Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols including natural products

Abstract

(2-Bromo-3,4-dimethoxyphenyl) (3,4-dimethoxyphenyl)methanone (10) and its derivatives with Br, one dibromide and isomeric three tribromides, were synthesized. Demethylation of these compounds afforded a series of new bromophenols. Inhibition of human cytosolic carbonic anhydrase II (hCA II) isozyme by these new bromophenols and naturally occurring 3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)benzene-1,2-diol (3), and 5,5′-methylenebis(3,4,6-tribromo-benzene-1,2-diol) (4) was investigated. The synthesized compounds showed carbonic anhydrase inhibitory capacities with IC₅₀ values in the range of 0.7–372 μM against hCA II. Some bromophenols investigated here showed effective hCA II inhibitory activity and might be used as leads for generating novel carbonic anhydrase inhibitors which are valuable drug candidates for the treatment of glaucoma, epilepsy, gastric and duodenal ulcers, neurological disorders, or osteoporosis.

Keywords::

• Bromophenols
• diphenylmethane
• carbonic anhydrase
• glaucoma
• enzyme inhibition

Introduction

Naturally occurring bromophenols, frequently isolated from red algae of the family Rhodomelaceae, have prominent biological activities^1,2. Of these natural compounds, 5,5′-methylenebis(3,4-dibromobenzene-1,2-diol) (1) and 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(methoxymethyl)benzyl)benzene-1,2-diol (2) exhibit enzyme inhibition, e.g. isocitrate lyase³ cytotoxicity⁴, feeding deterrent⁵, and microbial^6,7 activities, while 3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)benzene-1,2-diol (3) and 5,5′-methylenebis(3,4,6-tribromo-benzene-1,2-diol) (4) exhibit significant aldose reductase inhibitory activity⁸. Additionally, it was reported that bromophenol 1 is an inhibitor of protein tyrosine phosphatase⁹. Antioxidant activities of 1 and 4 have also been reported^10,11. Recently, we have achieved an alternative synthesis of 1¹⁰, first total synthesis of 2, 3, 4^12,13, and a series of diphenylmethanone like bromophenols 5¹⁰. We have reported that compound 1 and a series of 5 show high antioxidant and radical scavenging activities¹⁰. Compound 6 and its derivatives with different number of bromines are also diphenylmethanone like compounds which are similar to 5 (Figure 1).

Figure 1.  Some naturally occurring bromophenols.

The carbonic anhydrases (CA; Carbonate hydrolyase, EC 4.2.1.1) are a ubiquitous family of zinc-containing enzymes that classically participate in the maintenance of pH homeostasis in human body, catalyzing the reversible hydration of carbon dioxide in a two-step reaction to yield bicarbonate and protons¹⁴. Sixteen isozymes have been described so far, that differ in their subcellular localization, catalytic activity and susceptibility to different classes of inhibitors. Some of these isozymes 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 primates, but it is abundant in rodents and other higher vertebrates^15–17. CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation^15,18,19. The two major CA isozymes (CA I and CA II) are present at high concentrations in the cytosol in erythrocytes, and CA II has the highest turnover rate among all CAs. Many of the CA isozymes involved in these processes are important therapeutic targets with the potential to be inhibited to treat a range of disorders including oedema, glaucoma, obesity, cancer, epilepsy and osteoporosis^18–20.

Interaction of most CA isozymes with several types of phenols, such as simple phenol and its substituted derivatives, clioquinol, salicyclates and some of their derivatives, has been recently investigated^20–23. Here, we extend these earlier investigations to a novel series of bromophenols.

Chemicals are generally known to activate or inhibit several enzymes in vivo and affect metabolic pathways. Inhibitory effects of different anions, metal ions, drugs, phenols and sulfonamides, which are specific inhibitors, have been so far investigated against many CAs^21,24–28. CA II inhibitors are used for several purposes, in particular for the treatment of glaucoma, epilepsy, and as diuretics or antitumor agents/diagnostic tools^18,19,29.

