Accepted Manuscript N-Acylsulfonamides Strongly Inhibit Human Carbonic Anhydrase Isoenzymes I and II Alper Yıldırım, Ufuk Atmaca, Ali Keskin, Meryem Topal, Murat Çelik, İlhami Gülçin, Claudiu T. Supuran PII: DOI: Reference:

S0968-0896(14)00912-2 http://dx.doi.org/10.1016/j.bmc.2014.12.054 BMC 11986

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

5 September 2014 18 December 2014 22 December 2014

Please cite this article as: Yıldırım, A., Atmaca, U., Keskin, A., Topal, M., Çelik, M., Gülçin, İ., Supuran, C.T., NAcylsulfonamides Strongly Inhibit Human Carbonic Anhydrase Isoenzymes I and II, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.054

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N-Acylsulfonamides Strongly Inhibit Human Carbonic Anhydrase Isoenzymes I and II

Alper Yıldırım1, Ufuk Atmaca1, Ali Keskin1, Meryem Topal2, Murat Çelik1,*, İlhami Gülçin1,3,* Claudiu T. Supuran4

1

Faculty of Science, Department of Chemistry, Atatürk University, 25240-Erzurum, Turkey 2

Gumushane University, Vocational School of Health Services, Department of Medical Services and Techniques, Gumushane, Turkey

3 4

Department of Zoology, College of Science, King Saud University, Riyadh-Saudi Arabia i e i deg i

di di Fi e e, Neurofarba Department, Rm. 66, Via U. Schiff 6, I-50019 Sesto Fiorentino (Firenze), Italy

*Correspondence: Prof. Dr. Murat Çelik, Department of Chemistry, Faculty of Science, Ataturk University, 25240 Erzurum, Turkey E-mail : [email protected] Phones : +90 442 231 4390 : +90 533 396 5218

ABSTRACT

Sulfonamides represent a significant class of biologically active compounds that inhibit carbonic anhydrase (CA, EC.: 4.2.1.1) isoenzymes involved in different pathological and physiological events. Sulfonamide CA inhibitors are used therapeutically as diuretic, antiglaucoma, antiobesity and anticancer agents. A series of new sulfonamides were synthesized using imides and tosyl chloride as starting materials. These N-acylsulfonamides efficiently inhibited the cytosolic human carbonic anhydrase isoenzymes I, and II (hCA I, and II), with nanomolar range inhibition constants ranging between 36.4±6.0-254.6±18.0 and 58.3±0.6-273.3±2.5 nM, respectively. Keywords: Sulfonamide; Imide; N-acylsulfonamide; Carbonic Anhydrase; Enzyme Purification; Enzyme inhibition

1. INTRODUCTION N-Aclysulfonamides have attracted the attention of bioorganic chemists due to their importance in the pharmaceutical industry. Some N-acylsulfonamide derivatives have been used as precursors of he ape ic age

fo A eime ’ di ea e and as antibacterial inhibitors

and anti-proliferative agents.1 Primary sulfonamidees have been known for decades to act as potent carbonic anhydrase enzyme (CA, EC 4.2.1.1) inhibitors,2,3 whereas secondary and tertiary sulfonamides were only recently discovered to possess this biological activity.3,4 Considering a number of recent findings,5-7 in the present study, we investigated whether substituted bicyclic NAcylsulfonamides derivatives 1-5 possess activity against CA isoforms. For this reason we investigated attachment of the sulfonamide moiety to bicyclic ring systems by using the high reactivity of imides towards arylsulfonyl chlorides.8-11 Thus, we thus developed a method for the synthesizing bicyclic N-acylsulfonamides. Preparation of meso-imides (1a-5a), which are key precursors, was performed via cycloaddition of imides with different dienes (Table 1). Table-1 here The Diels-alder [4+2] cyclo addition synthesis method was studied as a first step for achieving the desired product, and in a short time, cyclo addition products were synthesized with high yield. Insert Scheme 1 here The tosyl group was attached to a nitrogen atom in the imide to allow sulfonimide ring opening. The ring-opening reaction was performed using benzyl alcohol. Bicyclic double bonds on ring-opening products were dehydroxylated with the reagent OsO4\NMO. Hydrolysis of the benzyl group was performed using K2CO3/MeOH. Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that catalyse the interconversion of carbon dioxide (CO2) and water (H2O) to bicarbonate (HCO3-) and proton (H+).12-17 CA appears to be almost ubiquitously expressed in living organisms. Thus far, six genetically distinct CA families are known, including he α-, β-, γ-, δ-, δ-, and η-class enzymes.18-21 These CA classes differ in their three-dimensional fold in the protein backbone and in their preference for the metal ion used within the active site for the catalysis process.2226

