Synthesis and carbonic anhydrase isoenzymes I and II inhibitory effects of novel benzylamine derivatives

Abstract

Synthesis and carbonic anhydrase inhibitory properties of novel diarylmethylamines 22–25 and sulfonamide derivatives 26–28 were investigated. Acylation of methoxy-substituted benzenes with benzene carboxylic acids, reduction of ketones with NaBH₄, conversion of alcohols to azides, Pd-C catalyzed hydrogenation of azides afforded title compounds 22–25. Compounds 22, 24 and 25 were converted to sulfonamide derivatives 26–28 with MeSO₂Cl. The inhibitory effects of novel benzylamine derivatives 22–28 were tested on human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes hCA I and II. The results demonstrated that compound 28 was found to be the best inhibitor against both hCA I (K_i: 3.68 µM) and hCA II (K_i: 9.23 µM).

• Benzylamine
• carbonic anhydrase
• enzyme inhibition
• sulfonamide

Introduction

Sulfonamides are important biologically active compounds. There are many drugs containing this functional group in their structures1. Anti-glaucoma agent dorzolamide (1), commercially known as trusopt, is a carbonic anhydrase (CA) inhibitor2. Brinzolamide (2) is a CA inhibitor used to lower intraocular pressure in patients with open-angle glaucoma or ocular hypertension3. CA inhibitory properties of dichlorophenamide (3) have been reported by Alterio and co-workers4. A CA inhibitor drug acetazolamide (AZA) (4) is used to treat glaucoma5 and idiopathic intracranial hypertension6 (Figure 1).

Figure 1.  Structures of some biologically active sulfonamides derivatives.

CA (EC 4.2.1.1.) is a pH regulatory/metabolic enzyme in all life kingdoms, found in organisms all over the phylogenetic tree3,7,8 catalyzing the hydration of carbon dioxide (CO₂) to bicarbonate () and the corresponding dehydration of in acidic medium with regeneration of CO₂ following a three-step catalytic mechanism3.

In the hydration direction, the first step is the nucleophilic attack of a Zn²⁺-bound hydroxide ion on CO₂ (Equation (1)) with consequent formation of (Equation (3)), which is then displaced from the active site by a water molecule (Equation (2)). The third step, which is rate limiting, regenerates the catalytically active Zn²⁺-bound hydroxide ion through a proton transfer reaction from the Zn²⁺-bound water molecule to an exogenous proton acceptor or to an active site residue, generically represented by B (Equation (3)).

Recently, the structure of the human carbonic anhydrase (hCA) II/ complex was also provided9. The lies in the same plane defined by the CO₂ molecule and the Zn²⁺-bound OH⁻ and is tetrahedrally coordinated to the catalytic metal ion through one of its oxygen atoms. The availability of both the hCA II/CO₂ and hCA II/ complex structures made it possible to obtain for the first time a detailed description of the first step of the enzymatic reaction providing new perspectives in the structural studies of this extremely interesting enzyme family3.

This metalloenzyme family is ubiquitous and present in majority of living organisms and divided to five unrelated gene families: the α-, β-, γ-, δ- and ζ-carbonic anhydrases (CAs). The α-, β- and δ-CAs contain a Zn²⁺ at the active site, the γ-CAs are almost certainly Fe²⁺ enzymes but they are active also with bound Zn²⁺ or Co²⁺, while the metal ion is usually replaced by cadmium in the ζ-CAs2,10. All hCAs belong to the α-class; up to this time, 16 isozymes have been identified, which differ by molecular features, oligomeric arrangement, cellular localization, distribution in organs and tissues, expression levels, kinetic properties and response to different classes of inhibitors3,11. Some of these are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA and CA VB) and one is secreted in saliva (CA VI). It has been reported that CA XV isoform is not expressed in humans or in other primates12,13. Twelve isoforms (CAs I–IV, VA–VB, VI–VII, IX, and XII–XIV) show a variable degree of enzymatic activity, whereas three isoforms (VIII, X and XI), the so-called CA-related proteins, are devoid of any catalytic activity3,14. Several studies demonstrated the important roles of CAs in a variety of physiological processes, such as acid–base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation. In addition, CAs demonstrated that the abnormal levels or activities of these enzymes have been frequently associated with different human diseases7,8,15.

