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Original article
12 (
8
); 2655-2667
doi:
10.1016/j.arabjc.2015.05.006

Design, synthesis and antiproliferative activity against human cancer cell lines of novel benzo-, benzofuro-, azolo- and thieno-1,3-thiazinone resorcinol hybrids

, , , , , , , ,
a Department of Chemistry, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland
b Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland
c Department of Experimental Oncology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland
d Medical University of Lublin, Department of Medicinal Chemistry, Jaczewskiego 4, 20-090 Lublin, Poland
e Institute of Rural Health in Lublin, Department of Medical Biology, Jaczewskiego 2, 20-090, Poland
f Maria Curie-Skłodowska University in Lublin, Department of Virology and Immunology, Akademicka 19, 20-033 Lublin, Poland

⁎Corresponding author. joanna.matysiak@up.lublin.pl (Joanna Matysiak)

Received: , Accepted: ,
© The Author(s) 2015
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In this paper we report the design and synthesis of novel derivatives of the 4H-3,1-benzothiazinone type and heterocyclic analogues, i.e. benzofuro-, azolo- and thieno-1,3-thiazin-4-ones possessing 2,4-dihydroxyphenyl substituent. The compounds were obtained by the one-step reaction of aminobenzamides or heterocyclic aminocarboxamides with aryl-modified sulfinylbis[(2,4-dihydroxyphenyl)methanethione]. Evaluation of their antiproliferative potency against human cancer cell lines showed that the activity of some analogues was similar to that of cisplatin. The highest activity and low toxicity were found for 6-tert-butyl-2-(5-chloro-2,4-dihydroxyphenyl)-4H-thieno[3,2-d][1,3]thiazin-4-one. The structure–activity elucidation reveals that the most active compounds are those with a thienothiazin-4-one and benzofuro[3,2-d][1,3]thiazin-4-one skeleton and the presence of the hydrophobic substituent (Et, Cl) in the benzenediol moiety increases their antiproliferative potency. The ADMET properties of selected compounds including metabolic stability and toxicity profile were estimated in silico.

Keywords

4H-3,1-Benzothiazin-4-ones
Heterocyclic resorcinols
SAR
Antiproliferative activity
ADMET
In silico
1

1 Introduction

Heterocyclic resorcinols are special group of compounds useful for the anticancer drug design and development. They exhibit a broad spectrum of antitumour activity and a low risk of drug resistance induction is assumed (Pratt and Toft 2003; Uehara 2003). They act as heat-shock protein 90 (Hsp90) inhibitors (Blagg and Kerr 2006; Jhaveri et al., 2012). The most important in this area are pyrazole (I) (Fig. 1) (McDonald et al., 2006; Smith et al., 2006) and isoxazole scaffold based resorcinols (Davenport et al., 2010; Hartmann et al., 2013). Luminespib (NVP-AUY922) (II) (Fig. 1). Isoxazole resorcinol derivative was entered in the phase I/II clinical trials for the patients with advanced solid tumours and multiple myeloma (Davenport et al., 2010). Recently 1,3,5-trisubstituted 1,2,3-triazole scaffold based compounds with resorcinol moiety were described as the small-molecule Hsp90 inhibitors of potent anticancer activity (Taddei et al., 2014). The most promising ganetespib (III) (Fig. 1) is developed for treating haematological cancers and multiple solid tumours (Zagouri et al., 2013). Two ring fused heterocycles benzisoxazoles (IV) (Gopalsamy et al., 2008) and 2,3-dihydrobenzoimidazol-2-ones (V) (Fig. 1) with 2,4-dihydroxyphenyl substituent are also known as compounds of potent anticancer potency (Jensen et al., 2008). X-ray crystallographic studies and SAR elucidation showed that the presence of hydrophobic substituents in position 5 of resorcinol moiety improves their anticancer potency by creating additional hydrophobic interactions in the binding site (Yang et al., 2011).

The heterocyclic benzenediols showing anticancer potency.
Figure 1
The heterocyclic benzenediols showing anticancer potency.

The research carried out by our team demonstrated antiproliferative activity of other heterocyclic resorcinols. It includes derivatives with one ring system of 1,3,4-thiadiazole (Matysiak et al., 2006) and two fused rings of 4H-3,1-benzothiazine (VI) (Fig. 1) (Niewiadomy et al., 2011), 1,3-thiazolo[5,4-b]pyridine and 1H-benzimidazole (Karpińska et al., 2011). The compounds showed high activity against the human derived cancer cells. An antiproliferative potency of some derivatives was similar to that of cisplatin. The SAR studies exhibited that the presence of a chlorine atom or Et substituents in position 5 of the resorcinol ring has a beneficial effect on the activity. Based on the promising data of preliminary studies of 2-(2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-4-one we decided to focus on other skeletons fused heterocycles to thiazinone as potential anticancer agents (Matysiak 2006).

Benzo/heterocyclo-1,3-thiazin-4-ones have been relatively weakly studied in the area of synthesis and medicinal chemistry. Besson and co-workers transformed anthranilic acid to 4-oxo-4H-3,1-benzothiazine-2-carbonitrile using 4,5-dichloro-1,2,3-dithiazolium chloride or PPh3 in CH2Cl2 (Besson et al., 1995a; Besson et al., 1995b; Besson et al., 1996). 2-Substituted derivatives were obtained from methyl 2-(3-alky/arylthioureido)benzoates, or 2-thioureidobenzamides, or 2-thioureidobenzonitriles using concentrated H2SO4 or HCl as cyclizing reagent (Gütschow et al., 1991; Neuman and Gütschow 1995). The synthesis of thieno-1,3-thiazin-4-ones was described from 2-thioureidothiophene-3-carbonitriles and the corresponding acid anhydride in the medium of concentrated sulphuric acid (Gütschow et al., 1992). In other reactions methyl 2-(3-aroylthioureido)-3-thiophenecarboxylates and ethyl 2-(3-benzylthioureido)-3-thiophenecarboxylates were applied as initial reagents (Güetschow et al., 2012; Leistner et al., 1988; Leistner et al., 1987). A two-step synthesis of the thieno[2,3-d][1,3]thiazin-4-one skeleton with 2-acyl substituent was also presented (Gütschow and Leistner 1995). Pyrazolo[3,4-d][1,3]thiazin-4-ones and pyrazolo[1,5-c][1,3,5]thiadiazine-4-one were obtained in the reaction of trichloromethylchloroformate with N-(1-alkyl/aryl-5-pyrazolyl)thiocarboxamides or N-(3-methyl-5-pyrazolyl)thiobenzamide respectively (Vicentini et al., 1994). The Lawesson reagent and 5(4)-substituted amino-4(5)-ethoxycarbonyl-1(3H)-imidazoles were used for the synthesis of imidazo[4,5-d][1,3]thiazine-7(3H)-thiones (Hara et al., 1992; Kaneko et al., 1991). 4,5-Diaryl-2,3-dihydro-2-mercaptoimidazoles in the reaction with bromopropionic acid create 6,7-diaryl imidazo[2,1-b]-1,3-thiazin-4-ones of considerable antimicrobial activity (Salama and Almotabacani 2004). The developed method allowed to obtain only a few analogues with limited reactivity of substrates.

In this paper we present the design, synthesis and biological evaluation of a series of novel 4H-3,1-benzothiazin-4-ones and of heterocyclic analogues: benzofuro-, azolo- and thienothiazin-4-ones. The activity of the compounds was additionally differentiated by the presence of a modified 2,4-dihydroxyphenyl moiety. Because physico-chemical properties and toxicity of compounds are a major reason for drug candidate failure in trials, therefore in silico ADMET properties of the obtained compounds were evaluated.

2

2 Experimental

2.1

2.1 Analytical studies

Melting points (mp) were determined using a BÜCHI B-540 (Flawil, Switzerland) melting point apparatus. The elemental analysis (C, H, N) was performed on Perkin-Elmer 2400. The IR spectra were measured with a Perkin–Elmer FT-IR 1725X spectrophotometer (in KBr) or a Varian 670-IR FT-IR spectrometer (ATR) in the range of 600–4000 cm−1. NMR spectra were recorded in DMSO-d6 using a Bruker DRX 500 instrument. Chemical shifts (δ, ppm) were described in relation to tetramethylsilane (TMS). The MS spectra (EI, 70 eV) were recorded using the apparatus AMD-604.

2.2

2.2 Synthesis of compounds

2.2.1

2.2.1 A general procedure for the synthesis of compounds 1a–9a

A mixture of the corresponding 2-amino-N-phenylbenzamide (2a, 3a–9a) or 2-amino-N-methylbenzohydrazide (1a, 3a, 4a) (1.5 mmol) and electrophile STB (1.5 mmol) in MeOH (8 mL) was treated to reflux for 3–3.5 h (Scheme 1). The hot mixture was filtered via a Büchner funnel. The filtrate was concentrated and the formed solid was filtered off (compounds 1a, 2a, 3a, 7a, 8a, 9a) or the solid formed during the synthesis was combined with that obtained after the filtrate concentration (4a). In the case of compounds 5a and 6a the reaction mixture was left at room temperature (24 h) and filtered. The compounds were crystallized from MeOH (5 mL) (compounds 1a, 2a, 4a–9a) or from MeOH/H2O (1:1, 5 mL) (3a).