The mammalian e yme be o g o he α-CA family and consist of sixteen active members

that have different functions, kinetic parameters, inhibitory properties, and cell and tissue localization.19,24-27 The α-CA family is the best-known group, although recent studies and reviews have shown rapid advancement in the knowledge of other CA families.25-29 The α-CA

family participates in several pathological and physiological processes in humans, including pH and CO2 homeostasis, biosynthetic reactions such as gluconeogenesis, respiration, the transport of CO2 and HCO3- between metabolizing tissues, lung electrolyte secretion, ureagenesis, lipogenesis, bone resorption, the production of biological fluids, cell adhesion and proliferation, tumorigenicity, and calcification, and in the growth and virulence of various fungal or bacterial pathogens.21,29-33 A total of 16 isozymes have been previously described as members of the α-CA family, and the α-CA family can be classified according to their subcellular localization. CAs I, II, III, VII, and XIII are cytosolic isoenzymes,21,30,34,35 CAs VA and VB are localized in the mitochondria,36-38 CA VI is a unique secreted isozyme,37,38 CAs IX, XII, and XIV are transmembrane proteins,39,40 and CAs IV and XV are GPI-anchored to the cell membrane.41,42 CA inhibition plays an important role in therapeutic applications as a diuretic and an antiglaucoma, anticonvulsant, and anticancer agent,2,15,43,44 Additionally, CA inhibitors (CAIs) are emerging targets for the design of such as antifungal and antibacterial agents16,45,46. CAIs are a class of pharmaceuticals that suppress the activity of carbonic anhydrase. Sulfonamides, the basis of several groups of drugs, belonging to many structural types, were reported to have significant inhibitory activity against many CA classes, but they were mostly investigated as inhibitors of the mammalian isoforms.16,45 However a critical barrier to the design of CAIs as therapeutic agents is the high number of CA isoforms in humans, their rather diffuse localization in many tissues and organs, and the lack of isoenzyme selectivity of the currently available inhibitors of sulfonamide or sulfamates.47,48 In this present study, we synthesized some N-acylsulfonamide derivatives and determined the inhibition properties of these N-acylsulfonamide derivatives on the CA I, and CA II isoenzymes.

2. RESULTS AND DISCUSSION 2.1. Chemistry Sulfonamides are highly versatile functional synthons. This property has led to comprehensive research into finding new sulfonamide derivatives. Here, we report the first example of a diol containing sulfonamides in good yields (Table 4). The structures of all synthesized compounds were characterized by 1H-, 13C-NMR.

2.2. Biochemistry Enzymes are synthesized by living cells and accelerate up chemical reactions during the metabolism in living organisms.49,50 In contrast, isoenzymes have different amino acid sequences but catalyze the same biochemical reactions. Isoenzymes usually display different kinetic parameters such as IC50 and KM and they possess different regulatory properties.50 The best-known example of an isoenzyme is glucokinase. This isoenzyme is a variant of hexokinase that is not inhibited by glucose 6-phosphate. In most cases, isoenzymes are encoded by homologous genes that have diverged over time. These isoenzymes are different in their affinity for substrates, cofactors and inhibitors. Furthermore, each isoenzyme has different isoelectric point and electrophoretic mobility. CA I, and II isoenzymes, which are examined in this study, have different activities. In mammals, CA II generally exists in red blood cells in a lower concentration than CA I, but with much higher specific CO2 hydration activity. In fact CA II is one of the fastest enzymes known while CA I, which is usually present in red cells at several fold higher concentration than CA II, demonstrates only approximately one-tenth of the activity of CA II.50 In recent decades, the CA isoenzymes have become an interesting target for the design of inhibitors or activators for biomedical applications including treatments for epilepsy, glaucoma, idiopathic intracranial hypertension, and altitude sickness.50-53 With this purpose, in this present study, we have investigated the inhibition effects of the N-acylsulfonamide derivatives (1c-5c and 1d-5d) toward both cytosolic human CA isoenzymes (hCA I and II). The compounds reported here were investigated for the inhibition of two cytosolic human CA isoforms, involved in crucial physiologic processes in mammals, the cytosolic, widespread hCA I and II. Inhibition data with N-acylsulfonamide derivatives (1c-5c and 1d-5d), as well as the sulfonamide in clinical use acetazolamide as standard compound (AZA) are reported in Table 5. Both of these isoenzymes, particularly CA II have proven to be strongly and inhibited by a large spectrum of chemicals, such as aromatic and heterocyclic sulfamides and sulfonamides.

Sulfonamides

and

their

2,18,24,43,54

pharmacologically relevant CAIs.

derivatives

represent

the

main

class

of

The sulfonamide moiety is responsible for a 2+

coordination bond with the zinc ion (Zn ) of CA isoenzymes. It was reported that benzenesulfonamide has interesting isoform-selective CA inhibitory action, and its X-ray crystal structure in complex with hCA II was reported by our group.55,56 Based of the above considerations, we investigated a new class of CAIs based on the N-acylsulfonamide derivatives. In our study, the inhibition properties of some N-acylsulfonamide derivatives (1c-

