Synthesis, molecular modelling and QSAR study of new N-phenylacetamide-2-oxoindole benzensulfonamide conjugates as carbonic anhydrase inhibitors with antiproliferative activity

Abstract

In continuation of our previous studies to optimise potent carbonic anhydrase inhibitors, two new series of isatin N-phenylacetamide based sulphonamides were synthesised and screened for their human (h) carbonic anhydrase (EC 4.2.1.1) inhibitory activities against four isoforms hCA I, hCA II, hCA IX and hCA XII. The indole-2,3-dione derivative 2h showed the most effective inhibition profile against hCAI and hCA II (K_I = 45.10, 5.87 nM) compared to acetazolamide (AAZ) as standard inhibitor. Moreover, 2h showed appreciable inhibition activity against the tumour-associated hCA XII, similar to AAZ showing K_I of 7.91 and 5.70 nM, respectively. The analogs 3c and 3d showed good cytotoxicity effects, and 3c revealed promising selectivity towards lung cell line A549. Molecular docking was carried out for 2h and 3c to predict their binding conformations and affinities towards the hCA I, II, IX and XII isoforms.

Graphical abstract

Keywords:

• Carbonic anhydrase inhibitors
• benzenesulfonamides
• molecular docking
• 2D-QSAR study

1. Introduction

The simple and fundamental interconversion of crucial ion species such as carbon dioxide and bicarbonate (CO₂/HCO₃⁻) occurs during biochemical processes in all living organisms. The CO₂/HCO₃^- interconversion is catalysed by a superfamily of metalloenzymes called carbonic anhydrases (CAs, EC 4.2.1.1). The enzymes belonging to the α-CA family tightly coordinate a Zn²⁺ ion within their active sites, that interacts with a water molecule and histidine residues in a tetrahedral geometry of the metal ion. The zinc ion binds and activates a water molecule to catalyse the hydration reaction of carbon dioxide to bicarbonate and proton ions by a metal hydroxide nucleophilic mechanism. Consequently, CAs act among the major physiological buffering systems^1–4.

Human CAs (hCAs) are represented by 15 isoforms which include the cytosolic (I, II, III, VII, XIII), mitochondrial (VA, VB), secreted (VI), and membrane associated (IV, IX, XII, XIV) in addition to the catalytic isoforms of carbonic anhydrase related proteins located in cytosol (VIII, X, XI)^5–8. Fluctuation in CA expression levels can lead to hypertension, glaucoma, epilepsy, neuropathic pain, cancer, etc. Accordingly, many hCA isoforms are considered as attractive therapeutic targets for many pharmacological applications^9,10. Recently, CA inhibitors (CAIs) were adopted as a combination therapy for solid tumours upon acknowledging the overexpression of the membrane-associated isoenzymes hCA IX and hCA XII in that kind of tumours. Those isoenzymes were discovered to play pivotal role in microenvironment pH regulation thus being important in the survival, proliferation, metastasis and development of drug resistance of malignant cells^11,12.

Known CAIs are generally primary aromatic or heteroaromatic sulphonamides in which the sulphonamide moiety coordinates the Zn⁺² ion in the enzyme active site with their terminal deprotonated nitrogen atom simulating the known non-selective CAI Acetazolamide (AAZ) I (Figure 1)^13–15. Sulphonamide based CAIs have versatile pharmacological activities such as diuretic¹⁶, antiglaucoma¹⁷, antiepileptic¹⁸, antimicrobial¹⁹, anti-inflammatory²⁰, antioxidant²¹ and anticancer^22–24. These activities made them attractive candidates for developing novel agents with varying biological activities through minor structural alterations^9,10.

Figure 1. Chemical structures of AAZ (I), Sunitinib (II) and reported CAIs incorporating sulphonamide moieties III-VIII.

Moreover, isatin (indole-2,3-dione) is considered as a crucial core in many anticancer drugs in the market as sunitinib II²⁵. Furthermore, isatin-benzenesulfonamide conjugates were reported to have promising anticancer activity as well as carbonic anhydrase inhibition^26–28. For example, 4-(2-(1-ethyl-2-oxoindolin-3-ylidene)hydrazine-1-carbonyl)benzenesulfonamide (III) revealed K_I = 8.9 nM against hCA IX²⁶. Additionally, isatin hydrazinyl benzenesulfonamide derivative IV exhibited K_I= 7.8 nM against hCA IX isoform²⁷. Based on this knowledge, the present study was designed to develop potent and selective isatin-based sulphonamide CAIs utilising the “tail approach”. This approach investigated the effect of adding different molecular fragments to the isatin scaffold on the binding interaction with the enzyme active site which in turn would modulate the potency and selectivity^29,30.

Previously reported unsubstituted isatin Schiff’s bases V and VI showed promising CA inhibitory activity²⁹ (Figure 1), then the developed N-benzoyl candidates showed high potency and selectivity towards hCA XII (compounds VII, VIII)³⁰ (Figure 1). Motivated by these results, we developed here new isatin-based sulphonamide candidates by incorporation of phenylacetamide moiety at the isatin nitrogen as a new structural modification. The attached phenylacetamide moiety was evaluated as unsubstituted as well as substituted by groups of diverse size, lipophilicity, and electronic property (such as Cl, Br, CH₃, OCH₃, SO₂NH₂) to investigate the interaction of the new adopted tail fragment with the CA active site. Additionally, the isatin scaffold was hybridised with the benzene sulphonamide through a hydrazinyl (compounds 3a–g), or hydrazinyl-2-oxoethylamino spacers (compounds 4a–g) in order to study the effect of this structure elongation along with the tail fragment on the CA inhibitory activity and selectivity (Figure 2). All new synthesised candidates (2h, 3a–g, 4a–g) were screened for their CA inhibitory activity against four hCA isoforms (I, II, IX, and XII), and the most active ones were selected for further in vitro evaluation for their anti-proliferative activity. Furthermore, to rationalise their significant in vitro biological results, molecular docking simulation was carried out for the most active derivatives to study their binding pattern and affinity to the hCA II, IV, IX, and XII active sites. Furthermore, 2D-QSAR study was performed using all reported derivatives of our previous work^29,30 in addition to the newly synthesised derivatives to obtain two validated models for the tumour associated isoforms hCA IX and XII elucidating the descriptors controlling the activity, therefore they can be used for prediction of activity of further isatin-based derivatives.

Figure 2. The rational of the newly synthesised isatin-based sulphonamide derivatives 2h, 3a-g and 4a-g.

2. Experimental

2.1. Chemistry

All chemicals were purchased from VWR International Merck, or Sigma-Aldrich, Germany. Melting points are uncorrected and were measured by open capillary tube method using Stuart SMP3 melting point apparatus. Elemental microanalyses were carried out at the Regional Centre for Mycology and Biotechnology, Al-Azhar University. Infra-red spectra were recorded on Shimadzu spectrometer IR Affinity-1 (FTIR- 8400S-Kyoto-Japan) and expressed in wave number (cm⁻¹). ¹H NMR and ¹³C NMR Spectra were recorded on Bruker High Performance Digital FT-NMR Spectrometer Avance III 400 MHz, ¹³C, 100 MHz NMR spectrometer. Chemical shifts were expressed in δ units and were related to that of the solvents. Mass Spectra were recorded using ISQLT Thermo Scientific Mass spectrometer at The Regional Centre for Mycology and Biotechnology, Al-Azhar University. All the reactions were monitored by TLC using silica gel F254 plates (Sigma-Aldrich), using chloroform: methanol 9.5:0.5 as eluting system and were visualised by UV-lamp. Compounds 1a–g^31,32, 2a–f^33–35, and the reagents 4-hydrazinylbenzenesulfonamide hydrochloride³⁶ and 4-(2-hydrazinyl-2-oxoethylamino)benzenesulfonamide³⁷ were prepared according to their reported methods.

2.1.1. General procedure for the synthesis of compounds 2a-h

A mixture of isatin (indole-2,3-dione) (0.29 g, 2 mmol) and potassium carbonate (0.82 g, 6 mmol) was stirred for 15 min. in DMF (5 ml), then an appropriate 2-chloro-N-phenylacetamide (1a-h) (2 mmol) was added. The reaction was stirred at room temperature for 4 h, then poured on ice water, the precipitated solid was filtered, washed with water and crystallised form methanol.

2.1.1.1. N-(2-Bromophenyl)-2–(2,3-dioxoindolin-1-yl)acetamide (2 g)

Yellow crystals, yield 80%, m.p. 221–222 °C; IR (KBr, ʋ cm⁻¹): 3244 (NH), 3036 (CH aromatic), 2931 (CH aliphatic), 1736, 1670 (3 C=O), 1612 (NH bending), 1589, 1531 (C=C); ¹H NMR (CDCl₃, 400 MHz): δ 4.62 (s, 2H, NCH₂), 7.17–7.20 (m, 3H, aromatic H), 7.38 (t, 1H, J = 7.64 Hz, aromatic H), 7.50 (d, J = 7.04 Hz, 1H, aromatic H), 7.63 (d, J = 6.60 Hz, 1H, aromatic H), 7.66–7.73 (m, 2H, aromatic H), 9.94 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (CDCl₃, 100 MHz): δ 44.7 (CH₂), 110.8, 117.8, 122.1, 124.7, 125.8, 126.1, 128.5, 132.4, 134.6, 138.8, 149.9 (aromatic carbons), 158.3, 163.8, 181.9 (3 C=O); MS, m/z [%]: 361.10 [M⁺+2, 3.27], 359.10 [M⁺, 3.87]; Anal. Calcd. for C₁₆H₁₁BrN₂O₃ (359.17): C, 53.50; H, 3.09; N, 7.80. Found: C, 53.74; H, 3.25; N, 8.07.

