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Journal of Enzyme Inhibition and Medicinal Chemistry Volume 38, 2023 - Issue 1
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Research Paper

Derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide as selective inhibitors of human carbonic anhydrases IX and XII over the cytosolic isoforms I and II

Jekaterīna Ivanovaa Latvian Institute of Organic Synthesis, Riga, LatviaORCID Iconhttps://orcid.org/0000-0001-8845-0193View further author information
ORCID Icon,
Morteza Abdolib Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, LatviaORCID Iconhttps://orcid.org/0000-0003-1647-4262View further author information
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Alessio Nocentinic Neurofarba Department, Università degli Studi di Firenze, Florence, ItalyORCID Iconhttps://orcid.org/0000-0003-3342-702XView further author information
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Raivis Žalubovskisa Latvian Institute of Organic Synthesis, Riga, Latvia;b Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, LatviaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-9471-1342View further author information
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Claudiu T. Supuranc Neurofarba Department, Università degli Studi di Firenze, Florence, ItalyORCID Iconhttps://orcid.org/0000-0003-4262-0323View further author information
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Article: 2170370 | Received 01 Dec 2022, Accepted 13 Jan 2023, Published online: 31 Jan 2023

Abstract

A series of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides with various substituents in 5, 6 or 7 positions was obtained from corresponding 2’-hydroxyacetophenones in their reaction with sulphamoyl chloride. 6- and 7-aryl substituted 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides were obtained from aryl substituted 2’-hydroxyacetophenonesprepared from 4- or 5-bromo-2’-hydroxyacetophenones via two-step protocol. 4-Methyl-1,2,3-benzoxathiazine-2,2-dioxides were investigated as inhibitors of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, off-target cytosolic hCA I and II, and target transmembrane, tumour-associated hCA IX and XII. Twenty derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide were obtained. With one exception (compound2a), they mostly act as nanomolar inhibitors of target hCA IX and XII. Basically, all screened compounds express none or low inhibitory properties towards off-target hCA I. hCA II is inhibited in micromolar range. Overwhelming majority of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides express excellent selectivity towards CA IX/XII over hCA I as well as very good selectivity towards CA IX/XII over hCA II.

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes found in all living organisms. They catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. This physiological reaction is essential for variety of normal and pathological processes such as CO2 and pH homeostasis, respiration, gluconeogenesis, calcification, bone resorption, fluid secretion and tumorigenesisCitation1–7.

Sixteen different α-CA isoforms have been identified in mammals so farCitation1,Citation3. hCA I and II are widely distributed isoforms which may serve as targets for some diseases and off-targets for othersCitation7. hCA IX and XII are two transmembrane, multi-domain, hypoxia-induced tumour-associated proteins discovered by Pastorek et al.Citation8 in 1994 and Türeci et al.Citation9 in 1998 that have received attention as diagnostic markers and potential drug targets for cancerCitation10,Citation11.

Tumour growth, angiogenesis, proliferation and metastasis are ascribed to the overexpressed levels of hCA IX and XII. This can be used as a strategy for targeting of these enzymes as a new approach in cancer treatment. Selective inhibition of the tumour-associated hCA IX and XII over the other isoforms, especially the most prevalent cytosolic hCA I and II is important and will result in cancer treatment with fewer side effectsCitation7.

hCA IX is not significantly expressed in the majority of normal tissues and is present only in the stomach and gallbladder epithelia, but is overexpressed in variety of solid tumorsCitation10,Citation11. On the other hand, hCA XII is abundant in many healthy tissues, like kidney, prostate, pancreas, intestine and lymphocytes, but overexpressed in a certain number of malignant tumours, and associated with less-aggressive, well-differentiated tumour phenotypes as compared to the hCA IX expressing tumours thus CA IX is the preferred isoform for pharmacological interventionCitation7,Citation10–12. hCA IX has been considered as a valuable marker for cancer, and the development of hCA IX inhibitors with selectivity over ubiquitous isoforms hCA I/II is a potential strategy for designing anticancer agentsCitation3,Citation12.

Classical CA inhibitors (CAIs) usually have a sulphonamide moiety as a zinc-binding group (ZBG), such as clinically used CAIs acetazolamide and methazolamide. On the other hand, the non-classical CAIs do not rely on ZBGCitation13,Citation14. Non-classical CAIs such as coumarins, carboxylic acids, phenols, polyamines inhibit the catalytic activity of CA by different mechanisms rather than coordinating to the zincCitation12. Coumarin derivatives were first reported as a non-classical type of CAIs in 2009 by Supuran and coworkersCitation15. Since then a number of structurally similar compounds (coumarin derivatives and their bioisosteres) were reported as CAIs, such as sulphocoumarinsCitation16–22, 3H-1,2-benzoxathiepine 2,2-dioxidesCitation23–26, benzoxepinonesCitation27, isocoumarinsCitation28. In our previous work we described the synthesis and CA inhibitory activity of new class of CAIs – nitrogen-containing analogues of 1,2-benzoxathiine 2,2-dioxides, namely 1,2,3-benzoxathiazine-2,2-dioxidesCitation29. In this work we continue the research and extend the range of 1,2,3-benzoxathiazine-2,2-dioxide derivatives focussing on 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides to provide more data about their CA inhibition properties ().

Figure 1. Structures of 1,2,3-benzoxathiazine-2,2-dioxide and its methyl derivative.

Figure 1. Structures of 1,2,3-benzoxathiazine-2,2-dioxide and its methyl derivative.

Materials and methods

Chemistry

Reagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatography was performed on silica gel, spots were visualised with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Bruker Avance Neo (400 MHz) spectrometer with chemical shifts values (δ) in ppm relative to TMS using the residual CDCl3 signal (1H 7.26; 13C 77.16) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique. Elemental analyses were performed on Carlo Erba CHNS-O EA-1108 apparatus. GC-MS analyses were performed on Agilent Technologies 7890 A gas chromatograph, column – HP-5HS (df = 0,25 μm, ID = 0,25 mm, length − 30 m) with Agilent Technologies 5975 C masselective detector.

Sulphamoyl chlorideCitation30

Chlorosulphonylisocyanate (3.80 ml, 43.3 mmol) was cooled to +5 °C. Formic acid (1.67 ml, 43.3 mmol) was added dropwise in temperature range between +5 and +13 °C. Reaction mixture was stirred at 0 °C for additional 45 min, then allowed to warm to room temperature. Toluene (30 ml) was added to the reaction mixture, precipitate was filtered, and filtrate was evaporated. Product was obtained as yellow oily solid (6.58 g, 98%). Mp 36–37 °C.

General procedure for the preparation of 1,2,3-benzoxathiazine 2,2-dioxides 2a-j

The derivative of acetophenone (1 equiv) was dissolved in dry DMA (6 ml). Reaction mixture was cooled to 0 °C. Sulphamoyl chloride (2.5 equiv) was slowly added to the reaction mixture. The stirring was continued at room temperature under argon atmosphere for 24–72 h. Reaction mixture was then poured into ice-water (25 ml), extracted with DCM (3 × 25 ml), washed with satd. NaHCO3 (3 × 25 ml) and satd. NaCl (3 × 25 ml). Organic phase was dried over Na2SO4, filtered, evaporated. Product was purified by column chromatography on silica gel with PE/EtOAc (2:1) followed by recrystallization from EtOH.

5-Methoxy-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2a)Citation31

Obtained from 1–(2-hydroxy-6-methoxyphenyl)ethanone (0.20 g, 1.20 mmol) and sulphamoyl chloride (0.35 g, 3.00 mmol). Reaction mixture was stirred for 72 h. Purified by column chromatography with PE/EtOAc (1:1). Obtained as light yellow solid (0.12 g, 44%). Mp 91–92 °C.

