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Discovery of novel isatin-based sulfonamides with potent and selective inhibition of the tumor-associated carbonic anhydrase isoforms IX and XII

Özlen Güzel-Akdemir a, Atilla Akdemir *b, Nilgün Karalı a and Claudiu T. Supuran *c
aIstanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
bBezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093, Fatih, Istanbul, Turkey. E-mail: aakdemir@bezmialem.edu.tr
cUniversita degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019, Sesto Fiorentino (Florence), Italy. E-mail: claudiu.supuran@unifi.it

Received 7th April 2015 , Accepted 27th April 2015

First published on 27th April 2015


A series of 2/3/4-[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamides, obtained from substituted isatins and 2-, 3- or 4-aminobenzenesulfonamide, showed low nanomolar inhibitory activity against the tumor associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII – recently validated antitumor drug targets, being much less effective as inhibitors of the off-target cytosolic isoforms CA I and II.


Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes with catalytic versatility, having as substrates CO2, COS, CS2, cyanamide, carboxylic, phosphoric and thiocarboxylic esters.1–4 Although their main physiological functions are related to the inter-conversion between CO2, bicarbonate and protons, they play crucial functions in pH regulation, electrolyte secretion, biosynthetic reactions, and carcinogenesis, among others.5–8 Of the 15 isoforms described in humans (hCA I–XIV), many are drug targets for pharmacological agents such as diuretics,9 antiglaucoma drugs,10 antiepileptics,11 antiobesity12 and ultimately, antitumor drugs/cancer diagnostic agents.13 The transmembrane isoforms CA IX and XII are overexpressed in hypoxic tumors as a consequence of the HIF-1α (hypoxia inducible factor-1α) activation pathway, and their inhibition by small molecules/antibodies was recently shown to lead to significant antitumor action.13,14 Furthermore, as these enzymes are present in normal tissues in a very small concentration compared to the tumors, they can also be used for imaging hypoxic tumors.15 Recently, a sulfonamide CA inhibitor (CAI) targeting CA IX and XII entered phase I clinical trials for the treatment of advanced metastatic solid tumors.16

Results and discussion

Chemistry

Sulfonamides (RSO2NH2) constitute the most important and investigated class of CAIs.17

A large number of structurally diverse sulfonamides were investigated for their CA inhibitory properties.17,18 However the main problem with sulfonamides is their promiscuous behavior as strong inhibitors of many of the 15 CA isoforms of human (h) origin.17,18 As isoforms hCA I and II are widespread and play important physiological functions,1–3 it is of great interest to design inhibitors targeting the tumor-associated isoforms hCA IX and XII, which, at the same time, show weak affinity for the off-target isoforms hCA I and II. Some Schiff bases incorporating sulfonamide moieties were among the first types of CAIs showing selective inhibition of some CA isoforms of interest for medicinal chemistry applications,19,20 and this is the reason why we explore here these types of compounds which incorporate substituted isatin moieties (Scheme 1). Reaction of isatins with aromatic sulfonamides was in fact investigated earlier by our and other groups,21–25 and a limited number of such compounds have been reported. Here we extend the previous studies, reporting a series of 23 such derivatives which incorporate orthanilamide, metanilamide or sulfanilamide moieties, as well as isatin or N-methyl-isatins substituted with methyl, halogens, nitro or trifluoromethyloxy moieties at the heterocyclic ring. We have chosen these substitution patterns at the isatin fragment of the molecule in order to investigate the structure–activity relationship (SAR) for the inhibition of four CA isoforms (hCA I, II, IX and XII) with this class of derivatives.26


image file: c5ob00688k-s1.tif
Scheme 1 Preparation of 2/3/4-[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamides.

