New coumarin derivatives as carbonic anhydrase inhibitors

Abstract

In the current study, a series of 4-chloromethyl-7-hydroxy-coumarin derivatives containing imidazolium, benzimidazolium, bisbenzimidazolium and quaternary ammonium salts were synthesized, characterized and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these coumarins were confirmed by FT-IR, (1)H NMR, (13)C NMR and LC–MS analyses. Structure activity relationship study showed that 3d (IC_50: 79 μM for hCA I and 88 μM for hCA II) performed higher inhibitory activity than others.

Keywords::

• benzimidazolium
• carbonic anhydrase
• coumarin
• imidazolium
• inhibition

Introduction

Coumarins are members of a class of compounds called benzopyrones and show various biological and pharmacological properties (Anand et al. 2012). Besides the numerous activities of coumarin compounds, they are reported as inhibitors of metalloenzyme carbonic anhydrase (Maresca et al. 2009, Maresca et al. 2010, Supuran 2010, Carta et al. 2012). Imidazole and their derivatives are generally used in the preparation of N-heterocyclic carbenes (NHC). Besides using in the synthesis of NHC's, antimicrobial and inhibitory properties of imidazolium salts and imidazole derivatives were reported (Hindi et al. 2009, Khan et al. 2012). The quaternary ammonium salts exhibit antimicrobial activity against a wide range of bacteria, fungi and viruses (Tischer et al. 2012).

The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) catalyzes a very simple but critically important physiological reaction: the involvement of the Carbonic anhydrase (CA) enzyme family, which catalyzes the physiological hydration of CO₂ to yield bicarbonate and a proton, in many physiological/pathological processes opens up widespread opportunities for the development of diverse, specific inhibitors for clinical application (Supuran 2008, Thiry et al. 2006, McKiernan et al. 1997, Scozzafava et al. 2006).

The active site of most CAs contains a zinc ion (Zn²⁺), which is essential for catalysis. The CA reaction is involved in many physiological and pathological processes, including respiration and transport of CO₂ and bicarbonate between metabolizing tissues and lungs; pH and CO₂ homeostasis; electrolyte secretion in various tissues and organs; biosynthetic reactions such as gluconeogenesis, lipogenesis and ureagenesis; bone resorption; calcification; and tumorigenicity (Supuran et al. 2003, Smith and Ferry 2000, Stams and Christianson 2000, Pastorekova et al. 2004, Nishimori et al. 2007a, 2007b, Vullo et al. 2004, Nishimori et al. 2005a, Vullo et al. 2005a). Many of the CA isoenzymes involved in these processes are important therapeutic targets with the potential to be inhibited, to treat a range of disorders including edema, glaucoma, obesity, cancer, epilepsy and osteoporosis (Nishimori 2004, Vullo et al. 2003, Vullo et al. 2005b, Lehtonen et al. 2004, Nishimori et al. 2005b, Ashida and Brey 1995). Given the physiological importance of the CA, the metabolic impact of chemicals for crop production should receive greater importance.

The present study was carried out in order to synthesize, characterize and evaluate the biological activity of a new series of coumarin derivatives. In the current study, our main aim is to check whether the new derivatives may show higher inhibitory effect on CA enzymes with respect to the compounds previously investigated. The synthesized molecules have been characterized by various techniques including NMR, FT-IR and LC–MS. For in vitro inhibition studies, human erythrocyte carbonic anhydrase (CA I and CA II) isoenzymes were purified using Sepharose-4B l-tyrosine-sulphanilamide affinity chromatography. The inhibitory effects of novel compounds on the activities of purified human erythrocyte CA I and CA II isozymes were investigated.

Materials and methods

All reactions for the preparation of imidazolium and quaternary ammonium salts were carried out under argon using standard Schlenk-type flasks. Chemicals were obtained by Sigma Aldrich. DMF used as a solvent in the synthesis of imidazolium salts was dried using P₂O₅. N-methyl imidazole (2g), N-butyl imidazole (2h), N,N-dimethyl isopropyl amine (2i) and N,N-dimethyl benzyl amine (2j) were supplied commercially, used in the synthesis of imidazolium and quaternary ammonium salts and used without further purification.

