Potential inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis and docking studies of new 1,3,4-thiadiazole derivatives

Bioorganic & Medicinal Chemistry 25 (2017) 3547–3554

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Potential inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis and docking studies of new 1,3,4-thiadiazole derivatives Mehlika Dilek Altıntop a,⇑, Belgin Sever a, Ahmet Özdemir a, Kaan Kucukoglu b, Hicran Onem c, Hayrunnisa Nadaroglu c,d, Zafer Asım Kaplancıklı a a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisßehir, Turkey Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bülent Ecevit University, Zonguldak, Turkey c Department of Nano-Science and Nano-Engineering, Faculty of Engineering, Atatürk University, 25240 Erzurum, Turkey d Department of Food Technology, Erzurum Vocational Training School, Atatürk University, 25240 Erzurum, Turkey b

a r t i c l e

i n f o

Article history: Received 9 March 2017 Revised 29 April 2017 Accepted 3 May 2017 Available online 5 May 2017 Keywords: Carbonic anhydrase Furan Hydrazone Thiadiazole Molecular docking

a b s t r a c t In the last years, inhibition of carbonic anhydrase (CA) has emerged as a promising approach for pharmacologic intervention in a variety of disorders such as glaucoma, epilepsy, obesity, and cancer. As a consequence, the design of CA inhibitors (CAIs) is a highly dynamic field of medicinal chemistry. Due to the therapeutic potential of thiadiazoles as CAIs, new 1,3,4-thiadiazole derivatives were synthesized and investigated for their inhibitory effects on hCA I and hCA II. Although the tested compounds did not carry a sulfonamide group, an important pharmacophore for CA inhibitory activity, it was a remarkable finding that most of them were more effective on hCAs than acetazolamide (AAZ), the reference agent. Among these compounds, N0 -((5-(4-chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol2-yl)thio)acetohydrazide (3) was found to be the most effective compound on hCA I with an IC50 value of 0.14 nM, whereas N0 -((5-(2-chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (1) was found to be the most potent compound on hCA II with an IC50 value of 0.15 nM. According to molecular docking studies, all compounds exhibited high affinity and good amino acid interactions similar to AAZ on the both active sites of hCA I and hCA II enzymes. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Carbonic anhydrases (CAs; EC 4.2.1.1) are ubiquitous zinc-containing metalloenzymes involved in the catalysis of a simple but essential physiological reaction: the reversible hydration of carbon dioxide to bicarbonate ion and proton.1,2 These enzymes play a pivotal role in crucial physiological processes related to respiration and acid-base regulation, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions (e.g. gluconeogenesis, lipogenesis and ureagenesis). Several studies have showed that abnormal levels or activities of CAs have been often associated with different human diseases. As a consequence, CA isozymes have been identified as potential drug targets for the design of inhibitors or activators with clinical applications.1–10 Inhibition of CAs has emerged as a promising approach for the treatment of a variety of disorders such as glaucoma, epilepsy, ⇑ Corresponding author. E-mail address: [email protected] (M.D. Altıntop). http://dx.doi.org/10.1016/j.bmc.2017.05.005 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

obesity, and cancer. Recent studies have also pointed out the importance of CAs for the design of anti-infective agents with a novel mechanism of action.1–10 Thiadiazole has attracted a great deal of interest as a privileged scaffold due to its significant therapeutic potential.11–15 The sulfur atom of thiadiazole ring imparts improved liposolubility and the mesoionic nature of 1,3,4-thiadiazoles also allows these compounds to cross cellular membranes and interact with biological targets with distinct affinities.11,12 Thiadiazoles exhibit a broad spectrum of biological activities through the inhibition of CAs, cyclooxygenases (COXs), neutral endopeptidase (NEP), aminopeptidase N (APN), matrix metalloproteinases (MMPs), phosphodiesterases (PDEs) and c-Src/Abl tyrosine kinase.13 In particular, medicinal chemists have focused on the inhibitory effects of 1,3,4-thiadiazoles on CAs.11–24 Among thiadiazole derivatives, acetazolamide (AAZ) (N-(5-sulfamoyl-1,3,4-thiadiazole-2-yl)acetamide), a potent carbonic anhydrase inhibitor, is used in the treatment of glaucoma, acute mountain sickness, epileptic seizures.15,24 On the other hand, furan-based hydrazone

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5-(phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B) with ethyl chloroacetate in the presence of potassium carbonate. The treatment of the ester (C) with hydrazine hydrate gave the corresponding hydrazide (D). Finally, the nucleophilic addition-elimination reaction of the hydrazide (D) with 5-arylfurfurals afforded compounds 1–10. The 1H NMR and 13C NMR data were consistent with the assigned structures. In the 1H NMR spectra of compounds 1–10, the SACH2 protons were observed at 4.03–4.44 ppm. The N@CH proton appeared at 7.86–8.16 ppm. The NHAN@ proton was observed at 11.69–11.82 ppm. The Ph-NH-Ar proton was observed at 10.32–10.40 ppm. In the 1H NMR spectra of most compounds, SACH2, N@CH and NAH protons gave rise to two singlet peaks in accordance with the presence of the E and Z isomers.26 All other protons were observed within the expected regions. In their 13C NMR spectra, the signal due to the N@CH carbon was observed in the region 146.26–150.28 ppm, whereas the signal due to the C@O carbon appeared at 165.13–168.69 ppm. In the mass spectra of the compounds, [M+H]+ peaks were observed in agreement with their molecular formula. Compounds 1–10 were investigated for their in vitro inhibitory effects on hCA I and hCA II and IC50 values were calculated for all derivatives (Table 1). AAZ was used as the reference agent.