Enzyme inhibition data

hCA I was inhibited moderately by the reported compounds irrespective of the substitution pattern, with KIs ranging between 146 and 816 nM. The same was true for the cytosolic dominant isoform hCA II; for which the inhibition constants were in the range of 101–728 nM (Table 1). All KI values were in the lower nanomolar region, in a narrow range, for both tumor-associated hCA isozymes (hCA IX: 1.0–15.6 nM; hCA XII: 2.8–53.8 nM; Table 1). The KI values for the widespread hCA I/II were significantly larger (hCA I: 146–816 nM; hCA II: 101–728 nM) and thus, the new compounds showed a discrete selectivity for the tumor-associated isozymes (Table 1). The difference in KI values is relatively small for the tumor-associated isozymes (hCA IX: ∼15-fold; hCA XII: ∼19-fold) and a conclusive SAR analysis is difficult to perform. Compound 4b drew our attention since it shows very low KI values for the tumor-associated isozymes (hCA IX: 1.1 nM; hCA XII: 3.3 nM) and it shows the highest selectivity for the tumor-associated isozymes compared to the widely distributed hCA I and II (Table 1).
Table 1 hCA I, II, IX and XII inhibition data of compounds 3, 4 and 5 by a stopped-flow CO2 hydrase assay26
Compounds K I (nM) Selectivity ratio
# R1 R2 I II IX XII I/IX II/IX I/XII II/XII
3a H H 816 728 9.3 53.8 88 78 15 14
3b CH3 H 600 711 4.2 49.7 143 169 12 14
3c F H 652 428 1.2 41.3 543 357 16 10
3d Cl H 778 652 5.7 52.5 136 114 15 12
3e OCF3 H 742 683 10.3 53.0 72 66 14 13
4a H H 579 618 8.4 43.9 69 74 13 14
4b CH3 H 510 565 1.1 3.3 464 514 155 171
4c F H 426 264 1.3 30.8 328 203 14 9
4d Cl H 490 547 4.8 41.1 102 114 12 13
4e OCF3 H 539 484 9.8 36.5 55 49 15 13
4f NO2 H 378 250 5.7 3.1 66 44 122 81
5a H H 422 523 6.5 29.3 65 80 14 18
5b H CH3 586 549 5.9 6.7 99 93 87 82
5c CH3 H 414 454 1.0 18.5 414 454 22 25
5d CH3 CH3 536 491 4.1 39.7 131 120 14 12
5e F H 375 468 4.0 32.5 94 117 12 14
5f F CH3 259 432 1.6 36.4 162 270 7 12
5g Cl H 249 309 3.3 25.2 75 94 10 12
5h Cl CH3 368 462 4.7 44.6 78 98 8 10
5i Br CH3 457 514 15.6 37.8 29 33 12 14
5j OCF3 H 295 236 3.9 3.8 76 61 78 62
5k NO2 H 229 164 4.9 2.8 47 33 82 59
5l NO2 CH3 146 101 4.2 4.3 35 24 34 23
AZ 250 12 25.0 5.7 10 0 44 2


Molecular modelling studies

Compound 4b has one of the lowest measured KI values for hCA IX and shows the highest selectivity towards hCA IX compared to the other isozymes (Table 1). Molecular modelling studies were applied to suggest a rationale for this selectivity. Available crystal structures of hCA isozymes with sulfonamide-containing ligands such as acetazolamide bound to their active site indicate that the sulfonamide moiety is oriented in a very similar way to the Zn2+-ion of the hCA active sites. The nitrogen atom of the SO2NH group is coordinated to the Zn2+-ion and forms a hydrogen bond with the side-chain of Thr199, whereas one of the sulfonamide oxygen atoms forms a hydrogen bond with the backbone NH of the same residue. A similar orientation and binding-interactions were enforced upon the ligands in our docking studies.
Docking studies on hCA IX. The docked pose of compound 4b in the active site of hCA IX reveals that the vicinal nitrogen and carbonyl group of the indole ring form hydrogen bonds with the side-chains of Gln67 and Gln92, respectively (Fig. 1). The other analogs with sulfonamide groups on the meta position of the phenyl ring (compound series 4) adopted similar docked poses and the range of KI values was 1.1–9.8 nM. The various substituents on the isatin ring did not form any additional interactions except for compound 4f, which has a NO2 group capable of forming a hydrogen bond with Trp5.
image file: c5ob00688k-f1.tif
Fig. 1 The docked poses of compounds 4b (magenta) and 5c (green) in the active site of hCA IX. Interactions of the sulfonamide group with the protein are not shown for clarity. Hydrogen bonds are indicated with red dashed lines. The Zn2+-ion is indicated with a turquoise sphere.

Compound 5c is very similar to 4b, except for the fact that the sulfonamide is substituted on the para position instead of the meta position. This reorients the isatin fragment to form hydrogen bonds with Gln92 via the imine group between the 2-indolinone and the phenyl ring (Fig. 1). In addition, hydrophobic interactions were observed between the isatin moiety and the sidechain of Val131. The analogs with a sulfonamide in the para position (compound series 5) showed a similar docked pose. Their range of KI values is 1.0–15.6 nM and the varying substituents do not form additional interactions with the active site, as they point towards the solvent.

