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Journal of Enzyme Inhibition and Medicinal Chemistry logoLink to Journal of Enzyme Inhibition and Medicinal Chemistry
. 2017 Jun 23;32(1):885–892. doi: 10.1080/14756366.2017.1337759

Carbonic anhydrase I, II, IV and IX inhibition with a series of 7-amino-3,4-dihydroquinolin-2(1H)-one derivatives

Murat Bozdag a, Silvia Bua a, Sameh M Osman b, Zeid AlOthman b, Claudiu T Supuran a,✉
PMCID: PMC6445181  PMID: 28644059

Abstract

A series of new derivatives was prepared by derivatisation of the 7-amino moiety present in 7-amino-3,4-dihydroquinolin-2(1H)-one, a compound investigated earlier as CAI. The derivatisation was achieved by: i) reaction with arylsulfonyl isocyanates/aryl isocyanates; (ii) reaction with fluorescein isothiocyanate; (iii) condensation with substituted benzoic acids in the presence of carbodiimides; (iv) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate; (v) reaction with methylsulfonyl chloride and (vi) reaction with maleic anhydride. The new compounds were assayed as inhibitors of four carbonic anhydrases (CA, EC 4.2.1.1) human (h) isoforms of pharmacologic relevance, the cytosolic hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX. hCA IX was the most inhibited isoform (KIs ranging between 243.6 and 2785.6 nm) whereas hCA IV was not inhibited by these compounds. Most derivatives were weak hCA I and II inhibitors, with few of them showing KIs < 10 µm. Considering that the inhibition mechanism with these lactams is not yet elucidated, exploring a range of such derivatives with various substitution patterns may be useful to identify leads showing isoform selectivity or the desired pharmacologic action.

Keywords: Carbonic anhydrase, inhibitor, coumarin, dihydroquinolinone, sulfonamide

Introduction

CO2, bicarbonate and protons are essential molecules/ions in important physiologic processes in the three life kingdoms (Bacteria, Archaea and Eukarya), and for this reason, relatively high amounts of the enzymes carbonic anhydrases (CAs, EC 4.2.1.1) are present in different tissues/cell compartments of most investigated organisms1–11. The α-CAs are present in vertebrates, protozoa, algae and cytoplasm of green plants and in some Bacteria1–19, the β-CAs are predominantly found in Bacteria, algae and chloroplasts of both mono- as well as dicotyledons, but also in many fungi and some Archaea1–11. The γ-CAs were found in plants, Archaea and Bacteria1–11, whereas the δ-, ζ- and θ-CAs seem to be present only in marine diatoms11. The η-CA class has been discovered in protozoa such as those belonging to the genus Plasmodium20. In many organisms, these enzymes are involved in crucial physiological processes connected with respiration and transport of CO2/bicarbonate, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (e.g. gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, tumourigenicity and many other physiologic or pathologic processes (thoroughly studied in vertebrates)1–11,21–26, whereas in algae, plants and some bacteria they play an important role in photosynthesis and other biosynthetic reactions8,11. In diatoms δ- and ζ-CAs play a crucial role in carbon dioxide fixation11. Many such enzymes from vertebrates, fungi and bacteria are well-known drug targets, with inhibitors and activators possessing various pharmacologic applications23–42.

Sulfonamides are the most important class of CA inhibitors CAIs1,4–12, with several compounds in clinical use for many years, as diuretics1,26,28, antiglaucoma agents1,27,33, antiepileptics30–34 and more recently as anticancer agents1,2,12. Although a large number of isoform-selective sulfonamide CAIs were reported ultimately, mostly by using the tail approach for their synthesis16–23,26, a large variety of other chemotypes were investigated for their interaction with these enzymes, which led to the development of a large number of non-classic CAIs, belonging to various classes14,33. Here, we report a new series of such derivatives which incorporate the 7-amino-3,4-dihydroquinolin-2(1H)-one scaffold43.

Materials and methods

Chemistry

Anhydrous solvents and all reagents were purchased from Sigma-Aldrich (Milan, Italy). All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance (1H-NMR, 13C-NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; dd, double of doublet. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O.

General procedure for the preparation of compounds 2–20

A solution of 7-amino-3,4-dihydroquinolin-2(1H)-one (1) in dry dimethylformamide (3–5 ml) was treated with a stoichiometric amount of appropriate isocyanates/isothiocyanate. The mixture was stirred at room temperature until the consumption of starting materials (TLC monitoring). The reaction was quenched with a 1.0 M aqueous solution of HCl to give a precipitate that was washed with diethyl ether (3 × 5 ml), filtered and dried under vacuum (compounds 2–19) or extracted with ethyl acetate (3 × 15 ml), the combined organic layers were washed with H2O (3 × 15 ml), dried over Na2SO4, filtered, and concentrated (compound 20) to afford the title compounds 2–20.

