SYNTHESIS AND IN VITRO ANTICANCER ACTIVITY OF NEW THIADIAZOLINES AND THIAZOLINONES CONTAINING A CHROMENYL
SCAFFOLD
I. IONUŢa*, C. NASTASĂa, J. T. NDONGOb, C. BRUYÈREc, H. LECLERCQZc, B. TIPERCIUCa, F. LEFRANCd, A. PÎRNĂUe, R. KISSc, O. ONIGAa
aIuliu Haţieganu University of Medicine and Pharmacy, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, 41 Victor Babeş Street, RO- 400010 Cluj-Napoca, România
bDépartement de Chimie, Ecole Normale Supérieure de Yaoundé, B.P. 47, Université de Yaoundé I, Cameroun
cLaboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, 1050 Bruxelles, Belgique
dService de Neurochirurgie, Hôpital Erasme, ULB, Bruxelles, Belgique
eNational Institute for Research and Development of Isotopic and Molecular Technologies, RO-400293 Cluj-Napoca, Romania
A series of 14 derivatives of chromenyl-dihydro-thiadiazole and chromenyl-methylene- thiazolin-4-one were synthesized and screened (using the MTT colorimetric assay) for their in vitro growth inhibition capacity in six human cancer cell lines. Three cell lines displayed various levels of resistance to pro-apoptotic stimuli, while two cell lines were sensitive to pro-apoptotic stimuli. Structure Activity Relationship (SAR) analyses indicate that, of the two compound series that were investigated, chromenyl-thiadiazolines displayed higher in vitro anticancer activity than the thiazolinone derivatives. With respect to the thiadiazoline derivatives (3a-l compounds), the substitution of the exocyclic amine with a substituted-phenyl moiety increased the in vitro growth inhibition of cancer cells, and the 3-trifluoromethyl-phenyl-derivatives (3h, 3l) displayed the highest antiproliferative activity. In addition, compounds 3h, 3k and 3l overcame the intrinsic resistance of cancer cells to pro-apoptotic stimuli by induction of either cytostatic (3h, 3l) or cytotoxic (3k) effects, which was determined by quantitative videomicroscopy.
(Received August 21, 2013; Accepted October 31, 2013)
Keywords: Thiadiazoline; Thiazolinone; Chromone; Antitumour; Videomicroscopy
1. Introduction
Malignant tumours represent one of the most serious threats against human health in the world, and the clinical prognosis remains relatively poor [1]. In 2008, cancer accounted for approximately eight million deaths worldwide [2], and the number of cases is expected to increase by more than 45% in the next 20 years [3]. Genotoxic agents have long targeted apoptotic cell death as a primary means of treating cancer [4,5]. However, the presence of cellular defects in many cancers has contributed to an acquired resistance to apoptotic cell death [6,7], thus lowering the effectiveness of chemo- and radiotherapies [4,5]. Resistance to anoikis, a programmed cell death that occurs upon cell detachment from the extracellular matrix [8], is emerging as a hallmark of metastatic cancers that are resistant to conventional therapies [9]. Therefore, it is necessary to identify novel types of compounds that overcome the intrinsic resistance of cancer cells to pro- apoptotic stimuli by the induction of non-apoptotic cell death (such as autophagy or necrosis [4]) or by cytostatic effects [10]. For example, glioma is naturally resistant to pro-apoptotic stimuli
*Corresponding author: [email protected]
[11], and therefore resistant to conventional therapies: the pro-autophagic alkylating agent temozolomide has significant therapeutic benefits for glioma patients [12,13]. Similar observations can be made for melanoma [14,15] and oesophageal cancers [16].
Molecules containing nitrogen- and sulphur-related heterocycles (thiazole, thiazolidine, thiazolinone, thiadiazoline) are considered important pharmacophores as they can possess interesting biological activities. For example, thiadiazolines have antihelmintic, antihypertensive, anticancer, anti-inflammatory, antibacterial, analgesic, and tyrosinase inhibitory activities [17].
Thiazolinones display hypoglycaemic, antibacterial, antifungal, antituberculous, anti-HIV and also antitumoural activities [18].