Many chemical substances and synthesized drugs affect metabolisms by changing enzyme activities^30–33. As CA II inhibitors are valuable molecules for therapeutical and pharmacological applications, we have synthesized novel bormophenols in the current research and evaluated their potency to be novel carbonic anhydrase inhibitors.

Materials and methods

All chemicals and solvents are commercially available and were used after distillation or treatment with drying agents. Column chromatography (CC): silica gel (SiO₂; 60 mesh, Merck, Darmstadt, Germany). Preparative thick layer chromatography: 1 mm of SiO₂ 60 PF (Merck) on glass plates. Mp: cap. melting-point apparatus (BUCHI 530: Flawil, Switzerland); uncorrected. IR Spectra: solns. in 0.1 mm cells with a Mattson 1000 FT-IR spectrophotometer (Cambridge, England). ¹H- and ¹³C- NMR spectra: 200 (50) and 400 (100)-MHz Varian spectrometer (Danbury, CT); δ in ppm; Me4Si as the internal standard. Elemental analyses: Leco CHNS-932 apparatus (MI, USA). Antioxidant activities of samples were determined in a spectrophotometer (UV-1208, Shimadzu, Japan).

Synthesis of (2-bromo-3,4-dimethoxyphenyl)(3,4-dimethoxyphenyl)methanone (10)

Polyphosphoric acid (PPA), prepared from conc. H₃PO₄ (85%, 2.63 g) and P₂O₅ (4.72 g, 33.2 mmol), was heated to 80°C in a beaker (100 mL). To this mixture were added 8 (0.84 g, 4.6 mmol) and 9³⁶ (1.0 g, 4.6 mmol) quickly. The mixture was stirred with a glass stick at 80°C for 45 min and was then carefully poured onto 35 mL of ice/water. The organic phase was extracted with EtOAc (2 × 125 mL). The combined organic layers were dried over Na₂SO₄ and the solvent was evaporated. Monobromide 10 (85%) was the sole product and was crystallized from ethyl acetate as white crystals. Mp 166–167°C; ¹H-NMR (400 MHz, CDCl₃): δ 7.54 (d, J = 2.2 Hz, 1 H), 7.23 (dd, A part of AB-system, J = 8.3 Hz, 2.2 Hz, 1 H), 7.07 (d, A part of AB-system, J = 8.4 Hz, 1 H), 6.93 (d, B part of AB-system, J = 8.4 Hz, 1 H), 6.82 (d, B part of AB-system, J = 8.4 Hz, 1 H), 3.93 (s, methoixde, 6 H), 3.92 (s, methoxide, 3 H), 3.88 (s, methoixde, 3 H); ¹³C-NMR (100 MHz, CDCl₃): δ 194.24 (CO), 154.89 (C), 154.05 (C), 149.45 (C), 146.95 (C), 134.35 (C), 129.95 (C), 126.51 (CH), 124.78 (CH), 116.18 (C), 111.46 (CH), 111.18 (CH), 110.14 (CH), 60.84 (OCH₃), 56.41 (OCH₃), 56.31 (OCH₃), 56.25 (OCH₃); IR (CH₂Cl₂, cm⁻¹): 3003, 2938, 2839, 1657, 1586, 1512, 1487, 1463, 1417, 1394, 1341, 1294, 1274, 1240, 1217, 1171, 1135, 1032, 991, 904, 879, 813, 796, 759, 729, 636, 569; Anal. Calcd for C₁₇H₁₇BrO₅: C, 53.56; H 4.49. found: C, 53.52; H 4.40.

Bromination of compound 10

To a stirring solution of monobromide 10 (2.0 g, 5.2 mmol) in CHCl₃ (50 mL) was added a solution of bromine (5.0 g, 31.2 mmol, 6 eq.) in CHCl₃ (30 mL) drop wise at room temperature (RT) over 10 min. After the reaction mixture was stirred at RT for 3 days, the solvent was evaporated. Chromatography of the residue (2.71 g) on silica gel (SiO₂, 100 g) with ethyl acetate/hexane (5:95) gave dibromide 11 (0.68 g, 28%), 14 (0.32 g, 12%), 13 (0.71 g, 26%) and 12 (0.76 g, 27%), respectively.