It was reported that none of the current clinically used CA inhibitors displayed selectivity for a specific isoenzyme3,16. Therefore, developing isozyme-specific CA inhibitors is extremely important in order to obtain novel classes of drugs devoid of various undesired side effects. Consequently, in recent years CA isozymes have become an interesting target for the design of inhibitors or activators with biomedical applications3,17–19. Recently, numerous structural studies have provided a scientific basis for the rational drug design of more selective enzyme inhibitors20. Nevertheless, although X-ray crystal structures are available for the majority of the 12 catalytically active members of the hCA family21–23, most of the reported complexes with inhibitors regards just isozyme II, which is the most thoroughly characterized CA isoform2.

In the current research, the first synthesis of a number of novel diarylmethylamine hydrochloride salts 22–25 and their sulfonamide derivatives 26–28 was studied. CA inhibitors are valuable molecules for therapeutic and pharmacological applications.

Experimental section

General information

All chemicals and solvents are commercially available. All solvents were distilled and dried according to standard procedures. Silica gel (SiO₂, 60 mesh; Merck, Darmstadt, Germany) was used for column chromatography. About 1 mm of SiO₂ 60 PF (Merck) on glass plates was used for preparative thick layer chromatography. Melting point (m.p.) of all compounds was determined with cap melting-point apparatus (BUCHI 530, Flawil, Switzerland) and uncorrected. Infrared (IR) spectra were recorded as solutions in 0.1 mm cells with a Mattson 1000 FT-IR spectrophotometer (Cambridge, England). ¹H- and ¹³C-NMR spectra were recorded on 400 (100)-MHz Varian spectrometer (Danbury, CT) in deuterated solvents (CDCl₃ and D₂O) with tetramethylsilane (SiMe₄) as an internal standard for protons and solvent signals, as internal standard for carbon spectra. Chemical shift values were mentioned δ in ppm. Elemental analyses were recorded on Leco CHNS-932 apparatus (Saint Joseph, MI). CA inhibitory properties of samples were determined on a spectrophotometer (UV-1208, Shimadzu, Kyoto, Japan).

Syntheses of compounds (1024, 1125, 1226, 1327, 1428, 1528, 1626, 1729 and 2230) have been reported previously.

General procedure for synthesis of azides 18–21

4,4′-(Azidomethylene)bis(1,2-dimethoxybenzene) (18)

To a stirred solution of alcohol 14 (3.12 g, 10.25 mmol) in CH₂Cl₂ (50 mL) were added NEt₃ (3.11 g, 30.76 mmol) and MeSO₂Cl (2.94 g, 25,63 mmol) at room temperature (r.t.) for 4 h. After the solvent was evaporated, the residue was dissolved in DMF (60 mL). NaN₃ (2 g, 30.76 mmol) was added to this solution and the mixture was refluxed at 60–70 °C for 18 h. H₂O (100 mL) and EtOAc (80 mL) were added to the cooled mixture. The organic layer was separated and washed with H₂O (3 × 60 mL). After drying the organic layer over Na₂SO₄ and evaporation of the solvent, chromatography of the liquid residue on silica gel (SiO₂, 50 g) with ethyl acetate/hexane (1:4) afforded oily azide 18 (2.38 g, 71%). ¹H-NMR (400 MHz, CDCl₃) δ 6.86–6.78 (m, 6H), 5.61 (s, 1H), 3.86 (s, 6H, 2OCH₃), 3.83 (s, 6H, 2OCH₃). ¹³C-NMR (100 MHz, CDCl₃) δ 149.4 (2C), 149.1 (2C), 132.5 (2C), 120.1 (2CH), 111.2 (2CH), 110.7 (2CH), 68.4 (CHN₃), 56.14 (2OCH₃), 56.13 (2OCH₃). IR (CH₂Cl₂, cm⁻¹) 2997, 2936, 2836, 2098, 1600, 1592, 1515, 1464, 1418, 1252, 1234, 1158, 1026 and 855. Anal. Calcd for C₁₇H₁₉N₃O₄: C 62.00, H 5.81 and N 12.76; Found: C 61.96, H 5.80 and N 12.74.

