Novel pyrazole-3,4-dicarboxamides bearing biologically active sulfonamide moiety as potential carbonic anhydrase inhibitors

Samet Mert; Zuhal Alım; Mehmet Mustafa İşgör; Barış Anıl; Rahmi Kasımoğulları; Şükrü Beydemir

doi:10.1016/j.arabjc.2015.05.020

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Original article

12 (

); 2740-2748

doi:

10.1016/j.arabjc.2015.05.020

Novel pyrazole-3,4-dicarboxamides bearing biologically active sulfonamide moiety as potential carbonic anhydrase inhibitors

Samet Mert^{^a}, Zuhal Alım^{^b}, Mehmet Mustafa İşgör^{^b}, Barış Anıl^{^b}, Rahmi Kasımoğulları^{^a,⁎}, Şükrü Beydemir^{^b,⁎}

a Chemistry Department, Faculty of Arts and Sciences, Dumlupinar University, 43100 Kutahya, Turkey

b Chemistry Department, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey

⁎Corresponding authors. rahmikasimoglu@hotmail.com (Rahmi Kasımoğulları), beydemir@atauni.edu.tr (Şükrü Beydemir)

Received: 2014-12-11, Accepted: 2015-5-23, Published: 2015-12-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

In this study a series of pyrazole-3,4-dicarboxamide (3–10) derivatives bearing sulfonamide moiety were synthesized starting from 1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylic acid (1). The structures of synthesized molecules were characterized by FT-IR, ¹H NMR, ¹³C NMR, and elemental analysis methods. Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified separately from erythrocyte cells by the Sepharose-4B-L-tyrosine-sulfanilamide affinity column chromatography and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied as in vitro. The K_i values of compounds were found in the range of 0.056–110.400 μM for hCA I and 0.057–533.400 μM for hCA II. Compound 4 has the highest inhibitory effect for hCA I and hCA II while compound 5 showed lowest inhibition. The structure–activity relationships for the inhibition of these isoforms with the pyrazole-sulfonamides reported here were also elucidated.

Keywords

Pyrazole

Pyrazole-3,4-dicarboxamide

Synthesis

Reduction

Carbonic anhydrase

Enzyme inhibition

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1 Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that catalyze a very simple but essential physiological reaction, carbon dioxide hydration to bicarbonate and protons (Supuran, 2010, 2012; Alterio et al., 2012; Güzel-Akdemir et al., 2013; Marini et al., 2012 ). In mammals, there are 16 different isoforms of these metalloenzymes among which several are cytosolic (CA I–III, CA VII and CA XIII), five are membrane-bound (CA IV, CA IX, CA XII, CA XIV, and CA XV), two are mitochondrial (CA VA and VB), and one (CA VI) is secreted into saliva/milk (Supuran, 2008a, 2009; Winum et al., 2009; Supuran et al., 2004; Pastorek et al., 2008; Hassan et al., 2013; Brzozowski et al., 2010 ). Three acatalytic forms (CA VIII, CA X and CA XI) are also known (Winum et al., 2009; Hen et al., 2011). Among them, human CA isoforms I and II are the object of major interest as druggable targets for antiglaucoma, anticonvulsants, and diuretics (Güzel-Akdemir et al., 2013; Supuran, 2008b; Balseven et al., 2013; Tanc et al., 2013 ).

Up to now myriad CA inhibitors have been synthesized or isolated as sulfonamide and non-sulfonamide (such as phenols, thiols, coumarins, polyamines, and dithiocarbamates) inhibitors (Kasımoğulları et al., 2011; Göcer et al., 2017; Bilginer et al., 2014; Akbaba et al., 2013; Carta et al., 2010, 2012, 2013; Almajan et al., 2005; Davis et al., 2013 ). Therefore, the synthetic studies continue at a great pace today for the synthesis of new CAIs that have a good inhibitory profile with minimal side effects.

Pyrazole compounds are unique molecules and pyrazole-fused ring systems are found in numerous applications in drug discovery efforts (Yet, 2008). Pyrazole derivatives exhibit broad spectrum of biological activities such as analgesic, antiinflammatory, antipyretic, antibacterial, antifungal, and antiproliferative (Kuo et al., 1984; Bekhit et al., 2010; Schegol’kov et al., 2006; Tanitame et al., 2004; Li et al., 2006; Kostakis et al., 2002 ). Among them, some are used as phosphodiesterase inhibitors, COX-2 inhibitors, and CB1 cannabinoid receptors such as Sildenafil, Celecoxib and Rimonabant, respectively (Terrett et al., 1996; Dale et al., 2000; Penning et al., 1997; Lan et al., 1999; Francisco et al., 2002; Katoch-Rouse et al., 2003 ) (see Fig. 1). In continuation of our research efforts of the discovery of novel pyrazole-sulfonamide derivatives (Balseven et al., 2013; Kasımoğulları et al., 2011; Şen et al., 2013 ), herein we describe synthesis, characterization, and evaluation of the inhibition effects of sulfonamide bearing pyrazole-3,4-dicarboxamides (3–10) on CA I and CA II isozymes. We use two free sulfonamide derivatives (4-aminobenzenesulfonamide and 5-amino-1,3,4-thiadiazole-2-sulfonamide) and two sterically hindered sulfonamide derivatives (sulfadiazine and sulfisoxazole) in the synthesis of new inhibitors. And four of our compounds (3–6) have 3-nitrophenyl moiety bonded the first position of the pyrazole ring. We reduced the nitro groups and we obtained four new derivatives (7–10) which have 3-aminophenyl moiety in same position (see Scheme 1 and Table 1). Based on this information, this article has two main aims. First, we aimed to make a comparison between the activities of our inhibitors that contain free SO₂NH₂ groups (3, 4, 7, and 8) and sterically hindered derivatives (4, 5, 9, and 10). Second we aimed to make a comparison between the activities of the derivatives that have nitro and amine groups. Third we aimed to discuss the selective inhibition effects of our compounds on human carbonic anhydrase isozymes I and II.