N-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)carbamoyl)benzenesulfonamide (2)

Beige solid, yield 89%; m.p.: 272–273 °C; silica gel TLC Rf = 0.16 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.42 (2H, d, J 6.8), 2.81 (2H, d, J 6.8), 6.84 (1H, dd, J 2.0, 8.4), 7.02 (1H, d, J 2.0), 7.06 (1H, d, J 8.4), 7.67 (2H, t, J 8.0), 7.73 (1H, t, J 8.0), 8.00 (2H, d, J 8.0), 8.91 (1H, s, exchange with D2O, NH), 10.03 (1H, s, exchange with D2O, NH), 10.70 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.7, 106.2, 112.9, 117.2, 127.7, 128.3, 129.1, 131.9, 139.2, 140.4, 145.2, 171.2; m/z (ESI negative) 344.0 [M − H]−.

4-Methyl-N-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)carbamoyl)benzenesulfonamide (3)

White solid, yield 60%; m.p.: 260–261 °C; silica gel TLC Rf = 0.16 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.43 (5H, m), 2.81 (2H, t, J 7.8), 6.84 (1H, dd, J 2.0, 8.0), 7.01 (1H, d, J 2.0), 7.06 (1H, d, J 8.0), 7.46 (2H, d, J 8.4), 7.87 (2H, d, J 8.4), 8.82 (1H, s, exchange with D2O, NH), 10.03 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 22.0, 25.1, 31.5, 106.8, 113.4, 119.3, 128.4, 128.8, 130.4, 137.9, 138.1, 139.5, 144.8, 150.1, 171.1; m/z (ESI negative) 358.0 [M − H]−.

2-Methyl-N-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)carbamoyl)benzenesulfonamide (4)

White solid, yield 79%, m.p.: 285–286 °C; silica gel TLC Rf = 0.43 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.42 (2H, d, J 7.6), 2.66 (3H, s), 2.80 (2H, t, J 7.6), 6.81 (1H, d, J 8.0), 7.05 (2H, m), 7.47 (2H, m), 7.61 (1H, m), 8.01 (1H, d, J 7.6), 8.69 (1H, s, exchange with D2O, NH), 10.02 (1H, s, exchange with D2O, NH), 10.58 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 20.6, 25.1, 31.5, 106.7, 113.3, 119.3, 127.2, 128.8, 131.0, 133.3, 134.3, 137.6, 137.8, 138.8, 139.6, 149.8, 171.1; m/z (ESI negative) 358.0 [M − H]−.

4-Chloro-N-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)carbamoyl)benzenesulfonamide (5)

White solid, yield 67%; m.p.: 253–254 °C; silica gel TLC Rf = 0.35 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.43 (2H, t, J 6.8), 2.81 (2H, t, J 6.8), 6.85 (1H, dd, J 2.0, 8.4), 7.01 (1H, d, J 2.0), 7.06 (1H, d, J 8.4), 7.75 (2H, d, J 8.8), 8.01 (2H, d, J 8.8), 8.94 (1H, s, exchange with D2O, NH), 10.03 (1H, s, exchange with D2O, NH), 10.81 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.1, 31.5, 107.0, 113.5, 119.5, 128.8, 130.1, 130.4, 137.8, 139.2, 139.6, 139.8, 150.1, 171.1; m/z (ESI negative) 378.0 [M − H]−.

4-Fluoro-N-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)carbamoyl)benzenesulfonamide (6)

White solid, yield 68%; m.p.: 245–246 °C; silica gel TLC Rf = 0.23 (Ethyl acetate/n-hexane 80% v/v); δH (400 MHz, DMSO-d6) 2.42 (2H, t, J 7.6), 2.81 (2H, t, J 7.6), 6.85 (1H, dd, J 1.8, 8.1), 7.02 (1H, d, J 1.8), 7.06 (1H, d, J 8.1), 7.51 (2H, m), 8.06 (2H, m), 8.92 (1H, s, exchange with D2O, NH), 10.04 (1H, s, exchange with D2O, NH), 10.77 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.1, 31.5, 106.9, 113.4, 117.2 (d, 2JC–F 23), 119.4, 128.8, 131.6 (d, 3JC–F 10), 137.2 (d, 4JC–F 3), 137.8, 139.5, 150.1, 165.6 (d, 1JC–F 250), 171.1; δF (376 MHz, DMSO-d6) −105.1 (1 F, s); m/z (ESI negative) 362.0 [M − H]−.

