Design, synthesis, cytotoxic evaluation and molecular docking of novel 1, 3, 4-thiadiazole sulfonamides with azene and coumarin moieties as carbonic anhydrase inhibitors

2.1.1 Synthesis of thiadiazole sulfonamides-based azene fragments (5, 7, 9, and 11)

Initially, the 4-(5-(1-hydrazonoethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfonamide 2 as a commencing material was prepared via the condensation of 1,3,4-thiadiazoline 1 with hydrazine hydrate in boiling ethanol. Subsequently, the hydrazone 2 was subjected to a diazotization reaction with nitrous acid, generated in situ from the treatment of sodium nitrite with HCl, to afford the non-isolable intermediate 4-(5-(1-chlorotriaz-2-enylidene)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfonamide 3. The diversity-oriented synthesis of 1,3,4-thiadiazole incorporating a triazene/tetrazene moiety 5, 7, 9, and 11 were realized using 3 as a precursor. Coupling 3 with 1,3-dihydroxybenzene 4 in a basic medium gave 4-(5-(1-((2,4-dihydroxyphenyl)triaz-2-en-1-ylidene)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfon amide 5. Similarly, the reaction of 3 with 2-hydroxynaphthalene 6 led to 4-(5-(1-((2-hydroxy naphthalene-1-yl)triaz-2-en-1-ylidene)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfon amide 7 (Scheme 1). In this connection, the scope of the reaction of 3 was extended to perform with amines (vide Scheme 1). Asymmetrical tetrazenes appended to 1,3,4-thiadiazole sulfonamides 9 and 11 were efficiently prepared via the treatment of 3 with sulfanilamide 8 and 4-aminoantipyrine 10, respectively, in a basic medium.

The spectral and analytical data provided confirmatory evidence for structures 5 and 7. The IR spectrum of 5 showed absorption bands at 3299–3260, 3107, 1624, 1587, and 1488 cm⁻¹ related to NH₂, NH, C = N, conjugated C = C, and N = N functions, respectively. The ¹H NMR spectrum of 5 revealed a singlet signal at δ 2.38 ppm due to CH₃, two singlet signals at 9.49 and 9.70 ppm due to imine and hydroxy protons, and a multiplet signal at region 7.34–8.50 ppm for the aromatic protons, NH₂ and hydroxyl protons. Its ¹³C NMR spectrum exhibited 14 carbon peaks which agree with its molecular structure. The signals that appeared at δ 13.89, 157.91, and 158.56 ppm were attributed to CH₃, C = N, and thiadiazole-C₂, respectively. The mass spectra of 5 and 7 showed parent ion peaks at m/z = 433 and 467, respectively, and exhibited several identical peaks at m/z = 324, 296, 255, 197, 172, 156, and 57. For example, the major fragmentation peaks in the MS of compound 15 can be accounted for by the cleavage in the 1,2,3-triazene side chain (Fig. 3).

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Fig. 3

Mass fragmentation pattern of compound 5.

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The structures of tetrazene derivatives 9 and 11 were established according to their spectral data. The IR spectrum of 11, as an example, showed characteristic bands at 3303–3249, 3145, 3114, 1686, 1588 and 1488 cm⁻¹ related to NH₂, two NH, amidic C = O, C = N, and N = N groups, respectively. The ¹H NMR spectrum of compound 11 showed, besides signals for the aromatic proton, six singlet signals at δ 2.45, 2.74, 3.19, 7.41, 9.48, 9.71 ppm to assigned two CH₃, CH₃N, NH₂, imine NH, and NH protons, respectively. Its ¹³C NMR spectrum displayed the existence of 17 carbon signals. The Sp³-hybridized C-atoms appeared at δ 14.06, 15.56, 38.75 ppm due to three methyl carbons. The characteristic signals of the amidic C = O and the imine carbon (C = NH) resonate at δ 161.87 and 158.56 ppm, respectively. The ESI MS of 11 showed the molecular ion (m/z 526), which is in agreement with a molecular formula (C₂₁H₂₂N₁₀O₃S₂).

