Discovery of 2,4-thiazolidinedione-tethered coumarins as novel selective inhibitors for carbonic anhydrase IX and XII isoforms.

Different 2,4-thiazolidinedione-tethered coumarins 5a-b, 10a-n and 11a-d were synthesised and evaluated for their inhibitory action against the cancer-associated hCAs IX and XII, as well as the physiologically dominant hCAs I and II to explore their selectivity. Un-substituted phenyl-bearing coumarins 10a, 10 h, and 2-thienyl/furyl-bearing coumarins 11a-c exhibited the best hCA IX (KIs between 0.48 and 0.93 µM) and hCA XII (KIs between 0.44 and 1.1 µM) inhibitory actions. Interestingly, none of the coumarins had any inhibitory effect on the off-target hCA I and II isoforms. The sub-micromolar compounds from the biochemical assay, coumarins 10a, 10 h and 11a-c, were assessed in an in vitro antiproliferative assay, and then the most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells to provide more insights into the anticancer activity of these compounds.

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes found in all living organisms and are responsible for the catalysis of the biologically crucial reversible hydration of carbon dioxide to bicarbonate and proton. This is a simple but pivotal physiological reaction which is essential for normal and pathological processes such as CO2 and pH homeostasis, respiration, gluconeogenesis, calcification, bone resorption, fluid secretion and tumorigenesis [1,2]. CAs are grouped into different families, amongst them α-CAs which are present in all vertebrates and are further sub-classified into fifteen isoforms (herein referred to as human CAs or hCAs) that differ by molecular features, expression levels, kinetic properties and cellular distribution in the different tissues [3]. Of note, only twelve hCAs are catalytically active (I–IV, VA, VB, VI, VII, IX, XII–XIV) with an active site containing three histidine residues in a triple coordination with a zinc ion [4]. Regarding the subcellular distribution of the catalytically active hCAs, they can be categorised into different subsets: cytosolic (I, II, III, VII and XIII), trans-membrane (IV, IX, XII, and XIV), mitochondrial (VA and VB), while VI is secreted in saliva and milk [5]. The over-expressed levels and/or dysfunctions of hCAs can lead to many disorders, hence CA inhibitors (CAIs) are utilised for the treatment of glaucoma (targeting hCA II, IV and XII), edoema (targeting hCA II, IV and XIV), mental disorders (targeting hCA II, VII and XIV) and obesity (targeting hCA VA and VB) [4,6,7].

It should be stressed that the trans-membranous hCA IX and XII are hypoxia-induced tumours-associated isozymes and overexpressed in most cancer cells compared to the normal ones [8]. While the overexpressed hCA IX isozyme is mainly linked to cancer poor prognosis and limited to hypoxic tumours, hCA XII can be found in some normal tissues like kidney and colon alongside the hypoxic tumours [9,10]. Interestingly, the tumour growth, angiogenesis, proliferation and metastasis are attributed to the overexpressed levels of hCA IX and XII suggesting a strategy for targeting of such enzymes as a new approach in cancer chemotherapy [8,11]. In this context, selective inhibition of the tumour-associated hCA IX and XII isozymes over the other isoforms, particularly the most prevalent cytosolic hCA I and II is highly desirable and will result in cancer treatment with fewer side effects [12].

In view of this, intensive efforts are being conducted for the development of hCA IX/XII selective inhibitors as a validated approach for cancer treatment [13-15]. hCA IX and XII can be inhibited by different strategies such as coordination to the zinc ion situated in the catalytic active site. Molecules in this class are exemplified by sulfonamide-derived hCAIs and their bioisosters. In addition, the occlusion of the catalytic active cleft is explored and this approach has been explored using coumarins as a newly discovered hCAIs class [16,17].

Coumarin I is a naturally-derived, privileged heterocyclic scaffold and molecules containing it show numerous biological properties such as inhibition of CK2, EGFR and PI3K-AKT-mTOR signalling. Moreover, coumarins are known to have anticoagulation, monoamine oxidase inhibition, anti-infective, antioxidant, anti-inflammatory and anticancer activities [14,18-20]. Coumarins are recently discovered as a novel class of hCAIs with inhibitory mechanism different from the sulfonamide-based inhibitors. Coumarinacts as prodrug undergoing hydrolysis by the esterase activity of CA to yield 2-hydroxycinnamic acid derivative II which can bind to the active site cleft occluding its entrance, Figure 1 [15,21]. Since coumarins binding sites are the most heterologous region of the active site between all CAs isoforms, it not surprising that these chemotypes displaying very high selectivity for specific CAs isoforms. Furthermore, the chemical simplicity of coumarins permits the facile incorporation of diverse substituents, leading to generation of a large number of derivatives with interesting biological profiles [22]. Consequently, many ongoing efforts have focussed on developing novel coumarin derivatives as selective hCAs IX/XII inhibitors that could be used for cancer therapy. For instance, diverse coumarins III-X have been reported as selective hCAs IX/XII inhibitors with nanomolar Ki, Figure 1 [8,10,13,17,21-24].

Figure 1.

Some reported coumarins acting as selective CA IX/XII inhibitors.