Differences in active sites between hCA IX and hCA XII. Gln67 and Gln92 are involved in hydrogen bonding to compound 4b and are believed to be responsible for the low KI value observed for this compound. Gln67 is not conserved amongst the other hCA isozymes (Table 2). hCA XII has a Lys67 instead of the Gln67 of hCA IX and the ligand cannot form the same interactions as observed in Fig. 1. Gln92 is conserved in both structures and the backbone is located at a similar position, but the sidechain conformation is slightly different. As such, no hydrogen bond is observed in the docking, but it should be possible after sidechain reorientation.
Table 2 The differences and similarities in the active site lining of the investigated hCA isozymes
hCA IX hCA XII hCA I hCA II
a This residue is conserved but has a different orientation in at least one crystal structure.
Trp5a Trp5 Trp5 Trp5
Gln67 Lys67 His67 Asn67
Gln92a Gln92 Gln92 Gln92
Val131 Ala131 Leu131 Phe131
Thr199 Thr199 His200 Thr199


Differences in active sites between hCA IX and hCA I. The bulky His67 is present in hCA I instead of the Gln67 of hCA IX (Table 2). In addition, Val131 and Thr200 of hCA IX are replaced by the larger Leu131 and His200 in hCA I (Table 2). The presence of His200 forces a reorientation of Trp5, which enters the active site more deeply and sterically interferes with the docked ligands. These changes in the binding site do not allow for the adoption of similar poses as observed in Fig. 1.
Differences in active sites between hCA IX and hCA II. Asn67 and Phe131 are present in hCA II (Table 2). Asn67 is shorter than its Gln67 counterpart observed in hCA IX, while Phe131 points into the active site to a larger degree compared to Val131.

