New 1,2,3-triazole/1,2,4-triazole hybrids linked to oxime moiety as nitric oxide donor selective COX-2, aromatase, B-RAF^V600E and EGFR inhibitors celecoxib analogs: design, synthesis, anti-inflammatory/anti-proliferative activities, apoptosis and molecular modeling study

Abstract

A new series of bis-triazole 19a-l was synthesised for the purpose of being hybrid molecules with both anti-inflammatory and anti-cancer activities and assessed for cell cycle arrest, NO release. Compounds 19c, 19f, 19h, 19 l exhibited COX-2 selectivity indexes in the range of 18.48 to 49.38 compared to celecoxib S.I. = 21.10), inhibit MCF-7 with IC₅₀ = 9–16 μM compared to tamoxifen (IC₅₀ = 27.9 μM). and showed good inhibitory activity against HEP-3B with IC₅₀ = 4.5–14 μM compared to sorafenib (IC₅₀ = 3.5 μM) (HEP-3B). Moreover, derivatives 19e, 19j, 19k, 19 l inhibit HCT-116 with IC₅₀ = 5.3–13.7 μM compared to 5-FU with IC₅₀ = 4.8 μM (HCT-116). Compounds 19c, 19f, 19h, 19 l showed excellent inhibitory activity against A549 with IC₅₀ = 3–4.5 μM compared to 5-FU with IC₅₀ = 6 μM (A549). Compounds 19c, 19f, 19h, 19 l inhibit aromatase (IC₅₀ of 22.40, 23.20, 22.70, 30.30 μM), EGFR (IC₅₀ of 0.112, 0.205, 0.169 and 0.066 μM) and B-RAF^V600E (IC₅₀ of 0.09, 0.06, 0.07 and 0.05 μM).

Keywords:

• Aromatase inhibition
• bis-Triazole
• B-RAF^V600E
• inhibition
• cyclooxygenase-2 inhibition
• (EGFR) inhibition
• nitric oxide donor

Introduction

Breast cancer (BC) represents the highest incidence rate responsible for mortality and morbidity in women all over the world especially metastatic breast cancer¹. About 80% of all breast cancers are oestrogen dependent “ER-positive” that means cancer cells grow in response to the hormone estrogen². The therapeutic strategies designed for treatment of breast cancer include the use of drugs which either act as antiestrogens, that block oestrogen receptors and preventing oestrogens from binding to their active site and performing their action, these are known as competitive inhibitors as tamoxifen (1), a selective oestrogen receptor modulator (SERM)^3,4.

Another principle of endocrine therapy for treatment of breast cancer is the use of aromatase inhibitors (AIs), which inhibit the conversion of androgens such as androstenedione (2) to oestrogens by inhibition of aromatase activity⁵. Aromatase is a member of the cytochrome P450 enzyme that possesses an iron-porphyrin group (Fe²⁺-containing heme) at its active site⁶. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer that are sub-classified into steroidal aromatase inhibitors which decrease the oestrogen biosynthesis by competing with the substrate androstenedione for binding to the active site of the enzyme⁷. Exemestane (3) is a hormone therapy drug used to treat breast cancer in women who have gone through menopause and is the most representative member of this class, with an irreversible mechanism of inhibition⁸. Non-steroidal aromatase inhibitors like letrozole (4) and anastrozole (5) act by a reversible mechanism of inhibition by formation of coordinating bond with heme-iron in the catalytic site of aromatase⁹. Nonsteroidal aromatase inhibitors have two primary elements in their structure, azole part containing nitrogen atom that interacts with heme-iron atom of aromatase, while the bulky aryl part resembles the substrate’s steroid ring¹⁰. These triazole derivatives are very potent, specific and selective aromatase inhibitors that allow almost complete oestrogen suppression^11,12. However, unwanted severe side effects due to complete inhibition of oestrogen synthesis by their prolonged use like increased risk of heart disease and osteoporosis restrict their domain of clinical application¹³, leading to continue exploration of new strategies for treatment.

Receptor tyrosine kinase (RTK) or HER receptor family (also called ErbB or epidermal growth factor receptor EGFR family) consists of four structurally close receptor: EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4)¹⁴. Their structure shows three domains responsible for their activity and subsequent phosphorylation of the key tyrosine residues namely, an extracellular ligand binding domain, a transmembrane domain and an intracellular tyrosine kinase domain^15–19.

HER2 (human epidermal growth factor receptor 2) participates in 20% of breast cancer cases called HER2-positive breast cancer, is unique within the EGFR family in that it does not have known ligands and cannot form ligand-dependent homodimers²⁰. Thus, to initiate downstream signalling, it must form heterodimers with other EGFR proteins once their specific ligands have bound¹⁹. HER dimerisation inhibitors are one of chemotherapeutic management that blocks the ability of HER2 receptor to bind to other EGFR receptor family members²¹. Another chemotherapeutic tool in treatment of breast cancer is the utilisation of EGFR tyrosine kinase inhibitors²². Many compounds possessing sulphonamide moiety have been reported as highly effective kinase inhibitors such as vemurafenib (6), the first kinase inhibitor for treatment of late-stage melanoma by inhibition of EGFR signalling through selectively blocking mutant B-RAF^V600E associated proliferative disorders²³. However, evident reports supported EGFR overexpression in numerous triple negative breast cancer (TNBC) patients, identified by lacking of oestrogen receptor (ER), progesterone receptor (PR) and HER-2 receptors, suggested that inhibition of EGFR is beneficial in these patients^24,25. Encorafenib (7) is another kinase inhibitors carrying both sulphonamide and pyrazole backbone which has been recently approved by the FDA to be used as combined kinases inhibitor cancer treatment²⁶. Currently, medications with a “single-target single drug” are becoming less effective in treating cancer as it is heterogeneous, complicated and multi-genic^27,28. Multi-target cancer chemotherapy as an alternative to combination therapy not only provides greater therapeutic anticancer benefits but it also reduces the risk of drug resistance²⁹. It was noticed that, following EGFR inhibitor treatment acquired BRAF^V600E mutation was suggested as a mechanism of resistance^30–32. B-RAF^V600E inhibition can also activate EGFR, resulting in recurring tumour proliferation, so in order to resolve this problem; a B-RAF^V600E/EGFR combination inhibitor was utilized³³.

Most of the clinically employed non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit the two isoforms of cyclooxygenase (COX) enzyme: COX-1 (constitutive form) and COX-2 (inducible form)^34–38. Concomitant use of non-selective NSAIDs such as indomethacin (8) which act on both COX isoforms results in an increased risk of gastrointestinal complications^39–41. In contrast, selective COX-2 inhibitors such as celecoxib (9); a pyrazole-based containing sulphonamide moiety, act as anti-inflammatory without unwanted side effects on G.I. and renal systems⁴². There is an evidence to suggest the role of COX-2 in carcinogenesis, as the resulted prostaglandins due to activation of COX-2 are involved in key processes of cancer development including proliferation, mutagenesis, resistance to apoptosis, angiogenesis, immune suppression and invasion^43,44. It has been proposed that COX-2 expression in ductal carcinoma in situ (DCIS) has a critical role in the development of breast cancer, as it plays a role in the destruction of the basal membrane and in formation of new blood vessels allowing tumour growth so the use of COX-2 inhibitors has been associated with reduced breast cancer risk^45–49.

In addition, non-steroidal anti-inflammatory drugs (NSAIDs) are used as auxiliary therapy with chemotherapeutics in order to cure pain caused by cancer or its treatment^50–52. Nitric oxide (NO) contributes to the modulation of several key physiological functions⁵³. One of the most important strategies used to overcome (GI-NSAIDs) side effects is designing nitric oxide-donating NSAIDs (NO-NSAIDs), which are capable of generating the radical bio-mediator and gastro protective NO⁵⁴. Moreover, NO can prevent tumour cells from metastasising and assists macrophage to kill tumour cells⁵⁵. Furthermore, evidences showed that low levels (nanomolar) of NO promote tumour cell proliferation, angiogenesis and metastasis, while higher concentrations sensitivity of tumours to chemotherapy and overcome drug resistance⁵⁶. Therefore combination of high concentrations NO donors and cancer therapy increases its efficiency⁵⁷.

Our previously synthesised compound (10) have been reported as anti-inflammatory/anti-cancer agent having 1,2,4 triazole core linked to nitric oxide donating tail and to pyrazole scaffold as celecoxib analogue and it was the most active derivative against EGFR having sulphonamide group⁵⁸. Recently, we also reported aldoxime derivative (11) with oxime as nitric oxide donor moiety acting as COX-2/aromatase inhibitor with both anti-inflammatory/anticancer activities⁵⁹. In addition, many triazoles and bistriazoles possess clinical importance (e.g. antibacterial, antifungal, anti-inflammatory, and anticancer)^60–62. Derivatives (12a,b) were reported as 1,2,4-triazoles bearing oxime as nitric oxide donor moiety and having B-RAF^V600E inhibitory activity as anti-cancer agents⁶³. Beside the exploration of new targets, there is another approach of combining two or more pharmacophores in a single molecule^64,65.

Based on the aforementioned data and as a continuation of our research interest in the scope of synthesis and biological evaluation of new anti-inflammatory and anti-cancer hybrid agents here we describe, synthesis, in vitro evaluation as COX-1/COX-2 inhibitors, in vitro anti-cancer activity on MCF7, Hep3B, HCT116, A549, normal fibroblasts F180 cell lines, cell cycle analysis, apoptosis, mechanistic study on aromatase, and EGFR & BRAF^V600E enzymes inhibition and NO release for new series of bis-triazole scaffold as celecoxib analogue with nitric oxide donor activity 19a-l. The target derivatives 19a-l are hybrid molecules, their design was coming from lead compounds celecoxib (9) and previously reported compounds 10,11,12 in which some modifications in the structure of these compounds were carried out to trigger COX-2/Aromatase/BRAF/EGFR selective inhibition as: (1) Y-shape structure of celecoxib was maintained while pyrazole ring is replaced by 1,2,3-triazole moiety linked to sulphonamide moiety which is important pharmacophore for COX-2 inhibition (2) the presence of oxime tail as nitric oxide donor moiety linked to 1,2,4-triazole linker was incorporated in our designed compounds 19a-l to maintain the aromatase/EGFR/COX-2 inhibition as in previously reported derivatives 10 & 11 and to maintain the selective B-RAF^V600E inhibition as in previously reported derivatives 12a,b (Figures 1 and 2).

Figure 1. Structure of selective oestrogen receptor blocker Tamoxifen (1), Steroidal aromatase inhibitors Exmestane (2), nonsteroidal aromatase inhibitor letrozole (4), anastrozole (5) Kinase inhibitors, Vemurafenib (6), Encorafenib (7), Non selective NSAIDs, Indomethacin (8), selective COX-2 inhibitors celecoxib (9), and 1,2,4 triazoles with anti-cancer activities (10) and anti-cancer 1,2,4 triazoles (10), (11), and B-RAF^V600E inhibitor (12).

Figure 2. structural hyperdization of designed compounds from COX-2 inhibitors celecoxib (9) and anti-cancer 1,2,4 triazoles (10), (11), B-RAF^V600E inhibitors (12a&b).