Structures of Sildenafil (an oral therapy drug for erectile dysfunction), Celecoxib (non-steroidal antiinflammatory drug), and Rimonabant (anorectic antiobesity drug).

Synthetic route for the target compounds (3–10).

Table 1 K_i values of the compounds (3–10) on the both isoenzymes (CA I and CA II).

Compound			K_i (μM)
Compound	R′	R″	CA I	CA II
3		—NO₂	0.331	0.163
4		—NO₂	0.056	0.057
5		—NO₂	No inhibition	533.400
6		—NO₂	14.283	211.000
7		—NH₂	0.306	0.121
8		—NH₂	5.108	0.599
9		—NH₂	110.400	45.470
10		—NH₂	23.940	54.470

2 Experimental protocols

2.1

2.1 Material and methods

The chemicals were purchased from the commercial venders and the solvents were purified by using appropriate purifying agents and distillation. Tetrahydrofuran (THF) was distilled from sodium/benzophenone prior to use. All reactions were monitored and purity of the product was checked by analytical thin-layer chromatography (TLC) on 0.25 mm precoated Kieselgel 60F 254 plates (E. Merck Co., Darmstadt, Germany) and compounds were visualized by TLC devices (Camag, Upland, CA, USA) UV (254 and 366 nm). And the solvent system was chloroform/methanol (20:1) for all compounds. Melting points (°C, uncorrected) were taken in open capillaries on a Barnstead Electrothermal 9200 melting point apparatus (Electrothermal Co, Essex, UK). Infrared (IR) spectra were recorded by Bruker Optics, Vertex 70 Fourier Transform Infrared Spectrometer (FT-IR) equipped with an ATR (Attenuated Total Reflection) device and the data were reported in reciprocal centimeters (cm⁻¹) (Bruker Optik GmbH, Ettlingen, Germany). ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ solutions at 298 K using a Bruker Ultrashield-400 spectrometer operating at 400.00 MHz for ¹H and 101.00 MHz for ¹³C (Bruker BioSpin GmbH Silberstreifen D-76287, Rheinstetten, Germany). The center of the peaks of DMSO-d₆ [δ (ppm): 2.50 (¹H) and δ (ppm): 39.5 (¹³C)] was used as an internal reference in a 5-mmNMR tube (Wilmad, No. 528-PP). Elemental analyses (C, H, N, and S) were performed on a Leco CHNS-932 elemental analyser (LECO Corporation, Saint Joseph, Michigan, USA).

2.2

2.2 Synthesis of target inhibitors

2.2.1

2.2.1 General procedure for the synthesis of pyrazole-3,4-dicarboxamides (3–6)

A mixture of acid dichloride (2) (1 mmol), and appropriate sulfonamide derivative (4 mmol) was heated under reflux condition in freshly distilled THF (30 ml) for 5 h. The solvent was evaporated in vacuo and residue was washed with water. The precipitated crude product was filtered and crystallized from appropriate solvent.

2.2.2

2.2.2 1-(3-Nitrophenyl)-5-phenyl-N³,N⁴-bis(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3,4-dicarboxamide (3)

Synthesized from 2 (0.39 g, 1 mmol) and 5-amino-1,3,4-thiadiazole-2-sulfonamide (0.72 g, 4 mmol) according to the general procedure. The crude product was purified by crystallization from ethanol. Yield: 92%; mp: 230–232 °C; IR (υ, cm⁻¹): 3367 and 3280 (NH), 3089 (ArCH), 1654 (C⚌O, amide), 1614–1450 (C⚌C and C⚌N), 1525 and 1351 (NO₂), 1378 and 1172 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 13.97 (br, s, 2H, 2CONH), 8.37 and 8.35 (s, 4H, 2SO₂NH₂), 8.43–7.44 (m, 9H, ArH); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 164.80 and 164.57 (C⚌O, amide), 161.15, 161.06, 160.79 and 160.72 (thiadiazole C-2 and C-5), 148.87 (C—NO₂), 147.93 (pyrazole C-3), 146.06 (pyrazole C-5), 120.31 (pyrazole C-4), 143.02, 138.73, 131.18, 130.58, 130.20, 128.76, 126.64, 123.59, 116.88. Anal. Calcd. for C₂₁H₁₅N₁₁O₈S₄: C, 37.22; H, 2.23; N, 22.74; S, 18.93; Found: C, 37.13; H, 2.27; N, 22.81; S, 19.02.