IR (KBr) cm−1 = 1374 (S = O), 1194 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.60 (1H, t, J = 8.4 Hz), 6.85–6.89 (2H, m), 3.99 (3H, s), 2.77 (3H, s) ppm 13C NMR (100 MHz, CDCl3)δ = 178.7, 160.0, 154.6, 137.2, 111.1, 108.8, 56.6, 29.2 ppm Analysis calculated for C9H9NO4S (227.23): C, 47.57; H, 3.99; N 6.16. Found: C, 48.17; H, 4.14; N, 6.02. GC-MS (m/z, %): 120 (60), 133 (37), 134 (24), 162 (26), 227 (100).

4,6-Dimethyl-1,2,3-benzoxathiazine 2,2-dioxide (2 b)Citation32

Obtained from 1–(2-hydroxy-5-methylphenyl)ethanone (0.20 g, 1.33 mmol) and sulphamoyl chloride (0.38 g, 3.33 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.18 g, 65%). Mp 125–126 °C. IR (KBr) cm−1= 1373 (S = O), 1187 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.55–7.58 (1H, m), 7.49–7.53 (1H, m), 7.18 (1H, d, J = 8.4 Hz), 2.70 (3H, s), 2.44 (3H, s) ppm 13C NMR (100 MHz, CDCl3)δ = 177.6, 151.7, 138.1, 136.2, 128.6, 119.0, 116.4, 23.9, 21.1 ppm. Analysis calculated for C9H9NO3S (211.24): C, 51.17; H, 4.29; N 6.63. Found: C, 51.46; H, 4.56; N, 6.47. GC-MS (m/z, %): 77 (27), 78 (57), 106 (20), 118 (31), 146 (54), 211 (100).

6-Fluoro-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2c)Citation32

Obtained from 1–(5-fluoro-2-hydroxyphenyl)ethanone (0.20 g, 1.30 mmol) and sulphamoylchloride (0.38 g, 3.25 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.15 g, 55%). Mp 147–148 °C. IR (KBr) cm−1 = 1379 (S = O), 1199 (S = O), 1178 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.40–7.50 (2H, m), 7.30 (1H, dd, J = 9.0, 4.3 Hz), 2.71 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.4 (d, J = 2.3 Hz), 159.1 (d, J = 248.4 Hz), 149.6 (d, J = 2.5 Hz), 124.4 (d, J = 24.0 Hz), 121.1 (d, J = 8.1 Hz), 117.2 (d, J = 7.7 Hz), 114.6 (d, J = 25.1 Hz), 23.9 ppm. Analysis calculated for C8H6FNO3S (215.20): C, 44.65; H, 2.81; N 6.51. Found: C, 44.73; H, 2.82; N, 6.40. GC-MS (m/z, %): 81 (24), 82 (99), 96 (30), 108 (28), 122 (33), 151 (46), 215 (100).

6-Chloro-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2d)Citation32

Obtained from 1–(5-chloro-2-hydroxyphenyl)ethanone (0.20 g, 1.17 mmol) and sulphamoyl chloride (0.34 g, 2.93 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.19 g, 69%). Mp 151–152 °C. IR (KBr) cm−1= 1375 (S = O), 1186 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.76 (1H, d, J = 2.4 Hz), 7.67 (1H, dd, J = 8.8, 2.4 Hz), 7.26 (1H, d, J = 8.8 Hz), 2.72 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.3, 152.0, 137.0, 131.5, 128.1, 120.8, 117.4, 23.9 ppm. Analysis calculated for C8H6ClNO3S (231.65): C, 41.48; H, 2.61; N 6.05. Found: C, 41.63; H, 2.60; N, 5.92. GC-MS (m/z, %): 63 (54), 98 (33), 104 (44), 124 (23), 167 (64), 169 (21), 231 (100).

6-Bromo-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2e)Citation32

Obtained from 1–(5-bromo-2-hydroxyphenyl)ethanone (0.20 g, 0.93 mmol) and sulphamoyl chloride (0.27 g, 2.33 mmol). Reaction mixture was stirred for 72 h. Purified by column chromatography with PE/EtOAc (4:1). Obtained as white solid (0.06 g, 23%). Mp 153–154 °C. IR (KBr) cm−1 = 1385 (S = O), 1193 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.91 (1H, d, J = 2.3 Hz), 7.81 (1H, dd, J = 8.8, 2.3 Hz), 7.20 (1H, d, J = 8.8 Hz), 2.72 (3H, s) ppm. 13C NMR (100 MHz, CDCl3)δ = 176.2, 152.5, 139.9, 131.1, 121.0, 118.6, 117.8, 23.9 ppm. Analysis calculated for C8H6BrNO3S (276.10): C, 34.80; H, 2.19; N 5.07. Found: C, 37.04; H, 2.77; N, 4.77. GC-MS (m/z, %): 62 (24), 63 (74), 104 (43), 170 (21), 211 (39), 277 (100).

4,7-Dimethyl-1,2,3-benzoxathiazine 2,2-dioxide (2f)Citation32

Obtained from 1–(2-hydroxy-4-methylphenyl)ethanone (0.19 ml, 0.20 g, 1.33 mmol) and sulphamoyl chloride (0.38 g, 3.33 mmol). Reaction mixture was stirred for 48 h. Obtained as white solid (0.18 g, 63%). Mp 134–135 °C. IR (KBr) cm−1 = 1378 (S = O), 1368 (S = O), 1189 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.66 (1H, d, J = 8.1 Hz), 7.16–7.20 (1H, m), 7.08–7.11 (1H, m), 2.69 (3H, s), 2.48 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.3, 153.7, 149.7, 128.4, 127.0, 119.3, 114.3, 23.7, 22.2 ppm. Analysis calculated for C9H9NO3S (211.24): C, 51.17; H, 4.29; N 6.63. Found: C, 51.21; H, 4.36; N, 6.57. GC-MS (m/z, %): 77 (20), 78 (39), 118 (43), 146 (35), 211 (100).

7-Methoxy-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2 g)Citation33

Obtained from 1–(2-hydroxy-4-methoxyphenyl)ethanone (0.20 g, 1.20 mmol) and sulphamoyl chloride (0.35 g, 3.00 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.08 g, 30%). Mp 163–164 °C. IR (KBr) cm−1 = 1384 (S = O), 1352 (S = O), 1186 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.69 (1H, d, J = 8.9 Hz), 6.87 (1H, d, J = 8.9, 2.5 Hz), 6.73 (1H, d, J = 2.5 Hz), 3.92 (3H, s), 2.66 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.7, 166.7, 156.1, 130.2, 113.5, 110.0, 103.0, 56.4, 23.7 ppm. Analysis calculated for C9H9NO4S (227.23): C, 47.57; H, 3.99; N 6.16. Found: C, 47.90; H, 4.08; N, 6.04. GC-MS (m/z, %): 106 (21), 120 (32), 148 (61), 227 (100).

7-Fluoro-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2 h)Citation33

Obtained from 1–(4-fluoro-2-hydroxyphenyl)ethanone (0.20 g, 1.30 mmol) and sulphamoyl chloride (0.38 g, 3.25 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.07 g, 24%). Mp 107–108 °C. IR (KBr) cm−1 = 1380 (S = O), 1374 (S = O), 1123 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.83 (1H, dd, J = 8.9, 5.8 Hz), 7.11 (1H, ddd, J = 8.9, 7.8, 2.5 Hz), 7.02 (1H, dd, J = 8.4, 2.5 Hz), 2.71 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.5, 167.2 (d, J = 262.6 Hz), 155.4 (d, J = 13.4 Hz), 131.0 (d, J = 11.1 Hz), 114.1 (d, J = 22.4 Hz), 113.4 (d, J = 3.4 Hz), 107.1 (d, J = 25.9 Hz), 24.0 ppm. Analysis calculated for C8H6FNO3S (215.20): C, 44.65; H, 2.81; N 6.51. Found: C, 44.83; H, 2.89; N, 6.37. GC-MS (m/z, %): 82 (26), 96 (29), 108 (24), 123 (36), 151 (32), 215 (100).