Synthesis scheme of 4H-3,1-benzothiazin-4-ones (a) and 4H-3,1-benzothiazine-4-thiones (b). The appropriate STB reagents were used.
Scheme 1
Synthesis scheme of 4H-3,1-benzothiazin-4-ones (a) and 4H-3,1-benzothiazine-4-thiones (b). The appropriate STB reagents were used.

2.2.1.1
2.2.1.1 2-(2,4-Dihydroxy-3-methylphenyl)-4H-3,1-benzothiazin-4-one (1a)

Yield: 82%, mp: 224–227 °C. 1H NMR (500 MHz, DMSO-d6) δ: 13.85 (s, 1H, C(2′)-OH), 10.47 (s, 1H, C(4′)-OH), 8.11 (dd, J = 7.95 and 1.55 Hz, 1H, C(5)-H), 7.94 (td, J = 8.47 and 1.58 Hz, 1H, C(7)-H), 7.85 (dd, J = 8.11 and 1.00 Hz, 1H, C(8)-H), 7.61 (td, J = 6.85 and 1.17 Hz, 1H, C(6)-H), 7.45 (d, J = 8.87 Hz, 1H, C(6′)-H), 6.54 (d, J = 8.9 Hz, 1H, C(5′)-H), 2.04 (s, 3H, Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 182.4, 164.7, 161.6, 160.0, 146.7, 137.2, 129.6, 129.1, 126.5, 124.9, 119.8, 111.6, 110.6, 108.4, 8.5 ppm; IR (KBr): 3567 (OH), 3468 (OH), 3067 (CH), 2922 (CH), 1660 (C⚌O), 1610 (C⚌N), 1539 (C⚌C), 1501 (C⚌C), 1470, 1369, 1311, 1286, 1250 (C—OH), 1154, 1111, 1075, 1058, 963, 923, 880, 791, 774, 707 cm−1; EI-MS m/z (%): 285 (M+, 100), 225 (44), 202 (7), 196 (9), 170 (3), 167 (2), 122 (8), 102 (2), 94 (6), 77 (6), 65 (4), 50 (3), 39 (4). Anal. Calcd for C15H11NO3S (285.05): C, 63.14; H, 3.89; N, 4.91; Found: C, 63.10; H, 3.88; N, 4.95.

2.2.1.2
2.2.1.2 2-(2,4-Dihydroxy-5-methylphenyl)-4H-3,1-benzothiazin-4-one (2a)

Yield: 83%, mp: 225–228 °C. 1H NMR (500 MHz, DMSO-d6) δ: 13.85 (s, 1H, C(2′)-OH), 10.49 (s, 1H, C(4′)-OH), 8.12 (dd, J = 7.93 and 1.37 Hz, 1H, C(5)-H), 7.94 (td, J = 6.99 and 1.55 Hz, 1H, C(7)-H), 7.85 (d, J = 8.07 Hz, 1H, C(8)-H), 7.61 (td, J = 7.83 and 1.05 Hz, 1H, C(6)-H), 7.46 (s, 1H, C(6′)-H), 6.56 (s, 1H, C(3′)-H), 2.04 (s, 3H, Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 181.8, 164.1, 161.1, 159.5, 146.2, 136.5, 129.0, 128.4, 125.8, 124.3, 119.2, 111.0, 110.1, 107.9, 8.0 ppm; IR (KBr): 3477 (OH), 3082 (OH), 2922 (CH), 2854 (CH), 1661 (C⚌O), 1611 (C⚌N), 1540 (C⚌C), 1501 (C⚌C), 1471, 1449, 1374, 1309, 1284, 1250 (C—OH), 965, 923, 789, 775, 706 cm−1; MS (EI) m/z (%): 285 (M+, 100), 271 (2), 239 (2), 225 (51), 202 (10), 196 (14), 168 (54), 151 (2), 148 (2), 122 (4), 94 (8), 65 (3), 39 (2). Anal. calc. for C15H11NO3S (285.05): C, 63.14; H, 3.89; N, 4.91; Found: C, 63.23; H, 3.88; N, 4.86.

2.2.1.3
2.2.1.3 2-(5-Ethyl-2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-4-one (3a)

Yield: 83%, mp: 245–247 °C. 1H NMR (500 MHz, DMSO-d6) δ: 10.50 (s, 1H, C(2′)-OH), 10.12 (s, 1H, C(4′)-OH), 7.85 (dd, J = 7.87 and 1.5 Hz, 1H, C(5)-H), 7.57 (m, 1H, C(7)-H), 7.43 (s, 1H, C(6′)-H), 7.31 (d, J = 7.82 Hz, 1H, C(8)-H), 7.22 (t, J = 7.57 Hz, 1H, C(6)-H), 6.61 (s, 1H, C(3′)-H), 2.50 (q, J = 7.5 Hz, 2H, CH2Me), 1.16 (t, J = 7.5 Hz, 3H, CH2Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 182.0, 163.5, 161.3, 159.5, 146.5, 136.5, 129.2, 128.3, 127.4, 124.2, 123.3, 119.1, 110.3, 112.8, 22.0, 14.0 ppm; IR (KBr): 3421 (OH), 3078 (OH), 2963 (CH), 2934 (CH), 2871 (CH), 1655 (C⚌O), 1618 (C⚌N), 1553 (C⚌C), 1488, 1469, 1446, 1433, 1407, 1343, 1296, 1264, 1245 (C—OH), 1204, 1186, 1161, 1123, 1101, 1061, 1041, 1026, 965, 942, 917, 893, 867, 842, 788, 760, 718 cm−1; EI-MS m/z (%): 299 (M+, 5), 283 (M+-Me, 8), 268 (100), 253 (3), 239 (7), 224 (18), 211 (4), 198 (11), 183 (4), 168 (3), 148 (7), 120 (7), 104 (2), 92 (5), 77 (7), 65 (5), 51 (4), 39 (3). Anal. Calcd for C16H13NO3S (299.34): C, 64.20; H, 4.38; N, 4.68; Found: C, 64.28; H, 4.36; N, 4.62.

2.2.1.4
2.2.1.4 2-(5-Chloro-2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-4-one (4a)

Yield: 83 %, mp: 272–274 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.81 (s, 1H, C(2′)-OH), 11.50 (s, 1H, C(4′)-OH), 8.15 (dd, 1H, J = 6.55 and 1.3 Hz, C(5)-H), 7.96 (m, 1H, C(7)-H), 7.92 (d, J = 7.15 Hz, 1H, C(8)-H), 7.77 (s, 1H, C(6′)-H), 7.67 (m, 1H, C(6)-H), 6.73 (s, 1H, C(3′)-H) ppm; IR (KBr): 3349 (OH), 2946 (CH), 2834 (CH), 1658 (C⚌C), 1641 (C⚌N), 1598 (C⚌C), 1531 (C⚌C), 1461, 1448, 1413, 1255 (C—OH), 1197, 1113, 1023, 778 cm−1; EI-MS m/z (%): 305 (M+, 100), 278 (5), 247 (42), 214 (9), 187 (15), 153 (11), 109 (6), 77 (4), 65 (8), 39 (6), 33 (14). Anal. Calcd for C14H8ClNO3S (305.74): C, 55.00; H, 2.64; N, 4.58; Found: C, 54.91; H, 2.66; N, 4.62.

2.2.1.5
2.2.1.5 2-(2,4-Dihydroxyphenyl)-6-fluoro-4H-3,1-benzothiazin-4-one (5a)

Yield: 71%, mp: 288–289 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.92 (s, 1H, C(2′)-OH), 10.56 (s, 1H, C(4′)-OH), 8.00 (m, 1H, C(5)-H), 7.87 (m, 2H, C(7,8)-H), 7.66 (d, J = 8.85 and 2.34 Hz, 1H, C(6′)-H), 6.45 (dd, J = 8.85 and 2.34 Hz, 1H, C(5′)-H), 6.38 (d, J = 2.32 Hz, 1H, C(3′)-H) ppm; IR (KBr): 3406 (OH), 2946 (CH), 1667 (C⚌O), 1628 (C⚌N), 1552 (C⚌C), 1514 (C⚌C), 1483, 1447, 1362, 1329, 1275, 1238 (C—OH), 1219, 1187, 1140, 1106, 988, 944, 856, 738 cm−1; EI-MS m/z (%): 289 (M+, 100), 229 (49), 201 (13), 172 (10), 108 (19), 94 (4), 69 (4). Anal. Calcd for C14H8FNO3S (289.28): C, 58.13; H, 2.79; N, 4.84; Found: C, 58.23; H, 2.75; N, 4.92.