4-[Azido(2,4-dimethoxyphenyl)methyl]-1,2-dimethoxybenzene (19)

The general procedure for the synthesis of 18 was applied to 15 (0.5 g, 1.64 mmol) to give an oily azide 19 (332.0 mg, 61%). ¹H-NMR (400 MHz, CDCl₃) δ 7.09 (d, 1H, J = 8.1 Hz), 6.88–6.81 (m, 3H), 6.48–6.45 (m, 2H), 6.05 (s, 1H), 3.85 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C-NMR (100 MHz, CDCl₃) δ 160.9 (C), 158.0 (C), 149.1 (C), 148.6 (C), 132.5 (C), 129.2 (CH), 121.0 (C), 119.8 (CH), 111.1 (CH), 110.7 (CH), 104.6 (CH), 98.7 (CH), 62.1 (CHN₃), 56.1 (2OCH₃), 55.7 (OCH₃), 55.6 (OCH₃). IR (CH₂Cl₂, cm⁻¹) 2998, 2936, 2837, 2098, 1681, 1611, 1588, 1515, 1464, 1419, 1256, 1209, 1182, 1158, 1140, 1115, 1030 and 926. Anal. Calcd for C₁₇H₁₉N₃O₄: C 62.00, H 5.81 and N 12.76; found: C 61.95, H 5.86 and N 12.82.

1-[Azido(3,4-dimethoxyphenyl)methyl]-4,5-dimethoxy-2-methylbenzene (20)

The general procedure for the synthesis of 18 was applied to 16 (3.25 g, 10.21 mmol). After crystallization from MeOH, azide 20 (2.48 g, 71%) was obtained as colorless block crystals. M.p. 102–104 °C. ¹H-NMR (400 MHz, CDCl₃) δ 69.92 (s, 1H), 6.82 (d, 1H, J = 8.6 Hz), 6.78–6.76 (m, 2H), 6.68 (s, 1H), 5.78 (s, 1H), 3.87 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 2.18 (s, 3H, CH₃). ¹³C-NMR (100 MHz, CDCl₃) δ 149.3 (C), 148.9 (C), 148.5 (C), 147.3 (C), 131.8 (C), 129.5 (C), 128.2 (C), 120.2 (CH), 114.0 (CH), 111.1 (CH), 111.0 (CH), 110.7 (CH), 65.6 (CHN₃), 56.3 (OCH₃), 56.1 (3OCH₃), 19.2 (CH₃). IR (CH₂Cl₂, cm⁻¹) 3429, 3003, 2958, 2835, 2098, 1646, 1515, 1464, 1417, 1399, 1342, 1261, 1234, 1212, 1183, 1140, 1098, 1027, 1002 and 848. Anal. Calcd for C₁₈H₂₁N₃O₄: C 62.96, H 6.16 and N 12.24; Found: C 63.01, H 6.12 and N 12.26.

3,5′-(Azidomethylene)bis(1,2-dimethoxybenzene) (21)

Compound 21 was synthesized according to the general procedure given for 18. From 17 (1.22 g, 4.01 mmol), azide 21 (0.986 g, 75%) was obtained, after crystallized from CH₂Cl₂. Colorless block crystals. M.p. 78–80 °C. ¹H-NMR (400 MHz, CDCl₃) δ 7.08 (t, 1H, J = 8.1 Hz), 6.96 (d, 1H, J = 7.7 Hz), 6.89–6.80 (m, 4H), 6.10 (s, 1H), 3.86 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.70 (s, 3H, OCH₃). ¹³C-NMR (100 MHz, CDCl₃) δ 152.8 (C), 149.2 (C), 148.8 (C), 146.6 (C), 133.9 (C), 132.5 (C), 124.4 (CH), 120.0 (CH), 119.7 (CH), 112.3 (CH), 111.1 (CH), 110.7 (CH), 62.6 (CHN₃), 61.0 (OCH₃), 56.12 (OCH₃), 56.10 (OCH₃), 56.0 (OCH₃). IR (CH₂Cl₂, cm⁻¹) 3423, 2829, 2098, 1645, 1516, 1480, 1415, 1271, 1234, 1140, 1072, 1027 and 1001. Anal. Calcd for C₁₇H₁₉N₃O₄: C 62.00, H 5.81 and N 12.76; Found: C 61.90, H 5.83 and N 12.77.