Inspired by these findings, we prepared a series of 2,4-thiazolidinedione-tethered coumarins, compounds 5a–b, 10a–n and 11a–d, and evaluated their inhibitory action against the cancer-associated hCAs IX and XII, and selectivity over inhibition of the physiologically dominant hCAs I and II to explore their selectivity in order to the cancer-related isoforms. Moreover, the efficient hCAs IX/XII inhibitors 10a, 10h and 11a–c were subjected to in vitro antiproliferative assay under hypoxic conditions and most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells furnishing more insights on the anticancer activity of such compounds.

Experimental

Chemistry

General

The NMR spectra were recorded by Bruker 400 MHz spectrometer. 1H and 13 C spectra were run at 400 and 100 MHz, respectively, in deuterated dimethylsulphoxide (DMSO-d) or deuterated triflouroacetic acid. All coupling constant (J) values are given in hertz. IR spectra were recorded with a Bruker FT-IR spectrophotometer. Reaction courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F254 Merck plates. Unless otherwise mentioned, all reagents and solvents are commercially available and have been used without further purification. Compounds 2 and 3 are previously reported [25,26].

General procedures for synthesis of 3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione derivatives (5a–b)

To a stirred solution of 3–(2-bromoacetyl)-2H-chromen-2-one 3a (0.27 g, 1.0 mmol) or 6-bromo-3–(2-bromoacetyl)-2H-chromen-2-one 3b (0.34 g, 1.0 mmol) in DMF (7 ml), thiazolidine-2,4-dione 4 (0.12 g, 1.0 mmol), anhydrous K2CO3 (0.28 g, 2.0 mmol) and KI (cat.) were added. The reaction mixture was heated on a water bath for 8h. then was poured over crushed ice. The precipitate was filtered, dried, and crystalsized from hot ethanol to give the corresponding key intermediates 5a-b, respectively.

3–(2-Oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 5a

Yellow crystals (yield, 80%); m. p. = 180–181 °C (reported: 161–163 °C [27]); 1H NMR (400 MHz, Trifluoroacetic acid-d): δ 8.79 (s, 1H, H-4 coumarin moiety), 8.01 (d, J = 8.0 Hz, 1H, H-5 coumarin moiety), 7.81 (t, J = 7.2 Hz, 1H, H-7 coumarin moiety), 7.51 (d, J = 8.0 Hz, 1H, H-8 coumarin moiety), 7.46 (t, J = 7.6 Hz, 1H, H-6 coumarin moiety), 5.13 (s, 2H, CH2, –N–CH2–CO–), 4.10 (s, 2H, CH2, –S–CH2–CO–); 13 C NMR (101 MHz, Trifluoroacetic acid-d1) δ 189.47, 172.32, 159.16, 155.25, 149.25, 135.73, 131.67, 125.63, 122.60, 118.53, 116.75, 50.43 (–N–CH2–), 34.54 (–S–CH2–); Anal. Calcd. for C14H9NO5S (303.29): C, 55.44; H, 2.99; N, 4.62; Found: C, 55.68; H, 3.01; N, 4.56.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)thiazolidine-2,4-dione 5b

Yellow crystals (yield, 75%); m. p.= 239–241 °C (reported: 171–173 °C [27]); 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H, H-4 coumarin), 8.20 (s, 1H, H-5 coumarin), 7.88 (d, J = 8.8 Hz, 1H, H-7 coumarin), 7.44 (d, J = 8.8 Hz, 1H, H-8 coumarin), 5.14 (s, 2H, CH2), 4.11 (s, 2H, CH2); 13 C NMR (101 MHz, TFA-deuterated) δ 195.41, 158.46, 154.08, 146.09, 137.04, 132.10, 125.85, 120.52, 118.86, 116.82, 60.22 (–N–CH2–), 30.49 (–S–CH2–); Anal. Calcd. for C14H8BrNO5S (382.18): C, 44.00; H, 2.11; N, 3.66; Found: C, 43.85; H, 2.12; N, 3.69.

General procedures for preparation of the intermediates 8a–g and 9a–b

To a solution of thiazolidine-2,4-dione 4 (0.12 g, 1 mmol) in glacial acetic acid (5 ml), anhydrous sodium acetate (0.08 g, 1 mmol) and the appropriate aldehyde derivative (6a–g and 7a–b) were added. The resulting reaction mixture was allowed to stir under reflux for 3h. The precipitated solid was collected by filtration while hot, washed with cold ethanol and water, and dried to afford intermediates 8a–g and 9a–b.

General procedures for preparation of coumarins 10a–n and 11a–d

The appropriate benzylidine derivative 8a–g (2 mmol) was added to a hot stirred mixture of 3-(bromoacetyl)coumarin derivatives 3a–b (2 mmol), K2CO3 (0.55 g, 4 mmol), KI (2 mmol) in DMF (8 ml), then the resulting mixture was stirred under reflux for 8h. The formed precipitates were collected by filtration, washed with water, dried and recrystalslized from DMF/water to yield the final target coumarin-based CAIs 10a–n and 11a–d.

5-Benzylidene-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10a

Grey crystals (yield, 70%); m. p. = 249–251 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H, ArH), 8.27 (s, 1H, –CH=), 8.01–7.97 (m, 2H, ArH), 7.70–7.61 (m, 7H, ArH), 5.64 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, TFA-deuterated) δ 173.27, 172.54, 168.52, 168.28, 153.27, 139.52, 139.52, 137.19, 132.00, 131.79, 131.19, 130.47, 129.01, 126.36, 118.57, 116.70, 115.75, 54.32 (–N–CH2–); Anal. Calcd. for C21H13NO5S (391.40): C, 64.44; H, 3.35; N, 3.58; Found: C, 64.71; H, 3.37; N, 3.52 [28].