2.2.1.6
2.2.1.6 2-(2,4-Dihydroxyphenyl)-5-fluoro-4H-3,1-benzothiazin-4-one (6a)

Yield: 71%, mp: 285–286 °C. 1H NMR (500 MHz, DMSO-d6) δ: 13.07 (s, 1H, C(2′)-OH), 10.62 (s, 1H, C(4′)-OH), 7.93 (m, 1H, C(7)-H), 7.71 (m, 1H, C(8)-H), 7.69 (d, J = 8.12 Hz, 1H, C(6)-H), 7.64 (d, J = 8.86 Hz, 1H, C(6′)-H), 6.48 (dd, J = 8.87 and 2.37 Hz, 1H, C(5′)-H), 6.38 (d, J = 2.38 Hz, 1H, C(3′)-H) ppm; IR (KBr): 3412 (OH), 2928 (CH), 1674 (C⚌O), 1633 (C⚌N), 1548 (C⚌C), 1526 (C⚌C), 1509 (C⚌C), 1492, 1450, 1369, 1341, 1282, 1267, 1244 (C—OH), 1208, 1176, 1137, 1125, 993, 950, 863, 742, 709 cm−1; EI-MS m/z (%): 289 (M+, 100), 261 (5), 229 (33), 204 (7), 201 (17), 172 (15), 108 (30), 94 (6), 80 (5), 69 (7), 52 (4), 39 (4). Anal. Calcd for C14H8FNO3S (289.28): C, 58.13; H, 2.79; N, 4.84; Found: C, 58.08; H, 3.00; N, 4.79.

2.2.1.7
2.2.1.7 6-Chloro-2-(2,4-dihydroxy-3-methylphenyl)-4H-3,1-benzothiazin-4-one (7a)

Yield: 88%, mp: 233–234 °C. 1H NMR (500 MHz, DMSO-d6) δ: 13.61 (s, 1H, C(2′)-OH), 10.53 (s, 1H, C(4′)-OH), 8.05 (d, J = 2.41 Hz, 1H, C(5)-H), 8.00 – 7.93 (m, 2H, C(7,8)-H), 7.48 (d, J = 8.9 Hz, 1H, C(6′)-H), 6.51 (d, J = 8.48 Hz, 1H, C(5′)-H), 2.04 (s, 3H, Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 181.0, 165.1, 160.4, 159.9, 146.9, 135.0, 130.4, 127.6, 126.0, 123.3, 122.4, 114.6, 110.3, 106.7, 8.13 ppm; IR (KBr): 3386 (OH), 2955 (CH), 2861 (CH), 1653 (C⚌O), 1618 (C⚌N), 1556 (C⚌C), 1519 (C⚌C), 1462, 1408, 1361, 1324, 1278, 1236 (C—OH), 1221, 1207, 1190, 1137, 1062, 1045, 978, 890, 875, 840, 821, 794, 776, 751, 728 cm−1; EI-MS m/z (%): 319 (M+, 100), 261 (21), 259 (53), 236 (6), 230 (10), 122 (6), 94 (12), 75 (3). Anal. Calcd for C15H10ClNO3S (319.76): C, 56.34; H, 3.15; N, 4.38; Found: C, 56.43; H, 3.15; N, 4.46.

2.2.1.8
2.2.1.8 6-Chloro-2-(5-ethyl-2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-4-one (8a)

Yield: 88%, mp: 254–255 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.79 (s, 1H, C(2′)-OH), 10.57 (s, 1H, C(4′)-OH), 8.05 (d, J = 2.50 Hz, 1H, C(5)-H), 7.96 (dd, J = 8.70 and 2.52 Hz, 1H, C(7)-H), 7.89 (d, J = 8.74 Hz, 1H, C(8)-H), 7.46 (s, 1H, C(6′)-H), 6.43 (s, 1H, C(3′)-H), 2.49 (q, J = 7.45 Hz, 2H, CH2Me), 1.13 (t, J = 7.47 Hz, 3H, CH2Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 182.1, 164.3, 162.0, 159.8, 146.0, 136.7, 132.9, 132.1, 128.3, 124.1, 123.8, 120.6, 111.1, 103.3, 22.5, 14.6 ppm; IR (KBr): 3397 (OH), 2967 (CH), 2934 (CH), 2875 (CH), 1642 (C⚌O), 1624 (C⚌N), 1558 (C⚌C), 1526 (C⚌C), 1467, 1402, 1345, 1318, 1283, 1248 (C—OH), 1220, 1205, 1183, 1138, 1059, 977, 939, 888, 876, 831, 813, 798, 770, 742, 721 cm−1; EI-MS m/z (%): 333 (M+, 100), 300 (M+-Me, 74), 273 (5), 258 (33), 167 (3), 148 (7), 69 (4). Anal. Calcd for C16H12ClNO3S (333.68): C, 57.57; H, 3.62; N, 4.20; Found: C, 57.66; H, 3.58; N, 4.26.

2.2.1.9
2.2.1.9 6-Chloro-2-(5-chloro-2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-4-one (9a)

Yield: 86%, mp: 307–309 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.46 (s, 1H, C(2′)-OH), 11.36 (s, 1H, C(4′)-OH), 8.07 (d, J = 2.31 Hz, 1H, C(5)-H), 8.02 (m, 2H, C(7,8)-H), 7.81 (s, 1H, C(6′)-H), 6.63 (s, 1H, C(3′)-H) ppm; IR (KBr): 3453 (OH), 3099 (OH), 2921 (CH), 2852 (CH), 1679 (C⚌O), 1608 (C⚌N), 1530 (C⚌C), 1475, 1413, 1259, 1194, 1026, 835, 740 cm−1; EI-MS m/z (%): 339 (M+, 100), 305 (5), 283 (10), 279 (64), 251 (11), 216 (9), 188 (10), 169 (7), 142 (23), 114 (5), 75 (10), 40 (6). Anal. Calcd for C14H7Cl2NO3S (340.18): C, 49.43; H, 2.06; N, 4.12; Found: C, 49.51; H, 2.05; N, 4.18.

2.2.2

2.2.2 A general procedure for the synthesis of compounds 1b, 2b

A mixture of 2-amino-N-phenylbenzothioamide (1.5 mmol) and the corresponding electrophile STB (1.5 mmol) in MeOH (8 mL) was treated to reflux for 3.5 h (Scheme 1). The hot mixture was filtered via a Büchner funnel and the filtrate was concentrated. The compounds were crystallized from MeOH (5 mL).

2.2.2.1
2.2.2.1 2-(2,4-Dihydroxyphenyl)-4H-3,1-benzothiazine-4-thione (1b)

Yield: 71%, mp: 195–196 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.76 (s, 1H, C(2′)-OH), 10.54 (s, 1H, C(4′)-OH), 8.61 (dd, J = 8.2 and 1.2 Hz, 1H, C(8)-H), 7.97 (td, J = 8.21 and 1.4 Hz, 1H, C(7)-H), 7.82 (d, J = 8.09 Hz, 1H, C(5)-H), 7.75 (d, J = 8.86 Hz, 1H, C(6′)-H), 7.62 (td, J = 7.09 and 1.16 Hz, 1H, C(6)-H), 6.47 (dd, J = 8.88 and 2.37 Hz, 1H, C(5′)-H), 6.39 (d, J = 2.4 Hz, 1H, C(3′)-H) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 210.2, 164.3, 163.8, 161.5, 140.6, 137.6, 130.5, 129.9, 129.6, 126.8, 126.0, 110.6, 109.4, 103.7 ppm; IR (KBr): 3413 (OH), 1625 (C⚌N), 1595 (C⚌N), 1561 (C⚌C), 1502 (C⚌C), 1459, 1439, 1345, 1325, 1242 (C—OH), 1196, 1160, 1124, 1017 (C⚌S), 982, 937, 828, 762 cm−1; EI-MS m/z (%): 287 (M+, 100), 254 (11), 243 (3), 223 (6), 218 (24), 211 (18), 186 (7), 183 (14), 154 (14), 152 (9), 120 (6), 108 (29), 77 (11), 69 (10), 50 (7), 45 (5), 39 (6). Anal. Calcd for C14H9NO2S2 (287.36): C, 58.52; H, 3.16; N, 4.87; Found: C, 58.62; H, 3.15; N, 4.91.

2.2.2.2
2.2.2.2 2-(5-Ethyl-2,4-dihydroxyphenyl)-4H-3,1-benzothiazine-4-thione (2b)

Yield: 71%, mp: 220–221 °C. 1H NMR (500 MHz, DMSO-d6) δ: 10.58 (s, 1H, C(2′)-OH), 10.04 (s, 1H, C(4′)-OH), 8.61 (dd, J = 8.2 and 1.38 Hz, 1H, C(8)-H), 7.98 (m, 1H, C(7)-H), 7.82 (d, J = 7.7 Hz, 1H, C(5)-H), 7.61 (m, 1H, C(6)-H), 7.58 (s, 1H, C(6′)-H), 6.46 (s, 1H, C(3′)-H), 2.35 (m, 2H, CH2Me), 1.14 (t, J = 7.47 Hz, 3H, CH2Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 210.0, 163.79, 161.43, 159.21, 140.32, 137.10, 129.94, 128.97, 127.51, 126.28, 123.55, 109.60, 102.91, 22.04, 14.31, 14.12 ppm; IR (KBr): 3201, 3173, 2963, 1616 (C⚌N), 1571 (C⚌C), 1521 (C⚌C), 1459, 1418, 1381, 1322, 1244 (C—OH), 1208, 1140, 1015 (C⚌S), 937, 874, 762 cm−1; EI-MS m/z (%): 315 (M+, 77), 300 ([M-Me]+, 97), 296 (45), 281 (100), 267 (5), 251 (15), 239 (6), 224 (41), 180 (5), 162 (10), 148 (14), 119 (7), 108 (7), 91 (5), 77 (15), 69 (14), 65 (9), 40 (30), 36 (8). Anal. Calcd for C16H13NO2S2 (315.41): C, 60.93; H, 4.15; N, 4.44; Found: C, 61.02; H, 4.14; N, 4.47.