Experimental

Synthetic procedures

Melting points were estimated with a Buchi 540 melting point apparatus in open capillaries and are uncorrected. Elemental analyses were performed on a Thermo Finnigan Flash EA 1112 elemental analyzer. IR spectra were recorded on KBr discs, using a Perkin-Elmer Model 1600 FT-IR spectrometer. 1H-NMR, D2O-exch., HSQC and HMBC spectra were obtained on VarianUNITY INOVA 500 and Bruker Avance DPX 400 spectrophotometers using DMSO-d6.
Synthesis of 2/3/4-[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamides (3a–e, 4a–f, 5a–l). Equimolar quantities of 1H-indole-2,3-diones (1) (0.01 mol) and 2-aminobenzenesulfonamide/3-aminobenzenesulfonamide/4-aminobenzenesulfonamide (2) were refluxed in glacial acetic acid (10 ml) for 6 h. The reaction mixture was allowed to stand for 24 h at room temperature. The product was filtered and recrystallized from ethanol.21
2-[(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (3a). Yellow powder, yield 66%; m.p. 249–250 °C; IR (KBr) (ν, cm−1): 3291, 3180 (NH), 1735 (C[double bond, length as m-dash]O), 1337, 1151 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.68–8.25 (10H, m, Ar–H, SO2NH2), 10.58, 10.96 (1H, 2s, indole NH). Anal. Calcd for C14H11N3O3S (301.32): C, 55.80, H, 3.68; N, 13.95; S, 10.64. Found: C, 55.51; H, 3.94; N, 13.84; S, 10.50.
2-[(5-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (3b). Orange powder, yield 24%; m.p. 235–236 °C; IR (KBr) (ν, cm−1): 3274, 3192 (NH), 1732 (C[double bond, length as m-dash]O), 1327, 1160 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 1.94, 2.29 (3H, 2s, 5-CH3), 6.09–7.65 (6H, m, Ar–H), 6.94, 7.03 (2H, 2s, SO2NH2), 7.80, 7.95 (1H, 2d, J = 7.81 Hz, phenyl C6-H), 10.68, 10.86 (1H, 2s, indole NH). Anal. Calcd for C15H13N3O3S (315.34): C, 57.13, H, 4.16; N, 13.33; S, 10.17. Found: C, 57.55; H, 4.44; N, 13.14; S, 9.90.
2-[(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (3c). Orange powder, yield 85%; m.p. 269–271 °C; IR (KBr) (ν, cm−1): 3290, 3187 (NH), 1731 (C[double bond, length as m-dash]O), 1338, 1168 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.81 (1H, d, J = 7.32 Hz, phenyl C3–H), 6.83 (1H, t, J = 7.81 Hz, phenyl C5–H), 6.86 (1H, dd, J = 8.29, 4.39 Hz, indole C7–H), 7.15 (1H, dt, J = 9.26, 2.93 Hz, indole C6–H), 7.32–7.36 (2H, m, indole C4–H, phenyl C4–H), 7.51 (1H, dd, J = 7.81, 0.98 Hz, phenyl C6–H), 7.64, 8.36 (2H, 2s, SO2NH2, D2O exch.), 10.64 (1H, s, indole NH, D2O exch.). HSQC (DMSO-d6) δ (ppm): 111.73 (d, J = 7.66 Hz, indole C7), 114.36 (d, J = 25.87 Hz, indole C4), 117.07 (phenyl C5), 117.53 (d, J = 23.48 Hz, indole C6), 118.29 (phenyl C3), 123.42 (phenyl C6), 123.95 (phenyl C1), 130.98 (d, J = 8.15 Hz, indole C3a), 133.81 (phenyl C4), 138.10 (indole C7a), 138.11 (indole C3), 143.81 (phenyl C2), 158.50 (d, J = 237.71, indole C5), 174.00 (indole C2). Anal. Calcd for C14H10FN3O3S (319.31): C, 52.66, H, 3.16; N, 13.16; S, 10.04. Found: C, 52.52; H, 3.13; N, 13.12; S, 10.11.
2-[(5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (3d). Orange powder, yield 40%; m.p. 248–250 °C; IR (KBr) (ν, cm−1): 3266, 3197 (NH), 1731 (C[double bond, length as m-dash]O), 1328, 1156 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.82 (1H, t, J = 7.81 Hz, phenyl C5-H), 6.89 (1H, d, J = 8.29 Hz, indole C7–H), 7.35 (1H, dd, J = 7.81, 1.46 Hz, phenyl C3–H), 7.36 (1H, dd, J = 8.29, 2.44 Hz, indole C6–H), 6.86, 8.41 (2H, 2s, SO2NH2, D2O exch.), 7.52 (1H, dd, J = 7.81, 2.44 Hz, phenyl C4–H), 7.54 (1H, d, J = 2.44 Hz, phenyl C6–H), 7.64 (1H, s, indole C4–H), 10.75 (1H, s, indole NH, D2O exch.). Anal. Calcd for C14H10ClN3O3S (335.76): C, 50.08, H, 3.00; N, 12.51; S, 9.55. Found: C, 49.77; H, 3.12; N, 12.35; S, 9.48.
