Isoform-selective inhibitory profile of 2-imidazoline-substituted benzene sulfonamides against a panel of human carbonic anhydrases

Abstract

A series of novel benzene sulfonamides (previously evaluated as selective cyclooxygenase-2 inhibitors) has been profiled against human carbonic anhydrases I, II, IV and VII in an attempt to observe the manifestation of the well established “tail” approach for designing potent, isoform-selective inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1). The compounds displayed an excellent (pKi 7–8) inhibitory profile against CA II (a cytosolic anti-glaucoma and anti-edema biological target) and CA VII (also a cytosolic target believed to be involved in epilepsy and neuropathic pain) and a marked (1–2 orders of magnitude) selectivity against cytosolic isoform CA I and membrane-bound isoform CA IV. The separation of the CA II and CA IV (both of which are catalytically active isoforms, highly sensitive to sulfonamide-type inhibitors) is particularly remarkable and is adding significantly to the global body of data on the chemical biology of carbonic anhydrases.

Keywords:

• Carbonic anhydrases
• cyclooxygenase-2 inhibitors
• dual active site architecture
• 2-imidazolines
• isoform selectivity
• lipophilic appendages
• tail approach

Introduction

The goal of developing isoform-selective inhibitors of human carbonic anhydrases (hCA) has stimulated extensive medicinal chemistry activity worldwide1. Selective inhibition of a particular isoform (in contrast to nonselective carbonic anhydrase inhibitors (CAIs), such as acetazolamide2) is likely to improve therapeutic benefit over side-effects and toxicity for a well-established hCA-disease relationship (e.g. hCA II for glaucoma)3. Enhancing the selectivity of CAIs may even validate CAs in fundamentally new therapeutic areas. For instance, selective targeting of membrane-bound isoforms hCA IX and XII has been proposed as a novel approach to halt the growth of solid tumors by suppressing tumor survival mechanism in hypoxic environment4. A significant body of pre-clinical proof-of-concept data5 as well as one front-runner compound (SLC-0111) in clinical trials for cancer6 offers much hope that selective hCA isoform targeting will receive clinical validation in the future.

A well-established, rational approach to designing isoform-selective CAIs exists in perhaps the most investigated class of inhibitors, namely, benzenesulfonamides. Anchoring of the sulfonamide group in this class of compounds to the prosthetic zinc ion defines the position of the organic portion of the molecule within the CA binding cavity7 and offers significant opportunity to build, somewhat serendipitously, additional interactions with the protein environment where two distinct parts have been delineated8. By grafting appendages of various nature (hydrophilic and lipophilic alike) onto the benzenesulfonamide scaffold, it has become possible to impart significant potency and selectivity to the resulting CAIs. The effectiveness of building contacts with the lipophilic side of the CA active site has been shown to correlate with the potency of the CAIs (exemplified by compounds 1–3)9. A well-designed and informative comparison of hydrophilic versus lipophilic (as well as mixed) appendage design by the so-called “tail approach”10 (illustrated by compounds 4–6) has been recently published by Poulsen and colleagues11.

Inspired by these findings, we became interested in assessing CAI profile of a series of benzene sulfonamides decorated with 2-aryl-2-imidazoline appendages (7). These compounds have been recently described in the context of selective cyclooxygenase-2 inhibition12. We were interested to see if these compounds (somewhat akin to ureido compounds 1–3)9 would be effective as CAIs in principle (Figure 1). Furthermore, assessing their isoform selectivity profile against CAs with COX-2 inhibition data12 would offer an opportunity to consider developing dual-action therapeutic agents (e.g. endowed with antiglaucoma and anti-inflammatory activity manifested via inhibition of CAs and COX-2, respectively)13. This article describes the details of our findings in this area.

Figure 1. Structures of benzenesulfonamide CAIs containing hydrophobic ureido tails (1–3), the “dual-tail” compounds (4–6) and the design rationale for 2-imidazoline-substituted compounds 7.