2.2.3

2.2.3 A general procedure for the synthesis of compounds 1c–4c

A mixture of the corresponding aminothiophenecarboxamide (1.8 mmol) and electrophile STB (1.8 mmol) in MeOH (8 mL) (Scheme 2) was treated to reflux for 2.5–3 h. The hot mixture was filtered via a Büchner funnel. The compounds were crystallized from MeOH (5 mL).

2.2.3.1
2.2.3.1 2-(5-Chloro-2,4-dihydroxyphenyl)-4H-thieno[3,2-d][1,3]thiazin-4-one (1c)

Yield: 81%, mp: 293–294 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.11 (s, 1H, C(2′)-OH), 11.25 (s, 1H, C(4′)-OH), 8.43 (d, J = 5.21 Hz, 1H, C(6)-H), 7.84 (s, 1H, C(6′)-H), 7.73 (d, J = 5.24 Hz, 1H, C(7)-H), 6.62 (s, 1H, C(3′)-H) ppm; IR (KBr): 3332 (OH), 3089 (OH), 2889 (CH), 1659 (C⚌O), 1620 (C⚌N), 1511 (C⚌C), 1490, 1429, 1392, 1351, 1243, 1221 (C—OH), 1196, 1090, 1039, 1026, 959, 832, 767, 747, 705 cm−1; EI-MS m/z (%): 311 (M+, 100), 85 (5), 283 (12), 276 (21), 251 (29), 234 (6), 188 (6), 160 (9), 142 (16), 114 (9), 110 (7), 82 (6), 69 (19), 45 (6), 39 (4). Anal. Calcd for C12H6ClNO3S2 (311.76): C, 46.23; H, 1.94; N, 4.49; Found: C, 46.18; H, 1.94; N, 4.56.

2.2.3.2
2.2.3.2 6-tert-Butyl-2-(5-chloro-2,4-dihydroxyphenyl)-4H-thieno[3,2-d][1,3]thiazin-4-one (2c)

Yield: 78%, mp: 272–274 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.18 (s, 1H, C(2′)-OH), 11.29 (s, 1H, C(4′)-OH), 7.82 (s, 1H, C(3′)-H), 7.61 (s, 1H, C(7)-H), 6.61 (s, 1H, C(6′)-H), 1.43 (s, 9H, Me) ppm; 13C NMR (125 MHz, DMSO-d6, δ): 173.9, 167.6, 166.6, 158.4, 158.0, 156.0, 129.0, 123.7, 117.6, 112.9, 112.2, 104.3, 35.3, 31.3 (3C) ppm; IR (KBr): 3385 (OH), 2965 (CH), 2830 (CH), 1641 (C⚌O), 1613 (C⚌N), 1595 (C⚌C), 1519 (C⚌C), 1486, 1450, 1391, 1366, 1247 (C—OH), 1208, 1174, 1096, 1031, 916, 835, 811, 751, 714 cm−1; EI-MS m/z (%): 367 (M+, 100), 332 (28), 324 (43), 316 (17), 292 (11), 197 (8), 155 (4), 148 (3), 123 (3), 77 (2), 69 (12), 41 (6). Anal. Calcd for C16H14ClNO3S2 (367.87): C, 52.24; H, 3.81; N, 3.82; Found: C, 52.34; H, 3.79; N, 3.83.

2.2.3.3
2.2.3.3 2-(5-Ethyl-2,4-dihydroxyphenyl)-5-methyl-4H-thieno[2,3-d][1,3]thiazin-4-one (3c)

Yield: 87%, mp: 272–273 °C. 1H NMR (500 MHz, DMSO-d6) δ: 11.65 (s, 1H, C(2′)-OH), 10.49 (s, 1H, C(4′)-OH), 7.53 (s, 1H, C(6)-H), 7.32 (s, 1H, C(6′)-H), 6.44 (s, 1H, C(3′)-H), 2.48 (s, 2H, CH2Me), 2.46 (s, 3H, Me), 1.13 (t, J = 7.46 Hz, 3H, CH2Me) ppm; IR (KBr): 3418 (OH), 2959 (CH), 1626 (C⚌O), 1531(C⚌N), 1510 (C⚌C), 1498 (C⚌C), 1460, 1391, 1321, 1276, 1237 (C—OH), 1211, 1184, 1126, 1014, 977, 903, 874, 852, 784, 768 cm−1; 13C NMR (125 MHz, DMSO-d6, δ): 171.1, 167.1, 165.4, 161.7, 158.7, 134.2, 128.7, 124.0, 119.5, 119.2, 111.9, 103.2, 22.5, 17.6, 14.6 ppm; EI-MS m/z (%): 319 (M+, 100), 305 (M+-Me, 48), 261 (17), 256 (24), 244 (10), 181 (10), 165 (13), 128 (8), 69 (5), 64 (17). Anal. Calcd for C15H13NO3S2 (319.40): C, 56.41; H, 4.10; N, 4.45; Found: C, 54.97; H, 3.63; N, 4.65.

2.2.3.4
2.2.3.4 2-(5-Chloro-2,4-dihydroxyphenyl)-5-methyl-4H-thieno[2,3-d][1,3]thiazin-4-one (4c)

Yield: 83%, mp: 273–275 °C. 1H NMR (500 MHz, DMSO-d6) δ: 11.52 (s, 1H, C(2′)-OH), 11.18 (s, 1H, C(4′)-OH), 7.86 (s, 1H, C(6′)-H), 7.38 (d, J = 1.16 Hz, 1H, C(6)-H), 6.62 (s, 1H, C(3′)-H), 2.47 (s, 3H, Me) ppm; IR (KBr, ν, cm−1): 3399 (OH), 3320 (OH), 3149 (OH), 2830 (CH), 1618 (C⚌O), 1532 (C⚌N), 1500 (C⚌C), 1441 (C⚌C), 1386, 1346, 1232 (C—OH), 1208, 1179, 1059, 1032, 1018, 871, 848, 780, 757; 13C NMR (125 MHz, DMSO-d6, δ): 186.5, 167.4, 157.4, 154.4, 145.3, 135.3, 132.4, 123.7, 118.5, 114.4, 112.0, 103.8, 16.7 ppm; EI-MS m/z (%): 325 (M+, 100), 292 (19), 267 (30), 189 (10), 187 (25), 173 (15), 171 (43), 155 (14), 139 (10), 131 (6), 95 (6), 69 (10), 45 (8). Anal. Calcd for C13H8ClNO3S2 (325.79): C, 47.93; H, 2.48; N, 4.30; Found: C, 48.02; H, 2.44; N, 4.36.

2.2.4

2.2.4 A general procedure for the synthesis of compounds 1d, 1e–4e

A mixture of 4-amino-1H-imidazole-5-carboxamide (1d) or the corresponding aminopyrazolecarboxamide (1e–4e) and electrophile STB (1.5 mmol) in MeOH (8 mL) (Scheme 3) was treated to reflux for 2.5–3 h. The hot mixture was filtered via a Büchner funnel. The filtrate was concentrated and the formed solid was filtered off (compounds 1d, 1e, 3e, 4e) or the reaction mixture was left at room temperature (24 h) and filtered (2e). The compounds were crystallized from MeOH (5 mL).

2.2.4.1
2.2.4.1 5-(2,4-Dihydroxyphenyl)imidazo[4,5-d][1,3]thiazin-7(1H)-one (1d)

Yield: 81%, mp: 334–335 °C. 1H NMR (500 MHz, DMSO-d6) δ: 13.44 (s, 1H, NH), 11.74 (s, 1H, C(2′)-OH), 10.23 (s, 1H, C(4′)-OH), 8.23 (d, J = 9.1 Hz, 1H, C(6′)-H), 7.48 (s, 1H, C(2)-H), 6.39 (m, 2H, C(3′,5′-H) ppm; IR (KBr): 3478 (OH), 3372 (OH), 3127 (OH), 1643 (C⚌O), 1585 (C⚌N), 1500 (C⚌C), 1459, 1390, 1353, 1272 (C—OH), 1214, 1115, 1058, 989, 965, 940, 826, 801, 779, 723 cm−1; 13C NMR (125 MHz, DMSO-d6) δ: 165.4, 163.0, 156.7, 143.5, 136.5, 134.0, 130.6, 120.7, 117.0, 108.8, 102.6 ppm; EI-MS m/z (%): 261 (M+, 100), 244 (18), 2334 (9), 228 (7), 203 (9), 172 (7), 153 (100), 143 (9), 137 (22), 126 (9), 120 (11), 110 (14), 97 (11), 81 (7), 69 (10), 53 (7), 44 (8), 39 (5). Anal. Calcd for C11H7N3O3S (261.18): C, 50.58; H, 2.68; N, 16.08; Found: C, 50.64; H, 2.71; N, 16.02.