1-(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)-3-phenylurea (7)

White solid, yield 85%; m.p.: 255–256 °C (dec.); silica gel TLC Rf = 0.65 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, d, J 7.6), 2.83 (2H, d, J 7.6), 6.99 (2H, m), 7.08 (2H, m), 7.31 (2H, d, J 7.9), 7.47 (2H, d, J 7.9), 8.60 (1H, s, exchange with D2O, NH), 8.66 (1H, s, exchange with D2O, NH), 10.09 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.7, 106.1, 112.7, 117.9, 119.0, 122.7, 128.8, 129.7, 139.5, 139.6, 140.6, 153.3, 171.2; m/z (ESI positive) 282.0 [M + H]+.

1-(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)-3-(p-tolyl)urea (8)

White solid, yield 88%; m.p.: 276–277 °C; silica gel TLC Rf = 0.48 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.28 (3H, s), 2.46 (2H, t, J 7.6), 2.83 (2H, t, J 7.6), 7.00 (1H, dd, J 2.0, 8.4), 7.09 (4H, m), 7.35 (2H, d, J 8.4), 8.48 (1H, s, exchange with D2O, NH), 8.60 (1H, s, exchange with D2O, NH), 10.07 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 21.2, 25.1, 31.6, 106.0, 112.6, 117.7, 119.1, 128.7, 130.0, 131.5, 138.0, 139.5, 139.7, 153.3, 171.2; m/z (ESI positive) 296.0 [M + H]+.

1-(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)-3-(o-tolyl)urea (9)

White solid, yield 90%; m.p.: > 300 °C; silica gel TLC Rf = 0.47 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.27 (3H, s), 2.46 (2H, t, J 6.8), 2.83 (2H, t, J 6.8), 6.97 (1H, t, J 7.2), 7.07 (3H, m), 7.18 (2H, m), 7.89 (2H, m, 1H exchange with D2O, NH), 9.01 (1H exchange with D2O, NH), 10.11 (1H exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 25.1, 31.7, 105.9, 112.5, 117.7, 121.6, 123.4, 127.1, 128.1, 128.8, 131.1, 138.4, 139.5, 139.8, 153.4, 171.2; m/z (ESI positive) 296.0 [M + H]+.

1-(4-Chlorophenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (10)

White solid, yield 97%; m.p.: 249–250 °C; silica gel TLC Rf = 0.55 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.6), 2.83 (2H, t, J 7.6), 7.00 (1H, dd, J 2.0, 8.4), 7.08 (2H, m), 7.35 (2H, d, J 9.2), 7.50 (2H, d, J 9.2), 8.08 (1H, s, exchange with D2O, NH), 8.88 (1H, s, exchange with D2O, NH), 10.09 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.7, 106.2, 112.8, 118.0, 120.5, 126.1, 128.8, 129.5, 139.5, 139.5, 139.7, 153.3, 171.2; m/z (ESI positive) 316.0 [M + H]+.

1-(2-Chlorophenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (11)

White solid, yield 83%; m.p.: > 300 °C; silica gel TLC Rf = 0.50 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, d, J 7.2), 2.84 (2H, t, J 7.2), 7.08 (4H, m), 7.33 (1H, t, J 8.0), 7.49 (1H, d, J 8.0), 8.20 (1H, d, J 8.0), 8.30 (1H, s, exchange with D2O, NH), 9.41 (1H, s, exchange with D2O, NH), 10.14 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.1, 31.6, 106.0, 112.6, 118.2, 122.0, 122.7, 124.1, 128.5, 128.9, 130.1, 136.9, 139.3, 139.6, 152.9, 171.2; m/z (ESI positive) 316.0 [M + H]+.

1-(4-Fluorophenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (12)

White solid, yield 98%; m.p.: 257–258 °C; silica gel TLC Rf = 0.59 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.45 (2H, t, J 7.8), 2.83 (2H, t, J 7.8), 7.00 (1H, dd, J 2.0, 8.8) 7.08 (2H, m), 7.14 (2H, m), 7.48 (2H, m), 8.62 (1H, s, exchange with D2O, NH), 8.64 (1H, s, exchange with D2O, NH), 10.08 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.6, 106.2, 112.8, 116.1 (d, 2JC–F 22), 117.9, 120.7 (d, 3JC–F 8), 128.8, 137.0 (q, JC–F 2), 139.5, 139.6, 153.4, 158.5 (d, 1JC–F 237), 171.2; δF (376 MHz, DMSO-d6) −121.5 (1 F, s); m/z (ESI positive) 300.0 [M + H]+.

1-(4-Fluoro-3-methylphenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (13)

White solid, yield 89%; m.p.: > 300 °C; silica gel TLC Rf = 0.47 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.24 (3H, d, J 1.5), 2.45 (2H, t, J 7.6), 2.82 (2H, t, J 7.6), 7.00 (1H, dd, J 2.0, 8.10), 7.07 (3H, m), 7.27 (1H, m), 7.38 (1H, m), 8.55 (1H, exchange with D2O, NH), 8.64 (1H, s, exchange with D2O, NH), 10.07 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 15.3 (d, JC–F 3), 25.2, 31.7, 106.2, 112.8, 115.8 (d, 2JC–F 23), 117.9, 118.2 (d, 3JC–F 8), 122.1 (d, 3JC–F 4), 125.1 (d, 2JC–F 18), 128.8, 136.6 (d, 4JC–F 3), 139.5, 139.7, 153.5, 157.0 (d, JC–F 236), 171.3; δF (376 MHz, DMSO-d6) −125.9 (1 F, s); m/z (ESI positive) 314.0 [M + H]+.

