Table S4 of the Supporting Information.

The present investigation establishes an expanded in vivo pharmacological profile. Compounds 5b and 5d demonstrated potent and selective COX-2 inhibition together with significant hCA II inhibitory activity, indicating a well-balanced dual-target mechanism. In addition, they exhibited a promising anti-inflammatory, analgesic, with reduced gastric toxicity relative to ibuprofen. Notably, compound 5b also exhibited significant intraocular pressure-lowering activity in vivo. Collectively, these findings highlight a broader therapeutic potential compared with previously reported pyrazole–sulfonamide derivatives, integrating efficacy with improved safety.

2.3. X-ray Crystallographic Analysis

We solved the X-ray crystal structure of hCA II bound to derivative 10d to understand the molecular basis of the CA inhibition. Initial refinement of the structure revealed a well-defined electron density for the inhibitor inside the active site (Figure S54), demonstrating that the benzenesulfonamide group engages the zinc ion in its characteristic deprotonated coordination, typical of this class of inhibitors (Figure ). , In addition, this moiety is stabilized by a hydrogen bond to the Thr199 backbone and hydrophobic contacts with Val121 and Leu198. , The amino group in the pyrazole ring contributes to stabilizing the interaction with a hydrogen bond with Thr200. Finally, the thioallyl tail, which exhibits a weaker and less defined electron density, likely because of increased conformational flexibility, is directed into a hydrophobic pocket through an interaction with Phe20.

7.

X-ray crystal structure of hCA II bound with compound 10d (green, PDB: 9T4S). Residues involved in the binding of inhibitors are also shown; the gray sphere represents the zinc ion in the active site of the proteins. Van der Waals interactions are shown in blue, and hydrogen bonds are shown in red.

2.4. Molecular Modeling

2.4.1. Molecular Docking

Molecular docking was conducted to assess the binding interactions of 5b and 5d within hCA II and COX-2 binding sites using the Glide docking tool. These two compounds were selected for modeling to provide qualitative, hypothesis-generating support for their experimental inhibitory activity. The docking approach was validated by demonstrating its capacity to reproduce the binding modes of the cocrystallized hCA II inhibitor (furosemide in PDB 1Z9Y) and the COX-2 inhibitor (rofecoxib in PDB 5KIR). This was performed by superimposing the crystallographic poses with their top-scored docking poses. The docked poses of furosemide and rofecoxib successfully resembled their crystallographic binding modes with RMSD values of 0.95 and 0.27 Å, respectively, providing impetus to initiate the docking of acetazolamide, celecoxib, 5b, and 5d using the same settings. The Glide docking scores of the top-ranked poses of acetazolamide, celecoxib, 5b, and 5d docked into hCA II and COX-2 are summarized in Table .

5. Glide Docking Scores Calculated for Top-Ranked Poses in hCA II and COX-2 Active Sites.

	Glide docking scores (kcal/mol)
Compound	hCA II	COX-2
Acetazolamide	–7.30	NA
Celecoxib	NA	–9.60
5b	–8.05	–9.18
5d	–7.48	–10.10

Notably, compounds 5b and 5d demonstrated docking scores comparable to those of the reference ligands in both of the enzymes. These docking results were consistent with the observed in vitro inhibitory activity; for instance, compound 5b, which showed relatively stronger inhibition of hCA II, also yielded a more favorable docking score compared to 5d. Conversely, compound 5d demonstrated a superior docking score in COX-2, in agreement with its favorable in vitro COX-2 inhibitory activity. These computational predictions support the experimental inhibitory data (Table ).

Figure illustrates the docked poses of acetazolamide in hCA II and celecoxib in COX-2, compared with the top-ranked poses of compounds 5b and 5d in both proteins. In hCA II, the Zn²⁺ ion exhibited a tetrahedral coordination with the deprotonated sulfonamide nitrogen of acetazolamide, 5b, and 5d as well as His94, His96, and His119 through their imidazole nitrogen atoms. Additionally, one sulfonamide oxygen atom in all three compounds formed bifurcated hydrogen bonds with the backbone NH groups of Thr199 and Thr200. The deprotonated NH of acetazolamide was anchored to the hydroxyl group of the Thr199 side-chain via a hydrogen bond, while the thiadiazole moiety was positioned near the hydroxyl group of Thr200, suggesting the possible formation of an additional hydrogen bond. However, in compounds 5b and 5d, the pyrazole nitrogen was oriented toward Gln92, forming a hydrogen bond with its side-chain amide NH, while the nitrogen atom of the neighboring oxadiazole ring established an additional hydrogen bond with the side-chain amide NH of Asn67. Furthermore, the phenyl ring attached to the pyrazole moiety in both compounds was positioned adjacent to Phe131, suggesting π–π stacking interactions comparable to that observed for the furan ring of furosemide.

8.

Detailed view of amino acid interactions within the binding sites of hCA II (PDB ID: 1Z9Y) and COX-2 (PDB ID: 5KIR) with reference ligands, 5b and 5d. (A) hCA II–acetazolamide, (B) hCA II–5b, (C) hCA II–5d, (D) COX-2–celecoxib, (E) COX-2–5b, and (F) COX-2–5d. Hydrogen bonds (≤3.0 Å) are depicted as yellow dashed lines, while Zn²⁺ coordination is depicted as magenta dashed lines.