2.1.2 Synthesis of thiadiazole sulfonamides-based coumarin fragments (13, 15, and 17)

The primary step for the preparation of new functionalized 1,3,4-thiadiazole-coumarin conjugates building is the synthesis of 4-(5-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzene sulfonamide 12. Compound 12 was prepared in spectacular yield (88%) from the condensation reaction of 5-acetylthiadiazoline 1 with cyanoacetohydrazide in refluxing ethanol in the presence of hydrochloric acid as a catalyst (Scheme 2). The spectral analyses of compound 12 supported the proposed chemical structure, whereas the IR spectrum exhibited bands at 3303–3251, 3197, 3115, 2270, 1687, 1595 cm⁻¹ attributable to NH₂, NH, imine NH, CN, amidic C = O, and C = N functions, respectively. Meanwhile, the ¹H NMR spectrum showed, besides the assigned signals of aromatic protons and NH₂ protons (δ 7.24–8.25 ppm), four singlet signals at 2.26, 4.14, 9.40, 11.60 ppm assignable to CH₃, CH₂, imine NH, and amide-NH protons, respectively. After profound explication of the ¹H NMR spectrum, the author was rationally able to demonstrate that compound 12 existed as a diastereomeric mixture, due to restricted rotation about azomethine bond (C = N), in the ratio 70:30 (E/Z), which was proved by the existence of extra four singlet signals at δ 2.29, 3.94, 9.42, 11.25 ppm due to CH₃, CH₂, imine NH, and amide-NH protons for Z-isomer as showed in Fig. 4. Further, the ¹³C NMR spectrum of 12 displayed the presence of two sets of 11 carbon peaks that support the existence of a mixture of E/Z-diastereoisomers. The signals resonate at about δ 12.18, 24.94, 115.72, 165.88 ppm assigned to CH₃, CH₂, CN, and amidic C = O carbons, respectively. The preferential formation of the E-isomer may be attributed to its stability due to less steric hindrance and low energy than Z-isomer. Additionally, the assignment of E/Z-isomer is based on comparing the chemical shift values of NH and CH₂ protons in amide 12 with their similar compounds reported earlier in the literature (A. Swain 2021).

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Fig. 4

Chemical structures of both E-and Z-isomers of derivative 12.

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The conduct of cyanoacetamide moiety of compound 12 towards a variety of substituted 2-hydroxybenzaldehydes was explored as a scaffold for the development of novel coumarins based on 1,3,4-thiadiazole. Thus, the Knoevenagel condensation of amide 12 with each of 2-hydroxybenzaldehyde 13 and 2-hydroxy-3-methyoxybenzaldehyde 15 in boiling acetic acid containing ammonium acetate yielded the respective 4-(2-imino-5-(1-(2-(2-oxo-2H-chromene-3-carbonyl)hydrazono)ethyl)-1,3,4-thiadiazol-3(2H)-yl)benzene sulfonamide 14 and 4-(2-imino-5-(1-(2-(8-methoxy-2-oxo-2H-chromene-3-carbonyl)hydrazono)ethyl)-1,3,4-thiadiazol-3(2H)-yl) benzenesulfonamide 16 (Scheme 2).