General procedure for synthesis amine hydrochloride salts 23–25

(2,4-Dimethoxyphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (23)

Pd–C (100 mg) and 4-(azido(2,4-dimethoxyphenyl)methyl)-1,2-dimethoxybenzene (19) (0.42 g, 1.28 mmol) in MeOH (40 mL) and CHCl₃ (6 mL) were placed in a 100 mL two-necked flask. A balloon filled with H₂ gas (3 L) was fitted to the flask. The mixture was deoxygenated by flushing with H₂ and then hydrogenated at r.t. for one day. The catalyst was removed by filtration. Recrystallization of the residue from MeOH–Et₂O yielded (2,4-dimethoxyphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (23) (0.378 g, 87%) as a white solid. M.p. 230–232 °C. ¹H-NMR (400 MHz, D₂O) δ 6.93 (d, 1H, J = 8.8 Hz), 6.88 (d, 1H, 8.1 Hz), 6.84–6.81 (m, 2H), 6.52 (s, 1H), 6.42 (qd, 1H, J = 8.4), 5.51 (s, 1H), 4.64 (bs, OH of D₂O and NH₃), 3.68 (s, 6H, 2OCH₃), 3.66 (s, 3H, OCH₃), 3.64 (s, 3H, OCH₃). ¹³C-NMR (100 MHz, CDCl₃) δ 161.2 (C), 157.9 (C), 148.4 (C), 129.8 (CH), 129.2 (2C), 120.1 (CH), 117.2 (C), 111.9 (CH), 110.6 (CH), 105.4 (CH), 99.1 (CH), 55.8 (CH–NH₃Cl), 55.75 (OCH₃), 55.6 (2OCH₃), 54.4 (OCH₃). Anal. Calcd for C₁₇H₂₂ClNO₄: C 60.09, H 6.53 and N 4.12; Found: C 60.02, H 6.59 and N 4.13.

(4,5-Dimethoxy-2-methylphenyl) (3,4-dimethoxyphenyl) methanamine hydrochloride (24)

The general procedure was carried out with 1-(azido(3,4-dimethoxyphenyl)methyl)-4,5-dimethoxy-2-methylbenzene (20) (1.34 g, 3.90 mmol). Recrystallization of the residue from MeOH–Et₂O afforded (4,5-dimethoxy-2-methylphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (24) (1.28 g, 93%) as a white solid. M.p. 235–237 °C. ¹H-NMR (400 MHz, CDCl₃) δ 6.93 (s, 1H), 6.87–6.77 (m, 4H), 5.61 (s, 1H), 4.64 (bs, OH of D₂O and NH₃), 3.72 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 2.04 (s, 3H, CH₃). 13C-NMR (100 MHz, CDCl₃) δ 148.9 (C), 148.8 (C), 148.3 (C), 146.9 (C), 129.8 (C), 129.4 (C), 127.3 (C), 120.8 (CH), 115.0 (CH), 112.3 (CH), 111.4 (CH), 109.7 (CH), 56.4 (CH–NH₃Cl), 56.1 (2OCH₃), 56.0 (OCH₃), 54.6 (OCH₃), 18.0 (CH₃). Anal. Calcd for C₁₈H₂₄ClNO₄: C 61.10, H 6.84 and N 3.96; Found: C 61.07, H 6.86 and N 3.94.

(2,3-Dimethoxyphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (25)

The general procedure was carried out with 3,5′-(azidomethylene)bis(1,2-dimethoxybenzene) (21) (2.14 g, 6.50 mmol). Recrystallization of the residue from MeOH–Et₂O yielded (2,3-dimethoxyphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (25) (2.02 g, 92%) as a white solid. M.p. 221–223 °C. ¹H-NMR (400 MHz, D₂O) δ 7.08–7.06 (m, 1H), 6.98–6.96 (m, 1H), 6.90–6.82 (m, 4H), 5.66 (s, 1H), 4.64 (bs, OH of D₂O and NH₃), 3.69 (s, 3H, OCH₃), 3.68 (s, 3H, OCH₃), 3.65 (s, 6H, 2OCH₃). 13C-NMR (100 MHz, CDCl₃) δ 152.7 (C), 148.7 (C), 148.5 (C), 145.6 (C), 130.4 (C), 129.4 (C), 125.4 (CH), 120.5 (CH), 118.7 (CH), 114.1 (CH), 112.0 (CH), 110.9 (CH), 60.8 (CH–NH₃Cl), 55.9 (OCH₃), 55.8 (2OCH₃), 53.6 (OCH₃). Anal. Calcd for C₁₇H₂₂ClNO₄: C 60.09, H 6.53 and N 4.12; Found: C 60.15, H 6.57 and N 4.09.