2.2.4.2
2.2.4.2 6-(2,4-Dihydroxyphenyl)pyrazolo[3,4-d][1,3]thiazin-4(2H)-one (1e)

Yield: 72%, mp: 271–272° C. 1H NMR (500 MHz, DMSO-d6) δ: 14.26 (s, 1H, NH), 12.11 (s, 1H, C(2′)-OH), 10.57 (s, 1H, C(4′)-OH), 8.18 (s, 1H, C(3)-H), 7.70 (d, J = 8.58 Hz, 1H, C(6′)-H), 7.63 (d, J = 8.56 Hz, 1H, C(5′)-H), 6.47 (d, J = 8.36 Hz, 1H, C(3′)-H) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 165.3, 165.2, 163.9, 161.1, 149.8, 134.0, 130.8, 111.6, 109.5, 107.7, 101.2 ppm; IR (KBr): 3443 (OH), 3182 (OH), 3112 (OH), 2923 (CH), 2851 (CH), 1657 (C⚌O), 1573 (C⚌N), 1531 (C⚌C), 1481, 1331, 1248 (C—OH), 1198, 1168, 1136, 1076, 983, 936, 888, 848, 778, 754, 706 cm−1; EI-MS m/z (%): 261 (M+, 100), 244 (5), 233 (6), 203 (10), 192 (25), 153 (11), 137 (19), 125 (6), 108 (8), 97 (5), 80 (3), 69 (8), 52 (6), 45 (3), 39 (7). Anal. Calcd for C11H7N3O3S (261.26): C, 50.57; H, 2.70; N, 16.08; Found: C, 50.65; H, 2.66; N, 15.99.

2.2.4.3
2.2.4.3 6-(2,4-Dihydroxyphenyl)-1-phenylpyrazolo[3,4-d][1,3]thiazin-4(1H)-one (2e)

Yield: 81%, mp: 300–303° C. 1H NMR (500 MHz, DMSO-d6) δ: 11.79 (s, 1H, C(2′)-OH), 10.64 (s, 1H, C(4′)-OH), 8.47 (s, 1H, C(3)-H), 7.85 (m, 2H, C(Ar)-H), 7.75 (d, J = 8.89 Hz, 1H, C(6′)-H), 7.65 (t, J = 7.6 Hz, 2H, C(Ar)-H), 7.55 (t, J = 7.40 Hz, 1H, C(Ar)-H), 6.47 (dd, J = 8.82 and 2.25 Hz, 1H, C(5′)-H), 6.36 (d, J = 2.24 Hz, 1H, C(3′)-H) ppm; IR (KBr): 3420 (OH), 3315 (OH), 2638 (CH), 2822 (CH), 1641 (C⚌O), 1628 (C⚌N), 1572 (C⚌N), 1523 (C⚌C), 1511 (C⚌C), 1487, 1440, 1365, 1269, 1236 (C—OH), 1193, 1168, 1147, 1118, 1038, 966, 934, 920, 895, 847, 786, 760, 739, 711 cm−1; EI-MS m/z (%): 337 (M+, 100), 304 (6), 279 (13), 268 (31), 201 (21), 172 (5), 153 (12), 137 (21), 103 (5), 91 (7), 77 (20), 69 (5), 51 (10), 44 (24), 40 (62). Anal. Calcd for C17H11N3O3S (337.35): C, 60.52; H, 3.29; N, 12.45; Found: C, 60.62; H, 3.23; N, 12.51.

2.2.4.4
2.2.4.4 6-(5-Ethyl-2,4-dihydroxyphenyl)-1-phenylpyrazolo[3,4-d][1,3]thiazin-4(1H)-one (3e)

Yield: 71%, mp: 217–219° C. 1H NMR (500 MHz, DMSO-d6,) δ: 11.63 (s, 1H, C(2′)-OH), 10.65 (s, 1H, C(4′)-OH), 8.45 (s, 1H, C(3)-H), 7.85 (m, 2H, C(Ar)-H), 7.65 (m, 3H, C(Ar)-H), 7.55 (m, 1H, C(Ar)-H), 6.64 (s, 1H, C(3′)-H), 2.48 (q, 2H, CH2Me), 1.13 (t, 3H, J = 7.48 Hz, CH2Me) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 176.7, 166.6, 160.8, 160.0, 149.7, 139.4, 138.7, 129.8 (2C), 127.5, 124.7, 123.5 (2C), 115.6, 112.5, 104.9, 98.0, 22.3, 14.3 ppm; IR (KBr): 3251 (OH), 2954 (CH), 1661 (C⚌O), 1629 (C⚌N), 1596 (C⚌N), 1520 (C⚌C), 1391, 1334, 1279, 1243 (C—OH), 1214, 1128, 1032, 944, 890, 871, 838, 806, 774, 756, 728 cm−1; EI-MS m/z (%): 365 (M+, 100), 350 (M-Me, 70), 308 (3), 296 (15), 290 (2), 203 (3), 165 (5), 149 (2), 103 (2), 91 (3), 77 (12), 51 (3). Anal. Calcd for C19H15N3O3S (365.41): 62.45; H, 4.14; N, 11.50; Found: C, 62.55; H, 4.08; N, 11.57.

2.2.4.5
2.2.4.5 6-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylpyrazolo[3,4-d][1,3]thiazin-4(1H)-one (4e)

Yield: 87%, mp: 308–309 °C. 1H NMR (500 MHz, DMSO-d6) δ: 11.68 (s, 1H, C(2′)-OH), 11.60 (s, 1H, C(4′)-OH), 7.92 (s, 1H, C(3)-H), 7.91 (s, 1H, C(6′)-H), 7.57 (m, 4H, C(Ar)-H), 7.38 (t, J = 7.2 Hz, 1H, C(6)-H, 6.64 (s, 1H, C(3′)-H) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 176.7, 169.7, 159.0, 158.8, 152.2, 138.0, 135.9, 130.3, 129.8 (2C), 129.0, 124.6 (2C), 114.3, 112.9, 105.4, 104.6 ppm; IR (KBr): 3439 (OH), 3324 (OH), 3138 (OH), 2960 (CH), 2835 (CH), 1635 (C⚌O), 1612 (C⚌N), 1567 (C⚌N), 1550 (C⚌C), 1514 (C⚌C), 1501 (C⚌C), 1462, 1438, 1339, 1254, 1220 (C—OH), 1181, 1152, 1104, 1021, 953, 927, 891, 845, 779, 753, 731 cm−1; EI-MS m/z (%): 371 (M+, 100), 336 (19), 313 (4), 294 (8), 268 (3), 201 (10), 187 (8), 171 (13), 147 (4), 103 (4), 91 (5), 77 (20), 51 (7). Anal. Calcd for C17H10ClN3O3S (371.80): C, 54.92; H, 2.71; N, 11.30; Found: C, 55.02; H, 2.71; N, 11.23.

2.2.5

2.2.5 A general procedure for the synthesis of compounds 1f-3f

A mixture of 3-aminobenzofuran-2-carboxamide (1.4 mmol) and the corresponding electrophile STB (1.4 mmol) in MeOH (4 mL) (Scheme 4) was treated to reflux for 2.5–3.5 h. The hot mixture was filtered via a Büchner funnel and the solid formed during the synthesis was combined with that obtained after the filtrate concentration (1e). In the case of compounds 2e and 3e the reaction mixture was left at room temperature (24 h) and filtered. The compounds were crystallized from MeOH (4 mL) (compounds 1e, 2e) or from MeOH/H2O (1:1, 4 mL) (3e).

2.2.5.1
2.2.5.1 2-(2,4-Dihydroxyphenyl)-4H-benzofuro[3,2-d][1,3]thiazin-4-one (1f)

Yield: 73%, mp: 279–281 °C. 1H NMR (500 MHz, DMSO-d6) δ: 11.62 (s, 1H, C(2′)-OH), 10.23 (s, 1H, C(4′)-OH), 8.20 (d, J = 9.44 Hz, 1H, C(Ar)-H), 7.82 (m, 2H, C(Ar)-H), 7.65 (m, 2H, C(Ar)-H), 7.48 (m, 1H, C(Ar)-H, 6.40 (m, 2H, C(3′,5′)-H) ppm; 13C NMR (125 MHz, DMSO-d6) δ: 170.3, 169.4, 163.5, 160.3, 160.2, 155.0, 143.4, 134.0, 131.5, 130.3, 124.8, 122.4, 121.6, 112.9, 111.8, 109.2, 103.1 ppm; IR (KBr): 3444 (OH), 3334 (OH), 3167 (OH), 1653 (C⚌O), 1631 (C⚌N), 1593 (C⚌N), 1578 (C⚌C), 1493, 1469, 1428, 1370, 1349, 1331, 1283, 1234 (C—OH), 1193, 1128, 987, 948, 915, 849, 791, 744 cm−1; EI-MS m/z (%): 371 (M+, 100), 296 (4), 283 (9), 251 (10), 242 (18), 222 (10), 195 (6), 176 (5), 153 (4), 141 (6), 137 (6), 120 (25), 103 (5), 88 (6), 76 (5), 62 (5). 51 (4), 39 (7). Anal. Calcd for C17H10ClN3O3S (371.80): C, 61.73; H, 2.91; N, 4.50; Found: C, 61.80; H, 2.89; N, 4.56.