5–(4-Methylbenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10b

Yellow crystals (yield, 85%); m. p. = 227–229 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H, ArH), 8.04 (d, J = 8.0 Hz, 1H, ArH), 7.79 (t, J = 7.6 Hz, 1H, H-7 ArH), 7.72 (s, 1H, –CH=), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.47 (d, J = 8.0 Hz, 2H, ArH), 7.48 (t, J = 7.6 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 2H, ArH), 5.13 (s, 2H, CH2, –N–CH2–CO–), 2.41 (s, 3H, CH3); Anal. Calcd. for C22H15NO5S (405.42): C, 65.18; H, 3.73; N, 3.45; Found: C, 64.95; H, 3.76; N, 3.51.

5–(4-Methoxybenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10c

Yellow crystals (yield, 85%); m. p. = 257–259 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H, ArH), 8.24 (s, 1H, –CH=), 8.00–7.96 (m, 2H, ArH), 7.71 (d, J = 8.8 Hz, 2H, ArH), 7.65–7.61 (m, 2H, ArH), 7.23 (d, J = 8.8 Hz, 2H, ArH), 5.63 (s, 2H, CH2), 4.10 (s, 3H, OCH3); 13 C NMR (101 MHz, TFA-deuterated) δ 172.54, 168.38, 162.43, 162.00, 161.56, 161.13, 153.19, 139.03, 137.03, 132.98, 131.07, 126.39, 126.00, 118.52, 118.05, 116.05, 115.71, 112.90, 110.08, 54.90 (OCH3), 51.47 (–N–CH2–); Anal. Calcd. for C22H15NO6S (421.42): C, 62.70; H, 3.59; N, 3.32; Found: 62.88; H, 3.62; N, 3.26 [28].

5–(2,5-Dimethoxybenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10d

Grey crystals (yield, 82%); m. p. = 238–240 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H, ArH), 8.28 (s, 1H, –CH=), 7.76–7.73 (m, 2H, ArH), 7.42–7.39 (m, 2H, ArH), 7.08–6.93 (m, 3H, ArH), 5.36 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 3.82 (s, 3H, OCH3); 13 C NMR (101 MHz, DMSO) δ 206.65, 189.23, 167.42, 165.61, 159.17, 153.66, 153.59, 152.95, 149.34, 135.63, 131.61, 129.04, 122.59, 122.52, 122.23, 121.89, 117.80, 116.65, 113.64, 56.31 (OCH3), 55.73 (OCH3), 50.67 (–N–CH2–); Anal. Calcd. for C23H17NO7S (451.45): C, 61.19; H, 3.80; N, 3.10; Found: 60.94; H, 3.83; N, 3.14.

5–(4-Nitrobenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10e

Yellow crystals (yield, 77%); m. p. = 263–265 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H, ArH), 8.36 (d, J = 8.8 Hz, 2H, ArH), 8.13 (s, 1H, –CH=), 8.00 (d, J = 8.4 Hz, 1H, ArH), 7.93 (d, J = 8.4 Hz, 2H, ArH), 7.86–7.79 (m, 2H, ArH), 7.44 (t, J = 8.0 Hz, 1H, ArH), 5.24 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 189.33, 165.32, 159.25, 155.31, 149.42, 148.23, 139.55, 135.84, 131.73, 131.65, 131.27, 128.47, 125.66, 124.72, 122.55, 118.57, 116.79, 50.99 (–N–CH2–); Anal. Calcd. for C21H12N2O7S (436.39): C, 57.80; H, 2.77; N, 6.42; Found: C, 58.03; H, 2.75; N, 6.49.

5–(2-Chlorobenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10f

Yellow crystals (yield, 77%); m. p. = 208–211 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H, ArH), 8.06 (s, 1H, –CH=), 8.01 (d, J = 8.0 Hz, 1H, ArH), 7.84 (d, J = 8.0 Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 1H, ArH), 7.71 (d, J = 8.0 Hz, 1H, ArH), 7.66–7.59 (m, 2H, ArH), 7.51 (d, J = 8.4 Hz, 1H, ArH), 7.44 (t, J = 8.4 Hz, 1H, ArH), 5.23 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 189.39, 167.21, 165.20, 159.24, 149.43, 135.84, 134.17, 133.07, 132.88, 132.04, 131.74, 129.69, 129.24, 125.79, 125.67, 125.19, 122.57, 118.56, 116.79, 55.39 (–N–CH2–); Anal. Calcd. for C21H12ClNO5S (425.84): C, 59.23; H, 2.84; N, 3.29; Found: C, 59.43; H, 2.82; N, 3.25.