2-[(2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (3e). Orange powder, yield 30%; m.p. 261–263 °C; IR (KBr) (ν, cm−1): 3289, 3187 (NH), 1733 (C[double bond, length as m-dash]O), 1339, 1163 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.82 (1H, d, J = 8.29 Hz, indole C7–H), 6.86 (1H, d, J = 7.81 Hz, phenyl C3–H), 6.95 (1H, d, J = 8.29 Hz, indole C6–H), 7.32 (1H, dd, J = 8.29, 1.95 Hz, indole C4–H), 7.36 (1H, dd, J = 7.81, 1.46 Hz, phenyl C5–H), 7.52 (2H, dd, J = 7.81, 1.46 Hz, phenyl C4,6–H), 7.66, 8.46 (2H, 2s, SO2NH2), 10.81 (1H, s, indole NH). HMBC (DMSO-d6) δ (ppm): 111.86 (indole C7), 117.10 (phenyl C5), 118.41 (indole C4), 119.49 (OCF3), 120.57 (indole C6), 123.37 (phenyl C3), 124.07 (phenyl C6), 124.46 (phenyl C1), 130.99 (indole C3a), 132.69 (indole C5), 133.86 (phenyl C4), 141.09 (indole C3), 143.73 (phenyl C2), 143.85 (indole C7a), 174.05 (indole C2). Anal. Calcd for C15H10F3N3O4S (385.32): C, 46.76, H, 2.62; N, 10.91; S, 8.32. Found: C, 46.44; H, 2.84; N, 10.56; S, 8.32.
3-[(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4a). Yellow powder, yield 24%; m.p. 263–264 °C; IR (KBr) (ν, cm−1): 3361, 3259 (NH), 1725, 1746 (C[double bond, length as m-dash]O), 1330, 1147 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.32–7.69 (8H, m, Ar–H), 7.33, 7.41 (2H, 2s, SO2NH2), 10.88, 10.98 (1H, 2s, indole NH). Anal. Calcd for C14H11N3O3S (301.32): C, 55.80, H, 3.68; N, 13.95; S, 10.64. Found: C, 55.89; H, 4.06; N, 13.64; S, 10.65.
3-[(5-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4b). Orange powder, yield 32%; m.p. 243–245 °C; IR (KBr) (ν, cm−1): 3269, 3175 (NH), 1731 (C[double bond, length as m-dash]O), 1326, 1154 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 1.95, 2.24 (3H, 2s, 5-CH3), 6.16–7.70 (7H, m, Ar–H), 7.33, 7.43 (2H, 2s, SO2NH2), 10.77, 10.88 (1H, 2s, indole NH). Anal. Calcd for C15H13N3O3S (315.34): C, 57.13, H, 4.16; N, 13.33; S, 10.17. Found: C, 56.83; H, 4.14; N, 12.82; S, 10.01.
3-[(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4c). Orange crystals, yield 43%; m.p. 247–250 °C; IR (KBr) (ν, cm−1): 3274, 3180 (NH), 1731 (C[double bond, length as m-dash]O), 1329, 1156 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 5.97–7.33 (4H, m, indole C4,6–H, phenyl C2,4–H), 6.90, 6.92 (1H, 2dd, J = 8.30, 4.39 Hz, indole C7–H), 7.34, 7.44 (2H, 2s, SO2NH2), 7.48, 7.66 (1H, 2t, J = 7.80 Hz, phenyl C5–H), 7.56, 7.71 (1H, 2d, J = 7.80 Hz, phenyl C6–H), 10.90, 11.02 (1H, 2s, indole NH). Anal. Calcd for C14H10FN3O3S (319.31): C, 52.66, H, 3.16; N, 13.16; S, 10.04. Found: C, 52.29; H, 3.58; N, 13.18; S, 10.38.
3-[(5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4d). Orange powder, yield 43%; m.p. 252–254 °C; IR (KBr) (ν, cm−1): 3284, 3173 (NH), 1733 (C[double bond, length as m-dash]O), 1325, 1148 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.87–7.73 (7H, m, Ar–H), 7.35, 7.46 (2H, 2s, SO2NH2), 11.10, 11.12 (1H, 2s, indole NH). Anal. Calcd for C14H10ClN3O3S (335.76): C, 50.08, H, 3.00; N, 12.51; S, 9.55. Found: C, 49.96; H, 3.01; N, 12.32; S, 9.79.
3-[(2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4e). Orange powder, yield 25%; m.p. 205–207 °C; IR (KBr) (ν, cm−1): 3338, 3282 (NH), 1667, 1733 (C[double bond, length as m-dash]O), 1327, 1150 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 7.31 (2H, s, SO2NH2), 7.42–7.71 (6H, m, Ar–H), 8.13 (1H, s, indole C4–H), 10.19 (1H, s, indole NH). Anal. Calcd for C15H10F3N3O4S (385.32): C, 46.76, H, 2.62; N, 10.91; S, 8.32. Found: C, 46.97; H, 2.32; N, 10.76; S, 8.27.