Chromones are a group of naturally occurring compounds that are ubiquitous, especially in plants [19,20]. In addition to forming the basic nucleus of an entire class of natural products, the flavones, the chromone moiety forms an important component of the pharmacophores found in many molecules with medicinal significance: tyrosine and protein kinase C inhibitors, antifungal, antiallergenic, antiviral, antitubulin, antihypertensive, and anticancer agents. Consequently, considerable attention is being devoted to the isolation of these compounds from natural resources and partial versus complete syntheses of chromone derivatives as well as to the evaluation of their biologic activity [19,20].
Herein we report the synthesis and in vitro growth inhibition characterisation of new thiadiazoline and thiazolinone derivatives substituted with a chromen-3-yl or 3-chromenyl- methylene moiety. We screened six human cancer cell lines with various levels of resistance to pro-apoptotic stimuli to investigate whether the compounds in the study are able to overcome this resistance. Three cell lines display various levels of resistance to pro-apoptotic stimuli, i.e., the A549 non-small-cell-lung cancer (NSCLC) [21,22], the SKMEL-28 melanoma [23] and the U373 glioma [24,25] cell lines. Two of the cell lines display actual sensitivity to pro-apoptotic stimuli, i.e., the Hs683 glioma [25,26] and the MCF-7 breast carcinoma [27] cell lines. The in vitro antiproliferative activity of each compound in the study has been determined using the MTT colorimetric assay [23-26]. The three most potent compounds were further analysed using computer-assisted phase-contrast microscopy, i.e., quantitative videomicroscopy [28,29].
2. Experimental Chemistry
Solvents were obtained from commercial sources. Analytical thin layer chromatography was carried out on precoated Silica Gel 60F254 sheets using UV absorption for visualisation. The melting points were taken with Electrothermal and MPM-H1 Schorpp melting point meters and are uncorrected. The 1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker Avance NMR spectrometer operating at 500 MHz and were in accordance with the assigned structures. Chemical shift values were reported relative to tetramethylsilane (TMS) as the internal standard. The samples were prepared by dissolving the powder of the synthesised compounds in DMSO-d6 (δH= 2.51 ppm) and the spectra were recorded using a single excitation pulse of 12 µs (1H NMR). GC-MS analyses were performed with an Agilent gas chromatograph 6890 equipped with an apolar Macherey Nagel Permabond SE 52 capillary column. Elemental analysis was measured with a Vario El CHNS instrument.
Synthesis of chromenyl-thiosemicarbazone derivatives 2a-l (General procedure)
In a flask equipped with a reflux condenser, a mixture of 3-formyl-chromone 1a,b (50 mmol) and various N4-substituted-thiosemicarbazides (50 mmol) was reacted in 50 ml of absolute ethanol with catalytic sulphuric acid. The reaction mixture was heated under reflux for 3 h, where upon the solid product partially crystallised out. The solution was left to cool and the solid product was filtered off, washed with water, dried, and recrystallized from ethanol.
N-methyl-2-((6-methyl-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2a)
Yield 88%. Yellow powder, mp: 250 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.46 (s, 3H, chromone-CH3); 3.43 (s, 3H, N-CH3); 7.62 (d, 1H, C8-chromone-H); 7.71 (d, 1H, C7-chromone- H); 7.93 (s, 1H, C2-chromone-H); 8.12 (s, 1H, C5-chromone-H); 8.40 (s, 1H, CH=N); 11.26 (s, 1H,
NHCS); 11.68 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 20.5 (chromone-CH3);
33.3 (NH-CH3); 113.2 (chromone-C3); 116.1 (chromone-C8); 121.3 (chromone-C4a); 127.3 (chromone-C5); 133.4 (CH=N); 136.5 (chromone-C6); 138.2 (chromone-C7); 151.4 (chromone- C8a); 163.5 (chromone-C2); 172.6 (C=S); 177.9 (chromone-C4). Anal. Calcd. (%) for C13H13N3O2S (275.33): C, 56.71; H, 4.76; N, 15.26; S, 11.65. Found: C, 56.46; H, 4.54; N, 14.84; S, 11.34. MS (EI, 70 eV): m/z 276 [M+].