Materials and methods

Chemical syntheses – general

All chemicals and solvents were used as received from the suppliers. The crude reaction mixtures were concentrated under reduced pressure by removing organic solvents on rotary evaporator. Column chromatography was performed using silica gel 60 (particle size 0.040–0.063 mm, 230–400 mesh ASTM). Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F₂₅₄ aluminum sheets. Proton and carbon-13 nuclear magnetic resonance (¹H NMR and ¹³C NMR) spectra were recorded on a Varian Unity INOVA 500 MHz spectrometer (NMR Laboratory, Urbana, IL). Chemical shifts for nuclear magnetic resonance (NMR) spectra were reported in parts per million (ppm, δ) using the residual solvent peak(s) as internal standard. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br) and doublets of doublet (dd). High-resolution mass spectra (HRMS) were acquired using a Bruker Daltonics Fourier Transform Ion Cyclotron Resonance Spectrometer (Bruker Daltonics Inc., Billerica, MA) fitted with an electrospray source operating in positive ion mode.

General procedure 1 for preparation of 2-imidazolines 6a–o14

Ethylenediamine (10.5 mmol, 1.05 equiv.) was added to a stirred solution of aldehyde (10.0 mmol, 1.0 equiv.) in CH₂Cl₂ (50 mL) at 0 °C, and the resulting solution stirred for 20 min. N-bromosuccinimide (10.5 mmol, 1.05 equiv.) was then added in portions over 10 min. The reaction mixture was warmed to room temperature and stirred for an additional 12 h, during which time a precipitate formed. A solution of KOH (1.0 M, 50 mL, 50 mmol, 5.0 equiv.) was added causing the precipitate to disappear and a clear biphasic solution was formed. The organic layer was separated, dried over anhydrous MgSO₄, filtered, and concentrated at reduced pressure to afford the crude product. The analytically pure imidazolines 6 were obtained by crystallization from EtOAc/hexane. All imidazolines 6a–o used in subsequent Pd-catalyzed N-arylation are known compounds12.

General procedure 2 for preparation of compounds 5a–o12

A thick-glass screw-capped pressure tube (50 mL) was charged with a suspension of starting imidazoline (3.0 mmol), 4-bromo-N,N-bis(2,4-dimethoxybenzyl)benzenesulfonamide15 (3.0 mmol), Cs₂CO₃ (3.0 mmol), and toluene (15 mL). Pd(OAc)₂ (0.12 mmol) and BINAP (0.24 mmol) were weighed as solids into a 10 mL screw-capped vial and toluene (8 mL) was added. The resulting suspension was heated at 110 °C until a clear purple solution was evident. The solution was rapidly transferred, while still hot, to the screw-capped pressure tube using a Pasteur pipette. The reaction vessel was filled with argon, sealed, and the reaction mixture vigorously stirred at 110 °C for 20 h. The reaction mixture was cooled to ambient temperature and filtered through a Celite pad (Beaver Chemicals, Burlington Ltd., Ontario, Canada), washed with copious amounts of EtOAc, and the combined filtrate and washings evaporated to dryness. The crude material was fractionated on silica (CH₂Cl₂:MeOH, 95:5) and the fractions containing the N-aryl 2-imidazolines were collected and evaporated to dryness. The residue was taken forward to the next step without further purification.

TFA (3 mL) was added drop wise to a solution of DMB-protected sulfonamides (obtained as described above) in CH₂Cl₂ (10 mL) at 0 °C and stirring continued at the same temperature for 30–60 min. The excess TFA was neutralized with sat. aq. NaHCO₃, the organic layer separated and dried over anhydrous MgSO₄. The solution was filtered and concentrated in vacuo. The resulting crude product was purified by column chromatography on silica using an appropriate gradient of MeOH in CH₂Cl₂ as eluent to provide sulfonamide products 5a–o.

4-(2-(4-Fluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5a)

Prepared according to General Procedure 2 from 2–(4-fluorophenyl)-4,5-dihydro-1H-imidazole. Yield 700 mg (73%), light yellow solid, m. p. 94–96 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.69 (d, J = 8.8 Hz, 2 H), 7.50–7.45 (m, 2 H), 7.07–7.02 (m, 2 H), 6.75 (d, J = 8.8 Hz, 2 H), 4.91 (br s, 2 H), 4.15–4.03 (m, 4 H); ¹³C NMR (125 MHz, CDCl₃) δ 163.9 (d, J_C-F = 251.4 Hz), 160.1, 146.2, 134.9, 130.6 (d, J_C-F = 8.6 Hz), 127.4, 126.8 (d, J_C-F = 3.4 Hz), 120.5, 115.8 (d, J_C-F = 21.9 Hz), 53.13, 53.11; HRMS m/z calcd for C₁₅H₁₅FN₃O₂S (M + H)⁺320.0864 found 320.0860.