derivatives were also reported to exhibit notable inhibitory effects on hCA I.25 Prompted by the afore-mentioned findings and in the continuation of our ongoing research in the field of design, synthesis and biological evaluation of thiadiazole derivatives as hCA inhibitors,23 herein we reported the synthesis and inhibitory effects of a new series of N’-(5-arylfuran-2-yl)methylene-2-[(5-(phenylamino)1,3,4-thiadiazol-2-yl)thio]acetohydrazide derivatives on hCA I and hCA II. Ultimately, all compounds were docked to the active sites of hCA I and hCA II with AAZ in order to speculate the possible binding modes of these molecules in the active sites of these enzymes. 2. Results and discussion The synthesis of compounds 1–10 followed the general pathway outlined in Scheme 1. Initially, 4-phenylthiosemicarbazide (A) was obtained by the reaction of phenyl isothiocyanate with hydrazine hydrate. 5-(Phenylamino)-1,3,4-thiadiazole-2(3H)thione (B) were synthesized via the ring closure reaction of 4phenylthiosemicarbazide (A) with carbon disulfide in the presence of potassium hydroxide. Ethyl 2-[[5-(phenylamino)-1,3,4thiadiazol-2-yl]thio]acetate (C) was obtained by the reaction of

N

C

S

H

S

N

C

NHNH 2 H

a

N

b

N H

A

N

S

S

B

c

N

N

N

d

N

NHNH2 N H

OEt

S

S

N H

S

S

O

O

C

D e

N

Compound 1 2 3 4 5 6 7 8 9 10

N H N

N H

S

N

S O

O

1-10

R 2-Cl 3-Cl 4-Cl 2-NO2 3-NO2 4-NO2 4-Br 2,4-diCl 3,4-diCl 4-Cl-2-NO2

R

Scheme 1. The synthetic route for the preparation of the thiadiazole derivatives (1–10). Reagents and conditions: (a) NH2NH2H2O, ethanol, rt, 5 h; (b) (1) CS2/KOH, ethanol, reflux, 10 h; (2) HCl, pH 4–5; (c) ClCH2COOEt, K2CO3, acetone, reflux, 10 h; (d) NH2NH2H2O, ethanol, rt, 3 h; (e) 5-arylfurfural, ethanol, reflux, 6 h.

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M.D. Altıntop et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3547–3554 Table 1 The results obtained from regression analysis graphs for hCA I and hCA II in the presence of the compounds. Compound

hCA I Inhibition

hCA II Inhibition

hCA I/hCA II

IC50 (nM)

Ki (nM)

Type of inhibition

IC50 (nM)

Ki (nM)

Type of inhibition

1 2 3 4 5 6 7 8 9 10

0.77 0.57 0.14 0.21 0.56 0.29 4.21 0.29 0.45 0.28

1.12 ± 0.11 0.77 ± 1.12 0.12 ± 0.16 0.12 ± 0.18 0.90 ± 0.03 0.63 ± 0.21 6.23 ± 0.23 0.37 ± 0.06 0.69 ± 0.63 0.90 ± 0.51

Competitive Competitive Competitive Non-Competitive Non-Competitive Non-Competitive Competitive Competitive Competitive Competitive

0.15 0.53 0.19 0.34 0.47 – 0.41 0.39 – –

0.64 ± 1.05 0.61 ± 0.36 0.27 ± 1.25 0.88 ± 0.25 0.66 ± 0.36 – 5.12 ± 0.86 0.66 ± 0.39 – –

Competitive Competitive Competitive Non-Competitive Non-Competitive – Competitive Competitive – –

Acetazolamide

5.8

8.89 ± 2.01

6.7

10.70 ± 4.23

The compounds were more effective on hCA I than AAZ (IC50 = 5.8 nM). The compounds showed notable inhibitory effects on hCA I with IC50 values ranging between 0.14 and 4.21 nM. In particular, compounds 3 and 4 were found to be the most potent hCA I inhibitors with IC50 values of 0.14 and 0.21 nM, respectively. The inhibitory effects of compounds 6, 8 and 10 on hCA I were fairly close. The effects of the chloro and nitro substituents on hCA I inhibitory activity revealed the following potency order: p-Cl > o-NO2 > 4-Cl-2-NO2 > p-NO2 > 2,4-diCl > 3,4-diCl > m-NO2 > m-Cl > o-Cl. Thus it can be concluded that the position of chloro and nitro substituents is important for hCA I inhibitory activity. Although bromo-substituted compound 7 was more effective than AAZ on hCA I, the compound showed less inhibitory activity than other derivatives against hCA I. It is noteworthy to indicate that p-bromo substituent significantly decreased hCA I inhibitory activity when compared with p-chloro substituent. It can be attributed to the steric hindrance caused by the bulky bromo substituent. Compounds 6, 9 and 10 showed no inhibitory activity against hCA II, whereas the other compounds were significantly more

5.25 1.07 0.76 0.28 1.19 – 10.29 0.76 – – 0.87

effective on hCA II than AAZ (IC50 = 6.7 nM). The compounds showed notable inhibitory effects on hCA II with IC50 values ranging between 0.15 and 0.53 nM. In particular, chloro-substituted compounds 1 and 3 showed notable inhibitory effects on hCA II with the IC50 values of 0.15 and 0.19 nM, respectively. Although compound 2 bearing m-chloro substituent was more effective than AAZ on hCA II, the compound showed less inhibitory activity than other derivatives against hCA II. 2,4-Dichloro substituted compound 8 exhibited inhibitory effect on hCA II with an IC50 value of 0.39 nM, whereas 3,4-dichloro substituted compound 9 did not show any inhibitory activity against hCA II. The results pointed out the importance of the position of chloro substituent for hCA II inhibitory activity. On the other hand, nitro-substituted compounds 4 and 5 showed inhibitory effects on hCA II with IC50 values of 0.34 and 0.47 nM, respectively. But compound 6 bearing p-nitro group did not show any inhibitory activity against hCA II. This outcome pointed out the significance of the position of nitro group for hCA II inhibitory activity.