General procedure for the synthesis of sulfonamides 26–28

N-(bis(3,4-dimethoxyphenyl)methyl)methanesulfonamide (26)

To a solution of bis(3,4-dimethoxyphenyl)methanamine hydrochloride (22) (0.197 g, 0.580 mmol) in MeOH (15 mL) was added 6 M NaOH (40 mL). The mixture was stirred at r.t. for 3 h. After most of the MeOH was evaporated, H₂O (20 mL) and CH₂Cl₂ (50 mL) were added to the residue. Organic phase was separated and H₂O phase was extracted with CH₂Cl₂ (3 × 30 mL). Combined organic layers were dried over Na₂SO₄ and the solvent was evaporated. After the residue was dissolved in CH₂Cl₂ (30 mL), NEt₃ (70 mg, 0.696 mmol) and MeSO₂Cl (86 mg, 0.754 mmol) were added to this solution at r.t. for 20 h. The solvent was evaporated and to the residue were added H₂O (20 mL) and EtOAc (30 mL). The organic layer was separated and H₂O layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over Na₂SO₄ and the solvent was evaporated. The chromatography of the residue on silica gel (SiO₂, 60 g) with ethyl acetate/hexane (1:9) gave a yellowish solid N-(bis(3,4-dimethoxyphenyl)methyl)methanesulfonamide (26) (0.202 g, 91%). Solidified; m.p. 123–125 °C. ¹H-NMR (400 MHz, CDCl₃) δ 6.83–6.78 (m, 6H), 5.60 (d, 1H, CH, J = 8.1 Hz), 5.57 (d, 1H, NH, J = 8.1 Hz), 3.83 (s, 6H, 2OCH₃), 3.80 (s, 6H, OCH₃), 2.60 (s, 3H, CH₃). 13C-NMR (100 MHz, CDCl₃) δ 149.5 (2C), 148.9 (2C), 133.5 (2C), 119.8 (2CH), 111.2 (2CH), 110,7 (2CH), 61.0 (CH-NH), 56.2 (2OCH₃), 56.1 (2OCH₃), 42.0 (CH₃). IR (CH₂Cl₂, cm⁻¹) 3565, 3261, 2829, 2090, 1645, 1515, 1464, 1418, 1318, 1252, 1189, 1140, 1063, 1025, 1063, 1025 and 979. Anal. Calcd for C₁₈H₂₃NO₆S: C 56.68, H 6.08, N 3.67 and S 8.41; Found: C 56.73, H 6.02, N 3.62 and S 8.40.

N-((4,5-dimethoxy-2-methylphenyl) (3,4-dimethoxyphenyl)methyl) methane sulfonamide (27)

The general procedure for 26 was applied to (4,5-dimethoxy-2-methylphenyl)(3,4-dimethoxyphenyl)methanamine hydrochloride (24) (0.250 g, 0.707 mmol) to give yellowish solid 27 (0.260 g, 93%). M.p. 104–106 °C. ¹H-NMR (400 MHz, CDCl₃) δ 6.84 (bs, 1H), 6.83 (s, 1H), 6.78 (d, 1H, J = 8.3 Hz), 6.75 (qd, 1H, J = 8.3 Hz), 6.68 (s, 1H), 5.83 (d, 1H, CH, J = 7.2 Hz), 5.24 (d, 1H, NH, J = 7.2), 3.86 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.81 (s, 6H, 2OCH₃), 2.63 (s, 3H, CH₃), 2.28 (s, 3H, CH₃). 13C-NMR (100 MHz, CDCl₃) δ 149.5 (C), 148.9 (C), 148.5 (C), 147.6 (C), 132.9 (C), 130.5 (C), 128.5 (C), 119.9 (CH), 114.2 (CH), 111.3 (CH), 111.0 (CH), 110.7 (CH), 57.7 (CH-NH), 56.4 (OCH₃), 56.2 (OCH₃), 56.11 (OCH₃), 56.10 (OCH₃), 42.2 (CH₃), 19.1 (CH₃). IR (CH₂Cl₂, cm⁻¹) 3618, 3161, 2061, 1635, 1514, 1457, 1320, 1242, 1208, 1149, 1102, 1027 and 979. Anal. Calcd for C₁₉H₂₅NO₆S: C 57.70, H 6.37, N 3.54 and S 8.11; Found: C 57.75 H 6.32, N 3.51 and S 8.09.