5–(2-Bromobenzylidene)-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10g

Yellow crystals (yield, 82%); m. p. = 230–232 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H, ArH), 8.64 (s, 1H, –CH=), 8.00–7.97 (m, 2H, ArH), 7.74–7.72 (m, 1H, ArH), 7.66–7.63 (m, 3H, ArH), 7.53–7.56 (m, 2H, ArH), 5.65 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, TFA-deuterated) δ 190.49, 172.12, 167.88, 163.26, 162.48, 162.04, 161.61, 161.17, 155.14, 136.60, 130.43, 121.61, 119.96, 118.49, 118.05, 115.68, 112.86, 110.05, 50.72 (–N–CH2–); Anal. Calcd. for C21H12BrNO5S (470.29): C, 53.63; H, 2.57; N, 2.98; Found: C, 53.81; H, 2.58; N, 3.01

5-Benzylidene-3–(2-(6-bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)thiazolidine-2,4-dione 10h

Yellow crystals (yield, 78%); m. p. = 252–254 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.21 (d, J = 2.4 Hz, 1H, ArH), 7.87 (d, J = 7.6 Hz, 1H, ArH), 7.78 (s, 1H, –CH=), 7.43–7.61 (m, 6H, ArH), 5.14 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 168.61, 168.30, 146.11, 137.06, 134.29, 133.60, 133.42, 133.01, 131.94, 130.81, 130.45, 129.78, 125.88, 124.43, 120.54, 118.88, 117.04, 116.83, 50.83(–N–CH2–); Anal. Calcd. for C21H12BrNO5S (470.29): C, 53.63; H, 2.57; N, 2.98; Found: C, 53.78; H, 2.56; N, 3.00.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(4-methylbenzylidene)thiazolidine-2,4-dione 10i

Yellow crystals (yield, 86%); m. p. = 224–225 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.20 (s, 1H, ArH), 7.86 (d, J = 8.8 Hz, 1H, ArH), 7.78 (s, 1H, –CH=), 7.47 (d, J = 8.0 Hz, 2H, ArH), 7.42 (d, J = 8.8 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 2H, ArH), 5.13 (s, 2H, –N–CH2–CO–), 2.35 (s, 3H, CH3); Anal. Calcd. for C22H14BrNO5S (484.32): C, 54.56; H, 2.91; N, 2.89; Found: C, 54.75; H, 2.89; N, 2.90.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(4-methoxybenzylidene)thiazolidine-2,4-dione 10j

Grey crystals (yield, 72%); m. p. = 245–247 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.21 (d, J = 2.4 Hz, 1H, ArH), 7.87 (d, J = 8.0 Hz, 1H, ArH), 7.73 (s, 1H, –CH=), 7.54 (d, J = 8.8 Hz, 2H, ArH), 7.43 (d, J = 8.0 Hz, 1H, ArH), 7.08 (d, J = 8.8 Hz, 2H, ArH), 5.20 (s, 2H, –N–CH2–CO–), 3.82 (s, 3H, OCH3); Anal. Calcd. for C22H14BrNO6S (500.32): C, 52.81; H, 2.82; N, 2.80; Found: C, 53.04; H, 2.83; N, 2.80.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(2,5 -dimethoxybenzylidene) thiazolidine-2,4-dione 10k

Yellow crystals (yield, 78%); m. p. = 204–206 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H, ArH), 8.21 (d, J = 2.4 Hz, 1H, ArH), 7.90 (s, 1H, –CH=), 7.87 (d, J = 8.0 Hz, 1H, ArH), 7.42 (d, J = 8.0 Hz, 1H, ArH), 7.08–7.07 (m, 2H, ArH), 6.91 (d, J = 8.0 Hz, 1H, ArH), 5.19 (s, 2H, –N–CH2–CO–), 3.83 (s, 3H, OCH3), 3.75 (s, 3H, OCH3); 13 C NMR (101 MHz, TFA-deuterated) δ 162.46, 162.02, 161.74, 161.59, 161.15, 154.65, 152.12, 134.08, 122.06, 120.32, 118.49, 115.74, 115.68, 113.18, 112.86, 110.05, 56.32, 55.38. Anal. Calcd. for C23H16BrNO7S (530.35): C, 52.09; H, 3.04; N, 2.64; Found: C, 51.89; H, 3.07; N, 2.63.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(4- nitrobenzylidene)thiazolidine-2,4-dione 10 l

Yellow crystals (yield, 82%); m. p. = 229–231 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.93 (dd, J = 8.8, 2.0 Hz, 1H, ArH), 7.88 (s, 1H, –CH=), 7.84 (d, J = 8.8 Hz, 2H, ArH), 7.49 (d, J = 8.8 Hz, 1H, ArH), 5.13 (s, 2H, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 189.24, 168.05, 167.94, 166.89, 165.28, 158.81, 154.31, 148.23, 147.88, 139.95, 139.52, 131.65, 131.35, 129.28, 128.90, 125.63, 124.73, 123.59, 120.40, 117.06, 50.98. Anal. Calcd. for C21H11BrN2O7S (515.29): C, 48.95; H, 2.15; N, 5.44; Found: C, 49.11; H, 2.14; N, 5.48.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(2-chlorobenzylidene)thiazolidine-2,4-dione 10m