7-Chloro-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2i)Citation34

Obtained from 1–(4-chloro-2-hydroxyphenyl)ethanone (0.15 ml, 0.20 g, 1.17 mmol) and sulphamoyl chloride (0.34 g, 2.93 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.13 g, 48%). Mp 160–161 °C. IR (KBr) cm−1 = 1383 (S = O), 1375 (S = O), 1193 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.73 (1H, d, J = 8.5 Hz), 7.37 (1H, dd, J = 8.5, 2.0 Hz), 7.32 (1H, d, J = 2.0 Hz), 2.71 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.6, 154.0, 143.3, 129.5, 126.6, 119.6, 115.0, 23.9 ppm. Analysis calculated for C8H6ClNO3S (231.65): C, 41.48; H, 2.61; N 6.05. Found: C, 42.29; H, 2.77; N, 5.88. GC-MS (m/z, %): 63 (50), 104 (53), 124 (24), 139 (39), 167 (42), 231 (100), 233 (38).

7-Bromo-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (2j)Citation32

Obtained from 1–(4-bromo-2-hydroxyphenyl)ethanone (0.20 g, 0.93 mmol) and sulphamoyl chloride (0.27 g, 2.33 mmol). Reaction mixture was stirred for 72 h. Obtained as white solid (0.05 g, 21%). Mp 193–194 °C. IR (KBr) cm−1 = 1379 (S = O), 1373 (S = O), 1189 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.65 (1H, d, J = 8.5 Hz), 7.53 (1H, dd, J = 8.5, 1.8 Hz), 7.49 (1H, d, J = 1.8 Hz), 2.70 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.7, 153.8, 131.7, 129.5, 129.4, 122.6, 115.4, 23.9 ppm. Analysis calculated for C8H6BrNO3S (276.10): C, 34.80; H, 2.19; N 5.07. Found: C, 35.90; H, 2.41; N, 4.82. GC-MS (m/z, %): 63 (69), 104 (67), 183 (26), 211 (25), 277 (100).

General procedure for the preparation of 2’-hydroxyacetophenones 4a-j

The derivative of acetophenone (1 equiv), the derivative of benzeneboronic acid (1.3 equiv), K2CO3 (2.5 equiv) and Pd(PPh3)4 (0.05 equiv) were suspended in toluene/water (5:1, 20 ml) mixture in a pressure tube. Reaction mixture was heated to 90 °C and stirred for 24 h, then cooled to room temperature, filtered through celite. EtOAc (30 ml) was added, reaction mixture was washed with NaHCO3 (satd., 3 × 25 ml) and brine (2 × 25 ml). Organic phase was dried over Na2SO4, filtered, evaporated. The product was purified by column chromatography on silica gel with PE/EtOAc (10:1) followed by recrystallization from EtOH.

4-Hydroxy-1,1'-biphenyl-3-carbaldehyde (4a)Citation35

Obtained from 5′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), benzeneboronic acid (0.37 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as light brown solid (0.37 g, 74%). Mp 60–61 °C. IR (KBr) cm−1 = 3038 (OH), 1643 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.27 (1H, s), 7.92 (1H, d, J = 2.3 Hz), 7.72 (1H, dd, J = 8.6, 2.3 Hz), 7.52–7.57 (2H, m), 7.43–7.49 (2H, m), 7.33–7.39 (1H, m), 7.07 (1H, d, J = 8.6 Hz), 2.70 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.7, 161.9, 140.1, 135.5, 132.5, 129.2, 129.1, 127.4, 126.8, 119.9, 119.0, 26.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H13O2) 213.0916. Found 213.0911. GC-MS (m/z, %): 115 (22), 197 (100), 212 (72).

4'-Fluoro-4-hydroxy-1,1'-biphenyl-3-carbaldehyde (4b)Citation36

Obtained from 5′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-fluorobenzeneboronic acid (0.42 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as light yellow solid (0.45 g, 84%). Mp 129–130 °C. IR (KBr) cm−1 = 1640 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.26 (1H, s), 7.85 (1H, d, J = 8.6, 2.3 Hz), 7.66 (1H, dd, J = 8.6, 2.3 Hz), 7.49 (2H, dd, J = 8.9, 5.2 Hz), 7.14 (2H, t, J = 8.7 Hz), 7.06 (1H, d, J = 8.6 Hz), 2.70 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.6, 162.5 (d, J = 246.4 Hz), 161.9, 136.3 (d, J = 3.3 Hz), 135.4, 131.6, 129.0, 128.4 (d, J = 8.1 Hz), 119.9, 119.1, 116.0 (d, J = 21.5 Hz), 26.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H12O2F) 231.0821. Found 231.0816. GC-MS (m/z, %): 159 (21), 215 (100), 230 (75).

4-Hydroxy-4'-methoxy-1,1'-biphenyl-3-carbaldehyde (4c)Citation36

Obtained from 5′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-methoxybenzeneboronic acid (0.46 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.50 g, 89%). Mp 112–113 °C. IR (KBr) cm−1 = 2958 (OH), 1653 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.21 (1H, s), 7.86 (1H, d, J = 2.3 Hz), 7.67 (1H, dd, J = 8.6, 2.3 Hz), 7.46 (2H, d, J = 8.8 Hz), 7.04 (1H, d, J = 8.6 Hz), 6.99 (2H, d, J = 8.8 Hz), 3.86 (3H, s), 2.69 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.7, 161.5, 159.2, 135.2, 132.7, 132.2, 128.7, 127.9, 119.9, 118.9, 114.5, 55.5, 26.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C15H15O3) 243.1021. Found 243.1023. GC-MS (m/z, %): 227 (63), 242 (100).

3',4'-Dichloro-4-hydroxy-1,1'-biphenyl-3-carbaldehyde (4d)

Obtained from 5′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 3,4-dichlorobenzeneboronic acid (0.58 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.39 g, 59%). Mp 145–146 °C. IR (KBr) cm−1 = 3060 (OH), 1646 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.32 (1H, s), 7.86 (1H, d, J = 2.3 Hz), 7.66 (1H, dd, J = 8.7, 2.2 Hz), 7.61 (1H, d, J = 2.2 Hz), 7.51 (1H, d, J = 8.3 Hz), 7.36 (1H, dd, J = 2.2 Hz, J = 8.3 Hz), 7.08 (1H, d, J = 8. 7 Hz), 2.71 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.5, 162.5, 140.1, 135.1, 133.2, 131.5, 131.0, 130.0, 129.1, 128.6, 126.1, 120.0, 119.4, 26.9 ppm. HRMS (ESI) [M - H]-: m/z calcd for (C14H9O2Cl2) 278.9980. Found 278.9976. GC-MS (m/z, %): 139 (21), 202 (26), 265 (100), 267 (66), 280 (72), 281 (12).