1-(2,4-Difluorophenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (14)

White solid, yield 95%; m.p.: 240–241 °C; silica gel TLC Rf = 0.42 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.8), 2.83 (2H, t, J 7.8), 7.07 (4H, m), 7.34 (1H, m), 8.13 (1H, m), 8.47 (1H, s, exchange with D2O, NH), 9.03 (1H, s, exchange with D2O, NH), 10.11 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.7, 104.7 (t, 2JC–F 24), 106.0, 111.9 (dd, 2JC–F 4, 22), 112.6, 118.1, 122.7, (dd, 3JC–F 3.0, 9.0), 125.1 (dd, 3JC–F 3.0, 10.0), 128.9, 139.4, 139.6, 153.1 (dd, 1JC–F 12.0, 244.0), 153.2, 157.7 (dd, 1JC–F 12.0, 240.0), 171.3; δF (376 MHz, DMSO-d6) −124.3 (1 F, d, J 3.0), −118.2 (1 F, d, J 3.0); m/z (ESI positive) 318.0 [M + H]+.

1-(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)-3-(perfluorophenyl)urea (15)

White solid, yield 88%; m.p.: 297–298 °C; silica gel TLC Rf = 0.8 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.45 (2H, d, J 7.2), 2.83 (2H, t, J 7.2), 7.00 (1H, dd, J 2.0, 8.0), 7.09 (2H, m), 8.41 (1H, s, exchange with D2O, NH), 9.07 (1H, s, exchange with D2O, NH), 10.10 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.6, 106.5, 113.1, 115.0 (m, JC–F 15), 118.5, 128.8, 138.1 (m, JC–F 245), 139.1, 139.3 (m, JC–F 245), 139.6, 143.9 (m, JC–F 245), 152.8, 171.3; δF (376 MHz, DMSO-d6) –164.3 (1 F, t, J 22), −159.9 (2 F, t, J 23), −146.4 (2 F, d, J 20); m/z (ESI negative) 370.0 [M − H]−.

1-(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)-3-(4-(trifluoromethyl)phenyl)urea (16)

White solid, yield 72%; m.p.: 284–285 °C; silica gel TLC Rf = 0.55 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.6), 2.84 (2H, t, J 7.6), 7.02 (1H, dd, J 2.0, 8.0), 7.10 (2H, d, J 8.0), 7.67 (4H, m), 8.79 (1H, s, exchange with D2O, NH), 9.01 (1H, s, exchange with D2O, NH), 10.09 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.1, 31.6, 106.3, 112.9, 118.3, 118.7, 122.6 (q, 2JC–F 32), 125.4 (q, 1JC–F 270), 126.9 (q, 3JC–F 4), 128.8, 139.1, 139.5, 144.3 (q, 4JC–F 1), 153.0, 171.1; δF (376 MHz, DMSO-d6) −60.1 (3 F, s); m/z (ESI positive) 350.0 [M + H]+.

1-(2-Chloro-4-(trifluoromethyl)phenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (17)

White solid, yield 85%; m.p.: > 300 °C; silica gel TLC Rf = 0.58 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.47 (2H, t, J 7.2), 2.85 (2H, t, J 7.2), 7.10 (3H, m), 7.71 (1H, dd, J 1.6, 8.8), 7.91 (1H, d, J 1.6), 8.51 (1H, d, J 8.8), 8.61 (1H, s, exchange with D2O, NH), 9.61 (1H, s, exchange with D2O, NH), 10.16 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.6, 106.2, 112.8, 118.6, 121.0, 122.2, 123.7 (q, JC–F 4), 124.6 (q JC–F 271), 125.7 (q, JC–F 4), 127.2 (q, JC–F 4), 129.0, 138.9, 139.6, 140.8 (q, JC–F 40), 152.5, 171.2; δF (376 MHz, DMSO-d6) −60.4 (3 F, s); m/z (ESI positive) 384.0 [M + H]+.