Fig. 1. Docking positions of all synthesized compounds on hCA I (PDB code: 2FW4) and hCA II (PDB code: 5SZ5) together, respectively. Compounds 6, 9 and 10 were not docked to hCA II. (Ligand custom carbons are colored given as in the figure and the zinc atom is displayed in grey sphere).

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Ki constants were calculated between 0.12 and 6.23 nM for hCA I using Lineweaver-Burk plots and are presented in Table 1. Most of the compounds showed competitive inhibition, whilst the other compounds showed non-competitive inhibition. On the other hand, AAZ, showed a Ki value of 8.89 ± 2.01 nM against hCA I. The hCA I inhibitory effects of compounds 1–10 were found to be greater than that of AAZ, which was a clinically standard CA inhibitor. Compounds 1–10 showed Ki values varying from 0.27 to 5.12 nM against hCA II, the physiologically dominant isoform (Table 1). Thus, compound 1–10 were found to inhibit hCA II isozyme at high levels. The synthesized compounds probably interact with the distinct hydrophilic and hydrophobic halves of the active site of hCA II. Molecular docking studies were performed for the potent hCA inhibitors on the active sites of hCA I (PDB code: 2FW4) and hCA II (PDB code: 5SZ5) which were previously defined.27,28 AAZ, the potent carbonic anhydrase inhibitor, was chosen as a reference

ligand for docking studies due to its structural resemblance with the compounds. According to the results, all compounds showed good affinity to the active site of both enzymes as indicated in Fig. 1. The docking score, glide gscore and glide emodel results of the compounds were also detected similar to AAZ and each other as shown in Table 2. In particular, the residual interactions of compound 3 on hCA I and compound 1 on hCA II were demonstrated in Figs. 2 and 3. Generally the thiadiazole and the furan ring systems and the phenylamino, phenyl substitutions on these rings presented pi-pi stacking contacts with His64, His67, His 200 and Tyr204 residues on hCA I. However, the hydrazone group formed H-bonds with Gln92 and Tyr20 residues similar to the sulfonamide group of AAZ. Moreover, the nitro substituent in some compounds was engaged in salt-bridge formation with Asp72 on hCA I. As regards to hCA II, the thiadiazole, the furan ring systems and the phenylamino, phenyl substitutions on these rings presented pi-pi stacking and H-bonds with His4, His64, His94 and Trp5 residues.

Table 2 Docking score (kcal/mol), glide gscore (kcal/mol) and glide emodel (kcal/mol) results of all compounds for hCA I and hCA II. Compound

1 2 3 4 5 6 7 8 9 10 Acetazolamide

R

2-Cl 3-Cl 4-Cl 2-NO2 3-NO2 4-NO2 4-Br 2,4-diCl 3,4-diCl 4-Cl-2-NO2

hCA I

hCA II

Docking score

Glide gscore

Glide emodel

Docking score

Glide gscore

Glide emodel

0.78 1.22 3.08 2.94 1.14 1.17 3.23 3.21 2.30 2.93

0.78 1.22 3.08 2.94 1.14 1.17 3.23 3.21 2.30 2.93

39.87 37.21 41.20 43.26 38.71 37.50 44.28 45.15 36.81 41.40

4.50 3.84 3.74 3.81 4.02 – 4.12 3.87 – –

4.50 3.84 3.74 3.81 4.02 – 4.12 3.87 – –

65.08 60.96 54.96 60.38 60.94 – 59.35 58.07 – –

3.32

5.02

40.52

6.88

7.88

72.92

Fig. 2. Docking positions of compound 3 and AAZ on hCA I and interactions between compound 3 and hCA I, respectively. (Ligand custom carbons are colored in aquamarine for compound 3 and in dark green for AAZ; Green line: Pi-pi stacking and the zinc atom is displayed in grey sphere).

M.D. Altıntop et al. / Bioorganic & Medicinal Chemistry 25 (2017) 3547–3554

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Fig. 3. Docking positions of compound 1 and AAZ on hCA II and interactions between compound 1 and hCA II, respectively. (Ligand custom carbons are colored in cyan for compound 1 and in dark green for AAZ; Green line: Pi-pi stacking and the zinc atom is displayed in grey sphere).

3. Conclusion In this paper, N0 -(5-arylfuran-2-yl)methylene-2-[(5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio]acetohydrazide derivatives (1– 10) were synthesized and evaluated for their ability to inhibit human carbonic anhydrase isozymes (hCA I and hCA II). In order to speculate the exact positions and the possible binding modes of these molecules, the potent hCA inhibitors were docked to the active sites of hCA I and hCA II with AAZ. Although the synthesized compounds do not carry a sulfonamide group, an important pharmacophore for hCA inhibitory activity, most of them showed more significant inhibitory activity against hCAs than AAZ. The inhibitory effects of four derivatives (1, 2, 5, 7) were more significant on hCA II than hCA I, whereas the inhibitory effects of three derivatives (3, 4 and 8) were more significant on hCA I than hCA II. Compounds 6, 9 and 10 showed notable inhibitory effects on hCA I, whereas these compounds showed no inhibitory activity against hCA II. According to docking studies, the potent hCA inhibitors were detected at the active sites of hCA I and hCA II enzymes reasonably forming pi-pi stacking and H-bonds with proper residues. In particular, compound 3 was identified as a potential hCA I inhibitor with an IC50 value of 0.14 nM when compared with AAZ (IC50 = 5.8 nM), whilst compound 1 was found to be a potential hCA II inhibitor with an IC50 value of 0.15 nM when compared with AAZ (IC50 = 6.7 nM). These agents stand out as promising candidates for further in vivo studies.