2.2.3

2.2.3 1-(3-Nitrophenyl)-5-phenyl-N³,N⁴-bis(4-sulfamoylphenyl)-1H-pyrazole-3,4-dicarboxamide (4)

Synthesized from 2 (0.39 g, 1 mmol) and 4-aminobenzenesulfonamide (0.69 g, 4 mmol) according to the general procedure. The crude product was purified by crystallization from MeOH/H₂O (1:1). Yield: 90%; mp: 227–229 °C; IR (υ, cm⁻¹): 3254 (NH), 3038 (ArCH), 1628 (C = O, amide), 1591–1483 (C⚌C and C⚌N), 1526 (NO₂), 1316 and 1151 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 13.12 and 10.91 (s, 2H, 2CONH), 8.33–7.42 (m, 17H, ArH), 7.31 and 7.28 (s, 4H, 2SO₂NH₂); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 160.89 and 159.82 (C⚌O, amide), 147.90 (C—NO₂), 144.37 (pyrazole C-3), 144.17 (pyrazole C-5), 120.49 (pyrazole C-4), 143.59, 141.86, 141.22, 139.16, 139.01, 138.63, 131.70, 130.71, 129.80, 128.75, 127.41, 126.72, 126.51, 123.50, 120.32, 120.22, 118.79. Anal. Calcd. for C₂₉H₂₃N₇O₈S₂: C, 52.64; H, 3.50; N, 14.82; S, 9.69; Found: C, 52.70; H, 3.56; N, 14.77; S, 9.73.

2.2.4

2.2.4 1-(3-Nitrophenyl)-5-phenyl-N³,N⁴-bis(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-1H-pyrazole-3,4-dicarboxamide (5)

Synthesized from 2 (0.39 g, 1 mmol) and sulfadiazine (1 g, 4 mmol) according to the general procedure. The crude product was purified by crystallization from EtOH/DMF (2:1). Yield: 94%; mp: 149–151 °C; IR (υ, cm⁻¹): 3373 (NH), 3036 (ArCH), 1686 (C⚌O, amide), 1625–1435 (C⚌C and C⚌N), 1532 (NO₂), 1343 and 1152 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.34 (br, s, 2H, 2SO₂NH), 11.14 and 10.94 (s, 2H, 2CONH), 8.54 and 8.51 (d, J = 4.8 Hz, 2 × (2H), 4CH, symmetrical pyrimidine H4 and H6), 8.49–6.56 (m, 17H, ArH), 7.07–6.99 (m, 2 × (1H), 2CH, symmetrical pyrimidine H5); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 158.34 and 158.22 (C⚌O, amide), 157.21 (pyrimidine C2), 156.94 and 153.02 (pyrimidine C4 and C6), 148.09 (C—NO₂), 147.63 (pyrazole C3), 141.92 (pyrazole C5), 120.20 (pyrazole C4), 118.29 (pyrimidine C5), 132.26, 131.68, 131.19, 130.81, 130.52, 130.38, 130.29, 129.80, 129.40, 129.05, 128.93, 128.16, 128.04, 127.50, 124.85, 124.39, 121.04. Anal. Calcd. for C₃₇H₂₇N₁₁O₈S₂: C, 54.34; H, 3.33; N, 18.84; S, 7.84; Found: C, 54.29; H, 3.36; N, 18.90; S, 7.88.

2.2.5

2.2.5 N³,N⁴-bis(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxamide (6)

Synthesized from 2 (0.39 g, 1 mmol) and Sulfisoxazole (1.07 g, 4 mmol) according to the general procedure. The crude product was purified by crystallization from MeOH/H₂O (3:1). Yield: 93%; mp: 233–235 °C; IR (υ, cm⁻¹): 3381 and 3359 (NH), 3029 (ArCH), 2980 (Aliphatic CH), 1687 and 1660 (C⚌O, amide), 1589–1442 (C⚌C and C⚌N), 1530 and 1343 (NO₂), 1370 and 1158 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.19 and 11.02 (s, 2H, 2CONH), 10.99 (br, s, 2H, SO₂NH), 8.34–7.42 (m, 17H, ArH), 2.10 (s, 6H, 2CH₃(3) isoxazole), 1.68 and 1.67 (s, 6H, 2CH₃(4) isoxazole); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 161.42 (isoxazole C3), 161.06 and 159.90 (C⚌O, amide), 155.49 (isoxazole C5), 147.91 (C—NO₂), 145.83 (pyrazole C3), 144.48 (pyrazole C5), 120.14 (pyrazole C4), 105.16 and 105.00 (isoxazole C4), 10.32 (isoxazole-CH₃(3)), 5.90 (isoxazole-CH₃(4)), 144.11, 143.21, 142.68, 138.98, 134.57, 134.08, 131.73, 130.71, 129.81, 128.77, 127.96, 127.73, 127.32, 123.54, 120.54, 119.12, 117.53. Anal. Calcd. for C₃₉H₃₃N₉O₁₀S₂: C, 54.99; H, 3.90; N, 14.80; S, 7.53; Found: C, 55.04; H, 3.88; N, 14.85; S, 7.55.