Ethyl 3'-formyl-4'-hydroxy-1,1'-biphenyl-4-carboxylate (4e)

Obtained from 5′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-(ethoxycarbonyl)benzeneboronic acid (0.65 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.41 g, 74%). Mp 145–146 °C. IR (KBr) cm−1 = 2998 (OH), 1716 (C = O), 1646 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.37 (1H, s), 8.12 (2H, d, J = 8.6 Hz), 7.96 (1H, d, J = 2.3 Hz), 7.75 (1H, dd, J = 8.6, 2.3 Hz), 7.60 (2H, d, J = 8.6 Hz), 7.09 (1H, d, J = 8.6 Hz), 4.41 (2H, q, J = 7.2 Hz), 2.72 (3H, s), 1.42 (3H, t, J = 7.2 Hz) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.6, 166.5, 162.6, 144.3, 135.4, 131.2, 130.4, 129.4, 129.3, 126.6, 120.0, 119.3, 61.2, 26.9, 14.5 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C17H17O4) 285.1127. Found 285.1127. GC-MS (m/z, %): 207 (80), 239 (58), 241 (24), 269 (91), 281 (27), 284 (100).

3-Hydroxy-1,1'-biphenyl-4-carbaldehyde (4f)Citation37

Obtained from 4′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), benzeneboronic acid (0.37 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.45 g, 74%). Mp 92–93 °C. IR (KBr) cm−1 = 3039 (OH), 1623 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.36 (1H, s), 7.80 (1H, d, J = 8.3 Hz), 7.60–7.65 (2H, m), 7.38–7.50 (3H, m), 7.22 (1H, d, J = 1.8 Hz), 7.15 (1H, dd, J = 8.3, 1.8 Hz), 2.66 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.2, 162.8, 149.3, 139.5, 131.3, 129.1, 128.8, 127.4, 118.7, 118.0, 116.6, 26.8 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H13O2) 213.0916. Found 213.0920. GC-MS (m/z, %): 197 (100), 212 (45).

4'-Fluoro-3-hydroxy-1,1'-biphenyl-4-carbaldehyde (4g)Citation38

Obtained from 4′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-fluorobenzeneboronic acid (0.42 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as light yellow solid (0.31 g, 58%). Mp 94–95 °C. IR (KBr) cm−1 = 3071 (OH), 1648 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.35 (1H, s), 7.79 (1H, d, J = 8.3 Hz), 7.59 (2H, dd, J = 8.9, 5.3 Hz), 7.12–7.18 (3H, m), 7.09 (1H, dd, J = 8.3, 1.8 Hz), 7.66 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.1, 163.3 (d, J = 248.5 Hz), 162.8, 148.2, 135.6 (d, J = 3.3 Hz), 131.4, 129.1 (d, J = 8.3 Hz), 118.7, 117.8, 116.4, 116.1 (d, J = 21.6 Hz), 26.7 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H12O2F) 231.0821. Found 231.0826. GC-MS (m/z, %): 215 (100), 230 (45).

3-Hydroxy-4'-methoxy-1,1'-biphenyl-4-carbaldehyde (4h)Citation39

Obtained from 4′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-methoxybenzeneboronic acid (0.46 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.31 g, 74%). Mp 124–125 °C. IR (KBr) cm−1 = 2969 (OH), 1635 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.37 (1H, s), 7.76 (1H, d, J = 8.3 Hz), 7.58 (2H, d, J = 8.9 Hz), 7.17 (1H, d, J = 1.8 Hz), 7.12 (1H, dd, J = 8.3, 1.8 Hz), 6.99 (2H, d, J = 8.9 Hz), 3.86 (3H, s), 2.65 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.0, 162.9, 160.4, 148.9, 131.8, 131.3, 128.5, 118.3, 117.5, 115.7, 114.5, 55.5, 26.7 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C15H15O3) 243.1021. Found 243.1028. GC-MS (m/z, %): 227 (100), 242 (56).

3',4'-Dichloro-3-hydroxy-1,1'-biphenyl-4-carbaldehyde (4i)

Obtained from 4′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 3,4-dichlorobenzeneboronic acid (0.58 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.41 g, 61%). Mp 166–167 °C. IR (KBr) cm−1 = 1634 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.3 (1H, s), 7.81 (1H, d, J = 8.3 Hz), 7.70 (1H, dd, J = 8.3, 2.1 Hz), 7.53 (1H, d, J = 8.3 Hz), 7.44 (1H, dd, J = 8.3, 2.1 Hz), 7.16 (1H, d, J = 1.9 Hz), 7.08 (1H, dd, J = 8.3, 1.9 Hz), 2.67 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.2, 162.8, 146.5, 139.5, 133.3, 133.1, 131.5, 131.0, 129.2, 126.5, 119.2, 117.7, 116.6, 26.8 ppm. HRMS (ESI) [M - H]-: m/z calcd for (C14H9O2Cl) 278.9980. Found 278.9980. GC-MS (m/z, %): 265 (100), 267 (65), 280 (37).

Ethyl 4'-formyl-3'-hydroxy-1,1'-biphenyl-4-carboxylate (4j)

Obtained from 4′-bromo-2′-hydroxyacetophenone (0.50 g, 2.33 mmol), 4-(ethoxycarbonyl)benzeneboronic acid (0.65 g, 3.02 mmol), K2CO3 (0.80 g, 5.81 mmol) and Pd(PPh3)4 (0.13 g, 0.12 mmol). Obtained as white solid (0.41 g, 61%). Mp 106–107 °C. IR (KBr) cm−1 = 2996 (OH), 1704 (C = O), 1642 (C = O). 1H NMR (400 MHz, CDCl3) δ = 12.30 (1H, s), 8.13 (2H, d, J = 8.4 Hz), 7.82 (1H, d, J = 8.4 Hz), 7.68 (2H, d, J = 8.4 Hz), 7.24 (1H, d, J = 1.8 Hz), 7.17 (1H, dd, J = 8.4, 1.8 Hz), 4.14 (2H, q, J = 7.2 Hz), 2.67 (3H, s), 1.42 (3H, t, J = 7.2 Hz) ppm. 13C NMR (100 MHz, CDCl3) δ = 204.2, 166.4, 162.8, 148.0, 143.7, 131.4, 130.6, 130.3, 127.3, 119.2, 118.1, 117.0, 61.3, 26.8, 14.5 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C17H17O4) 285.1127. Found 285.1135. GC-MS (m/z, %): 241 (27), 269 (10), 284 (56).

General procedure for the preparation of 1,2,3-benzoxathiazine 2,2-dioxides 5a-j

The derivative of 1–(2-hydroxyphenyl)ethanone 4a-j (1 equiv) was dissolved in dry DMA (6 ml). Reaction mixture was cooled to 0 °C. Sulphamoyl chloride (2.5 equiv) was slowly added to the reaction mixture. The stirring was continued at room temperature under argon atmosphere for 24 h. Reaction mixture was then poured into ice-water (25 ml), extracted with DCM (3 × 25 ml), washed with satd. NaHCO3 (3 × 25 ml) and satd. NaCl (3 × 25 ml). Organic phase was dried over Na2SO4, filtered, evaporated. The product was purified by column chromatography with PE/EtOAc (2:1) followed by recrystallization from EtOH.

4-Methyl-6-phenyl-1,2,3-benzoxathiazine 2,2-dioxide (5a)

Obtained from 4-hydroxy-1,1′-biphenyl-3-carbaldehyde (4a)(0.20 g, 0.94 mmol) and sulphamoyl chloride(0.27 g, 2.36 mmol). Obtained as light brown (0.08 g, 29%). Mp 147–148 °C. IR (KBr) cm−1 = 1383 (S = O), 1197 (S = O), 1187 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.88–7.93 (2H, m), 7.42–7.58 (5H, m), 7.37 (1H, dd, J = 8.4, 0.4 Hz), 2.78 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.5, 152.8, 139.7, 138.6, 135.9, 129.4, 128.6, 127.3, 126.9, 119.5, 116.7, 23.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H12NO3S) 274.0538. Found 274.0540. GC-MS (m/z, %): 139 (87), 140 (45), 209 (88), 273 (100).