2.1.1.2. 2–(2,3-Dioxoindolin-1-yl)-N-(4-sulfamoylphenyl)acetamide (2 h)

Yellow orange crystals, yield 78%, m.p. 237–238 °C; IR (KBr, ʋ cm⁻¹): 3325, 3267, 3205 (NH₂, NH), 3067 (CH aromatic), 2935 (CH aliphatic), 1740, 1697 (3 C=O), 1612 (NH bending), 1597, 1543 (C=C), 1308, 1153 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.63 (s, 2H, NCH₂), 7.16–7.18 (m, 2H, aromatic H), 7.28 (s, 2H, NH₂, D₂O exchangeable), 7.62–7.73 (m, 4H, aromatic H), 7.78 (d, J = 8.64 Hz, 2H, aromatic H), 10.59 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.7 (CH₂), 111.5, 118.0, 119.6, 124.0, 125.0, 127.2, 138.8, 139.4, 141.7, 151.3 (aromatic carbons), 158.9, 166.0, 183.4 (C=O); MS, m/z [%]: 360.15 [M⁺+1, 5.37], 359.20 [M⁺, 22.48]; Anal. Calcd. for C₁₆H₁₃N₃O₅S (359.36): C, 53.48; H, 3.65; N, 11.69. Found: C, 53.69; H, 3.80; N, 11.88.

2.1.2. General procedure for the synthesis of compounds 3a–g

A mixture of equimolar amounts of (2a–g) (1 mmol) and 4-hydrazinylbenzenesulfonamide hydrochloride (0.22 g, 1 mmol) in methanol (5 ml) was stirred at room temperature for 4 h. The obtained solid was filtered, washed with ethanol and recrystallized from ethanol.

2.1.2.1. 2–(2-Oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl)-N-phenylacetamide (3a)

Yellow crystals, yield 78%, m.p. 294–295 °C; IR (KBr, ʋ cm⁻¹): 3364, 3329 (NH₂), 3197, 3140 (2NH), 3059 (CH aromatic), 2974 (CH aliphatic), 1700, 1678 (2 C=O), 1605 (NH bending), 1570, 1551 (C=C), 1300, 1157 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.65, 4.70 (2 s, 2H, NCH₂), 7.05–7.10, 7.13–7.19 (2 m, 3H, aromatic H), 7.25, 7.27 (2 s, 2H, NH₂, D₂O exchangeable), 7.31–7.36, 7.37–7.43 (2 m, 4H, aromatic H), 7.59, 7.68 (2d, J = 7.72 Hz, 2H, aromatic H), 7.62, 7.68 (2d, J = 7.72 Hz, 2H, aromatic H), 7.82, 7.84 (2d, J = 7.30 Hz, 2H, aromatic H), 10.34, 10.38 (2 s, 1H, NH, D₂O exchangeable), 10.86, 12.68 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.1 (NCH₂), 109.7, 114.5, 115.1, 119.4, 122.3, 123.1, 124.1, 127.6, 129.3, 129.7, 138.3, 139.0, 141.9, 145.6 (aromatic carbons), 161.7, 165.5 (C=O); MS, m/z [%]: 449 [M⁺, 40]; Anal. Calcd. for C₂₂H₁₉N₅O₄S (449.48): C, 58.79; H, 4.26; N, 15.58. Found: C, 59.03; H, 4.42; N, 15.79.

2.1.2.2. N-(4-Chlorophenyl)-2–(2-oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl)acetamide (3 b)

Yellow crystals, yield 75%, m.p. 297–298 °C; IR (KBr, ʋ cm−1): 3282 (NH₂, 2 NH), 3067 (CH aromatic), 2932 (CH aliphatic), 1693, 1674 (2 C=O), 1601 (NH bending), 1570, 1539 (C=C), 1331, 1153 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.65, 4.70 (2 s, 2H, NCH₂), 7.09–7.14, 7.16–7.20 (2 m, 4H, aromatic H), 7.24, 7.26 (2 s, 2H, NH₂, D₂O exchangeable), 7.61–7.64, 7.66–7.69 (2 m, 4H, aromatic H), 7.81–7.85 (m, 3H, aromatic H), 8.31 (d, J = 7.68 Hz, 1H, aromatic H), 10.49, 10.86 (2 s, 1H, NH, D₂O exchangeable), 10.52, 12.67 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.0, 43.3 (NCH₂), 109.7, 110.1, 114.5, 115.1, 116.0, 119.5, 120.6, 121.2, 121.3, 122.4, 123.2, 124.8, 127.6, 127.7, 128.0, 129.1, 129.2, 129.7, 131.1, 137.9, 138.0, 138.3, 141.8, 143.3, 145.6, 147.2 (aromatic carbons), 161.7, 164.7, 165.8, 166.0 (2 C=O); MS, m/z [%]: 486.15 [M⁺+2, 13], 484.17 [M⁺+1, 24]; Anal. Calcd. for C₂₂H₁₈ClN₅O₄S (483.93): C, 54.60; H, 3.75; N, 16.85. Found: C, 54.87; H, 3.92; N, 14.68.

2.1.2.3. N-(4-Bromophenyl)-2–(2-oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl)acetamide (3c)

Yellow crystals, yield 75%, m.p. 300–302 °C; IR (KBr, ʋ cm⁻¹): 3333, 3267, 3155 (NH₂, 2NH), 3059 (CH aromatic), 2931 (CH aliphatic), 1712, 1674 (2 C=O), 1605 (NH bending), 1570, 1539 (C=C), 1296, 1157 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.65, 4.70 (2 s, 2H, NCH₂), 7.10 (d, J = 7.92 Hz, 1H, aromatic H), 7.15 (t, J = 7.34 Hz, 1H, aromatic H), 7.25 (s, 2H, NH₂, D₂O exchangeable), 7.40 (t, J = 7.68 Hz, 1H, aromatic H), 7.50 (d, J = 8.92 Hz, 2H, aromatic H), 7.56 (d, J = 8.92 Hz, 2H, aromatic H), 7.65 (d, J = 8.84 Hz, 2H, aromatic H), 7.84 (d, J = 8.72 Hz, 2H, aromatic H), 8.30 (d, 1H, J = 7.64 Hz, aromatic H), 10.49, 10.53 (2 s, 1H, NH, D₂O exchangeable), 10.87, 12.67 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.14, 43.33 (NCH₂), 109.7, 110.1, 114.5, 115.1, 115.6, 115.7, 116.0, 119.5, 120.6, 121.6, 121.7, 122.4, 123.1, 124.8, 127.7, 127.9, 129.1, 129.7, 131.2, 138.04, 138.3, 141.8, 143.3, 145.6, 147.2 (aromatic carbons), 161.7, 164.6, 165.8, 166.1 (2 C=O); MS, m/z [%]: 528.02 [M⁺, 25]; Anal. Calcd. for C₂₂H₁₈BrN₅O₄S (528.38): C, 50.01; H, 3.43; N, 13.25. Found: C, 50.12; H, 3.67; N, 13.49.

2.1.2.4. 2–(2-Oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl)-N-p-tolylacetamide (3d)

Yellow crystals, yield 73%, m.p. 298–299 °C; IR (KBr, ʋ cm⁻¹): 3341, 3171 (NH₂, 2NH), 3059 (CH aromatic), 2939 (CH aliphatic), 1713, 1682 (2 C=O), 1605 (NH bending), 1570, 1547 (C=C), 1296, 1157 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.26 (s, 3H, CH₃), 4.63, 4.68 (2 s, 2H, NCH₂), 7.09–7.19 (m, J = 8.08 Hz, 4H, aromatic H), 7.25, 7.27 (2 s, 2H, NH₂, D₂O exchangeable), 7.41 (t, J = 7.74 Hz, 1H, aromatic H), 7.47 (d, J = 8.28 Hz, 2H, aromatic H), 7.66 (d, 2H, J = 8.72 Hz, aromatic H), 7.84 (d, J = 8.60 Hz, 2H, aromatic H), 8.31 (d, J = 7.64 Hz, 1H, aromatic H), 10.27, 10.31 (2 s, 1H, NH, D₂O exchangeable), 10.87, 12.68 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 20.9 (CH₃), 43.0, 43.2 (NCH₂), 109.7, 110.3, 114.4, 115.1, 116.0, 119.4, 119.6, 119.7, 120.5, 122.3, 123.1, 124.8, 127.6, 127.8, 129.1, 129.6, 131.1, 131.2, 132.9, 133.0, 136.5, 136.6, 138.0, 138.3, 141.9, 143.4, 145.6, 147.1 (aromatic H), 161.7, 164.6, 165.2, 165.6 (C=O); MS, m/z [%]: 463.51 [M⁺, 39]; Anal. Calcd. for C₂₃H₂₁N₅O₄S (463.51): C, 59.60; H, 4.57; N, 15.11. Found: C, 59.84; H, 4.73; N, 15.38.