Yellow crystals (yield, 77%); m. p. = 192–194 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H, ArH), 8.12 (s, 1H, ArH), 8.05 (d, J = 8.8 Hz, 1H, ArH), 7.75–7.51 (m, 6H, ArH), 5.64 (s, 2H, –N–CH2–CO–); 13 C NMR (101 MHz, TFA-deuterated) δ 162.52, 162.09, 161.66, 161.22, 151.67, 139.52, 138.48, 135.27, 133.00, 132.66, 132.42, 130.36, 128.68, 127.03, 119.33, 118.51, 115.69, 112.88, 110.07. Anal. Calcd. for C21H11BrClNO5S (504.74): C, 49.97; H, 2.20; N, 2.78; Found: C, 50.13; H, 2.21; N, 2.80.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5–(2-bromobenzylidene)thiazolidine-2,4-dione 10n

Yellow crystals (yield, 72%); m. p. = 208–210 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H, ArH), 8.22 (s, 1H, ArH), 7.87 (d, J = 8.8 Hz, 1H, ArH), 7.79 (s, 1H, –CH=), 7.51–7.59 (m, 3H, ArH), 7.43 (d, J = 8.8 Hz, 1H, ArH), 7.34 (t, J = 8.0 Hz, 1H, ArH), 5.14 (s, 2H, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 207.46, 158.94, 158.47, 154.43, 154.09, 146.11, 137.06, 134.21, 134.16, 133.92, 133.01, 131.75, 129.88, 129.61, 129.39, 129.23, 128.93, 120.54, 118.88, 116.83, 30.50. Anal. Calcd. for C21H11Br2NO5S (549.19): C, 45.93; H, 2.02; N, 2.55; Found: C, 46.09; H, 2.03; N, 2.53.

General procedures for preparation of target coumarins 11a–d

5-(Thiophen-2-ylmethylene)thiazolidine-2,4-dione 7a (15 mmol) and/or 5-((5-methylfuran-2-yl)methylene)thiazolidine-2,4-dione 7b (15 mmol) was added to a hot stirred solution of 3-(bromoacetyl) coumarin derivatives 3a,b (15 mmol) in DMF (10 ml), K2CO3 (15 mmol), KI (15 mmol), then the resulting mixture was stirred under reflux for 8h. The precipitates were collected by filtration, washed with water, dried and recrystalslized from hexane/ethanol to yield the final target compounds 11a-d.

3–(2-Oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)-5-(thiophen-2-ylmethylene)thiazolidine-2,4-dione 11a

Yellow crystals (yield, 83%); m. p. = 250–251 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H, ArH), 8.44 (s, 1H, –CH=), 7.97–7.91 (m, 3H, ArH), 7.68–7.61 (m, 3H, ArH), 7.33 (t, J = 4.8 Hz, 1H, ArH), 5.62 (s, 2H, CH2, –N–CH2–CO–); 13 C NMR (101 MHz, TFA-deuterated) δ 190.74, 162.45, 162.02, 161.58, 161.14, 153.33, 137.00, 136.28, 134.41, 131.79, 131.08, 128.63, 126.38, 119.93, 118.48, 115.67, 112.85, 110.04, 50.74. Anal. Calcd. for C19H11NO5S2 (397.42): C, 57.42; H, 2.79; N, 3.52; Found: C, 57.26; H, 2.78; N, 3.55.

5-((5-Methylfuran-2-yl)methylene)-3–(2-oxo-2–(2-oxo-2H- chromen-3-yl)ethyl)thiazolidine-2,4-dione 11b

Yellow crystals (yield, 70%); m. p. = 227–229 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H, ArH), 7.96–7.90 (m, 3H, –CH = and ArH), 7.62–7.60 (m, 2H, ArH), 7.09 (d, J = 4.0 Hz, 1H, ArH), 6.40 (d, J = 4.0 Hz, 1H, ArH), 5.60 (s, 2H, CH2, –N–CH2–CO–), 2.54 (s, 3H, CH3); 13 C NMR (101 MHz, TFA-deuterated) δ 162.45, 162.02, 161.57, 161.14, 153.32, 147.87, 136.96, 131.05, 126.35, 124.21, 118.47, 118.02, 116.58, 115.66, 112.84, 110.55, 110.03, 110.03, 50.50, 12.05; Anal. Calcd. for C20H13NO6S (395.39): C, 60.76; H, 3.31; N, 3.54; Found: C, 60.92; H, 3.34; N, 3.51.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5-(thiophen-2-ylmethylene)thiazolidine-2,4-dione 11c

Yellow crystals (yield, 76%); m. p. = 255–257 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.27 (d, J = 2.4 Hz, 1H, ArH), 8.04 (s, 1H, –CH=), 7.99 (d, J = 5.2 Hz, 1H, ArH), 7.93 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.48 (d, J = 3.6 Hz, 1H, ArH), 7.27 (t, J = 3.6 Hz, 1H, ArH), 5.20 (s, 2H, –N–CH2–CO–); 13 C NMR (101 MHz, DMSO) δ 189.40, 167.97, 167.84, 165.35, 158.79, 147.90, 146.11, 137.84, 137.41, 136.00, 134.92, 133.42, 129.39, 127.63, 125.40, 121.86, 120.42, 117.04, 50.93. Anal. Calcd. for C19H10BrNO5S2 (476.32): C, 47.91; H, 2.12; N, 2.94; Found: C, 47.79; H, 2.13; N, 2.96.