N-phenyl-2-((6-methyl-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2b)
Yield 90%. White powder, mp: 242 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.43 (s, 3H, chromone-CH3); 7.36 (m, 5H, Ar-H); 7.64 (d, 1H, C8-chromone-H); 7.73-7.74 (dd, 1H, C7- chromone-H); 7.89 (s, 1H, C2-chromone-H); 8.08 (s, 1H, C5-chromone-H); 8.36 (s, 1H, CH=N);
11.06 (s, 1H, NHCS); 11.59 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.6 (chromone-CH3); 113.4 (chromone-C3); 118.6 (chromone-C8); 122.1 (chromone-C4a); 126.4 (phenyl-C2, C6); 127.7 (CH=N); 128.4 (phenyl-C3, C5); 128.9 (phenyl-C4); 129.6 (chromone-C5);
135.6 (chromone-C6); 138.4 (chromone-C7); 140.5 (phenyl-C1); 152.7 (chromone-C8a); 162.4 (chromone-C2); 175.46 (C=S); 178.47 (chromone-C4). Anal. Calcd. (%) for C18H15N3O2S (337.40):
C, 64.08; H, 4.48; N, 12.45; S, 9.50. Found: C, 63.92; H, 4.70; N, 12.82; S, 9.27. MS (EI, 70 eV):
m/z 338 [M+].
N-allyl-2-((6-methyl-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2c)
Yield 83%. Yellow powder, mp: 239 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.43 (s, 3H, chromone-CH3); 4.58-4.66 (m, 2H, N-CH2); 5.12-5.18 (m, 2H, CH=CH2); 5.87-6.02 (m, 1H, CH);
7.61-7.63 (d, 1H, C8-chromone-H); 7.67-7.69 (dd, 1H, C7-chromone-H); 7.79-7.80 (s, 1H, C2- chromone-H); 8.08 (s, 1H, C5-chromone-H); 8.33 (s, 1H, CH=N); 11.06 (s, 1H, NHCS); 11.49 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 20.6 (chromone-CH3); 46.1 (allyl-CH2);
115.4 (chromone-C3); 116.9 (CH2=); 117.5 (chromone-C8); 120.7 (chromone-C4a); 129.6 (chromone-C5); 134.1 (CH=N); 135.4 (chromone-C6); 135.8 (CH=); 137.9 (chromone-C7); 152.6 (chromone-C8a); 163.8 (chromone-C2); 172.9 (C=S); 179.7 (chromone-C4). Anal. Calcd. (%) for C15H15N3O2S (301.36): C, 59.78; H, 5.02; N, 13.94; S, 10.64. Found: C, 59.59; H, 4.92; N, 13.55;
S, 10.88. MS (EI, 70 eV): m/z 302 [M+].
N-methyl-2-((6-chloro-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2d)
Yield 85%. White powder, mp: 260 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 3.27 (s, 3H, N- CH3); 7.59-7.62 (d, 1H, C8-chromone-H); 7.65-7.72 (d, 1H, C7-chromone-H); 8.00 (s, 1H, C2- chromone-H); 8.02 (s, 1H, C5-chromone-H); 8.22 (s, 1H, CH=N); 11.31 (s, 1H, NHCS); 11.68 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 33.3 (CH3); 113.2 (chromone-C3); 121.4 (chromone-C4a); 121.4 (chromone-C8); 124.9 (chromone-C5); 126.7 (chromone-C6); 135.3 (CH=N); 137.2 (chromone-C7); 154.4 (chromone-C8a); 162.4 (chromone-C2); 175.1(C=S); 181.2 (chromone-C4). Anal. Calcd. (%) for C12H10ClN3O2S (295.74): C, 48.73; H, 3.41; N, 14.21; S, 10.84. Found: C, 48.93; H, 3.24; N, 14.42; S, 11.19. MS (EI, 70 eV): m/z 296 [M+].
N-phenyl-2-((6-chloro-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2e)
Yield 91%. White powder, mp: 238 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 7.54-7.58 (m, 5H, Ar-H); 7.76-7.79 (d, 1H, C8-chromone-H); 7.88-7.93 (dd, 1H, C7-chromone-H); 8.03-8.06 (d, 1H, C2-chromone-H); 8.14 (s, 1H, C5-chromone-H); 9.01 (s, 1H, CH=N); 11.28 (s, 1H, NHCS); 11.40 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 114.6 (chromone-C3); 122.3 (chromone- C4a); 123.7 (chromone-C8); 125.4 (chromone-C5); 128.8 (phenyl-C2, C6); 129.7 (chromone-C6);
130.5 (CH=N); 131.1 (phenyl-C4); 133.4 (phenyl-C3, C5); 137.6 (chromone-C7); 138.3 (phenyl- C1); 155.2 (chromone-C8a); 163.5 (chromone-C2); 173.08 (C=S); 180.6 (chromone-C4). Anal.