The structures of 2-oxo-chromenes 14 and 16 were assured via their correct elemental analyses and spectral data. IR spectra were free of CN group and showed, in each case, characteristic bands at 3339–3239, 3196–3102, 1708–1706, and 1699–1670, 1608–1607 cm⁻¹ assignable to NH₂, two NH, coumarin C = O, amidic C = O, and C = N groups, respectively. The ¹H NMR spectrum of 14, as a typical example, showed, alongside phenyl proton signals, three singlet signals accompanied proton–deuterium (H/D) exchange at δ 11.96, 9.08, 7.49 ppm due to amidic-NH, imine-NH, and NH₂ protons, and two singlets appeared at δ 2.32, 8.70 ppm due to CH₃ and chromene-H₄, respectively. The highly downfield shift of the amidic-NH proton is attributed to the intramolecular H-bond between the amidic N–H and C = O group of the chromene ring. Its ¹³C NMR spectrum of 14 revealed the existence of 18C-signals. The Sp³-hybridized carbon atom resonated at δ 12.70 ppm due to the methyl carbon. The signals at δ 153.96, 156.40, 158.81, and 158.92 ppm were assigned to azomethine (C = N), imine carbon (C = NH), amidic C = O, and chromene C = O carbons, respectively. The ESI MS of 14 showed the molecular ion (m/z 484), which agrees with a molecular formula (C₂₀H₁₆N₆O₅S₂). Further, the mass fragmentation pattern of compound 14 is shown in Scheme 3.

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Scheme 3

Mass fragmentation pattern of compound 14.

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Similarly, treatment of amide 12 with 2-hydroxynapthaldehyde 17 in boiling acetic acid containing ammonium acetate yielded 4-(2-imino-5-(1-(2-(3-oxo-3H-benzo[f]-chromene-2-carbonyl)hydrazono)ethyl)-1,3,4-thiadi-azol-3(2H)-yl)benzene sulfonamide 18 as a sole product (Scheme 2). Elemental analyses and spectrum data were used to confirm the structure of compound 18. The ESI MS of 18 exhibited a parent peak at m/z = 534 (M⁺) corresponding to its formula (C₂₄H₁₈N₆O₅S₂). The IR spectrum of 18 showed new absorption bands at 3330–3239, 3195–3117, 1701, 1688, and 1584 cm⁻¹ assignable to NH₂, two NH, coumarin C = O, amidic C = O, and C = N functions, respectively. Its NMR spectrum exhibited five singlet signals at δ 1.93, 7.66, 8.66, 9.58, 11.95 ppm characteristic for the CH₃, NH₂, coumarin-H₄, imine NH, and amidic NH protons, respectively, alongside aromatic multiplets in the region δ 7.48–8.35 ppm. The ¹³C NMR spectrum of 18 displayed one methyl carbon peak at δ 12.67 ppm in addition to characteristic amidic (C = O) and coumarin (C = O) peaks at 158.63, 161.30 ppm among a total of 22 carbon signals.

2.2.1 Cytotoxicity evaluation

Considering the reported overexpression of hCAIX in case of colorectal, gastric, and breast cancers, the cytotoxic potential of the synthesized derivatives 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 was tested against three different human cancerous cell lines (SK. Chia 2001, J. Chen 2005, AM. Niemela 2007). The tested cell lines were hepatoblastoma cell line HepG-2, colorectal adenocarcinoma cell lines Caco2, breast ductal carcinoma MCF-7 and the diploid lung fibroblast cell lines WI 38 as normal cell line (L. Hayflick 1961) using staurosporine as a reference. Staurosporine is a natural indolocarbazole alkaloid with a proven cytotoxic effect against several cancerous cell lines (J. Boix 1997, A.L. McKeague 2003). The best cytotoxicity was attained by the hydrazonoethyl 2, benzochromene 18 and dihydroxyphenyl 5 derivatives (Table 1 and Fig. 5). Derivative 2 showed better cytotoxicity than staurosporine against HepG-2, Caco2 and MCF-7 with IC₅₀ 7.15, 5.28 and 1.18 μM, respectively compared to staurosporine IC₅₀ 13.60, 8.18 and 6.19 μM. Similarly, derivative 18, 5 accomplished better HepG-2 and Caco2 growth inhibition than staurosporine in addition to comparable inhibition of WI 38 growth.

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Fig. 5

The in vitro cytotoxic evaluation of derivatives 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 using four human cancerous cell lines presented as IC₅₀ in μM.