3-[(5-Nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (4f). Orange powder, yield 30%; m.p. 175–178 °C; IR (KBr) (ν, cm−1): 3311, 3262 (NH), 1748 (C[double bond, length as m-dash]O), 1307, 1154 (S[double bond, length as m-dash]O). 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.31, 7.42 (2H, 2s, SO2NH2), 6.97–8.65 (7H, m, Ar–H), 10.19, 11.26 (1H, 2s, indole NH). Anal. Calcd for C14H10N4O5S (346.32): C, 48.55, H, 2.91; N, 16.18; S, 9.26. Found: C, 48.74; H, 3.04; N, 16.18; S, 9.46.
4-[(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5a)22. Yellow powder, yield 68%; m.p. 278–279 °C; IR (KBr) (ν, cm−1): 3324, 3233 (NH), 1723, 1750 (C[double bond, length as m-dash]O), 1329, 1144 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.33, 7.60 (1H, 2d, J = 7.81 Hz, indole C7–H), 6.73, 7.06 (1H, 2t, J = 7.81 Hz, indole C6–H), 6.86, 6.89 (1H, 2d, J = 7.81 Hz, indole C4–H), 7.08, 7.15 (2H, 2d, J = 8.30 Hz, phenyl C3,5–H), 7.28, 7.35 (2H, 2s, SO2NH2), 7.35, 7.46 (1H, 2t, J = 7.81 Hz, indole C5–H), 7.73, 7.89 (2H, 2d, J = 8.30 Hz, phenyl C2,6–H), 10.89, 11.00 (1H, 2s, indole NH). Anal. Calcd for C14H11N3O3S (301.32): C, 55.80; H, 3.68; N, 13.95; S, 10.64. Found: C, 56.05; H, 4.05; N, 13.86; S, 10.53.
4-[(1-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5b)23. Orange powder, yield 62%; m.p. 252–253 °C; IR (KBr) (ν, cm−1): 3328, 3213 (NH), 1725 (C[double bond, length as m-dash]O), 1321, 1154 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.07, 3.19 (3H, 2s, 1–CH3), 6.36, 7.64 (1H, 2d, J = 7.81 Hz, indole C7–H), 6.80–7.16 (4H, m, indole C4,6–H, phenyl C3,5–H), 7.30, 7.36 (2H, 2s, SO2NH2), 7.46, 7.56 (1H, 2t, J = 7.81 Hz, indole C5–H), 7.74, 7.90 (2H, 2d, J = 8.29 Hz, phenyl C2,6–H). Anal. Calcd for C15H13N3O3S (315.34): C, 57.13, H, 4.16; N, 13.33; S, 10.17. Found: C, 57.37; H, 4.57; N, 13.20; S, 10.34.
4-[(5-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5c)24. Orange powder, yield 58%; m.p. 265–266 °C; IR (KBr) (ν, cm−1): 3323, 3232 (NH), 1723, 1751 (C[double bond, length as m-dash]O), 1320, 1147 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 2.24, 2.27 (3H, 2s, 5-CH3), 5.77, 6.11 (1H, 2s, indole C4–H), 6.57, 6.79 (2H, 2d, J = 8.79 Hz, phenyl C3,5–H), 6.85, 7.36 (2H, 2s, SO2NH2, D2O exch.), 7.07, 7.29 (1H, 2d, J = 8.29 Hz, indole C6–H), 7.14, 7.28 (1H, 2d, J = 8.29 Hz, indole C7–H), 7.38, 7.43 (2H, 2d, J = 8.79 Hz, phenyl C2,6–H), 10.78, 10.89 (1H, 2s, indole NH, D2O exch.). Anal. Calcd for C15H13N3O3S (315.34): C, 57.13, H, 4.16; N, 13.33; S, 10.17. Found: C, 56.89; H, 4.14; N, 12.95; S, 10.53.
4-[(1,5-Dimethyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5d). Orange powder, yield 20%; m.p. 223–225 °C; IR (KBr) (ν, cm−1): 3325, 3235 (NH), 1723, 1753 (C[double bond, length as m-dash]O), 1328, 1147 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 1.99, 2.31 (3H, 2s, 5-CH3), 3.04, 3.17 (3H, 2s, 1-CH3), 6.14, 7.47 (1H, 2s, indole C4–H), 6.98, 7.07 (1H, 2d, J = 8.30 Hz, indole C6–H), 7.01, 7.14 (2H, 2d, J = 8.30 Hz, phenyl C3,5–H), 7.18, 7.28 (1H, 2d, J = 8.30 Hz, indole C7–H), 7.29, 7.37 (2H, 2s, SO2NH2), 7.73, 7.89 (2H, 2d, J = 8.30 Hz, phenyl C2,6–H). Anal. Calcd for C16H15N3O3S (329.37): C, 58.34, H, 4.59; N, 12.76; S, 9.74. Found: C, 58.01; H, 4.90; N, 12.34; S, 9.65.
4-[(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5e). Orange powder, yield 20%; m.p. 275–277 °C; IR (KBr) (ν, cm−1): (C[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.08, 7.52 (1H, 2dd, J = 7.81, 4.32 Hz, indole C7–H), 6.95, 6.99 (1H, 2dd, J = 8.78, 2.44 Hz, indole C6–H), 7.18, 7.25 (2H, 2d, J = 8.30 Hz, phenyl C3,5–H), 7.34, 7.40 (1H, 2dd, J = 8.78, 2.44 Hz, indole C4–H), 7.37, 7.46 (2H, 2s, SO2NH2), 7.81, 7.98 (2H, 2d, J = 8.30 Hz, phenyl C2,6–H), 10.97, 11.11 (1H, 2s, indole NH). Anal. Calcd for C14H10FN3O3S (319.31): C, 52.66, H, 3.16; N, 13.16; S, 10.04. Found: C, 52.99; H, 3.17; N, 12.90; S, 9.85.