6–(4-Fluorophenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5 b)

Obtained from 4′-fluoro-4-hydroxy-1,1′-biphenyl-3-carbaldehyde (4 b) (0.25 g, 1.09 mmol) and sulphamoyl chloride (0.31 g, 2.72 mmol). Obtained as brown solid (0.14 g, 45%). Mp 175–176 °C. IR (KBr) cm−1 = 1378 (S = O), 1185 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.83–7.88 (2H, m), 7.51 (2H, dd, J = 8.9, 5.2 Hz), 7.37 (1H, dd, J = 8.4, 0.5 Hz), 7.19 (2H, t, J = 8.7 Hz), 2.78 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.4, 163.2 (d, J = 249.3 Hz), 152.8, 138.7, 135.8, 134.8 (d, J = 3.2 Hz), 129.0 (d, J = 8.2 Hz), 126.7, 119.7, 116.8, 116.4 (d, J = 21.8 Hz), 23.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H11NO3SF) 292.0444. Found 292.0445. GC-MS (m/z, %): 157 (80), 158 (49), 227 (91), 291 (100).

6–(4-Methoxyphenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5c)

Obtained from 4-hydroxy-4′-methoxy-1,1′-biphenyl-3-carbaldehyde (4c) (0.25 g, 1.03 mmol) and sulphamoyl chloride(0.30 g, 2.58 mmol). Obtained as yellow solid (0.13 g, 41%). Mp 121–122 °C. IR (KBr) cm−1 = 1386 (S = O), 1375 (S = O), 1189 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.83 (2H, m), 7.48 (2H, d, J = 8.9 Hz), 7.34 (1H, dd, J = 8.2, 0.8 Hz), 7.02 (2H, d, J = 8.9 Hz), 3.87 (3H, s), 2.77 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.6, 160.2, 152.3, 139.4, 135.5, 131.0, 128.4, 126.3, 119.5, 116.7, 114.8, 55.6, 23.9 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C15H14NO4S) 304.0644. Found 304.0654.

6–(3,4-Dichlorophenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5d)

Obtained from 3′,4′-dichloro-4-hydroxy-1,1′-biphenyl-3-carbaldehyde (4d) (0.22 g, 0.78 mmol) and sulphamoyl chloride(0.23 g, 1.96 mmol). Obtained as white solid (0.06 g, 22%). Mp 130–131 °C. IR (KBr) cm−1 = 1388 (S = O), 1190 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.83–7.89 (2H, m), 7.63 (1H, d, J = 2.2 Hz), 7.58 (1H, d, J = 8.4 Hz), 7.39 (1H, dd, J = 8.4, 0.5 Hz), 7.37 (1H, dd, J = 8.4, 2.2 Hz), 2.80 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.2, 153.3, 138.5, 137.2, 135.6, 133.7, 133.2, 131.4, 129.1, 126.8, 126.5, 120.0, 116.9, 24.0 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H8NO3SCl) 339.9602. Found 339.9608.

Ethyl 4–(4-methyl-2,2-dioxido-1,2,3-benzoxathiazin-6-yl)benzoate (5e)

Obtained from ethyl 3′-formyl-4′-hydroxy-1,1′-biphenyl-4-carboxylate (4e) (0.22 g, 0.77 mmol) and sulphamoyl chloride(0.22 g, 1.94 mmol). Obtained as white solid (0.10 g, 37%). Mp 190–191 °C. IR (KBr) cm−1 = 1384 (S = O), 1369 (S = O), 1193 (S = O), 1182 (S = O). 1H NMR (400 MHz, CDCl3) δ = 8.17 (2H, d, J = 8.6 Hz), 7.91–7.97 (2H, m), 7.62 (2H, d, J = 8.6 Hz), 7.40 (1H, d, J = 8.5 Hz), 4.42 (2H, q, J = 7.2 Hz), 2.80 (3H, s), 1.43 (3H, t, J = 7.2 Hz) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.3, 166.2, 153.3, 142.7, 138.5, 135.9, 130.5, 130.6, 127.2, 127.1, 119.8, 116.8, 61.4, 24.0, 14.5 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C17H16NO5S) 346.0749. Found 346.0747.

4-Methyl-7-phenyl-1,2,3-benzoxathiazine 2,2-dioxide (5f)

Obtained from 3-hydroxy-1,1′-biphenyl-4-carbaldehyde (4f) (0.20 g, 0.94 mmol) and sulphamoyl chloride (0.27 g, 2.36 mmol). Obtained as light yellow solid (0.11 g, 43%). Mp 181–182 °C. IR (KBr) cm−1 = 1378 (S = O), 1369 (S = O), 1188 (S = O), 1154 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.84 (1H, d, J = 8.3 Hz), 7.58–7.65 (3H, m), 7.45–7.55 (4H, m), 2.75 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.1, 154.1, 150.5, 137.9, 129.8, 129.5, 129.0, 127.4, 124.5, 117.2, 115.1, 23.8 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H12NO3S) 274.0538. Found 274.0536. GC-MS (m/z, %): 139 (45), 180 (25), 209 (30), 273 (100).

7–(4-Fluorophenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5g)

Obtained from 4′-fluoro-3-hydroxy-1,1′-biphenyl-4-carbaldehyde (4 g) (0.22 g, 0.96 mmol) and sulphamoyl chloride (0.28 g, 2.39 mmol). Obtained as white solid (0.14 g, 49%). Mp 201–202 °C. IR (KBr) cm−1 = 1375 (S = O), 1189 (S = O), 1157 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.84 (1H, d, J = 8.3 Hz), 7.61 (2H, dd, J = 8.9, 5.2 Hz), 7.55 (1H, dd, J = 8.3, 1.8 Hz), 7.44 (1H, d, J = 1.8 Hz), 7.20 (2H, t, J = 8.6 Hz), 2.75 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.1, 163.9 (d, J = 250.3 Hz), 154.1, 149.3, 134.1 (d, J = 3.4 Hz), 129.3 (d, J = 8.5 Hz), 129.1, 124.3, 117.0, 116.6 (d, J = 21.8 Hz), 115.1, 23.8 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C14H11NO3SF) 292.0444. Found 292.0457. GC-MS (m/z, %): 157 (41), 198 (25), 227 (28), 291 (100).

7–(4-Methoxyphenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5h)

Obtained from 3-hydroxy-4′-methoxy-1,1′-biphenyl-4-carbaldehyde (4 h) (0.22 g, 0.91 mmol) and sulphamoyl chloride (0.26 g, 2.27 mmol). Obtained as light brown solid (0.11 g, 38%). Mp 182–183 °C. IR (KBr) cm−1 = 1378 (S = O), 1363 (S = O), 1197 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.80 (1H, d, J = 8.3 Hz), 7.59 (2H, d, J = 8.9 Hz), 7.56 (1H, dd, J = 8.3, 1.8 Hz), 7.44 (1H, d, J = 1.8 Hz), 7.03 (2H, d, J = 8.8 Hz), 3.88 (3H, s), 2.73 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.1, 161.2, 154.2, 150.1, 130.1, 129.0, 128.7, 123.8, 116.2, 114.9, 114.5, 55.6, 23.8 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C15H14NO4S) 304.0644. Found 304.0656.