2.2.5.2
2.2.5.2 2-(5-Ethyl-2,4-dihydroxyphenyl)-4H-benzofuro[3,2-d][1,3]thiazin-4-one (2f)

Yield: 84%, mp: 286–287 °C. 1H NMR (500 MHz, DMSO-d6) δ: 12.13 (s, 1H, C(2′)-OH), 10.59 (s, 1H, C(4′)-OH), 8.16 (d, J = 7.8 Hz, 1H, C(9)-H), 8.10 (s, 1H, C(6′)-H), 7.88 (d, J = 8.48 Hz, 1H, C(6)-H), 7.79 (td, J = 7.23 and 1.16 Hz, 1H, C(8)-H), 7.58 (t, J = 7.27 Hz, 1H, C(7)-H), 6.49 (s, 1H, C(3′)-H), 2.54 (q, J = 7.5 Hz, 2H, CH2Me), 1.15 (t, J = 7.5 Hz, 3H, CH2Me) ppm; IR (KBr): 3295 (OH), 3206 (OH), 1662 (C⚌O), 1640 (C⚌N), 1588 (C⚌N), 1530 (C⚌C), 1502 (C⚌C), 1487, 1470, 1441, 1381, 1350, 1329, 1264, 1228 (C—OH), 1187, 1135, 996, 938, 867, 851, 735 cm−1; EI-MS m/z (%): 339 (M+, 100), 324 (M+ -Me, 80), 296 (12), 270 (23), 264 (23), 165 (5), 145 (5), 120 (10), 69 (10). Anal. Calcd for C18H13NO4S (339.37): C, 63.71; H, 3.86; N, 4.13; Found: C, 63.46; H, 3.84; N, 4.15.

2.2.5.3
2.2.5.3 2-(5-Chloro-2,4-dihydroxyphenyl)-4H-benzofuro[3,2-d][1,3]thiazin-4-one (3f)

Yield: 87%, mp: 291–293 °C. 1H NMR (500 MHz, DMSO-d6) δ: 11.83 (s, 1H, C(2′)-OH), 11.27 (s, 1H, C(4′)-OH), 8.23 (d, J = 7.59 Hz, 1H, C(9)-H), 8.04 (s, 1H, C(6′)-H), 7.88 (d, J = 8.48 Hz, 1H, C(6)-H), 7.79 (td, J = 7.25 and 1.21 Hz, 1H, C(8)-H), 7.58 (t, J = 7.23 Hz, 1H, C(7)-H), 6.67 (s, 1H, C(3′)-H) ppm; IR (KBr): 3228 (OH), 3257 (OH), 1661 (C⚌O), 1628 (C⚌N), 1583 (C⚌N), 1557 (C⚌C), 1481, 1470, 1419, 1367, 1322, 1273, 1229 (C—OH), 1198, 1125, 990, 953, 907, 853, 786, 732 cm−1; EI-MS m/z (%): 345 (M+, 100), 317 (14), 310 (25), 301 (9), 285 (12), 256 (10), 229 (5), 175 (9), 142 (6), 120 (24), 88 (8), 69 (9), 51 (4). Anal. Calcd for C16H8ClNO4S (345.76): C, 55.58; H, 2.33; N, 4.05; Found: C, 55.66; H, 2.34; N, 4.03.

2.3

2.3 Antiproliferative assay in vitro

There were applied the following human cell lines established in vitro: T47D (breast cancer), SW707 (rectal adenocarcinoma), A549 (nonsmall cell lung carcinoma) from the American Type Culture Collection (Rockville, Maryland, U.S.A.) and HCV29T (bladder cancer) from the Fibiger Institute, Copenhagen, Denmark. Twenty-four hours before the addition of the tested agents, the cells were plated in 96-well plates (Sarstedt Inc, Newton, NC, USA) at a density of 104 cells/well. All cell lines were maintained in the opti-MEM medium supplement with 2 mM glutamine (Gibco, Warsaw, Poland), streptomycin (50 μg/mL), penicillin (50 U/mL) (Polfa, Tarchomin, Poland) and 5% foetal calf serum (Gibco, Grand Island, USA). The cells were incubated at 37 °C in the humid atmosphere saturated with 5% CO2. The solutions of compounds (l mg/mL) were prepared ex tempore by dissolving the substance in 100 μL of DMSO completed with 900 μL of tissue culture medium. Afterwards, the compounds were diluted in the culture medium to reach the final concentrations ranging from 0.1 to 100 μg/mL. The solvent (DMSO) used at the highest concentration in the test did not reveal any cytotoxic activity. Cisplatin was applied as a test referential agent. The cytotoxicity assay was performed after 72 h exposure of the cultured cells at the concentration ranging from 0.1 to 100 μg/mL of the tested agents. The SRB test measuring the cell proliferation inhibition in the in vitro culture was applied. The cells attached to the plastic were fixed with cold 50% TCA (trichloroacetic acid, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) added on the top of the culture medium in each well. The plates were incubated at 4 °C for 1 h and then washed five times with tap water. The background optical density was measured in the wells filled with the medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (Chemie GmbH, Steinheim, Germany) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min. The unbound dye was removed by rinsing (four times) with 1% acetic acid, and the protein-bound dye was extracted with 10 mM unbuffered Tris base (tris(hydroxymethyl)aminomethane, POCh, Gliwice, Poland) for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Uniskan II (Labsystems, Helsinki, Finland). The compounds were tested in triplicates per experiment. The experiments were repeated at least 3 times. The IC50 values were calculated by Cheburator 0.9.0 software.

2.4

2.4 In silico ADMET evaluations

In silico ADMET evaluation of compounds was performed by the ADMET Predictor version 7.1 (Admet 2014a). Structures of the compounds were saved as the mol file and they were uploaded into the ADMET predictor software for further evaluations. All parameters were calculated at pH 7.4.

3

3 Results and discussion

3.1

3.1 Chemistry

4H-3,1-benzothiazin-4-ones as a leading group of compounds (a) were prepared from 2-amino-N-phenylbenzamides (2a, 5a–9a) or 2-amino-N-methylbenzohydrazide (1a, 3a, 4a) and the corresponding electrophile (STB) (Scheme 1). In the synthesis de novo of sulphur analogues 4H-3,1-benzothiazine-4-thiones (b) 2-amino-N-phenylbenzothioamide was applied (Scheme 1). The 13C NMR spectra of compounds a show a low field signal at 182–181 ppm which can be attributed to carbon atom of C⚌O group. In the case of compounds b C⚌S group is registered at 210 ppm. The IR spectra of compounds a exhibit a strong band in the range of 1674–1642 cm−1 of ν(C⚌O). Next we made an attempt to obtain some heterocyclic analogues of benzothiazin-4-one to find the influence on antiproliferative activity of molecule size, the presence and localization of additional heteroatoms in a heterocycle fused to thiazin-4-one. 4H-Thieno[3,2-d][1,3]thiazin-4-ones (1c and 2c) were obtained from 3-aminothiophene-2-carboxamide, but in a different manner configured 4H-thieno[2,3-d][1,3]thiazin-4-ones (3c and 4c) from properly substituted 2-aminothiophene-3-carboxamide (Scheme 2). In the 1HNMR spectra, the thienothiazinone ring proton of compounds 2c and 3c gives a singlet at 7.6 and 7.5 ppm, respectively. Two nitrogen atoms of various positions were introduced in the form of fused to thiazin-4-one either imidazole (d) or pyrazole (e) ring (Scheme 3). In compounds 2e–6e′, the acidic = NH proton of the pyrazole moiety was substituted by the phenyl ring or Me substituent (Scheme 3). In this way the compounds of higher lipophilicity were obtained. 1HNMR spectra give signals in the range of 14.26–13.44 ppm which is attributed to the NH proton. The CH proton of imidazo[4,5-d][1,3]thiazin-7(1H)-one and pyrazolo[1,3]thiazinones appears as a singlet at 7.5 and in the range of 8.5–7.9 ppm respectively. To increase significantly the size of the molecule and its shape as well as add another heteroatom we obtained 3 analogues of 4H-benzofuro[3,2-d][1,3]thiazin-4-one (f) (Scheme 4). The IR spectra of compounds f exhibit a strong band in the range of 1662–1654 cm−1 of ν(C⚌O). In the IR spectra of all compounds a broad strong band in the range of 3500–3100 cm−1 of ν(O-H) is registered. In the synthesis we concentrated on the compounds with Et or Cl substituents in benzenediol moiety because it is generally believed that this type of substitution has a beneficial effect on the anticancer activity (McDonald et al. 2006; Niewiadomy et al., 2011). Electrophiles (STB) (Schemes 1–4) as initial compounds were obtained from properly substituted resorcinols and SOCl2 in the diethyl ether medium (Matysiak and Niewiadomy, 2006). The compounds were obtained according to the elaborated by our team synthesis method preliminary used for the synthesis of 4H-3,1-benzothiazin-4-ones (Matysiak et al., 2012) and next applied for the preparations of some thienothiazinones (Matysiak et al., 2015a) and azolothiaziones (Matysiak et al., 2015b). At present we optimized synthesis conditions for a much broader spectrum of nucleophilic reagents and we concentrated on the ethyl and chlorine substituted benzenediols.