Experimental

Chemistry

General: Melting points were determined on a Thomas-Hoover capillary apparatus and are uncorrected. Infra-red (IR) spectra were recorded as films on KBr plates using a Nicolet 550 Series II Magna FT-IR spectrometer.¹H NMR and ¹³C NMR spectra were measured on a Bruker 400 MHz NMR Spectrophotometer, Faculty of Pharmacy, Cairo University, Egypt in dimethyl sulfoxide (DMSO)-d₆ with TMS as the internal standard, where J (coupling constant) values are estimated in Hertz (Hz). Mass spectra (MS) were recorded on a Water’s Micromass ZQ 4000 mass spectrometer using the electro-spray (ES) ionisation mode. Microanalyses were performed for C, H and N were carried out on Perkin-Elmer 2400 analyser (Perkin-Elmer, Norwalk, CT, USA) at the microanalytical unit of EL-Azhar University, Egypt. All compounds were within ± 0.4% of the theoretical values. Compounds 13, 15a⁶⁶ and 17a-f^67,68 were prepared according to previously reported procedures.

Experimental procedures and spectral data for compound 15a,b (supplementary data S3)

A mixture of acid hydrazide (13) (0.01 mole) and phenyl isothiocyanate or 4-methoxy phenyl isothiocyanate (14a,b) (0.01 mole) in absolute ethanol (60 ml) was heated under reflux for 4h. The formed precipitate on cooling was filtered, dried and crystallised from ethanol to give compounds 15a,b. Physical and spectral data for 15b is listed below:

N-(4-methoxyphenyl)-2-(5-methyl-1-(4-sulfamoylphenyl)-1H-1,2,3-triazole-4-carbonyl) hydrazinecarbothioamide (15b): Yield 80%; white solid; m.p. 218–220 °C; IR (KBr): 3282, 3224 (NH₂), 3147 (NH), 3043 (C–H aromatic), 2974 (C–H aliphatic), 1681 (C = O), 1550 (C = N), 1334, 1166 (SO₂NH₂), 1276 (C = S) cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.61(s, 3H, CH₃), 3.34 (s, 3H, OCH₃) 7.11 (s, 1H, NH, CSNHNH-, D₂O exchangeable D₂O exchangeable), 7.30 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-3, H-5), 7.52 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-2, H-6), 7.64 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.88 (d, 2H, J = 8.0 Hz, 4-aminosulfonylphenyl H-3, H-5), 7.96 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 9.79 (s, 1H, CSNH-phenyl, D₂O exchangeable), 10.62 (s, 1H, -CSNHNH-, D₂O exchangeable); MS m/z (ES⁺) 461.09 (M+) (100%). Anal. Calcd. for C₁₈H₁₉N₇O₄S₂: C, 46.84; H, 4.15; N, 21.24; Found; C, 46.62; H, 4.28; N, 21.46.

Experimental procedures and spectral data for compounds 16a,b (supplementary data S3)

A mixture of hydrazinecarbothioamide (15a,b) (0.01 mole) in an aqueous solution of sodium hydroxide (0.01 mole) was heated under reflux for 4h. After cooling the solution was acidified with hydrochloric acid and the formed precipitate was filtered, dried and crystallised from ethanol to give compounds 16a,b. Physical and spectral data are listed below:

4-(4-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (16a): Yield 65%; white solid; m.p. 244–246 °C; IR (KBr): 3360, 3278 (NH₂), 3249 (NH), 3070 (C–H aromatic), 2966 (C–H aliphatic), 1593 (C = N), 1280 (C = S), 1330, 1168 (SO₂NH₂) cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.61 (s, 3H, CH3), 7.41 (d, 2H, J = 8.0 Hz, phenyl H-2, H-6), 7.49 (m, 3H, phenyl H-3, H-4, H-5), 7.60 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.83 (d, 2H, J = 7.8 Hz, 4-aminosulfonylphenyl H-2, H-6), 8.02 (d, 2H, J = 7.8 Hz, 4-aminosulfonylphenyl H-3, H-5), 14.31 (s, 1H, NH-, D₂O exchangeable); MS m/z (ES⁺) 413.07 (M+) (100%). Anal. Calcd. For C₁₇H₁₅N₇O₂S₂; C, 49.38; H, 3.66; N, 23.71; Found; C, 49.56; H, 3.84; N, 23.90.

4-(4-(5-mercapto-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (16b): Yield 68%; white solid; m.p. 268–270 °C; IR (KBr): 3360, 3275 (NH₂), 3251 (NH), 3066 (C–H aromatic), 2970 (C–H aliphatic), 1593 (C = N), 1331, 1168, (SO₂NH₂), 1281 (C = S), cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.60 (s, 3H, CH₃), 3.34 (s, 3H, OCH₃), 7.40 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-3, H-5), 7.49 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-2, H-6), 7.60 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.80 (d, 2H, J = 7.8 Hz, 4-aminosulfonylphenyl H-2, H-6), 8.04(d, 2H, J = 7.8 Hz, 4-aminosulfonylphenyl H-3, H-5), 14.30 (s, 1H, NH-, D₂O exchangeable); MS m/z (ES⁺) 443.08 (M+) (100%). Anal. Calcd. For C₁₇H₁₅N₇O₂S₂; C, 48.75; H, 3.86; N, 22.11; Found; C, 48.88; H, 3.72; N, 22.18.

Experimental procedures and spectral data for compounds 18a-l (Supplementary data S3)

An equimolecular mixture of 16a,b, phenacyl bromide derivatives (17a-f) (0.01 mole) and triethyl amine (0.12 g, 0.012 mole) in acetonitrile (50 ml) was heated under reflux for 10 h. The reaction mixture was evaporated to dryness and the residue was crystallised from ethanol to give compounds 17a-f. Physical and spectral data are listed below:

4-(5-methyl-4-(5-((2-oxo-2-phenylethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18a): Yield 61%; white solid; m.p. 288 - 290 °C; IR (KBr): 3370, 3206 (NH₂), 3070 (C–H aromatic), 2981 (C–H aliphatic), 1674 (C = O), 1581 (C = N), 1323, 1161 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, CH₃), 4.99 (s, 2H, CH₂), 7.49 (d, 2H, J = 8.0 Hz, phenyl H-2, H-6), 6.58 (m, 7H, 4-phenyltriazole H-2, H-6, H-3, H-4, H-5, NH₂, D₂O exchangeable), 7.69 (m, 1H, phenyl H-4), 7.85 (d, 2H, J = 8.0 Hz phenyl H-3, H-5), 8.04 (m, 4H, 4-aminosulfonylphenyl H-2, H-6, H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.09, 126.00, 127.64, 128.13, 128.89, 129.33, 129.99, 130.38, 133.71, 134.20, 134.27, 135.79, 138.17, 145.51, 147.91, 152.06, 193.51; MS m/z (ES⁺) 531.11 (M+) (100%). Anal. Calcd. For C₂₅H₂₁N₇O₃S₂: C, 56.48; H, 3.98; N, 18.44; Found; C, 56.78; H, 4.12; N, 18.10.

4-(5-methyl-4-(5-((2-oxo-2-(p-tolyl)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18b): Yield 61%; white solid; m.p. 236- 238 °C; IR (KBr): 3367, 3267 (NH₂), 3066 (C–H aromatic), 2920 (C–H aliphatic), 1674 (C = O), 1597 (C = N), 1346, 1161 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.41 (s, 3H, phenyl-CH₃), 2.57 (s, 3H, triazole-CH₃), 4.96 (s, 2H, CH₂), 7.38 (d, 2H, J = 8.0 Hz, 4-methylphenyl H-3, H-5), 7.48 (d, 2H, J = 8.0 Hz, 4-methylphenyl H-2, H-6), 7.57 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.58 (m, 3H, phenyl H-3, H-4, H-5), 7.86 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 7.94 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.08, 21.69, 46.21, 126.00, 127.65, 128.12, 129.00, 129.87, 129.99, 130.39, 133.26, 133.70, 134.18, 135.81, 138.17, 144.85, 145.47, 147.89, 152.12, 193.01; MS m/z (ES⁺) 545.13 (M+) (100%). Anal. Calcd. For C₂₆H₂₃N₇O₃S₂: C, 57.23; H, 4.25; N, 17.97; Found; C, 57.54; H, 4.06; N, 18.14.

4-(4-(5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18c): Yield 71%; white solid; m.p. 296 - 298 °C; IR (KBr): 3347, 3209 (NH₂), 3070 (C–H aromatic), 2939 (C–H aliphatic), 1666 (C = O), 1597 (C = N), 1327, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, triazole-CH₃), 3.87 (s, 3H, phenyl-OCH₃), 4.93 (s, 2H, CH₂), 7.08 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-3, H-5), 7.48 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-2, H-6), 7.57 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.58 (m, 3H, phenyl H-3, H-4, H-5), 7.86 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 8.01 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.08, 46.20, 56.12, 114.55, 126.00, 127.65, 128.13, 128.61, 129.98, 130.37, 131.30, 133.71, 134.20, 135.81, 138.17, 145.48, 147.88, 152.17, 164.09, 191.84; MS m/z (ES⁺) 561.13 (M+) (100%). Anal. Calcd. For C₂₆H₂₃N₇O₄S₂: C, 55.60; H, 4.13; N, 17.46; Found; C, 55.88; H, 4.34; N, 17.22.

4-(4-(5-((2-(4-chlorophenyl)-2-oxoethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18d): Yield 67%; white solid; m.p. 236 - 238 °C; IR (KBr): 3348, 3251 (NH₂), 3066 (C–H aromatic), 2997 (C–H aliphatic), 1681 (C = O), 1589 (C = N), 1338, 1180 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, triazole-CH₃), 4.97 (s, 2H, CH₂), 7.48 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 7.57(s, 2H, SO₂NH₂, D₂O exchangeable), 7.58 (m, 3H, phenyl H-3, H-4, H-5), 7.65 (d, 2H, J = 8.2 Hz, 4-chlorophenyl H-2, H-6), 7.86 (d, 2H, J = 8.2 Hz, 4-chlorophenyl H-3, H-5), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.06 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.10, 25.95, 62.49, 126.00, 127.64, 128.12, 129.45, 129.99, 130.39, 130.82, 133.70, 134.17, 134.50, 135.81, 138.17, 139.18, 145.50, 147.94, 151.95, 192.67; MS m/z (ES⁺) 565.08 (M+) (100%). Anal. Calcd. For C₂₅H₂₀ClN₇O₃S₂: C, 53.05; H, 3.56; N, 17.32; Found; C, 53.22; H, 3.72; N, 17.56.

