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

ORIGINAL ARTICLE

Novel pyrazole-3,4-dicarboxamides bearing

biologically active sulfonamide moiety as potential

carbonic anhydrase inhibitors

Samet Mert

, Zuhal Alım

, Mehmet Mustafa _Isßgo¨r

, Barısß Anıl

,

Rahmi Kasımog˘ulları

_{, S}

_{ßu¨kru¨ Beydemir}

a

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

b

Chemistry Department, Faculty of Sciences, Atatu¨rk University, 25240 Erzurum, Turkey

Received 11 December 2014; accepted 23 May 2015 Available online 11 June 2015

KEYWORDS Pyrazole; Pyrazole-3,4-dicarboxamide; Synthesis; Reduction; Carbonic anhydrase; Enzyme inhibition

Abstract In this study a series of pyrazole-3,4-dicarboxamide (3–10) derivatives bearing sulfon-amide 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,13_{C 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-sulfa nilamide affinity column chromatography and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied as in vitro. The Kivalues of compounds

were found in the range of 0.056–110.400 lM for hCA I and 0.057–533.400 lM for hCA II. Compound 4 has the highest inhibitory effect for hCA I and hCA II while compound 5 showed low-est inhibition. The structure–activity relationships for the inhibition of these isoforms with the pyrazole-sulfonamides reported here were also elucidated.

ª 2015 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that catalyze a very simple but essential phys-iological reaction, carbon dioxide hydration to bicarbonate and protons (Supuran, 2010, 2012; Alterio et al., 2012; Gu¨zel-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,

* Corresponding authors.

E-mail addresses:rahmikasimoglu@hotmail.com(R. Kasımog˘ulları),

beydemir@atauni.edu.tr(Sß. Beydemir).

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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 drug-gable targets for antiglaucoma, anticonvulsants, and diuretics (Gu¨zel-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) inhibi-tors (Kasımog˘ulları et al., 2011; Go¨cer 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 synthe-sis of new CAIs that have a good inhibitory profile with min-imal 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, anti-inflammatory, 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) (seeFig. 1). In continuation of our research efforts of the discovery of novel

pyrazole-sulfonamide derivatives (Balseven et al., 2013; Kasımog˘ulları et al., 2011; Sßen et al., 2013), herein we describe synthesis, characterization, and evaluation of the inhibition effects of sul-fonamide 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 (seeScheme 1andTable 1). Based on this informa-tion, this article has two main aims. First, we aimed to make a comparison between the activities of our inhibitors that con-tain free SO2NH2 groups (3, 4, 7, and 8) and sterically

hin-dered 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 anhy-drase isozymes I and II.

2. Experimental protocols

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

Sildenafil N N Cl Cl Cl NH O _N Rimonabant N N F3C S NH2 O O Celecoxib N N HN N O O S N N O O

Figure 1 Structures of Sildenafil (an oral therapy drug for erectile dysfunction), Celecoxib (non-steroidal antiinflammatory drug), and Rimonabant (anorectic antiobesity drug).

N N O Cl O Cl Ph NO2 4 R'-NH₂ reflux, 5h N N O NHR' O R'HN Ph NO2 2 3-6 _7-10 HCl Na₂SX9H2O N N O OH O HO Ph NO2 1 SOCl2,80 °C reflux, 5h N N O NHR' O R'HN Ph NH2

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 com-pounds. Melting points (C, uncorrected) were taken in open

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

Compound Ki(lM) R0 _R00 _{CA I CA II} 3 ANO2 0.331 0.163 4 ANO2 0.056 0.057 5 ANO2 No inhibition 533.400 6 ANO2 14.283 211.000 7 ANH2 0.306 0.121 8 ANH2 5.108 0.599 9 ANH2 110.400 45.470 10 ANH2 23.940 54.470

capillaries on a Barnstead Electrothermal 9200 melting point apparatus (Electrothermal Co, Essex, UK). Infrared (IR) spec-tra 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 (cm1) (Bruker Optik GmbH, Ettlingen, Germany).1H NMR and13C NMR spectra were recorded in DMSO-d6solutions at 298 K using a Bruker

Ultrashield-400 spectrometer operating at 400.00 MHz for1_H

and 101.00 MHz for13C (Bruker BioSpin GmbH Silberstreifen D-76287, Rheinstetten, Germany). The center of the peaks of DMSO-d6 [d (ppm): 2.50 (1H) and d (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 per-formed on a Leco CHNS-932 elemental analyser (LECO Corporation, Saint Joseph, Michigan, USA).