were recorded on a Shimadzu LCMS-8040 (Shimadzu, Kyoto, Japan). Elemental analyses were performed on a Perkin Elmer EAL 240 elemental analyser (Perkin-Elmer, Norwalk, CT, USA). 4.1.1. General procedure for the synthesis of the compounds 4.1.1.1. 4-Phenylthiosemicarbazide (A). A mixture of phenyl isothiocyanate (0.1 mol) and hydrazine hydrate (0.2 mol) in ethanol (30 mL) was stirred at room temperature for 5 hours and then filtered. The residue was crystallized from ethanol.29 4.1.1.2. 5-(Phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B). 4Phenylthiosemicarbazide (A) was dissolved in a solution of potassium hydroxide in ethanol. Carbon disulfide was then added while stirring and the reaction mixture was heated under reflux for 10 h. The solution was cooled and acidified to pH 4–5 with hydrochloric acid solution and crystallized from ethanol.29 4.1.1.3. Ethyl 2-[[5-(phenylamino)-1,3,4-thiadiazol-2-yl]thio]acetate (C). A mixture of 5-(phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B) (0.05 mol) and ethyl chloroacetate (0.05 mol) in the presence of potassium carbonate (0.05 mol) in acetone was refluxed for 10 h. The reaction mixture was cooled, filtered and the crude product was solved in water and then extracted with ether.29 4.1.1.4. 2-[[5-(Phenylamino)-1,3,4-thiadiazol-2-yl]thio]acetohydrazide (D). A mixture of the ester (C) (0.05 mol) and hydrazine hydrate (0.1 mol) in ethanol was stirred at room temperature for 3 h and then filtered.29

4. Experimental 4.1. Chemistry All reagents were purchased from commercial suppliers and were used without further purification. The melting points (M.p.) of the compounds were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on an IRPrestige21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). 1H NMR and 13C NMR spectra were recorded on a Bruker spectrometer (Bruker, Billerica, MA, USA). Mass spectra

4.1.1.5. N0 -(5-Arylfuran-2-yl)methylene-2-[(5-(phenylamino)-1,3,4thiadiazol-2-yl)thio]acetohydrazide derivatives (1–10). A mixture of the hydrazide (D) (0.01 mol) and 5-arylfurfural (0.01 mol) was refluxed in ethanol for 6 h, filtered and crystallized from ethanol. 4.1.1.5.1. N0 -((5-(2-Chlorophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (1). Vanilla colored solid. Yield: 80%; m.p. 207.1 °C. IR mmax (cm1): 3178.69 (NAH stretching), 3043.67 (Aromatic CAH stretching), 2927.94, 2902.87, 2858.51, 2825.72, 2785.21 (Aliphatic CAH stretching), 1678.07 (C@O stretching), 1600.92, 1570.06, 1531.48, 1496.76, 1462.04, 1435.04 (NAH bending, C@N and C@C stretching),