4-(5-methyl-4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18e): Yield 84%; white solid; m.p. 298 − 300 °C; IR (KBr): 3332, 3251 (NH₂), 3059 (C–H aromatic), 2994 (C–H aliphatic), 1685 (C = O), 1597 (C = N), 1338, 1172 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, triazole-CH₃), 5.03 (s, 2H, CH₂), 7.48 (d, 2H, J = 7.8 Hz phenyl H-2, H-6), 7.57 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.58 (m, 3H, phenyl H-3, H-4, H-5), 7.86 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-2, H-6), 8.03(d, 2H, J = 8.2 Hz, 4-nitrophenyl H-3, H-5), 8.27 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.38 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.08, 25.90, 62.52, 124.39, 126.00, 127.65, 128.08, 130.02, 130.34, 130.44, 133.64, 134.11, 135.85, 138.15, 140.49, 145.47, 147.99, 150.64, 151.83, 193.01; MS m/z (ES⁺) 576.10 (M+) (100%). Anal. Calcd. For C₂₅H₂₀N₈O₅S₂: C, 52.07; H, 3.50; N, 19.43; Found; C, 57.18; H, 3.34; N, 19.58.

4-(4-(5-((2-(4-bromophenyl)-2-oxoethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18f): Yield 79%; white solid; m.p. 274 - 276 °C; IR (KBr): 3344, 3248 (NH₂), 3066 (C–H aromatic), 2951 (C–H aliphatic), 1681 (C = O), 1585 (C = N), 1338, 1171 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, triazole-CH₃), 4.96 (s, 2H, CH₂), 7.47 (d, 2H, J = 7.8 Hz phenyl H-2, H-6), 7.57 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.58 (m, 3H, phenyl H-3, H-4, H-5), 7.79 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-2, H-6), 7.85 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-3, H-5), 8.02 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.05 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.07, 25.91, 46.03, 126.00, 127.65, 128.09, 128.41, 130.01, 130.41, 130.89, 132.40, 133.66, 134.13, 134.79, 135.83, 138.15, 145.47, 147.93, 151.96, 192.90; MS m/z (ES⁺) 609.03 (M+) (100%). Anal. Calcd. For C₂₅H₂₀BrN₇O₃S₂: C, 49.18; H, 3.30; N, 16.06; Found; C, 49.44; H, 3.52; N, 16.28.

4-(4-(4-(4-methoxyphenyl)-5-((2-oxo-2-phenylethyl)thio)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18 g): Yield 75%; white solid; m.p. 294 - 296 °C; IR (KBr): 3340, 3224 (NH₂), 3062 (C–H aromatic), 2966 (C–H aliphatic), 1681 (C = O), 1597 (C = N), 1300, 1168 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, CH₃), 3.34 (s, 3H, OCH₃) 4.98 (s, 2H, CH₂), 7.08 (d, 2H, J = 8.4 Hz, 4-methoxyphenyl H-2, H-6), 7.40 (d, 2H, J = 8.4 Hz, 4-methoxyphenyl H-3, H-5), 6.56 (m, 4H, 4-phenyltriazole H-3, H-5, NH₂, D₂O exchangeable), 7.69 (m, 1H, phenyl H-4), 7.86 (d, 2H, J = 7.6 Hz phenyl H-2, H-6), 8.03 (d, 2H, J = 8.4 Hz, 4-aminosulfonylphenyl H-2, H-6), 8.05 (d, 2H, J = 8.4 Hz, 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.07, 55.96, 115.03, 125.99, 126.60, 127.65, 128.88, 129.34, 129.47, 133.78, 134.29, 135.76, 138.18, 145.45, 148.15, 152.50, 160.46, 193.58; MS m/z (ES⁺) 561.13 (M+) (100%). Anal. Calcd. For C₂₆H₂₃N₇O₄S₂: C, 55.60; H, 4.13; N, 17.46; Found; C, 55.76; H, 4.32; N, 17.12.

4-(4-(4-(4-methoxyphenyl)-5-((2-oxo-2-(p-tolyl)ethyl)thio)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18h): Yield 80%; white solid; m.p. 286- 288 °C; IR (KBr): 3390, 3116 (NH₂), 3016 (C–H aromatic), 2920 (C–H aliphatic), 1681 (C = O), 1597 (C = N), 1361, 1145 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.41 (s, 3H, phenyl-CH₃), 2.56 (s, 3H, triazole-CH₃), 3.84 (s, 3H, phenyl-OCH₃) 4.94 (s, 2H, CH₂), 7.08 (d, 2H, J = 8.4 Hz, 4-methoxyphenyl H-3, H-5), 7.37 (d, 2H, J = 8.4 Hz, 4-methoxphenyl H-2, H-6), 7.41 (d, 2H, J = 8.2 Hz, 4-methylphenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.86 (d, 2H, J = 8.2 Hz 4-methylphenyl H-2, H-6), 7.93 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.08, 21.69, 55.95, 115.02, 125.98, 126.62, 127.66, 129.00, 129.47, 129.87, 133.26, 133.80, 135.76, 138.20, 144.84, 145.46, 148.14, 152.54, 160.45, 193.07; MS m/z (ES⁺) 575.14 (M+) (100%). Anal. Calcd. For C₂₇H₂₅N₇O₄S₂: C, 56.33; H, 4.38; N, 17.03; Found; C, 56.58; H, 4.16; N, 16.86.

4-(4-(4-(4-methoxyphenyl)-5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18i): Yield 70%; white solid; m.p. 242 - 244 °C; IR (KBr): 3425, 3223 (NH₂), 3116 (C–H aromatic), 2962 (C–H aliphatic), 1743 (C = O), 1593 (C = N), 1369, 1145 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.57 (s, 3H, triazole-CH₃), 3.84 (s, 3H, phenyl-OCH₃), 3.87 (s, 3H, 4-phenyl-triazole-OCH₃), 4.92 (s, 2H, CH₂), 7.08 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-3, H-5), 7.10 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-2, H-6), 7.86 (d, 2H, J = 8.2 Hz, 4-(4-methoxyphenyl)triazole H-2, H-6), 7.59 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.86 (d, 2H, J = 8.2 Hz, 4-(4-methoxyphenyl)triazole H-3, H-5), 8.01 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.07, 25.89, 55.95, 56.11, 114.55, 115.02, 125.99, 126.61, 127.66, 128.59, 129.48, 131.31, 133.79, 135.77, 138.19, 145.44, 148.13, 152.60, 160.45, 164.08, 191.90; MS m/z (ES⁺) 591.14 (M+) (100%). Anal. Calcd. For C₂₇H₂₅N₇O₅S₂: C, 54.81; H, 4.26; N, 16.57; Found; C, 54.62; H, 4.12; N, 16.84.

4-(4-(5-((2-(4-chlorophenyl)-2-oxoethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18j): Yield 77%; white solid; m.p. 214 - 216 °C; IR (KBr): 3325, 3174 (NH₂), 3066 (C–H aromatic), 2962 (C–H aliphatic), 1674 (C = O), 1589 (C = N), 1346, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.56 (s, 3H, triazole-CH₃), 3.84 (s, 3H, OCH₃), 4.95 (s, 2H, CH₂), 7.08 (d, 2H, J = 8.0 Hz 4-methoxyphenyl H-3, H-5), 7.39 (d, 2H, J = 8.0 Hz 4-methoxyphenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.64 (d, 2H, J = 8.0 Hz 4-chlorophenyl H-2, H-6), 7.86 (d, 2H, J = 8.2 Hz, 4-chlorophenyl H-3, H-5), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.05 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.07, 25.90, 55.95, 115.03, 125.99, 126.57, 127.66, 129.44, 130.82, 133.76, 134.46, 135.78, 138.18, 139.19, 145.45, 148.18, 152.38, 160.46, 192.73; MS m/z (ES⁺) 595.09 (M+) (100%). Anal. Calcd. For C₂₆H₂₂ClN₇O₄S₂: C, 52.39; H, 3.72; N, 16.45; Found; C, 52.12; H, 3.54; N, 16.20.

4-(4-(4-(4-methoxyphenyl)-5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18k): Yield 89%; white solid; m.p. 310 – 312 °C; IR (KBr): 3344, 3248 (NH₂), 3088 (C–H aromatic), 2997 (C–H aliphatic), 1681 (C = O), 1585 (C = N), 1338, 1172 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.56 (s, 3H, triazole-CH₃), 3.84 (s, 3H, OCH₃), 5.01 (s, 2H, CH₂), 7.08 (d, 2H, J = 7.8 Hz 4-methoxyphenyl H-3, H-5), 7.40 (d, 2H, J = 7.8 Hz 4-methoxyphenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.86 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-2, H-6), 8.02(d, 2H, J = 8.2 Hz, 4-nitrophenyl H-3, H-5), 8.27 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.38 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.07, 46.26, 55.96, 115.04, 124.38, 125.99, 126.53, 127.66, 129.45, 130.35, 133.72, 135.80, 138.17, 140.47, 145.45, 148.23, 150.63, 152.23, 160.47, 193.06; MS m/z (ES⁺) 576.10 (M+) (100%). Anal. Calcd. For C₂₆H₂₂N₈O₆S₂: C, 51.48; H, 3.66; N, 18.47; Found; C, 51.26; H, 3.56; N, 18.72.

4-(4-(5-((2-(4-bromophenyl)-2-oxoethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (18 l): Yield 86%; white solid; m.p. 230 - 232 °C; IR (KBr): 3367, 3263 (NH₂), 3012 (C–H aromatic), 2974 (C–H aliphatic), 1674 (C = O), 1585 (C = N), 1338, 1166 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.56 (s, 3H, triazole-CH₃), 2.84 (s, 3H, OCH₃), 4.94 (s, 2H, CH₂), 7.07 (d, 2H, J = 7.8 Hz 4-methoxyphenyl H-3, H-5), 7.39 (d, 2H, J = 7.8 Hz 4-methoxyphenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.79 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-2, H-6), 7.39 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-3, H-5), 7.86 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-3, H-5), 7.97 (d, 2H, J = 8.4 Hz 4-aminosulfonylphenyl H-2, H-6), 8.02 (d, 2H, J = 8.4 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.09, 55.96, 115.02, 125.99, 126.60, 127.64, 128.38, 129.47, 130.91, 132.39, 133.78, 134.81, 135.75, 138.19, 145.49, 148.18, 152.34, 160.46, 192.96; MS m/z (ES⁺) 639.04 (M+) (100%). Anal. Calcd. For C₂₆H₂₂BrN₇O₄S₂: C, 48.75; H, 3.46; N, 15.31; Found; C, 48.52; H, 3.50; N, 15.22.