2.2. Synthesis of target inhibitors

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 sul-fonamide derivative (4 mmol) was heated under reflux condi-tion 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. 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-thiadia zole-2-sulfonamide (0.72 g, 4 mmol) according to the general procedure. The crude product was purified by crystallization from ethanol. Yield: 92%; mp: 230–232C; IR (t, cm1): 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) d (ppm):

13.97 (br, s, 2H, 2CONH), 8.37 and 8.35 (s, 4H, 2SO2NH2),

8.43–7.44 (m, 9H, ArH);13_{C NMR (101 MHz, DMSO-d} 6) d

(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 (CANO2), 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. 1-(3-Nitrophenyl)-5-phenyl-N3,N4 -bis(4-sulfamoylpheny-l)-1H-pyrazole-3,4-dicarboxamide (4)

Synthesized from 2 (0.39 g, 1 mmol) and 4-aminobenzene-sulfonamide (0.69 g, 4 mmol) according to the general proce-dure. The crude product was purified by crystallization from MeOH/H2O (1:1). Yield: 90 %; mp: 227–229C; IR (t,

cm1): 3254 (NH), 3038 (ArCH), 1628 (C = O, amide), 1591–1483 (C‚C and C‚N), 1526 (NO2), 1316 and 1151

(S‚O);1_{H NMR (400 MHz, DMSO-d}

6) d (ppm): 13.12 and

10.91 (s, 2H, 2CONH), 8.33–7.42 (m, 17H, ArH), 7.31 and 7.28 (s, 4H, 2SO2NH2);

13

C NMR (101 MHz, DMSO-d6) d

(ppm): 160.89 and 159.82 (C‚O, amide), 147.90 (CANO2),

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. 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 pro-duct was purified by crystallization from EtOH/DMF (2:1). Yield: 94%; mp: 149–151C; IR (t, cm1_{): 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) d (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) d (ppm):

158.34 and 158.22 (C‚O, amide), 157.21 (pyrimidine C2), 156.94 and 153.02 (pyrimidine C4 and C6), 148.09 (CANO2),

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. N3,N4 -bis(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)p- henyl)-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3,4-dicarboxa-mide (6)

Synthesized from 2 (0.39 g, 1 mmol) and Sulfisoxazole (1.07 g, 4 mmol) according to the general procedure. The crude pro-duct was purified by crystallization from MeOH/H2O (3:1).

Yield: 93 %; mp: 233–235C; IR (t, cm1): 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) d (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); 13

C NMR (101 MHz, DMSO-d6) d (ppm): 161.42 (isoxazole

C3), 161.06 and 159.90 (C‚O, amide), 155.49 (isoxazole C5), 147.91 (CANO2), 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. 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 com-pound (3–6) (2 mmol) in methanol. The reaction mixture was boiled for 1 h. Then the reaction mixture was cooled and acid-ified with concentrated HCl. The precipitated product was fil-tered 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. 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: 260–262C; IR (t, cm1_{): 3332 and 3284 (NH}

and NH2), 3061 (ArCH), 1667 (C‚O, amide), 1605–1411

(C‚C and C‚N), 1343 and 1166 (S‚O); 1_{H NMR}

(400 MHz, DMSO-d6) d (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) d

(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 (CANH2), 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. 1-(3-Aminophenyl)-5-phenyl-N3,N4 -bis(4-sulfamoylphen-yl)-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: 270–272C; IR (t, cm1_{): 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) d (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); 13

C NMR (100 MHz, DMSO-d6) d (ppm): 161.54 and 161.34 (C‚O,

amide), 149.38 (CANH2), 146.20 (pyrazole C-3), 142.63

(pyra-zole C-5), 120.93 (pyra(pyra-zole 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. 1-(3-Aminophenyl)-5-phenyl-N3_,N4 -bis(4-(N-(pyrimidi-n-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: 224–226C; IR (t, cm1): 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); 1_{H NMR}

(400 MHz, DMSO-d6) d (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); 13

C NMR (101 MHz, DMSO-d6) d (ppm): 158.34 and 158.21

(C‚O, amide), 157.20 (pyrimidine C2), 156.94 and 153.00 (pyrimidine C4 and C6), 149.58 (CANH2), 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. 1-(3-Aminophenyl)-N3_,N4

-bis(4-(N-(3,4-dimethylisox- azol-5-yl)sulfamoyl)phenyl)-5-phenyl-1H-pyrazole-3,4-dicarbo-xamide (10)

Synthesized according to the general procedure and the crude product was purified by crystallization from methanol. Yield: 87%; mp: 226–228C; IR (t, cm1): 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); 1_{H NMR (400 MHz, DMSO-d}

6) d (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); 13

C NMR (101 MHz, DMSO-d6) d (ppm): 161.73 and 160.10

(C‚O, amide), 161.34 (isoxazole C3), 155.89 and 155.83 (isox-azole C5), 149.59 (CANH2), 143.57 (pyrazole C3), 143.29

(pyrazole C5), 120.34 (pyrazole C4), 104.56 and 104.53 (isoxa-zole C4), 10.33 (isoxa(isoxa-zole-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. Biological activity evaluation

2.3.1. Hemolysate preparation

Erythrocytes were purified from fresh human blood, which was obtained from the University Hospital Blood Center of Erzurum Atatu¨rk University. Following low speed centrifuga-tion (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 cen-trifugation (20,000 rpm for 30 min.) at 4C and the pH of the hemolysate adjusted to 8.7 with solid Tris.