3–(2-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-5-((5-methylfuran-2-yl)methylene)thiazolidine-2,4-dione 11d

Yellow crystals (yield, 79%); m. p. = 236–238 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.22 (d, J = 2.4 Hz, 1H, ArH), 7.99 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 7.74 (s, 1H, –CH=), 7.43 (d, J = 8.8 Hz, 1H, ArH), 6.99 (d, J = 4.0 Hz, 1H, ArH), 6.39 (d, J = 4.0 Hz, 1H, ArH), 5.17 (s, 2H, –N–CH2–CO–), 2.38 (s, 3H, CH3); 13 C NMR (101 MHz, DMSO) δ 189.51, 169.37, 167.80, 157.74, 148.41, 147.87, 146.11, 137.80, 137.06, 133.40, 133.01, 122.00, 120.69, 119.25, 119.05, 118.80, 111.03, 110.72, 50.58, 14.22. Anal. Calcd. for C20H12BrNO6S (474.28): C, 50.65; H, 2.55; N, 2.95; Found: C, 50.52; H, 2.57; N, 2.98.

Biological evaluation

The experimental procedures for CA stopped-flow [29-31], MTT cell viability [32,33], cell cycle [34] and Annexin V-FITC/PI [35] assays are included in the Supporting Information.

Results and discussion

The proposed synthetic routes to obtain the target coumarins are depicted in Scheme 1 and Scheme 2. First, condensation of 2-hydroxybenzaldehydes 1a–b with ethyl 3-oxobutanoate in refluxing absolute ethanol in the presence of a few drops of piperidine yielded 3-acetylcoumarins 2a–b. These were subjected to bromination via reaction with Br2 in glacial acetic acid to yield the key 3-(bromoacetyl)coumarin intermediates 3a–b, which were subsequently treated with thiazolidine-2,4-dione 4 in refluxing DMF using anhydrous K2CO3 as base and KI as a nucleophilic catalyst to afford 3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione derivatives 5a–b (Scheme 1).

Scheme 1.

Reagents and conditions: (i) Abs. Ethanol, piperidine, reflux, 2 h.; (ii) bromine 99%, glacial acetic acid, r.t., 6 h.; (iii) anhydrous DMF, potassium carbonate, potassium iodide, heating on a water bath, 8 h.

Scheme 2.

Reagents and conditions: (i) glacial acetic acid, reflux 3 h.; (ii) DMF, potassium carbonate, potassium iodide, reflux 8 h.

Synthesis of compounds 8a–g and 9a–b (Scheme 2) was achieved via refluxing of thiazolidine-2,4-dione 4 with benzaldehyde derivatives 6a–g and 7a–b in glacial acetic acid and anhydrous sodium acetate. Treatment of 8a–g and 9a–b with the key intermediates 3a–b in refluxing DMF using anhydrous K2CO3 and KI furnished the corresponding final targets 5-benzylidene-3–(2-oxo-2–(2-oxo-2H-chromen-3-yl)ethyl)thiazolidine-2,4-dione 10a–n and 11a–d, respectively. Proposed structures for the synthesised coumarins were in agreement with their various spectroscopic and analytical data.

Carbonic anhydrase inhibition

The inhibitory influence of all the synthesised coumarins 5a–b, 10a–n and 11a–d was investigated against hCA I, II IX and XII isoforms using a stopped flow CO2 hydrase assay and a well-known hCAI, acetazolamide (AAZ) as control [36]. From the resulting inhibition constants (KI) shown in Table 1, certain structure activity relationship (SAR) can be inferred.

Table 1.

Inhibition data for hCA I, II, IX and XII isoforms with 2,4-thiazolidinedione-tethered coumarins (5a–b, 10a–n and 11a–d) and AAZ.

Cmpd	R1	R2	KI (μM)a,b
			CA I	CA II	CA IX	CA XII
5a	H	–	>100	>100	0.12	0.15
5b	Br	–	>100	>100	0.24	0.31
10a	H		>100	>100	0.82	0.75
10b	H		>100	>100	4.3	4.0
10c	H		>100	>100	5.8	4.5
10d	H		>100	>100	8.4	6.2
10e	H		>100	>100	12.3	8.0
10f	H		>100	>100	2.2	3.8
10g	H		>100	>100	2.3	4.1
10h	Br		>100	>100	0.93	0.87
10i	Br		>100	>100	6.2	2.3
10j	Br		>100	>100	8.9	4.9
10k	Br		>100	>100	16.4	6.6
10l	Br		>100	>100	18.2	10.4
10m	Br		>100	>100	2.9	3.2
10n	Br		>100	>100	3.4	2.8
11a	H		>100	>100	0.48	0.83
11b	H		>100	>100	0.79	1.1
11c	Br		>100	>100	0.59	0.44
11d	Br		>100	>100	0.91	0.82
AAZ	–	–	250	12.5	25.0	5.7

aMean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5–10% of the reported values); bincubation time of 6 h.

Coumarins 5a–b, 10a–n and 11a–d are devoid of significant inhibition towards the off-target ubiquitous hCA I and the physiologically dominant hCA II (KIs > 100 µM) isoforms, Table 1. In contrast, these coumarins inhibited the cancer-related hCA IX with inhibition constants spanning a range between 0.12 and 18.2 µM, (Table 1). Regarding the unsubstituted thiazolidinedione-bearing coumarins 5a–b, the absence of substitution at 6-position of coumarin scaffold furnished the most effective hCA IX inhibitor in this work displaying KI of 0.12 µM, whereas, the 6-bromination decreased the hCA IX inhibitory power 2-folds (KI = 0.24 µM).