4-[(5-Fluoro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5f). Orange powder, yield 44%; m.p. 247–249 °C; IR (KBr) (ν, cm−1): 3327, 3225 (NH), 1734 (C[double bond, length as m-dash]O), 1322, 1155 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.06, 3.19 (3H, 2s, 1-CH3), 6.03–7.52 (5H, m, indole C4,6,7–H, phenyl C3,5), 7.31, 7.40 (2H, 2s, SO2NH2), 7.75, 7.92 (2H, 2d, J = 8.29 Hz, phenyl C2,6–H). Anal. Calcd for C15H12FN3O3S (333.33): C, 54.05, H, 3.63; N, 12.61; S, 9.62. Found: C, 54.06; H, 3.71; N, 12.46; S, 9.70.
4-[(5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5g)25. Yellow powder, yield 61%; m.p. 271–272 °C; IR (KBr) (ν, cm−1): 3330, 3259 (NH), 1723, 1745 (C[double bond, length as m-dash]O), 1328, 1151 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 6.28–7.50 (3H, m, indole C4,6,7–H), 7.11, 7.17 (2H, 2dd, J = 6.83, 1.95 Hz, phenyl C3,5–H), 7.29, 7.40 (2H, 2s, SO2NH2), 7.74, 7.91 (2H, 2dd, J = 6.83, 1.95 Hz, phenyl C2,6–H), 11.03, 11.14 (1H, 2s, indole NH). Anal. Calcd for C14H10ClN3O3S (335.76): C, 50.08, H, 3.00; N, 12.51; S, 9.55. Found: C, 49.96; H, 3.24; N, 12.48; S, 9.20.
4-[(5-Chloro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5h). Orange crystals, yield 40%; m.p. 221–223 °C; IR (KBr) (ν, cm −1): 3341, 3235 (NH), 1678, 1727 (C[double bond, length as m-dash]O), 1332, 1157 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.07, 3.20 (3H, 2s, 1-CH3), 6.30–7.62 (5H, m, indole C4,6,7–H, phenyl C3,5–H), 7.30, 7.41 (2H, 2s, SO2NH2), 7.75, 7.92 (2H, 2dd, J = 8.78, 1.95 Hz, phenyl C2,6–H). Anal. Calcd for C15H12ClN3O3S (349.79): C, 51.51, H, 3.46; N, 12.01; S, 9.17. Found: C, 49.05; H, 3.93; N, 11.56; S, 9.53.
4-[(5-Bromo-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5i). Orange crystals, yield 16%; m.p. 235–237 °C; IR (KBr) (ν, cm−1): 3336, 3234 (NH), 1676, 1729 (C[double bond, length as m-dash]O), 1331, 1156 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.19 (3H, s, 1-CH3), 7.11 (1H, 2dd, J = 8.78, 3.42 Hz, indole C6–H), 7.17 (1H, d, J = 8.78 Hz, indole C7–H), 7.30 (1H, s, indole C4–H), 7.41 (2H, s, SO2NH2), 7.75 (2H, d, J = 8.30 Hz, phenyl C3,5–H), 7.92 (2H, d, J = 8.30 Hz, phenyl C2,6–H). Anal. Calcd for C15H12BrN3O3S (394.24): C, 45.70, H, 3.07; N, 10.66; S, 8.13. Found: C, 45.72; H, 3.24; N, 10.96; S, 8.17.
4-[(2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5j). Yellow powder, yield 20%; m.p. 193–195 °C; IR (KBr) (ν, cm−1): 3370, 3289, 3212 (NH), 1736 (C[double bond, length as m-dash]O), 1331, 1158 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.19 (2H, s, SO2NH2), 7.65 (4H, s, Ar–H), 6.55–7.89 (3H, m, indole C4,6,7–H), 10.24 (1H, s, indole NH). Anal. Calcd for C15H10F3N3O4S (385.32): C, 46.76, H, 2.62; N, 10.91; S, 8.32. Found: C, 46.66; H, 2.85; N, 10.78; S, 8.50.
4-[(5-Nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5k)25. Orange powder, yield 15%; m.p. 257–259 °C; IR (KBr) (ν, cm−1): 3339, 3263 (NH), 1752 (C[double bond, length as m-dash]O), 1335, 1153 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.32, 7.43 (2H, 2s, SO2NH2), 7.05–8.37 (7H, m, Ar–H), 11.59, 11.70 (1H, 2s, indole NH). Anal. Calcd for C14H10N4O5S (346.32): C, 48.55, H, 2.91; N, 16.18; S, 9.26. Found: C, 48.56; H, 3.28; N, 16.27; S, 9.56.
4-[(1-Methyl-5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)amino]benzenesulfonamide (5l). Yellow powder, yield 27%; m.p. 230–232 °C; IR (KBr) (ν, cm−1): 3312, 3241 (NH), 1678, 1741 (C[double bond, length as m-dash]O), 1339, 1157 (S[double bond, length as m-dash]O); 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.16, 3.20 (3H, 2s, 1-CH3), 7.17–8.47 (9H, m, SO2NH2 and Ar–H). Anal. Calcd for C15H10N4O5S (360.34): C, 50.00, H, 3.36; N, 15.55; S, 8.90. Found: C, 50.48; H, 3.84; N, 15.53; S, 9.03.