Experimental procedures and spectral data for compounds 19a-l (supplementary data S3)

A mixture of the appropriate ketones 18a-l (0.01 mole), hydroxylamine hydrochloride (0.01 mole) and sodium acetate (0.01 mole) in acetonitrile (30 ml) was heated under reflux for 10h. The formed precipitate upon cooling was filtered off, washed with sodium carbonate solution (10%) and distilled water, dried and recrystallized from ethanol to give compounds 19a-l. Physical and spectral data are listed below:

(E)-4-(4-(5-((2-(hydroxyimino)-2-phenylethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19a): Yield 54%; white solid; m.p. 308 - 310 °C; IR (KBr): 3618 (OH), 3330, 3240 (NH₂), 3062 (C–H aromatic), 2920 (C–H aliphatic), 1597 (C = N), 1342, 1161 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.60 (s, 3H, CH₃), 4.46 (s, 2H, CH₂), 7.40 (m, 5H, phenyl H-2, H-3, H-4, H-5, H-6), 7.51 (m, 3H, 4-phenyltriazole H-3, H-4, H-5), 7.59 (s, 2 H, NH₂, D₂O exchangeable), 7.68 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 7.86 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5), 11.83 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.13, 26.02, 126.01, 126.37, 127.67, 128.25, 129.02, 129.67, 129.81, 130.28, 133.75, 134.22, 134.74, 135.89, 138.17, 145.49, 148.28, 151.80, 152.40; MS m/z (ES⁺) 546.13 (M+) (100%). Anal. Calcd. For C₂₅H₂₂N₈O₃S₂: C, 54.93; H, 4.06; N, 20.50; Found; C, 54.78; H, 4.22; N, 20.68.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(p-tolyl)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19b): Yield 67%; white solid; m.p. 302- 304 °C; IR (KBr): 3607 (OH), 3347, 3224 (NH₂), 3066 (C–H aromatic), 2958 (C–H aliphatic), 1597 (C = N), 1323, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.60 (s, 3H, phenyl-CH₃), 3.78 (s, 3H, triazole-CH₃), 4.44 (s, 2H, CH₂), 6.95 (d, 2H, J = 8.0 Hz, 4-methylphenyl H-3, H-5), 7.40 (d, 2H, J = 8.0 Hz, 4-methylphenyl H-2, H-6), 7.51 (m, 3H, phenyl H-3, H-4, H-5), 7.53 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.61 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 7.86 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 11.60 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.12, 25.89, 55.68, 114.44, 126.00, 127.10, 127.66, 127.78, 128.24, 129.82, 130.28, 133.76, 134.21, 135.87, 138.17, 145.59, 148.25, 151.95, 160.54; MS m/z (ES⁺) 560.14 (M+) (100%). Anal. Calcd. For C₂₆H₂₄N₈O₃S₂: C, 55.70; H, 4.31; N, 19.99; Found; C, 55.92; H, 4.52; N, 20.16.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(4-methoxyphenyl)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19c): Yield 69%; white solid; m.p. 300 - 302 °C; IR (KBr): 3609 (OH), 3340, 3244 (NH₂), 3062 (C–H aromatic), 2990 (C–H aliphatic), 1597 (C = N), 1346, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.32 (s, 3H, triazole-CH₃), 2.60 (s, 3H, phenyl-OCH₃), 4.44 (s, 2H, CH₂), 7.20 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-3, H-5), 7.39 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-2, H-6), 7.41 (m, 3H, phenyl H-3, H-4, H-5), 7.53 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.55 (d, 2H, J = 8.0 Hz phenyl H-2, H-6), 7.86 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 11.71 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.12, 21.28, 25.93, 126.00, 126.26, 127.67, 128.24, 129.60, 129.82, 130.28, 131.93, 133.76, 134.21, 135.88, 138.17, 139.26, 148.26, 151.87, 152.24; MS m/z (ES⁺) 576.14 (M+) (100%). Anal. Calcd. For C₂₆H₂₄N₈O₄S₂: C, 54.15; H, 4.20; N, 19.43; Found; C, 54.38; H, 4.42; N, 19.54.

(E)-4-(4-(5-((2-(4-chlorophenyl)-2-(hydroxyimino)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19d): Yield 72%; white solid; m.p. 292 - 294 °C; IR (KBr): 3611 (OH), 3332, 3244 (NH₂), 3070(C–H aromatic), 2990 (C–H aliphatic), 1583 (C = N), 1342, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.60 (s, 3H, triazole-CH₃), 4.43 (s, 2H, CH₂), 7.40 (d, 2H, J = 8.2 Hz phenyl H-2, H-6), 7.42 (d, 2H, J = 8.0 Hz, 4-chlorophenyl H-2, H-6), 7.49 (m, 3H, phenyl H-3, H-4, H-5), 7.53 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.69 (d, 2H, J = 8.0 Hz, 4-chlorophenyl H-3, H-5), 7.86 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 11.93 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.13, 25.94, 62.49, 126.00, 127.65, 128.17, 128.26, 129.03, 129.80, 130.27, 133.63, 134.23, 134.32, 135.88, 138.16, 145.51, 148.31, 151.51, 151.55; MS m/z (ES⁺) 580.09 (M+) (100%). Anal. Calcd. For C₂₅H₂₁ClN₈O₃S₂: C, 51.68; H, 3.64; N, 19.28; Found; C, 51.78; H, 3.86; N, 19.52.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(4-nitrophenyl)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19e): Yield 73%; white solid; m.p. 274 – 276 °C; IR (KBr): 3618 (OH), 3330, 3244 (NH₂), 3070 (C–H aromatic), 2908 (C–H aliphatic), 1597 (C = N), 1342, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.59 (s, 3H, triazole-CH₃), 4.47 (s, 2H, CH₂), 7.40 (d, 2H, J = 8.2 Hz phenyl H-2, H-6), 7.42 (m, 3H, phenyl H-3, H-4, H-5), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.85 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-2, H-6), 7.94 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-3, H-5), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.25 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5), 12.35 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.11, 25.95, 124.15, 126.00, 127.58, 127.67, 128.26, 129.80, 130.28, 133.72, 134.21, 135.93, 138.15, 141.01, 145.49, 148.02, 148.38, 151.19, 151.38; MS m/z (ES⁺) 591.11 (M+) (100%). Anal. Calcd. For C₂₅H₂₁N₉O₅S₂: C, 50.75; H, 3.58; N, 21.31; Found; C, 50.58; H, 3.74; N, 21.08.

(E)-4-(4-(5-((2-(4-bromophenyl)-2-(hydroxyimino)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19f): Yield 80%; white solid; m.p. 290 - 292 °C; IR (KBr): 3602 (OH), 3344, 3251 (NH₂), 3097 (C–H aromatic), 2927 (C–H aliphatic), 1583 (C = N), 1338, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.60 (s, 3H, triazole-CH₃), 4.42 (s, 2H, CH₂), 7.40 (d, 2H, J = 7.8 Hz phenyl H-2, H-6), 7.42 (m, 3H, phenyl H-3, H-4, H-5), 7.51 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-2, H-6), 7.53 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.59 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-3, H-5), 7.86 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5), 11.94 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.14, 25.87, 123.04, 126.00, 127.66, 128.26, 128.44, 129.80, 130.27, 130.90, 131.95, 132.40, 133.75, 133.99, 134.23, 135.88, 138.16, 145.51, 148.31, 151.50, 151.; MS m/z (ES⁺) 624.04 (M+) (100%). Anal. Calcd. For C₂₅H₂₁BrN₈O₃S₂: C, 48.00; H, 3.38; N, 17.91 Found; C, 47.82; H, 3.12; N, 18.08.

(E)-4-(4-(5-((2-(hydroxyimino)-2-phenylethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19 g): Yield 82%; white solid; m.p. 240 - 242 °C; IR (KBr): 3600 (OH), 3336, 3232 (NH₂), 3070 (C–H aromatic), 2974 (C–H aliphatic), 1597 (C = N), 1346, 1168 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.59 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃) 4.46 (s, 2H, CH₂), 7.02 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-2, H-6), 7.31 (d, 2H, J = 8.0 Hz, 4-methoxyphenyl H-3, H-5), 7.41 (m, 3H, 4-phenyltriazole H-3, H-4, H-5), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.68 (d, 2H, J = 7.6 Hz phenyl H-2, H-6), 7.87 (d, 2H, J = 8.4 Hz, 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.4 Hz, 4-aminosulfonylphenyl H-3, H-5), 11.83 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.14, 55.90, 114.86, 125.99, 126.38, 126.67, 127.65, 129.00, 129.57, 129.65, 133.86, 134.78, 135.80, 138.20, 145.49, 148.51, 152.22, 152.41, 160.37; MS m/z (ES⁺) 576.14 (M+) (100%). Anal. Calcd. For C₂₆H₂₄N₈O₄S₂: C, 54.15; H, 4.20; N, 19.43; Found; C, 54.34; H, 4.48; N, 19.70.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(p-tolyl)ethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19h): Yield 81%; white solid; m.p. 212- 214 °C; IR (KBr): 3599 (OH), 3340, 3217 (NH₂), 3020 (C–H aromatic), 2915 (C–H aliphatic), 1512 (C = N), 1342, 1168 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.32 (s, 3H, phenyl-CH₃), 2.59 (s, 3H, triazole-CH₃), 3.81 (s, 3H, phenyl-OCH₃) 4.44 (s, 2H, CH₂), 7.02 (d, 2H, J = 8.4 Hz, 4-methoxyphenyl H-3, H-5), 7.20 (d, 2H, J = 8.4 Hz, 4-methoxphenyl H-2, H-6), 7.31 (d, 2H, J = 8.2 Hz, 4-methylphenyl H-2, H-6), 7.56 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.86 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 11.71 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.11, 21.29, 55.90, 114.86, 125.99, 126.29, 126.66, 127.64, 128.55, 129.58, 129.87, 132.14, 133.26, 133.80, 135.76, 139.22, 144.84, 145.46, 148.14, 152.28, 160.36; MS m/z (ES⁺) 590.15 (M+) (100%). Anal. Calcd. For C₂₇H₂₆N₈O₄S₂: C, 54.90; H, 4.44; N, 18.97; Found; C, 55.26; H, 4.68; N, 19.30.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(4-methoxyphenyl)ethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19i): Yield 75%; white solid; m.p. 270 - 272 °C; IR (KBr): 3610 (OH), 3371, 3262 (NH₂), 3059 (C–H aromatic), 2966 (C–H aliphatic), 1516 (C = N), 1300, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.59 (s, 3H, triazole-CH₃), 3.78 (s, 3H, phenyl-OCH₃), 3.82 (s, 3H, 4-phenyl-triazole-OCH₃), 4.43 (s, 2H, CH₂), 6.95 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-3, H-5), 7.02 (d, 2H, J = 8.2 Hz, 4-methoxyphenyl H-2, H-6), 7.32 (d, 2H, J = 8.2 Hz, 4-(4-methoxyphenyl) triazole H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.61 (d, 2H, J = 8.2 Hz, 4-(4-methoxyphenyl)triazole H-3, H-5), 7.86 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.0 Hz 4-aminosulfonylphenyl H-3, H-5), 11.60 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.11, 25.76, 55.68, 55.70 114.42, 114.86, 125.99, 126.64, 127.11, 127.65, 127.78, 129.56, 133.84, 135.80, 138.19, 145.47, 148.47, 151.96, 152.36, 160.36, 160.53; MS m/z (ES⁺) 606.15 (M+) (100%). Anal. Calcd. For C₂₇H₂₆N₈O₅S₂: C, 53.45; H, 4.32; N, 18.47; Found; C, 53.26; H, 4.64; N, 18.66.