For compound 5b, the closest atom–centroid distance is 3.8 Å and the centroid–centroid distance is 5.0 Å, whereas for compound 5d, the corresponding distances are 3.9 Å and 5.3 Å, respectively. In both compounds, the angle between the ring planes is approximately 80°. These metrics support the formation of T-shaped (edge-to-face) π–π interactions.

In COX-2, the two sulfonamide oxygen atoms of celecoxib, 5b, and 5d engaged Arg513 and Phe518 in hydrogen bonding interactions. Additionally, the sulfonamide NH₂ group of the three compounds formed bifurcated hydrogen bonds with Gln192 and Leu352. In addition, the pyrazole ring of celecoxib as well as the oxadiazole ring of 5b and 5d was positioned in proximity to Arg120, indicating possible π–cation interactions. Finally, in all three compounds, the phenyl ring attached to the pyrazole moiety was located within a hydrophobic pocket defined by Leu352, Tyr385, and Val523, suggesting hydrophobic interactions and π–π stacking stabilization.

2.4.2. Molecular Dynamics Simulations

To evaluate the stability of the predicted binding modes obtained from molecular docking, the docking poses in complex with their corresponding receptor were simulated for 200 ns using the Desmond package. Analysis of the simulation trajectories focused on monitoring the Root Mean Square Deviation (RMSD) values and interaction occupancy rates, as illustrated in Figures and . The RMSD values of the Cα atoms of hCA II and COX-2 fluctuated between 1 and 2.5 Å in all simulated runs. Acetazolamide and celecoxib stabilized at RMSD values of around 1.5–2 Å upon superposition onto the Cα atoms of their cognate receptors. Compound 5b and 5d in complex with hCA II showed RMSD values ranging between 1 and 2 Å, with few short peaks reaching 3 Å, observed in 5d. In COX-2, 5b also maintained RMSD fluctuations between 1 and 2 Å, whereas 5d displayed slightly higher RMSD values of nearly 2–3 Å (Figure ).

9.

RMSD values throughout 200 ns MD simulations. (A) hCA II–acetazolamide, (B) hCA II–5b, (C) hCA II–5d, (D) COX-2–celecoxib, (E) COX-2–5b, and (F) COX-2–5d.

10.

Interaction occupancy rates throughout 200 ns MD simulations. (A) hCA II–acetazolamide, (B) hCA II–5b, (C) hCA II–5d, (D) COX-2–celecoxib, (E) COX-2–5b, and (F) COX-2–5d.

The key interactions with hCA II and COX-2 pocket residues observed in molecular docking were largely preserved throughout the simulation trajectories. The Zn²⁺ coordination by the three hCA II histidine residues and the deprotonated sulfonamide nitrogen was maintained, while additional coordination interactions were established with the carboxylate oxygen of Glu106. Furthermore, a sustained coordination interaction with the Zn²⁺ atom was observed via one sulfonamide oxygen of compounds 5b and 5d, whereas the second sulfonamide oxygen maintained the bifurcated hydrogen bond with Thr199 and Thr200, with occupancy rates between 34% and 98%. In acetazolamide, the two hydrogen bonds formed of the sulfonamide oxygen and the NH group with Thr199 were preserved for 92% and 38% of the simulation time, respectively. In addition, the pyrazole nitrogen and its attached amide NH adopted a favorable orientation toward Thr200 and Thr201, forming two hydrogen bonds that lasted for 98% and 66% of the simulation time, respectively. Similarly, the hydrogen bond with Gln92, mediated by the oxadiazole nitrogen atoms of compounds 5b and 5d, was maintained for 86% and 98% of the simulation time, respectively. However, the direct hydrogen bond with Asn67 observed in both compounds was disrupted during the simulation and replaced by two water-mediated hydrogen bonds involving Asn67 and Glu69. Additionally, the π–π stacking interaction with Phe131 observed in all three compounds was not consistently maintained during the simulation, whereas a hydrophobic interaction with Leu198 was observed and remained stable (Figure A,B).

In COX-2, the interactions of celecoxib, 5b, and 5d with Leu352, Ser353, Ile517, Phe518 (hydrogen bonds), and Arg120 (π–cation) were preserved throughout the simulation runs, with occupancy rates ranging from 40% to 100%. In compound 5b, two additional hydrogen bonds formed between the carboxylate oxygens of the tail and Arg120 were observed during the simulation. In both compounds, a π–π stacking interaction was established between the phenyl ring of the benzenesulfonamide moiety and Tyr355 and was maintained for approximately half of the simulation time (Figure D–F). These simulation results suggest that the proposed binding modes of compounds 5b and 5d within the hCA II and COX-2 active sites are stable and offer a molecular explanation for their observed structure–activity relationships.