5c and 1d-5d) were measured for off-target cytosolic hCA I, and II effects by analysis of the hydration activity of CA.21 N-acylsulfonamides of 1d-5d were derived from Nacylsulfonamides 1c-5c. For both N-acylsulfonamide derivative groups, hCA I, and II inhibition constants (Ki) were in the low nanomolar range. All N-acylsulfonamide derivatives (1c-5c and 1d-5d) were found to inhibit hCA I, with Ki values ranging from 36.4±6.0254.6±18.0 nM. Additionally, all N-acylsulfonamides (1c-5c and 1d-5d) inhibited hCA II with Ki values ranging from 61.21±6.13-273.31±2.56 nM. In contrast, AZA, which was used as a positive standard inhibitor for CA isoenzymes, showed less inhibition with Ki values of 184.34 nM for hCA I and 61.12 nM for hCA II. Furthermore, N-acylsulfonamide derivative of 5c and 5d, which have six chlorine atoms and gemind dichloro atoms, were found to be the best inhibitors for both cytosolic isoenzymes. Also, it was demonstrated that there was an impact of the differing acyl groups on key biopharmaceutical properties, confirming that this group protected carbohydrate-based sulfonamides and have potential as prodrugs for selectively targeting extracellular cancer-associated CA enzymes.57 It was also reported that the tumor associated hCA IX was effectively inhibited by new synthesized acetylated derivatives.58 The cytosolic widespread isoforms hCA I, and II were effectively inhibited by compounds 5c and 5d. The inhibition constants (Kis) for compounds 5c and 5d against hCA I were 36.4±6.0 and 51.7±9.0 nM, respectively. These inhibition constants for hCA II were 48.09±3.92 nM and 58.27±0.634 nM, respectively. The presence of benzene or a pyridine in the heterocyclic scaffolds both of leads to effective CAIs. Moreover, the most effective inhibitors were those bearing a methoxy or chlorine group in the inhibitor moieties.16 Because of these properties, the best hCA I, and II inhibitors in this series were the N-acylsulfonamide derivatives of 5c and 5d. We estimate that these N-acylsulfonamide derivatives bind to the enzyme on acyl groups. For this purpose, an example of possible coupling structure of enzyme and acyl groups of newly synthesized N-acylsulfonamide derivatives is shown in Figure 2. Enzymes are only specific to their substrates. The fragmentation effects of the enzymes on another bond or a group are not possible because of enzymatic selectivity and steric hindrance of active side of enzymes. Also, it was reported that when the anionic inhibitor such as hydrogen sulfide (HS−) and bisulfite (HSO3−) lacks a protonated ligand atom, three different binding modes have been observed so far. In addition anions such as bromide (Br−) and azide (N3−) coordinate the Zn2+

with a distorted tetrahedral geometry.59 On the contrary, anions such as formate (HCOO−), which is the simplest carboxylate anion, and thiocyanate (SCN−), which known as rhodanide bind to the CA active site by addition to the metal coordination sphere generating a trigonal bipyramidal species. Finally, a last group of anions, including nitrate (NO3−), which produced by a number of species of nitrifying bacteria, cyanide (CN−) and cyanate (OCN−), seems not to coordinate the zinc ion but to be located in a hydrophobic cavity near the Zn2+ but these studies were not confirmed by others.44,59 Considering that this information, both of Nacylsulfonamide derivatives of 5c and 5d, which are the most active molecules, had six chlorine groups (Cl−). Chlorine (Cl−) groups can coordinate the Zn2+ ions with a distorted tetrahedral geometry like bromide (Br−) and azide (N3−). This coordination, which favor of inhibition properties is inevitable.59

3. CONCLUSIONS In summary, the meso-imides are obtained using addition reaction of Diels-Alder 4+2 addition reaction. The tosyl group, which comprises a sulfur bond and is classified as an electron-withdrawing group, is connected to the imides via nitrogen bonds that open the imides. After contributing to increase the activity, an opening reaction is conducted with diols (Scheme 3). The structures of all synthesized compounds were characterized by 1H-,

13

C-

NMR. N-acylsulfonamide derivatives (1c-5c and 1d-5d) are selective hCA I, and II inhibitors with selectivity ratios in the range of 36.4±6.0-273.31±2.56 nM. Additionally, these findings indicate that novel N-acylsulfonamide derivatives (1c-5c and 1d-5d) may be used as leads for generating potent hCA I, and II isoenzyme inhibitors eventually targeting other isoforms, which have not been assayed yet for their interactions with such agents. In addition to the established role of CAIs, N-acylsulfonamide derivatives (1c-5c and 1d-5d) can be potent candidates for diuretics and antiglaucoma drugs. These compounds have potential for use the treatment of idiopathic intracranial hypertension (glaucoma), epilepsy, and altitude sickness and as anticonvulsant, antiobesity, anticancer, and anti-infective drugs.

4. EXPERIMENTAL All chemicals and solvents are commercially available and were used after purification. Melting points were established in a capillary melting apparatus (BUCHI 530) and are uncorrected. IR spectra were observed from solutions in 0.1 mm cells with a Perkin-Elmer spectrophotometer. The 1H and 13C NMR spectra were recorded with 400 (100)-MHz Varian and 400 (100)-MHz Bruker spectrometers. Elemental analyses were performed with a Leco CHNS-932 apparatus. All column chromatography was performed in silica gel (60-mesh, Merck). PLC is preparative thin-layer chromatography: 1 mm of silica gel 60 PF (Merck) on glass plates.

4.1. General Procedure of [4+2] Diels-Alder Reactions: 4.1.1. Standard procedure for the synthesis of [4+2] Diels-Alder Cycloaddition: (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dionel (1a)

1,3-cyclohexadiene (0.96 mL, 0.01 mol) was added to a solution of maleimide (1 g, 0.01 mol) in diethyl ether (50 mL). The reaction mixture was stirred to room temperature for 18 h. The solvent was evaporated (1.7 g, 93%) (1a) as a white solid.10,11 [4+2] Diels-Alder products 1a-5a were also synthesized by this procedure with yields of 93% (1a), 90% (2a), 98% (3a), 97% (4a) and 98% (5a). White solid. 1H NMR (400 MHz, CDCl3 δ=1.37 (dd, 2H, J=7.3, 1.2 Hz), 1.57 (dd, 2H, J=7.3, 1.1 Hz), 2.86 (s, 2H), 3.12 (d, 2H, J=1.1 Hz) 6.2 (dd, 2H, J=4.5,3.3 Hz), 8.5 (bs, 1H).