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1411.89, 1379.10, 1332.81, 1294.24 (CAH bending), 1251.80, 1226.73, 1197.79, 1165.00, 1138.00, 1082.07, 1041.56, 1022.27 (CAN, CAO stretching and aromatic CAH in plane bending), 927.76, 883.40, 869.90, 813.96, 798.53, 785.03, 746.45, 729.09, 713.66, 688.59, 675.09, 653.87, 592.15 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.42 (2H, s + s, SACH2), 6.96–7.11 (2H, m, ArAH), 7.24–7.60 (8H, m, ArAH), 7.89–7.91 (1H, m, ArAH), 7.95 + 8.16 (1H, s + s, CH@N), 10.35 + 10.39 (1H, s + s, NH), 11.74 + 11.78 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.50 and 36.47 (CH2), 113.11 (CH, d, J = 8.9 Hz), 114.93 (CH), 115.86 (CH), 117.38 (2CH, d, J = 3.2 Hz), 121.96 (CH, d, J = 6.4 Hz), 127.65 (2CH), 128.13 (CH, d, J = 16.7 Hz), 129.02 (CH, d, J = 5.1 Hz), 129.43 (CH, t, J = 8.0, 14.1, 22.4 Hz), 130.81 (C), 133.31 (C), 136.68 (C), 140.33 (C), 148.88 (CH, d, J = 5.7 Hz), 150.57 and 150.88 (C), 151.78 and 152.27 (C), 163.47 and 165.03 (C), 165.17 and 168.55 (C). MS (ESI) (m/z): 470 [M+H]+, 471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2 Calculated: C, 53.67; H, 3.43; N, 14.90. Found: C, 53.63; H, 3.47; N, 14.91. 4.1.1.5.2. N0 -((5-(3-Chlorophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (2). White solid. Yield: 82%; m.p. 211.6 °C. IR mmax (cm1): 3251.98, 3196.05 (NAH stretching), 3138.18, 3082.25, 3010.88 (Aromatic CAH stretching), 2870.08 (Aliphatic CAH stretching), 1672.28 (C@O stretching), 1614.42, 1602.85, 1560.41, 1489.05, 1458.18, 1446.61 (NAH bending, C@N and C@C stretching), 1429.25, 1404.18, 1338.60, 1313.52 (CAH bending), 1217.08, 1130.29, 1093.64, 1078.21, 1026.13, 1002.98 (CAN, CAO stretching and aromatic CAH in plane bending), 948.98, 933.55, 871.82, 779.24, 746.45, 711.73, 686.66, 673.16 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.44 (2H, s + s, SACH2), 6.96–7.06 (2H, m, ArAH), 7.22–7.58 (7H, m, ArAH), 7.71–7.81 (2H, m, ArAH), 7.93 + 8.13 (1H, s + s, CH@N), 10.35 + 10.40 (1H, s + s, NH), 11.74 + 11.77 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.56 and 36.47 (CH2), 109.66 (CH, d, J = 8.4 Hz), 115.52 (CH), 116.41 (CH), 117.38 (2CH, d, J = 4.4 Hz), 121.99 (CH, d, J = 7.7 Hz), 122.40 (CH, d, J = 12.1 Hz), 123.41 (CH), 127.92 (CH, d, J = 10.9 Hz), 129.07 (2CH, d, J = 6.4 Hz), 130.94 and 131.41 (C), 133.40 and 133.87 (C), 136.70 (C), 140.35 (C), 149.28 (CH, d, J = 7.1 Hz), 151.84 and 152.35 (C), 152.83 and 153.18 (C), 163.49 and 165.03 (C), 165.17 and 168.58 (C). MS (ESI) (m/z): 470 [M+H]+, 471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2 Calculated: C, 53.67; H, 3.43; N, 14.90. Found: C, 53.63; H, 3.47; N, 14.89. 4.1.1.5.3. N0 -((5-(4-Chlorophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (3). White solid. Yield: 86%; m.p. 234.8 °C. IR mmax (cm1): 3277.06, 3192.05 (NAH stretching), 3138.18, 3086.11, 3041.74 (Aromatic CAH stretching), 2978.09, 2914.44 (Aliphatic CAH stretching), 1670.35 (C@O stretching), 1651.07, 1616.35, 1600.92, 1552.70, 1485.19, 1475.54, 1446.61 (NAH bending, C@N and C@C stretching), 1409.96, 1396.46, 1328.95, 1305.81 (CAH bending), 1251.80, 1215.15, 1149.57, 1089.78, 1043.49, 1024.20, 1001.06 (CAN, CAO stretching and aromatic CAH in plane bending), 925.83, 831.32, 794.67, 750.31, 729.09, 692.44, 648.08 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.44 (2H, s + s, SACH2), 6.96–7.05 (2H, m, ArAH), 7.14 + 7.17 (1H, d + d, J = 4 Hz + J = 3.2 Hz, ArAH), 7.29–7.35 (2H, m, ArAH), 7.49–7.58 (4H, m, ArAH), 7.76–7.79 (2H, m, ArAH), 7.92 + 8.12 (1H, s + s, CH@N), 10.35 + 10.39 (1H, s + s, NH), 11.70 + 11.75 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.49 and 36.47 (CH2), 108.95 (CH, d, J = 9.0 Hz), 115.73 (CH), 116.53 (CH), 117.37 (2CH, d, J = 3.9 Hz), 121.98 (CH, d, J = 6.4 Hz), 125.59 (2CH, d, J = 12.2 Hz), 128.28 (CH, d, J = 3.8 Hz), 129.07 (2CH, d, J = 4.5 Hz), 132.61 and 132.70 (C), 133.43 (C), 136.72 (C), 140.34 (C), 148.99 (CH, d, J = 9.6 Hz), 151.82 and 152.36 (C), 153.34 and 153.70 (C), 163.43 and 165.00 (C), 165.17 and 168.50 (C). MS (ESI) (m/z):