Synthesis scheme of 4H-thieno[1,3]thiazin-4-ones (c). The appropriate STB reagents were used. For STB structure, see Scheme 1. The compounds 1c′, 2c′, 3c′ have been described previously.
Scheme 2
Synthesis scheme of 4H-thieno[1,3]thiazin-4-ones (c). The appropriate STB reagents were used. For STB structure, see Scheme 1. The compounds 1c′, 2c′, 3c′ have been described previously.
Synthesis scheme of imidazo[1,3]thiazinones (d) and pyrazolo[1,3]thiazinones (e). The appropriate STB reagents were used. For STB structure, see Scheme 1. The compounds 1d′, 1e′, 5e′, 6e′ have been described previously.
Scheme 3
Synthesis scheme of imidazo[1,3]thiazinones (d) and pyrazolo[1,3]thiazinones (e). The appropriate STB reagents were used. For STB structure, see Scheme 1. The compounds 1d′, 1e′, 5e′, 6e′ have been described previously.
Synthesis scheme of 4H-benzofuro[3,2-d][1,3]thiazin-4-ones (f). The appropriate STB reagents were used. For STB structure, see Scheme 1.
Scheme 4
Synthesis scheme of 4H-benzofuro[3,2-d][1,3]thiazin-4-ones (f). The appropriate STB reagents were used. For STB structure, see Scheme 1.

3.2

3.2 Antiproliferative activity

The compounds presented in Schemes 1–4 have been evaluated for their antiproliferative activities against human bladder cancer HCV29T cells. For this purpose, the SRB assay was used (Skehan et al., 1990). The cytotoxic activity in vitro was expressed as IC50 – the concentration of a compound to inhibit proliferation rate of the tumour cells by 50% as compared to the untreated cells. The results of screening are collected in Table 1. The selected compounds of the highest activity against HCV29T were also tested against human non-small cell lung carcinoma A549, human breast cancer T47D and human rectal adenocarcinoma cells SW707 (Table 2). The results show that the derivatives of the 4H-thieno[3,2-d][1,3]thiazin-4-one (2c) and 1-phenylpyrazolo[3,4-d][1,3]thiazin-4(1H)-one (3e, 4e) type were the most active compounds against all studied cells. In particular interesting is 2-(5-chloro-2,4-dihydroxyphenyl)-4H-benzofuro[3,2-d][1,3]thiazin-4-one (3f) of very high activity towards HCV29T cells (IC50 = 2.05 μM). The antiproliferative action of the most active compounds is comparable or stronger than that of cisplatin evaluated comparatively.

Table 1 Antiproliferative activity of compounds against human bladder cancer cell line HCV29T expressed as IC50a.
No. IC50 (μM) No. IC50 (μM)
1a 54.83 ± 7.51 1c′ 66.49 ± 6.45
2a 46.98 ± 23.99 3c′ 69.95 ± 8.48
3a 35.14 ± 11.59 1d 322.27 ± 57.66
4a 24.37 ± 7.78 1d′ 106.91 ± 3.97
5a 74.53 ± 14.58 1e 71.33 ± 29.86
6a n.a.b 1e′ 34.81 ± 7.22
1b 68.48 ± 14.48 5e′ 26.06 ± 7.81
Cisplatin 6.43 ± 1.20 1f 20.37 ± 4.78
a IC50 indicates the compound concentration (μM) that inhibits the proliferation rate of tumour cells by 50% as compared to the untreated control cells. The values are the means ± SD of 9 independent experiments.
b n.a. – activity above studied concentrations.
Table 2 Antiproliferative activity of selected compounds against the human cancer cells: bladder HCV29T, non-small lung carcinoma A549, breast T47D and rectal adenocarcinoma SW707 expressed as IC50a.
No. Line/IC50 (μM)
HCV 29T A549 T47D SW 707
7a 16.01 ± 3.94 21.20 ± 1.72 13.10 ± 3.38 21.00 ± 8.69
8a 19.60 ± 1.59 12.86 ± 0.75 9.98 ± 3.96 29.37 ± 1.71
9a 14.46 ± 8.17 16.40 ± 9.58 8.05 ± 2.52 15.40 ± 4.03
2b 16.36 ± 3.58 21.24 ± 10.21 14.00 ± 0.38 14.84 ± 2.44
1c 10.52 ± 1.44 15.65 ± 3.59 10.33 ± 1.83 16.62 ± 4.30
2c 5.79 ± 0.76 8.59 ± 0.71 5.38 ± 2.01 8.45 ± 4.05
4c 15.16 ± 7.52 9.64 ± 1.72 16.21 ± 4.39 13.72 ± 1.96
2c′ 15.38 ± 0.72 19.13 ± 9.58 13.71 ± 6.32 9.66 ± 2.19
2e 18.91 ± 4.30 16.39 ± 4.83 9.46 ± 1.57 21.37 ± 4.83
3e 8.98 ± 2.52 9.55 ± 2.96 12.62 ± 5.83 9.41 ± 3.94
4e 6.29 ± 2.34 11.22 ± 3.33 6.64 ± 0.75 7.77 ± 2.04
6e′ 17.60 ± 2.89 13.52 ± 1.22 9.23 ± 7.79 21.13 ± 7.64
2f 9.19 ± 2.30 10.25 ± 5.36 13.94 ± 2.59 7.25 ± 0.50
3f 2.05 ± 0.90 11.94 ± 0.46 5.61 ± 0.64 9.40 ± 3.35
Cisplatin 6.43 ± 1.20 11.60 ± 0.77 14.89 ± 3.90 13.30 ± 1.71
a IC50 indicates the compound concentration (μM) that inhibits the proliferation rate of tumour cells by 50% as compared to the untreated control cells. The values are the means ± SD of 9 independent experiments.

3.3

3.3 QSAR analysis

For more comprehensive structure–activity elucidation some previously described compounds were also studied under the same biological condition and included in the analysis (Matysiak et al., 2015a; Matysiak et al., 2015b). The results show that the activity of compounds significantly depends on the kind of heterocyclic skeleton as well as on substitution pattern of both heterocyclic and phenyl rings. The analysis shows, that in the group of benzothiazin-4-ones (a), the beneficial influence on the activity is exhibited by a chlorine atom in the heterocyclic ring (Table 1) (7a, 8a, 9a). The lowest antiproliferative effect for the compounds with a fluorine atom (5a and 6a) was detected. In the group of the analogues with unsubstituted benzothiazin-4-one ring, the presence of Et or chlorine atom in the benzenediol moiety increases their antiproliferative potency (3a, 4a). This effect of compound 9a against all studied cells was also observed. Replacing of C⚌O group with C⚌S one gave compounds of higher activities against all studied cell lines (3a and 2b). For compound with the Et substituent more beneficial properties were found compared to the unsubstituted analogue, according to the overall trend. Considering replacement of the benzothiazin-4-one ring with the thienothiazinone one, it can be observed that this modification is beneficial for anticancer potency (4a compared to 1c). In the group of differently configured thienothiazinones, the most active ones are compounds with the tert-butyl substituent (2c and 2c′). In this group also a beneficial influence of the presence of chlorine atom in the benzenediol moiety on the antiprolifertive potency is observed (1c′, 3c′ against HCV29T, 2c′ against all studied cell lines). Replacement of the benzothiazin-4-one ring by imidazothiazinone one produced the compounds of the lowest potency in the considered group of analogues (1d, 1d′), however, a beneficial effect on activity of Et substituent insertion was confirmed. Pyrazolothiazinones showed a low activity (1e and 1e′) but higher compared to imidazo[4,5-d][1,3]thiazin-7(1H)-one. N-alkyl(aryl) substitution on the pyrazole ring produced compounds of significantly higher activity, especially in the case of the presence of a phenyl ring (compounds 2e–4e). In the group of pyrazolothiazinones all ethyl analogues were more active than that unsubstituted ones. The chlorine analogue (4e) was the most active. In the group of 4H-benzofuro[3,2-d][1,3]thiazin-4-ones (f) the compound with unsubstituted benzenediol moiety is also significantly less active. The activity of other compounds was higher and in some cases similar to that of cisplatin. 3f is the most active compound against HCV29T cells.