13

C

NMR (100 MHz, CDCl3) δ=179.5, 132.6, 45.8, 31.7, 23.7.IR (CH2Cl2, cm-1): 3234, 2962, 2946, 1752, 1706, 1348, 1316, 1187.10

4.1.2. (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione (2a) White solid. 1H NMR (400 MHz, CDCl3) δ=AB system: 1.5 (d, 1H, B part of AB, J=6.7 Hz), AB system: 1.71 (d, 1H, A part of AB, J=8.7 Hz), 3.2 (dd, 2H, J=1.4, 1.5, 2.9 Hz), 3.32 (m, 2H), 3.34 (m, 2H), 6.16 (s, 2H), 8.5 (bs, 2H). -1

13

C NMR (100 MHz, CDCl3) δ=178.5, 134.9,

52.49, 47.54, 45.14.IR (CH2Cl2, cm ): 3159, 3062, 2990, 2954, 1755, 1700, 1354, 1295, 1227, 1188, 1122.60

4.1.3. 3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (3a) White solid. 1H NMR (400 MHz, CDCl3 (m, 2H), 9.04 (bs, 1H,). 13C NMR (100 MHz, CDCl3

, 40.3, 23.3. IR (CH2Cl2,

cm-1): 3234, 2962, 2946, 1752, 1706, 1348, 1316, 1187.61

4.1.4. 5-methyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (4a) White solid. 1H NMR (400 MHz, CDCl3

(bs, 1H), 5.5 (m, 1H), 3.08 (m, 2H), 2.4 (m,

1H), 1.7 (s, 3H. 13C NMR (100 MHz, CDCl3 24.1, 23.7. IR (CH2Cl2, cm-1): 3284, 2972, 2968, 1752, 1719, 1343, 1313, 1107.62 4.1.5.(3aR,4R,7R,7aS)-4,5,6,7,8,8-hexachloro-3a,4,7,7a-tetrahydro-1H-4,7methanoisoindole-1,3(2H)-dione (5a) White solid. 1H NMR (400 MHz, CDCl3 MHz, CDCl3

13

C NMR (100

169.9, 131.1, 53.4, 31.1. IR (CH2Cl2, cm-1): 3534, 3061, 2968, 1605, 1564

1348, 1316, 1187.63

4.2. General Procedure of N-Tosylation Reactions:

Asolution of 4-methylbenzene-1-sulfonyl chloride (2.52 g, 0,01 mol) and DMAP (1.62 g, 0.01 mol) in acetonitrile (20 mL) was added to a magnetically-stirred, ice-bathed, solution containing the substituted 3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (3a) (2.13 g, 0.01 mol) in acetonitrile (30 mL). The reaction was stirred at room temperature during 12 hours. There action mixture was evaporated under reduced pressure. The precipitated crude products were purified by recrystallization from dichloromethane/hexane. The product 2-tosyl3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (3b) (3 g, 74%) was formed as a white solid.

4.2.1. 2-tosyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (1b) White solid. 1H NMR (400 MHz, CDCl3 Hz), 5.79 (s, 2H), 3.12 (m, 2H), 2.52 (d, 2H, J=1.72 Hz), 2.48 (s, 3H), 2.22 (d, 2H, J=3.52 Hz).

13

C NMR (100 MHz, CDCl3

(CH2Cl2, cm-1): 3650, 3563, 3036, 2964, 2907, 2845, 2375, 2257, 1924, 1795, 1752, 1596, 1440, 1381, 1308, 1174, 1088, 879. Anal. Calcd. for (C17H17NO4S) C:61.61, H:5.17, N:4.23, S:9.68. Found C:61.59, H:5.19, N:4.25, S:9.66.

4.2.2. (4R,7S)-2-tosyl-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione (2b) White solid. 1H NMR (400 MHz, CDCl3) δ=1.3 (m, 2H), 1.5 (m, 2H), 2.4 (s, 3H), 2.9 (d, 2H, J=1.4 Hz), 3.1 (d, 2H, J=1.46 Hz), 6 (dd, 2H, J=3.3, 4.4 Hz), 7.3 (d, 2H, J=8.05 Hz), 8 (d, 2H, J=8.4 Hz). 13C NMR (100 MHz, CDCl3) δ=174, 146.3, 135.1, 133, 130, 129, 45, 32.1, 24, 22. IR (CH2Cl2, cm-1): 2953, 1739, 1597, 1384, 1239, 1190, 1178, 1089. Anal. Calcd. for (C16H15NO4S) C:60.55, H:4.76, N:4.41, S:10.10. Found C:60.52, H:4.74, N:4.42, S:10.14.

4.2.3 (3aR,4S,7R,7aS)-2-tosyl-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione (3b) White solid. 1H NMR (400 MHz, CDCl3) δ=1.45 (d, 2H, J=8.78 Hz), 1.61 (d, 2H, J=9.1 Hz), 2.45 (s, 3H), 3.28 (d, 2H, J=2.83 Hz), 3.38 (s, 2H), 5.79 (s, 2H), 7.33 (d, 2H, J=6.05 Hz), 7.92 (d, 2H, J=8.05 Hz). 13C NMR (100 MHz, CDCl3) δ=172.7, 146.4, 134.8, 129.8, 128.8, 51.9, 46.3, 46, 21.9. IR (CH2Cl2, cm-1): 2992, 1792, 1740, 1596, 1381, 1336, 1268, 1190, 1117, 1089. Anal. Calcd. for (C15H15NO4S) C:59.00, H:4.95, N4.59, S10.50. Found C:59.06, H:5.00, N:4.57, S:10.42.