Melting points were determined by Electrothermal-9200 melting point apparatus. FT-IR spectra were recorded on ATR unit in the range 400–4000 cm^{− 1} with Perkin Elmer Spectrum 100 Spectrophotometer. ¹H-NMR and ¹³C-NMR were recorded using a Bruker AC300P FT spectrometer operating at 300.13 MHz (¹H) and 75.47 MHz (¹³C). Chemical shifts are given in ppm relative to TMS, and coupling constants (J) in Hz. Elemental Analyses and LC–MS analyses were performed by IBTAM (Inonu University Scientific and Technological Research Central).

Synthesis of 4-chloromethyl-7-hydroxy-2H-chromen- 2-one (1)

4-chloromethyl-7-hydroxy-2H-chromen-2-one was synthesized by the procedure of Gumus et al. (2010). Crude product was washed with diethyl ether and dichloromethane and dried under vacuum. Yellow solid, yield: 54%, mp:173–175°C; IR (cm^{− 1}): 3207 (Ar-OH), 1682 (−C = O), 1603 (−C = C−); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm:10.72 (1H, s, −OH) 7.69–7.67 (d, J = 8.8 Hz, 1H, CHC(OH)CH′CH″,), 6.85 (dd, 1H, CHC(OH)CH’CH″, J_HH’ = 2.2 Hz, J_H’H″ = 8.8 Hz), 6.80 (d, J = 2.2 Hz, 1H, CHC(OH)CH’CH″), 6.40 (s, 1H, −C = CH), 4.95 (s, 2H, −CH₂Cl); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 161.8, 160.6, 155.7, 151.4, 127.0, 114.1, 112.6, 109.8, 104.1, 42.5.

General procedure for preparation of 1-alkylbenzimidazole and bisbenzimidazole compounds (2a–f)

1-alkylbenzimidazole and bisbenzimidazole compounds were synthesized by the procedure of Ozdemir et al. (2005). With this procedure N-methyl benzimidazole (2a), N-butyl benzimidazole (2b), N-benzyl benzimidazole (2c), N-(3,4,5-trimethoxybenzyl) benzimidazole (2d), 1,1’-(1,4-butanediyl)bis(benzimidazole) (2e) and 1,1’-(1,5pentanediyl)bis(benzimidazole) (2f) were synthesized.

General procedure for preparation of coumarin bearing benzimidazolium salts (3a–d)

Ten millimoles of 1-alkylbenzimidazole (2a–d) was dissolved in dried DMF (5 mL) and 10 mmol of 4-chloromethyl-7-hyrdroxy-2H-chromen-2-one was added into the solution, and the mixture was heated for 48 h at 90°C. After 48 h, the mixture was cooled to room temperature and diethyl ether was added to the mixture and the precipitates were collected by filtration. Crude product was washed with acetone and ethanol and dried under vacuum.

1-methyl-3-(4-methyl-7-hydroxy-2H-chromen-2-one) benzimidazolium chloride (3a)

White solid, yield; 77%, mp:277–279°C; anal.cal.for.:C₁₈H₁₅ClO₃N₂; C:63.07, H:4.41, N:8.17, found: C:63.14, H:4.45, N:8.0; IR (cm^{− 1}): 3240 (Ar-OH), 1700(−C = O), 1573 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.07 (s, 1H, Ar-OH), 9.93 (s, 1H, NCHN), 8.13–6.88 (m, 7H, ArH), 6.17 (s, 2H,-CH₂N), 5.72 (s, 1H, −CH = C−), 4.14 (s, 3H, −CH₃); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm:162.5, 160.4, 155.4, 149.6, 144.3, 132.6, 131.5, 127.4, 127.2, 126.5, 114.4, 114.1, 113.8, 109.5, 108.6, 103.0, 46.8, 34.1. LC–MS (m/z):345.2 [MH⁺].