2.1.2.5. N-(4-Methoxyphenyl)-2–(2-oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl) acetamide (3e)

Yellow crystals, yield 70%, m.p. 294–295 °C; IR (KBr, ʋ cm⁻¹): 3333, 3213, 3171 (NH₂, 2NH), 3040 (CH aromatic), 2978, 2935 (CH aliphatic), 1713, 1678 (2 C=O), 1609 (NH bending), 1570, 1547 (C=C), 1300, 1157 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.72 (s, 3H, OCH₃), 4.61, 4.62 (2 s, 2H, NCH₂), 6.89 (d, J = 8.88 Hz, 2H, aromatic H), 7.09 (d, J = 7.88 Hz, 1H, aromatic H), 7.17 (t, J = 7.58 Hz, 1H, aromatic H), 7.25, 7.27 (2 s, 2H, NH₂, D₂O exchangeable), 7.41 (t, J = 7.74 Hz, 1H, aromatic H), 7.49 (d, J = 8.88 Hz, 2H, aromatic H), 7.65 (d, J = 8.80 Hz, 2H, aromatic H), 7.84 (d, J = 8.64 Hz, 2H, aromatic H), 8.30 (d, J = 7.64 Hz, 1H, aromatic H), 10.20, 10.21 (2 s, 1H, NH, D₂O exchangeable), 10.86, 12.69 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.0, 43.2 (NCH₂), 55.6 (OCH₃), 109.7, 110.1, 114.4, 114.5, 115.1, 116.0, 119.5, 120.6, 121.2, 121.3, 122.4, 123.1, 124.8, 127.7, 127.9, 129.2, 129.7, 131.1, 131.3, 132.1, 132.2, 138.0, 138.3, 141.9, 143.4, 145.6, 147.2, 155.8, 155.9 (aromatic carbons), 161.7, 164.6, 165.0, 165.3 (C=O); MS, m/z [%]: 479.47 [M⁺, 26]; Anal. Calcd. for C₂₃H₂₁N₅O₅S (479.51): C, 57.61; H, 4.41; N, 14.61. Found: C, 57.49; H, 4.63; N, 14.88.

2.1.2.6. N-(2-Chlorophenyl)-2–(2-oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl) acetamide (3f)

Yellow crystals, yield 78%, m.p. 275–276 °C; IR (KBr, ʋ cm⁻¹): 3363, 3333, 3136 (NH₂, 2NH), 3059 (CH aromatic), 2974, 2935 (CH aliphatic), 1701 (2 C=O), 1601 (NH bending), 1570, 1512 (C=C), 1296, 1153 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.73, 4.78 (2 s, 2H, NCH₂), 6.93 (d, J = 7.20 Hz, 1H, aromatic H), 7.13–7.23 (m, 2H, aromatic H), 7.27 (s, 2H, NH₂, D₂O exchangeable), 7.30–7.41 (m, 2H, aromatic H), 7.51 (d, J = 8.40 Hz, 1H, aromatic H), 7.62 (d, J = 8.80 Hz, 2H, aromatic H), 7.68 (t, J = 8.40 Hz, 1H, aromatic H), 7.82 (d, J = 8.80 Hz, 2H, aromatic H), 8.30 (d, J = 8.00 Hz, 1H, aromatic H), 9.99, 11.10 (2 s, 1H, NH, D₂O exchangeable), 12.66 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.8 (NCH₂), 110.1, 114.2, 114.4, 119.4, 120.6, 123.1, 126.6, 127.2, 127.6, 127.8, 129.1, 129.6, 130.0, 134.8, 138.3, 141.7, 145.6 (aromatic carbons), 161.6, 166.2 (2 C=O); MS, m/z [%]: 485.30 [M⁺+2, 17.85], 483.30 [M⁺, 48.45]; Anal. Calcd. for C₂₂H₁₈ClN₅O₄S (483.93): C, 54.60; H, 3.75; N, 14.47. Found: C, 54.51; H, 3.89; N, 14.70.

2.1.2.7. N-(2-Bromophenyl)-2–(2-oxo-3–(2-(4-sulfamoylphenyl)hydrazono)indolin-1-yl) acetamide (3 g)

Yellow crystals, yield 73%, m.p. 279–280 °C; IR (KBr, ʋ cm⁻¹): 3364, 3333, 3155 (NH₂, 2NH), 3059 (CH aromatic), 2974, 2935 (CH aliphatic), 1697 (2 C=O), 1597 (NH bending), 1570, 1539 (C=C), 1300, 1153 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.71, 4.76 (2 s, 2H, NCH₂), 7.10 (d, J = 7.84 Hz, 1H, aromatic H), 7.14–7.19 (m, 2H, aromatic H), 7.27, 7.25 (2 s, 2H, NH₂, D₂O exchangeable), 7.36–7.43 (m, 2H, aromatic H), 7.57–7.69 (m, 4H, aromatic H), 7.82 (d, J = 8.48 Hz, 2H, aromatic H), 8.31 (d, J = 7.60 Hz, 1H, aromatic H), 9.93, 10.87 (2 s, 1H, NH, D₂O exchangeable), 9.97, 12.66 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.7, 43.0 (NCH₂), 109.7, 110.2, 114.5, 115.1, 116.0, 118.6, 119.4, 120.6, 122.5, 123.2, 127.7, 127.9, 128.6, 129.2, 129.7, 131.2, 133.2, 136.2, 138.0, 138.3, 141.7, 143.1, 145.6, 147.2 (aromatic carbons), 161.6, 164.5, 166.1, 166.2 (C=O); MS, m/z [%]: 530 [M⁺+2, 13.24], 528 [M⁺, 13.20]; Anal. Calcd. for C₂₂H₁₈BrN₅O₄S (528.38): C, 50.01; H, 3.43; N, 13.25. Found: C, 50.28; H, 3.61; N, 13.19.

2.1.3. General procedure for the synthesis of compounds 4a-g

A mixture of equimolar amounts of (2a-g) (1 mmol) and 4-((2-hydrazinyl-2-oxoethyl)amino)benzenesulfonamide (0.24 g, 1 mmol) was dissolved in methanol (5 ml) in the presence of glacial acetic acid (0.5 ml) and stirred at room temperature for 3 h. The obtained precipitate was filtered, washed with ethanol and recrystallized from ethanol.

2.1.3.1. 2–(2-Oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono)indolin-1-yl)-N-phenylacetamide (4a)

Yellow crystals, yield 80%, m.p. 272–273 °C; IR (KBr, ʋ cm⁻¹): 3379, 3283, 3237, 3144 (NH₂, 3NH), 3071 (CH aromatic), 2978, 2939 (CH aliphatic), 1720, 1693, 1663 (3 C=O), 1601 (NH bending), 1550, 1516 (C=C), 1312, 1150 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.38 (s, 2H, NHCH₂), 4.64 (s, 2H, NCH₂), 6.71–6.79 (m, 3H, aromatic H), 6.95 (s, 2H, NH₂, D₂O exchangeable), 7.06–7.15 (m, 3H, aromatic H), 7.32 (t, J = 7.90 Hz, 2H, aromatic H), 7.47 (t, J = 7.63 Hz, 1H, aromatic H), 7.54–7.59 (m, 4H, aromatic H), 8.13 (s, 1H, NH, D₂O exchangeable), 10.35 (s, 1H, NH, D₂O exchangeable), 11.41 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.2, 43.3 (2CH₂), 110.2, 111.8, 115.2, 119.7, 123.8, 124.1, 126.3, 127.8, 133.1, 138.9, 139.0, 143.8, 144.9, 151.5 (aromatic carbons), 164.1, 165.2, 165.5 (3 C=O); MS, m/z [%]: 507.79 [M⁺+1, 15]; Anal. Calcd. for C₂₄H₂₂N₆O₅S (506.53): C, 56.91; H, 4.38; N, 16.59. Found: C, 57.12; H, 4.60; N, 16.78.