4.2.4. 5-methyl-2-tosyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (4b) White solid. 1H NMR (400 MHz, CDCl3) δ=7.94 (d, 2H, J=8.05 H ), 7.33 (d, 2H, J=8.05 H ), 5.37 (m, 1H), 3.07 (m, 2H), 2.43 (s, 3H), 2.36 (m, 2H), 2.13 (m, 2H), 1.52 (s, 3H). 13C NMR (100 MHz, CDCl3) δ=175.2, 175.1, 146.4, 136.4, 135.0, 129.9, 120.1, 40.1, 39.8, 28.7, 24.4, 23.4. IR (CH2Cl2, cm-1): 3645, 3453, 2936, 2864, 2845, 2364, 2250, 1950, 1800, 1750, 1556, 1446, 1380, 1160, 1080, 814. Anal. Calcd. for (C16H17NO4S) C:60.17, H:5.37, N:4.39, S:10.04. Found C:60.12, H:5.40, N:4.42, S:10.01.

4.2.5

(3aR,4R,7R,7aS)-4,5,6,7,8,8-hexachloro-2-tosyl-3a,4,7,7a-tetrahydro-1H-4,7-

methanoisoindole-1,3(2H)-dione (5b) White solid. 1H NMR (400 MHz, CDCl3) δ=7.93 (m, 2H), 7.39 (m, 2H), 3.91 ( , 2H), 2.47 (s, 3H).

13

C NMR (100 MHz, CDCl3) δ=165.2, 147.5, 130.4, 130.2, 129.3, 127.2, 53.4, 52.2,

22.1, 22.0. IR (CH2Cl2, cm-1): 3658, 3098, 2997, 2879, 1749, 1593, 1378, 1227, 1172, 1069,

913, 743. Anal. Calcd. for (C16H9Cl6NO4S) C:36.67, H:1.73, N:2.67, S:6.12. Found C:36.58, H:1.94, N:2.71, S:6.2.

4.3. General Procedure of Reactions:

A solution of benzylalcohol (0.354 g, 3.27 mmol) and NEt3 (4.56 ml, 3.27 mmol) in toluene (10 mL) was added to a magnetically-stirred, ice-bathed, solution containing the substituted (4R,7S)-2-tosil-3a,4,7,7a-tetrahidro-1H-4,7-etanoisoindole-1,3(2H)-dion (3b) (1 g, 3.27 mmol) toluene (50 mL), There action was stirred at room temperature for 3 days. Then, the mixture was cooled to room temperature and EtOAc (200 mL) and cold 3 M aqueous HCl (200 mL) were added. The organic phase was separated and the aqueous phase was extracted with EtOAc (200 mL). The combined organic phases were washed with water (100 mL), dried (Na2SO4) and concentrated in a vacuum. The residue was chromatographed by using silica gel (n-hexane/EtOAc 1:4) yielding 1.2 g (88%) benzyl 6-(tosylcarbamoyl)cyclohex-3enecarboxylate (3c)

4.3.1. (1S,4R)-benzyl 3-(tosylcarbamoyl)bicyclo[2.2.2]oct-5-ene-2-carboxylate (1c) White solid. Mp: 210-212 °C 1H NMR (400 MHz, CDCl3) δ=1.3 (m, 4H,), 2.3 (s, 3H), 3 (m, 4H), 4.4 (d, 1H, J=12.4, Hz), 4.99 (d, 2H, J=12.4 Hz), 6.2 (t, 1H, J=7.3 Hz), 6.5 (t, 2H, J=7.3 Hz), 7.3 (m, 7H), 7.92 (d, 2H, J=8.05 Hz), 8.45 (bs, 2H).

13

C NMR (100 MHz, CDCl3)

δ=172.4, 170.2, 145, 136, 136, 131, 129.6, 128.8, 128.7, 128.4, 128.3, 66.7, 50.5, 48.1, 33.3, 31.9, 24.9, 24.4, 21.8. IR (CH2Cl2, cm-1): 3433, 3440, 3350, 3010, 1643, 1656, 1645, 1172, 1087, 950. Anal. Calcd. for (C24H25NO5S) C: 65.59, H: 5.73, N: 3.19, S: 7.29, Found C: 65.61, H: 5.79, N: 3.21, S: 7.25.

4.3.2. (1S,2R,4R)-benzyl 3-(tosylcarbamoyl)bicyclo[2.2.1]hept-5-ene-2-carboxylate (2c) White solid. Mp: 207-209 °C, 1H NMR (400 MHz, CDCl3) δ=1.25 (d, 1H, J=8.8 Hz), 1.42 (d, 2H, J=8.8), 2.38 (s, 3H), 3.12 (d, 2H, J=7.7 Hz), 3.21 (s, 2H), 4.62 (d, 1H, J=12.5 Hz), 4.71 (s, 2H), 5.03 (d, 1H, J=12.1 Hz), 5.98 (dd, 1H, J=2.9, 5.13 Hz), 6.32 (dd, 1H, J=2.9, 5.13 Hz), 7.2 (m, 7H), 7.92 (d, 2H, J=8.05), 9.03 (bs, 1H).