1-butyl-3-(4-methyl-7-hydroxy-2H-chromen-2-one) benzimidazolium chloride (3b)

White solid, yield: 68%, mp: 231–233°C; anal.cal.for.: C₂₁H₂₁ClO₃N₂; C:65.54, H:5.50, N:7.28, found: C:65.61, H:5.58, N:7.18; IR (cm^{− 1}): 3245 (Ar-OH), 1709 (−C = O), 1567 (C-N); ¹ H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.03 (s, 1H, Ar-OH), 10.01 (s, 1H, NCHN), 8.05–6.87 (m, 7H, ArH), 6.14 (s, 2H, -CH₂N), 7.75 (s, 1H, −CH = C), 4.55 (t, J = 7.2 Hz, 2H, −CH₂CH₂CH₂CH₃), 1.99 (five, J = 7.3 Hz, 2H, CH₂CH₂CH₂CH₃), 1.38 (six, J = 7.5 Hz, 2H, CH₂CH₂CH₂CH₃), 0.97 (t, J = 7.3 Hz, 3H, CH₂CH₂CH₂CH₃); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.8, 162.5, 160.4, 155.5, 149.4, 143.7, 131.8, 131.7, 127.4, 127.3, 126.4, 114.5, 114.1, 113.8, 109.5, 108.9, 103.1, 47.3, 46.9, 30.8, 19.6, 13.9. LC–MS (m/z): 387.2 [MH⁺].

1-benzyl-3-(4-methyl-7-hydroxy-2H-chromen-2-one) benzimidazolium chloride (3c)

White solid, yield: 62%, mp: 210–213°C, anal.cal.for: C₂₄H₁₉ ClO₃N₂; C:68.82, H:4.57, N:6.69, found: C:68.95, H:4.60, N:6.59; IR (cm^{− 1}): 3230 (Ar-OH), 1705 (−C = O), 1563 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.39 (s, 1H, Ar-OH), 11.00 (s, 1H, NCHN), 8.10–6.72 (m, 12H, ArH), 6.15 (s, 2H, -CH₂N), 5.82 (s, 2H, Ph-CH₂-N), 5.79 (s, 1H, −CH = C); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm:162.5, 160.4, 160.1, 155.5, 149.4, 144.1, 134.2, 132.0, 131.6, 129.5, 129.3, 129.0, 128.0, 127.6, 127.4, 126.4, 114.6, 114.4, 113.8, 109.5, 109.0, 103.1, 56.5, 47.1. LC–MS (m/z):421.2 [MH⁺].

1-(3,4,5-trimethoxybenzyl)-3-(4-methyl-7-hydroxy-2H-chromen-2-one) benzimidazolium chloride (3d)

White solid, yield: 68%, mp:267–268°C, anal.cal.for: C₂₇H₂₅ ClO₆N₂; C:63.72, H:4.95, N:5.50, found: C63.74, H:4.96, N:5.47; IR (cm^{− 1}): 3220 (Ar-OH), 1731 (−C = O), 1562 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.04 (s, 1H, Ar-OH), 10.08 (s, 1H, NCHN), 8.21–6.87 (m, 9H, ArH), 6.16 (s, 2H, -CH₂N), 5.74 (s, 1H, -CH = C), 5.72 (s, 2H, Ph-CH₂-N), 3.77 (s, 6H, Ar-OCH₃(m-position)), 3.65 (s, 3H, Ar-OCH₃ (p- position)); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.8, 162.5, 160.4, 155.5, 153.7, 149.5, 143.8, 138.2, 131.9, 131.6, 129.3, 127.6, 127.4, 126.4, 114.7, 114.3, 113.8, 109.5, 108.9, 107.0, 103.1, 60.5, 56.5, 51.0, 47.0, 36.3. LC–MS (m/z):511.2 [MH⁺].

General procedure for preparation of coumarin bearing bisbenzimidazolium salts (3e, f)

Five millimoles of bisbenzimidazole (2e, f) was dissolved in dried DMF (5 mL) and 10 mmol of 4-chloromethyl- 7-hyrdroxy-2H-chromen-2-one was added into the solution, and the mixture was heated for 72 h at 90°C. After 72 h, the mixture was cooled to room temperature and diethyl ether was added to the mixture, and the precipitates were collected by filtration. Crude product was washed with acetone and ethanol and dried under vacuum.