(E)-4-(4-(5-((2-(4-chlorophenyl)-2-(hydroxyimino)ethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19j): Yield 86%; white solid; m.p. 233 - 235 °C; IR (KBr): 3607 (OH), 3340, 3217 (NH₂), 3070 (C–H aromatic), 2978 (C–H aliphatic), 1512 (C = N), 1346, 1168 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.59 (s, 3H, triazole-CH₃), 3.82 (s, 3H, OCH₃), 4.43 (s, 2H, CH₂), 7.02 (d, 2H, J = 8.0 Hz 4-methoxyphenyl H-3, H-5), 7.32 (d, 2H, J = 8.0 Hz 4-methoxyphenyl H-2, H-6), 7.46 (d, 2H, J = 8.0 Hz 4-chlorophenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.69 (d, 2H, J = 8.2 Hz, 4-chlorophenyl H-3, H-5), 7.86 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 11.92 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.12, 25.79, 55.90, 114.84, 125.99, 126.66, 127.65, 128.18, 129.03, 129.57, 133.64, 133.84, 134.32, 135.82, 138.19, 145.50, 148.54, 151.59, 151.94, 160.36; MS m/z (ES⁺) 610.10 (M+) (100%). Anal. Calcd. For C₂₆H₂₃ClN₈O₄S₂: C, 51.10; H, 3.79; N, 18.34; Found; C, 51.36; H, 3.98; N, 18.70.

(E)-4-(4-(5-((2-(hydroxyimino)-2-(4-nitrophenyl)ethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19k): Yield 83%; white solid; m.p. 288 – 290 °C; IR (KBr): 3614 (OH), 3321, 3298 (NH₂), 3066 (C–H aromatic), 2982 (C–H aliphatic), 1516 (C = N), 1342, 1172 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.58 (s, 3H, triazole-CH₃), 3.81 (s, 3H, OCH₃), 4.46 (s, 2H, CH₂), 7.01 (d, 2H, J = 8.2 Hz 4-methoxyphenyl H-3, H-5), 7.32 (d, 2H, J = 8.2 Hz 4-methoxyphenyl H-2, H-6), 7.58 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.86 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-2, H-6), 7.94 (d, 2H, J = 8.2 Hz, 4-nitrophenyl H-3, H-5), 8.03 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-2, H-6), 8.24 (d, 2H, J = 8.2 Hz 4-aminosulfonylphenyl H-3, H-5), 12.34 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.12, 25.85, 55.89, 114.82, 124.14, 125.98, 126.68, 127.57, 127.66, 129.59, 133.84, 135.84, 138.18, 141.02, 145.51, 148.01, 148.61, 151.37, 151.58, 160.34; MS m/z (ES⁺) 621.12 (M+) (100%). Anal. Calcd. For C₂₆H₂₃N₉O₆S₂: C, 50.23; H, 3.73; N, 20.28; Found; C, 50.38; H, 3.54; N, 20.52.

(E)-4-(4-(5-((2-(4-bromophenyl)-2-(hydroxyimino)ethyl)thio)-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (19 l): Yield 83%; white solid; m.p. 243 - 245 °C; IR (KBr): 3613 (OH), 3367, 3244 (NH₂), 3066 (C–H aromatic), 2935 (C–H aliphatic), 1512 (C = N), 1333, 1165 (SO₂NH₂) cm¹; ¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.59 (s, 3H, triazole-CH₃), 2.82 (s, 3H, OCH₃), 4.42 (s, 2H, CH₂), 7.02 (d, 2H, J = 8.2 Hz 4-methoxyphenyl H-3, H-5), 7.32 (d, 2H, J = 8.2 Hz 4-methoxyphenyl H-2, H-6), 7.58 (d, 2H, J = 8.2 Hz, 4-bromophenyl H-2, H-6), 7.62 (s, 2H, SO₂NH₂, D₂O exchangeable), 7.64 (d, 2H, J = 8.4 Hz, 4-bromophenyl H-3, H-5), 7.86 (d, 2H, J = 8.4 Hz 4-aminosulfonylphenyl H-2, H-6), 8.03 (d, 2H, J = 8.4 Hz 4-aminosulfonylphenyl H-3, H-5); ¹³C NMR (DMSO-d₆ 100 MHz, δ ppm): 10.12, 25.76, 55.90, 114.84, 123.04, 125.99, 126.64, 127.68, 128.45, 129.56, 131.12, 131.94, 133.83, 135.84, 138.18, 145.47, 148.53, 151.69, 151.93, 160.36; MS m/z (ES⁺) 654.05 (M+) (100%). Anal. Calcd. For C₂₆H₂₃BrN₈O₄S₂: C, 47.64; H, 3.54; N, 17.09; Found; C, 47.88; H, 3.74; N, 17.40.

Biological evaluation

In-vitro anti-inflammatory activity

The in vitro COX-1/COX-2 inhibition was determined 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 potency of the tested compounds was determined as the concentration causing 50% enzyme inhibition (IC₅₀). In addition, the COX-2 selectivity indexes (S.I values) which are calculated as IC₅₀ (COX-1)/IC₅₀ (COX-2) were carried out and compared with that of the standard drugs celecoxib⁶⁹.

In-vitro cytotoxicity screening and IC₅₀ determination

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) cell viability assay was done in all the three cancer cell lines MCF7, HCT116, Hep3b, A549, and F180. In brief, cells were seeded at 1 × 10⁴ density per well in 96-well plate, in the next day cell were treated with a single dose of 10 µM from each compound. The wells without treatment were used as a control. After 48 h, media was removed and fresh media which contains 0.5 mg/mL thiazolyl blue tetrazolium bromide reagent (Sigma-Aldrich- St. Louis, MO, USA, M2128) was added. The cells where incubated for 3 h at 37 °C and 5% CO₂, then DMSO (Sigma-Aldrich- St. Louis, MO, USA) was added to solubilise the formed formazan crystals and was taken at 570 nm using Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA) microplate spectrophotometer. The half-maximal inhibitory concentration (IC50) of the most potent compounds was assessed using dose-response curve (GraphPad Prism software, version 8.00, Graph Pad software, Inc)^70,71.

Cell cycle analysis

The effect of compounds 19c, 19f, 19h, 19 l on cell growth was determined using flow cytometry according to reported procedure.⁷² Compounds 19c, 19f, 19h, 19 l were added on breast MCF7 cells with (IC₅₀ = 14, 16, 15, and 9 μM, respectively) for 24h. Rinsed breast cells with phosphate buffer saline (PBS), centrifuged, 70% ethanol was added, cells were washed with PBS and suspended with 0.1 mg/mL RNase. Moreover, cells were marked with 40 mg/mL propidium iodide (PI) and cell cycle distribution was determined using FACS Calibur flow cytometer (Becton Dickinson). (Table 1)

Table 1. Cell cycle analysis of compounds 19c, 19f, 19h, 19 l and control for breast MCF7 cells.

Cell cycle phase	%G0-G1	%S	%G2-M
19c	37.88	35.14	26.98
19f	69.07	26.89	4.04
19h	45.44	28.17	26.39
19l	63.69	23.42	12.62
Cont. MCF7	59.04	29.83	11.13

Determination of apoptosis using Annexin-V

The apoptosis of compounds 19c, 19f, 19h, 19 l was determined using Annexin-V-FITC technique.^73,74 Where these final compounds were added on MCF7 breast cells at their IC₅₀ = 14, 16, 15, 9 μM, respectively, cells were incubated for 24h., collected, rinsed twice with PBS and marked with mixture of fluorescein isothiocyanate (FITC), Annexin-V and PI (propidium iodide) are left for 30 min at room temperature in the dark. Analysis was carried out using FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) according to the following procedure:

(1) Transfer the previously prepared cells from 4 °C to the bench-top and equilibrate to room temperature. (2) Gently re-suspend cells by inverting the tube or by gentle up and down pipetting. (3) Pellet the cells at 500 × g for 5 min. Carefully aspirate the supernatant without disrupting the pellet. It is better to incompletely remove the supernatant than to accidently aspirate part of the pellet and lose cells. (4) Wash the cells by gently suspending in 1 ml 1X PBS. Again, pellet the cells at 500 x g for 5 min and carefully remove the supernatant. (5) Gently suspend the cell pellet in 200 µL 1X Propidium Iodide + RNase Staining Solution. (6) Incubate at 37 °C in the dark for 20 – 30 min. (7) Place tubes on ice (still in the dark). (8) Gently suspend cells that settled during the incubation by pipetting up and down, pass cells through an appropriate filter to remove cell aggregates. (9) Run samples on flow cytometer, set appropriate FSC vs. SSC gates to exclude debris and cell aggregates. Collect Propidium iodide fluorescence intensity on FL2 of a flow cytometer and 488 nM laser excitation (Table 2).

Table 2. Apoptosis of compounds 19c, 19f, 19h, 19 l and control against breast MCF7 cells.

	Total	Early	Late	Necrosis
19c	41.55	21.85	12.36	7.34
19f	39.18	24.15	12.61	2.42
19h	42.21	22.54	14.91	4.76
19l	44.76	12.91	24.74	7.11
Cont. MCF7	1.88	0.41	0.27	1.2

Aromatase inhibitory activity

Aromatase inhibitory activity was determined using aromatase (CYP19A) inhibitor screening Kit-K984 (Biovision, S. Milpitas Blvd., Milpitas, CA 95035 USA). IC₅₀ of compounds 19c, 19f, 19h, 19 l were determined in MCF7 cells according to the following procedure⁵⁹, to create stock solutions, dissolve test substances in the appropriate solvent. Prepare a 5X solution of each required test concentration for each test substance by dilution in aromatase assay buffer. Reconstitute the Recombinant Human Aromatase stock (2X) with 1 ml of aromatase assay buffer to prepare it. To create a uniform solution, thoroughly vortex the contents before transferring the mixture to a 15 ml conical tube. Aromatase assay buffer should be used to raise the volume to 24.50 l, then 50 l of the NADPH generating system (100X) should be added for a final volume of 2.5 ml. Prepare reaction wells with test substances, corresponding no inhibitor controls (which may also serve as solvent controls), a background control (containing no fluorogenic aromatase substrate), and (if desired) a positive inhibition control using 5 M letrozole (5X solution, 1 M final concentration). Let the test ligands to interact with the aromatase by incubating the plate at 37 °C for at least 10 min. With the exception of the background control, add 30 l of the Aromatase Substrate/NADP+ (3X) mixture to each well to begin the reaction. This will result in a final reaction volume of 100 l/well. Measure the fluorescence at Ex/Em = 488/527 nm in kinetic mode for 60 min right away (within 1 s).