4-(2-(4-Fluorophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5b)

Prepared according to General Procedure 2 from 2-(4-fluorophenyl)-4-methyl-4,5-dihydro-1H-imidazole. Yield 360 mg (36%), yellow solid, m. p. 157–159 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.86–7.82 (m, 2 H), 7.57–7.53 (m, 2 H), 7.26–7.19 (m, 4 H), 4.63–4.53 (m 2 H), 4.14–4.06 (m 1 H), 1.56–1.51 (m, 3 H), NH₂ protons in exchange. ¹³C NMR (125 MHz, CD₃OD) δ 166.7 (d, J_C-F = 254.0 Hz), 164.2, 143.3, 142.8, 133.1 (d, J_C-F = 9.4 Hz), 128.6, 125.5, 117.5 (d, J_C-F = 22.6 Hz), 61.3, 55.8, 20.9. HRMS m/z calcd for C₁₆H₁₇FN₃O₂S (M + H)⁺334.1020 found 334.1016.

4-(2-(4-Chlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5c)

Prepared according to General Procedure 2 from 2-(4-chlorophenyl)-4,5-dihydro-1H-imidazole. Yield 663 mg (66%), yellow solid, m. p. 141–143 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.81 (d, J = 8.8 Hz, 2 H), 7.54–7.46 (m, 4 H), 7.08 (d, J = 8.8 Hz, 2 H), 4.36 (t, J = 9.9 Hz, 2 H), 4.14 (t, J = 9.9 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 163.9, 145.0, 140.7, 1387, 131.5, 130.1, 128.4, 128.3, 123.7, 54.3, 51.1; HRMS m/z calcd for C₁₅H₁₅ClN₃O₂S (M + H)⁺336.0568 found 336.0564.

4-(2-(4-Chlorophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5d)

Prepared according to General Procedure 2 from 2-(4-chlorophenyl)-4-methyl-4,5-dihydro-1H-imidazole. Yield 534 mg (51%), light brown solid, m. p. 160–162 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.85 (d, J = 8.7 Hz, 2 H), 7.53–7.47 (m, 4 H), 7.26 (d, J = 8.7 Hz, 2 H), 4.67–4.58 (m, 2 H), 4.17–4.09 (m, 1 H), 1.58–1.50 (m, 3 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 164.3, 143.5, 142.5, 140.5, 132.0, 130.6, 128.6, 125.7, 124.5, 61.3, 55.6, 20.8; HRMS m/z calcd for C₁₆H₁₇ClN₃O₂S (M + H)⁺350.0724 found 350.0719.

4-(2-(3-Bromophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5e)

Prepared according to General Procedure 2 from 2-(3-bromophenyl)-4,5-dihydro-1H-imidazole. Yield 409 mg (36%), white solid, m. p. 194–194 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.73 (d, J = 8.8 Hz, 2 H), 7.68–7.64 (m, 2 H), 7.40–7.36 (m, 1 H), 7.31 (t, J = 7.8 Hz, 1 H), 6.98 (d, J = 8.8 Hz, 2 H), 4.20 (td, J = 9.3, 1.2 Hz, 2 H), 4.05 (td, J = 9.3, 1.2 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 162.6, 146.2, 139.4, 134.8, 134.0, 132.3, 131.5, 128.5, 128.1, 123.5, 122.6, 54.1, 53.3; HRMS m/z calcd for C₁₅H₁₅BrN₃O₂S (M + H)⁺380.0063 found 380.0057.

4-(2-(3-Bromophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5f)

Prepared according to General Procedure 2 from 2-(3-bromophenyl)-4-methyl-4,5-dihydro-1H-imidazole. Yield 755 mg (64%), white solid, m. p. 180 °C (decomp.); ¹H NMR (500 MHz, CD₃OD) δ 7.91 (d, J = 8.7 Hz, 2 H), 7.86–7.83 (m, 1 H), 7.81–7.78 (m, 1 H), 7.48–7.37 (m, 4 H), 4.75–4.68 (m, 2 H), 4.32–4.24 (m, 1 H), 1.63–1.60 (m, 3 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 164.7, 145.1, 141.0, 138.0, 133.2, 132.2, 129.3, 128.8, 126.7, 125.9, 124.0, 51.5, 54.0, 20.4; HRMS m/z calcd for C₁₆H₁₇BrN₃O₂S (M + H)⁺394.0219 found 394.0214.