Calcd. (%) for C17H12ClN3O2S (357.81): C, 57.06; H, 3.38; N, 11.74; S, 8.96. Found: C, 56.42; H, 3.31; N, 11.39; S, 8.63. MS (EI, 70 eV): m/z 358 [M+].
N-allyl-2-((6-chloro-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2f)
Yield 85%. White powder, mp: 229 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 4.58-4.65 (m, 2H, N-CH2); 5.19-5.23 (m, 2H, CH=CH2); 6.02-6.06 (m, 1H, CH); 7.48-7.54 (d, 1H, C8-chromone-H);
7.68-7.76 (dd, 1H, C7-chromone-H); 7.89 (s, 1H, C2-chromone-H); 8.05 (s, 1H, C5-chromone-H);
8.46 (s, 1H, CH=N); 11.11 (s, 1H, NHCS); 11.55 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 46.3 (allyl-CH2); 112.6 (chromone-C3); 115.5 (CH2=); 122.3 (chromone-C4a); 123.6 (chromone-C8); 126.1 (chromone-C5); 127.9 (chromone-C6); 133.6 (CH=N); 134.2 (CH=); 136.8
(chromone-C7); 154.2 (chromone-C8a); 162.3 (chromone-C2); 170.6 (C=S); 182.1 (chromone-C4).
Anal. Calcd. (%) for C14H12ClN3O2S (321.78): C, 52.26; H, 3.76; N, 13.06; S, 9.96. Found: C, 52.13; H, 3.81; N, 12.80; S, 9.64. MS (EI, 70 eV): m/z 322 [M+].
N-(4-methoxyphenyl)-2-((6-methyl-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2g)
Yield 84%. White powder, mp: 230 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.43 (s, 3H, chromone-CH3); 3.77 (s, 3H, OCH3); 7.22-7.25 (d, 2H, Ar-H); 7.39-7.43 (d, 2H, Ar-H); 7.46-7.50 (d, 1H, C8-chromone-H); 7.62-7.64 (dd, 1H, C7-chromone-H); 7.85 (d, 1H, C2-chromone-H); 7.99 (s, 1H, C5-chromone-H); 8.30 (s, 1H, CH=N); 11.07 (s, 1H, NHCS); 11.59 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.9 (chromone-CH3); 55.6 (OCH3); 111.5 (chromone-C3);
118.8 (phenyl-C2,C5); 119.3 (chromone-C8); 123.2 (chromone-C4a); 127.3 (phenyl-C2, C6); 129.4 (CH=N); 130.9 (chromone-C5); 131.8 (phenyl-C1); 134.2 (chromone-C6); 137.8 (chromone-C7);
154.2 (chromone-C8a); 156.4 (phenyl-C4); 163.3 (chromone-C2); 174.7 (C=S); 179.7 (chromone- C4). Anal. Calcd. (%) for C19H17N3O3S (367.42): C, 62.11; H, 4.66; N, 11.44; S, 8.73. Found: C, 62.39; H, 4.79; N, 11.28; S, 8.49. MS (EI, 70 eV): m/z 368 [M+].
N-(3-(trifluoromethyl)phenyl)-2-((6-methyl-4-oxo-4H-chromen-3-yl)methylene) hydrazinecarbothioamide (2h)
Yield 80%. White powder, mp: 210 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.45 (s, 3H, chromone-CH3); 7.44-7.48 (d, 1H, C8-chromone-H); 7.59-7.63 (dd, 1H, C7-chromone-H); 7.72- 7.78 (m, 1H, Ar-H); 7.83-7.87 (m, 3H, Ar-H); 8.03 (d, 1H, C2-chromone-H); 8.07 (s, 1H, C5- chromone-H); 8.34 (s, 1H, CH=N); 11.27 (s, 1H, NHCS); 11.67 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.0 (chromone-CH3); 112.3 (chromone-C3); 116.4 (chromone-C8);
119.1 (phenyl-C2); 121.3 (chromone-C4a); 122.9 (CF3); 124.4 (phenyl-C4); 124.7 (phenyl-C6);
129.1 (CH=N); 131.3 (phenyl-C5); 132.0 (chromone-C5); 132.2 (phenyl-C3); 136.3 (chromone- C6); 139.1 (chromone-C7); 150.3 (phenyl-C1); 154.8 (chromone-C8a); 164.1 (chromone-C2); 174.6 (C=S); 179.3 (chromone-C4). Anal. Calcd. (%) for C19H14F3N3O2S (405.39): C, 56.29; H, 3.48; N, 10.37; S, 7.91. Found: C, 56.01; H, 3.43; N, 10.09; S, 7.71. MS (EI, 70 eV): m/z 406 [M+].