7–(3,4-Dichlorophenyl)-4-methyl-1,2,3-benzoxathiazine 2,2-dioxide (5i)

Obtained from 3′,4′-dichloro-3-hydroxy-1,1′-biphenyl-4-carbaldehyde (4i) (0.22 g, 0.78 mmol) and sulphamoyl chloride(0.23 g, 1.96 mmol). Obtained as white solid (0.11 g, 42%). Mp 202–203 °C. IR (KBr) cm−1 = 1370 (S = O), 1196 (S = O), 1178 (S = O). 1H NMR (400 MHz, CDCl3) δ = 7.86 (1H, d, J = 8.4 Hz), 7.72 (1H, d, J = 2.2 Hz), 7.59 (1H, d, J = 8.4 Hz), 7.55 (1H, dd, J = 8.3, 1.8 Hz), 7.44–7.48 (2H, m), 2.76 (3H, s) ppm. 13C NMR (100 MHz, CDCl3) δ = 176.9, 154.1, 147.7, 137.8, 134.3, 133.9, 131.5, 129.3, 129.2, 126.6, 124.3, 117.2, 115.8, 23.9 ppm. HRMS (ESI) [M - H]-: m/z calcd for (C14H8NO3SCl2) 339.9602. Found 339.9600.

Ethyl 4–(4-methyl-2,2-dioxido-1,2,3-benzoxathiazin-7-yl)benzoate (5j)

Obtained from ethyl 4′-formyl-3′-hydroxy-1,1′-biphenyl-4-carboxylate (4j) (0.22 g, 0.77 mmol) and sulphamoyl chloride (0.22 g, 1.94 mmol). Obtained as white solid (0.10 g, 38%). Mp 151–152 °C. IR (KBr) cm−1 = 1387 (S = O), 1198 (S = O). 1H NMR (400 MHz, CDCl3) δ = 8.18 (2H, d, J = 8.7 Hz), 7.88 (1H, d, J = 8.3 Hz), 7.69 (2H, d, J = 8.7 Hz), 7.63 (1H, dd, J = 8.3, 1.7 Hz), 7.52 (1H, d, J = 1.7 Hz), 4.42 (2H, q, J = 7.2 Hz), 2.76 (3H, s), 1.43 (3H, t, J = 7.2 Hz) ppm. 13C NMR (100 MHz, CDCl3) δ = 177.0, 166.0, 154.1, 149.2, 142.0, 131.6, 130.6, 129.1, 127.4, 124.7, 117.5, 115.7, 61.5, 23.9, 14.5 ppm. HRMS (ESI) [M + H]+: m/z calcd for (C17H16NO5S) 346.0749. Found 346.0759.

Ca inhibitory assay

An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activityCitation40.

Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5), and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed 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 rate. The uncatalysed 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 – deionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay in order to allow for the formation of the E – I complex. Data from were obtained after 6 h incubation of enzyme and inhibitor. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng – Prusoff equation, as reported earlierCitation41–46 and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlierCitation15,Citation47–55.

Table 1. Inhibition data of human CA isoforms hCA I, II, IX, XII with compounds 2a-j and 5a-j reported here and the standard inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.

Results and discussion

Chemistry

A series of 4-methylbenzo[1,2,3]oxathiazine-2,2-dioxides 2a-j was prepared from corresponding aryl 2-hydroxybenzaldehydes 1a-j in their reaction with sulphamoyl chlorideCitation47,Citation56 that was prepared from chlorosulphonyl isocyanate (Scheme 1). The yields of the products were moderate ().

Scheme 1. Reagents and conditions: (i). HCOOH, 0 °C, 15 min, then RT, 45 min. (ii). ClSO2NH2, DMA, RT, 24 h.

Scheme 1. Reagents and conditions: (i). HCOOH, 0 °C, 15 min, then RT, 45 min. (ii). ClSO2NH2, DMA, RT, 24 h.

5-, 7- and 8-aryl substituted 4-methylbenzo[1,2,3]oxathiazine-2,2-dioxides 5a-j were obtained from aldehydes 4a-j (Scheme 2). The first step was Suzuki-Miyaura coupling of compounds 3 with various boronic acids followed by cyclisation using sulfamoylphchloride. Yields of the intermediates were from moderate to high but yields of the products are quite low due to the loss during purification process.

Scheme 2. Reagents and conditions: (i). K2CO3, Pd(PPh3)4, toluene/H2O (5:1), 90 °C, 24 h. (ii). ClSO2NH2, DMA, RT, 24 h.

Scheme 2. Reagents and conditions: (i). K2CO3, Pd(PPh3)4, toluene/H2O (5:1), 90 °C, 24 h. (ii). ClSO2NH2, DMA, RT, 24 h.

Carbonic anhydrase inhibition

20 derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide were screened for the inhibition of four human CA isoforms – the cytosolic off-target hCA I and II as well as transmembrane, tumour-associated hCA IX and XII that are anticancer drug targetsCitation15,Citation57–62. Inhibition data of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides 2a-j and 5a-j (as well as acetazolamide AAZ as standard) against hCA I, II, IX and XII, after 6 h of incubation period of the enzyme and inhibitor solutions have been reported ()Citation40.

None of screened derivatives showed significant inhibitory activity towards off-target hCA I. 7-Aryl substituted compounds 5f-j as well as some others (2a, 2e, 2j, 5d) do not inhibit hCA I at all, whereas other ones showed micromolar inhibitory activity (KI = 10 – 82 µM).

Compound 2a was the only one which did not inhibit cytosolic off-target hCA II. Other ones showed slight inhibitory properties. In general, aryl substituted derivatives inhibited hCA II less effectively than compounds 2a-j with small substituents in positions 5, 6 or 7.

Target transmembrane isoenzyme hCA IX was significantly inhibited by most derivatives in nanomolar range (KI = 7.0 − 99.8 nM), except compound 2a which KI = 75 µM.

Another target tumour-associated isoenzyme hCA XII was effectively inhibited by most derivatives (KI = 10.5 – 390 nM) as well, although compound 2a did not express hCA XII inhibitory properties also in this case.

Compound 2a was the only one which showed very low or did not show any inhibitory activity against target as well as non-target hCAs. This is probably due to the sterical hindrance provided by the substituent in position 5. Perhaps 5-methoxy group limits binding of the compound 2a to the enzyme.

In general 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides exhibited excellent selectivity towards hCA IX/XII over hCA I and very good selectivity towards hCA IX/XII over hCA II except compound 2a. Some of the most promising derivatives that express selectivity towards both off-target hCA isoforms are aryl substituted 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides 5c, 5e, 5i, as well as Br- substituted ones (2e, 2j).

Conclusions

Here we report a series of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxide derivatives 2a-j with small substituents (Me, MeO, F, Br) in 5, 6 or 7 position prepared by straightforward synthesis from corresponding 2-hydroxyacetophenones in their reaction with sulphamoyl chloride. A series of 6- or 7-aryl substituted 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides 5a-j was obtained in two-step protocol from aryl substituted 2-hydroxyacetophenones in Suzuki-Miyaura reaction with various boronic acids followed by cyclisation using sulphamoyl chloride.

The derivatives were assayed as inhibitors of four hCA isoforms, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX and XII.

Only compound 2a did not inhibit any of investigated hCA isoforms.

4-Methyl-1,2,3-benzoxathiazine-2,2-dioxides generally do not inhibit or show low inhibitory activity towards cytosolic off-target hCA I.

Off-target hCA II was slightly inhibited by the derivatives. Better inhibition of hCA II was observed in the case of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides without aryl substituents.

Transmembrane target isoform hCA IX was significantly inhibited by most derivatives in nanomolar range.

Another tumour-associated target isoform hCA XII was effectively inhibited by almost all derivatives as well.

Excellent selectivity towards hCA IX/XII over hCA I and very good selectivity towards hCA IX/XII over hCA II was observed in most cases of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxide derivatives.

Disclosure statement

No potential conflict of interest was reported by all author(s) except CTS. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The work was supported by the European Regional Development Fund [ERDF, project no. 1.1.1.2/VIAA/3/19/576].