1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (18)

White solid, yield 15%; m.p.; 253–254 °C; silica gel TLC Rf = 0.50 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.47 (2H, t, J 7.2), 2.84 (2H, t, J 7.2), 7.05 (1H, dd, J 2,8), 7.12 (2H, m), 7.42 (1H, m), 7.53 (1H, m), 8.66 (1H, m), 9.18 (1H, exchange with D2O, NH), 9.61 (1H, s, exchange with D2O, NH), 10.09 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.6, 106.1, 112.6, 117 (d, JC–F 21), 117.3 (t, JC–F 3), 118.5, 120.0 (m), 123.5, 126.3 (td, JC–F 3, 32), 128.9, 129.7 (d, JC–F 11), 138.9, 139.6, 152.9, 154.3 (d, JC–F 248), 171.2; δF (376 MHz, DMSO-d6) −60.7 (3 F, s), −124.5 (1 F, s); m/z (ESI positive) 368.0 [M + H]+.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)urea (19)

White solid, yield 30%; m.p.; 278–279 °C; silica gel TLC Rf = 0.70 (Ethyl acetate 100% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.5), 2.85 (2H, t, J 7.5), 7.05 (2H, m), 7.17 (1H, m), 7.67 (1H, s), 8.16 (2H, s), 9.00 (1H, s, exchange with D2O, NH), 9.32 (1H, s, exchange with D2O, NH), 10.08 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.2, 31.6, 106.8, 113.3, 115.2 (m), 118.6, 118.8 (m), 124.2 (q, 1JC–F 270), 128.8, 131.6 (q, 2JC–F 32), 138.9, 139.6, 142.8, 153.2, 171.2; δF (376 MHz, DMSO-d6) −61.7 (6 F, s); m/z (ESI negative) 416.0 [M − H]−.

2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-(3-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)thioureido)benzoic acid (20)

Red solid, yield 75%; m.p.: 189–190 °C; silica gel TLC Rf = 0.23 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.49 (2H, t, J 7.6), 2.89 (2H, t, J 7.6), 6.62 (4H, m), 6.71 (2H, d, J 2.0), 7.04 (2H, m), 7.18 (1H, d, J 8.4), 7.24 (1H, d, J 8.4), 7.86 (1H, dd, J 2.0, 8.4), 8.22 (1H, d, J 2.0), 10.09 (1H, s, exchange with D2O, NH), 10.12 (1H, s, exchange with D2O NH), 10.16 (2H, s, exchange with D2O, OH), 10.17 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.4, 31.4, 103.2, 110.6, 111.7, 113.6, 118.4, 118.5, 121.2, 124.8, 127.4, 128.7, 130.0, 131.4, 138.9, 139.4, 142.3, 152.8, 160.5, 169.4, 171.1, 180.5; m/z (ESI negative) 550.0 [M − H]−.

2-((2,3-Dimethylphenyl)amino)-N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)benzamide (21)

A solution of 1 (1.2 mmol) was treated with mefenamic acid (2.4 mmol) in dry N,N-Dimethylformamide (DMF) (5 ml) then N,N′-Dicyclohexylcarbodiimide (DCC) (2.0 equiv.) and catalytic amount of 4-(Dimethylamino)pyridine (DMAP) were added to reaction mixture. The reaction continued until the consumption of starting materials (TLC monitoring), quenched with 1 M aqueous HCl solution and extracted with ethyl acetate (3 × 15 ml). The combined organic layers were washed with H2O (3 × 15 ml), dried over Na2SO4, filtered, and concentrated to obtain a residue which was purified by silica gel column chromatography eluting with ethyl acetate/n-hexane 50% v/v to afford titled compound.

White solid, yield 20%; m.p.: 220–221 °C; silica gel TLC Rf = 0.18 (Ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6) 2.15 (3H, s), 2.31 (3H, s), 2.48 (2H, t, J 7.6), 2.87 (2H, t, J 7.6), 6.87 (2H, m), 6.98 (1H, m), 7.13 (3H, m), 7.23 (1H, dd, J 2.0, 8.0), 7.34 (1H, td, J 2.0, 7.8), 7.42 (1H, d, J 2.0), 7.81 (1H, dd, J 2.0, 8.0), 9.15 (1H, s, exchange with D2O, NH), 10.16 (1H, s, exchange with D2O, NH), 10.32 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 14.5, 21.2, 25.3, 31.5, 108.9, 115.1, 115.5, 117.9, 118.8, 120.0, 120.8, 126.2, 126.8, 128.5, 130.3, 130.4, 133.2, 138.6, 138.7, 139.3, 140.1, 147.1, 168.8, 171.2; m/z (ESI negative) 384.0 [M − H]−.

2′,4′-Difluoro-4-hydroxy-N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-[1,1′-biphenyl]-3-carboxamide (22)

A solution of 1 (1.0 mmol) was treated with diflunisal (1.0 mmol) in dry N,N-Dimethylacetamide (DMA) (4 ml) then N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) (2.0 mmol), 1-Hydroxy-7-azabenzotriazole (HOAT) (2.0 mmol), N,N-Diisopropylethylamine (DIPEA) (3.0 mmol) were added to reaction mixture. The reaction continued until the consumption of starting materials (TLC monitoring), quenched with 1 M aqueous HCl solution and extracted with ethyl acetate (3 × 15 ml). The combined organic layers were washed with H2O (3 × 15 ml), dried over Na2SO4, filtered, and concentrated to obtain a residue which was purified by silica gel column chromatography eluting with ethyl acetate/n-hexane 50% v/v to afford titled compound.