3,3’-bis(4-methyl-7-hydroxy-chromen-2-one)- 1,1'buthylenedi(benzimidazolium)dichloride (3e)

White-yellow solid, yield: 48%, mp: 303–304°C; anal.cal.for: C₃₈H₃₂Cl₂O₆N₄; C:64.14, H:4.53, N:7.87, found: C:64.15, H:4.55, N:7.93; IR (cm^{− 1}): 3244 (Ar-OH), 1722 (−C = O), 1567 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.03 (s, 1H, Ar-OH), 10.22 (s, 1H, NCHN), 8.24–6.88 (m, 7H, ArH), 6.19 (s, 2H, −CH₂N), 5.74 (s, 1H, −CH = C), 4.67, (t, 2H, −NCH₂CH₂−), 2.11 (t, 2H, −NCH₂CH₂−); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.5, 160.4, 155.4, 144.0, 131.8, 131.7, 127.5, 127.3, 126.5, 114.5, 114.3, 113.8, 109.5, 108.7, 103.1, 47.0, 36.3, 25.8. LC–MS (m/z):713.2 [MH⁺].

3,3’-bis(4-methyl-7-hydroxy-chromen-2-one)-1,1'penthylenedi(benzimidazolium)dichloride (3f)

White-yellow solid, yield: 51%, mp: 252–255°C; anal.cal.for: C₃₉H₃₄Cl₂O₆N₄; C:64.55, H:4.72, N:7.72, found: C:64.57, H:4.72, N:7.81; IR (cm^{− 1}): 3205 (Ar-OH), 1722 (−C = O), 1568 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.05 (s, 1H, ArOH), 10.27 (s, 1H, NCHN), 8.21–7.79 (m, 7H, ArH), 6.02 (s, 2H, −CH2N), 5.74 (s, 1H, −CH = C), 4.59 (t, 2H, −NCH₂CH₂CH₂−), 1.95 (2H, −NCH₂CH₂CH₂−), 1.45 (2H, −NCH₂CH₂CH₂−); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.5, 160.4, 155.5, 143.9, 131.8, 131.6, 127.5, 127.3, 126.5, 114.5, 114.2, 113.8, 109.5, 108.8, 103.1, 47.3, 36.3, 31.2, 23.4. LC–MS (m/z):727.6 [MH⁺].

General procedure for preparation of coumarin bearing imidazolium salts (3g, h)

Ten millimoles of 1-alkylimidazole (2g, h) was dissolved in dried DMF (5 mL) and 10 mmol of 4-chloromethyl-7- hydroxy-2H-chromen-2-one was added into the solution, and the mixture was heated for 24 h at 90°C. After 24 h, the mixture was cooled to room temperature and diethyl ether was added to the mixture, and the precipitates were collected by filtration. The crude product was washed with acetone and ethanol and dried under vacuum.

1-methyl-3-(4-methyl-7-hydroxy-2H-chromen-2-one) imidazolium chloride (3g)

White solid, yield: 72%, mp: 262–264°C, anal.cal.for: C₁₄H₁₃ClO₃N₂; C:57.44, H:4.48, N:9.57, found: C:57.55, H:4.51, N:9.50; IR (cm^{− 1}): 3310 (Ar-OH), 1705 (−C = O), 1575 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.09 (s, 1H, Ar-OH), 9.36 (s, 1H, NCHN), 7.89–6.87 (m,5H, ArH, N-CH = CH-N), 5.81 (s, 3H), 3.91 (s, 3H, −CH₃); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.8, 162.5, 160.4, 155.4, 150.2, 138.2, 126.2, 124.7, 123.5, 113.9, 109.5, 103.1, 48.8, 36.6. LC–MS (m/z):295.1 [MH⁺].

1-butyl-3-(4-methyl-7-hydroxy-2H-chromen-2-one) imidazolium chloride (3h)

White solid, yield: 63%, mp: 180–184°C, anal.cal.for: C₁₇H₁₉ClO₃N_2; C: 60.99, H:5.72, N:8.37, found: C:61.04, H:5.77, N:8.29. IR (cm^{− 1}): 3240 (Ar-OH), 1710 (−C = O), 1567 (C-N); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 11.03 (s, 1H, Ar-OH), 9.44 (s, 1H, NCHN), 7.93–6.85 (m, 5H, ArH, N-CH = CH-N), 5.82 (s, 1H, −CH = C), 5.78 (s, 2H, −CH₂N), 4.22 (t, J = 7.3 Hz, 2H, −CH₂CH₂CH₂CH₃), 1.81 (five, J = 7.3 Hz, 2H, −CH₂CH₂CH₂CH₃), 1.24 (six, J = 7.5 Hz, 2H −CH₂CH₂CH₂CH₃), 0.91 (t, J = 7.3 Hz, 3H, CH₂CH₂CH₂CH₃); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.5, 160.4, 155.5, 149.9, 137.7, 126.2, 123.7, 123.5, 113.8, 109.9, 109.4, 103.1, 49.4, 48.9, 34.6, 19.4, 13.8. LC–MS (m/z):345.2 [MH⁺].