References

  • Elbadawi MM, Eldehna WM, Nocentini A, Somaa WR, Al-Rashood ST, Elkaeed EB, El Hassab MA, Abdel-Aziz HA, Supuran CT, Fares M. Development of 4-((3-oxo-3-phenylpropyl)amino)benzenesulfonamide derivatives utilizing tail/dual-tail approaches as novel carbonic anhydrase inhibitors. Eur J Med Chem. 2022;238:114412.
  • Singh P, Sridhar Goud N, Swain B, Kumar Sahoo S, Choli A, Angeli A, Singh Kushwah B, Madhavi Yaddanapudi V, Supuran CT, Arifuddin M. Synthesis of a new series of quinoline/pyridine indole-3-sulfonamide hybrids as selective carbonic anhydrase IX inhibitors. Bioorg Med Chem Lett. 2022;70:128809.
  • Siwach K, Kumar A, Panchal H, Kumar R, Bhardwaj JK, Angeli A, Supuran CT, Sharma PK. Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis. J Enzyme Inhib Med Chem. 2022;37(1):1454–1463.
  • Angeli A, Carta F, Nocentini A, Winum JY, Zalubovskis R, Akdemir A, Onnis V, Eldehna WM, Capasso C, Simone G, et al. Carbonic Anhydrase Inhibitors Targeting Metabolism and Tumor Microenvironment. Metabolites. 2020;10(10):412.
  • Nocentini A, Angeli A, Carta F, Winum JY, Zalubovskis R, Carradori S, Capasso C, Donald WA, Supuran CT. Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase. J Enzyme Inhib Med Chem. 2021;36(1):561–580.
  • Buabeng ER, Henary M. Developments of small molecules as inhibitors for carbonic anhydrase isoforms. Bioorg Med Chem. 2021;39:116140.
  • Eldehna WM, Taghour MS, Al-Warhi T, Nocentini A, Elbadawi MM, Mahdy HA, Abdelrahman MA, Alotaibi OJ, Aljaeed N, Elimam DM, et al. Discovery of 2,4-thiazolidinedione-tethered coumarins as novel selective inhibitors for carbonic anhydrase IX and XII isoforms. J Enzyme Inhib Med Chem. 2022;37(1):531–541.
  • Pastorek J, Pastoreková S, Callebaut I, Mornon JP, Zelník V, Opavský R, Zat’ovicová M, Liao S, Portetelle D, Stanbridge EJ, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene. 1994;9:2877–2888.
  • Türeci O, Sahin U, Vollmar E, Siemer S, Göttert E, Seitz G, Parkkila AK, Shah GN, Grubb JH, Pfreundschuh M, et al. Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers. Proc Natl Acad Sci U S A. 1998;95(13):7608–7613.
  • Kumar A, Siwach K, Supuran CT, Sharma PK. A decade of tail-approach based design of selective as well as potent tumor associated carbonic anhydrase inhibitors. Bioorg Chem. 2022;126:105920.
  • Becker HM, Deitmer JW. Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases. IJMS. 2021;22(6):3171.
  • Tawfik HO, Shaldam MA, Nocentini A, Salem R, Almahli H, Al-Rashood ST, Supuran CT, Eldehna WM. Novel 3-(6-methylpyridin-2-yl)coumarin-based chalcones as selective inhibitors of cancer-related carbonic anhydrases IX and XII endowed with anti-proliferative activity. J Enzyme Inhib Med Chem. 2022;37(1):1043–1052.
  • Lomelino CL, Supuran CT, McKenna R. Non-classical inhibition of carbonic anhydrase. IJMS. 2016;17(7):1150.
  • Aimene Y, Eychenne R, Rodriguez F, Mallet-Ladeira S, Saffon-Merceron N, Winum J-Y, Nocentini A, Supuran CT, Benoist E, Seridi A. Synthesis, crystal structure, inhibitory activity and molecular docking of coumarins/sulfonamides containing triazolyl pyridine moiety as potent selective carbonic anhydrase IX and XII inhibitors. Crystals. 2021;11(9):1076.
  • Maresca A, Temperini C, Vu H, Pham NB, Poulsen SA, Scozzafava A, Quinn RJ, Supuran CT. Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors. J Am Chem Soc. 2009;131(8):3057–3062.
  • Tars K, Vullo D, Kazaks A, Leitans J, Lends A, Grandane A, Zalubovskis R, Scozzafava A, Supuran CT. Sulfocoumarins (1,2-benzoxathiine-2,2-dioxides): a class of potent and isoform-selective inhibitors of tumor-associated carbonic anhydrases. J Med Chem. 2013;56(1):293–300.
  • Grandane A, Tanc M, Zalubovskis R, Supuran CT. Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII. Bioorg Med Chem. 2014;22(5):1522–1528.
  • Grandane A, Tanc M, Zalubovskis R, Supuran CT. 6-Triazolyl-substituted sulfocoumarins are potent, selective inhibitors of the tumor-associated carbonic anhydrases IX and XII. Bioorg Med Chem Lett. 2014;24(5):1256–1260.
  • Grandane A, Tanc M, Žalubovskis R, Supuran CT. Synthesis of 6-aryl-substituted sulfocoumarins and investigation of their carbonic anhydrase inhibitory action. Bioorg Med Chem. 2015;23(7):1430–1436.
  • Grandane A, Tanc M, Di Cesare Mannelli L, Carta F, Ghelardini C, Žalubovskis R, Supuran CT. 6‑Substituted sulfocoumarins are selective carbonic anhdydrase IX and XII inhibitors with significant cytotoxicity against colorectal cancer cells. J Med Chem. 2015;58(9):3975–3983.
  • Grandāne A, Nocentini A, Domračeva I, Žalubovskis R, Supuran CT. Development of oxathiino[6,5-b]pyridine 2,2-dioxide derivatives as selective inhibitors of tumor-related carbonic anhydrases IX and XII. Eur J Med Chem. 2020;200:112300.
  • Krasavin M, Žalubovskis R, Grandāne A, Domračeva I, Zhmurov P, Supuran CT. Sulfocoumarins as dual inhibitors of human carbonic anhydrase isoforms IX/XII and of human thioredoxin reductase. J Enzyme Inhib Med Chem. 2020;35(1):506–510.
  • Pustenko A, Stepanovs D, Žalubovskis R, Vullo D, Kazaks A, Leitans J, Tars K, Supuran CT. 3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors. J Enzyme Inhib Med Chem. 2017;32(1):767–775.
  • Pustenko A, Nocentini A, Balašova A, Alafeefy A, Krasavin M, Žalubovskis R, Supuran CT. Aryl derivatives of 3H-1,2-benzoxathiepine 2,2-dioxide as carbonic anhydrase inhibitors. J Enzyme Inhib Med Chem. 2020;35(1):245–254.
  • Pustenko A, Nocentini A, Balašova A, Krasavin M, Žalubovskis R, Supuran CT. 7-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides as new isoform-selective carbonic anhydrase IX and XII inhibitors. J Enzyme Inhib Med Chem. 2020;35(1):650–656.
  • Podolski-Renić A, Dinić J, Stanković T, Jovanović M, Ramović A, Pustenko A, Žalubovskis R, Pešić M. Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance. Eur J Pharm Sci. 2019;138:105012.
  • Grandane A, Nocentini A, Werner T, Zalubovskis R, Supuran CT. Benzoxepinones: A new isoform-selective class of tumor associated carbonic anhydrase inhibitors. Bioorg Med Chem. 2020;28(11):115496.
  • Onyılmaz M, Koca M, Bonardi A, Degirmenci M, Supuran CT. Isocoumarins: a new class of selective carbonic anhydrase IX and XII inhibitors. J Enzyme Inhib Med Chem. 2022;37(1):743–748.