13

C NMR (100 MHz, CDCl3) δ=172.3,

169.9, 145, 136.7, 136, 133.7, 129.7, 128.8, 128.7, 128.6, 128.5, 128.3, 127.9, 127.2, 66.7, 65.6, 50.1, 49.2, 48.9, 47.5, 46.3, 21.8. IR (CH2Cl2, cm-1): 3141, 2879, 1710, 1596, 1497, 1445, 1368, 1343. Anal. Calcd for (C23H23NO5S) C: 64.92, H: 5.45, N: 3.29, S: 7.53, Found C:65.00, H: 5.47, N: 3.32, S: 7.59. 4.3.3. Benzyl 6-(tosylcarbamoyl)cyclohex-3-enecarboxylate (3c) White solid. 1H NMR (400 MHz, CDCl3) δ=7.85 (d, 2H, J=8.3 H ), 7.32 (m, 5H), 7.20 (d, 2H, J=8.12), 5.92 (d, 1H, J=3 Hz), 5.61 (s, 1H), 4.9 (m, 2H), 3.10 (m, 1H), 2.96 (m, 1H), 2.56 (m, 2H), 2.33 (s, 3H), 2.224 (m, 2H). 13C NMR (100 MHz, CDCl3) δ=180.8, 174.0, 143.4, 135.8, 129.5, 129.1, 128.1, 127.9, 127.7, 127.6, 126.3, 125.1, 125.0, 66.4, 45.5, 41.5, 40.2, 26.2, 25.8, 21.5, 21.4, 8.3. IR (CH2Cl2, cm-1): 3226, 3033, 2980, 2946, 2851, 2767, 2254, 1916, 1770, 1713, 1597, 1566, 1454, 1337, 1173, 1088, 1010, 913, 738. Anal. Calcd. for (C22H23NO5S) C:63.91, H:5.61, N:3.39, S:7.75. Found C:63.94, H:5.64, N:3.37, S:7.71.

4.3.4. Benzyl 4-methyl-6-(tosylcarbamoyl)cyclohex-3-enecarboxylate (4c) White solid. Mp: 211-213 °C, 1H NMR (400 MHz, CDCl3) δ=7.84 (d, 2H, J=8.41), 5.27 (s, 1H), 5.01 (m, 2H), 3.43 (s, 3H), 2.99 (s, 1H), 2.96 (s, 1H), 2.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ=174.7, 174.4, 135.8, 135.7, 132.8, 132.4, 129.4, 128.7, 128.4, 128.2, 128.0, 127.8, 127.2, 119.3, 66.9, 65.4, 40.4, 30.6, 26.3, 26.2. IR (CH2Cl2, cm-1): 3242, 2889, 1895, 1680, 1495, 1455, 1370, 1335, 1330, 941. Anal. Calcd. for (C23H25NO5S) C:64.62, H:5.89, N:3.28, S:7.50. Found C:64.68, H:5.80, N:3.30, S:7.45.

4.3.5. (1R,2R,4R)-benzyl 1,4,5,6,7,7-hexachloro-3-(tosylcarbamoyl)bicyclo[2.2.1]hept-5-ene2-carboxylate (5c) White solid. Mp: 270-272 °C, 1H NMR (400 MHz, CDCl3) δ=7.83 (d, 2H, J=11.6 H ), 7.24 (m, 7H), 5.06 (d, 1H, J=12.4 Hz), 4.76 (d, 1H, J=12 Hz), 3.90 (s, 2H), 3.44 (s, 3H). 13C NMR (100 MHz, CDCl3) δ=168.0, 150.8, 134.8, 132.1, 131.4, 129.5, 129.4, 128.8, 128.7, 128.76, 128.71, 128.6, 128.1, 127.8, 127.5, 127.2, 68.5, 68.1, 55.4, 55.0, 51.8, 50.9, 21.7. IR (CH2Cl2, cm-1): 3390, 3365, 3250, 1700, 1565, 1460, 1371, 1331, 1320, 950, 923. Anal. Calcd. for (C23H17Cl6NO5S) C:43.70, H:2.71, N:2.22, S:5.07. Found C:43.65, H:2.73, N:2.23, S:5.08.

4.4. General Procedure of Diol Reactions

To a mixture of benzyl 6-(tosylcarbamoyl)cyclohex-3-enecarboxylate (3c) (1 mmol) in H2Oacetone (2:8) (20 mL) was NMO (1 equiv) and the reaction was stirred. The reaction mixture was filtered and the solvent was concentrated under reduced pressure to produce a predominantly aqueous solution. The product was extracted into ethyl acetate and the organic layer was concentrated to produce a crude product, diolin good yield. Then solution of 500 mg (rac)-diol (4.3 mmol), which was dissolved in 8.2 mL acetic acid anhydride (86 mmol), was mixed with 8.2 mL pyridine. After 12 h of incubation at room temperature, the mixture was cooled to room temperature and EtOAc (200 mL) and cold 3 M aqueous HCl (200 mL) were added. The organic phase was separated and the aqueous phase was extracted with EtOAc (200 mL). The combined organic layers were washed with 10 mL of a saturated NaHCO3 solution and then dried with anhydrous Na2SO4, filtered, and evaporated. In a roundbottomed flask (25 mL) equipped with a magnetic stirrer, a solution of -diacetoxy (0.2 g, 1 mmol) in MeOH (10 mL) was prepared. K2CO3 (1.5 mmol) was then added and the reaction mixture was stirred for 30 min at room temperature. After completion of the reaction, the mixture was acidified with HCl (1 N). The organic phase was evaporated and the mixture was extracted with Et2O (3×5 mL). The combined their phases were dried over anhydrous Na2SO4 and evaporated with 4,5-dihydroxy-2-(tosylcarbamoyl)cyclohexanecarboxylic acid (3d) (0.09 g, 85%)