N-((2,3-dimethoxyphenyl)(3,4-dimethoxyphenyl)methyl)methanesulfonamide (28)

The general procedure described above was applied to 25 (0.300 g, 0.883 mmol) to yield an oily N-((2,3-dimethoxyphenyl)(3,4-dimethoxyphenyl)methyl)methanesulfonamide (28) (0.328 g, 97%). ¹H-NMR (400 MHz, CDCl₃) δ 7.08 (t, 1H, J = 7.9 Hz), 6.93–6.90 (m, 3H), 6.76 (bs, 2H), 5.77 (d, 1H, CH, J = 9.1 Hz), 5.71 (d, 1H, NH, J = 9.1 Hz), 3.85 (OCH₃), 3.82 (OCH₃), 3.51 (OCH₃), 2.68 (CH₃). 13C-NMR (100 MHz, CDCl₃) δ 153.4 (C), 149.2 (C), 148.5 (C), 146.6 (C), 135.0 (C), 134.0 (C), 124.5 (CH), 120.8 (CH), 119.0 (CH), 112.8 (CH), 111.0 (CH), 110.4 (CH), 60.7 (CH-NH), 58.0 (OCH₃), 56.2 (OCH₃), 56.1 (OCH₃), 56.0 (OCH₃), 41.7 (CH₃). IR (CH₂Cl₂, cm⁻¹) 3713, 3279, 3003, 2938, 2837, 1589, 1516, 1481, 1463, 1323, 1272, 1235, 1147, 1086, 1046, 1027, 1003, 982. Anal. Calcd for C₁₈H₂₃NO₆S: C 56.68, H 6.08, N 3.67 and S 8.41; Found: C 56.75, H 6.04, N 3.65 and S 8.42.

Purification of CA isoenzymes from human erythrocytes by affinity chromatography

CA isoenzymes were purified via a simple single-step method using sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography, described previously31,32. The serum was obtained from fresh human blood acquired from the Blood Centre of the Research Hospital at Atatürk University. The blood samples were centrifuged at 5000 rpm for 15 min and the precipitant was removed and the serum was isolated. The pH was adjusted to 8.7 with solid Tris. sepharose-4B-tirozyne-sulfanylamide 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 hCA VI isozyme was eluted with 0.25 M H₂NSO₃H/25 mM Na₂HPO4 (pH 6.7). All procedures were performed at 4 °C33,34.

Hydratase activity assay

The hydratase activities of hCA I and II were assayed by following the hydration of CO₂ according to the method described by Wilbur and Anderson35. The activity of CO₂-hydratase in enzyme units (EU) was calculated by using the equation (t_o – t_c/t_c) where t_o and t_c are the times for of pH change of the non-enzymatic and the enzymatic reactions, respectively36.

Esterase activity assay

CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (Shimadzu, UVmini-1240 UV-VIS spectrophotometer) according to the method described by Verpoorte et al.37 The enzymatic reaction contained 1.4 mL 0.05 M Tris–SO₄ buffer (pH 7.4), 1 mL 3 mM 4-NPA, 0.5 mL H₂O and 0.1 mL enzyme solution (total volume, 3.0 mL). A reference measurement was obtained by preparing the mixture without the enzyme solution. All measurements were recorded in triplicate. The K_i values were determined from a series of experiments using three different bromophenols concentrations and 4-NPA as the substrate at five different concentrations to construct Lineweaver–Burk curves38.

Protein determination

The yield of protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method39, using bovine serum albumin as the standard40.

SDS polyacrylamide gel electrophoresis

The purity of the enzymes was confirmed using SDS polyacrylamide gel electrophoresis. The running and stacking gels contained 10% and 3% acrylamide, respectively, and 0.1% SDS, according to the Laemmli procedure41 described previously12. A 20 mg sample was applied to the electrophoresis medium. Gels were stained for 1.5 h in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, then distained with several changes of the same solvent without the dye.