3. Conclusion

A series of novel sulfonamide derivatives 5a–g and 10a–e were designed, synthesized, and biologically evaluated for their anti-inflammatory activity. In vitro cyclooxygenase (COX-1 and COX-2) inhibitory assays were performed using celecoxib as a reference drug, and most of the synthesized compounds demonstrated enhanced COX-2 selectivity over COX-1. Notably, compounds 5b, 5c, 5d, 5g, 10c, and 10e exhibited promising COX-2 inhibitory activity (IC₅₀ = 0.05–0.17 μM) compared to celecoxib (IC₅₀ = 0.05 μM). Among them, compounds 5b and 5d emerged as the most promising candidates, displaying high selectivity indices (SI = 9.25 and 12.02, respectively). Moreover, carbonic anhydrase inhibition studies demonstrated that compound 5b acted as a selective inhibitor of the hCA II isoform. Based on their superior in vitro profiles, compounds 5b and 5d were further evaluated in vivo, and the results demonstrated promising analgesic and anti-inflammatory activities, characterized by a relatively rapid onset and prolonged duration of action. Importantly, ulcerogenic studies demonstrated a markedly improved gastric safety profile for 5b and 5d; however, these findings are considered as preliminary and require further pharmacological confirmation. Collectively, compounds 5b and 5d demonstrated encouraging pharmacological profiles under the current experimental conditions. Nevertheless, additional comprehensive studies are necessary to validate these preliminary findings and to further establish their therapeutic potential, safety profile, and underlying mechanisms of action. Furthermore, in a rabbit model of glaucoma, compound 5b exhibited a statistically significant and sustained reduction in intraocular pressure, surpassing that of compound 5d and underscoring its potential as an ocular hypotensive agent. However, the absence of experimental evaluation against the hCA IV isoform, which is known to contribute to aqueous humor regulation and represents a relevant target in glaucoma therapy, should be acknowledged as a limitation of the present study. Therefore, the antiglaucoma interpretation and the proposed ocular hypotensive mechanism of compound 5b require further validation through hCA IV inhibition studies.

Finally, integrated molecular docking and molecular dynamics studies were performed to gain insights into the binding conformations and key molecular interactions between the synthesized compounds and the target enzymes. In summary, compound 5b exhibited favorable efficacy and safety profiles, with enhanced antiglaucoma activity.

4. Experimental Section

4.1. Chemistry

All reactions were monitored by TLC using silica gel F₂₅₄ plates (Merck, Germany), and the spots were observed using a UV light with a wavelength of 254 nm. The NMR spectra were recorded on Bruker in deuterated chloroform CDCl₃ or deuterated dimethylsulfoxide (DMSO-d ₆) at 400 MHz for ¹H NMR and 100 MHz for ¹³C MR on Bruker AVANCE III 400 MHz FT-NMR spectrometers, the Microanalytical Unit, Faculty of Pharmacy, Cairo University. Chemical Shifts were quoted in δ as parts per million (ppm). As for the proton magnetic resonance, D₂O was carried out for NH and OH exchangeable protons. HPLC purity was measured by UV absorbance at 254 nm using MeOH/H₂O/0.05% TFA. The HPLC consisted of a LiChrosorb RP-18 (5 μm) 100-4.6 Merck column (Merck, Darmstadt, Germany), two LC-10AD pumps, an SPD-M10A VP PDA detector, and an SIL-HT autosampler, all from the manufacturer Shimadzu (Kyoto, Japan). All compounds are >95% pure by HPLC analysis. The absorption spectra were recorded with an SPD-M10A diode array detector Shimadzu spectrophotometer (Kyoto, Japan). HRMS-ESI (high-resolution mass spectrometry) was measured on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).

4.1.1. General

The following intermediates have been synthesized using the previously mentioned procedures: , ethyl 2-benzoyl-3-(dimethylamino)­acrylate (2), ethyl 5-phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carboxylate (3), 4-(4-(hydrazinecarbonyl)-5-phenyl-1H-pyrazol-1-yl)­benzenesulfonamide (4), ethyl 2-cyano-3-ethoxyacrylate (7), ethyl 5-amino-1-(4-sulfamoylphenyl)-1H-pyrazole-4-carboxylate (8), and 4-(5-amino-4-(hydrazinecarbonyl)-1H-pyrazol-1-yl)­benzenesulfonamide (9). All instruments and spectral data are included in the Supporting Information (S7–S64).

4.1.2. General Procedure for Synthesis of Compounds 5a–g and 10a–e

In a 50 mL round flask, acid hydrazide 4 or 9 (1.0 mmol) was added to DMF (2.0 mL), followed by the addition of carbon disulfide (3.0 mmol), and stirred for 15 min at room temperature. Then, the reaction mixture temperature was raised to 70 °C for 4 h until the oxadiazole ring closure was complete; derivative 5g was afforded. After that, the reaction mixture was cooled to room temperature, and triethylamine (4 mmol) was added. The alkyl halide or bromoacetic acid (1.2 mmol) was then added and stirred overnight at room temperature. The reaction was monitored using TLC with the elution system dichloromethane/methanol. The reaction mixture was poured on ice/water, and the precipitate formed was filtered and recrystallized from methanol.