(2-Bromo-3,4-dimethoxyphenyl)(2-bromo-4,5-dimethoxyphenyl)methanone (11)

Mp 122–123°C as white crystals; ¹H-NMR (400 MHz, CDCl₃) δ 7.21 (d, A part of AB-system, J = 8.4 Hz, 1 H), 7.06 (s, 1 H), 7.03 (s, 1 H) 6.88 (d, B part of AB-system, J = 8.4 Hz, 1 H) 3.93 (s, methoxide, 3 H), 3.92 (s, methoxide, 3 H), 3.86 (s, methoxide, 3 H), 3.85 (s, methoxide, 3 H); ¹³C-NMR (100 MHz, CDCl₃) δ 194.19 (CO), 156.31 (C), 152.18 (C), 148.49 (C), 147.26 (C), 133.38 (C), 131.96 (C), 127.67 (CH), 117.97 (C), 116.56 (CH), 114.19 (CH), 113.61 (C), 110.83 (CH), 60.80 (OCH₃), 56.56 (OCH₃), 56.45 (OCH₃), 56.37 (OCH₃); IR (CH₂Cl₂, cm⁻¹): 3005, 2964, 2842, 2591, 1668, 1584, 1505, 1486, 1463, 1445, 1399, 1375, 1336, 1271, 1211, 1171, 1159, 1059, 1030, 994, 919, 867, 820, 785, 735, 702, 647, 584; Anal. Calcd for C₁₇H₁₆Br₂O₅: C, 44.38, H 3.51 found: C, 44.38; H 3.52.

(2-Bromo-3,4-dimethoxyphenyl)(2,3-dibromo-4,5-dimethoxyphenyl)methanone (12)

Mp 100–101°C as pale yellow crystals; ¹H-NMR (400 MHz, CDCl₃) δ 7.24 (d, A part of AB-system, J = 8.8 Hz, 1 H), 6.98 (s, 1 H), 6.86 (d, part of AB-system, J = 8.8 Hz, 1 H), 3.93 (s, methoxide, 3 H), 3.91 (s, methoxide, 3 H), 3.87 (s, methoxide, 3 H), 3.86 (s, methoxide, 3 H); ¹³C-NMR (100 MHz, CDCl₃) δ 193.60 (CO), 157.03 (C), 152.76 (C), 147.67 (C), 138.11 (C), 131.33 (C), 129.07 (CH), 126.18 (C), 123.27 (C), 118.75 (C), 114.70 (C), 113.20 (CH), 110.54, (CH), 60.93 (OCH₃), 60.81 (OCH₃), 56.62 (OCH₃), 56.41 (OCH₃); IR (CH₂Cl₂, cm⁻¹): 3003, 2938, 1673, 1588, 1564, 1507, 1464, 1403, 1337, 280, 1262, 1217, 1166, 1141, 1070, 1032, 996, 924, 865, 837, 790, 733, 681, 609. Anal. Calcd for C₁₇H₁₆Br₃O₅: C, 37.88, H 2.80 found: C, 37.93; H 2.85.

(2-Bromo-4,5-dimethoxyphenyl)(2,6-dibromo-3,4-dimethoxyphenyl)methanone (13)