EGFR Kinase inhibitory activity

EGFR (Catalog # 40321) Kinase Assay Kit was used to determine EGFR kinase inhibitory activity of compounds 19c, 19f, 19h, 19 l according to reported procedure⁷⁵: (1) We add ATP, Poly (Glu:Tyr 4:1) and PTK substrate (10 mg/ml) in 5x Kinase Buffer 1.0 to achieve a 10 mM concentration then adding DTT to 5x Kinase Buffer 1.0; (2) The master mixture (25 l per well) was prepared as follows, 6 l (5x Kinase Buffer 1), 1 l of ATP (500 M), 1 l of the PTK substrate Poly (Glu:Tyr 4:1) (10 mg/ml), and 17 l of water are divided among N wells then Pour 25 l into each well, finally, Add 5 l of the "Test Inhibitor" well’s inhibitor solution. (3) We add 5 l of the identical solution devoid of an inhibitor to the "Positive Control" and "Blank" samples (Inhibitor buffer). (4) By combining 2400 ml of water with 600 ml of 5x Kinase Buffer 1, we can make 3 ml of 1x Kinase Buffer 1. For 100 reactions, 3 ml of 1x Kinase Buffer 1 are sufficient. The "Blank" wells should have 20 l of 1x Kinase Buffer 1 added to them. (5) On ice, we thaw the EGFR enzyme, when the tube containing the enzyme initially defrosts, quickly spin it to recover its entire contents and determine how much EGFR is needed for the test, then we diluted the enzyme with 1x Kinase Buffer 1 to 1 ng/l. At −80 °C, aliquot the remaining undiluted enzyme. (6) We Add 20 l of diluted EGFR enzyme to the "Positive Control" and "Test Inhibitor Control" wells to start the reaction. (7) We incubate for 40 min at 30 °C. Defrost the Kinase-Glo max solution, 50 l of the Kinase-Glo Max reagent should be added to each well following the 40 min reaction. (8) We Covered the plate with aluminium foil and incubated for 15 min at room temperature. (9) Finally, we use the microplate reader to measure luminescence.

B-RAF ^V600E kinases inhibitory activity

BRAF^V600E (Catalog # 40533) Kinase Assay Kit was used to assess BRAF^V600E kinase inhibitory activity of compounds 19c, 19f, 19h, 19 l according to the reported procedure⁷⁶: Defrost the ATP, 5X Raf substrate, and 5X Kinase Buffer. (Optional) To achieve a 10 mM concentration, add DTT to 5x Kinase Buffer 1 (e.g. add 10 l of 1 M DTT to 1 ml of 5x Kinase Buffer 1). the master mixture (25 l per well) as follows: 6 l of 5x Kinase Buffer 1, 1 l of ATP (500 mM), 10 l of 5X Raf substrate, and 8 l of water are divided among N wells. Pour 25 l into each well. Add 5 l of the "Test Inhibitor" well’s inhibitor solution. Add 5 l of the identical solution devoid of an inhibitor to the "Positive Control" and "Blank" samples (Inhibitor buffer). By combining 2400 ml of water with 600 ml of 5x Kinase Buffer 1, you can make 3 ml of 1x Kinase Buffer 1. For 100 reactions, 3 ml of 1x Kinase Buffer 1 are sufficient. The "Blank" wells should have 20 l of 1x Kinase Buffer 1 added to them. On ice, defrost the B-Raf(V600E) or B-Raf(WT) enzyme. When the tube containing the enzyme initially defrosts, quickly spin it to recover its entire contents. Determine how much B-Raf is needed for the test, and then diluted the enzyme with 1x Kinase Buffer 1 to 2 ng/l for B-Raf ^V600E or 2.5 ng/l for B-Raf ^WT. At −80 °C, aliquot the remaining undiluted enzyme. The freeze/thaw cycle is sensitive to B-Raf enzymes. Reduce the number of freeze-thaw cycles. Reusing thawed aliquots or diluted enzyme is not advised. Add 20 l of diluted B-Raf ^V600E or B-Raf ^WT enzyme to the wells labelled "Positive Control" and "Test Inhibitor Control" to start the reaction. 45 min at 30 °C of incubation. Defrost the Kinase-Glo Max solution. Add 50 l of the Kinase-Glo Max reagent to each well once the 45-min reaction has finished. Plate should be covered with aluminium foil and incubated for 15 min at room temperature. Use the microplate reader to measure luminescence.

Nitric oxide release measurement

To 2 ml of a 1:1 v/v combination of 50 mM phosphate buffer (pH 7.4) with methanol containing 5 × 1 0 ⁻⁴ M of N-acetyl-cysteine, compounds 19c, 19f, 19h, and 19 l were dissolved in DMSO (20 ml). The medication had a final concentration of 1 0 ⁻⁴ M. The tested solutions were maintained in an incubator at 37 °C for 1 h, and then 1 ml of the reaction mixture was treated with 250 ml of Griess reagent, which is composed of sulphanilamide (2 g), N-naphthylethylenediamine dihydrochloride (0.2 g), and 85% phosphoric acid in distilled water (final volume: 100 ml). The absorbance at a maximum wavelength of 540 nm was measured after 10 min. at room temperature. To create the calibration curve, sodium nitrite standard solutions (10–80 nmol/mL) were employed. The target compounds 19c, 19f, 19h, and 19 l were reacted with in various solutions using the identical conditions, but with 0.1 N HCl of pH 1 instead of pH 7.4 phosphate buffer. The percentage of NO released in relation to a theoretical maximum release of 1 mol NO/mol of test chemical was used to express the results⁷⁷.

Molecular docking study

Docking study was out using Molecular Operating Environment (MOE) program (2014) and all docking studies were carried out using enzyme downloaded from Protein Data Bank (PDB). The enzymes included in study were COX-2, EGFR, aromatase, and B-RAF^V600E enzymes. The crystal structure of the reference drug celecoxib bound at the COX-2 active site obtained from protein data bank at Research Collaboration for Structural Bioinformatics (RSCB) protein data bank [PDB] (entry 3LN1), EGFR enzyme (PDB, ID: 1M17). Aromatase enzyme (PDB, ID: 3EQM) and B-RAF^V600E enzyme (PDB, ID: 1UWJ). The preparation of the target derivatives for docking was done via their 3D structure built by MOE. Before docking, 3D protonation of the structures, running conformational analysis using systemic search, selecting the least energetic conformer and applying the same docking protocol used with ligand were done.

Results and discussion

Chemistry

The general reactions used for the preparation of the final target 1,2,4-triazole-NO hybrids 19a-l are outlined in Scheme 1. The 1,2,3-triazole acid hydrazide 13 and 1,2,3-triazole-N-phenylhydrazinecarbothioamides derivative 15a were synthesised according to previously reported procedure⁶⁶. The addition reaction of 4-substituted phenylisothiocyanate 14b to acid hydrazide 13 in ethanol resulted in formation of 1,2,3-triazole-N-phenyl hydrazinecarbothioamides derivative 15b. The two 1,2,3-triazole-N-phenyl hydrazinecarbothioamides derivative 15a,b underwent cyclisation into the cornerstone intermediates 5-mercapto-1,2,4-triazole 16a,b through heating in alcoholic basic medium. The characteristic NH appeared as a sharp peak at 3249–3251 cm⁻¹ in IR spectra, also characteristic acidic SH proton appeared at 14.30–14.31 ppm in ¹H NMR. The target triazole-NO hybrids 19a-l were synthesised from the key intermediates 18a-l through 2 steps; the first one includes reaction of 5-mercapto-1,2,4-triazole 16a,b derivatives with 4-substituted phenacyl bromide 17a-l for preparation of benzene sulphonamide derivatives 18a-l. The second step was through heating of intermediates 18a-l with hydroxylamine hydrochloride in acetonitrile. The structures of the intermediates (18a-l) and the target hybrids (19a-l) were fully confirmed using IR,¹H NMR, DEPT-¹³C NMR, EI-MS and elemental analyses. For intermediates 18a-l, the characteristic CH₂ protons signal in ¹H NMR spectra of both the intermediate derivatives 18a-l and the final compounds 19a-l appeared at δ 4.92–5.03 ppm, 4.42–4.47 ppm, respectively. While final compounds 19a-l showed characteristic D₂O exchangeable OH proton of oxime at 11.60–12.35 ppm, also OH group appeared in IR spectrum of final compounds 19a-l at 3618–2599 cm⁻¹ with the disappearance of carbonyl peak of the intermediates 18a-l. In addition, DEPT-¹³C NMR spectra, showed the disappearance of the deshielded quaternary carbon of the intermediates 18a-l carbonyl group at 191.84–193.58 ppm and appearance of more shielded signal at 151.44–160.54 ppm corresponding to C = N of oximes 19a-l.

Scheme 1. Reagents and conditions: (a) ethanol 95% reflux 4h., (b) alcoholic NaOH reflux 4h., (c) acetonitrile, triethylamine, reflux 10h., (d) acetonitrile, hydroxylamine hydrochloride, sod. acetate, reflux, 10h.

Biological evaluation

In-vitro anti-inflammatory activity

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) were used. The potency of the screened compounds was estimated as the concentration causing 50% enzyme inhibition (IC₅₀). The results (Table 3) displayed that compounds 19a, 19b, 19d, 19e, 19f, 19 g, 19h, 19i, 19j, 19k had good inhibitory activities against COX-1 isozyme (IC₅₀ = 3.43–8.60 μM range). On the other hand, Compounds 19c, 19f, 19h, 19 l exhibited excellent COX-2 isozyme inhibitory activities (IC₅₀ = 0.243–0.329 μM range). From the above results, it can be concluded that compounds 19c, 19f, 19h, 19 l have outstanding COX-2 selectivity indexes in the range of 18.48 to 49.38 when compared to celecoxib (the COX-2 selective reference drug) (COX-1 IC₅₀ = 8.78 μM, COX-2 IC₅₀ = 0.416 μM and S.I. = 21.10) (Supplementary data S1).

Table 3. In vitro COX-1 and COX-2 inhibitory activity of triazole derivatives 19a-l and reference drug celecoxib.

Compound	COX-1 IC50 (μM)a ± S.E.	COX-2 IC50 (μM)^a± S.E.	COX-2 S.I.b
19a	4.84 ± 0.020	0.343 ± 0.070	14.11
19b	4.42 ± 0.021	0.485 ± 0.048	9.11
19c	10.70 ± 0.025	0.309 ± 0.041	34.62
19d	4.63 ± 0.023	0.437 ± 0.037	10.59
19e	4.39 ± 0.033	0.442 ± 0.058	9.93
19f	6.08 ± 0.016	0.329 ± 0.049	18.48
19g	4.76 ± 0.034	0.394 ± 0.031	12.08
19h	8.60 ± 0.027	0.309 ± 0.071	27.83
19i	4.60 ± 0.048	0.363 ± 0.061	12.67
19j	3.43 ± 0.026	0.397 ± 0.025	8.63
19k	4.76 ± 0.025	0.377 ± 0.040	12.62
19l	12.00 ± 0.027	0.243 ± 0.011	49.38
Celecoxib	8.78 ± 0.030	0.416 ± 0.010	21.10

The bold values indicates the highest S.I. of the most potent compounds.

^aThe concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme.

^bThe in vitro COX-2 selectivity index (COX-1/COX-2).

In-vitro cytotoxicity screening and IC₅₀ determination

Cell culture methedology: One cell line from four different types of cancers were used as models, Hep3b hepatocellular carcinoma cell line and MCF7 human breast adenocarcinoma cell line, both were purchased from Sigma-Aldrich (Labco). While HCT116 colorectal cancer, A549 non-small cell lung cancer and normal fibroblast F180 (bought from American type culture collection (ATCC) cell line was bought from American type culture collection (ATCC). HCT116 and MCF7 cells were cultured in RPMI-1640 medium (Sigma-Aldrich- St. Louis, MO, USA) supplied with 10% foetal bovine serum (Sigma-Aldrich- St. Louis, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich- St. Louis, MO, USA). Hep3b was maintained in DMEM medium (Sigma-Aldrich- St. Louis, MO, USA), in which 10% foetal bovine serum (Sigma-Aldrich- St. Louis, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich- St. Louis, MO, USA) were added to the medium. All cells were incubated at 37 °C and 5% CO₂. were used to evaluate the anti-cancer activity of the newly synthesised compounds 19a–l.