2.2.6

2.2.6 General procedure for reduction of nitro groups

Sodium polysulfide solution was prepared from Na₂S.9H₂O (2.5 g) with powdered sulfur (0.7 g) dissolved in approximately 100 ml boiling water. Hot sodium polysulfide solution was then added dropwise to a stirred boiling solution of nitro compound (3–6) (2 mmol) in methanol. The reaction mixture was boiled for 1 h. Then the reaction mixture was cooled and acidified with concentrated HCl. The precipitated product was filtered and dissolved in hot acetone. The hot solution was filtered to remove unreacted sulfur. Then acetone was evaporated in vacuo to dryness. Finally the crude product was crystallized from appropriate solvent.

2.2.7

2.2.7 1-(3-Aminophenyl)-5-phenyl-N³,N⁴-bis(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3,4-dicarboxamide (7)

Synthesized according to the general procedure and the crude product was purified by crystallization from EtOH/H₂O (2:1). Yield: 86%; mp: 260–262 °C; IR (υ, cm⁻¹): 3332 and 3284 (NH and NH₂), 3061 (ArCH), 1667 (C⚌O, amide), 1605–1411 (C⚌C and C⚌N), 1343 and 1166 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 13.72 (br, s, 2H, 2CONH), 8.44 and 8.40 (s, 4H, 2SO₂NH₂), 8.30–7.31 (m, 9H, ArH), 3.88 (br, s, 2H, Ar-NH₂); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 164.98 and 164.63 (C⚌O, amide), 161.26, 160.95, 160.72 and 160.11 (thiadiazole C-2 and C-5), 147.92 (C—NH₂), 145.80 (pyrazole C-3), 142.71 (pyrazole C-5), 120.24 (pyrazole C-4), 138.68, 131.13, 130.64, 130.15, 129.61, 128.82, 126.45, 123.61, 117.07. Anal. Calcd. for C₂₁H₁₇N₁₁O₆S₄: C, 38.94; H, 2.65; N, 23.79; S, 19.80; Found: C, 38.88; H, 2.62; N, 23.81; S, 19.77.

2.2.8

2.2.8 1-(3-Aminophenyl)-5-phenyl-N³,N⁴-bis(4-sulfamoylphenyl)-1H-pyrazole-3,4-dicarboxamide (8)

Synthesized according to the general procedure and the crude product was purified by crystallization from EtOH/DMF (3:1). Yield: 84%; mp: 270–272 °C; IR (υ, cm⁻¹): 3341 and 3248 (NH and NH₂), 3040 (ArCH), 1661 (C⚌O, amide), 1620–1494 (C⚌C and C⚌N), 1314 and 1152 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.53 and 10.50 (s, 2H, 2CONH), 7.11 and 7.04 (s, 4H, 2SO₂NH₂), 7.91–6.25 (m, 17H, ArH), 3.55 (br, s, 2H, Ar-NH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 161.54 and 161.34 (C⚌O, amide), 149.38 (C—NH₂), 146.20 (pyrazole C-3), 142.63 (pyrazole C-5), 120.93 (pyrazole C-4), 142.34, 141.39, 139.48, 139.34, 138.55, 130.18, 129.55, 129.50, 128.91, 128.47, 127.15, 127.08, 119.54, 118.69, 115.10, 114.03, 111.89. Anal. Calcd. for C₂₉H₂₅N₇O₆S₂: C, 55.14; H, 3.99; N, 15.52; S, 10.15; Found: C, 55.21; H, 4.02; N, 15.57; S, 10.09.

2.2.9

2.2.9 1-(3-Aminophenyl)-5-phenyl-N³,N⁴-bis(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-1H-pyrazole-3,4-dicarboxamide (9)

Synthesized according to the general procedure and the crude product was purified by crystallization from EtOH/DMF (2:1). Yield: 83%; mp: 224–226 °C; IR (υ, cm⁻¹): 3356 and 3257 (NH and NH₂), 3039 (ArCH), 1689 and 1628 (C⚌O, amide), 1580–1439 (C⚌C and C⚌N), 1323 and 1151 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.44 (br, s, 2H, 2SO₂NH), 11.02 and 10.81 (s, 2H, 2CONH), 8.51 and 8.48 (d, J = 4.5 Hz, 2 × (2H), 4CH, symmetrical pyrimidine H4 and H6), 8.02–6.01 (m, 17H, ArH), 7.02 (t, J = 5.4 Hz, 2 × (1H), 2CH, symmetrical pyrimidine H5), 5,46 (br, s, 2H, ArNH₂); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 158.34 and 158.21 (C⚌O, amide), 157.20 (pyrimidine C2), 156.94 and 153.00 (pyrimidine C4 and C6), 149.58 (C—NH₂), 143.42 (pyrazole C3), 142.97 (pyrazole C5), 119.91 (pyrazole C4), 118.53 (pyrimidine C5), 142.42, 139.32, 134.75, 134.31, 129.80, 129.29, 128.84, 128.55, 128.42, 124.86, 115.76, 115.49, 113.00, 112.12, 111.02. Anal. Calcd. for C₃₇H₂₉N₁₁O₆S₂: C, 56.41; H, 3.71; N, 19.56; S, 8.14; Found: C, 56.48; H, 3.74; N, 19.57; S, 8.16.