4-(2-(3-Fluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5g)

Prepared according to General Procedure 2 from 2–(3-fluorophenyl)-4,5-dihydro-1H-imidazole. Yield 584 mg (61%), white solid, m. p. 225–227 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.77–7.75 (m, 2 H), 7.48–7.44 (m, 1 H), 7.29 (t, J = 7.4 Hz, 2 H), 7.25 (d, J = 8.0 Hz, 1 H), 6.97–6.96 (m, 2 H), 4.24 (t, J = 9.6 Hz, 2 H), 4.09 (t, J = 9.6 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 164.7 (d, J_C-F = 244.6 Hz), 163.6, 147.1, 140.2, 134.9 (d, J_C-F = 7.8 Hz), 132.6 (d, J_C-F = 8.1 Hz), 128.9, 126.5 (d, J_C-F = 3.2 Hz), 123.5, 117.4 (d, J_C-F = 23.1 Hz), 119.4 (d, J_C-F = 21.1 Hz), 54.9, 54.2; HRMS m/z calcd for C₁₅H₁₅FN₃O₂S (M + H)⁺320.0864 found 320.0854.

4-(2-(3-Chlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5h)

Prepared according to General Procedure 2 from 2-(3-chlorophenyl)-4,5-dihydro-1H-imidazole. Yield 794 mg (79%), white solid, m. p. 188–190 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.78–7.76 (m, 2 H), 7.54–7.56 (m, 2 H), 7.44–7.38 (m, 2 H), 6.98–6.96 (m, 2 H), 4.25 (t, J = 9.4 Hz, 2 H), 4.10 (t, J = 9.7 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 163.5, 147.1, 140.2, 136.5, 134.7, 132.6, 132.2, 130.5, 129.0, 128.9, 123.5, 55.0, 54.2; HRMS m/z calcd for C₁₅H₁₅ClN₃O₂S (M + H)⁺336.0568 found 336.0560.

4-(2-m-Tolyl-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5i)

Prepared according to General Procedure 2 from 2-(m-tolyl)-4,5-dihydro-1H-imidazole. Yield 350 mg (37%), cream solid, m. p. 244–246 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.73 (d, J = 8.8 Hz, 2 H), 7.37–7.26 (m, 3 H), 7.22–7.18 (m, 1 H), 6.99 (d, J = 8.8 Hz, 2 H), 4.28 (t, J = 10.3 Hz, 2 H), 4.07 (t, J = 10.3 Hz, 2 H), 2.33 (s, 3H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 164.7, 145.5, 140.1, 140.0, 133.1, 130.3, 130.2, 129.8, 128.1, 126.9, 123.1, 54.1, 51.4, 21.3; HRMS m/z calcd for C₁₆H₁₈N₃O₂S (M + H)⁺316.1114 found 316.1107.

4-(2-(3-Fluoro-4-methoxyphenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5j)

Prepared according to General Procedure 2 from 2-(3-fluoro-4-methoxyphenyl)-4,5-dihydro-1H-imidazole. Yield 712 mg (68%), yellow solid, m. p. 195–197 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.78–7.76 (m, 2 H), 7.25 (t, J = 7.3 Hz, 2 H), 7.14 (t, J = 7.6 Hz, 1 H), 7.00–6.98 (m, 2 H), 4.22 (t, J = 9.6 Hz, 2 H), 4.06 (t, J = 9.6 Hz, 2 H), 3.95 (s, 3H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 163.6, 153.9 (d, J_C-F = 245.3 Hz), 152.0 (d, J_C-F = 10.6 Hz), 147.5, 140.1, 128.9, 127.4 (d, J_C-F = 3.5 Hz), 124.8 (d, J_C-F = 7.0 Hz), 123.7, 118.1 (d, J_C-F = 20.0 Hz), 115.3, 57.6, 55.1, 53.8; HRMS m/z calcd for C₁₆H₁₇FN₃O₃S (M + H)⁺350.0969 found 350.0960.