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Structurally, changing the acetyl terminus 1 with an unsubstituted diazene 5 significantly improved the cytotoxic activity among the three tested cancerous cell lines (Fig. 5). However, adding cyanoacetyl moiety 12 to the free diazene group dropped its cellular growth inhibitory potential over the four cell lines. Moreover, a slight reduction of cytotoxic activity was established upon adding benzochromene moiety to the diazene tail 18, yet; it still showed better cytotoxic activity than staurosporine. Comparably, the 8-methoxy chromene 16 exhibited better cytotoxic activity against the three cancerous cell lines than its parent unsubstituted chromene 14 tail (Fig. 6). In contrast, switching the acetyl terminus of 1 into a triazene linkage connected to a phenolic moiety like dihydroxyphenyl 5 or hydroxynaphthalene 7 improved its cytotoxicity among the three cancerous cell lines. Nonetheless, the dihydroxyphenyl derivative 5 displayed better activity than its hydroxyphenyl analogue 7 almost 2–5 times amongst the tested cell lines. In the same context, the tetrazene containing tails 9 and 11 displayed improved cytotoxicity in the case of HepG-2 than their acetyl parent derivative 1. Furthermore, additional benzenesulfonamide moiety 9 enhanced the cytotoxic effect of the tetrazene analogues in HepG-2 and Caco2 without significant change on WI 38 in contrast to its heterocyclic benzopyrazole analogue 10. Further explanation of those derivatives' binding conformation was discussed in the molecular docking section.

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Fig. 6

Schematic representation of structure activity relationship of derivatives 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 based on their in vitro cancerous cell lines cytotoxicity.

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2.2.2 In silico molecular docking discussion

To further investigate the achieved in vitro cancerous cell lines inhibition results, molecular docking was used to test derivatives 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 abilities to inhibit the human tumor-associated carbonic anhydrase (hCA) isoforms IX and XII. The X-ray crystals of the tumor-associated hCAIX and hCAXII were downloaded from the Protein Data Bank https://www.rcsb.org using PDB: 3IAI (V. Alterio 2009) and 1JD0 (D.A. Whittington 2001) for hCAIX and hCAXII, respectively. The molecular docking protocol was validated before commencing the actual docking of 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 by self-docking the corresponding co-crystallized ligand AAZ to get the lowest RMSD value with the same interaction pattern with the lowest possible binding energy score. The achieved RMSD of hCAIX was 0.37 Å with an energy score of −8.83 Kcal/mol in contrast to 0.96 Å RMSD of hCAXII with anenergy score of −9.01 Kcal/mol (Fig. S1 at supplementary material).

The achieved molecular docking results of the targeted thiadiazole derivatives revealed the capability of the tested derivatives to coordinate with zinc inside both hCA isoforms catalytic sites by their oxygen and/or deprotonated nitrogen of the sulfonamide moiety (Figs. 7-12 and table S1 at supplementary material). The terminal sulfonamide amine was reported to be deprotonated at the physiological pH exposing a negative charge to coordinate with the positive zinc ion and hindering its cofactor role (K. Kanamori 1983, N. Chiaramonte 2018). The produced zinc coordination of the evaluated derivatives had an average distance of 1.90–3.21 Å. Moreover, all derivatives attained binding energy scores surpassed the co-crystallized AAZ with the best values achieved by 9 and 16 for hCAIX and hCAXII, respectively (Table 2), which translated into strong inhibitory effect on HepG2, Caco2 and MCF-7 (Table 1). Furthermore, the aliphatic tailed 11 had the privilege over AAZ to form H-bonds with one of the catalytic histidine triad His94 of hCAIX with a distance 3.48 Å (Table S1) (Supuran 2010). Interestingly, all derivatives formed one or more H-bonds with the active site Thr199 of both isoforms with a maximum distance of 3.10 Å in a similar way to AAZ. Similarly, derivatives 9, 12, 14, 16 and 18 formed H-bonds and hydrophobic interaction with Leu198 with an average bond distance of 3.8 Å in the case of hCAIX. Comparably, all derivatives formed a hydrogen bond with Leu198 in the case of hCAXII with maximum bond distance of 3.59 Å (Table 2 and Table S1 at supplementary material).