4.1.2.1. 4-{4-[5-(Benzylthio)-1,3,4-oxadiazol-2-yl]-5-phenyl-1H-pyrazol-1-yl}­benzenesulfonamide (5a)

Yield: 72%; ¹H NMR (400 MHz, DMSO-d ₆): δ 4.36 (s, 2H, –CH₂−), 7.29 (s, 5H, Ar-H), 7.45–7.52 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.81 (d, J = 8.52 Hz, 2H, Ar-H), 8.42 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 36.29 (−CH₂−), 107.35, 126.12 (2C), 127.05 (2C), 128.05, 128.22 (2C), 129.01, 129.04 (2C), 129.33, 130.33, 130.93 (2C), 132.99, 136.88, 140.65, 141.49, 142.98, 143.91, 160.73, 162.54 (Ar-Cs); For C₂₄H₁₉N₅O₃S₂ (489.57) Calc.: C, 58.88; H, 3.91; N, 14.31. Found: C, 59.04; H, 4.08; N, 14.56; HRMS (ESI) for C₂₄H₂₀N₅O₃S₂: calcd, 490.1002; found, 490.1003 [M + H]⁺. HPLC: rt 14.03 min (purity 96.66%).

4.1.2.2. 2-{(5-[5-Phenyl-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl]-1,3,4-oxadiazol-2-yl)­thio}­acetic Acid (5b)

Yield: 63%; ¹H NMR (400 MHz, DMSO-d ₆): δ 3.79 (s, 2H, –CH₂), 7.42–7.47 (m, 10H, 7H of Ar-H and 3H of OH and NH₂, D₂O exchangeable), 7.81 (d, J = 8.0 Hz, 2H, Ar-H), 8.42 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 45.69 (−CH₂−), 107.50, 126.09 (2C), 127.04 (2C), 128.04, 128.95 (2C), 130.23, 130.89 (2C), 140.63, 141.54, 142.84, 143.88, 160.04, 163.96 (Ar-Cs), 168.93 (CO); For C₁₉H₁₅N₅O₅S₂ (457.48) Calc.: C, 49.88; H, 3.30; N, 15.31. Found: C, 50.06; H, 3.47; N, 15.54; HRMS (ESI) for C₁₉H₁₆N₅O₅S₂: calcd, 458.0587; found, 458.0590 [M + H]⁺. HPLC: rt 10.46 min (purity 94.55%).

4.1.2.3. 4-{5-Phenyl-4-[5-(propylthio)-1,3,4-oxadiazol-2-yl]-1H-pyrazol-1-yl}­benzenesulfonamide (5c)

Yield: 86%; ¹H NMR (400 MHz, DMSO-d ₆): δ 0.90 (t, J = 7.3 Hz, 3H, –SCH₂CH₂CH ₃), 1.60 (h, J = 7.2 Hz, 2H, −SCH₂CH ₂CH₃), 3.05 (t, J = 7.1 Hz, 2H, −SCH ₂CH₂CH₃), 7.41–7.49 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.81 (d, J = 8.6 Hz, 2H, Ar-H), 8.43 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 13.18 (propyl-C), 22.93 (propyl-C), 34.27 (propyl-C), 107.49, 126.07 (2C), 127.05 (2C), 128.10, 128.99 (2C), 130.26, 130.86 (2C), 140.6, 141.5, 142.93, 143.90, 160.52, 163.19 (Ar-Cs); For C₂₀H₁₉N₅O₃S₂ (441.52) Calc.: C, 54.41; H, 4.34; N, 15.86. Found: C, 54.60; H, 4.58; N, 16.02; HRMS (ESI) for C₂₀H₂₀N₅O₃S₂: calcd, 442.1002; found, 442.1004 [M + H]⁺. HPLC: rt 12.89 min (purity 95.08%).

4.1.2.4. 4-{4-[5-(Allylthio)-1,3,4-oxadiazol-2-yl]-5-phenyl-1H-pyrazol-1-yl}­benzenesulfonamide (5d)

Yield: 74%; ¹H NMR (400 MHz, DMSO-d ₆): δ 3.74 (d, J = 6.92 Hz, 2H, −SCH ₂CHCH₂), 5.07–5.12 (m, $1\frac{1}{2}\mathrm{H}$ , –SCH₂CHCH ₂), 5.16 (d, J = 0.96 Hz, 1/2H, –SCH₂CHCH ₂), 5.80–5.87 (m, 1H, −SCH₂CHCH₂), 7.44–7.48 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.81 (d, J = 8.6 Hz, 2H, Ar-H), 8.45 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 35.06 (allyl-C), 107.42, 119.58 (allyl-C), 126.11 (2C), 127.05 (2C), 128.08, 129.0 (2C), 130.30, 130.91 (2C), 133.14 (allyl-C), 140.64, 141.50, 142.98, 143.92, 160.79, 162.41 (Ar-Cs); For C₂₀H₁₇N₅O₃S₂ (439.51) Calc.: C, 54.65; H, 3.90; N, 15.93. Found: C, 54.92; H, 3.69; N, 16.09; HRMS (ESI) for C₂₀H₁₈N₅O₃S₂: calcd, 440.0845; found, 440.0848 [M + H]⁺. HPLC: rt 12.34 min (purity 96.39%).