White solid, yield 15%, m.p.: 281–282 °C; silica gel TLC Rf = 0.23 (Ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6) 2.49 (2H, d, J 7.8), 2.89 (2H, t, J 7.8), 7.12 (1H, d, J 7.6), 7.23 (3H, m), 7.41 (2H, m), 7.65 (2H, m), 8.13 (1H, m), 10.18 (1H, s, exchange with D2O, NH), 10.47 (1H, s, exchange with D2O, NH), 12.04 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 25.3, 31.5, 105.4 (t, JC–F 26), 108.9, 112.9 (dd, JC–F 4, 21), 115.6, 118.5 (d, JC–F 19), 120.5, 125.0 (dd, JC–F 4, 14), 126.0 (d, JC–F 1), 128.7, 130.0 (d, JC–F 2), 132.6 (dd, JC–F 5, 10), 134.8 (d, JC–F 3), 137.9, 139.5, 158.7 (d, JC–F 12), 159.1, 161.1 (dd, JC–F 3, 12), 163.6 (d, JC–F 12), 167.1, 171.2; δF (376 MHz, DMSO-d6) −113.8 (1 F, d, J 7), −111.5 (1 F, d, J 7); m/z (ESI negative) 393.0 [M − H]−.

(Z)-4-oxo-4-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)amino)but-2-enoic acid (23)

A solution of compound 1 (1.0 mmol) was treated with maleic anhydride (1.05 mmol) in dry DMF then heated up to 150 °C. The reaction continued until the consumption of starting materials, quenched with 1 M aqueous HCl solution to obtain a precipitate which was washed with Et2O (3 × 5 ml) and dried under vacuum to obtain desired product.

White solid, yield 30%; m.p.: > 300 °C; δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.6), 2.86 (2H, t, J 7.6), 6.67 (1H, d, J 15.3), 7.16 (2H, m), 7.23 (1H, dd, J 1.8, 8.0), 7.34 (1H, d, J 1.8), 10.20 (1H, s, exchange with D2O, NH), 10.51 (1H, s, exchange with D2O, NH), 13.03 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 25.2, 31.4, 107.3, 113.9, 120.2, 128.8, 131.4, 138.1, 138.4, 139.5, 162.3, 167.1, 171.1; m/z (ESI positive) 261.0 [M + H]+.

N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)methanesulfonamide (24)

Compound 1 (1.2 mmol) was treated with methanesulfonyl chloride (1.01 mmol) in dry THF (3.0 ml) followed by addition of Et3N (1.1 mmol). The reaction continued until the consumption of starting materials (TLC monitoring) then quenched with 1 M aqueous HCl solution. Excess of solvents were removed under reduced pressure to obtain a residue which was filtered, washed with Et2O (3 × 5 ml) and dried under vacuum to afford titled compound.

White solid, yield 57%; m.p.: 236–237 °C; silica gel TLC Rf = 0.37 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.46 (2H, t, J 7.6), 2.85 (2H, t, J 7.6), 2.98 (3H, s), 6.79 (1H, dd, J 2.4, 8.0), 6.84 (1H, d, J 2.4), 7.14 (1H, d, J 2.4), 9.66 (1H, s, exchange with D2O, NH), 10.13 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.1, 31.4, 39.9, 108.0, 114.5, 120.2, 129.2, 138.2, 139.9, 171.1; m/z (ESI negative) 239.0 [M − H]−.

N-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)methanesulfonamide (25)

A solution of 24 (0.4 mmol) was treated with iodomethane (0.4 mmol) in dry DMF (3.0 ml) at 0 °C, followed by addition of K2CO3 (0.4 mmol) then warmed up to rt. The reaction continued until the consumption of starting materials and quenched with slush, acidified with 1 M aqueous HCl solution to obtain a precipitate which was collected, washed with Et2O (3 × 5 ml) and dried under vacuum to obtain desired product.

White solid; 80% yield; m.p.: 226–227 °C; silica gel TLC Rf = 0.59 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.49 (2H, t, J 7.6), 2.90 (2H, t, J 7.6), 2.97 (3H, s), 3.22 (3H, s), 6.91 (1H, d, J 2.2), 7.01 (1H, dd, J 2.2, 8.0), 7.24 (1H, d, J 8.0), 10.15 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 25.3, 31.1, 35.8, 38.8, 114.6, 119.9, 123.5, 129.1, 139.7, 141.5, 171.0; m/z (ESI positive) 255.0 [M + H]+.