Mp 115–117°C as pale yellow crystals; ¹H-NMR (400 MHz, CDCl₃) δ 7.35 (s, 1 H), 7.11 (s, 1 H), 7.02 (s, 1 H) 3.94 (s, methoxide, 3 H), 3.91 (s, methoxide, 3 H), 3.87 (s, methoxide, 3 H), 3.84 (s, methoxide, 3 H); ¹³C-NMR (100 MHz, CDCl₃) δ 192.88 (CO), 153.66 (C), 152.80 (C), 152.14 (C), 148.59 (C), 137.82 (C), 130.58 (C), 129.52 (CH), 117.00 (C), 116.81 (C), 116.74 (CH), 114.36 (CH), 114.23 (C), 61.39 (OCH₃), 61.25 (OCH₃), 56.62 (OCH₃), 56.50 (OCH₃); IR (CH₂Cl₂, cm⁻¹): 2938, 2841, 1671, 1579, 1541, 1512, 1464, 1419, 1399, 1366, 1300, 1270, 1212, 1185, 1142, 1080, 1032, 1004, 918, 804, 775, 734, 665; Anal. Calcd for C₁₇H₁₆Br₃O₅: C, 37.88, H 2.80 found: C, 37.86; H 2.84.

(2-Bromo-4,5-dimethoxyphenyl)(2,5-dibromo-3,4-dimethoxyphenyl)methanone (14)

Mp 138–139°C as colourless crystals; ¹H-NMR (400 MHz, CDCl₃) δ 7.42 (s, 1 H), 7.10 (s, 1 H), 7.09 (s, 1 H) 3.86 (s, methoxide, 3 H), 3.85 (s, methoxide, 3 H), 3.85 (s, methoxide, 3 H), 3.84 (s, methoxide, 3 H); ¹³C-NMR (100 MHz, CDCl₃) δ 191.38 (CO), 154.50 (C), 153.46 (C), 148.58 (C), 146.65 (C), 134.96 (C), 128.23 (C), 117.55 (CH), 116.61 (C), 116.29 (CH), 115.80 (C), 115.02 (CH), 114.60 (C), 60.97 (OCH₃), 56.63 (2 OCH₃), 56.43 (OCH₃); IR (CH₂Cl₂, cm⁻¹): 3003, 2936, 2841, 1679, 1655, 1586, 1508, 1476, 1442, 1380, 1338, 1298, 1262, 1212, 1158, 1065, 1027, 992, 931, 844, 815, 785, 756, 733, 701, 588; Anal. Calcd for C₁₇H₁₆Br₃O₅: C, 37.88, H 2.80 found: C, 37.66; H 2.82.

Standard procedure for demethylation of compounds with OMe by ether cleavage (2-bromo-3,4-dihydroxyphenyl)(3,4-dihydroxyphenyl)methanone (6)

A solution of monobromide 10 (0.43 g, 1.32 mmol) in CH₂Cl₂ (15 mL) was cooled to 0°C and then a solution of BBr₃ (0.9 mL) in CH₂Cl₂ (10.0 mL) was added drop wise under N₂(g) over 5 min. After the cold bath was removed, the mixture was stirred at RT and under N₂ for 1 day. Methanol (35 mL) was slowly added over 15 min and then the solvent was evaporated. After water (45 mL) and EtOAc (2 × 40 mL) were added, the mixture was shaken. The organic phase was separated and the water phase was extracted with EtOAc (2 × 30 mL). The combined organic phases were dried over Na₂SO₄ and the solvent was evaporated. Bromophenol 6 (0.40 g, 93%) was obtained as pale yellow amorphous. Mp 77–78°C; ¹H-NMR (400 MHz, CD₃COCD₃) δ 9.06 (m, 1 OH), 8.77 (m, 1 OH), 8.43 (m, 1 OH) 8.28 (m, 1 OH), 7.32 (d, J = 2.2 Hz, 1 H), 7.17 (dd, A part of AB-system, J = 8.1, 2.2 Hz, 1 H), 6.94 (d, A part of AB-system, J = 8.1 Hz, 1 H), 6.90 (d, B part of AB-system, J = 8.1 Hz, 1 H), 6.74 (d, B part of AB-system, J = 8.1 Hz, 1 H); ¹³C-NMR (100 MHz, CD₃COCD₃) δ 193.59 (CO), 150.79 (C), 146.86 (C), 145.12 (C), 143.33 (C), 133.88 (C), 129.86 (C), 124.26 (CH), 120.21 (CH), 116.69 (CH), 115.08 (CH), 113.89 (CH), 107.53 (C); IR (CH₂Cl₂, cm⁻¹): 3434, 2967, 2075, 1638, 1595, 1524, 1442, 1388, 1300, 1201, 1120, 1032, 1015, 943, 816, 782, 763; Anal. Calcd for C₁₃H₉BrO₅: C, C, 48.03; H 2.79 found: C, 48.01; H 2.80.