2-((6-Methyl-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2i)
Yield 91%. Light yellow powder, mp: 231 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.45 (s, 3H, chromone-CH3); 7.62-7.63 (d, 1H, C8-chromone-H); 7.66-7.68 (dd, 1H, C7-chromone-H); 7.90 (s, 1H, C2-chromone-H); 8.09 and 8.25 (2 br s, 1H each, NH2); 8.19 (s, 1H, C5-chromone-H); 9.14 (s, 1H, CH=N); 11.53 (s, 1H, NHCS). 13C NMR (DMSO-d6, 500 MHz, ppm): 20.3 (chromone- CH3); 112.8 (chromone-C3); 118.1 (chromone-C8); 121.6 (chromone-C4a); 131.2 (chromone-C5);
135.0 (CH=N); 136.4 (chromone-C6); 138.9 (chromone-C7); 155.1 (chromone-C8a); 163.9 (chromone-C2); 174.8 (C=S); 180.4 (chromone-C4). Anal. Calcd. (%) for C12H11N3O2S (261.30):
C, 55.16; H, 4.24; N, 16.08; S, 12.27. Found: C, 54.88; H, 4.13; N, 15.74; S, 12.68. MS (EI, 70 eV): m/z 262 [M+].
2-((6-Chloro-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2j)
Yield 83%. Light yellow powder, mp: 244 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 7.69-7.72 (d, 1H, C8-chromone-H); 7.74-7.80 (dd, 1H, C7-chromone-H); 7.92-8.02 (d, 1H, C2-chromone-H);
8.05 and 8.08 (2 br s, 1H each, NH2); 8.11 (s, 1H, C5-chromone-H); 8.36 (s, 1H, CH=N); 11.30 (s, 1H, NHCS). 13C NMR (DMSO-d6, 500 MHz, ppm): 112.2 (chromone-C3); 123.2 (chromone- C4a); 123.7 (chromone-C8); 127.1 (chromone-C5); 128.9 (chromone-C6); 135.4; (CH=N); 138.2 (chromone-C7); 156.3 (chromone-C8a); 164.5 (chromone-C2); 175.1 (C=S); 180.1 (chromone-C4).
Anal. Calcd. (%) for C11H8ClN3O2S (281.72): C, 46.90; H, 2.86; N, 14.92; S, 11.38. Found: C, 46.69; H, 2.52; N, 14.63; S, 11.61. MS (EI, 70 eV): m/z 282 [M+].
N-(4-methoxyphenyl)-2-((6-chloro-4-oxo-4H-chromen-3-yl)methylene)hydrazinecarbothioamide (2k)
Yield 90%. White powder, mp: 227 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 3.81 (s, 3H, OCH3); 7.04-7.09 (d, 2H, Ar-H); 7.36-7.41 (d, 2H, Ar-H); 7.66-7.69 (d, 1H, C8-chromone-H);
7.85-7.89 (dd, 1H, C7-chromone-H); 8.03 (s, 1H, C2-chromone-H); 8.11 (s, 1H, C5-chromone-H);
8.35 (s, 1H, CH=N); 11.11 (s, 1H, NHCS); 11.47 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 56.3 (OCH3); 111.9 (chromone-C3); 117.1 (phenyl-C3, C5); 122.9 (chromone-C4a); 123.4 (chromone-C8); 127.3 (chromone-C5); 127.9 (chromone-C6); 128.3 (phenyl-C2, C6); 131.1
(CH=N); 131.5 (phenyl-C1); 137.4 (chromone-C7); 156.1 (chromone-C8a); 157.9 (phenyl-C4);
164.7 (chromone-C2); 174.9 (C=S); 179.7 (chromone-C4). Anal. Calcd. (%) for C18H14ClN3O3S (387.84): C, 55.74; H, 3.64; N, 10.83; S, 8.27. Found: C, 55.52; H, 3.75; N, 10.68; S, 7.89. MS (EI, 70 eV): m/z 388 [M+].