In the context of hCA IX inhibition constants of the benzylidene counterparts 10a–n, it was found that 6-unsubsituted coumarins 10a–g showed effective inhibition (KIs ranged from 0.82 to 12.3 µM) compared to the 6-bromo analogues 10h–n (KIs spanned between 0.93 and 18.2 µM). In the term of coumarins with the benzylidene moiety, 10a–g, it is worth stressing that appending an unsubstituted aryl ring to the thiazolidinedione moiety 10a provided the most effective hCA IX inhibitor within this series (KI = 0.82 µM). Indeed, the incorporation of ortho-chloro or ortho-bromophenyl (10f and 10 g, respectively) decreased the inhibition constants to low micromolar values (KIs = 2.2 and 2.3 µM, respectively). Regrettably, the remaining phenyl substitution pattern were similarly poorer hCA IX inhibitors with KIs equalling 4.3 µM (p-methyl, 10b), 5.8 µM (p-methoxy, 10c), 8.4 µM (2,5-dimethoxy, 10d), 12.3 µM (p-nitro, 10e).

In a similar fashion, the appending of unsubstituted aryl ring to the 6-bromocoumarins 10h–n produced the most potent hCA IX inhibitor within this series (10h; KI = 0.93 µM), whereas ortho chlorination or bromination reduced the inhibition constants to low micromolar values (10m and 10n; KIs = 2.9 and 3.4 µM, respectively). Similar to the 6-unsubstituted coumarins 10a-g, it was noted that in the 6-bromocoumarin series, inclusion of other phenyl substituents(compounds10h–n) lowered the hCA IX inhibition constants showing KIs in the range 6.2–18.2 µM. Superiorly, the replacement of six-membered phenyl with five-membered 2-thienyl ring potentially elevated the inhibition constants for both 6-unsubstituted and 6-bromocoumarins (11a and 11c; KIs = 0.48 and 0.59 µM, respectively) compared to their phenyl counterparts (10a and 10h; KIs = 0.82 and 0.93 µM, respectively). Notably, utilising 2-furyl functionality in place of phenyl/thienyl group did not result in significant change in potency (11b and 11d; KIs = 0.79 and 0.91 µM, respectively).

Collectively, the deduced SAR for hCA IX inhibition suggests that applying of unsubstituted thiazolidinedione (5a–b) is more favoured than their substituted analogues (10a–n and 11a–d) affording the most potent hCA IX inhibitors in this study. Furthermore, the lack of substitution at 6-position of coumarin (5a, 10a–g and 11a–b) is more advantageous for such type of activity relative to 6-bromo counterparts (5b, 10h–n and 11c–d). Additionally, appending of unsubstituted phenyl ring (10a, 10h) is the most beneficial pattern within all tested benzylidene counterparts (10a–n), while replacement of phenyl with 2-thienyl moiety gave the most potent hCA IX inhibitors (11a and 11c) within all arylidene derivatives (10a–n and 11a–d).

Finally, the inhibition profiles (Table 1) revealed that the cancer-related hCA XII isoform was inhibited by the coumarins 5a–b, 10a–n and 11a–d displaying a range of inhibition constants from submicromolar level to low micromolar values (KIs ranged from 0.15 to 10.4 µM). The unsubstituted thiazolidinedione-bearing coumarins 5a-b emerged as the most effective hCA XII inhibitors, with submicromolar inhibition constants (5a; KI = 0.15 µM and 5b; KI = 0.31 µM).

It should be pointed out that bromination at 6-position of coumarin 5b led to 2-fold diminished inhibition for hCA XII relative to the unsubstituted analogue 5a, in a similar manner observed in the SAR for hCA IX inhibition, Table 1. Concerning the benzylidene derivatives 10a–n, it was observed that appending a phenyl to thiazolidinedione moiety resulted in the most potent hCA XII inhibitors (at submicromolar level) within this series (10a; KI = 0.75 µM and 10h; KI = 0.87 µM). The incorporation of different substituents to the phenyl group reduced the inhibitory potential affording KIs spanning between 2.3 and 10.4 µM. Furthermore, the absence of substitution at 6-position of coumarin is beneficial for inhibition (10a; KI = 0.75 µM), whereas 6-bromination decreased the activity (10h; KI = 0.87 µM), Table 1. It was noted that replacement of the phenyl group (10a; KI = 0.75 µM and 10h; KI = 0.87 µM) with 2-thienyl or 2-furyl functionalities reduced KIs for the 6-unsubstituted coumarins (11a; KI = 0.83 µM and 11b; KI = 1.1 µM, respectively), while raised KIs for the 6-bromocoumarins (11c; KI = 0.44 µM and 11d; KI = 0.82 µM, respectively). This is unlike the pattern in hCA IX inhibition profile and points to a potential future avenue of exploration towards selectivity of hCA XII over hCA IX.