Synthesis of N-(4-methyl-7-hydroxy-2H-chromen-2-one)-N-isopropyl-N,N-dimethylammonium chloride (3i)

4-chloromethyl-7-hydroxy-2H-chrome-2-one (2.5 mmol) was dissolved in CH₃CN (10 mL) and N,N-dimethyl-N-isopropyl amine (2.5 mmol) was added into the solution, and the mixture was refluxed for 72 h. CH₃CN was evaporated under vacuum. The crude product was extracted with hot water which was evaporated under vacuum. Brown solid, yield: 10%, mp:204–207°C; anal.cal.for: C₁₅H₂₀ClO₃N; C:60.50, H:6.77, N:4.70, found: C:60.59, H:6.87, N:4.59; IR (cm^{− 1}): 3345 (Ar-OH), 1705 (−C = O), 1605 (−C = C−); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 7.99–7.96 (d, J = 8.8 Hz, 1H, CHC(OH)CH’CH″,), 6.91 (dd, 1H, CHC(OH)CH’CH″, J_HH’ = 2.2 Hz, J_H’H″ = 8.8 Hz), 6.84 (s, J = 2.2 Hz, 1H, CHC(OH)CH’CH″), 4.64 (s, 2H, −CH₂N), 4,08(seven, J = 6.5 Hz, 1H NCH(CH₃)₂), 2.96 (s, 6H, N(CH₃)₂), 1.46 (d, J = 6.5 Hz, 6H, NCH(CH₃)₂). ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.3, 160.0, 155.9, 142.8, 127.7, 119.7, 113.7, 111.8, 103.2, 68.5, 58.2, 47.6, 16.9. LC–MS (m/z):300.2 [MH⁺].

Synthesis of N-(4-methyl-7-hydroxy-2H-chromen-2- one)-N-benzyl-N,N-dimethylammonium chloride (3j)

4-chloromethyl-7-hydroxy-2H-chromen-2-one (3.75 mmol) was dissolved in DMF (10 mL) and N,N-dimethyl-N-benzyl amine (3.75 mmol) was added into the solution, and the mixture was heated for 24 h at 60°C. DMF was evaporated under vacuum. The crude product was extracted using distilled water which was evaporated under vacuum, too. The oily crude product was washed with THF and precipitated. White solid, yield: 16%, mp:194–196°C. anal.cal.for: C₁₉H₂₀ClO₃N: C:65.99, H:5.83, N:4.05, found: C:66.07, H:5.89, N:3.96; IR (cm^{− 1}): 3357 (Ar-OH), 1704 (−C = O), 1615 (−C = C−); ¹H-NMR (DMSO-d₆, 300 MHz) δ/ppm: 10.99 (s, 1H, Ar-OH), 8.21–6.85 (m, 8H, ArH), 6.62 (s, 1H, −CH = C), 4.90 (s, 2H, −CH₂N), 4.86 (s, 2H, Ph-CH₂N), 3.03 (s, 6H, N(CH₃)₂); ¹³C-NMR (DMSO-d₆, 75 MHz) δ/ppm: 162.0, 160.1, 156.0, 142.5, 133.7, 131.5, 129.9, 129.4, 129.2, 128.4, 113.6, 112.0, 103.2, 67.5, 61.8, 49.5.