N-(3-(trifluoromethyl)phenyl)-2-((6-chloro-4-oxo-4H-chromen-3- yl)methylene)hydrazinecarbothioamide (2l)
Yield 84%. Yellow powder, mp: 235 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 7.73-7.80 (m, 2H, chromone-H); 7.87-7.93 (m, 3H, Ar-H); 8.02-8.03 (d, 1H, Ar-H); 8.05 (d, 1H, C2-chromone- H), 8.12 (s, 1H, C5-chromone-H); 8.42 (s, 1H, CH=N); 11.04 (s, 1H, NHCS); 11.45 (s, 1H, CSNH). 13C NMR (DMSO-d6, 500 MHz, ppm): 112.1 (chromone-C3); 118.1 (phenyl-C2); 121.6 (phenyl-C4); 122.2 (chromone-C4a); 122.4 (phenyl-C6); 122.6 (chromone-C8); 126.2 (chromone- C5); 127.7 (chromone-C6); 128.3 (CF3); 129.4 (CH=N); 131.3 (phenyl-C5); 132.3 (phenyl-C3);
136.9 (chromone-C7); 149.2 (phenyl-C1); 155.6 (chromone-C8a); 163.3 (chromone-C2); 174.2 (C=S); 178.9 (chromone-C4). Anal. Calcd. (%) for C18H11ClF3N3O2S (425.81): C, 50.77; H, 2.60;
N, 9.87; S, 7.53. Found: C, 50.64; H, 2.47; N, 9.63; S, 7.42. MS (EI, 70 eV): m/z 426 [M+].
Synthesis of chromenyl-1,3,4-thiadiazoline derivatives 3a-l (General procedure)
For synthesis, 5 mmol of the correspondent chromenyl-thiosemicarbazone 2a-l was refluxed with 10 ml of acetic anhydride and 0.5 ml of pyridine for 4 h. After cooling, the mixture was poured into water and stirred for 30 minutes at room temperature. The resultant precipitate was filtered, washed with water and recrystallized from absolute ethanol.
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- methylacetamide (3a)
Yield 72%. White powder, mp: 285 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.27 (s, 6H, N3- thiadiazoline-COCH3, NCOCH3); 2.47 (s, 3H, chromone-CH3); 3.46 (s, 3H, N-CH3); 6.64 (s, 1H, C2-thiadiazoline-H); 7.58-7.60 (d, 1H, J = 8.5, C8-chromone-H); 7.66-7.68 (d, 1H, J = 8.0, C7- chromone-H); 7.88 (s, 1H, C2-chromone-H); 8.11 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 20.4 (chromone-CH3); 21.4 (N3-thiadiazoline-acetyl-CH3); 23.6 (N-acetyl- CH3); 32.2 (N-CH3); 53.1 (thiadiazoline-C2); 116.2 (chromone-C8); 119.2 (chromone-C3); 123.6 (chromone-C4a); 129.5 (chromone-C5); 135.3 (chromone-C6); 137.7 (chromone-C7); 153.3 (chromone-C8a); 155.6 (chromone-C2); 158.9 (thiadiazoline-C5); 168.9 (N-C=O); 170.1 (N3- thiadiazoline-C=O); 178.5 (chromone-C4). Anal. Calcd. (%) for C17H17N3O4S (359.40): C, 56.81;
H, 4.77; N, 11.69; S, 8.92. Found: C, 56.98; H, 4.56; N, 11.32; S, 8.58. MS (EI, 70 eV): m/z 360 [M+].