2.2.10

2.2.10 1-(3-Aminophenyl)-N³,N⁴-bis(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxamide (10)

Synthesized according to the general procedure and the crude product was purified by crystallization from methanol. Yield: 87%; mp: 226–228 °C; IR (υ, cm⁻¹): 3399 and 3338 (NH and NH₂), 3035 (ArCH), 2933 (Aliphatic CH), 1694 and 1661 (C⚌O, amide), 1589–1476 (C⚌C and C⚌N), 1333 and 1152 (S⚌O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.20 and 10.89 (s, 2H, 2CONH), 11.01 (br, s, 2H, SO₂NH), 8.09–6.38 (m, 17H, ArH), 3,57 (br, s, 2H, ArNH₂), 2.09 (s, 6H, 2CH₃(3) isoxazole), 1.67 and 1.66 (s, 6H, 2CH₃(4) isoxazole); ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 161.73 and 160.10 (C⚌O, amide), 161.34 (isoxazole C3), 155.89 and 155.83 (isoxazole C5), 149.59 (C—NH₂), 143.57 (pyrazole C3), 143.29 (pyrazole C5), 120.34 (pyrazole C4), 104.56 and 104.53 (isoxazole C4), 10.33 (isoxazole-CH₃(3)), 5.95 and 5.91 (isoxazole-CH₃(4)), 142.92, 142.76, 139.19, 134.63, 134.17, 129.32, 128.44, 127.95, 127.88, 127.62, 119.45, 119.00, 114.19, 113.02, 112.64, 111.05, 99.51. Anal. Calcd. for C₃₉H₃₅N₉O₈S₂: C, 56.99; H, 4.29; N, 15.34; S, 7.80; Found: C, 57.08; H, 4.33; N, 15.30; S, 7.78.

2.3

2.3 Biological activity evaluation

2.3.1

2.3.1 Hemolysate preparation

Erythrocytes were purified from fresh human blood, which was obtained from the University Hospital Blood Center of Erzurum Atatürk University. Following low speed centrifugation (1500 rpm for 15 min) and removal of plasma and buffy coat, the red blood cells were isolated, washed twice with 0.9% NaCl and hemolyzed with 1.5 volumes of ice-cold water. Ghost and intact cells were then removed by high-speed centrifugation (20,000 rpm for 30 min.) at 4 °C and the pH of the hemolysate adjusted to 8.7 with solid Tris.

2.3.2

2.3.2 Purification of carbonic anhydrase isozymes from human erythrocytes by affinity chromatography

Sepharose-4B L-tyrosine affinity chromatography column was prepared according to our previous studies (Balseven et al., 2013; Kasımoğulları et al., 2011). The pH-adjusted human erythrocyte hemolysate was applied to the Sepharose 4B-L-tyrosine-sulfanilamide affinity column pre-equilibrated with 25 mM Tris–HCl/0.1 M Na₂SO₄ (pH 8.7). The affinity gel was washed with 25 mM tris–HCl/22 mM Na₂SO₄ (pH = 8.7). The human carbonic anhydrase isozymes (hCA I and hCA II) were eluted with 1.0 M NaCl/25 mM Na₂HPO₄ (pH 6.3) and 0.1 M NaCH₃COO/0.5 M NaClO₄ (pH 5.6), respectively.

During purification procedures of hCA I and hCA II, the absorbency was measured at 280 nm to monitor protein elution by affinity chromatography. CO₂-hydratase activity was determined in eluted fractions and the active fractions were collected.

2.3.3

2.3.3 Hydratase and esterase activity assay

2.3.3.1

2.3.3.1 Hydratase activity assay

Carbonic anhydrase catalyzes the hydration of carbon dioxide to form bicarbonate and a proton. ${CO}_{2} + H_{2} O ⇌ H^{+} + {HCO}_{3}^{-}$

Carbonic anhydrase activity was assayed by following the hydration of CO₂ according to the method described by Wilbur and Anderson (1948).

2.3.3.2

2.3.3.2 Esterase activity assay

Carbonic anhydrase enzymes catalyze some non-physiological reactions under in vitro conditions. For instance, it was observed that the purified enzyme has esterase activity under in vitro conditions.

Esterase activity of human erythrocyte carbonic anhydrase was assayed by following the change in absorbance at 348 nm of 4-nitrophenyl acetate to 4-nitrophenolate ion over a period of 3 min at 25 °C using a spectrophotometer (Verpoorte et al., 1967).