Results and discussion

Synthesis

Polyphosphoric acid (PPA) prepared from phosphoric acid and P₂O₅ catalyzed acylation or alkylation of aromatic compounds has been used in the literature42–45. Following this procedure, acylation of electron rich aromatic compounds 5–7 with carboxylic acid 8 gave diarylmethanone derivatives 1046, 1125 and 1226. By a similar approach, the reaction of 5 with acid 9 afforded 1346. Reduction of diarylmethanones with NaBH₄ in MeOH at 0–25 °C by the procedure described by Lantano et al.47 furnished diarylmethanols 14, 1528, 1626 and 1729 in excellent yields. Alcohols can be converted to their azide derivatives by Mitsunobu reaction48 or via the substitution of the corresponding mesylate esters with NaN₃49. Here, the later procedure was chosen. For this purpose, esterification of alcohols 14–17 with MeSO₂Cl in CH₂Cl₂ in the presence of Et₃N at 25 °C and then substitution of the corresponding mesylate esters with NaN₃ in DMF at 60–70 °C afforded four novel diarylmethylazides 18–21 in good yields. The most critical step of our synthesis was the reduction of azides to amine hydrochloride salts. Due to the dibenzylic position of the azide groups, the Pd--C catalyzed hydrogenation could have possibly failed by leading to a deamination reaction. Fortunately, the reduction of azide groups to the corresponding amine derivatives was carried out without encountering any problems. The reduction of azides 18–21 with Pd-C catalyzed hydrogenation in the presence of CHCl₃ (for in situ production of HCl)49 in MeOH yielded amine hydrochlorides 22–25 from which only 22 has previously been reported30. Amine hydrochloride salts 22, 24 and 25 were converted to their amines with 6 M NaOH. Then the reaction of these amines with MeSO₂Cl in the presence of Et₃N gave new sulfonamide derivatives 26–28 in high yields (Scheme 1). The structures of all synthesized compounds were characterized by ¹H-, ¹³C-NMR and azide functional groups were characterized by IR. Especially, in the 13C-NMR spectra, carbonyl carbons of diarylmethanones 10–13 resonating at 194.5–196.8 which displayed positive evidence for the formation of the ketone group. In this context, diarylcarbinol carbons of compounds 14–17 resonating at 71.8–76.0 ppm are in good agreement with the structure. On the other hand; benzylic carbons of the azides resonated at upper field (62.1–68.4 ppm) than the corresponding alcohols as expected. Similarly, characteristic IR bands of N₃ groups at 2098 cm⁻¹ were good evidence for the presence of azide functional groups. Benzylic carbons of amine hydrochlorides resonating at 55.8–60.8 ppm were in agreement with the structures.

Scheme 1. Reagents and conditions: (i) PPA, 80 °C, 1 h; (ii) NaBH₄, THF/MeOH, 30 min 0 °C, then 4 h, r.t.; (iii) CH₂Cl₂, NEt₃, MeSO₂Cl, r.t., 4 h, then DMF, NaN₃, 60–70 °C, 18 h; (iv) Pd–C, MeOH/CHCl₃, H₂, r.t., 1 d and (v) MeOH, 6 M NaOH r.t., 3 h then CH₂Cl₂, NEt₃, MeSO₂Cl r.t., 20 h.

CA purification and activity assay

In this study, CA isoenzymes I and II (hCA I and hCA II) were purified from human erythrocytes (Table 1). The purification of both CA isozymes was performed using a simple one-step method with a sepharose-4B-thyrosine-sulfanilamide affinity column chromatography. Human erythrocyte CA I isoenzyme was purified, 312.2-fold with a specific activity of 626 EU/mg and overall yield of 60.7%; CA II isoenzyme was purified, 296-fold with a specific activity of 594 EU/mg and an overall yield of 44.1%. We report here the first study on the inhibitory effects of novel benzylamine derivatives (22–28) on the esterase activity of hCA I and II. The inhibitor concentrations that caused 50% inhibition (IC₅₀) were determined from activity versus (%)-[benzylamines] plots and the K_i values were calculated from the Lineweaver–Burk plots (Table 2). The data of Table 2 show the following inhibition of hCA I and II with novel benzylamine derivatives (22–28), by an esterase assay, with 4-NPA as substrate:

Table 1. Summary of purification procedure for hCA I and hCA II isoenzymes by a sepharose-4B-thyrosine-sulfanilamide affinity column chromatography.