4.1.2.5. 4-{4-[5-(Butylthio)-1,3,4-oxadiazol-2-yl]-5-phenyl-1H-pyrazol-1-yl}­benzenesulfonamide (5e)

Yield: 82%; ¹H NMR (400 MHz, DMSO-d ₆): δ 0.85 (t, J = 6.72 Hz, 3H, –SCH₂CH₂CH₂CH ₃), 1.30–1.35 (m, 2H, –SCH₂CH₂CH ₂CH₃), 1.58 (t, J = 6.56 Hz, 2H, −SCH₂CH ₂CH₂CH₃), 3.07 (t, J = 6.44 Hz, 2H, −SCH ₂CH₂CH₂CH₃), 7.43–7.47 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.81 (d, J = 7.8 Hz, 2H, Ar-H), 8.43 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 13.75 (butyl-C), 21.41 (butyl-C), 31.49 (butyl-C), 32.1 (butyl-C), 107.47, 126.07 (2C), 127.05 (2C), 128.08, 128.98 (2C), 130.28, 130.85 (2C), 140.57, 141.50, 142.92, 143.88, 160.51, 163.22 (Ar-Cs); For C₂₁H₂₁N₅O₃S₂ (455.55) Calc.: C, 55.37; H, 4.65; N, 15.37. Found: C, 55.29; H, 4.59; N, 15.58; HRMS (ESI) for C₂₁H₂₂N₅O₃S₂: calcd, 456.1158; found, 456.1160 [M + H]⁺. HPLC: rt 13.66 min (purity 100%).

4.1.2.6. 4-{4-[5-(Isobutylthio)-1,3,4-oxadiazol-2-yl]-5-phenyl-1H-pyrazol-1-yl}­benzenesulfonamide (5f)

Yield: 88%; ¹H NMR (400 MHz, DMSO-d ₆): δ 0.90 (d, J = 6.12 Hz, 6H, –SCH₂CH­(CH ₃)₂), 1.81–1.85 (m, 1H, −SCH₂CH­(CH₃)₂), 2.97 (d, J = 6.24 Hz, 2H, −SCH ₂CH­(CH₃)₂), 7.43–7.47 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.81 (d, J = 7.88 Hz, 2H, Ar-H), 8.44 (s, 1H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 21.57 (isobutyl-2C), 28.56, 40.58 (isobutyl-2C), 107.45, 126.09 (2C), 127.04 (2C), 128.08, 129.01 (2C), 130.30, 130.86 (2C), 140.59, 141.49, 142.94, 143.89, 160.50, 163.33; For C₂₁H₂₁N₅O₃S₂ (455.55) Calc.: C, 55.37; H, 4.65; N, 15.37. Found: C, 55.48; H, 4.74; N, 15.60; HRMS (ESI) for C₂₁H₂₂N₅O₃S₂: calcd, 456.1158; found, 456.1161 [M + H]⁺. HPLC: rt 13.54 min (purity 100%).

4.1.2.7. 4-{4-(5-Mercapto-1,3,4-oxadiazol-2-yl)-5-phenyl-1H-pyrazol-1-yl}­benzenesulfonamide (5g)

Yield: 86%; ¹H NMR (400 MHz, DMSO-d ₆): δ 7.40–7.46 (m, 9H, 7H of Ar-H and 2H of NH ₂, D₂O exchangeable), 7.80 (d, J = 8.6 Hz, 2H, Ar-H), 8.41 (s, 1H, Ar-H), 14.51 (s, 1H, SH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d ₆): δ 106.80, 126.18 (2C), 127.05 (2C), 127.76, 129.02 (2C), 130.36, 130.82 (2C), 140.62, 141.41, 143.09, 143.99, 156.47, 177.13 (Ar-Cs); For C₁₇H₁₃N₅O₃S₂ (399.44) Calc.: C, 51.12; H, 3.28; N, 17.53. Found: C, 51.35; H, 3.40; N, 17.79. HPLC: rt 10.58 min (purity 95.37%).

4.1.2.8. 4-{5-Amino-4-[5-(benzylthio)-1,3,4-oxadiazol-2-yl]-1H-pyrazol-1-yl}­benzenesulfonamide (10a)

Yield: 72%; ¹H NMR (400 MHz, DMSO-d ₆): δ 4.50 (s, 2H, −CH ₂−), 6.52 (s, 2H NH ₂, D₂O exchangeable), 7.23–7.36 (m, 3H, Ar-H), 7.43 (d, J = 7.5 Hz, 2H, Ar-H), 7.50 (s, 2H, NH ₂, D₂O exchangeable), 7.79 (dd, J = 8.6, 2.3 Hz, 2H, Ar-H), 7.94 (td, J = 7.7, 2.2 Hz, 3H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 36.52 (−CH₂−), 89.19, 123.90 (2C), 127.52 (2C), 128.22, 129.06 (2C), 129.52 (2C), 137.21, 139.04, 140.83, 143.10, 147.52, 160.31, 161.58 (Ar-Cs); For C₁₈H₁₆N₆O₃S₂ (428.49) Calc.: C, 50.45; H, 3.76; N, 19.61. Found: C, 50.32; H, 3.94; N, 19.89; HRMS (ESI) for C₁₈H₁₇N₆O₃S₂: calcd, 429.0798; found, 429.0801 [M + H]⁺. HPLC: rt 12.82 min (purity 97.21%).