2.3.4

2.3.4 In vitro inhibition studies

In inhibition studies carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 °C using a spectrophotometer according to the method described by Verpoorte et al. (1967). The enzymatic reaction, in a total volume of 1 ml, contained 50 mM Tris–SO₄ buffer (pH = 7.4), 3 mM 4-nitrophenylacetate and enzyme solution. A reference measurement was done by the same cuvette without enzyme solution. The inhibitory effects of newly synthesized sulfonamide derivatives on carbonic anhydrase enzyme activity purified from human erythrocyte were tested in triplicate at each concentration used. CA activities were measured in the presence of different substrate concentrations. Control activity in the absence of inhibitor was assumed to be 100%. For each compound, a percent activity versus inhibitor concentration graph was plotted. K_i values of the compounds were calculated by measuring enzyme activity at three different inhibitor concentrations with five different substrate concentrations. Lineweaver–Burk curves (see Fig. 2) were used for the determination of K_i and inhibition type (Balseven et al., 2013; Kasımoğulları et al., 2011).

Lineweaver–Burk graphs of the compound 4 on CA I and CA II.

3 Results and discussion

3.1

3.1 Chemistry

Recently it has been reported that Celecoxib, pyrazole and sulfonamide containing antiinflammatory agent, was a strong inhibitor of CA IX and CA II (Weber et al., 2004; Gluszok et al., 2010). When administered Celecoxib and Valdecoxib orally to glaucomatous rabbits, it was observed these drugs lowered the intraocular pressure, suggesting that they may have utility in the treatment of this disorder (Weber et al., 2004; Di Fiore et al., 2006). Also some modification studies have made over pyrazole ring for a good selectivity CA IX versus CA II (Gluszok et al., 2010; Rogez-Florent et al., 2013). Based on these informations, our study was conducted in order to design synthesize, characterize and evaluate the biological activity of novel pyrazole-3,4-dicarboxamides (3–10) which bearing two sulfonamide scaffolds in the 3 and 4 position of the pyrazole ring.

Firstly 1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxylic acid (1) was synthesized according to our previous studies and its carboxyl groups were activated (2) with SOCl₂ (Mert et al., 2014). Then novel nitro substitute pyrazole-3,4-dicarboxamides 3–6 were synthesized from the reaction of acid dichloride (2) with the 4-aminobenzene sulfonamide (sulfanilamide), 5-amino-1,3,4-thiadiazole-2-sulfonamide, sulfadiazine, and Sulfisoxazole, respectively (see Scheme 1 and Table 1). Acid chloride was reacted with sulfonamide derivatives in the molar range of 1:4, at reflux in THF, leading to the nitro derivatives (3–6). Then the subsequent reduction of the nitro group of 3–6 with sodium-polysulfide hydrogenation (Na₂S/S/H₂O) afforded four novel amine derivatives (7–10). The general synthetic route shown in Scheme 1 and Table 1 was used to prepare the pyrazole-sulfonamide derivatives 3–10, as reported earlier by our group (Balseven et al., 2013; Kasımoğulları et al., 2011).

The synthesized molecules have been characterized by various spectral and analytical techniques including FT-IR, ¹H NMR, ¹³C NMR and Elemental analysis. The infrared (IR) spectra of all pyrazole-sulfonamide (3–10) derivatives showed NH or NH₂ stretching bands at 3399–3248 cm⁻¹ and showed sharp peaks for the carbonyl groups in the region between 1694 and 1628 cm^−l. S⚌O asymmetric and symmetric stretching bands were observed at 1378–1314 cm^−l and 1172–1151 cm^−l, respectively. The compounds 3–6 exhibited asymmetric and symmetric stretching bands at around ∼1525 cm⁻¹ and ∼1350 cm⁻¹ that belong to NO₂ groups. Aromatic C—H stretching bands were observed in the region between 3097 and 3036 cm⁻¹. The aliphatic C—H stretching bands of 6 and 10 were observed at 2980 cm⁻¹ and 2933 cm⁻¹, respectively. The absorption values of other functional groups appeared in the expected regions and they were consistent with the literature (Kasımoğulları et al., 2011; Sener et al., 2004; Jennings and Lovely, 1991 ) (see Experimental for details).

In the ¹H NMR spectrum of 3–10 CONH protons were observed between 13.97 and 10.50 ppm. SO₂NH₂ peaks appeared at around ∼8.4 ppm and ∼7.2 ppm in thiadiazole and sulfanilamide containing carboxamides, respectively. Also SO₂NH peaks appeared at around ∼11 ppm in sterically hindered sulfonamide containing carboxamides. Signals for the aromatic protons were observed in the range of 8.49–6.01 ppm. NH₂ protons were observed between 5.46 and 3.55 ppm. In sulfadiazine containing carboxamides, H4 and H6 protons of the pyrimidine rings were observed between 8.54 and 8.48 ppm as dublets. Also H5 protons of the pyrimidine rings appeared at around ∼7 ppm as triplet or multiplets. In sulfisoxazole containing carboxamides, the methyl protons bonded the third and fourth position of the isoxazole ring showed singlet peaks at around ∼2.1 and ∼1.7 ppm, respectively.