2.1.3.2. N-(4-Chlorophenyl)-2–(2-oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono) indolin-1-yl)acetamide (4 b)

Yellow crystals, yield 78%, m.p. 270–271 °C; IR (KBr, ʋ cm⁻¹): 3333, 3294, 3206, 3136 (NH₂, 3NH), 3063–3032 (CH aromatic), 2978, 2947 (CH aliphatic), 1716, 1693, 1659 (3 C=O), 1612 (NH bending), 1554, 1512 (C=C), 1319, 1149 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.37 (s, 2H, NHCH₂), 4.58, 4.64 (2 s, 2H, NCH₂), 6.61 (d, J = 8.52 Hz, 2H, aromatic H), 6.72 (d, J = 8.48 Hz, 1H, aromatic H), 6.94, 6.95 (2 s, 2H, NH₂, D₂O exchangeable), 7.11, 7.20 (m, 2H, aromatic H), 7.38 (d, J = 8.76 Hz, 2H, aromatic H), 7.45 (d, J = 7.60 Hz, 1H, aromatic H), 7.51–7.63 (m, 3H, aromatic H), 7.67 (t, J = 7.74 Hz, 1H, aromatic H), 8.12 (s, 1H, NH, D₂O exchangeable), 10.36, 10.49 (2 s, 1H, NH, D₂O exchangeable), 11.37 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.6, 45.0 (2CH₂), 111.4, 115.2, 117.9, 121.3, 121.6, 124.1, 125.0, 127.7, 129.2, 131.2, 137.7, 138.9, 144.7, 151.1, 158.9 (aromatic carbons), 165.7, 169.3, 183.4 (3 C=O); MS, m/z [%]: 542.94 [M⁺+2, 32.72], 540.88 [M⁺, 40]; Anal. Calcd. for C₂₄H₂₁ClN₆O₅S (540.98): C, 53.28; H, 3.91; N, 15.53. Found: C, 53.61; H, 4.08; N, 15.79.

2.1.3.3. N-(3-Bromophenyl)-2–(2-oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono) indolin-1-yl)acetamide (4c)

Orange yellow crystals, yield 78%, m.p. 260–262 °C; IR (KBr, ʋ cm⁻¹): 3329, 3294, 3236, 3198 (NH₂, 3NH), 3067, 3032 (CH aromatic), 2947, 2897 (CH aliphatic), 1725, 1697, 1650 (3 C=O), 1600 (NH bending), 1550, 1512 (C=C), 1315, 1149 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.36 (s, 2H, NHCH₂), 4.58, 4.64 (2 s, 2H, NCH₂), 6.61, 6.71 (2d, J = 8.44 Hz, 2H, aromatic H), 6.91, 7.11 (2d, J = 8.10 Hz, 1H, aromatic H), 6.94, 6.95 (2 s, 2H, NH₂, D₂O exchangeable),7.03, 7.17 (2t, J = 7.68 Hz, 1H, aromatic H), 7.45, 7.67 (2t, J = 7.70 Hz, 1H, aromatic H), 7.47–7.56 (m, 6H, aromatic H), 7.62 (d, J = 7.36 Hz, 1H, aromatic 8.12 (s, 1H, NH, D₂O exchangeable), 10.37, 10.49 (2 s, 1H, NH, D₂O exchangeable), 11.37 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.4, 43.7, 45.0 (2CH₂), 111.5, 111.7, 115.7, 115.9, 117.9, 121.3, 121.6, 121.9, 122.2, 122.8, 124.0, 125.0, 126.3, 126.5, 127.7, 127.8, 131.2, 132.1, 138.1, 138.3, 138.8, 143.7, 144.7, 151.2, 151.4, 151.5, 158.9 (aromatic carbons), 165.5, 169.2, 183.3 (C=O); MS, m/z [%]: 583.20 [M⁺-2, 1.84]; Anal. Calcd. For C₂₄H₂₁BrN₆O₅S (585.43): C, 49.24; H, 3.62; N, 14.36. Found: C, 49.52; H, 3.83; N, 14.59.

2.1.3.4. 2–(2-Oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono)indolin-1-yl)-N-m-tolylacetamide (4d)

Yellow crystals, yield 75%, m.p. 273–274 °C; IR (KBr, ʋ cm⁻¹): 3344, 3294 (NH₂, 3NH), 3032 (CH aromatic), 2974, 2897 (CH aliphatic), 1717, 1697, 1659 (3 C=O), 1597 (NH bending), 1512 (C=C), 1315, 1145 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.26 (s, 3H, CH₃), 4.37 (s, 2H, NHCH₂), 4.62 (s, 2H, NCH₂), 6.61, 6.71 (2d, J = 8.60 Hz, 2H, aromatic H), 6.94, 6.95 (2 s, 2H, NH₂, D₂O exchangeable), 7.10–7.14 (m, 4H, aromatic H), 7.44 (d, J = 8.04 Hz, 2H, aromatic H), 7.49 (d, J = 8.76 Hz, 2H, aromatic H), 7.55 (d, J = 8.72 Hz, 2H, aromatic H), 8.12 (s, 1H, NH, D₂O exchangeable), 9.16 (s, 1H, NH, D₂O exchangeable), 10.25 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 20.9 (CH₃), 43.3, 45.0 (2CH₂), 110.2, 111.6, 111.7, 115.2, 119.8, 122.8, 126.3, 127.8, 129.7, 131.2, 133.0, 136.5, 143.8, 144.9, 151.5 (aromatic carbons), 165.3, 169.2, 173.1 (3 C=O); MS, m/z [%]: 522.38 [M⁺+2, 18], 520.41 [M⁺, 19]; Anal. Calcd. For C₂₅H₂₄N₆O₅S (520.56): C, 57.68; H, 4.65; N, 16.14. Found: C, 57.96; H, 4.82; N, 16.36.

2.1.3.5. N-(3-Methoxyphenyl)-2–(2-oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl) hydrazono)indolin-1-yl)acetamide (4e)

Yellow crystals, yield 70%, m.p. 278–279 °C; IR (KBr, ʋ cm⁻¹): 3379, 3329, 3290, 3233 (NH₂, 3NH), 3074 (CH aromatic), 2974, 2901 (CH aliphatic), 1720, 1693, 1659 (3 C=O), 1601 (NH bending), 1512 (C=C), 1311, 1149 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.72 (s, 3H, OCH₃), 4.37 (s, 2H, NHCH₂), 4.60 (s, 2H, NCH₂), 6.70–6.73 (m, 2H, aromatic H), 6.89 (d, J = 9.00 Hz, 2H, aromatic H), 6.95, 6.97 (2 s, 2H, NH₂, D₂O exchangeable), 7.10–7.21 (m, 3H, aromatic H), 7.45–7.50 (m, 3H, aromatic H), 7.55 (d, J = 7.64 Hz, 2H, aromatic H), 8.12 (s, 1H, NH, D₂O exchangeable), 10.19 (s, 1H, NH, D₂O exchangeable), 11.40 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.1, 43.3 (2CH₂), 55.6 (OCH₃), 110.2, 111.8, 114.4, 115.2, 121.3, 122.8, 123.7, 126.3, 127.8, 132.1, 133.0, 143.8, 144.9, 151.4, 155.9 (aromatic carbons), 164.0, 164.5, 165.0 (3 C=O); MS, m/z [%]: 536.20 [M⁺, 40]; Anal. Calcd. For C₂₅H₂₄N₆O₆S (536.56): C, 55.96; H, 4.51; N, 15.66. Found: C, 56.13; H, 4.67; N, 15.89.

2.1.3.6. N-(2-Chlorophenyl)-2–(2-oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono) indolin-1-yl)acetamide (4f)

Yellow crystals, yield 65%, m.p. 206–208 °C; IR (KBr, ʋ cm⁻¹): 3379, 3283, 3256 (NH₂, 3NH), 3066 (CH aromatic), 2927, 2835 (CH aliphatic), 1740, 1720, 1674 (3 C=O), 1597 (NH bending), 1539, 1520 (C=C), 1312, 1150 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.69, 3.70 (2 s, 2H, NHCH₂), 4.26, 4.32 (2s, 1H, NH, D₂O exchangeable), 4.46, 4.71 (2 s, 2H, NCH₂), 6.61 (d, J = 8.68 Hz, 2H, aromatic H), 6.70 (d, J = 8.72 Hz, 2H, aromatic H), 6.93 (s, 2H, NH₂, D₂O exchangeable), 7.08 (d, J = 7.68 Hz, 1H, aromatic H), 7.11 (d, J = 7.56 Hz, 1H, aromatic H), 7.23 (t, J = 6.96 Hz, 1H, aromatic H), 7.33 (t, J = 7.36 Hz, 1H, aromatic H), 7.42–7.46 (m, 1H, aromatic H), 7.51 (d, J = 8.72 Hz, 2H, aromatic H), 7.70 (d, J = 7.12 Hz, 1H, aromatic H), 9.15 (s, 1H, NH, D₂O exchangeable), 9.96 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.03, 46.07 (2CH₂), 111.64, 111.83, 122.84, 127.6, 127.8, 128.0, 128.3, 130.0, 131.2, 131.7, 132.1, 133.5, 134.8, 135.3, 138.1, 151.5 (aromatic carbons), 159.2, 169.2 (C=O); MS, m/z [%]: 541.10 [M⁺+1, 1.63], 539.10 [M⁺-1, 2.34]; Anal. Calcd. For C₂₄H₂₁ClN₆O₅S (540.98): C, 53.28; H, 3.91; N, 15.53. Found: C, 53.44; H, 4.07; N, 15.76.