2,4,6-Trimethyl-1-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)pyridin-1-ium perchlorate (26)

A solution of 1 (2.0 mmol) was treated with 2,4,6-trimethylpyrylium tetrafluoroborate (2.4 mmol) in dry methanol (10 ml) then the solution was refluxed for 5 h. Solvent was partially removed, the mixture was cooled down to room temperature and treated with a 1.0 M aqueous solution of HClO4 (3.0 equiv.). The precipitate formed was collected by filtration, and crystallised from water to afford the desired product.

Pale yellow solid, yield 40%; m.p.: 280–281 °C; silica gel TLC Rf = 0.10 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 2.37 (6H, s), 2.59 (2H, d, J 7.8), 2.63 (3H, s), 3.06 (2H, t, J 6.8), 6.97 (1H, d, J 2.4), 7.13 (1H, dd, J 2.4, 8.0) 7.56 (1H, d, J 8.0), 7.94 (2H, s), 10.50 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 22.2, 22.4, 25.5, 30.8, 112.6, 119.8, 127.6, 128.1, 130.9, 138.0, 141.3, 155.6, 159.8, 171.1; m/z (ESI positive) 267.0 [M]+.

CA assay

A stopped-flow method44 has been used for assaying the CA catalysed CO2 hydration activity with Phenol red as an indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalysed CO2 hydration reaction for 10–100 s. For each inhibitor, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.01 mm) were prepared in distilled-deionised water with 5% DMSO and dilutions up to 0.1 nm were done thereafter with the assay buffer. Enzyme and inhibitor were incubated for 6 h45–48. The inhibition constant (KI) was obtained by considering the classical Michaelis–Menten equation which has been fitted by using non-linear least squares with PRISM 3 (La Jolla, CA). All CA isozymes used in the experiments were purified, recombinant proteins obtained as reported earlier by our group49–59.

Results and discussion

Chemistry

In a previous report from this group43, we showed that 7-amino-3,4-dihydroquinolin-2(1H)-one (1) (Scheme 1) possesses interesting CA inhibitory properties against many human isoforms such as hCA VII, IX, XII and XIV, some of which are important drug targets for various applications of the CAIs. The lactam 1 was investigated as a CAI due to its structural similarity with the coumarins, a class of CAIs reported by this group45–48. Indeed, unlike other classes of such pharmacological agents, the coumarins act as prodrug inhibitors, being hydrolysed by the CA esterase activity to substituted 2-hydroxy-cinnamic acids, which thereafter bind at the entrance of the active site cavity, far away from the catalytic Zn(II) ion with which most CAIs interact13,45. That region is the most variable among the 15 human CAs, and this explains why coumarins and their derivatives are among the most isoform-selective CAIs reported so far1,13,45–48. In fact, a large number of substitution patterns at the coumarin ring, isosteric replacements or various other modifications were done on this chemotype, leading to a large number of CAIs possessing interesting properties13,45–48. Thus, the rationale of this work was to derivatise the 7-amino moiety of the lead 1, by reacting it with a variety of agents used earlier for the design of sulfonamide or dithiocarbamate CAIs (Scheme 1)13–16,22–25,35–37,60,61.

Scheme 1.

Scheme 1.

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Synthesis of compounds 2–26.

As shown in Scheme 1, a multitude of derivatisations of the amino moiety of compound 1 were achieved, such as: (i) reaction with arylsulfonyl isocyanates (leading to arylsulfonylureido derivatives 2–6); (ii) reaction with isocyanates, leading to ureas 7–19; (iii) reaction with fluoresceine isothiocyanate, leading to the fluorescent thiourea 20; (iv) condensation with substituted benzoic acids in the presence of carbodiimides, leading to the amides 21 and 22; (v) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate, leading to the pyridinium salt 26; (vi) reaction with methylsulfonyl chloride leading to the secondary sulfonamide 24, which was subsequently methylated with methyl iodide, leading to the 1-N-methyl derivative 25, and (vii) reaction with maleic anhydride leading to the monoamide 23 (Scheme 1). All these compounds were thoroughly characterised by physicochemical procedures which confirmed their structures (see “Materials and methods” for details).

CA inhibition

Compounds 2–26 were assayed for their CA inhibitory activity by a stopped-flow, CO2 hydrase method44 against four isoforms of pharmacologic relevance, the cytosolic human (h) hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX (Table 1). The following structure-activity relationship can be observed from the inhibition data of Table 1:

Table 1.

Inhibition data of hCA I, hCA II, hCA IV, hCA IX with compounds 2–26 reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO2 hydrase assay.