4-(2-(4-Fluoro-3-methoxyphenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5k)

Prepared according to General Procedure 2 from 2-(4-fluoro-3-methoxyphenyl)-4,5-dihydro-1H-imidazole. Yield 691 mg (66%), white solid, m. p. 248–249 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.79–7.76 (m, 2 H), 7.24 (dd, J = 8.1, 1.8 Hz, 1 H), 7.14 (dd, J = 13.7, 8.3 Hz, 1 H), 7.01–7.04 (m, 1 H), 7.00 (d, J = 8.8 Hz, 2 H), 4.25 (t, J = 9.4 Hz, 2 H), 4.09 (t, J = 10.3 Hz, 2 H), 3.83 (s, 3 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 164.1, 155.8 (d, J_C-F = 249.1 Hz), 150.0 (d, J_C-F = 11.3 Hz), 147.3, 140.1, 130.7, 128.8, 123.7, 123.6, 118.0 (d, J_C-F = 19.0 Hz), 116.2 (d, J_C-F = 1.8 Hz), 57.6, 54.9, 53.8; HRMS m/z calcd for C₁₆H₁₇FN₃O₃S (M + H)⁺350.09693 found 350.0960.

4-(2-(3-Chloro-4-methoxyphenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5l)

Prepared according to General Procedure 2 from 2-(3-chloro-4-methoxyphenyl)-4,5-dihydro-1H-imidazole. Yield 712 mg (63%), white solid, m. p. 255–257 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.78 (d, J = 8.6 Hz, 2 H), 7.55 (s, 1 H), 7.38 (d, J = 8.6 Hz, 1 H), 7.11 (d, J = 8.4 Hz, 1 H), 6.99 (d, J = 8.6 Hz, 2 H), 4.23 (t, J = 9.6 Hz, 2 H), 4.06 (t, J = 9.2 Hz, 2 H), 3.97 (s, 3 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 163.5, 159.3, 147.5, 140.1, 132.3, 130.8, 129.0, 125.4, 124.6, 123.6, 114.0, 57.7, 55.1, 53.8; HRMS m/z calcd for C₁₆H₁₇ClN₃O₃S (M + H)⁺366.0674 found 366.0664.

4-(2-(3,4-Difluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5m)

Prepared according to General Procedure 2 (alternatively, the recently reported microwave-assisted protocol was used from 2-(3,4-difluorophenyl)-4,5-dihydro-1H-imidazole. Yield 465 mg (46%), white solid, m. p. 177–179 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.78 (d, J = 7.9 Hz, 2 H), 7.46–7.44 (dd, J = 10.6, 8.9 Hz, 1 H), 7.38–7.30 (m, 2 H), 6.98 (d, J = 8.0 Hz, 2 H), 4.24 (t, J = 9.8 Hz, 2 H), 4.09 (t, J = 9.7 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 162.9, 153.8 (dd, J_C-F = 251.8, 12.5 Hz), 152.2 (dd, J_C-F = 248.6, 12.8 Hz), 147.1, 140.3, 129.8 (dd, J_C-F = 6.3, 4.2 Hz), 129.0, 127.7 (dd, J_C-F = 6.7, 3.7 Hz), 123.7, 119.9 (d, J_C-F = 19.0 Hz), 119.7 (d, J_C-F = 18.0 Hz), 55.1, 54.2; HRMS m/z calcd for C₁₅H₁₄F₂N₃O₂S (M + H)⁺338.0769 found 338.0760. Alternatively, the recently reported microwave-assisted protocol15 was used to obtain the same compound in 68% yield.

4-(2-(3,4-Dichlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5n)

Prepared according to General Procedure 2 from 2-(3,4-dichlorophenyl)-4,5-dihydro-1H-imidazole. Yield 886 mg (80%), white solid, m. p. 232–234 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.79–7.78 (m, 2 H), 7.70 (s, 1 H), 7.59–7.58 (m, 1 H), 7.36–7.38 (m, 1 H), 6.99–6.98 (m, 2 H), 4.29–4.19 (m, 2 H), 4.15–4.03 (m, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 155.8, 147.0, 140.5, 136.7, 134.7, 133.0, 132.8, 132.6, 130.3, 129.1, 123.7, 55.2, 54.3; HRMS m/z calcd for C₁₅H₁₄Cl₂N₃O₂S (M + H)⁺370.0178 found 370.0169.