2.1.3.7. N-(2-Bromophenyl)-2–(2-oxo-3–(2-(2–(4-sulfamoylphenylamino)acetyl)hydrazono) indolin-1-yl)acetamide (4 g)

Buff crystals, yield 70%, m.p. 216–217 °C; IR (KBr, ʋ cm⁻¹): 3379, 3345, 3283 (NH₂, 3NH), 3062, 3032 (CH aromatic), 2920, 2850 (CH aliphatic), 1728, 1720, 1662 (3 C=O), 1601 (NH bending), 1520 (C=C), 1315, 1149 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.15, 4.16 (2 s, 2H, NHCH₂), 4.62, 4.66 (2 s, 2H, NCH₂), 6.33, 8.99 (2 s, 1H, NH, D₂O exchangeable), 6.61 (d, J = 8.68 Hz, 2H, aromatic H), 6.69 (d, J = 8.72 Hz, 2H, aromatic H), 6.89, 6.93 (2 s, 2H, NH₂, D₂O exchangeable), 6.98, 8.36 (2d, J = 7.32 Hz, 1H, aromatic H), 7.12–7.20 (m, 2H, aromatic H), 7.38 (t, J = 7.62 Hz, 1H, aromatic H), 7.51 (d, J = 8.48 Hz, 2H, aromatic H), 7.61–7.70 (m, 2H, aromatic H), 9.21, 9.59 (2 s, 1H, NH, D₂O exchangeable), 9.81, 10.02 (2 s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.85, 45.05 (2CH₂), 111.5, 115.2, 118.8, 122.0, 125.1, 127.3, 127.6, 127.7, 128.6, 130.2, 131.2, 133.2, 136.4, 151.8 (aromatic carbons), 167.0, 173.9, 181.3 (3 C=O); MS, m/z [%]: 585.20 [M⁺, 0.90], 583.20 [M⁺-2, 1.38]; Anal. Calcd. For C₂₄H₂₁BrN₆O₅S (585.43): C, 49.24; H, 3.62; N, 14.36. Found: C, 49.45; H, 3.74; N, 14.63.

2.2. Carbonic anhydrase inhibitory activity

An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO₂ hydration activity³⁸. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na₂SO₄ (for mainaining constant the ionic strength), following the initial rates of the CA-catalysed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentration ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been use for determining the initial velocity. The un catalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together at room temperature (15 min) prior to assay, in order to allow for the formation of the E-I complex. Data from Table 1 were obtained after 15 min incubation of enzyme and inhibitor, as for the sulphonamides reported earlier³⁹. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier^40,41 and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier⁴².

Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with 2,3-dioxoindole sulfamoylphenyl acetamide 2h, 2-oxindole based benzenesulfonamides 3a-g, 4a-g and the standard inhibitor Acetazolamide (AAZ) using a stopped flow CO₂ hydrase assay.

Compound ID	R	KI^a (nM)				Selectivity ratio
		hCA I	hCA II	hCA IX	hCA XII	I/IX	II/IX	I/XII	II/XII
2h	SO2NH2	45.1	5.87	67.0	7.91	0.673	0.087	5.701	0.742
3a	H	3971	260	216	3649	18.384	1.203	1.088	0.071
3b	4-Cl	892	452	123	474	7.252	3.674	1.881	0.953
3c	4-Br	762	56.4	56.9	612	13.392	0.991	1.245	0.092
3d	4-CH3	656	79.9	85.5	710	7.672	0.934	0.923	0.112
3e	4-OCH3	776	180	598	599	1.297	0.301	1.295	0.300
3f	2-Cl	2695	804	673	3295	4.004	1.194	0.817	0.244
3g	2-Br	708	420	401	766	1.765	1.047	0.924	0.548
4a	H	4639	804	808	6792	5.741	0.995	0.683	0.118
4b	4-Cl	8429	4586	3922	5045	2.149	1.169	1.670	0.909
4c	4-Br	2352	732	654	2285	3.596	1.119	1.029	0.320
4d	4-CH3	8509	458	574	6025	14.824	0.797	1.412	0.076
4e	4-OCH3	9318	6722	3151	1522	2.957	2.133	6.122	4.416
4f	2-Cl	4391	2969	5358	754	0.819	0.554	5.823	3.937
4g	2-Br	6088	2001	7336	843	0.829	0.272	7.221	2.373
AAZ	—	250.0	12.0	25.0	5.7	10	0.48	43.859	2.105

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

2.3. Anti-proliferative activity

Cancer cell lines were obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin and 10% of heat-inactivated foetal bovine serum in humidified, 5%(v/v) CO₂ atmosphere at 37 °C.

Cell viability assay was assessed by SRB assay. Aliquots of 100 μL cells suspension (5x10³cells) were in 96 well plates and incubated in complete media for 24 h. Cells were treated with another aliqot of 100 μL media containing drugs at various concentrations. After 72 h of drug exposure, cells were fixed by replacing media with 150 μL of 10%TCA and incubated at 4 °C for 1 h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 μL SRB solution (0.4%w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3times with 1% acetic acid and allowed to air-dry overnight. Then, 150 μL of TRIS (10 mM) was added to dissolve protein-bound SRB stain; the absorbance was measured at 540 nm using a BMGLABTECH^® -FLUO star Omega microplate reader (Ortenberg, Germany)^43,44. All experiments were performed in triplicate wells for each condition. IC₅₀ values were calculated from concentration-response curves by Sigma Plot software, version 12.0 (System Software, San Jose, CA, USA), using an E-max model equation. Also selectivity index was determined.

2.4. Molecular docking

Molecular Operating Environment MOE^® 2014.0901 was used as the computational software throughout the docking study. The best quality chains of the four PDB files (3W6H, 3HS4, 3IAI and 1JD0 for hCA I, II, IX and XII, respectively) were selected where the unnecessary ions, ligands and water molecules were removed in a radius of 4.0 Å. The 3D protein structures were protonated using their corresponding pH and temperature conditions. The selected compounds 2h and 3c were constructed then deprotonated in-silico using amber:10EHT forcefield followed by energy minimisation at a gradient of 0.01 RMSD. On the fly-docking protocol was implemented at the same forcefield using triangle matcher, London dG and GBVI/WSA dG as placement and rescoring function 1 and 2, respectively.

For accurate molecular docking protocol, validation was done by self-docking the co-crystallised ligands to get the lowest root mean square deviation (RMSD) and the highest energy score (S).

2.5. 2D-QSAR study

The compounds were constructed, and energy minimised using MOE^® 2014.0901 MMFF94x forcefield at the default parameters with RMS gradient 0.1 Kcal/mol/A² then divided into training and testing sets for model generation and validation, respectively. For hCA IX, 34 compounds were used as the training set and 5 as test set of compounds while for hCA XII, 34 and 6 compounds were used as the training and test set, respectively. A variety of 413 1D and 2D descriptors were calculated using Chemopy^® descriptors from ChemDes^® open-source webserver. Thereafter, descriptors with constant values across the training set or equal to zero were excluded. RapidMiner^® Studio 9.10 program was used for descriptors filtration through the forward selection optimisation algorithm against the dependent variable log K_I then the best descriptors were transferred back to MOE^® for a linear model generation using the partial least square (PLS) method.

3. Results and discussion

3.1. Chemistry

Scheme 1 illustrates the synthesis of the target 2-oxindole benzenesulfonamide analogs 3a-g and 4a-g. Known 2-chloro-N-[(un)sub)]phenylacetamides (1a-h) were prepared according to the reported method by alkylation of the appropriate substituted aniline derivatives with chloroacetylchloride^31,32. The reported key intermediates (2a-f)^33–35 and the new ones (2g, h) were prepared according to the conventional method by the reaction of the commercially available isatin (indole-2,3-dione) with the corresponding 2-chloro-N-phenacetamide (1a-h) in dry N, N-dimethyl formamide (DMF) in the presence of potassium carbonate. Then, 2a-g were condensed with the prepared 4-hydrazinylbenzenesulfonamide hydrochloride³⁶ at room temperature to give new 4-[2–(2-oxindolin-3-ylidene)hydrazinyl]benzenesulfonamide (3a-g). Similarly, the condensation of 2a-g with the prepared 4–(2-hydrazinyl-2-oxoethylamino)benzenesulfonamide³⁷ in methanol at room temperature afforded 4–(2-oxindolin-3-ylidene)hydrazinyl-2-oxoethylamino benzenesulfonamide derivatives (4a-g). All new derivatives were confirmed by spectral and elemental analyses as detailed in the experimental part.

Scheme 1. Synthetic pathway for the target compounds 1–4. Reagents and reaction conditions. (i) Glacial acetic acid, sodium acetate, 0 °C. (ii) MeOH, r.t. (iii) MeOH, glacial acetic acid, r.t.

It is noteworthy that all target compounds 3a-g and 4a-g contain an exocyclic C=N imine bond which results in their presence as E/Z geometrical isomers. However, it was reported that E/Z ratio is dependent on each isomer stability and NMR solvent^28,45,46. This fact was observed in many compounds like 3a,b,f,g and 4b,c,f,g which were found in a mixture of E and Z isomers as observed from their ¹H NMR spectra that revealed additional signals due to both isomers. For instance, ¹H NMR spectrum of compound 3a had two singlets attributed to the methylene protons at 4.65 ppm (E-isomer) and 4.70 ppm (Z-isomer) with integration ratio 3.23:3.42, respectively, whereas compounds 3c,d,e showed both isomeric forms with integration ratio (6:1) for Z–isomer. On the other hand, compounds 4a,d,e were isolated mainly as Z isomers. (see the experimental section).