Synthesis of bromophenols 15–18 from the corresponding compounds 11–14, respectively.

The standard procedure^10,12,13,35 described above for the synthesis of 6 with BBr₃ was applied. From these reactions, bromophenols 15–18 were obtained.

(2-Bromo-3,4-dihydroxyphenyl)(2-bromo-4,5-dihydroxyphenyl)methanone (15)

It was crystallized from ethyl acetate/hexane as pale yellow crystals (0.382 g, 85%); mp 186–187°C; ¹H-NMR (400 MHz, CD₃COCD₃) δ 9.29 (s, 1 OH), 8.96 (s, 1 OH), 8.53 (s, 1 OH) 8.26 (s, 1 OH), 7.11 (s, 1H), 6.96 (s, 1H), 6.91 (d, A part of AB-system, J = 8.2 Hz, 1 H), 6.86 (d, B part of AB-system, J = 8,2 Hz, 1 H); ¹³C-NMR (100 MHz, CD₃COCD₃) δ 193.29 (CO), 149.15 (C), 148.58 (C), 144.52 (C), 143.70 (C), 132.57 (C), 131.49 (C), 123.31 (CH), 120.58 (CH), 118.66 (CH), 113.64 (CH), 111.01 (C), 108.98 (C); IR (CH₂Cl₂, cm⁻¹): 3368, 2947, 2834, 2526, 2041, 1655, 1594, 1452, 1419, 1295, 1115, 1032, 668; Anal. Calcd for C₁₃H₈Br₂O₅: C, 38.65; H 2.00. found: C, 38.64; H 2.01.

(2-Bromo-3,4-dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)methanone (16)

Yellow amorphous (0.34 g, 95%); mp 121–123°C; ¹H-NMR (400 MHz, CD₃COCD₃) δ 6.96 (s, 1 H), 6.91 (s, 1 H), 6.90 (s, 1 H); ¹³C-NMR (100 MHz, CD₃COCD₃), δ 193.05 (CO), 149.24 (C), 146.94 (C), 144.55 (C), 143.96 (C), 133.79 (C), 131.19 (C), 124.47 (CH), 116.19 (CH), 114.38 (C), 113.54 (CH), 113.37 (C), 109.43 (C); IR (CH₂Cl₂, cm⁻¹): 3400, 2950, 2839, 2076, 1648, 1452, 1396, 1295, 1114, 1019, 667; Anal. Calcd for C₁₃H₇Br₃O₅: C, 32.33; H 1.46. found: C, 32.11; H 1.45.

(2-Bromo-4,5-dihydroxyphenyl)(2,6-dibromo-3,4-dimethoxyphenyl)methanone (17)

Red amorphous; (0.47 g, 87%); mp 240–242°C′(its color was changed at ≥ 180°C); ¹H-NMR (400 MHz, CD₃COCD₃) δ 7.20 (s, 1 H), 7.15 (s, 2 H); ¹³C-NMR (100 MHz, CD₃COCD₃) δ 190.55 (CO), 150.60 (C), 147.04 (C), 144.49 (C), 143.42 (C), 133.90 (C), 122.01 (CH), 120.16 (CH), 118.75 (C), 118.29 (CH), 113.62 (C), 112.90 (C), 108.33 (C); IR (CH₂Cl₂, cm⁻¹): 3681, 2973, 2863, 2071, 1654, 1587, 1495, 1476, 1454, 1286, 1213, 1144, 1054, 1033; Anal. Calcd for C₁₃H₇Br₃O₅: C, 32.33; H 1.46. found: C, 32.33; H 1.45.