In ¹³C NMR spectra the peaks of C⚌O for amides (3–10) were observed between 165.98 and 158.21 ppm. Other characteristic peaks appeared at expected regions (see Experimental for details). Also we observed some characteristic peaks related to sulfonamides which include thiadiazole, pyrimidine and isoxazole rings. In ¹³C NMR spectra of 5 and 9, C2 carbons of pyrimidine ring appeared at 157 ppm. Also C3, C4 and C5 carbons were observed at 156, 118 and 153 ppm, respectively. Besides in the spectrum of 6 and 10; C3, C4 and C5 carbons of isoxazole ring give peaks at 161, 105 and 155 ppm, respectively. Finally the methyl carbons bonded the third and fourth position of the isoxazole ring give peaks at around 10.3 ppm and 5.9 ppm. The sum up, ¹H NMR, ¹³C NMR, FT-IR and elemental analysis results all of the synthesized compounds were in full agreement with the proposed structures (see Experimental and Table 1).

3.2

3.2 Biological activity results

CA inhibitors have been used as antiglaucoma, diuretics, anticonvulsants, antiobesity, antitumor agents and tumor diagnostic tools (Supuran, 2007). So designing novel CA inhibitors are very important for discovery of new pharmacological agents and understanding in detail protein–drug interactions at molecular level. In the current study, one of the major goal is to investigate in vitro inhibitory effect of novel sulfonamide derivatives of pyrazole-3,4-dicarboxamides (3–10) on CA isozymes (hCA I and hCA II). For this aim, CA isozymes (hCA I and II) were purified from human erythrocytes by Sepharose-4B-L-tyrosine affinity chromatography. The purification results for hCA I depicted a 67% yield, 670 U/mg specific activity and approximately 81 purification fold. hCA II was obtained as 84.6% yield, 4230 U/mg specific activity and approximately 512 purification fold. Additionally, purity of enzymes was controlled by using SDS–PAGE. Purified enzymes had single bands around 29 kDa. After the purification step, the effects of novel compounds (3–10) on the esterase activity of purified isozymes were investigated as in vitro. All compounds showed inhibitory effects on both hCA I and hCA II isoenzymes except compound 5. Compound 5 did not show any inhibition effects for hCA I while it showed lowest inhibition effects for hCA II.

Compound 4 has the strongest inhibitory effect on hCA I and hCA II activities. K_i value of this compound is almost in nanomolar concentration range (0.056 μM for hCA I and 0.057 μM for hCA II). Also compounds 3 and 7 depicted relatively strong inhibitory effects on both izoenzymes. According to decreasing inhibition potential on hCA I, compounds can be ranked as 4, 7, 3, 8, 6, 10 and 9. The inhibition decreasing list of hCA II is in the order of 4, 7, 3, 8, 9, 10, 6 and 5 (see Table 1).

Also structure activity relationships can be drawn with a comparison between functional groups of the synthesized compounds. In the nitro substituted derivatives (3–6) activity results can be ranked as 4 > 3 > 6 > 5 both for hCA I and hCA II. Besides in the amino substituted derivatives (7–10) activity results can be ranked as 7 > 8 > 10 > 9 for hCA I also 7 > 8 > 9 ∼ 10 for hCA II. Thus, as expected, the results obtained confirmed the free sulfonamide derivatives (3, 4, 7, and 8) have a higher inhibition potential on both isozymes. Furthermore, when comparing the results of the inhibition between nitro substituted and amino substituted derivatives generally we can say the reduced products more effective on the isozymes. If the sterically hindered sulfonamide derivatives were compared between themselves, the derivatives containing sulfisoxazole generally seen to have stronger inhibitory effects than the ones containing sulfadiazine. It can be thought that, sulfisoxazole ring has positive contribution to the inhibition effect.

If we mention the selectivity of the compounds against isoenzymes, 8 has approximately ten-fold stronger inhibition potential on hCA II than hCA I. Also compounds 3, 7, and 9 exhibit two-fold stronger inhibition potential on hCA II than hCA I. So we can say that above mentioned compounds are more selective inhibitors for hCA II. Unlike other derivatives, the ones containing sulfisoxazole ring (6 and 10) be more selective against hCA I is quite remarkable.

The inhibition type of the compounds was determined noncompetitively. In another study of our group some pyrazole sulfonamides were synthesized and the inhibition effects of these compounds on esterase activities of hCA I and II isoenzymes were investigated as in vitro. Our inhibition type results in this study are consistent with those of our previous study (Balseven et al., 2013).

4 Conclusions

Up to now, inhibitory effects of various different anions, metal ions, drugs, phenols and sulfonamides have investigated the activity of CA isozymes (Ekinci et al., 2007). Inhibition studies on carbonic anhydrase are important for clarification of the mechanisms of enzyme catalysis and designating new drugs. In this study novel pyrazole-3,4-dicarboxamide (3–10) derivatives bearing sulfonamide moiety were synthesized and inhibitory effects of these derivatives on esterase activities of these isoenzymes have been studied as in vitro. Compound 4 possess remarkable inhibition effects on hCA I and hCA II and may be used in the generation of potent CAIs.