3.2. Carbonic anhydrase inhibitory activity

The new candidates 2,3-dioxoindole sulfamoylphenyl acetamide 2h, 2-oxoindole derivatives (hydrazinyl benzenesulfonamide (3a-g) and hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) were evaluated for their ability to inhibit four physiologically relevant hCA isoforms (cytosolic isoforms; hCA I, hCA II and the transmembrane, tumour-associated isoforms hCA IX, hCA XII) through a stopped flow CO₂ hydrase assay^38–42. Their potency as K_I (nM) were compared to the standard CA inhibitor acetazolamide (AAZ) and these results were summarised in (Table 1). A brief study of SAR revealed the followings:

1. Regarding the ability of the tested compounds to inhibit hCA I, all tested compounds except 2h revealed relatively lower activity than other isoforms. It was noticed that 2,3-dioxoindole 2h showed an extremely high potency (K_I = 45.1 nM) compared to AAZ (K_I = 250 nM) and to other tested compounds that showed inhibitory activity ranged from 656 to 9318 nM. Derivatives linked to hydrazinyl benzenesulfonamide (3a-g) were more active (K_Is = 656-3971 nM) than their analogs linked to hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) (K_Is = 2353-9318 nM). Also, in hydrazinyl benzenesulfonamides (3a-g) substituted phenylacetamide moiety revealed activity better than unsubstituted congener, the order of activity was (4-CH₃> 4-Br> 4-OCH₃> 4- Cl> 2-Br> 2-Cl > H), thus, the inductive effect of substituents at the phenyl ring has no absolute impact on the activity. On the other hand, in hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) the order of activity was 4-Br> 2-Cl > H> 2-Br> 4-Cl> 4-CH₃> 4-OCH₃), that the presence of electron withdrawing groups is more favourable for activity than electron donating moieties.

2. Activity towards hCA II isoform was better with K_I ranged from 5.87 to 4586 nM; like hCA I, compound 2h was also the most potent one with K_I = 5.87 nM. Derivatives linked to hydrazinyl benzenesulfonamides (3a-g) were more active (K_Is = 56.4-804 nM) than their analogs linked to hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) (K_I = 458-4586 nM). In both series (3a-g) and (4a-g) it was obvious that substitution of phenylacetamide with 4-Br or 4-CH₃ was preferable for activity.

3. All the tested compounds except compounds 2h, and 4e-g represented a reasonable selectivity towards tumour isoform hCA IX compared to hCA I isoform. The hydrazinyl benzenesulfonamide (3a-g) were more active (K_Is = 56.9–673 nM) than their analogs linked to hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) (K_Is = 574–7336 nM). The 4-bromophenylacetamide derivative (3c) was the most active one. Similar to hCA II activity, in both series (3a-g) and (4a-g) it was obvious that substitution of phenylacetamide with 4-Br or 4-CH₃ was preferable for activity.

4. Regarding the hCA XII inhibition, all the tested compounds except compounds 2h, revealed very weak selectivity towards hCA XII. Compound 2h was relatively the most potent one with K_I = 7.91 nM. Hydrazinyl benzenesulfonamide (3a-g) were more active than their analogs linked to hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g). In hydrazinyl benzenesulfonamide (3a-g) it was obvious that substitution of phenylacetamide with 4-Br or 4-Cl was preferable for activity, while in hydrazinyl 2-oxoethylamino benzenesulfonamide congeners (4a-g) substitution at ortho position of phenylacetamide with Br or Cl was more favoured.

5. Finally, from our previous studies to develop isatin-based CA derivatives^29,30 we can find in the current study that the presence of substituted phenylacetamide increase the potency specially for hCA I and shifted the selectivity mainly towards hCA IX comparing to substituted benzoyl moiety in the previous study³⁰ that reflects the ability of these compounds to have promising anticancer activity. Also, in our current study increasing the length of the N-substitution from N-benzoyl to N-phenylacetamido group leading to decrease in potency mainly against all isoforms and slightly shifted the selectivity in case of 2-substituted phenylacetamide analogs.

3.3. Anti-proliferative activity

As CA IX is overexpressed in MCF-7 breast cancer cell line⁴⁷ and in A549 lung cancer cells⁴⁸, therefore, compounds 2h, 3c and 3d eliciting the best CA IX inhibition were selected for further evaluation for their anti-proliferative activity against these cancer cell lines. Moreover, they were tested for their cytotoxicity against normal human skin fibroblast cell line HSF to confirm their safety, the results are illustrated in Table 2 and Figures S1–S6. Compound 3d showed good cytotoxic activity against MCF-7 and A549 cell lines with IC₅₀ = 0.869 and 5.12 μM, respectively. On the other hand, 3c exerted only cytotoxic activity against A549 cell line with IC₅₀ = 5.90 μM. Furthermore, 3c showed promising safety margin towards normal cells through its selectivity towards A549 cells in contrast to doxorubicin and 3d which were unselectively cytotoxic to both normal and cancer cell lines. Unfortunately, compound 2h found to have no cytotoxic activity against both cell lines, thus, its anti-proliferative activity is not correlated to its CA IX inhibition, however, it can be considered as an adjuvant with an anti-cancer agent to enhance its cytotoxic effect due to its CA IX inhibition activity similar to acetazolamide.

Table 2. Cytotoxic activity and selectivity index of compounds 2h, 3c, and 3d on cancer and normal cell lines

Cell line	Compd.
	IC50 (μM)				Selectivity index
	Doxorubicin	2h	3c	3d	Doxorubicin	2h	3c	3d
MCF-7	0.63	>100	>100	0.869	0.349	1	1	1.68
A549	0.19	>100	5.90	5.12	1.157	1	16.94	0.28
HSF	0.22	>100	>100	1.46	—	—	—	—

3.4. Molecular docking study

Molecular docking was done for 2h and 3c to relate their in vitro biological results with their possible conformations for binding to the four CA isoforms. Hence, the crystal structure of each isoform co-crystallised with AAZ was downloaded from the Protein Data Bank (PDB) using PDB ID: 3W6H, 3HS4, 3IAI and 1JD0 for isoforms hCA I, II, IX and XII, respectively⁴⁹. Molecular docking method was validated using the co-crystallised AAZ by self-docking till getting the same interaction pattern with each isoform binding site residues as presented in Supplementary Figure S10a-d. Considering the importance of sulphonamide moiety deprotonation to coordinate with the positively charged zinc ion⁵⁰, the tested compounds sulphonamide group were in-silico deprotonated and energy minimised before docking.

The molecular docking of the most active derivative 2h augmented the achieved in vitro K_I values that surpassed AAZ using both hCA isoforms I and II. Considering hCA I (PDB ID 3W6H), it revealed the formation of the two characteristic co-ordinations with zinc ion using the deprotonated nitrogen of the sulphonamide moiety (Figure 3(a)) which translated into K_I value of 45.1 and 250.0 nM for 2h and AAZ, respectively. The achieved co-ordinations were distanced by 2.01 (zinc- nitrogen) and 2.65 Å (zinc – oxygen) from zinc which are consistent with many crystallographic findings (Figure 3(b)). Moreover, the sulphonamide negatively charged deprotonated nitrogen was able to interact with the basic His200, a unique hCA I isoform zinc ligand⁵¹. This interaction could support the conformation of the sulphonamide moiety to approach zinc and stabilised the outcome interaction. Furthermore, two arene-H bonds were formed with Leu198, and Pro202 by the two phenyl groups beside the sulphonamide oxygen adeptness to accept the H-bond from Thr199. Another explanation of the resulted lower K_I value of 2h than AAZ was that the capability of 2h to block most of the hCAI active site entrance by its isatin moiety (Figure 3(c)).

Figure 3. Molecular docking data of 2h on hCAI isoform using PDB ID 3W6H as 2D (a) and 3D (b) presentations showing 2h as green ball and stick model with the formed H-bonds and arene-H were shown in blue and black dotted lines, respectively with their distance in Å highlighting the interaction site. c) The overlaid 3D presentation of 2h (green) and AAZ (magenta) inside the CA I active site.

Similarly, 2h resulted in better K_i inhibitory value of hCA II isoform than AAZ exhibiting 5.87 and 12.0 nM, respectively. The binding mode of 2h inside the active site of PDB ID 3HS4 managed to explain the achieved in vitro results. Its sulphonamide moiety interacted with zinc, Thr199, Thr200 and Leu198 forming four H-bonds ranging 2.26–3.2 Å and zinc co-ordination at 1.98 Å. In addition, two arene-H interactions were formed with Val121 and Phe131 using phenyl moiety of sulphonamide terminus and acetamide linkage, respectively (Figure 4(a,b)). Similar to hCAI, 2h oriented in a conformation that blocked the entrance of hCA II active site by its isatin moiety (Figure 4(c)). The binding energy of 2h was better than AAZ calculated as −10.11 and −7.69 Kcal/mol, respectively which complemented the binding conformations and achieved K_I values.