4-(2-(4-Chloro-3-(trifluoromethyl)phenyl)-4,5-dihydroimidazol-1-yl)benzenesulfonamide (5o)

Prepared according to General Procedure 2 from 2-[4-chloro-3-(trifluoromethyl)phenyl]-4,5-dihydro-1H-imidazole. Yield 786 mg (65%), white solid, m. p. 215–217 °C; ¹H NMR (500 MHz, CD₃OD) δ 7.98 (s, 1 H), 7.79 (d, J = 8.6 Hz, 2 H), 7.66–7.58 (m, 2 H), 7.00 (d, J = 8.6 Hz, 2 H), 4.27 (t, J = 9.7 Hz, 2 H), 4.12 (t, J = 9.6 Hz, 2 H), NH₂ protons in exchange; ¹³C NMR (125 MHz, CD₃OD) δ 162.7, 147.0, 140.7, 136.2 (unresolved q, J_C-F = 1.9 Hz), 135.5, 133.9, 132.1, 130.5 (q, J_C-F = 31.8 Hz), 130.0 (q, J_C-F = 5.1 Hz), 129.1, 124.7 (q, J_C-F = 271.1 Hz), 123.9, 55.2, 54.4; HRMS m/z calcd for C₁₆H₁₄ClF₃N₃O₂S (M + H)⁺404.0442 found 404.0431.

Carbonic anhydrase inhibition assay

An Applied Photophysics Stopped-Flow instrument (Applied Photophysics Ltd., Leatherhead, Surrey, UK) has been used for assaying the CA catalyzed CO₂ hydration activity16. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.005 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were pre incubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house17–20.

Results and discussion

Compounds 5a–o were synthesized as outlined in Scheme 1 12. Imidazolines 6 were prepared by condensation of a series of benzaldehydes with ethylenediamine or 1,2-diaminopropane using a well-established protocol13. Pd-Catalyzed N-arylation of 6a–o with 8 proved facile and, without further purification, the DMB (2,4-dimethoxybenzyl) protecting group from 7a–o was removed with TFA and the target sulfonamides were obtained in fair to good yields (see Supplementary material).

Following our earlier reported synthesis of compounds 5a–o and their evaluation as selective COX-2 inhibitors12, we have since developed an operationally convenient protocol21 for microwave-assisted, Pd-catalyzed N-arylation of 2-imidazolines which significantly shortened reaction times and, occasionally, improved isolated product yields. In this work, we demonstrated the same microwave-promoted procedure to be applicable to the synthesis of a representative compound (5m) and to lead to a noticeable improvement of the isolated yield (Scheme 2).

Scheme 1. Synthesis of 2-imidazoline-substituted benzene sulfonamides 5a–o.

Scheme 2. Microwave-assisted synthesis of 5m.

When tested for CA I, II, IV and VII inhibition, compounds 5a–o, reassuringly, displayed an interesting inhibition profile (Table 1), which is likely a result of the sulfonamide moiety acting as a zinc-binding group (ZBG)22. A double-digit nanomolar inhibition was reliably achieved for CA II, which is a well-established biological target for anti-glaucoma and anti-edema drugs2. The same level of activity was also achieved against CA VII, while the lower activity against CA I is perhaps not very unusual considering the generally lower inhibitory potency of sulfonamides toward this isoform1. What constitutes an especially valuable finding in this work was, in our opinion, the marked selectivity (SI) between CA II and CA IV, which are both highly catalytically active CA isoforms and known to be very sensitive to sulfonamides. While the structural understanding of the observed separation of the CA II versus IV activity remains to be gained via crystallographic studies, the body of biological data found here contributes quite substantially to the global knowledge about isoform-selective CAIs.

Table 1. Inhibitory activity data for compound 5a–o obtained against hCA I, II, IV and VII.