Cell viability assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) cell viability assay was done in all the four cancer cell lines MCF7, HCT116, A549 non-small cell lung cancer and Hep3b. in brief, cells were seeded at 1 X 10⁴ density per well in 96-well plate, in the next day cell were treated with a single dose of 10 µM from each compound. The wells without treatment were used as a control. After 48 h, media was removed and fresh media which contains 0.5 mg/mL thiazolyl blue tetrazolium bromide reagent (Sigma-Aldrich- St. Louis, MO, USA, #M2128) was added. The cells where incubated for 3 h at 37 °C and 5% CO₂, then DMSO (Sigma-Aldrich- St. Louis, MO, USA) was added to solubilise the formed formazan crystals and OD was taken at 570 nm using Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA) microplate spectrophotometer. The half-maximal inhibitory concentration (IC50) of the most potent compounds was assessed using dose-response curve (GraphPad Prism software, version 8.00, GraphPad software, Inc). IC₅₀ of Compounds 19a–l was determined using the MTT cell viability assay (Figures 3–7). Compounds 19c, 19f, 19h, 19 l showed good inhibitory activity against MCF-7 with IC₅₀ = 9–16 μM compared to tamoxifen (IC₅₀ = 27.9 μM) (MCF-7). On the other hand, compounds 19f, 19j, 19k, 19 l showed inhibitory activity against HEP-3B with IC₅₀ = 4.5–14 μM compared to sorafenib with IC₅₀ = 3.5 μM (HEP-3B). Moreover, derivatives 19e, 19j, 19k, 19 l showed good inhibitory activity against HCT116 with IC₅₀ = 5.3–13.7 μM compared to 5-FU with IC₅₀ = 4.8 μM (HCT116). As working with EGFR, Non-small cell lung cancer (A549) was used. Compounds 19c, 19f, 19h, 19 l showed excellent inhibitory activity against A549 with IC₅₀ = 3–4.5 μM compared to 5-FU with IC₅₀ = 6 μM (A549) (Figure 3 and Table 4).

Figure 3. Cell viability % of compounds 19a-l and reference compound single dose (10 μM) against MCF-7, HEP-38, HCT-116 and A549 cancer cell line.

Figure 4. Compounds 19c, 19f, 19h, 19 l effect on DNA-ploidy flow cytometric analysis of breast MCF7 cells compared to negative control.

Figure 5. The percentage of Annexin-V-FITC-positive staining in breast MCF7 cells when treated with compounds 19c, 19f, 19h, 19 l compared to sorafenib and negative control.

Figure 6. IC₅₀% values of compounds 19c, 19f, 19h, 19 l compared to letrozole on aromatase against breast MCF7 cells.

Figure 7. IC₅₀ values of compounds 19c, 19f, 19h, 19 l compared to encorafenib on EGFR.

Table 4. In vitro MCF-7, HEP-3B, HCT-116, A549 and F180 inhibitory activity of triazole derivatives 19c,e,f,h,j-l and reference drugs doxorubicin, sorafenib, 5-F.U. and tamoxifen..

Compound	MCF-7 (μM)^a	HEP-3B (μM)^a	HCT-116 (μM)^a	A549 (μM)^a	F180 (μM)^a
19c	14	N.D.	N.D.	4.5	15.4
19e	N.D.	N.D.	4.8	N.D.	N.D.
19f	16	14	N.D.	3	10.4
19h	15	N.D.	N.D.	4	11.3
19j	N.D.	4.5	9.9	N.D.	N.D.
19k	N.D.	12	13.7	N.D.	N.D.
19l	9	12	8	3.3	16.5
Doxorubicin	0.3	—	—	—	4.28
Sorafenib	—	3.5	—	—	—
5-F.U.	—	—	4.8	6	---
Tamoxifen^78	27.9	—	—	—	—

^aThe concentration of test compound produce 50% inhibition of MCF-7, HEP-3B, HCT-116, A549 and F180 cell lines, the result is the mean of six values.

Cell cycle analysis

Cell cycle analysis of compounds 19c, 19f, 19h, 19 l were tested against breast cell line MCF7 at their IC₅₀ concentrations, cell cycle phases alterations were noticed upon compounds 19c, 19f, 19h, 19 l addition on breast MCF7 cells (Figure 4). It was noted that the percentage of cells upon adding compounds 19c, 19h at G0/G1 was decreased to 37.88% and 45.44%, respectively, in comparison to the control which showed 59.04%. While treating the same cells with 19f, 19 l caused in comparable increase in G0-G1 (69.07% and 63.69%, respectively). On the other hand, when treating breast cells MCF7 with compounds 19c, 19h, a dramatic increase in cells at G2/M phase was occurred to (26.98% and 26.39%, respectively) than control (11.13%). According to the displayed results, it can be concluded that compounds 19c, 19h showed cell cycle arrest at G2/M phase. On the other hand, compounds 19f, 19 l exerting their cell cycle arrest at G0-G1 phase (Figure 4).

Determination of apoptosis using Annexin-V

Treating MCF7 breast cells with compounds 19c, 19f, 19h, 19 l resulted in an increment of annexin-V positive apoptotic cells at the early and late apoptotic stages with 21.85, 24.15, 22.54, 12.91% (early) (53.29, 58.90, 54.97, 31.48 fold) and 12.36, 12.61, 14.91, 24.74% (late) (45.77, 46.70, 55.22, 91.62 fold), respectively, compared to control (0.41% (early) and 0.27% (late), respectively). Moreover, compounds 19c, 19f, 19h, 19 l induced a percentage of necrosis 7.34%, 2.42%, 4.76%, and 7.11%, respectively, which were considerably higher than control that showed only 1.2% necrosis (Figure 5).

Aromatase inhibitory activity

Aromatase (CYP19A) inhibitor screening Kit was used to determine aromatase inhibitory activity of compounds 19c, 19f, 19h, 19 l. The determination of IC₅₀ was carried out using five concentrations and letrozole was used as reference. It was noticed that compounds 19c, 19f, 19h, 19 l induced strong inhibitory activity against aromatase with IC₅₀ of 22.40, 23.20, 22.70 and 30.30 μM, respectively, which was comparable to that of letrozole (IC₅₀ = 18.70 μM) (Figure 6) (Supplementary data S1).

EGFR kinase inhibitory activity

EGFR (Catalog # 40321) Kinase Assay Kit was used to evaluate EGFR kinase inhibitory activity of compounds 19c, 19f, 19h, 19 l. Five concentrations were used in this assay and encorafenib was used as reference for the determination of IC₅₀. The results showed that compounds 19c, 19f, 19h, 19 l induced improved inhibitory activity against EGFR with IC₅₀ of 0.112, 0.205, 0.169, and 0.066 μM, respectively, which was comparable to that of encorafenib (IC₅₀ = 0.037 μM) (Figure 7) (Supplementary data S1).

B-RAF ^V600E kinases inhibitory activity

B-RAF^V600E (Catalog # 40533) Kinase Assay Kit was used to assess B-RAF^V600E kinase inhibitory activity of compounds 19c, 19f, 19h, 19 l. Five concentrations were used in this assay and encorafenib was used as reference for the determination of IC₅₀. It was noted that compounds 19c, 19f, 19h, 19 l induced improved inhibitory activity against B-RAF^V600E with IC₅₀ of 0.09, 0.06, 0.07, and 0.05 μM, respectively, which was comparable to that of encorafenib (IC₅₀ = 0.035 μM) (Figure 8) (Supplementary data S2).

Figure 8. IC₅₀ values of compounds 19c, 19f, 19h, 19 l compared to that of encorafenib on B-RAF^V600E.

Nitric oxide (NO) release

The percentage of NO released from the synthesised compounds 19a-l was determined using phosphate-buffered-saline (PBS at pH 7.4) through incubation (see data in Table 5). All tested compounds 19a-l released NO in a slow rate (0.73–3.36%). Furthermore, derivatives 19c, 19f, 19h, 19 l were the highest NO donor (3.18%, 2.94%, 2.87%, and 3.36%, respectively).

Table 5. % of NO release of the synthesised compounds.

Compound No.	% NO released
19a	1.52
19b	1.66
19c	3.18
19d	0.73
19e	1.76
19f	2.94
19g	2.14
19h	2.87
19i	2.55
19j	0.90
19k	0.88
19l	3.36

Molecular docking study

Molecular docking studies are used to predict protein-ligand interactions with different enzymes at the atomic level⁷⁹. In this study, molecular docking studies of 1,2,3 triazole derivatives are docked with different enzymes downloaded from protein data bank (PDB) such as COX-2 enzyme (PDB, ID: 3LN1)⁸⁰, EGFR enzyme (PDB ID: 1M17)⁸¹, aromatase enzyme (PDB, ID: 3EQM)⁵ and B-RAF ^V600E enzyme (PDB ID: 1UWJ)⁸¹.

Docking of triazole derivatives with COX-2 enzyme: The target triazole derivatives 19a-l were docked with COX-2 enzyme (PDB, ID; 3LN1). The native ligand Celecoxib showed an energy score − 9. 6 Kcal/Mol and the selected compounds 19c, 19f, 19h, 19l showed energy score of −8.1, −8.9, −8.5, and −8.2 Kcal/Mol, respectively. The higher selectivity index of such compounds may attributed to the presence of 1,2,3 triazole and 1,2,4 triazole which are hydrogen bond acceptor and sulphonamide moiety which is COX-2 selective moiety in addition these derivative contains NO₂ and OCH₃ moieties which can form hydrogen bond with amino acids of the target protein (Table 6 and Figure 9).

Figure 9. (A) Visual representation (3D) of celecoxib docked with 3LN1 active site (B) Visual representation (3D) of compound 19c docked with 3LN1active site, (C) Visual representation (3D) of compound 19f docked with 3LN1 active site (D) Visual representation (3D) of compound 19h docked with 3LN1 active site (E) Visual representation (3D) of compound 19 l docked with 3LN1 active site.