470 [M+H]+, 471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2 Calculated: C, 53.67; H, 3.43; N, 14.90. Found: C, 53.64; H, 3.45; N, 14.90. 4.1.1.5.4. N0 -((5-(2-Nitrophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (4). Yellow solid. Yield: 83%; m.p. 195.7 °C. IR mmax (cm1): 3325.28, 3273.20 (NAH stretching), 3118.90, 3074.53 (Aromatic CAH stretching), 2935.66, 2897.08 (Aliphatic CAH stretching), 1666.50 (C@O stretching), 1624.06, 1597.06, 1548.84, 1521.84, 1496.76, 1440.83 (NAH bending, C@N and C@C stretching), 1415.75, 1396.46, 1369.46, 1344.38, 1301.95 (CAH bending), 1230.58, 1192.01, 1072.42, 1028.06 (CAN, CAO stretching and aromatic CAH in plane bending), 999.13, 964.41, 947.05, 921.97, 893.04, 848.68, 802.39, 746.45, 686.66, 646.15, 626.87 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.38 (2H, s + s, SACH2), 6.96–7.08 (3H, m, ArAH), 7.29–7.35 (2H, m, ArAH), 7.54–7.65 (3H, m, ArAH), 7.71–7.78 (1H, m, ArAH), 7.86–8.14 (3H, m, ArAH and CH@N), 10.36 + 10.39 (1H, s + s, NH), 11.72 + 11.76 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.40 and 36.47 (CH2), 112.02 (CH, d, J = 19.3 Hz), 114.66 (CH), 115.44 (CH), 117.39 (2CH, d, J = 3.2 Hz), 121.97 (CH, d, J = 8.4 Hz), 124.06 (CH, d, J = 26.3 Hz), 128.93 (2CH, d, J = 32 Hz), 129.34 (CH), 129.76 (CH, d, J = 23.1 Hz), 132.39 (C), 132.86 (C), 136.67 and 140.37 (C), 147.02 (C), 149.21 and 149.64 (C), 150.28 (CH, d, J = 7.7 Hz), 151.85 and 152.34 (C), 163.53 and 165.08 (C), 165.15 and 168.62 (C). MS (ESI) (m/z): 481 [M+H]+. For C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49. Found: C, 52.45; H, 3.40; N, 17.50. 4.1.1.5.5. N0 -((5-(3-Nitrophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (5). Pale yellow solid. Yield: 85%; m.p. 218.1 °C. IR mmax (cm1): 3253.91, 3196.05 (NAH stretching), 3138.18, 3086.11 (Aromatic CAH stretching), 2991.59, 2862.36 (Aliphatic CAH stretching), 1670.35 (C@O stretching), 1610.56, 1562.34, 1525.69, 1489.05, 1446.61 (NAH bending, C@N and C@C stretching), 1348.24, 1309.67 (CAH bending), 1217.08, 1130.29, 1091.71, 1024.20, 1002.98 (CAN, CAO stretching and aromatic CAH in plane bending), 945.12, 891.11, 862.18, 792.74, 754.17, 736.81, 723.31, 711.73, 690.52, 680.87, 667.37 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.05 + 4.43 (2H, s + s, SACH2), 6.93–7.08 (2H, m, ArAH), 7.26–7.37 (3H, m, ArAH), 7.51 (1H, d, J = 8.0 Hz), 7.56 (1H, d, J = 8.8 Hz), 7.67–7.72 (1H, m, ArAH), 7.93–8.16 (3H, m, ArAH and CH@N), 8.43–8.46 (1H, m, ArAH), 10.32 + 10.39 (1H, s + s, NH), 11.75 + 11.81 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.56 and 36.49 (CH2), 110.56 (CH, d, J = 7.7 Hz), 115.41 (CH), 116.47 (CH), 117.37 (2CH, d, J = 6.4 Hz), 118.01 (CH), 121.96 (CH, d, J = 10.3 Hz), 122.41 (CH, d, J = 14.8 Hz), 129.04 (2CH, d, J = 9.0 Hz), 129.87 (CH, d, J = 18.6 Hz), 130.60 and 130.87 (C), 133.31 and 136.61 (C), 140.33 (C), 148.36 (C), 149.72 (CH, d, J = 7.1 Hz), 151.83 and 152.03 (C), 152.22 and 152.38 (C), 163.60 and 165.15 (C, d, J = 6.4 Hz), 168.67 (C). MS (ESI) (m/z): 481 [M+H]+. For C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49. Found: C, 52.46; H, 3.39; N, 17.49. 4.1.1.5.6. N0 -((5-(4-Nitrophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (6). Yellow solid. Yield: 90%; m.p. 237.4 °C. IR mmax (cm1): 3253.91, 3194.12 (NAH stretching), 3053.32 (Aromatic CAH stretching), 2914.44, 2821.86 (Aliphatic CAH stretching), 1672.28 (C@O stretching), 1622.13, 1595.13, 1573.91, 1508.33, 1498.69, 1462.04 (NAH bending, C@N and C@C stretching), 1417.68, 1390.68, 1325.10 (CAH bending), 1159.22, 1103.28, 1041.56, 1029.99 (CAN, CAO stretching and aromatic CAH in plane bending), 977.91, 923.90, 852.54, 800.46, 742.59, 694.37, 682.80, 655.80 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.04 + 4.43 (2H, s + s, SACH2), 6.95–7.01 (1H, m, ArAH), 7.10 + 7.13 (1H, d + d, J = 3.6 Hz + J = 3.6 Hz, ArAH), 7.28–7.35 (2H, m, ArAH), 7.42 + 7.45 (1H, d + d, J = 4.0 Hz + J = 3.2 Hz, ArAH), 7.52 (1H, d, J = 8.0 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.95–8.16 (3H, m, ArAH and