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Fig. 7

The 2D and 3D presentation of derivatives 2 (a and b) and 9 (c and d) binding conformations to hCAIX using PDB:3IAI. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds, illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared as a red dotted line with their corresponding distance in Å.

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Fig. 8

The 2D and 3D presentation of derivatives 5 (a and b) and 7 (c and d) binding conformations to hCAIX using PDB:3IAI. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds, illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared as a red dotted line with their corresponding distances in Å.

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Fig. 9

The 2D and 3D presentation of derivatives 16 (a and b) and 18 (c and d) binding conformations to hCAIX using PDB:3IAI. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds, illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared a red dotted line with their corresponding distances in Å.

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Fig. 10

The 2D and 3D presentation of derivatives 2 (a and b) and 9 (c and d) binding conformations to hCAXII using PDB:1JD0. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds, illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared as a red dotted line with their corresponding distance in Å.

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Fig. 11

The 2D and 3D presentation of derivatives 5 (a and b) and 7 (c and d) binding conformations to hCAXII using PDB:1JD0. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared as a red dotted line with their corresponding distance in Å.

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Fig. 12

The 2D and 3D presentation of derivatives 16 (a and b) and 18 (c and d) binding conformations to hCAXII using PDB:1JD0. The tested derivatives and the co-crystallized AAZ appeared as a green and blue stick model, respectively, with the H-bonds, illustrated as a dotted green line. In contrast, the hydrophobic interactions appeared as a red dotted line with their corresponding distance in Å.

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On the other hand, molecular docking managed to explain the astonishing cytotoxic effect of 2 with the hydrazone terminus on the tested cancerous cell lines, which in addition to its zinc coordination, one of its sulfonamide oxygen atoms accepted H-bonds from the active site Thr199 in both isoforms with additional H-bond formation with Thr200 and Leu198 for hCAIX and hCAXII, respectively (Fig. 7a-b and 10a-b). Those formed H-bonds could fix the molecule orientation in proximity to zinc and stabilize the formed coordination. Meanwhile, additional hydrophobic interaction was formed with Val121 in the case of hCAIX by its thiazole and Leu198 in the case of hCAXII by the phenyl moiety. Those extra hydrophobic interactions might assist in preserving 2 favored structural orientations inside the active site of both isoforms. Likewise, 9 with the tetraaz-2-ene benzenesulfonamide tail showed the usual zinc coordination along with multiple H- bond formation with eight and five residues of the active sites of hCAIX and hCAXII, respectively (Fig. 7c-d and 10c-d). Its calculated binding energy was −12.26 Kcal/mol and −11.60 Kcal/mol for hCAIX and hCAXII, respectively, which exceeded AAZ and could justify its interesting in vitro IC₅₀ values.

The triazene containing derivatives 5 and 7 demonstrated hydrogen bond formation with the crucial Thr199 of both isoforms with a maximum bond distance of 3.07 Å (Table 2), similar to AAZ. Moreover, they formed additional hydrogen bonds with hCAIX Thr200 and Gln92 (Fig. 8a and 7c) with the overall binding energy of −12.03 and −11.27 Kcal/mol for 5 and 7, respectively. The astonishing IC₅₀ values of 5 over 7 on the tested cell lines were rationalized by its multiple interactions and proper positioning within the binding sites of both hCA isoforms (Figs. 8 and 11). Furthermore, the dihydroxyphenyl moiety of 5 formed H-bond and arene-cation interaction with Thr91 and Lys67, respectively (Fig. 11a-b). In contrast, the hydroxynaphthalene moiety of 7 formed only one arene-H bond with Asn62 of slightly weak energy −0.60 Kcal/mol and 4.42 Å distance (Fig. 11c-d). Additionally, the binding energy of 5 was better than 7 in both isoforms, which matched the difference in their in vitro cytotoxicity.