3.4

3.4 In silico ADME/Toxicity evaluations

Not satisfactory ADME/Toxicity properties of compounds are a major reason for drug candidate failure in drug development. Therefore, there is a great interest of scientists in developing predictive in silico models which may be used in the initial stage of the drug candidate development (Guerra et al., 2010). For the evaluation of compounds ADMET properties, ADMET Predictor version 7.1 was used in this paper (Admet 2014a). The selected compounds, mainly of the highest activity were examined (Tables 3 and 4). For the evaluation of drug-like properties of compounds relatively simple parameters such: molecular weight (M), hydrophobicity: log D, log P, a number of hydrogen bonding atoms (HBD, HBA) and Lipinski’s rule of five were used (Lipinski et al., 1997). The results collected in Table 3 show, that all analysed compounds fulfil this criterion (rule of 5–0). Compounds obey the Oprea’s criteria. They possess 1–4 rotatable bonds (nRB) (less than 10), so they are conformationally stable and their polar surface area (PSA) is in the range of 53.4–99.1 Å2 (lower than 120 Å2) (Oprea et al., 2000). Additionally good water solubility (>10 μg/mL) for the compounds is predicted (except for 2b) (Kerns et al., 2008). The data exhibit that the analysed compounds are drug-like, and there is a significant probability that compounds might have favourable pharmacokinetics after oral administration (Lipinski 2000). The analysis of the more advanced parameters of adsorption process prediction: the human jejunal effective permeability (Peff) and apparent permeability (Papp) for Madin-Darby Canine Kidney (MDCK) shows that for most of the studied compounds a medium permeability is predicted (Table 3). Their MDCK is in the range of 25–500 × 10−7 cm/s and Peff is >4 × 10−4 cm/s (except for 1d and 1e) (Irvine et al., 1999). The unbd parameter, which reflects the overall fraction of a drug unbound in human blood plasma (in%) is rather low. Two derivatives have a very weak affinity for human blood proteins (1d, 1e). The calculated distribution volume (Vd) is in the range of 0.22–0.98 L/kg (Table 3). BBB filtering (qualitative likelihood high/low of crossing the blood–brain barrier (BBB)) and log BBB (logarithm of the brain/blood partition coefficient) were estimated to predict the BBB penetration. According to the calculated values (Table 3) all compounds show low brain penetration (BBB < 0.19) and they do not rather pass across the BBB. Taking this into account the compounds are not recommended as CNS active agents (Ma et al., 2005). For the prediction of compounds metabolism CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 enzymes in human were taken into consideration (Guengerich 2008). The data collected in Table 4 show, that all derivatives may be substrates of CYP 1A2 and the majority of 2C9. Intrinsic clearances (Clint) due to metabolism mediated by both enzymes are also calculated. Compounds show middle toxicity in the range of 474–1205 mg/kg predicted for rats after oral administration. In this area only for 4c not satisfactory result was calculated (Zhu et al., 2009). The global ADMET Risk of the compounds was also estimated (Table 4). The algorithm elaborated by Simulations Plus using a refined subset of WDI was applied (Admet 2014b). That characteristic obeys 24 different rules, including risk rules connected with oral absorption in human, toxicity, metabolic potency and additional two rules: low fraction unbound in plasma (Unbnd < [1,3]) and high steady-state volume of distribution (Vd > [3.7,5.7]). The considered compounds possess global ADMET Risk in the range of 1–5. It is greater than 6.5 for about 10% of the focused WDI (Admet 2014b). Clearly the lowest risk for 1d was predicted. Relatively high toxicity for active compounds 2e, 3e and 4e was estimated and slightly lower for 3f. For the most active compound 2c the lowest toxicity in the analysed group of derivatives was predicted. Its relatively high ADMET risk parameter is connected mainly with metabolism. Toxicities are the principle problem in the drug design and therefore, this compound may be subjected to further testing as a potential anticancer agent.

Table 3 Molecular descriptors and parameters characterizing adsorption and distribution processesa of the selected compounds estimated using ADMET Predictor software.
No. PSA (Å2) Log D Mlog P Rule of 5 nRB S (μg/mL) Peff (cm/s × 10−4) MDCK (cm/s × 10−7) Vd (L/kg) BBB Log BB Unbd (%)
7a 70.4 3.67 2.33 0 1 24.2 5.65 296.12 0.86 Low 0.17 2.16
8a 70.4 3.98 2.58 0 2 20.1 5.36 290.16 0.95 Low 0.19 1.9
9a 70.4 3.48 2.33 0 1 19.3 6.39 344.45 0.29 Low −0.21 0.46
2b 70.4 3.96 2.87 0 2 5.26 7.61 240.11 0.75 Low 0.16 2.36
2c 70.4 4.36 2.58 0 2 10.0 4.56 257.7 0.46 Low 0 0.64
3c 70.4 3.59 2.08 0 2 18.7 5.51 203.2 0.79 Low 0.03 2.43
4c 70.4 2.97 1.83 0 2 20.2 5.98 249.35 0.26 Low −0.38 0.68
1d 53.4 1.1 0.26 0 1 47.4 2.32 123.02 0.69 Low −0.43 10.09
2e 70.4 2.56 2.02 0 2 37.2 4.93 156.77 0.85 Low −0.12 3.6
3e 70.4 3.11 2.49 0 3 30.3 4.72 179.21 0.98 Low −0.14 2.72
4e 70.4 2.85 2.26 0 2 30.8 5.29 230.14 0.39 Low −0.31 0.84
2f 99.1 3.76 1.88 0 2 13.1 4.59 230.48 0.95 Low −0.12 1.77
3f 99.1 3.36 1.64 0 1 12.9 5.17 286.42 0.34 Low −0.46 0.52
a PSA – polar surface area; Log D – octanol–water distribution coefficient; M log P – log P according to Moriguchi model; nRB – number of rotatable bonds: S – water solubility: Peff – human jejunal effective permeability; MDCK – apparent permeability for Madin-Darby Canine Kidney (MDCK) cells; Vd – volume of distribution; BBB – qualitative likelihood high/low of crossing the blood–brain barrier; Log BB – logarithm of the brain/blood partition coefficient: unbd – overall fraction of a drug unbound in human blood plasma (in%).
Table 4 Characteristics of metabolism and toxicitya of the selected compounds in silico by the ADMET Predictor software.
No. Substr. of CYP1A2/clint (μL/min/mg) Substr. of CYP2C9/clint (μL/min/mg) TOX RAT (mg/kg) TOX Risk/TOX code ADMET Risk code ADMET Risk
7a Yes (63%) 32.7 Yes (58%) 92.6. 872.87 1/Mu ow, fu, Mu, 1A, C9 3.58
8a Yes (60%) 13.5 Yes (56%) 11.1 916.47 2/Hp, Mu ow, fu, Hp, Mu, C9 4.03
9a Yes (63%) 59.4 Yes (75%) 10.6 474.74 1/Hp fu, Hp, 1A, C9 4
2b Yes (80%) 10.9 Yes (75%) 16.7 1041.21 1.07/Xm, Hp ow, Sw, fu, Xm, Hp, C9 3.52
2c Yes (55%) 19.5 Yes (58%) 15.2 479.08 1/Hp ow, fu, Hp, 1A, C9 4.16
3c Yes (63%) 49.2 Yes (53%) 15.2 988.24 2/Hp, Mu ow, fu, Hp, Mu, C9 3.37
4c Yes (57%) 14.6 Yes (63%) 61.9 252.24 1.56/ra, Hp fu, ra, Hp, C9 3.56
1d Yes (63%) 43.2 Yes (63%) 12.4 1044.25 1/Hp Hp 1
2e Yes (57%) 26.8 No (65%) – 1205.85 3/Xm, Hp, Mu Xm, Hp, Mu 3
3e Yes (55%) 59.1 No (71%) – 1189.38 3/Xm, Hp, Mu fu, Xm, Hp, Mu 3.14
4e Yes (58%) 79.7 Yes (58%) 31.5 639.43 3/Xm, Hp, Mu fu, Xm, Hp, Mu, C9 5
2f Yes (55%) 51.3 No (73%) – 734.17 2/Hp, Mu ow, fu, Hp, Mu, ti 2.97
3f Yes (57%) 26.5 Yes (75%) 94.9 308.17 2.1/ra, Hp, Mu fu, ra, Hp, Mu, 1A, C9 4.87
a Xm – carcinogenicity in chronic mouse studies; Hp – hepatotoxicity; Mu – TOX MUT Risk > 1; Ra – TOX RAT < 320 (acute toxicity in rats); Xm – TOX BRM Mouse < 35 (carcinogenicity in chronic mouse studies); fu:% Unbnd < [1, 3] (low fraction unbound in plasma); Vd: Vd > [3.7, 5.7] (high steady-state volume of distribution); ow: log P > [4.5, 5.5] or log D > [3.5, 4.5] or Mlog P > [3.7, 4.3] (high log P o /w); 1A – MET 1A2 Km > 0.01 μM and MET 1A2 CLint > [15,30]; C9: MET 2C9 Km > 0.01 μM and MET 2C9 CLint > [15,30 μL/min/mg]; ti: MET 3A4 Ki tes < 1.0 and (MET 3A4 I tes = Yes or MET 3A4 Inh = Yes); SG – TOX SGOT = Toxic and TOX SGPT = Toxic (SGOT and SGPT elevation); Sw: S < [0.003, 0.010 g/mL] (low solubility).

4

4 Conclusion

New compounds of benzothiazin-4-one type and its heterocyclic analogues were obtained according to the one-step synthesis method elaborated by us. SAR analysis reveals that the most active compounds are those with a 4H-thieno[3,2-d][1,3]thiazin-4-one and benzofuro[3,2-d][1,3]thiazin-4-one skeleton. The presence of the hydrophobic substituents (Et, Cl) in the benzenediol moiety increases their antiproliferative potency. The evaluation in silico of the compounds showed that they possess significant drug-like characteristics, they are metabolically stable and possess low or medium toxicity profile. For the most active 6-tert-butyl-2-(5-chloro-2,4-dihydroxyphenyl)-4H-thieno[3,2-d][1,3]thiazin-4-one (2c) low toxicity was assumed and the compound can be further studied as a potential anticancer agent. Very promising ADMET properties were predicted for compound 1d which displays a low antiproliferative potency. Therefore, for this compound, it would be desirable to try to indicate other directions of biological activities than considered. Additionally, next studies including design, synthesis, and evaluation of novel groups of compounds with the modified 2,4-dihydroxyphenyl substituent as potential anticancer agents should be carried out.

Acknowledgement

This project was financed from the funds of the National Science Centre in Poland allocated on the basis of the decision number DEC-2011/01/B/NZ4/05005.

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