To summarise, the elicited SAR highlighted that unsubstituted thiazolidinedione derivatives (5a–b) exerted more superior potency relative to their substituted counterparts (10a–n and 11a–d) resulting in the most potent hCA XII inhibitors in this work (5a-b). Moreover, within all benzylidene derivatives 10a–n, the unsubstituted phenyl counterparts exerted the best inhibition profiles (10a and 10h), however the replacement of phenyl with 2-thienyl or 2-furyl along with 6-bromination at coumarin scaffold potentiated the inhibitory impact of compounds (11c and 11d). Overall, the herein reported coumarins emerge as selective inhibitors towards the tumour-related hCA IX and XII over the off-target hCA I and II that suggests their use as promising candidates for the development of more potent, selective hCA IX and XII inhibitors as anticancer agents.

Anticancer activity

In vitro antiproliferative activity against MCF-7 breast cancer cell line

The antiproliferative action of the most potent and selective hCA IX/XII inhibitors 10a, 10h and 11a–d was assessed against MCF-7 breast cancer cell line, since the overexpression of hCA IX is well-reported to be associated with poor prognosis of breast cancer [37] and the cell line has been previously used as a model in CA medicinal chemistry investigations. The antiproliferative potential was investigated using MTT assay [38] under hypoxic conditions employing staurosporine as a reference anticancer drug. The results are presented in Table 2 as median inhibitory concentration (IC50) which denotes the concentration of the tested drug required to produce 50% growth inhibition of the cancer cell compared to the negative control.

Table 2.

Anti-proliferative activities of 2,4-thiazolidinedione-tethered coumarins 10a, 10 h and 11a–c against MCF-7 cell line.

Compound	IC50 (μM)a (MCF-7)
10a	3.13 ± 0.18
10h	11.1 ± 0.65
11a	0.48 ± 0.03
11b	4.14 ± 0.24
11c	9.56 ± 0.56
11d	1.65 ± 0.1
Staurosporine	2.44 ± 0.14

aIC50 values are the mean ± SD of three experiments.

Investigation of the antiproliferative effects towards MCF-7 breast cancer cell line confirmed that the tested coumarins 10a, 10h and 11a–c exhibited moderate to excellent growth inhibitory influence (IC50 ranged between 0.48 and 11.1 µM). Of special interest, the 2-thienyl-bearing 6-unsubstitued coumarin 11a, that displayed potent hCA IX/XII inhibition at submicromolar level, exerted excellent antiproliferative action at submicromolar value (IC50 equals 0.48 µM). Likewise, the other tested coumarins 10a, 10h, 11b–d demonstrated moderate growth inhibitory action with IC50 values equal 3.13, 11.1, 4.14, 9.56 and 1.65 µM, respectively compared to staurosporine as reference drug (IC50 = 2.44 µM), Table 2.

Cell cycle analysis

The influence of 2-thienyl-bearing 6-unsubstitued coumarin 11a on the cell cycle progression was investigated by flow cytometric in MCF-7 breast cancer cells, at 24h following treatment at its IC50 value (0.48 ± 0.03 µM), Figure 2.

Figure 2.

Impact of the tested coumarin 11a on the progression of cell cycle of MCF-7 cells.

As illustrated in Figure 2, the flow cytometric results showed that the exposure of MCF-7 breast cancer cells to compound 11a gave rise to a significant rise in the cell populations at Sub-G1, which increased by 19.7 folds with concomitant decrease in G2-M phase by 2.6 folds compared to the control, in addition to decline in cell populations within S and G0-G1 phases. This observation strongly suggests coumarin 11a induces apoptosis in MCF-7 cells.

Annexin V-FITC/propidium iodide (AV/PI) apoptosis assay

Annexin V-FITC/propidium iodide (AnxV/PI) dual staining assay was employed to confirm the potential apoptotic impact of coumarin 11a on early and late apoptosis percentages in MCF-7 breast cancer cells (Figure 3 and Table 3). This flow cytometric analysis highlighted that compound 11a was able to induce apoptosis in MCF-7 cells as indicated by the significant elevation in the percentage of annexin V-FITC-stained apoptotic cells including early apoptosis (Figure 3, lower right) from 0.37 to 4.23% and late apoptosis, Figure 3, upper right) from 0.15 to 25.7%. This represents 57 folds total increase relative to the control in apoptotic cells.

Figure 3.

Effect of coumarin 11a on the percentage of AV positive staining in breast MCF-7 cells.

Table 3.

Distribution of AV-FITC/PI positive stained apoptotic MCF-7 cells.

Comp.	Apoptosis			Necrosis
	Total	Early	Late
11a	29.93	4.23	25.7	17.39
Control	0.52	0.37	0.15	1.14

Conclusions

In this study, different 2,4-thiazolidinedione-tethered coumarins 5a–b, 10a–n and 11a–d have been synthesised and evaluated for their inhibitory action against the cancer-associated hCAs IX and XII, in addition to the physiologically dominant hCAs I and II, in order to explore their selectivity. Interestingly, none of the coumarins had any inhibitory effect on off-target hCA I and II isoforms. Unsubstituted phenyl-bearing coumarins 10a, 10h, and 2-thienyl/furyl-bearing coumarins 11a–c exhibited the best hCA IX (KIs between 0.48 and 0.93 µM) and hCA XII (KIs between 0.44 and 1.1 µM) inhibitory actions. Coumarins 10a, 10h and 11a–c were subjected to an in vitro antiproliferative assay, and then the most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells furnishing more insights on the potential anticancer activity of such compounds.

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