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CH@N), 8.28–8.31 (2H, m, ArAH), 10.33 + 10.39 (1H, s + s, NH), 11.78 + 11.82 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.48 and 36.47 (CH2), 112.31 (CH), 115.74 (CH), 116.60 (CH), 117.36 (2CH, d, J = 7.1 Hz), 121.97 (CH, d, J = 9.0 Hz), 124.43 (CH), 124.54 (2CH, d, J = 13.5 Hz), 129.05 (2CH, d, J = 7.0 Hz), 133.13 (C), 135.10 (C, d, J = 4.4 Hz), 136.45 (C), 140.31 (C), 146.26 (CH, d, J = 7.7 Hz), 150.58 and 151.79 (C, d, J = 8.3 Hz), 152.19 and 152.52 (C), 163.59 and 165.13 (C, d, J = 9.0 Hz), 168.67 (C). MS (ESI) (m/ z): 481 [M+H]+. For C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49. Found: C, 52.45; H, 3.40; N, 17.50. 4.1.1.5.7. N0 -((5-(4-Bromophenyl)furan-2-yl)methylene)-2-((5(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (7). Beige solid. Yield: 85%; m.p. 234.0 °C. IR mmax (cm1): 3277.06, 3194.12 (NAH stretching), 3138.18, 3086.11, 3039.81 (Aromatic CAH stretching), 2978.09, 2914.44 (Aliphatic CAH stretching), 1670.35 (C@O stretching), 1649.14, 1616.35, 1600.92, 1550.77, 1485.19, 1473.62, 1444.68 (NAH bending, C@N and C@C stretching), 1406.11, 1305.81 (CAH bending), 1251.80, 1213.23, 1147.65, 1087.85, 1070.49, 1043.49, 1024.20, 1002.98 (CAN, CAO stretching and aromatic CAH in plane bending), 921.97, 894.97, 829.39, 796.60, 748.38, 690.52 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.04 + 4.44 (2H, s + s, SACH2), 6.96–7.04 (2H, m, ArAH), 7.13 + 7.15 (1H, d + d, J = 3.6 Hz + J = 3.6 Hz, ArAH), 7.29–7.34 (2H, m, ArAH), 7.54–7.71 (6H, m, ArAH), 7.93 + 8.13 (1H, s + s, CH@N), 10.34 + 10.38 (1H, s + s, NH), 11.69 + 11.74 (1H, s + s, NH). 13C NMR (100 MHz, DMSOd6): 35.50 and 36.49 (CH2), 108.95 (CH, d, J = 8.3 Hz), 115.62 (CH), 116.46 (CH), 117.37 (2CH, d, J = 4.5 Hz), 121.24 (CH, d, J = 9.7 Hz), 121.95 (CH, d, J = 7.1 Hz), 125.78 (CH, d, J = 12.2 Hz), 128.57 (CH, d, J = 3.8 Hz), 129.02 (2CH, d, J = 4.5 Hz), 131.89 (C), 133.43 (C), 136.74 (C), 140.33 (C), 149.01 (CH, d, J = 8.3 Hz), 151.77 and 152.30 (C), 153.36 and 153.72 (C), 163.42 and 165.01 (C), 165.18 and 168.48 (C). MS (ESI) (m/z): 514 [M+H]-, 516 [M+H]+. For C21H16BrN5O2S2 Calculated: C, 49.03; H, 3.14; N, 13.61. Found: C, 49.05; H, 3.13; N, 13.60. 4.1.1.5.8. N0 -((5-(2,4-Dichlorophenyl)furan-2-yl)methylene)-2((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (8). White solid. Yield: 83%; m.p. 231.7 °C. IR mmax (cm1): 3219.19, 3184.48 (NAH stretching), 3136.25, 3066.82, 3028.24 (Aromatic CAH stretching), 2956.87, 2825.72, 2748.56 (Aliphatic CAH stretching), 1662.64 (C@O stretching), 1598.99, 1560.41, 1496.76, 1456.26, 1435.04 (NAH bending, C@N and C@C stretching), 1400.32, 1386.82, 1357.89, 1323.17, 1301.95 (CAH bending), 1253.73, 1240.23, 1213.23, 1141.86, 1112.93, 1097.50, 1033.85 (CAN, CAO stretching and aromatic CAH in plane bending), 989.48, 912.33, 864.11, 819.75, 806.25, 792.74, 756.10, 738.74, 688.59, 671.23, 644.22, 601.79 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.42 (2H, s + s, SACH2), 6.95–7.01 (1H, m, ArAH), 7.04 + 7.08 (1H, d + d, J = 3.2 Hz + J = 3.6 Hz, ArAH), 7.25–7.34 (3H, m, ArAH), 7.51– 7.57 (3H, m, ArAH), 7.69 + 7.72 (1H, d + d, J = 2.0 Hz + J = 2.0 Hz, ArAH), 7.87 (1H, d, J = 8.4 Hz), 7.94 + 8.16 (1H, s + s, CH@N), 10.33 + 10.38 (1H, s + s, NH), 11.75 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6): 35.48 and 36.46 (CH2), 113.52 (CH, d, J = 9.6 Hz), 114.98 (CH), 115.89 (CH), 117.35 (2CH, d, J = 5.8 Hz), 121.94 (CH, d, J = 7.6 Hz), 126.60 (CH), 127.89 (CH), 129.01 (2CH, d, J = 5.8 Hz), 129.22 (C), 130.05 and 130.17 (C, d, J = 4.5 Hz), 132.93 and 133.10 (C, d, J = 7.7 Hz), 136.53 (C), 140.30 (C), 149.12 (CH, d, J = 7.7 Hz), 149.58 and 149.87 (C), 151.75 and 152.18 (C), 163.49 and 165.05 (C), 165.15 and 168.60 (C). MS (ESI) (m/z): 504 [M+H]+, 506 [M+H]+++. For C21H15Cl2N5O2S2 Calculated: C, 50.01; H, 3.00; N, 13.88. Found: C, 50.02; H, 3.00; N, 13.87. 4.1.1.5.9. N0 -((5-(3,4-Dichlorophenyl)furan-2-yl)methylene)-2((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (9). Ivory solid. Yield: 84%; m.p. 208.1 °C. IR mmax (cm1): 3259.70, 3194.12 (NAH stretching), 3051.39 (Aromatic CAH stretching),