4.4.1. Benzyl 4,5-dihydroxy-2-(tosylcarbamoyl)cyclohexanecarboxylate (3d) White solid. Mp: 240-242 °C 1H NMR (400 MHz, CD3OD) δ=7.75 (d, 2H, J=8.4 H ), 7.23 (d, 2H, J=8 Hz), 3.55 (m, 2H), 3.30 (m, 2H), 2.36 (s, 3H), 2.01 (m, 4H). 13C NMR (100 MHz, CD3OD) δ=181.2, 176.1, 141.3, 128.5, 128.1, 126.7, 68.5, 68.3, 48.6, 48.4, 47.1, 30.2, 29.9, 20.1. IR (CH2Cl2, cm-1): 3400, 2924, 2476, 1655, 1630, 1400, 1313, 1253, 1139, 1085, 1011, 835, 691. Anal. Calcd. for (C15H19NO7S) C:50.41, H:5.36, N:3.92, S:8.97. Found C:50.49, H:5.32, N:3.95, S:8.95.

4.4.2.

(1R,4S,5S,6R)-5,6-Dihydroxy-3-(tosylcarbamoyl)bicyclo[2.2.2]octane-2-carboxylic

acid (1d) White solid. Mp: 273-275 °C 1H NMR (400 MHz, CD3OD) δ=7.77 (d, 2H, J=2 H ), 7.23 (m, 2H), 4.59 (s, 2H), 3.51 (m, 2H), 3.34 (s, 2H), 2.35 (s, 3H), 1.88 (s, 2H) 1.27 (s, 2H). 13C NMR (100 MHz, CD3OD) δ=183.8, 181.9, 140.9, 140.8, 128.5, 126.6, 64.7, 64.1, 48.6, 47.1, 44.8, 41.6, 36.1, 29.5, 20.1. IR (CH2Cl2, cm-1): 3300, 2952, 2546, 1755, 1680, 1485, 1390, 1298, 1239, 1101, 956. Anal. Calcd. for (C17H21NO7S) C:53.25, H:5.52, N:3.65, S:8.36. Found C:53.30, H:5.49, N:3.70, S:8.30.

4.4.3.

(1R,2R,3S,4S,5S,6R)-5,6-Dihydroxy-3-(tosylcarbamoyl)bicyclo[2.2.1]heptane-2-

carboxylic acid (2d) White solid. Mp: 202-204 °C, 1H NMR (400 MHz, D2O) δ=7.65 (d, 2H, J=8.4 Hz), 7.23 (d, 2H, J=8.05 Hz), 3.52 (m, 2H), 3.29 (m, 2H), 2.87 (m, 1H), 2.33 (m, 2H), 2.22 (s, 3H), 1.66 (m, 1H), 1.26 (m, 2H). 13C NMR (100 MHz, D2O) δ=180.8, 177.5, 144.9, 136.9, 129.8, 127.4, 73.6, 70.1, 50.0, 48.3, 48.1, 47.0, 46.1, 32.1, 20.9. IR (CH2Cl2, cm-1): 3010, 2962, 2574, 1745, 1685, 1445, 1370, 1292. Anal. Calcd. for (C16H19NO7S) C:52.02, H:5.18, N:3.79, S:8.68. Found C:52.10, H:5.10, N:3.70, S:8.70.

4.4.4. 4,5-Dihydroxy-4-methyl-2-(tosylcarbamoyl)cyclohexanecarboxylic acid (4d) Obtained as colorless oil. 1H NMR (400 MHz, CD3OD) δ=7.74 (m, 2H), 7.45 (m, 2H), 4.86 (m, 1H), 4.66 (s, 1H), 3.68 (m, 1H), 3.67 (m, 1H), 3.56 (m, 2H), 3.38 (m, 2H), 2.42 (s, 3H), 1.95 (s, 3H).

13

C NMR (100 MHz, CD3OD) δ=181.2, 176.1, 141.3, 128.4, 128.1, 126.8,

126.7, 68.5, 68.3, 66.0, 64.0, 50.8, 30.3, 29.9, 20.1. IR (CH2Cl2, cm-1): 3410, 2930, 2456, 1650, 1400, 1445, 1250, 690.

4.4.5. (1R,2R,3S,4R,5R,6R)-1,4,5,6,7,7-Hexachloro-5,6-dihydroxy-3-(tosylcarbamoyl)bicycle [2.2.1] heptane-2-carboxylic acid (5d) White solid. Mp: 368-370 °C, 1H NMR (400 MHz, CDCl3) δ=7.5 (m, 2H), 7.2 (m, 2H), 4.41 (s, 2H), 3.32 (s, 3H). 13C NMR (100 MHz, CDCl3) δ=170.0, 165.5, 147.5, 147.0, 133.5, 131.0, 130.4, 130.2, 129.3, 127.2, 103.9, 79.4, 53.4, 52.2, 22.1, 22.0. Anal. Calcd. for (C16H13Cl6NO7S) C:33.36, H:2.27, N:2.43, S:5.57. Found C:33.40, H:2.20, N:2.45, S:5.51.