4.1.2.9. 2-{(5-[5-Amino-1-(4-sulfamoylphenyl)-1H-pyrazol-4-yl]-1,3,4-oxadiazol-2-yl)­thio}­acetic Acid (10b)

Yield: 63%; ¹H NMR (400 MHz, DMSO-d ₆): δ 4.18 (s, 2H, −CH ₂−), 6.53 (s, 2H, NH ₂, D₂O exchangeable), 7.50 (s, 2H, NH ₂, D₂O exchangeable), 7.82 (d, J = 8.7 Hz, 2H, Ar-H), 7.93–8.00 (m, 3H, Ar-H), 13.16 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d ₆): δ 34.79 (−CH₂−), 89.12, 123.91 (2C), 127.51 (2C), 139.0, 140.83, 143.12, 147.50, 160.32, 161.44 (Ar-Cs), 169.50 (CO); For C₁₃H₁₂N₆O₅S₂ (396.40) Calc.: C, 39.39; H, 3.05; N, 21.20. Found: C, 39.60; H, 3.21; N, 21.04; HRMS (ESI) for C₁₃H₁₃N₆O₅S₂: calcd, 397.0383; found, 397.0385 [M + H]⁺. HPLC: rt 9.04 min (purity 99.66%).

4.1.2.10. 4-{5-Amino-4-[5-(propylthio)-1,3,4-oxadiazol-2-yl]-1H-pyrazol-1-yl}­benzenesulfonamide (10c)

Yield: 86%; ¹H NMR (400 MHz, DMSO-d ₆): δ 1.01 (t, J = 7.4 Hz, 3H, –SCH₂CH₂CH ₃), 1.78 (h, J = 7.3 Hz, 2H, −SCH₂CH ₂CH₃), 3.26 (t, J = 7.1 Hz, 2H, −SCH ₂CH₂CH₃), 6.54 (s, 2H, NH ₂, D₂O exchangeable), 7.51 (s, 2H, NH ₂, D₂O exchangeable), 7.83 (d, J = 8.7 Hz, 2H, Ar-H), 7.96–8.02 (m, 3H, Ar-H); For C₁₄H₁₆N₆O₃S₂ (380.45) Calc.: C, 44.20; H, 4.24; N, 22.09. Found: C, 44.39; H, 4.53; N, 22.30; HRMS (ESI) for C₁₄H₁₇N₆O₃S₂: calcd, 381.0798; found, 381.0800 [M + H]⁺. HPLC: rt 13.68 min (purity 100%).

4.1.2.11. 4-{4-[5-(Allylthio)-1,3,4-oxadiazol-2-yl]-5-amino-1H-pyrazol-1-yl}­benzenesulfonamide (10d)

Yield: 74%; ¹H NMR (400 MHz, DMSO-d ₆): δ 3.94 (dt, J = 7.0, 1.1 Hz, 2H, SCH ₂CHCH₂), 5.17 (ddd, J = 9.9, 1.7, 0.9 Hz, 1H, SCH₂CHCH ₂), 5.34 (dq, J = 16.9, 1.4 Hz, 1H, SCH₂CHCH ₂), 5.99 (ddt, J = 17.0, 10.0, 7.0 Hz, 1H, SCH₂CHCH₂), 6.54 (s, 2H, NH ₂, D₂O exchangeable), 7.51 (s, 2H, NH ₂, D₂O exchangeable), 7.82 (d, J = 8.7 Hz, 2H, Ar-H), 7.98 (d, J = 8.7 Hz, 3H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 35.38 (allyl-C), 89.24, 119.72 (allyl-C), 123.89 (2C), 127.51 (2C), 133.31 (allyl-C), 139.04, 140.83, 143.11, 147.53, 160.14, 161.64 (Ar-Cs); For C₁₄H₁₄N₆O₃S₂ (378.43) Calc.: C, 44.43; H, 3.73; N, 22.21. Found: C, 44.67; H, 3.92; N, 22.47; HRMS (ESI) for C₁₄H₁₅N₆O₃S₂: calcd, 379.0641; found, 379.0643 [M + H]⁺. HPLC: rt 11.49 min (purity 99.34%).

4.1.2.12. 4-{5-Amino-4-[5-(ethylthio)-1,3,4-oxadiazol-2-yl]-1H-pyrazol-1-yl}­benzenesulfonamide (10e)

Yield: 86%; ¹H NMR (400 MHz, DMSO-d ₆): δ 1.41 (t, J = 7.3 Hz, 3H, −CH₂CH ₃), 3.28 (q, J = 7.3 Hz, 2H, −CH ₂CH₃), 6.54 (s, 2H, NH ₂, D₂O exchangeable), 7.51 (s, 2H, NH ₂, D₂O exchangeable), 7.80–7.86 (m, 2H, Ar-H), 7.94–8.00 (m, 3H, Ar-H); ¹³C NMR (100 MHz, DMSO-d ₆): δ 15.35 (ethyl-C), 27.25 (ethyl-C), 89.28, 123.85 (2C), 127.51 (2C), 139, 140.85, 143.09, 147.5, 160.84, 161.41 (Ar-Cs); For C₁₃H₁₄N₆O₃S₂ (366.42) Calc.: C, 42.61; H, 3.85; N, 22.94. Found: C, 42.83; H, 3.97; N, 23.17; HRMS (ESI) for C₁₃H₁₅N₆O₃S₂: calcd, 367.0641; found, 367.0643 [M + H]⁺. HPLC: rt 11.23 min (purity 98.45%).