Table 6. Docking data of triazoles compounds 19a-l and celecoxib with COX-2 active site (3LN1).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of bonds	Functional group
19a	−7.3	Pro 500 (2.61), Val509 (3.5)	H-bond	SO2NH2
19b	−6.3	Val509 (3.6), Ser516 (3.8)	H-bond	SO2NH2
19c	−8.1	Leu338(2.6), Val509 (2.7), Pro500 (2.5),	H-bond, hydrophobic	SO2NH2, OCH3
19d	−6.7	Ser516 (3.6), Trp343 (2.8)	H-bond	SO2NH2
19e	−7.9	Leu517 (2.4), Thr79 (2.7)	H-bond	SO2NH2
19f	−8.9	Pro500 (3.9), Val509 (2.7)	H-bond, hydrophobic	SO2NH2, NO2
19g	−7.6	Gly340 (2.8), Val509 (3.7)	H-bond	SO2NH2
19h	−8.5	Pro500 (2.3), Val509 (2.6)	H-bond, hydrophobic	SO2NH2, OCH3
19i	−7.5	Phe504 (2.1), Ser516 (2.9)	H-bond	SO2NH2
19j	−7.2	Leu338 (2.6), Gly340 (3.9)	H-bond	SO2NH2
19k	−6.2	Val102 (2.9), pro500 (2.3)	H-bond	SO2NH2
19l	−8.2	Arg499(2.4)	H-bond	SO2NH2, NO2
Celecoxib	−9.6	Ser339 (2.7), Gln178 (2.1), Arg499 (2.9).	H-bond	SO2NH2

Docking of triazole derivatives with EGFR enzyme: The 1,2,3 triazole 19a-l compounds and pyrazole EGFR inhibitor encorafenib were docked with EGFR enzyme (PDB, ID: 1M17). The ligand encorafenib showed an energy score − 7. 5 Kcal/Mol and the selected compounds 19c, 19f, 19h, 19l showed energy score of −8.7, −8.9, −8.8, and −8.8 Kcal/Mol respectively. The higher inhibitory activity of compounds 19c, 19f, 19h, 19 l to enzyme may attribute to the presence of higher number of nitrogen available for formation of hydrogen bond. They formed three or four H-bond interactions commonly with different amino acids (Lys721, Met769, Val702) amino acids residues through SO₂NH₂ or 1,2,3 triazole or 1,2,4 triazole or oxime nitrogen atoms. In addition to the formation of π … π stacking with aryl moieties or 1,2,3-triazole or 1,2,4-triazole rings of the synthesised compounds (Table 7 and Figure 10).

Figure 10. (ِA) Visual representation (2D) of compound 19c docked with 1M17 active site, (B) Visual representation (2D) of compound 19f docked with 1M17 active site (C) Visual representation (2D) of compound 19h docked with 1M17 active site (D) Visual representation (2D) of compound 19 l docked with 1M17 active site (E)Visual representation (2D) of encorafenib docked with 1M17 active site (F) Visual representation (2D) of 4-anilinoquinazline docked with 1M17 active site.

Table 7. Docking data of triazoles 19a-l and encorafenib with EGFR enzyme (1M17).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19a	−8.3	Pro770 (3.8), Met769 (3.7), Lys721 (4.1), Thr830 (3.2), Asp831 (2.9)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19b	−8.4	Asp831 (2.9), Pro770(3.1), Lys721 (3.9).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19c	−8.7	Asp830 (4.2), Met769 (3.5), Lys721(3.7), Thr830 (3.9).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19d	−8.3	Asp831 (3.7), Leu694 (3.9), Lys721(2.8).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19e	−8.2	Lys721(3.8), Met769 (3.7), Val702 (3.6)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19f	−8.9	Met742 (7.2), Asp831(3.1), Lys721 (3.4), Lys773 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19g	−8.1	Pro770 (3.2), Thr830 (3.7), Asp831 (4.1), Met769 (3.9), Lys721(3.5)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19h	−8.8	Met769v(3.8), vCys773v(3.7), Lys721(3.9), Leu694 (3.7), Gly772 (4.1)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19i	−8.5	Thr830 (4.1), Asp831(3.9), Lys692 (4.0), Lys721 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19j	−8.3	Asp831(3.7), Met769 (3.8), His781(3.7), Asp831(3.8), Lys721(3.9)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19k	−8.1	Asp831(3.8), Pro770 (3.2), Met742 (3.9), Lys721 (3.9), Cys773 (3.2)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19l	−8.8	Met742 (3.7), Cys773 (3.1), Lys721(3.5), pro770	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
encorafenib	−7.5	Lys721(2.3), Met769 (2.8), Arg857 (2.8)	H-bond π … π stacking	SO2NH- pyrazole
4-anilinoquinazoline	−8.5	Lys721(2.1), Meth761(2.6)	H-bond π … π stacking	Pyrimidine moiety Phenyl moiety

Docking of triazole derivatives with aromatase enzyme: The synthesised 1,2,3 triazole derivatives 19a-l and non-steroidal aromatase inhibitor letrozole were docked with aromatase enzyme (PDB, ID: 3EQM). The ligand letrozole showed an energy score − 7. 1 Kcal/Mol and the selected compounds 19c, 19f, 19h, 19l showed energy score of −11.3, −8.0, −10.2, and −8.8 Kcal/Mol, respectively. They formed three or four H bond interactions commonly with different amino acids (Met374, Leu372, Ala307) amino acids residues through SO₂NH₂ or 1,2,3 triazole or 1,2,4 triazole or oxime nitrogen atoms. In addition to the formation of π … π stacking with 1,2,3-triazole or 1,2,4- triazole rings of synthesised compounds with Hem 600 iron (Table 8 and Figure 11).

Figure 11. (A) Visual representation (3D & 2D) of letrozole docked with aromatase enzyme 3EQM active site (B) Visual representation (3D & 2D) of compound 19c docked with aromatase enzyme 3EQM active site, (C) Visual representation (3D & 2D) of compound 19f docked with aromatase enzyme 3EQM active site (D) Visual representation (3D & 2D) of compound 19h docked with aromatase enzyme 3EQM active site (E) Visual representation (3D & 2D) of compound 19 l docked with aromatase enzyme 3EQM active site.

Table 8. Docking data of triazoles 19a-l and letrozole with aromatase enzyme (3EQM).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19a	−10.8	Met447 (3.1), Met311(3.2), Arg115 (3.4), Cys437 (3.6)	H-bond π … π stacking	SO2NH2 1,2,4triazole 1,2,3 triazole
19b	−10.9	Met374 (3.9), Leu372 (3.8), Leu477 (3.7)	H-bond π … π stacking	SO2NH2, 1,2,4 triazole 1,2,3 triazole
19c	−11.3	Met374 (4.1), Ile133 (4.0), Ala438 (4.2)	H-bond π … π stacking	SO2NH2- 1,2,3 triazole 1,2,4triazole
19d	−10.1	Met311(3.8), Ile132 (3.9), Trp141 (3.5), Arg145 (3.5), Arg115 (3.6), Ala348(3.7)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19e	−8.6	Val369 (3.7), Arg115 (3.6), Arg435 (3.8), Ala306 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19f	−8.0	Met374 (3.5), Leu372 (3.8), Ala307 (3.9)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19g	−10.1	Met311 (3.7), Leu372 (3.5), Met374 (3.4), Phe430 (3.4)	H-bond π … π stacking	SO2NH2 1,2,4 triazole 1,2,3 triazole
19h	−10.2	Met303 (2.8), Met446 (2.9), Ala307 (2.7), Val370 (2.6)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19i	−7.7	Met447 (3.1), Ser314 (3.2), Leu372 (2.9)	H-bond π … π stacking	SO2NH2 1,2,4 triazole 1,2,3 triazole
19j	−6.5	Val370 (3.5), 369 (3.6), Met303(3.8), Ala438 (3.8)	H-bond π … π stacking	SO2NH2-1,2,3 triazole 1,2,4triazole
19k	−7.5	Met303 (3.8), Ala307 (3.7), Arg375 (3.7), Leu372 (3.9)	H-bond π … π stacking	SO2NH2-1,2,4 triazole 1,2,3 triazole
19l	−8.8	Met311 (2.4), Met446(2.6), Arg115 (2.7), Leu372 (2.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
letrozole	−7.1	Arg115 (2.9), Arg435(2.8)	H-bond π … π stacking	CN group 1,2,3 triazole

Docking of triazole derivatives with B-RAF ^V600E enzyme: The most active 1,2,3-triazole compounds 19c, 19f, 19h, 19 l were docked with kinase enzyme B-RAF^V600E (PDB, ID: 1UWJ) active site. The selected compounds showed good energy score range (-7.4 to −8.3) incomparable with pyrazole B-Raf inhibitor encorafenib (-7.4) and sorafenib (-7.8). They formed three to four H-bonds and π … π stacking interaction with B-Raf^V600E enzyme through 1,2,3 triazole, 1,2,4 triazole, oxime moiety, SO₂NH₂ and phenyl moieties. They formed H-bond with amino acids like Asp593, Glu500, Val470 and Ser535 (Table 9 and Figure 12).

Figure 12. (A) Visual representation (2D) of compound 19c docked with enzyme B-RAF^V600E (PDB entry 1UWJ) active site, (B) Visual representation (2D) of compound 19f docked with B-RAF^V600E (C) Visual representation (2D) of compound 19h docked with B-RAF^V600E (D) Visual representation (2D) of compound 19 l docked with B-RAF^V600E (E)Visual representation (2D) of encorafenib docked with B-RAF^V600E (F) Visual representation (2D) of Sorafenib docked with B-RAF^V600E.

Table 9. Docking data of the most active triazoles 19c,f,h,l and encorafenib kinase enzyme B-RAF^V600E (PDB, ID: 1UWJ) active site.

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19c	−8.3	Glu500 (2.9), Asp593 (3.0), Ser535 (2.9), Asp593 (3.3), Val470 (4.1)	H-bond π … π stacking	SO2NH2 1,2,3triazole Oxime moiety
19f	−7.7	Lys482 (2.3), Phe594 (2.9), Asp593 (2.7)	H-bond π … π stacking	1,2,3 triazole 1,2,4triazole
19h	−8.0	Phe594 (2.7), Lys482 (2.8), Asp593 (2.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole
19l	−7.4	Asp593 (2.9), Glu500 (2.9), Phe594 (2.8), Lys482 (2.7)	H-bond π … π stacking	SO2NH2 1,2,3triazole Oxime moiety
Encorafenib	−7.4	Asp593 (2.9), Glu500 (3.1), Val470 (2.7).	H-bond π … π stacking	Amide moiety Pyrimidine moiety
Sorafenib	−7.8	Glu500(2.9), Cys531(2.7), Asp593(2.9)	H-bond π … π stacking	Thiourea moiety Pyrimidine moiety

Statistical analysis

Differences between experimental groups were analysed using analysis of variance (ANOVA) followed by the Tukey’s test (p < 0.05) and all statistical procedures were done using Graph Pad Prism 7 for Mac OS X (Intuitive Software for Science, San Diego, CA, USA).

Authors’ contributions

Idea was created by Wael A. A. Fadaly. Synthesis of the compounds was carried out by Wael A. A. Fadaly and Mohamed T. M. Nemr. Discussion part was reedited by Fatma E. A. Mohamed and Marwa M. Abdelhakeem. Introduction part was reedited by Khaled R. A. Abdellatif. Docking studies were carried out by Taha H. Zidan. In vitro screening and IC₅₀ determinations were performed by Nour N. Abu Jayab and Hany A. Omar. All the authors shared in writing, reviewing the manuscript in its final form.

Acknowledgement

The authors thank Dr. Nada S. Abd El-Wahab, Pharmaceutical analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Egypt for carrying out nitric oxide release experiment.

Disclosure statement

The authors have declared no conflict of interest.

Additional information

Funding

The authors declare that they have no financial sources of support for this study.