Enzyme inhibition assay

A stopped-flow instrument (SX.18MV-R Applied Photophysics model) was used for assaying the CA-catalyzed CO2 hydration activity.26 Inhibitor and enzyme were preincubated for 15 min for allowing the complete formation of the enzyme–inhibitor adduct. IC50 values were obtained from dose response curves working at seven different concentrations of the test compound (from 0.1 nM to 50 μM), by fitting the curves using PRISM (http://www.graphpad.com) and non-linear least squares methods, the obtained values representing the mean of at least three different determinations. The inhibition constants (KI) were derived from the IC50 values by using the Cheng–Prusoff equation, as follows: KI = IC50/(1 + [S]/Km) where [S] represents the CO2 concentration at which the measurement was carried out, and Km the concentration of the substrate at which the enzyme activity is at half maximal. All enzymes used were recombinant, produced in E. coli as reported earlier.27–30 The concentrations of enzymes used in the assay were: hCA I, 12.4 nM; hCA II, 8.7 nM; hCA IX, 9.2 nM and hCA XII, 10.8 nM.

Molecular modelling studies

Preparation of ligand structures. The isatin structures 3, 4 and 5 were prepared in 3D with the MOE software package (v2013.08.02, Chemical Computing Group Inc., Montreal, Canada) and the ligands were energy minimized using a steepest-descent protocol (MMFF94x force field).
Preparation of hCA crystal structures for docking studies. The structures of hCA I (PDB: 3LXE, 1.90 Å), hCA II (PDB: 4E3D, 1.60 Å), hCA IX (PDB: 3IAI; 2.20 Å) and hCA XII (PDB: 1JD0; 1.50 Å) were obtained from the protein databank. The protein atoms and the active site zinc ions were retained and all other atoms were omitted. The remaining structure was protonated using the MOE software package and subsequently the obtained structure was energy-minimized (AMBER99 force field). Finally, the obtained protein models were superposed on the hCA I structure using the backbone Cα-atoms and all Zn2+-ions, zinc-binding histidines and the overall backbone atoms superposed well (RMSD value: 1.281 Å).
Docking of the compounds into the hCA structures. The GOLD Suite software package (v5.2, CCDC, Cambridge, UK) and the ChemScore scoring function were used to dock the compounds into the hCA structures (50 dockings per ligand). The binding pocket was defined as all residues within 13 Å of a centroid (x: −17.071, y: 35.081, 43.681; corresponding approximately to the position of the thiadiazole ring of acetazolamide in the 1JD0 structure). Position restraints were applied to the sulfur and nitrogen atoms of the acetazolamide sulfonamide tail of hCA XII (default settings) and were also applied to the other three hCA structures due to the low RMSD value of the superpositions.

Conclusions

We report here a panel of 23 new sulfonamides incorporating Schiff base moieties. They were obtained by reactions of variously substituted isatins with 2-, 3- and 4-amino-benzenesulfonamides. These new derivatives were tested as inhibitors of four physiologically relevant CA isoforms, involved in crucial physiological and pathological processes: the house-keeping cytosolic hCA I and II, as well as the transmembrane, tumor-associated hCA IX and XII, validated drug targets for theranostics for the management of hypoxic tumors. The new sulfonamides were moderate–weak hCA I/II inhibitors and highly potent, low nanomolar hCA IX/XII inhibitors. By using docking studies we also explained the differential inhibition of the four CA isoforms and the structural reasons connected with the selective inhibition of the transmembrane over the cytosolic isoforms. As a sulfonamide CA IX/XII inhibitor recently entered Phase I clinical trials for the management of metastatic solid tumors, compounds of the type reported here may be useful for designing different derivatives with such properties.

Acknowledgements

This project was in part financed by the Istanbul University Scientific Research Projects Department (Project Number: UDP-51361) and by an FP7 EU project (Dynano).

Notes and references

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