Figure 4. Molecular docking data of 2h on hCA II isoform using PDB ID 3HS4 as 2D (a) and 3D (b) presentations showing 2h as green ball and stick model with the formed H-bonds and arene-H were shown in blue and black dotted lines, respectively with their distance in Å highlighting the interaction site. c) The overlaid 3D presentation of 2h (green) and AAZ (magenta) inside the hCA II active site.

Regarding the tumour-associated hCA IX isoform, 2h molecular docking data using PDB ID 3IAI showed its interaction with zinc in resemblance to other isoforms with the sulphonamide oxygen and deprotonated nitrogen suggesting good inhibitory potential (Figure 5(a)). Moreover, the oxygen atoms of sulphonamide group and acetamide linkage acted as acceptors to three H-bonds with Thr199, Thr200 and Gln92, respectively. Despite its in silico interaction pattern, its binding energy score was not as favourable as AAZ giving −8.75 and −10.81 Kcal/mol, respectively which explained the superiority of the achieved AAZ Ki value. Nonetheless, 2h showed better K_Ivalue on the other tested tumour-associated hCA XII isoform comparing to AAZ showing 7.91 and 5.7 nM, respectively. Its binding mode using hCA XII PDB 1JD0 supported the achieved K_I value in which it interacted with zinc, Thr199, Thr200, Gln92 and Val131 (Figure 5(b)). However, AAZ surpassed 2h binding energy score giving −9.12 Kcal/mol comparing to −8.09 Kcal/mol of 2h which was translated into a slight difference in their K_I values.

Figure 5. The 2D presentation of 2h molecular docking study interacting with the tumour-associated isoforms hCA IX (a) and XII (b) using PDB ID 3IAI and 1JD0, respectively.

The following most promising derivative 3c showed equal sub-nanomolar K_I values on hCA II and IX giving 56.4 and 56.9 nM, respectively compared to AAZ with K_I 12.0 and 25.0 nM for both isoforms, respectively. Its molecular docking investigation using PDB ID 3HS4 and 3IAI for hCA II and hCA IX, respectively explained the achieved K_I values (Figure S11(a,b)). Zinc ion co-ordination was detected in both isoforms yet, the number of interactions of 3c with the active site residues were more at hCA IX than hCA II. This was translated into half the inhibitory activity in case of hCA IX compared to almost one-fifth the inhibition in case of hCA II in comparison to AAZ. Furthermore, the superiority of AAZ was clarified by its type of interactions with the crucial active site residues of hCA II and hCA IX (Figure S11(c,d)) together with its favourable binding energy −11.85 and −10.98 Kcal/mol compared to 3c with −8.45 and −9.98 Kcal/mol for both isoforms, respectively. In contrast, 3c did not show as promising inhibitory activity on the other two isoforms I and XII which were elucidated by its molecular docking data (Figure S12(a,b)).

3.5. 2D-QSAR study

As a useful manipulation of our current and previously achieved in vitro hCA inhibitory data of benzenesulfonamide derivatives as hCA inhibitors, (Figure 6) ^29,30, the tumour associated hCA IX and XII data were employed to build two 2D Quantitative Structure-Activity Relationship (2D-QSAR) models given their scaffold similarity, substitution variation and the unified in vitro assay method, these QSAR models were used to correlate the achieved inhibitory activity with their structural features for future structural optimisation. The collective biology data showed wide biology spectrum ranges from K_I = 14 to 7336 nM and 8 to 6025 nM for hCA IX and hCAXII, respectively. Different mathematical forms of the dependent variable were tried such as K_I, log K_I and Pk_I where log K_I were found to be linear with approximate r² value= 0.95 (Figures S13,14). Linear regression method was used for descriptors selection and optimisation by RapidMiner^® Studio then the model was generated using Partial Least Square (PLS) method by MOE^® program. In PLS method, the results of the fit are presented as the root mean square error (RMSE) which is the square root of the MSE function at the chosen descriptors value and the correlation coefficient (r²) which is equal to 1-MSE/YVAR where YVAR is the sample variance of the experimental values^52,53. The calculated 1D and 2D descriptors by Chemopy^®54 included different constitutional, topological, charge-related, thermodynamic and atom/bond-type descriptors properties of the compounds. An exclusion of descriptors with constant values amongst the training set or equal to zero was done before descriptors selection using the forward linear regression algorithm with leave 10 out validation method of RapidMiner^® Studio program. The selected descriptors co-linearity of both models was evaluated using correlation matrices to prevent model over-fitting.

Figure 6. Chemical structures of the reported benzenesulfonamide derivatives as hCA inhibitors included in the 2D-QSAR training set ^29,30.

3.5.1. 2D-QSAR model of hCA IX

For the tumour associated hCA IX, the 40 compounds were divided into training N = 34 and test n = 5 sets and their descriptors were calculated and filtered to 147 after excluding descriptors with fixed values. The selected linear descriptors values of the training set were presented in Table S1. The descriptors governed the activity were found to be the distance and adjacency matrix of the heavy atoms (BCUT_PEOE-0)⁵⁵, the partial equalisation of orbital electronegativities (PEOE_VSA-3)⁵², fractional positive polar van der Waals surface area (QVSA_FPPOS)⁵⁶, partition coefficient of subdivided surface area (SLogP_VSA 4)⁵⁷ and wiener polarity number (wienerPol)⁵⁸ according to the model presented in Equation (1)(1) . The generated model showed regression coefficient r²= 0.862 and RMSE= 0.269. $$\begin{array}{l}{\hspace{0.17em}}_{\text{Log Ki}}=\mathrm{ }29.73797\mathrm{ }+\mathrm{ }12.53594\mathrm{ }*\mathrm{ }\text{BCUT}\_\text{PEOE}\_0\mathrm{ }-\mathrm{ }0.11013\\ \mathrm{ }*\mathrm{ }\text{PEOE}\_\text{VSA}-3\mathrm{ }-\mathrm{ }0.04030\mathrm{ }*\mathrm{ }\text{Slog}P\_\text{VSA}4\mathrm{ }\\ +\mathrm{ }0.10445\mathrm{ }*\mathrm{ }\text{wienerPol}\mathrm{ }-\mathrm{ }1.29806\mathrm{ }*\mathrm{ }\mathrm{Q}\_\text{VSA}\_\text{FPPOS}\end{array}$$ (1) $$\mathrm{N}=\mathrm{ }32,{\mathrm{ }\mathrm{r}}^2=\mathrm{ }0.862,\mathrm{ }\text{RMSE}=\mathrm{ }0.269,{\text{ r}}^2{}_{\left(\text{LOO}\right)\mathrm{ }}=0.797,{\text{ r}}^2{}_{\left(\text{validation}\right)}=0.766,\mathrm{ }\mathrm{F}=33.039,{\text{ s}}^2=0.089$$

Examining the coefficient values of the independent variables displayed in Equation (1)(1) , it revealed that the distance and adjacency of the heavy atoms had the main proportional impact on the dependent inhibitory activity of hCA IX. Furthermore, electronegativity and polarity of the compound had appreciable effect on the predicted inhibitory action.

This model was capable of predicting the inhibitory activity of the most potent analogue 7a amongst all in-vitro tested derivatives of benzenesulfonamide with very small difference in log K_I value of −0.11. The predicted log K_I of 7a was 1.26 (K_I= 18.2 nM) compared to the actual value 1.146 (K_I= 13.9 nM).

The generated QSAR model was validated using leave one out (LOO) cross validation method which involved developing a number of models with one example excluded at a time and externally validated using the test set compounds. The achieved r²_(LOO) = 0.797 with a corresponding RMSE= 0.328 while test set predictive dependent variables showed r²_(validation) = 0.766. The predictive values of the dependent variables and their corresponding graphical presentation of both LOO and external validation were presented in Table 3 and Figures 7(a,b). As shown in Table 3, the model managed to predict the inhibitory activity of the test set of compounds that were compatible with the actual experimental values with small residuals. Moreover, the obtained Z-score during cross validation remained less than 2.4 suggesting compounds fitting to the generated model with no outliers. Additionally, Fisher-value test (F) and standard deviation (s²) were calculated for the model to confirm its statistical reliability and significance⁵⁹. This model revealed F = 33.039 and s² = 0.089 with the higher value of F relative to s² was an additional support of the model validation.

Figure 7. Graphical presentation between the experimental and predicative log K_i of hCAIX training set (LOO) validation (a) and test set (b) where the corresponding squared linear coefficient r² were displayed.

Table 3. The validation data of hCAIX 2D-QSAR model as calculated from LOO method and test set external validation.