Preparation of haemolysate and purification from blood red cells

Blood samples (25 mL) were taken from healthy human volunteers. They were anticoagulated with acid-citrate- dextrose, centrifuged at 2000 g for 20 min at 4°C and the supernatant was removed. The packed erythrocytes were washed three times with 0.9% NaCl and then haemolysed in cold water. The ghosts and any intact cells were removed by centrifugation at 2000 g for 25 min at 4°C, and the pH of the haemolysate was adjusted to pH 8.5 with solid Tris-base. The 25 mL haemolysate was applied to an affinity column containing L-tyrosine-sulphonamide-Sepharose-4B (Arslan et al. 1996) equilibrated with 25mMTris–HCl/0.1M Na₂SO₄ (pH 8.5). The affinity gel was washed with 50 mL of 25 mM Tris–HCl/22 mM Na₂SO₄ (pH 8.5). The human CA (hCA) isozymes were then eluted with 0.1 M NaCl/25 mM Na₂HPO₄ (pH 6.3) and 0.1 M CH₃COONa/0.5 M NaClO₄ (pH 5.6), which recovered hCA-I and hCA-II, respectively. Fractions of 3 mL were collected and their absorbance was measured at 280 nm.

CA enzyme assay

CA activity was measured by the Maren method which is based on determination of the time required for the pH to decrease from 10.0 to 7.4 due to CO₂ hydration (Maren 1960). The assay solution was 0.5 M Na₂CO₃/0.1 M NaHCO₃ (pH 10.0) and Phenol Red was added as the pH indicator. CO₂-hydratase activity (enzyme units (EU)) was calculated by using the equation t₀-tc/tc where t₀ and tc are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively.

In vitro inhibition studies

For the inhibition studies of coumarin, different concentrations of these compounds were added to the enzyme. Activity percentage values of CA for different concentrations of each coumarin were determined by regression analysis using Microsoft Office 2000 Excel. CA enzyme activity without a coumarin solution was accepted as 100% activity.

Results and discussion

For evaluating the CA inhibitory activity, all synthesized compounds (Scheme 1) were subjected to CA inhibition assay with CO₂ as substrate. The results showed that all synthesized compounds (3a–f) inhibited the hCA I and II enzyme activity. The inhibition values of 1–3f analogues against CAs are summarized in Table I. We have determined the IC₅₀ values of 79–620 μM for hCA I and 88–698 μM for hCA II, respectively. Among the compounds synthesized, 3d was found to be the most active one for CAs (IC_50:79 μM for hCA I and 88 μM for hCA II).

Scheme 1. Synthesized Salts. Reagents and Conditions; (i) 2a–d, DMF, 90°C, 48 h (Karatas et al. 2013). (ii) 2e–f, DMF, 90°C, 72 h. (iii) 2g–h, DMF, 90°C, 24 h. (iv) 2j, DMF, 60°C, 24 h. (v) 2j, CH₃CN, reflux, 72 h.

Table I. IC₅₀ values of novel coumarin compounds.

Compound	hCA I (μM)	hCA II (μM)
1	620	698
3a	138	137
3b	147	221
3c	153	154
3d	79	88
3e	81	94
3f	99	89
3g	264	327
3h	218	188
3i	198	167
3j	165	134

Coumarins/thiocoumarins may possess various tautomeric forms, such as the zwitterionic benzo (thio) pyrylium phenoxides, which may bind within the CA active site similarly to phenols (Ebbesen et al. 2009), that is, by anchoring to the zinc-bound water molecule/hydroxide ion (Nair et al. 1994). It was reported that the coumarin-binding site in CAs may interact with diverse compounds, such as the antiepileptic drug lacosamide, which inhibits mammalian CAs I–XV, with inhibition constants in the range of 331–4.56 μM (Temperini et al. 2010).

The natural product (Vu et al. 2008) coumarin 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one, as well as structurally related, simple coumarin derivatives possessing various substitution patterns at the heterocyclic ring, (Smith and Ferry 2000) was shown to be hydrolyzed within the CA active site with formation of 2-hydroxy- cinnamic acids, which possess significant CA inhibitory properties and bind in a completely unprecedented manner to the enzyme, not interacting with the Zn(II) ion as the sulphonamide/sulphamate/sulphamide type of inhibitors (Maren 1960). It was reported that the coumarin lacks classical zinc-binding functional groups and exhibits an unusual binding mode that may be exploited to design isoform–selective CA inhibitors. The inhibitor exhibits an extended two-arm conformation that effectively plugs the entrance to the active site (Vu et al. 2008). The present investigation reported that coumarin derivatives (3a–f) had potent inhibitory effects on the hCA I and II.