(2-Bromo-4,5-dihydroxyphenyl)(2,5-dibromo-3,4-dihydroxyphenyl)methanone (18)

It was crystallized from ethyl acetate/hexane as pale yellow crystals (0.382 g, 85%); mp 186–188°C (its color changed at ≥ 160°C); ¹H-NMR (400 MHz, CD₃COCD₃), 9.09 (bs, 1 OH), 9.05 (bs, 1 OH), 8.80 (bs, 1 OH) 8.58 (bs, 1 OH), δ 7.16 (s, 1 H), 7.13 (s, 1 H), 7.02 (s, 1 H); ¹³C-NMR (100 MHz, CD₃COCD₃), δ 192.02 (CO), 149.66 (C), 146.28 (C), 144.65 (C), 144.30 (C), 133.35 (C), 130.49 (C), 125.70 (CH), 120.78 (CH), 118.94 (CH), 111.33 (C), 108.45 (C), 108.15 (C); IR (CH₂Cl₂, cm⁻¹): 3436, 3225, 2076, 1638, 1285, 1033, 720; Anal. Calcd for C₁₃H₇Br₃O₅: C, 32.33; H 1.46. found: C, 32.33; H 1.49.

CA purification assay

The purification of the CA II isozyme was performed in a simple single-step method by means of Sepharose-4B-aniline-sulfanilamide affinity column chromatoghrapy³⁶. hCA II was purified 311-fold with a specific activity of 2500 EU mg⁻¹ and an overall yield of 16%. Erythrocytes were purified from fresh human blood obtained from the Blood Centre of the Research Hospital at Atatürk University. The blood samples were centrifuged at 1500 rpm for 15 min and the plasma and buffy coat were removed. The red cells were isolated and washed twice with 0.9% NaCl and hemolyzed with 1.5 volumes of ice-cold water. The ghost and intact cells were removed by centrifugation at 20,000 rpm for 30 min at 4°C. The pH of the hemolysate was adjusted to 8.7 with solid Tris. Firstly, Sepharose-4B was oxidized by KMnO₄ and subsequently activated by SOCl₂. Subsequently, aniline was attached to the activated gel as a spacer arm and finally diazotized sulfanilamide was clamped to the para position of aniline molecule as ligand. The hemolysate was applied to the prepared Sepharose 4B-aniline-sulfanylamide affinity column which had been 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 II (hCA II) isozyme was eluted with 0.1 M CH₃COONa/0.5 M NaClO₄ (pH 5.6). All procedures were performed at 4°C.

Hydratase activity assay

Carbonic anhydrase activity was assayed by following the hydration of CO₂ according to our previous studies^37,38. CO₂-hydratase activity as an enzyme unit was calculated by using the equation (t₀-tc/tc) where t₀ and tc are the times for pH change of the non-enzymatic and the enzymatic reactions, respectively.

Protein determination

Protein quantity was determined spectrophotometrically at 595 nm according to the Bradford method during the purification steps, using bovine serum albumin as the standard³⁹.

SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis was performed after purification of the enzymes. It was carried out in 10 and 3% acrylamide for the running and the stacking gel, respectively, containing 0.1% SDS according to Laemmli procedure. A 20-μg 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 destained with several changes of the same solvent without the dye⁴⁰.