Purification steps		Activity (EU/mL)	Total volume (mL)	Protein (mg/mL)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification fold
Hemolysate		67.14	27	33.5	904.2	1812	2.004	100	1
Sepharose-4B-thyrosine-sulfanilamide  affinity column chromatography	hCA I	182.8	6	0.292	1.752	1096.8	626.0	60.7	312.2
	hCA II	59.9	5	0.269	1.346	799.6	594	41.1	296.4

Table 2. hCA I and II isoenzymes inhibition data with some synthesized benzylamine derivatives (22–28) by an esterase assay with 4-NPA as substrate.

	Ki (µM)
Compounds	hCA I	hCA II
22	13.69 ± 1.48	15.6 ± 1.41
23	36.04 ± 2.81	34.32 ± 2.55
24	39.52 ± 3.22	47.64 ± 3.12
25	66.89 ± 4.85	73.31 ± 4.43
26	25.64 ± 2.56	18.11 ± 1.81
27	48.62 ± 3.25	18.95 ± 3.43
28	3.68 ± 1.28	9.23 ± 2.73
AZA*	36.2†	3.70†

*AZA was used as positive standard for hCA I and hCA II isoenzymes.

†Values taken from Öztürk Sarıkaya et al.31

CA isoenzymes inhibition effects

CA isoenzymes (hCA I and hCA II) inhibitory effects of the novel benzylamine derivatives (22–28) were tested under in vitro conditions and K_i values were calculated and given in Table 2.

The data in Table 2 show the following regarding the inhibition of hCA I and II by novel benzylamine derivatives 22–28. The strongest inhibitory activity has been observed with compound 28, investigated here for the inhibition of the rapid cytosolic isozymes hCA I and II (Table 2). According to these activities, the novel benzylamine derivative of 28 showed the strongest hCA I inhibitory activity with K_i of 3.68 ± 1.28 µM and hCA II inhibitory activity with K_i of 9.23 ± 2.73 µM (Table 2). Also, the remaining other benzylamine derivatives were quite effective cytosolic isozyme hCA I inhibitors with Ki-s within the range of 3.68–66.89 µM and hCA II inhibitors with Ki-s in the range of 9.23–73.31 µM (Table 2). It is widely known that amine compounds are CA isoenzyme50.

In our study, all of the novel benzylamine derivatives 22–28 had four methoxy moieties in both the aromatic rings. In addition, two of the novel benzylamine derivatives 24 and 27 have a methyl group in the aromatic ring. We suppose that these groups make aromatic rings electronically richer. Thus, these molecules easily connect to zinc ions in active sides of CA isoenzymes.

The synthesized novel benzylamine derivatives 22–28 have two aromatic rings. These new synthesized compounds have been investigated as CA izoenzymes inhibitors in this study. The rationale of investigating these compounds as CA inhibitors exists in the fact that the compounds with aromatic rings have been shown to be the only competitive inhibitor with CO₂ as the substrate for the main isoform of CA, i.e. hCA I and II. All new synthesized compounds 22, 23, 26 and 28 were much stronger CA I inhibition effects than the clinically used AZA included in the assays as a standard inhibitor. On the other hand, CA II inhibition effects of all new synthesized compounds 22–28 are weaker than that of AZA (Table 2).

Conclusion

In summary, diarylmethylamine hydrochloride salts 22–25 were synthesized starting from methoxy-substituted benzenes in five steps with high yields. Compounds 22, 24 and 25 were converted to their sulfonamide derivatives 26–28. All synthesized compounds can be important synthons for further synthetic and biological purposes. In addition, CA activities of the synthesized benzylamines 22–25 and their sulfonamide derivatives 26–28 were also investigated. All of the new synthesized compounds were found as effective CA I and CA II isoenzymes inhibitor. Further, these findings point out that these compounds 22–28 may potentially be used as leads for generating a useful CA isoenzymes inhibitor.

Declaration of interest

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

We thank Atatürk University for the financial supports of this work (BAP, Project no: 2009/84 for YÇ and BAP, Project no: 2009/251 for IG) and the Scientific and Technological Research Council of Turkey (TUBITAK, Project no: 109T241 for SG) and Prof Dr Hasan Seçen for his contribution.