  KI (nm)
Cmp hCA I hCA II hCA IV hCA IX
2 8241.0 7467.6 >10,000 2133.3
3 6813.4 6966.7 >10,000 1461.0
4 3690.4 6852.2 >10,000 1051.8
5 >10,000 6379.1 >10,000 2234.6
6 3202.4 4437.9 >10,000 1688.2
7 >10,000 >10,000 >10,000 2420.3
8 >10,000 >10,000 >10,000 >10,000
9 >10,000 >10,000 >10,000 2267.5
10 >10,000 >10,000 >10,000 >10,000
11 >10,000 >10,000 >10,000 1158.3
12 >10,000 >10,000 >10,000 2489.6
13 >10,000 >10,000 >10,000 2105.0
14 >10,000 >10,000 >10,000 1373.1
15 >10,000 7883.8 >10,000 243.6
16 >10,000 5724.1 >10,000 >10,000
17 5328.9 4973.1 3801.4 2165.2
18 8749.6 5490.4 >10,000 1524.5
19 >10,000 >10,000 >10,000 2386.7
20 >10,000 3378.5 >10,000 1941.1
21 >10,000 >10,000 >10,000 >10,000
22 >10,000 >10,000 >10,000 2516.7
23 >10,000 >10,000 >10,000 1473.3
24 >10,000 >10,000 >10,000 292.8
25 >10,000 >10,000 >10,000 2758.6
26 >10,000 >10,000 >10,000 2658.3
AAZ 250 12 74 25
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Errors were in the range of ±5–10% of the reported data, from three different assays.

  1. hCA I was poorly inhibited by most derivatives 2–26, with only seven of them showing KIs in the micromolar range (i.e. 3.20–8.75 µm), the remaining ones having KIs > 10 µm (Table 1). The more effective inhibitors were 2–4, 6, 17 and 18, which incorporate arylsulfonylureido and ureido moieties. The other substitution patterns led to compounds with much weaker hCA I inhibitory activity.

  2. hCA II, the dominant cytosolic isoform was generally also poorly inhibited by these derivatives (KIs > 10 µm) except the arysulfonylureido ones 2–6 (KIs of 4.43–7.46 µm) the ureas 15–18 (KIs of 4.97–7.88 µm) and the thiourea 20 (KI of 3.37 µm), which was the best hCA II inhibitor in the series.

  3. hCA IV was the least sensitive isoform to these compounds with only one of them (17, KI of 3.80 µm) having an activity <10 µm (Table 1). It is rather difficult to explain this result considering that the inhibition mechanism with these lactams is not yet elucidated.

  4. The tumour-associated hCA IX was the most inhibited isoform among the four investigated ones, with KIs ranging between 243.6 and 2758.6 nm (Table 1). Only four derivatives (8, 10, 16 and 21) had KIs > 10 µm, whereas the best hCA IX inhibitors were 15 and 24 with KIs of 243.6–292.8 nm. These compounds rather different as the first one is a urea incorporating a pentafluorophenyl moiety, whereas the second one has the secondary sulfonamide functionality. It should be noted that small variations in the structures of such derivatives (as the N1-methylation of 24 leading to 25) or the reduction of the number of fluorine atoms on the phenyl ring, as in 14, led to a rather important reduction of the hCA IX inhibitory power compared to 24 and 15, respectively. Generally, all other arylsulfonylureas/ureas 2–19 (except the two compounds mentioned above as weak inhibitors and 15 which is one of the best) showed a similar behaviour of medium potency hCA IX inhibitors with KIs of 1.05–2.48 µm.

  5. All the derivatives reported here showed much weaker CA inhibitory activity compared to the clinically used sulfonamide acetazolamide AAZ (Table 1).

Conclusions

A series of derivatives was prepared by derivatisation of the 7-amino moiety of 7-amino-3,4-dihydroquinolin-2(1H)-one, a compound investigated earlier as CAI. The derivatisation was achieved by: (i) reaction with arylsulfonyl isocyanates (ii) reaction with aryl isocyanates; (iii) reaction with fluoresceine isothiocyanate; (iv) condensation with substituted benzoic acids in the presence of carbodiimides; (v) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate; (vi) reaction with methylsulfonyl chloride and (vii) reaction with maleic anhydride. The new compounds were assayed as inhibitors of four CA human isoforms of pharmacologic relevance, the cytosolic hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX. hCA IX was the most inhibited isoform (KIs ranging between 243.6 and 2658.3 nm) whereas hCA IV was not inhibited by these compounds. Most derivatives were weak hCA I and II inhibitors, with few of them showing KIs < 10 µm. Considering that the inhibition mechanism with these lactams is not yet elucidated, exploring a large range of derivatives with various substitution patterns may be useful to identify leads showing isoform selectivity.

Acknowledgements

This work was financed in part by a Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

One author (CTS) declares conflict of interest, being author of several patents in the field of CA inhibitors/activators. The other authors do not declare conflict of interest.

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