Crystal structure determination

For the crystal structure determination, the single-crystal of 13 and 14 were used for data collection on a four-circle Rigaku R-AXIS RAPID-S diffractometer (equipped with a two-dimensional area IP detector). The graphite-monochromatized Mo K_α radiation (λ = 0.71073 Å) and oscillation scans technique with Δω = 5° for one image were used for data collection. The lattice parameters were determined by the least-squares method on the basis of all reflections with F² > 2σ (F²). Integration of the intensities, correction for Lorentz and polarization effects and cell refinement were performed using CrystalClear (Rigaku/MSC Inc., 2005) software⁴¹. The structures were solved by direct methods using SHELXS-97⁴² and refined by a full-matrix least-squares procedure using the program SHELXL-97. H atoms were positioned geometrically and refined using a riding model. The final difference Fourier maps showed no peaks of chemical significance. Crystal data for 13: C₁₇H₁₅O₅Br₃, crystal system, space group: triclinic, P-1; (no:2); unit cell dimensions: a = 8.2823(2), b = 10.2487(2), c = 12.7924(3) Å, α = 72.529(5), β = 68.923(5), γ = 87.664(7)°; volume: 963.71(6) ų; Z = 2; calculated density: 1.86 mg/m³; absorption coefficient: 6.302 mm⁻¹; F(000): 524; θ range for data collection 2.6–30.5°; refinement method: full-matrix least-square on F²; data/parameters: 3936/228; goodness-of-fit on F²: 1.267; final R indices [I > 2σ(I)]: R₁ = 0.089, wR₂ = 0.103; R indices (all data): R₁ = 0.137, wR₂ = 0.115; largest diff. peak and hole: 0.391 and −0.556 e Å⁻³; CCDC: 774839. Crystal data for 14: C₁₇H₁₅O₅Br₃, crystal system, space group: triclinic, P-1; (no:2); unit cell dimensions: a = 8.3610(5), b = 8.3972(5), c = 14.7542(7)Å, α = 98.097(2), β = 95.915(3), γ = 107.200(2)°; volume: 968.2(2) ų; Z = 2; calculated density: 1.85 mg/m³; absorption coefficient: 6.273 mm⁻¹; F(000): 524; θ range for data collection 2.6–30.5°; refinement method: full-matrix least-square on F²; data/parameters: 4293/230; goodness-of-fit on F²: 1.333; final R indices [I > 2σ(I)]: R₁ = 0.086, wR₂ = 0.135; R indices (all data): R₁ = 0.139, wR₂ = 0.146; largest diff. peak and hole: 0.367 and −0.662 e Å⁻³; crystallographic data were deposited in CSD under CCDC registration number 774807.

Results and discussion

We have synthesized natural product bromophenols 3 and 4 from corresponding materials by the known method as shown in Scheme 1¹³. Reactions of compound 7 with 1,4-dibromo-2,3-dimethoxybenzene and 1,2,5-tribromo-3,4-dimethoxybenzene in the presence of PPA at 80°C gave methylether substituted diarylmethanes in high yields as sole product. The ether cleavage reaction of these diarylmethanes with BBr₃ under mild conditions afforded naturally occurring bromophenols 3 and 4¹³.

Scheme 1.  (A) 1,4-dibromo-2,3-dimethoxybenzene, PPA/80°C, (B) BBr₃/CH₂Cl₂, 0–25°C, (C) 1,2,5-tribromo-3,4-dimethoxybenzene, PPA/80°C.

(2-Bromo-3,4-dimethoxyphenyl)(3,4-dimethoxyphenyl)methanone (10) was synthesized from the reaction of 3,4-dimethoxybenzoic acid (8) and 3-bromoveratrole (9) with PPA in 85% yield as sole product (Scheme 1). Bromination of monobromide 10 (in CHCl₃) with Br₂ (6 eq.) at RT for 3 days followed by CC allowed us to isolate four products 11–14 (Scheme 1). The NMR analysis of 13 and 14 did not allow determination of their structures. Therefore, the exact structures of them were determined by X-ray diffraction analysis (Figure 2).

Figure 2.  (A) The molecular structure of tribromide 13 showing the atom numbering scheme. (B) Packing diagram for 13. (C) The molecular structure of tribromide 14 showing the atom numbering scheme. (D) Packing diagram for 14.

Bromophenols derived from compounds 10–14 may be potential biologically active compounds, because they are similar to 5 with high antioxidant and radical scavenging activities¹⁰. Therefore, bromophenols 6 and 15–18 were synthesized from compounds 10–14 by ether cleavage reaction with BBr₃ in high yields (Figure 3). Spectroscopic data of 6 and 15–18 are consistent with the proposed structures.