3.5. Biochemistry The hCA I, and II isoenzymes were purified by Sepharose-4B-L tyrosine-sulfanilamide affinity chromatography in a single step.12,13,26,64 As a column material, Sepharose-4B-L tyrosine-sulfanilamide affinity gel was prepared according to a previous method.14,65-67 To this end, the pH of the homogenate was adjusted to 8.7 with a pH-meter using solid Tris. Subsequently, a 50 mL aliquot of the supernatant was transferred to a Sepharose-4B-L tyrosine-sulfanilamide affinity column. The proteins streams in the column eluates were spectrophotometrically monitored at 280 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both isoenzymes was performed after the Sepharose-4B-L tyrosine-sulfanilamide affinity chromatography purification step. The purity of both isoenzymes were visualized by SDS-PAGE, and a single band was observed for each the hCA I and II isoenzymes.68 This protein imaging method was previously described.69,70 For our application, the imaging method was performed out in 10% and 3% acrylamide for the running and the stacking gel, respectively, with 0.1% SDS.71-74 The hCA I and II isoenzymes activities were determined according to the method of Verpoorte et al.75 and was previously described.76-78 The change in absorbance at 348 nm for p-nitrophenylacetate (NPA) to p-nitrophenolate (NP) was recorded over a 3 min period at the room temperature (25 °C) using a spectrophotometer (Shimadzu, UV-VIS Spectrophotometer, UVmini-1240, Kyoto, Japan).79 The protein quantity was spectrophotometrically measured at 595 nm during the purification steps according to the Bradford method.80 As used in previous studies, bovine serum albumin (BSA) was used as the standard protein.73,74,81 For determining the inhibition effect of each newly synthesized N-acylsulfonamide derivative, an activity (%)[N-Acylsulfonamide] graph was drawn. As can given in literature,82 0.1 and 1% of Triton X100 were added to each N-Acylsulfonamide derivatives for excluding aggregation under the assay conditions. It was found that there is not found any inhibition or activation effect of 1% of Triton X-100 on CA I, and II activity determination. But, 0.1% of Triton X-100 addition on CA I slightly reduced inhibition effects of N-Acylsulfonamides derivatives CA I. To determine Ki values, three different N-acylsulfonamide derivative concentrations were tested. In these experiments, NPA was used as the substrate at five different concentrations and Lineweaver-Burk curves were drawn83 as previously described.84-90

Acknowledgments This study was financed by Ataturk University (TUBITAK 110T483). Additionally, İ hami Gülçin would like to extend his sincere appreciation to the Research Chairs Program at King Saud University for funding this research.

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Table 1 Diels-Alder reactions

Entry

Diene

Dienofil

Product

Yield (%)

1a

93

2a

90

3a

98

4a

97

5a

98

Table 2. N-Tosylation reactions

Entry

Product

Yield (%)

Table 3 Benzylation reactions

Entry

Product

Yield (%)

Table 4. Diol reactions Entry

Product

Yield (%)

Table 5. Inhibition constants for N-acylsulfonamide derivatives (1c-5c and 1d-5d) toward human carbonic anhydrase isoenzymes I, and II (hCA I, and II) determined using an esterase bioassay KI (nM)

IC50 (nM) Compounds hCA I

R2

hCA II

R2

hCA I

hCA II

1c

178.7

0.9932

121.5

0.9948

186.2±31.0

133.6±34.0

2c

132.9

0.8167

110.2

0.9678

190.6±25.0

111.7±23.0

3c

153.1

0.9948

110.7

0.9746

174.8±28.0

90.3±3.6

4c

178.9

0.9792

130.6

0.9932

122.8±16.0

138.5±43.0

5c

90.0

0.9825

70.1

0.9746

36.4±6.0

61.2±6.0

1d

133.5

0.9615

126.5

0.9939

97.3±11.0

150.9±4.3

2d

210.2

0.9361

110.9

0.9552

254.6±18.0

273.3±2.5

3d

209.9

0.9339

154.4

0.9776

144.7±13.0

138.0±2.0

4d

215.0

0.9653

144.1

0.9404

105.4±12.0

122.4±5.0

5d

170.8

0.9754

120.3

0.9637

51.7±9.0

58.3±0.6

AZA*

315.5

0.9642

123.5

0.9324

184.3±0.3

61.1±0.02

*AZA was used as appositive control

FIGURE LEGENDS

Figure 1. Some sulfamide drug examples

Figure 1. Possible coupling structure of CA isoenzyme and acyl groups of newly synthesised Nacylsulfonamide derivatives

Leu198 209Trp R

H

Val143 NHTs N

O H

Thr199

Val121

O O

H

Zn2+ His119

H

94His

His94

SCHEMES

Scheme 1. General synthesis of benzyl 4,5-dihydroxy-2-(tosylcarbamoyl)cyclohexane carboxylate

Graphical abstract

N-Acylsulfonamides Strongly Inhibit Human Carbonic Anhydrase Isoenzymes I and II