4.2. Biological Evaluation

4.2.1. Determination of the COX-1 and COX-2 Inhibitory Activities

The efficacy of the synthesized sulfonamide derivatives 5a–g and 10a–e to inhibit COX-1 and COX-2 was assessed in vitro following previously established protocols. ,

4.2.2. Carbonic Anhydrase (CA) Inhibition Assay

An Applied Photophysics stopped-flow instrument was used to evaluate the ability of the synthesized sulfonamide derivatives 5a–g and 10a–e to inhibit CA-catalyzed CO₂ hydration. Phenol red (0.2 mM) was used as a pH indicator and monitored at its absorbance maximum (557 nm). Measurements were performed in 20 mM HEPES buffer (pH 7.4) containing 20 mM Na₂SO₄ to maintain a constant ionic strength. The initial rates of the CA-catalyzed CO₂ hydration reaction were followed for a period of 10–100 s. CO₂ concentrations ranged from 1.7 to 17 mM for determining the kinetic parameters and inhibition constants. Enzyme concentrations ranged between 5 and 12 nM. For each inhibitor, at least six traces of the initial 5–10% of the reaction were carried out to determine the initial velocity. Uncatalyzed rates were assessed similarly and subtracted from the total observed rates. Stock solutions of the inhibitors (0.1 mM) were prepared in distilled–deionized water, and serial dilutions were subsequently performed in assay buffer to obtain final concentrations ranging down to 0.01 nM. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature to allow for the formation of the enzyme–inhibitor complex. Inhibition constants (K _i) were determined by nonlinear least-squares fitting using GraphPad Prism 9 (San Diego, CA, USA) and the Cheng–Prusoff equation and represent the mean from at least three different determinations. Human hCA I and hCA II were purchased from Sigma-Aldrich, whereas recombinant hCA IX was produced in-house as previously described.

4.2.3. In Vivo Studies

All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and in strict compliance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Ethical approval for the study was obtained from the Research Ethics Committee of the Faculty of Pharmacy, Cairo University, Egypt (REC-FOPCU; Approval No. PC3428) Study, design, and treatment are illustrated in the Supporting Information (S84–S94).

The experimental procedures for the hot plate test to assess analgesic activity, the paw edema assay to evaluate anti-inflammatory activity, the acute ulcerogenic assay, and IOP reduction in a rabbit model of induced glaucoma were conducted in accordance with previously reported methodologies. All animals were randomly allocated to the respective experimental groups prior to treatment administration, and all pharmacological evaluations, observations, and data analyses were conducted in a blinded manner to reduce the potential experimental bias and ensure the reliability of the obtained results.

4.3. Crystallization and X-ray Data Collection

Crystals of hCAII were obtained using the hanging drop vapor diffusion method using a 24-well Linbro plate. 2 μL of a 10 mg/mL solution of hCA II in Tris–HCl 20 mM pH 8.0 was mixed with 2 μL of a solution of 1.5 M sodium citrate and 0.1 M Tris pH 8.0 and equilibrated against the same solution at 296 K. The complex was prepared by soaking hCA II native crystals in the mother liquor solution containing the inhibitors at a concentration of 10 mM for 2 days. A crystal was flash-frozen at 100 K using a solution obtained by adding 15% (v/v) glycerol to the mother liquor solution as a cryoprotectant. Data on the 10d–CAII crystal was collected using synchrotron radiation at the ID30B beamline at ESRF (Grenoble, France) with a wavelength of 0.87313 Å and a EIGER2 X 9M detector. Data was integrated and scaled using the program XDS. Data processing statistics are shown in the Supporting Information (S65–S67).

4.3.1. Structure Determination

The crystal structure of hCA II (PDB accession code: 9T4S) without solvent molecules and other heteroatoms was used to obtain initial phases using Refmac5. 5% of the unique reflections were selected randomly and excluded from the refinement data set for the purpose of Rfree calculations. The initial |F _o – F _c| difference electron density maps unambiguously showed the inhibitor molecules. The inhibitor was introduced in the model with 1.0 occupancy. Refinements proceeded using normal protocols of positional and isotropic atomic displacement parameters alternating with manual building of the models using COOT. The quality of the final models was assessed with COOT and RAMPAGE. Crystal parameters and refinement data are summarized in the Supporting Information (S95–S102). Atomic coordinates were deposited in the Protein Data Bank (PDB accession code: 9T4S). Graphical representations were generated with Chimera.

4.4. Molecular Modeling

4.4.1. Protein and Ligand Preparation

The crystal structures of the hCA II and COX-2 were retrieved from the Protein Data Bank (PDB) and prepared using the protein preparation wizard module in the Schrödinger Suite (v2025). hCA II in complex with furosemide (PDB ID: 1Z9Y) and COX-2 in complex with rofecoxib (PDB ID: 5KIR) were selected to evaluate the binding modes of 5b and 5d within the binding sites of both proteins. The procedures of protein preparation involved adding hydrogen atoms, missing side chains, and missing loops while removing water molecules and ions (except zinc in the case of hCA II) from the protein structures. The protonation states and tautomeric forms of amino acids were optimized using the PROPKA tool at pH 7.0. Subsequently, a restrained energy minimization of the prepared protein structures was performed using the OPLS4 force field. The structures of the cognate inhibitors of hCA II and COX-2, along with acetazolamide, celecoxib, and the two test compounds (5b and 5d), were prepared using the LigPrep module in the Schrödinger Suite (v2025) and energy-minimized using the OPLS4 force field. Possible ionization states were generated at a physiological pH of 7.0 ± 1.0 using the Epik module.