Figure 3.  The new synthesized bromophenols.

Inhibitory effects of the compounds on CA II catalytic activity were tested under in vitro conditions; IC₅₀ values were calculated and are given in Table 1.

Table 1.  IC₅₀ values (concentration that causes 50% inhibition of the enzyme activity) for the molecules.

Inhibitor	IC50 (µM)
3	86.4
4	38.29
6	26.4
10	372
11	11.7
12	49.5
13	126
14	58
15	1.65
16	0.7
17	9.23
18	1.36

We report here the first study on the inhibitory effects of the bromophenols derivatives 3–6 and 10–18 on the hydratase activity of hCA II. The data in Table 1 show the following regarding the inhibition of hCA II by bromophenol derivatives.

The strongest inhibitory activity has been observed with compounds 11, 15–18, (Table 1). Three derivatives, 3, 10, 13, showed weak hCA II inhibitory activity with IC₅₀-s in the range of 86.4–372 μM, (Table 1), whereas the remaining four derivatives were quite effective hCA II inhibitors, with IC₅₀-s in the range of 26.4–58 µM, (Table 1). The best hCA II inhibitor in this series of derivatives was the bulky, (2-Bromo-3,4-dihyroxyphenyl)(2,3-dibromo-4,5-dihyroxyphenyl)methanone (16), with a IC₅₀ of 0.7 µM.

As revealed by a comparison of the inhibition ranges of molecules, 13 has a higher IC₅₀ value than those of its isomers 12 and 14, and, likewise, 17 has a higher IC₅₀ than those of 16 and 18.

The phenolic compounds have been investigated as CA inhibitors (CAIs) in this study. The rationale of investigating these compounds as CAIs lies in the fact that phenol has been shown to be the only competitive inhibitor with CO₂ as the substrate for the main isoform of CA, i.e., human CA II (hCA II)²⁰. In a very sound study, Christianson and colleagues reported on the X-ray crystal structure for the adduct of hCA II with phenol²⁰, showing this compound to bind to CA by anchoring its OH moiety to the zinc-bound water/hydroxide ion of the enzyme active site through a hydrogen bond as well as to the NH amide of Thr199, an amino acid conserved in all α-CAs and critically important for the catalytic cycle of these enzymes^18,19.

CAIs are a class of pharmaceuticals used as antiglaucoma agents, diuretics, antiepileptics, in the management of mountain sickness, gastric and duodenal ulcers, neurological disorders, or osteoporosis. Thus, discovery of novel CAIs is of great importance for pharmacological and medicinal approaches, and many inhibitors have been designed and synthesized in the literature. However, it is critically important to explore further classes of potent CAIs in order to detect compounds with a different inhibition profile when compared to sulfonamides and their bioisosteres, and to find novel applications for the inhibitors of these widespread enzymes.

Conclusions

Diphenylmethanone derivative 10 was obtained and its bromination gave dibromide 11 and tribromides 12–14. From these compounds, potential biological active bromophenols 6 and 15–18 were synthesized in high yields. The structures of the products were determined and characterized by spectroscopic methods. Bromophenol derivatives 1–13 used in this study affect the activity of CA II isozyme due to the presence of the different functional groups (OH, OCH₃) in their aromatic scaffold. It has been determined in our study that compounds 15, 16 and 18 are effective inhibitors for CA II when compared to Acetazolamide, which is used as the reference inhibitor for carbonic anhydrase. Our findings here indicate thus another class of possible CAIs of interest, in addition to the well-known sulfonamides/sulfamates/sulfamides. These findings point out that substituted phenolic compounds may be used for generation of potent CAIs.

Acknowledgment

The authors are indebted to the Department of Chemistry (Atatürk University) for research conditions.

Declaration of interest

This research was financed by grants from of TÜBİTAK (The Scientific and Technological Research Council of Turkey) (Project no: TBAG-107T348).