It has been reported that the novel coumarin derivatives containing sulphonamide were shown to exhibit inhibition effect against the hydratase activities of hCA-I, hCA-II (IC₅₀ values of 4.7, 9.6 and 25.3 μM) and dog CA (Vullo et al. 2004, Bayram et al. 2007). Our inhibition values were closer to theirs. Gupta and Kumaran (2005) reported that uncharged compounds cannot be made selective for cytosolic or membrane-bound isozyme since in both the cases the compounds appear to follow the same mechanism of inhibition. However, for the charged compounds, the polarizability of the molecule seems to greatly favour the inhibition of the membrane-bound enzyme, and hence they can be made selective for this enzyme by enhancing their polarizability, which is found to play no role in the inhibition of cytosolic enzymes (Gupta and Kumaran 2005). Barros et al. reported that the reaction of 20 aromatic/heterocyclic sulphonamides containing a free amino, imino, hydrazino or hydroxyl group, with 8-quinoline-sulphonyl chloride afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as inhibitors of the zinc enzyme CA, and more precisely of three of its isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form), involved in important physiological processes. Some of the best inhibitors synthesized were topically applied as 2% water solutions onto the eye of normotensive and glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. This new compound is quite water soluble as hydrochloride salt, behaves as a strong CA II inhibitor and fared better than the parent molecule in lowering IOP in experimental animals (Borras et al. 1999). Therefore, our new water-soluble coumarins can be effective inhibitors for CA isozymes.

Structure Activity Relationship (SAR)

From this study, some opinions about SAR were emerged. First, all salts showed higher carbonic anhydrase inhibitor activity than 4-chloromethyl-7-hydroxycoumarin obviously. When we investigate factors that effect the inhibitory activity of the synthesized compounds, two factors were revealed; molecule size and electronic effects. When compounds (3a–d) containing imidazolium salts are compared, it can be said that the inhibitory activity increased by the size of the molecule. When coumarin derivatives containing benzimidazolium salts (3a–d) and imidazolium salts (3g–h) were compared, benzimidazolium salts showed higher inhibitory activity than imidazolium salts (3a > 3g, 3b > 3h). When bisbenzimidazolium salts (3e, 3f) and benzimidazolium salts (3a, 3b) are compared, the effect of molecule size on the inhibitory activity again can be seen (3e, f > 3a, b). When benzimidazolium salts 3c and 3d are compared, the effect of molecule size on the inhibitory activity again can be seen. Electron-donating methoxy group-bearing benzimidazolium salt (3d) is the most active salt in synthesized compounds. In this study, we only synthesized 7-hydroxy coumarin derivatives. If we want to see the role of coumarin skeleton on inhibitory activity, we can compare synthesized salts with the literature. Recently our group synthesized 7,8-dihydroxycoumarin-bearing benzimidazolium salts and their inhibitory effect on CA (4, 5, 6, 7) was evaluated (Figure 1) (Karatas et al. 2013). If we compare 3a–d salts with their 7,8-dihydroxy analogues (4–7); 7,8 –dihydroxy coumarin derivatives showed higher inhibitory effect obviously (Table II).

Figure 1. 7,8-dihydroxy coumarin benzimidazolium salts synthesized by our group (Karatas et al. 2013).

Table II. IC₅₀ values of coumarin compounds (Karatas et al. 2013).

Inhibitors	hCA I (μM)	hCA II (μM)
4	28.55	49.4
5	49.32	51.45
6	25.63	40.01
7	33.1	20.33

In summary, enzyme inhibition is more important issue for drug design and biochemical applications (Aydemir and Kavrayan 2009, Gencer and Arslan 2011, Demir et al. 2012, Bytyqi-Damoni et al. 2012, Sayin et al. 2012, Cicek et al. 2012, Gokce et al. 2012, Sonmez et al. 2011, Arslan et al. 2012, Gencer et al. 2012a, 2012b, Demirel and Tarhan 2004). The results showed that the newly synthesized coumarin derivatives inhibited the hCA I and II enzyme activity. Therefore, our results suggested that new coumarin derivatives are likely to be adopted as candidates to treat glaucoma and may be taken for further evaluation in vivo studies.

Acknowledgments

Special thanks to Dr. Fatih Sonmez for his help and advice during the study.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.