  • Ivanova J, Abdoli M, Nocentini A, Žalubovskis R, Supuran CT. 1,2,3-Benzoxathiazine-2,2-dioxides – efective inhibitors of human carbonic anhydrases. J Enzyme Inhib Med Chem. 2023;38(1):225–238.
  • Appel R, Berger G. Hydrazinesulfonic acid amide. I. Chem Ber. 1958;91(6):1339–1341.
  • Chen Y-H, Lv X-J, You Z-H, Liu Y-K. Assymetric organocatalyzed reaction sequence to synthesize chiral bridged and spiro-bridged benzofused aminals via divergent pathways. Org Lett. 2019;21(14):5556–5561.
  • An Q, Shen J, Butt N, Liu D, Liu Y, Zhang W. Assymetric domino reaction of cyclic N-sulfonylimines and simple aldehydes with trans-perhydroindolic acid as an organocatalyst. Org Lett. 2014;16(17):4496–4499.
  • Fei J, Qian Q, Sun X, Gu X, Zou C, Ye J. Organocatalytic enantioselective formal [4 + 2] cycloaddition of enones with cyclic N-sulfonylimines and methylene chromene for chiral spirocyclic compounds. Org Lett. 2015;17(21):5296–5299.
  • Liu Q, Chen XY, Li S, Rissanen K, Enders D. N-Heterocyclic carbene catalyzed asymmetric synthesis of pentacyclic spirooxindoles via [3 + 3] annulations of isatin-derived enals and cyclic N-sulfonyl ketimines. Adv Synth Catal. 2019;361(9):1991–1994.
  • Cheetham KH, Hey DH. Migration reactions in polycyclic systems. II. The Fries rearrangement of 4-acetoxybiphenyl. J Chem Soc. 1937:770–772.
  • Fernandes C, Soares P, Gaspar A, Martins D, Gomes LR, Low JN, Borges F. Synthesis of 6-aryl/heteroaryl-4-oxo-4H-chromene-2-carboxylic ethyl ester derivatives. Tetrahedron Lett. 2016;57(27-28):3006–3010.
  • Bradsher CK, Brown FC, Porter HK. Synthesis and fungistatic activity of some 3-hydroxybiphenyl derivatives. J Am Chem Soc. 1954;76(9):2357–2362.
  • Sheelam K, Thota P, Kottawar S, Vinnakota S, Chidara S, Yennam S, Behera M. Greener route for the synthesis of chromone using Amberlyst15 via enaminones. ARKIVOC (Gainesville, FL, United States). 2022;2022(6):55–65.
  • Azimvand J. Synthesis of some new derivatives of 2-methyl-4H-4-chromenone. J Chem Pharm Res. 2012;4:3929–3933.
  • Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem. 1971;246(8):2561–2573.
  • Pustenko A, Nocentini A, Gratteri P, Bonardi A, Vozny I, Žalubovskis R, Supuran CT. The antibiotic furagin and its derivatives are isoform-selective human carbonic anhydrase inhibitors. J Enzyme Inhib Med Chem. 2020;35(1):1011–1020.
  • Nocentini A, Carta F, Tanc M, Selleri S, Supuran CT, Bazzicalupi C, Gratteri P. Deciphering the mechanism of human carbonic anhydrases inhibition with sulfocoumarins: computational and experimental studies. Chemistry. 2018;24(31):7840–7844.
  • Angeli A, Carta F, Nocentini A, Winum J-Y, Zalubovskis R, Onnis V, Eldehna WM, Capasso C, Carradori S, Donald WA, et al. Response to perspectives on the classical enzyme carbonic anhydrase and the search for inhibitors. Biophys J. 2021;120(1):178–181.,.
  • Bua S, Bozdag M, Del Prete S, Carta F, Donald WA, Capasso C, Supuran CT. Mono- and di-thiocarbamate inhibition studies of the δ-carbonic anhydrase TweCAd from the marine diatom Thalassiosira weissflogii. J Enzyme Inhib Med Chem. 2018;33(1):707–713.
  • Akocak S, Lolak N, Bua S, Supuran CT. Discovery of novel 1,3-diaryltriazene sulfonamides as carbonic anhydrase I, II, VII, and IX inhibitors. J Enzyme Inhib Med Chem. 2018;33(1):1575–1580.
  • Nocentini A, Bonardi A, Gratteri P, Cerra B, Gioiello A, Supuran CT. Steroids interfere with human carbonic anhydrase activity by using alternative binding mechanisms. J Enzyme Inhib Med Chem. 2018;33(1):1453–1459.
  • Ivanova J, Carta F, Vullo D, Leitans J, Kazaks A, Tars K, Žalubovskis R, Supuran CT. N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic anhydrases IX and XII. Bioorg Med Chem. 2017;25(13):3583–3589.
  • Supuran CT. Carbon- versus sulphur-based zinc binding groups for carbonic anhydrase inhibitors? J Enzyme Inhib Med Chem. 2018;33(1):485–495.
  • Briganti F, Pierattelli R, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Part 37. Novel classes of carbonic anhydrase inhibitors and their interaction with the native and cobalt-substituted enzyme: kinetic and spectroscopic investigations. Eur J Med Chem. 1996;31(12):1001–1010.
  • Pastorekova S, Casini A, Scozzafava A, Vullo D, Pastorek J, Supuran CT. Carbonic anhydrase inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX. Bioorg Med Chem Lett. 2004;14(4):869–873.
  • Carta F, Supuran CT, Scozzafava A. Sulfonamides and their isosters as carbonic anhydrase inhibitors. Future Med Chem. 2014;6(10):1149–1165.
  • Yıldırım A, Atmaca U, Keskin A, Topal M, Çelik M, Gülçin İ, Supuran CT. N-Acylsulfonamides strongly inhibit human carbonic anhydrase isoenzymes I and II. Bioorg Med Chem. 2015;23(10):2598–2605.
  • Davis RA, Vullo D, Maresca A, Supuran CT, Poulsen SA. Natural product coumarins that inhibit human carbonic anhydrases. Bioorg Med Chem. 2013;21(6):1539–1543.
  • Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008;7(2):168–181.
  • Maresca A, Scozzafava A, Supuran CT. 7,8-Disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II in the low nanomolar/subnanomolar range. Bioorg Med Chem Lett. 2010;20(24):7255–7258.
  • Yan Z, Wu B, Gao X, Chen M-W, Zhou Y-G. Enantioselective synthesis of α-amino phosphonates via Pd-catalyzed asymmetric hydrogenation. Org Lett. 2016;18(4):692–695.
  • Neri D, Supuran CT. Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov. 2011;10(10):767–777.
  • Supuran CT. Carbonic anhydrase inhibitors in the treatment and prophylaxis of obesity. Expert OpinTher Pat. 2003;13(10):1545–1550.
  • Supuran CT. Carbonic anhydrase inhibitors as emerging drugs for the treatment of obesity. Expert Opin Emerg Drugs. 2012;17(1):11–15.
  • De Simone G, Alterio V, Supuran CT. Exploiting the hydrophobic and hydrophilic binding sites for designing carbonic anhydrase inhibitors. Expert Opin Drug Disc. 2013;8(7):793–810.
  • Nishimori I, Minakuchi T, Kohsaki T, Onishi S, Takeuchi H, Vullo D, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors: The β-carbonic anhydrase from Helicobacter pylori is a new target for sulfonamide and sulfamate inhibitors. Bioorg Med Chem Lett. 2007;17(13):3585–3594.
  • Scozzafava A, Supuran CT, Carta F. Antiobesity carbonic anhydrase inhibitors: a literature and patent review. Expert Opin Ther Pat. 2013;23(6):725–735.
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