			Ki, nM				SI (hCAIV/hCAII)
Compound	Ar	R	hCA I	hCA II	hCA IV	hCA VII
5a	4-FC6H4	H	2 343.0	76.1	9 726.0	303.5	127.8
5b	4-FC6H4	Me	816.5	52.4	8 705.0	86.6	166.1
5c	4-ClC6H4	H	960.5	46.7	8 142.0	269.0	174.3
5d	4-ClC6H4	Me	897.9	54.1	6 785.0	69.6	125.4
5e	3-BrC6H4	H	1 844.0	53.0	5 010.0	204.7	94.5
5f	3-BrC6H4	Me	1 281.0	55.3	5 272.0	45.8	95.3
5g	3-FC6H4	H	3 230.0	75.9	>10 000	311.6	131.8
5h	3-ClC6H4	H	1 668.0	185.1	5 701.0	204.2	30.8
5i	3-MeC6H4	H	3 943.0	111.9	9 778.0	906.7	87.4
5j	3-F,4-MeOC6H3	H	1 231.0	80.7	8 266.0	709.4	102.4
5k	4-F,3-MeOC6H3	H	2 166.0	61.6	4 228.0	258.6	68.6
5l	3-Cl,4-MeOC6H3	H	3 987.0	227.1	>10 000	103.9	44.0
5m	3,4-diFC6H3	H	746.8	34.6	5 381.0	71.3	155.5
5n	3,4-diClC6H3	H	787.7	30.2	932.0	35.5	30.9
5o	4-Cl,3-CF3C6H3	H	3 685.0	84.3	3 845.0	32.6	45.6
Acetazolamide (AAZ)			250.0	12.0	74.0	2.5	6.2

Currently, the absence of the crystallographic information on the potent 2-imidazoline CAIs described herein does not allow drawing substantiated conclusions on the role of the 2-aryl-2-imidazoline tails grafted onto benzenesulfonamide pharmacophore. However, in our view, it appears unlikely that the polar 2-imidazoline moiety participates in active interactions with the protein target. It is rather the lipophilic aromatic tail responsible for interacting with the hydrophobic half of the CA binding side which is likely to result in the observed potency and selectivity. Should the polar 2-imidazoline core be contributing significantly (e.g. by building hydrogen-bonding interactions with the hydrophilic half of the binding site), the introduction of a methyl substituent on the 2-imidazoline scaffold (as in 5b, 5d and 5f) would be expected to disrupt this vital interaction. However, such a modification only seems to improve the CA VII affinity and virtually does not affect the CA I, II and IV inhibitory profile. This strongly argues for 2-imidazoline being a generally indifferent element of the periphery, responsible for a correct projection of the lipophilic periphery toward the target rather than building any specific interactions with the latter, in a direct analogy to 4-ureido benzenesulfonamides 1–3 (vide supra). The analogy is illustrated (Figure 2) quite well by the effective spatial superposition (in energy-minimized conformations) of compound 3, a potent CA II inhibitor (K_i = 96 nM) with compound 5a which inhibits CA II with roughly the same potency (K_i = 76 nM).

Figure 2. Spatial alignment of compound 5a (green) with its ureido analog 3 (red).

Interestingly, compound 5n which we had earlier identified as the lead selective COX-2 inhibitor12, turned out to be a rather potent CA II and CA VII inhibitor which confirms the potential of using this series as a dual CA/COX-2 inhibitors.

Conclusion

The polypharmacology of primary aromatic sulfonamides prompted us to evaluate the profile of compounds 5a–o (earlier described in the context of selective cyclooxygenase-2 inhibition) against a panel of hCA (CA I, II, IV and VII). The compounds showed a strong inhibition of CA II (a well-established cytosolic glaucoma target) and CA VII (also a cytosolic isoform believed to be involved in the onset of epilepsy). The absence of activity against CA I is perhaps unsurprising (considering the documented lower affinity of CA I toward sulfonamides). However, the selectivity established for some of compounds 5a–o with respect to CA II versus CA IV (both of which are highly active isoforms of the enzyme, strongly inhibited by sulfonamides in general) constitutes a very significant finding in light of isoform-selective CA targeting. For compound 5n, strong inhibition of COX-2 and some CA isoforms could be a basis for designing dual-action therapeutic agents.

Declaration of interest

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

Supplementary material available online

Acknowledgements

This research was supported by the Russian Scientific Fund (project grant 14-50-00069).