Training set (LOO validation)						Test set^a
Compd.	log KI	$PRED	$Residuals	$Z-SCORE	$XPRED	Compd.	log KI	$PRED	$ Residuals
7a	1.146	1.261	0.115	0.428	1.298	8a	1.929	1.883	−0.046
6d	1.204	1.171	−0.033	0.123	1.163	8c	2.161	2.102	−0.059
7c	1.204	0.974	−0.23	0.855	0.855	7d	2.418	1.994	−0.424
6a	1.322	1.134	−0.188	0.698	1.036	3e	2.777	2.502	−0.275
5c	1.477	1.483	0.006	0.023	1.488
7b	1.505	1.653	0.148	0.548	1.702
3c	1.756	2.388	0.632	2.348	2.463
2h	1.826	1.546	−0.28	1.041	1.426
9a	1.833	2.021	0.188	0.702	2.049
3d	1.935	2.184	0.249	0.927	2.209
9c	1.968	2.242	0.274	1.016	2.282
3b	2.09	2.383	0.293	1.088	2.414
5a	2.13	2.619	0.489	1.815	2.884
9b	2.155	2.233	0.078	0.289	2.245
9e	2.217	2.391	0.174	0.646	2.43
8b	2.236	2.095	−0.141	0.523	2.056
8e	2.283	2.241	−0.042	0.156	2.232
9d	2.294	2.249	−0.045	0.169	2.242
3a	2.334	2.154	−0.18	0.671	2.136
8d	2.423	2.108	−0.315	1.171	2.011
3g	2.603	2.509	−0.094	0.352	2.497
5e	2.752	2.854	0.102	0.378	2.895
4d	2.759	3.06	0.301	1.12	3.089
4c	2.816	3.248	0.432	1.609	3.299
5d	2.828	2.552	−0.276	1.024	2.48
3f	2.828	2.508	−0.32	1.19	2.473
4a	2.907	3.015	0.108	0.402	3.027
5b	2.923	2.825	−0.098	0.365	2.783
4e	3.498	3.379	−0.119	0.444	3.348
4b	3.594	3.242	−0.352	1.307	3.199
4f	3.729	3.358	−0.371	1.379	3.306
4g	3.865	3.362	−0.503	1.87	3.294

^aCompound 7e was excluded from test set as outlier to the model.

3.5.2. 2D-QSAR model of hCA XII

Similarly, the achieved hCA XII inhibitory data was used to construct a 2D-QSAR model to correlate with their physicochemical and topological properties. The compounds were classified into 34 and 6 as training and testing set, respectively where the descriptors selection was done in a similar way to hCA IX model complying with the usual linear regression method. The four descriptors governed the activity were depicted in Equation (2)(1) and Table S2 were the fractional positive polar van der Waals surface area (PEOE_VSA_FPPOS)⁵⁶, fractional hydrophobic van der Waals surface area (PEOE_VSA_ FHYD)⁵⁶, partition coefficient of subdivided surface area (SLogP_VSA 1)⁵⁷ and the total positive partial charge (Q_PC⁺)⁵⁶. The estimated model showed regression coefficient r²= 0.699 and RMSE= 0.408. $$\begin{array}{l}\hspace{0.17em}\text{Log}\hspace{0.17em}\mathrm{ }\text{Ki}\mathrm{ }=\mathrm{ }-8.31628\mathrm{ }+\mathrm{ }8.98857\mathrm{ }*\mathrm{ }\text{PEOE}\_\text{VSA}\_\text{FHYD}\mathrm{ }-\mathrm{ }4.02069\mathrm{ }\\ *\mathrm{ }\text{PEOE}\_\text{VSA}\_\text{FPPOS}\mathrm{ }+\mathrm{ }0.07244\mathrm{\hspace{0.17em}}*\mathrm{\hspace{0.17em}}\mathrm{Q}\_{\text{PC}}^{+}+\mathrm{ }0.04101\mathrm{\hspace{0.17em}}*\mathrm{\hspace{0.17em}}\text{Slog}P\_\text{VSA}1\end{array}$$ (2) $$\mathrm{N}=33,{\mathrm{ }\mathrm{r}}^2=\mathrm{ }0.699,\mathrm{ }\text{RMSE}=\mathrm{ }0.408,{\text{ r}}^2{}_{(\text{LOO})}=\mathrm{ }0.62,{\text{ r}}^2{}_{\left(\text{validation}\right)\mathrm{ }}=0.841,\mathrm{ }\mathrm{F}=16.27,{\text{ s}}^2=\mathrm{ }0.197$$

According to the shown coefficient value of each independent variable in Equation (2)(2) , it was suggested that both van der Waals hydrophobic and polar fractional surface area had the main influence on the inhibitory activity of hCA XII in direct and inverse proportional, respectively. Similar to hCA IX 2D-QSAR model, both fractional positive polar van der Waals surface area and partition coefficient of subdivided surface area had an appreciable effect on the inhibition of hCA XII.

The obtained model managed to predict the inhibitory activity of the most potent hCAXII inhibitor 2 h amongst the tested analogues with difference of 0.051. The model suggested a log K_I of 0.954 (K_I= 8.99 nM) compared to the actual log K_I of 0.903 (K_I= 7.91 nM).

Resembling the hCAIX inhibitors 2D-QSAR model validation parameters, the predicative log K_I values were presented in Table 4. This model displayed r² _(LOO) = 0.62 with RMSE= 0.46 however, its external validation using the test set revealed more promising r²_(validation) = 0.841 (Figure 8(a,b)) accompanied with F = 16.27 and s²= 0.197.

Figure 8. Graphical presentation between the experimental and predicative log K_I of hCAXII training set (LOO) validation (a) and test set (b) where the corresponding squared linear coefficient r² were displayed.

Table 4. The validation data of hCAXII 2D-QSAR model as calculated from LOO method and test set external validation.

Training set (LOO validation)						Test set
Compd.	Log KI	$PRED	$RES	$Z-SCORE	$XPRED	Compd.	Log KI	$PRED	$ Residuals
2h	0.903	0.954	−0.051	0.124	0.997	5b	1.531	1.035	0.496
9a	1.342	1.906	−0.563	1.378	1.968	5d	1.851	1.311	0.54
9e	1.342	2.065	−0.722	1.766	2.147	8d	1.949	2.181	−0.231
8e	1.491	2.16	−0.669	1.636	2.193	6c	2.666	2.183	0.482
8a	1.519	2.015	−0.497	1.214	2.038	4f	2.877	2.999	−0.122
8c	1.623	2.121	−0.498	1.217	2.145	4c	3.359	3.053	0.306
5e	1.623	1.546	0.077	0.188	1.511
8b	1.681	1.771	−0.09	0.22	1.777
6b	1.756	2.077	−0.321	0.785	2.125
7b	1.792	1.825	−0.032	0.079	1.826
5c	1.881	1.163	0.718	1.756	0.958
9b	1.898	1.698	0.2	0.489	1.663
5a	1.919	1.539	0.38	0.93	1.457
9c	1.934	2.015	−0.081	0.197	2.023
7c	1.964	1.941	0.023	0.056	1.94
9d	2.114	2.078	0.036	0.089	2.075
7d	2.283	2.081	0.202	0.494	2.067
6a	2.439	2.621	−0.182	0.445	2.685
7e	2.452	2.307	0.144	0.353	2.287
3b	2.676	3.095	−0.42	1.026	3.135
3e	2.777	3.127	−0.35	0.855	3.159
7a	2.78	2.357	0.424	1.036	2.275
3c	2.787	3.145	−0.359	0.877	3.184
3d	2.851	3.091	−0.24	0.587	3.114
3g	2.884	3.145	−0.261	0.639	3.174
4a	2.884	2.904	−0.02	0.05	2.907
4g	2.926	3.053	−0.127	0.31	3.067
6d	2.98	2.314	0.666	1.63	2.211
4e	3.182	3.041	0.142	0.347	3.021
3f	3.518	3.095	0.423	1.033	3.056
3a	3.562	3.006	0.557	1.361	2.96
4b	3.703	2.999	0.704	1.721	2.918
4d	3.78	2.995	0.785	1.92	2.907

Considering the formerly mentioned internal and external validation and statistical analysis of the calculated descriptors of both hCA IX and XII 2D-QSAR models, it was concluded that they showed sufficient predictive capability of the inhibitory activity with minimal error.

4. Conclusion

From our current study we can conclude that isatin N-phenylacetamide based sulphonamides could be considered as promising scaffold for future research to develop potent and selective carbonic anhydrase inhibitors along with anti-proliferative activity. It was observed that 2,3-dioxoindole 2h, hydrazinyl benzenesulfonamides 3c and 3d were relatively the most potent ones. Derivative 2h showed promising potency against all tested isoforms with the best inhibitory activity towards hCAI and hCA II (K_I = 45.10 and 5.87 nM, respectively) more than that of acetazolamide (AZZ) (250 and 12 nM, respectively). Moreover, compounds 3c and 3d showed good cytotoxicity where compound 3c revealed a promising selectivity towards human lung adenocarcinoma A549 (SI= 16.94). Docking simulation of the most active ones 2h and 3c predicted their binding pattern and binding affinity to the hCA II, IV, IX and XII active sites and rationalising their selectivity based on their docking binding patterns and scores. Furthermore, 2D-QSAR developed models using training set of the published data of our previous work of benzenesulfonamide derivatives as hCA IX and XII inhibitors showed sufficient predictive capability of the inhibitory activity with minimal error.

Disclosure statement

No potential conflict of interest was reported by all authors. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The carbonic anhydrase inhibition assay in this research was financed by the Italian Ministry for Education and Science (MIUR), grant PRIN: rot. 2017XYBP2R and by Ente Cassa di Risparmio di Firenze (ECRF), grant CRF2020.1395 to CTS.