Comparable binding energy to both hCA isoforms was achieved by 16 and 18 with an average of −12.02 Kcal/mol, which translated into analogous in vitro IC₅₀ values. Apart from the established zinc coordination, the aromatic moieties of 16 formed three hydrophobic bonds with the active site Leu198 and Pro202 (Fig. 9a-b) and one with Leu198 (Fig. 12a-b) in the case of hCAIX and hCAXII, respectively. In contrast, 18 showed several hydrophobic interactions with Val131, Asp132, Leu135 and Leu198 in the case of hCAIX (Fig. 9c-d) and with Leu198 and Pro202 in the case of hCAXII (Fig. 12c-d). Other hydrogen bonds were formed between the sulfonamide moiety of 18 and Thr199 in both isoforms, with additional interaction between its carbonyl and His64 and Lys67 in hCAXII. The illustrated multiple interactions of 18 over 16 with comparable binding energy in the case of both hCA isoforms were extrapolated into better IC₅₀ values of HepG-2, Caco2 and WI38.

To evaluate the possibility of the synthesized derivatives to interact with hCAI (Y. Takaoka 2013) and hCAII (A.H. Robbins 2009) as off-targets, molecular docking simulation was done for further investigation. The achieved simulation results demonstrated that despite possessing better binding energy than AAZ, the synthesized derivatives showed unfavorable interaction pattern (Table 3). The typical zinc coordination in both isoforms with sulfonamide terminus was observed among the derivatives with average distance 2.44 Å. Nonetheless, derivatives 2 and 18 with the highest cytotoxic activity formed H-bond with the crucial Thr199 in case of hCAI (Fig. 13a and 13b). On the other hand, both derivatives only demonstrated zinc coordination without further interactions with hCAII binding site residues (Fig. 13c and 13d). Consequently, the most potent cytotoxic derivatives 2 and 18 were expected to exhibit low off-target interactions.

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Fig. 13

The 2D interactions of derivatives 2 (a, c) and 18 (b, d) using hCAI (PDB 3W6H) and hCAII (PDB 3HS4), respectively.

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2.2.3 Physicochemical, pharmacokinetics properties and ADME prediction

The calculated physicochemical descriptors predicted pharmacokinetics properties and drug-likeness of 1, 2, 5, 7, 9, 11, 12, 14, 16, and 18 were determined by the Swiss Institute Bioinformatics free web SwissADME® (Table 4) (Bioinformatics 2017). As stated in Table 4, the calculated physicochemical values of the tested derivatives complied with Lipinski‘s rule of five, and thus these compounds could be administered orally (Lipinski. 2004). Fortunately, all the tested derivatives demonstrated predicted Log P_(o/w) values less than 5; however, their topological polar surface area TPSA exceeded 120 Å² which was reflected in their low gastrointestinal absorption and skin permeation (Kelder 1999, S. J. D. Veber 2002). Luckily, these TPSA values prevented their blood–brain barrier penetration, which could avoid undesirable CNS side effects. Moreover, all derivatives were unaffected by the drug efflux pump P-glycoprotein or the hepatic cytochrome enzymes.

3.1.1 Synthesis of (E)-4-(5-(1-hydrazineylideneethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl) benzenesulfonamide (2)