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; Kasımog˘ulları 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 elu-tion by affinity chromatography. CO2-hydratase activity was

determined in eluted fractions and the active fractions were collected.

2.3.3. Hydratase and esterase activity assay

2.3.3.1. Hydratase activity assay.Carbonic anhydrase catalyzes the hydration of carbon dioxide to form bicarbonate and a proton.

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. Esterase activity assay. Carbonic anhydrase enzymes catalyze some non-physiological reactions under in vitro condi-tions. 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 25C using a spectrophotometer (Verpoorte et al., 1967).

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 25C using a spectrophotometer according to the method described byVerpoorte et al. (1967). The enzy-matic 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 activ-ities were measured in the presence of different substrate con-centrations. Control activity in the absence of inhibitor was assumed to be 100%. For each compound, a percent activity versus inhibitor concentration graph was plotted. Ki values

of the compounds were calculated by measuring enzyme activ-ity at three different inhibitor concentrations with five different substrate concentrations. Lineweaver–Burk curves (seeFig. 2) were used for the determination of Ki and inhibition type

(Balseven et al., 2013; Kasımog˘ulları et al., 2011).

3. Results and discussion

3.1. Chemistry

Recently it has been reported that Celecoxib, pyrazole and sul-fonamide 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 ver-sus 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-dicarb-oxylic acid (1) was synthesized according to our previous stud-ies 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 (sulfanil-amide), 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 inScheme 1 andTable 1 was used to prepare the pyrazole-sulfonamide derivatives 3–10, as reported earlier by our group (Balseven et al., 2013; Kasımog˘ulları et al., 2011).

The synthesized molecules have been characterized by var-ious spectral and analytical techniques including FT-IR, 1H NMR, 13_{C NMR and Elemental analysis. The infrared (IR)}

spectra of all pyrazole-sulfonamide (3–10) derivatives showed NH or NH2stretching bands at 3399–3248 cm1and showed

sharp peaks for the carbonyl groups in the region between 1694 and 1628 cml. S‚O asymmetric and symmetric stretch-ing bands were observed at 1378–1314 cml and 1172– 1151 cml, respectively. The compounds 3–6 exhibited

CA I CA II 0 5 10 15 20 25 30 35 -2 0 2 4 6 8 1/V (μmol/min) -1 1/[S]x103_M-1 Control 30% inhibition 50% inhibition 70% inhibition 0 10 20 30 40 50 60 70 -2 3 8 1/V (μmol/min) -1 1/[S]x103_M-1 Control 30% inhibition 50% inhibition 70% inhibition

Figure 2 Lineweaver–Burk graphs of the compound 4 on CA I and CA II.

asymmetric and symmetric stretching bands at around 1525 cm1 and 1350 cm1 that belong to NO2 groups.

Aromatic CAH stretching bands were observed in the region between 3097 and 3036 cm1. The aliphatic CAH stretching bands of 6 and 10 were observed at 2980 cm1 and 2933 cm1, respectively. The absorption values of other func-tional groups appeared in the expected regions and they were consistent with the literature (Kasımog˘ulları et al., 2011; Sener et al., 2004; Jennings and Lovely, 1991) (see Experimental for details).

In the 1_{H 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 around11 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 pyrim-idine rings appeared at around7 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.

In13C NMR spectra the peaks of C‚O for amides (3–10) were observed between 165.98 and 158.21 ppm. Other charac-teristic 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. In13_{C 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, respec-tively. Besides in the spectrum of 6 and 10; C3, C4 and C5 car-bons 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 com-pounds were in full agreement with the proposed structures (see Experimental andTable 1).

3.2. Biological activity results

CA inhibitors have been used as antiglaucoma, diuretics, anti-convulsants, antiobesity, antitumor agents and tumor diagnos-tic 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 iso-zymes (hCA I and hCA II). For this aim, CA isoiso-zymes (hCA I and II) were purified from human erythrocytes by Sepharose-4B-L-tyrosine affinity chromatography. The purifi-cation results for hCA I depicted a 67% yield, 670 U/mg speci-fic activity and approximately 81 purispeci-fication 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 purifica-tion 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 low-est inhibition effects for hCA II.

Compound 4 has the strongest inhibitory effect on hCA I and hCA II activities. Kivalue of this compound is almost in

nanomolar concentration range (0.056 lM for hCA I and 0.057 lM for hCA II). Also compounds 3 and 7 depicted rel-atively strong inhibitory effects on both izoenzymes. According to decreasing inhibition potential on hCA I, com-pounds 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 (seeTable 1).

Also structure activity relationships can be drawn with a comparison between functional groups of the synthesized com-pounds. 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 selec-tive against hCA I is quite remarkable.

The inhibition type of the compounds was determined non-competitively. 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 isoen-zymes 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 Atatu¨rk 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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