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2937.59, 2823.79 (Aliphatic CAH stretching), 1672.28 (C@O stretching), 1624.06, 1600.92, 1573.91, 1502.55, 1462.04, 1440.83 (NAH bending, C@N and C@C stretching), 1417.68, 1402.25, 1392.61, 1381.03, 1357.89, 1317.38, 1280.73 (CAH bending), 1226.73, 1159.22, 1134.14, 1099.43, 1043.49, 1026.13 (CAN, CAO stretching and aromatic CAH in plane bending), 977.91, 929.69, 883.40, 808.17, 788.89, 740.67, 669.30, 655.80 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.04 + 4.43 (2H, s + s, SACH2), 6.95–7.04 (2H, m, ArAH), 7.21–7.34 (3H, m, ArAH), 7.52–7.71 (4H, m, ArAH), 7.91–8.14 (2H, m, ArAH and CH@N), 10.32 + 10.37 (1H, s + s, NH), 11.72 + 11.77 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.56 and 36.49 (CH2), 110.09 (CH, d, J = 9.6 Hz), 115.35 (CH), 116.26 (CH), 117.36 (2CH, d, J = 6.5 Hz), 121.93 (CH, d, J = 8.4 Hz), 123.73 (CH, d, J = 16.0 Hz), 125.33 (CH, d, J = 3.2 Hz), 129.49 (2CH, d, J = 7.1 Hz), 129.85 (C, d, J = 3.8 Hz), 130.27 and 130.39 (C), 131.11 and 131.88 (C, d, J = 2.5 Hz), 133.26 and 136.64 (C), 140.32 (C), 149.48 (CH, d, J = 7.7 Hz), 151.81 (C, d, J = 11.5 Hz), 152.21 (C, d, J = 2.6 Hz), 163.49 and 165.06 (C), 165.17 and 168.57 (C). MS (ESI) (m/z): 504 [M+H]+, 506 [M+H]+++. For C21H15Cl2N5O2S2 Calculated: C, 50.01; H, 3.00; N, 13.88. Found: C, 50.01; H, 3.01; N, 13.86. 4.1.1.5.10. N0 -((5-(4-Chloro-2-nitrophenyl)furan-2-yl)methylene)2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (10). Yellow solid. Yield: 82%; m.p. 222.4 °C. IR mmax (cm1): 3194.12 (NAH stretching), 3086.11, 3047.53 (Aromatic CAH stretching), 2954.95, 2920.23, 2873.94 (Aliphatic CAH stretching), 1668.43 (C@O stretching), 1620.21, 1600.92, 1573.91, 1562.34, 1531.48, 1500.62, 1463.97 (NAH bending, C@N and C@C stretching), 1417.68, 1363.67, 1342.46, 1317.38, 1261.45 (CAH bending), 1220.94, 1199.72, 1174.65, 1085.92, 1053.13, 1031.92 (CAN, CAO stretching and aromatic CAH in plane bending), 972.12, 933.55, 883.40, 875.68, 837.11, 796.60, 767.67, 746.45, 725.23, 686.66, 663.51, 653.87, 642.30 (aromatic CAH out of plane bending and CAS stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.38 (2H, s + s, SACH2), 6.96–7.08 (3H, m, ArAH), 7.29–7.34 (2H, m, ArAH), 7.53–7.58 (3H, m, ArAH), 7.77–7.83 (1H, m, ArAH), 7.87–8.14 (2H, m, ArAH and CH@N), 10.35 + 10.38 (1H, s + s, NH), 11.73 + 11.78 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.38 and 36.47 (CH2), 112.60 (CH, d, J = 19.9 Hz), 114.71 (CH), 115.37 (CH), 117.38 (2CH, d, J = 3.8 Hz), 120.52 and 120.94 (CH), 121.97 (CH, d, J = 6.4 Hz), 123.83 and 124.07 (CH), 129.22 (2CH, d, J = 3.8 Hz), 132.20 and 132.60 (C, d, J = 9.6 Hz), 133.38 and 133.63 (C), 136.55 (C), 140.35 (C), 147.05 (CH, d, J = 4.5 Hz), 148.07 and 148.43 (C), 150.49 and 150.61 (C), 151.82 and 152.27 (C), 163.58 and 165.12 (C), 165.15 and 168.69 (C). MS (ESI) (m/z): 515 [M +H]+, 517 [M+H]+++. For C21H15ClN6O4S2 Calculated: C, 48.98; H, 2.94; N, 16.32. Found: C, 48.96; H, 2.95; N, 16.34. 4.2. Biochemistry 4.2.1. Purification of human carbonic anhydrase isozymes (hCA I and hCA II) from human erythrocytes by affinity chromatography Fresh human blood was obtained from the blood center, Atatürk University, it was stored at 4 °C used within 2–3 days at most. The blood samples were centrifuged to separate erythrocytes at 2500 rpm for 15 min and plasma and buffy coat were carefully removed. Then, underlying erythrocytes were washed with 0.9% NaCl solution twice and upper portions were also discarded. The erythrocytes were hemolyzed with distilled water at 0 °C, it was stirred for half an hour at 4 °C. The hemolysate was centrifuged at 20,000 rpm for 30 min and cell membranes were separated. pH was adjusted to 8.7 with solid Tris. So, the hemolysate was recovered to be applied to the column.30,31 The affinity gel was prepared on Sepharose-4B matrix. After Sepharose-4B was activated with CNBr, L-tyrosine was covalently fitted. Then sulfanilamide was coupled to tyrosine with diazotiza-

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tion reaction as a ligand. The hemolysate was applied to the prepared Sepharose-4B-L-tyrosine-sulfanilamide affinity column 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 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6), respectively. All procedures were performed at 4 °C.32 4.2.2. Determination of CA activity 4.2.2.1. Hydratase activity. Carbonic anhydrase activity was determined using the Wilbur-Anderson Method which was modified by Rickli et al.33,34 This method, as a result hydration of CO2 is released H+ ions and the pH changes were determined by means of bromine thymol blue indicator, based on measuring the elapsed time. Enzyme Unit (EU) was calculated using the equation (to  tc/tc) where to and tc are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively. 4.2.2.2. Inhibition assays. The inhibitory effects of compounds 1–10 and AAZ on the hydratase activity of hCA I and hCA II enzymes were investigated. IC50 values were calculated for the compounds at different concentrations while maintaining a constant substrate concentration. The activities of enzymes in the medium without inhibitors were used as 100% activity. The activity% values of enzymes were calculated by measuring the hydratase activity in the presence of different concentrations of inhibitors. The IC50 value was calculated by utilizing graphs of activity%-[I] for each inhibitor. The Ki values and inhibition types of the compounds were determined by Lineweaver-Burk graphics.33–35 4.2.2.3. Docking studies. Compounds 1–10 were docked to the active site of hCA I and hCA II. Ligands were set to the physiological pH (pH = 7.4) at the protonation step and crystal structures of hCA I and hCA II were retrieved from Protein Data Bank server (PDB codes: 2FW4 for hCA I; 5SZ5 for hCA II). The structures of compounds 1–10 were submitted in protein preparation module of Schrodinger’s Maestro molecular modeling package. In molecular docking simulations: Glide/XP docking protocols were applied for the prediction of topologies of compounds 1–10 at the active sites of target structures.36,37 Acknowledgement This study was supported by Anadolu University Scientific Research Projects Commission under the grant no: 1605S318.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.05.005. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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