A solution of 1 (2.98 g, 0.01 mol) and N₂H₄ (1.5 mL, 0.03 mol) in EtOH (40 mL) containing AcOH (3 drops) under reflux for 6 h. The precipitate was filtered and recrystallized from EtOH to get product 2 as a brown powder (78%); M.P 207–210 °C. IR v_max/cm⁻¹ = 3416, 3285, 3233, 3119 (2NH₂), 3065 (NH), 1645 (C = N), 1585 (C = C), 1323, 1305 (SO₂); ¹H NMR (DMSO‑d₆): δ_ppm = 2.05 (s,3H, CH₃), 7.32 (s, 1H, ²N-H^b), 7.33 (s, 1H, ²N-H^a), 7.36 (s, 2H, NH₂), 8.26 (d, J = 8.5 Hz, 2H, Ar-H_2,6), 8.34 (d, J = 8.5 Hz, 2H, Ar-H_3,5), 9.49 (s, 1H, NH); ¹³C NMR (DMSO‑d₆): δ_ppm = 11.55 (CH₃), 120.24 (2CHAr-C_2,6), 126.80 (2CHAr-C_3,5), 131.35 (Ar-C₄), 139.75 (Ar-C₁), 145.89 (C = N), 151.28 (thiadiazole-C₅), 159.23 (thiadiazole-C₂); MS m/z (%): 312 (M⁺, 10.3), 270 (1.3), 251 (2.5), 227 (2.2), 205 (1.4), 193 (1.5), 176 (9.6), 158 (2.4), 145 (2.2), 129 (7.1), 111 (8.9), 88 (2.2), 65 (11.7), 57 (1 0 0); Anal. Calc. For C₁₀H₁₂N₆O₂S₂ (312.37): C, 38.45; H, 3.87; N, 26.90%; Found: C, 38.46; H, 3.86; N, 26.91%.

3.1.2 General procedure for the synthesis of triaz-2-en-1-ylidene)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzene-sulfonamide derivatives (5, 7)

A solution of compound 3 (1.07 g, 3.4 mmol) in DMF (5 mL) and concentrated HCl (0.75 mL, 7. 5 mmol) cooled to 0–5 °C was diazotized with sodium nitrite (0.26 g, 3.7 mmol) in water (5 mL). After 10 min, a solution of 1,3-dihydroxybenzene 4 or 2-hydroxynaphthalene 6 (3.4 mmol), dissolved in an aqueous solution of 10% sodium hydroxide (10 mL) was added to diazonium salt solution with stirring over 10 min. Then, the pH was adjusted to 7 with the addition of dilute acetic acid. After stirring the mixture for a further 1 h, the formed green precipitate was filtered off, washed with water, dried and recrystallized from ethanol to afford compounds 5 and 7.

3.1.3 General procedure for the synthesis of tetraaz-2-en-1-ylidene)ethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfonamide derivatives (9, 11)

A solution of compound 2 (1.07 g, 3.4 mmol) in DMF (5 mL) and conc. HCl (0.75 mL, 7. 5 mmol) was cooled to 0–5 °C and diazotized with NaNO₂ (0.26 g, 3.7 mmol) in water (5 mL). After 10 min, a solution of sulfanilamide 8 or 4-aminoantipyrine 10 (3.4 mmol) dissolved in 10% aq.NaOH (10 mL) was added to diazonium salt solution with stirring over 10 min. Then the reaction mixture pH was adjusted to 7 by adding dilute AcOH. After stirring for a further 1 h. The formed green precipitate was filtered off, washed with water, dried and recrystallized from EtOH to afford compounds 9 or 11, respectively.

3.1.4 Synthesis of 4-(5-(1-(2-(2-cyano-acetyl)-hydrazineylidene)-ethyl)-2-imino-1,3,4-thia diazol-3(2H)-yl)benzenesulfonamide (12)