Acknowledgments

The authors gratefully acknowledge the research support of the Dumlupınar University Research Fund (Grant No. 2010/14). Also they would like to thank Atatürk University Faculty of Sciences Department of Chemistry for providing spectroanalytical facilities and Dumlupınar University Faculty of Arts and Sciences Department of Physics for FT-IR measurements.

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Introduction

Experimental protocols

Material and methods
Synthesis of target inhibitors
Biological activity evaluation

Results and discussion

Chemistry
Biological activity results

Conclusions

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[1] Akbaba Y., Balaydın H.T., Menzek A., Göksu S., Şahin E., Ekinci D., . Synthesis and biological evaluation of novel bromophenol derivatives as carbonic anhydrase inhibitors. Arch. Pharm. Chem. Life Sci.. 2013;346:447-454.
[Google Scholar]

[2] Almajan G.L., Innocenti A., Puccetti L., Manole G., Barbuceanu S., Saramet I., Scozzafava A., Supuran C.T., . Carbonic anhydrase inhibitors. Inhibition of the cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-thiadiazole- and 1,2,4-triazole-thiols. Bioorg. Med. Chem. Lett.. 2005;15:2347-2352.
[Google Scholar]

[3] Alterio V., Di Fiore A., D’Ambrosio K., Supuran C.T., De Simone G., . Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem. Rev.. 2012;112:4421-4468.
[Google Scholar]

[4] Balseven H., İşgör M.M., Mert S., Alım Z., Beydemir Ş., Ok S., Kasımoğulları R., . Facile synthesis and characterization of novel pyrazole-sulfonamides and their inhibition effects on human carbonic anhydrase isoenzymes. Bioorg. Med. Chem.. 2013;21:21-27.
[Google Scholar]

[5] Bekhit A.A., Hymete A., Bekhit A.E.D.A., Damtew A., Aboul-Enein H.Y., . Pyrazoles as promising scaffold for the synthesis of anti-inflammatory and/or antimicrobial agent: a review. Mini Rev. Med. Chem.. 2010;10:1014-1033.
[Google Scholar]

[6] Bilginer S., Unluer E., Gul H.I., Mete E., Isik S., Vullo D., Ozensoy-Guler O., Beyaztas S., Capasso C., Supuran C.T., . Carbonic anhydrase inhibitors. Phenols incorporating 2- or 3-pyridyl-ethenylcarbonyl and tertiary amine moieties strongly inhibit Saccharomyces cerevisiae β-carbonic anhydrase. J. Enzyme Inhib. Med. Chem.. 2014;29:495-499.
[Google Scholar]

[7] Brzozowski Z., Slawinski J., Saczewski F., Innocenti A., Supuran C.T., . Carbonic anhydrase inhibitors: synthesis and inhibition of the human cytosolic isozymes I and II and transmembrane isozymes IX, XII (cancer-associated) and XIV with 4-substituted 3-pyridinesulfonamides. Eur. J. Med. Chem.. 2010;45:2396-2404.
[Google Scholar]

[8] Carta F., Aggarwal M., Maresca A., Scozzafava A., McKenna R., Masini E., Supuran C.T., . Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo. J. Med. Chem.. 2012;55:1721-1730.
[Google Scholar]

[9] Carta F., Temperini C., Innocenti A., Scozzafava A., Kaila K., Supuran C.T., . Polyamines inhibit carbonic anhydrases by anchoring to the zinc-coordinated water molecule. J. Med. Chem.. 2010;53:5511-5522.
[Google Scholar]

[10] Carta F., Vullo D., Maresca A., Scozzafava A., Supuran C.T., . Mono-/dihydroxybenzoic acid esters and phenol pyridinium derivatives as inhibitors of the mammalian carbonic anhydrase isoforms I, II, VII, IX, XII and XIV. Bioorg. Med. Chem.. 2013;21:1564-1569.
[Google Scholar]

[11] Dale D.J., Dunn P.J., Golightly C., Hughes M.L., Levett P.C., Pearce A.K., Searle P.M., Ward G., Wood A.S., . The chemical development of the commercial route to Sildenafil: a case history. Org. Process Res. Dev.. 2000;4:17-22.
[Google Scholar]

[12] Davis R.A., Vullo D., Maresca A., Supuran C.T., Poulsen S.A., . Natural product coumarins that inhibit human carbonic anhydrases. Bioorg. Med. Chem.. 2013;21:1539-1543.
[Google Scholar]

[13] Di Fiore A., Pedone C., D’Ambrosio K., Scozzafava A., De Simone G., Supuran C.T., . Carbonic anhydrase inhibitors: valdecoxib binds to a different active site region of the human isoform II as compared to the structurally related cyclooxygenase II ‘selective’ inhibitor celecoxib. Bioorg. Med. Chem. Lett.. 2006;16:437-442.
[Google Scholar]

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Novel pyrazole-3,4-dicarboxamides bearing biologically active sulfonamide moiety as potential carbonic anhydrase inhibitors

Abstract