4.4.2. Molecular Docking

Molecular docking into the binding sites of hCA II and COX-2 was carried out using the Standard Precision (SP) mode of the Glide ligand docking module. Receptor grid generation was performed using the cocrystallized ligandsfurosemide for hCAII and rofecoxib for COX-2as the centroids, with an inner grid box size of 10 × 10 × 10 Å. To assess the ability of the docking protocol to reproduce the experimentally observed binding mode, redocking of the cocrystallized inhibitors was conducted. The root-mean-square deviation (RMSD) values of the top-scoring poses, relative to the crystallographic conformations, were found to be below 1.0 Å, indicating a high level of accuracy in pose prediction. For each compound, a maximum of 10 docking poses were generated, with all other parameters maintained at their default settings. The top-scoring Glide poses in complex with their respective proteins were subsequently subjected to 200 ns molecular dynamics (MD) simulations to evaluate the stability of their binding modes.

4.4.3. Molecular Dynamics Simulations

The stability of the binding modes obtained from molecular docking was assessed by performing MD simulations for 200 ns. The Desmond simulation package was employed to set up the systems and run the MD simulations. The systems were solvated using the TIP3P water model in a periodic orthorhombic box with a 15 Å buffer distance from the protein surface in all directions and neutralized with either Na⁺ or Cl^– ions. Ions were placed 5 Å away from ligand atoms to avoid their interference in the ligand–protein interactions. For all the simulation runs, the OPLS4 force field and NVT and NPT (number of particles (N), volume (V), pressure (P), and temperature (T)) ensembles were utilized. Prior to performing the production simulation, the default Desmond protocol for energy minimization and model relaxation was applied. Initially, solvent molecules and ions were energy-minimized while restraining the protein–ligand complex, followed by minimization of the entire system. The system was then equilibrated in multiple stages for 12 ps per each stage: an initial NVT equilibration at 10 K with small restraints, followed by NPT equilibration with gradual restraint removal. A final unrestrained NPT equilibration ensured system stability before initiating the simulation production run. A cutoff of 9 Å was used to smoothly truncate the Lennard-Jones interactions and short-range Coulombic interactions. The Particle-Mesh Ewald (PME) summation was used to calculate the long-range electrostatic interactions. Finally, 200 ns MD simulations with a trajectory interval of 100 ps were carried out at a temperature of 300 K and a pressure of 1.01325 bar in the NPT ensemble using a Nose–Hoover chain thermostat and a Martyna–Tobias–Klein barostat. , The trajectories were saved at 2 fs intervals for further analysis using the Simulation Interaction Diagram (SID) tool implemented in the Desmond MD package. SID was used to analyze the interactions between the compound and the protein. The stability of the complexes was monitored by examining the protein Cα and ligand RMSD over time using frame zero as a reference.

Glossary

Abbreviations

Å

angstrom

AAZ

acetazolamide

CA

carbonic anhydrase

CAI

CA inhibitor

COX

cyclooxygenase

DMSO

dimethyl sulfoxide

IOP

intraocular pressure

RMSD

root-mean-square deviation

SI

selectivity index

ZBGs

zinc binding groups

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c01117.

• Spectral data (NMR, HRMS, and HPLC purity spectra); in vitro and in vivo biological assays data; X-ray crystallography; and molecular docking (PDF)

• Molecular formula strings of compounds celecoxib, SLC-558, II, III, IV, V, VI, 5a–g, 10a–e, AAZ, and ibuprofen (CSV)

• Co-crystallized ligand (10d) in CA II (PDB: 9T4S) (PDB)

• Docking pose for compound 5b and 5d within COX-2 (PDB ID: 5KIR) and CAII (PDB ID: 1Z9Y) (PDB)

• Docking pose for compound 5b within COX-2 (PDB ID: 5KIR) (PDB)

• Docking pose for compound 5d within COX-2 (PDB ID: 5KIR) (PDB)

‡‡.

M.A.F.E. and E.M.S. contributed equally. M.A.F.E., synthesis of the compounds, writing of the manuscript; E.M.S. synthesis of the compounds, writing of the manuscript, formal analysis, visualization; H.A.A., synthesis of the compounds, writing of the manuscript; H.N., molecular docking, data analysis, writing of the manuscript; M.F. and A.A., X-ray crystallography, data analysis, writing of the manuscript; M.S., data analysis; A.B. and C.T.S., in vitro enzymatic assay, writing of the manuscript; C.T.S. design of the concept; A.A.A., Y.T.T.K., R.A.R., and O.A.M.A.E.-G. designing and performing the in vivo biological experimental models; H.S.I., conceptualization, synthesis of the compounds, data analysis, writing of the manuscript; and all authors revised and approved the manuscript.

The authors declare no competing financial interest.