A mixture of compound 1 (2.00 g, 0.006 mol) and 2-cyanoacetohydrazide (0.66 g, 0.006 mol) in absolute EtOH (50 mL), containing five drops of conc. HCl was refluxed for 6 h. The resulting precipitate was filtered off, washed with EtOH, dried, and recrystallized from EtOH to produce compound 12 as orange powder (88%); M.P 265–267 °C; IR v_max/cm⁻¹ = 3303, 3251 (NH₂), 3197, 3115 (2NH), 2270 (CN), 1687 (C = O), 1595 (C = N), 1582 (C = C), 1328, 1305 (SO₂); ¹H NMR (DMSO‑d₆): δ_ppm = (anti-configuration) 2.26 (s, 3H, CH₃), 4.14 (s, 2H, CH₂), 7.24 (d, J = 8.5 Hz, 2H, Ar-H_2,6), 7.40 (s, 2H, NH₂), 8.25 (d, J = 8.5 Hz, 2H, Ar-H₃,₅), 9.40 (s, 1H, NH), 11.60 (s, 1H, amide-NH); (syn-configuration) 2.29 (s, 3H, CH₃), 3.94 (s, 2H, CH₂), 7.24 (d, J = 8.5 Hz, 2H, Ar-H_2,6), 7.40 (s, 2H, NH₂), 8.25 (d, J = 8.5 Hz, 2H, Ar-H₃,₅), 9.42 (s, 1H, NH), 11.25 (s, 1H, amide-NH); ¹³C NMR (DMSO‑d₆): δ_ppm = 12.18 (CH₃), 24.94 (CH₂), 115.72 (CN), 120.72 (2CH Ar-C_2,6), 126.47 (2CH Ar-C_3,5), 126.91 (Ar-C₄), 141.92 (Ar-C₁), 145.47 (thiadiazole-C₅), 148.51 (C = N), 159.90 (thiadiazole-C₂), 165.88 (amidic C = O); MS m/z (%): 379 (M⁺, 28.9), 378 (13.3), 352 (0.3), 323 (0.4), 311 (4.2), 298 (1.7), 283 (0.8), 270 (0.3), 247 (0.2), 213 (0.5), 197 (2.0), 191 (0.4), 175 (0.9), 156 (12.3), 133 (3.8), 108 (14.2), 89 (14.8), 67 (35.1), 57 (1 0 0); Anal. Calc. For C₁₃H₁₃N₇O₃S₂ (379.41): C, 41.15; H, 3.45; N, 25.84%; Found: C, 41.14; H, 3.47; N, 25.83%.

3.1.5 General procedure for the synthesis of coumarin derivatives (14, 16 and 18)

A mixture of compound 12 (0.7 g, 0.0018 mol) and 0.0018 mol of salicylaldehyde derivatives (13, 15 or 17) in AcOH (15 mL), containing (0.13 g, 0.0018 mol) of CH₃COONH₄, was refluxed for 7–10 h. The formed precipitate was filtered off, washed with EtOH, dried and recrystallized from EtOH to get the desired products 14, 16 and 18, respectively.

3.2.1 Cytotoxicity evaluation

A volume of 3 mL of the medium containing the tested cells in the log phase was placed in each well of the multi-well plate without phenol red and serum. The final count of the tested cells did not exceed 10⁶ cells/cm². Then a volume of MTT (M−5655) equivalent to 10% of the culture medium was added and incubated for 2–4 h. The formed formazan crystals were dissolved in the MTT solubilizing solution (M−8910) with occasional shaking equal to the original culture medium. The absorbance of the resulting solution was measured spectrophotometric at wavelength 570 nm. A blank experiment was performed using a medium without cells at every test (S. Bondock, 2023).

3.2.2 Molecular docking analysis

Human carbonic anhydrase IX and XII X-ray crystal structures were downloaded from the Protein Data Bank using PDB: 3IAI and 1JD0 for hCAIX and hCAXII, respectively (A. Sloboda 1964, J. Boix 1997). Their crystal structures were prepared by removing the unnecessary chains, ligands and water molecules, preserving zinc ions. They were corrected, and 3D protonated at cutoff 15 Å using amber10:EHT force field of Molecular Operating Environment (MOE 2014.0901) software. The binding site was selected at the co-crystallized ligand site at a radius of 4.5 Å then molecular docking was performed using Triangle Matcher, London dG, GBVI/WSA as the placement, rescoring function 1 and 2, respectively as the docking algorithm. The tested derivatives were drawn using Chemdraw Ultra 12.0, then transferred as smiles to the MOE builder window, added their hydrogens and energy minimized at the same forcefield.