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- phenylacetamide (3b)
Yield 66%. Light yellow powder, mp: 215 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.89 (s, 3H, N3-thiadiazoline-COCH3); 1.90 (s, 3H, NCOCH3); 2.46 (s, 3H, chromone-CH3); 6.69 (s, 1H, C2-thiadiazoline-H); 7.50-7.57 (m, 5H, Ar-H); 7.60-7.61 (d, 1H, C8-chromone-H); 7.67-7.69 (dd, 1H, C7-chromone-H); 7.90 (s, 1H, C2-chromone-H); 8.07 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.8 (chromone-CH3); 21.5 (N3-thiadiazoline-acetyl-CH3); 23.7 (N- acetyl-CH3); 54.6 (thiadiazoline-C2); 117.1 (chromone-C8); 119.7 (chromone-C3); 122.5 (chromone-C4a); 126.2 (phenyl-C2, C6); 128.3 (phenyl-C4); 130.4 (chromone-C5); 130.7 (phenyl- C3, C5); 134.8 (chromone-C6); 137.7 (phenyl-C1); 138.6 (chromone-C7); 152.1 (chromone-C8a);
158.9 (thiadiazoline-C5); 159.3 (chromone-C2); 164.3 (N-C=O); 167.9 (N3-thiadiazoline-C=O);
177.9 (chromone-C4). Anal. Calcd. (%) for C22H19N3O4S (421.47): C, 62.69; H, 4.54; N, 9.97; S, 7.61. Found: C, 63.12; H, 4.68; N, 10.2; S, 7.43. MS (EI, 70 eV): m/z 422 [M+].
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- allylacetamide (3c)
Yield 70%. White powder, mp: 217 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.23 (s, 3H, N3- thiadiazoline-COCH3); 2.26 (s, 3H, NCOCH3); 2.43 (s, 3H, chromone-CH3); 4.52-4.61 (m, 2H, N- CH2); 5.21-5.26 (m, 2H, CH=CH2); 6.00-6.05 (m, 1H, CH); 6.65 (s, 1H, C2-thiadiazoline-H); 7.58- 7.60 (d, 1H, C8-chromone-H); 7.66-7.68 (dd, 1H, C7-chromone-H); 7.87-7.88 (s, 1H, C2-
chromone-H); 8.08 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.9 (chromone-CH3); 21.5 (N3-thiadiazoline-acetyl-CH3); 22.9 (N-acetyl-CH3); 48.2 (allyl-CH2); 54.5 (thiadiazoline-C2); 116.9 (chromone-C8); 120.2 (chromone-C3); 120.8 (CH2=); 122.3 (chromone- C4a); 126.8 (CH=); 129.8 (chromone-C5); 134.2 (chromone-C6); 136.5 (chromone-C7); 152.4 (chromone-C8a); 157.1 (chromone-C2); 157.6 (thiadiazoline-C5); 163.4 (N-C=O); 167.2 (N3- thiadiazoline-C=O); 175.6 (chromone-C4). Anal. Calcd. (%) for C19H19N3O4S (385.44): C, 59.21;
H, 4.97; N, 10.90; S, 8.32. Found: C, 58.95; H, 4.83; N, 10.78; S, 8.22. MS (EI, 70 eV): m/z 386 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- methylacetamide (3d)
Yield 68%. White powder, mp: 300 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.26 (s, 3H, N3- thiadiazoline-COCH3); 2.27 (s, 3H, NCOCH3); 3.46 (s, 3H, N-CH3); 6.63 (s, 1H, C2-thiadiazoline- H); 7.76-7.78 (d, 1H, C8-chromone-H); 7.89-7.91 (dd, 1H, C7-chromone-H); 8.02-8.03 (s, 1H, C2- chromone-H); 8.21 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 21.7 (N3- thiadiazoline-acetyl-CH3); 23.7 (N-acetyl-CH3); 32.6 (N-CH3); 54.6 (thiadiazoline-C2); 118.6 (chromone-C3); 119.2 (chromone-C8); 123.4 (chromone-C4a); 125. 6 (chromone-C5); 135.3 (chromone-C6); 136.3 (chromone-C7); 155.1 (chromone-C8a); 157.0 (chromone-C2); 158.3 (thiadiazoline-C5); 167.6 (N-C=O); 169.4 (N3-thiadiazoline-C=O); 176.8 (chromone-C4). Anal.
Calcd. (%) for C16H14ClN3O4S (379.82): C, 50.60; H, 3.72; N, 11.06; S, 8.44. Found: C, 50.12; H, 3.79; N, 10.59; S, 8.21. MS (EI, 70 eV): m/z 380 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- phenylacetamide (3e)
Yield 53%. Light brown powder, mp: 251 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.90 (s, 6H, N3-thiadiazoline-COCH3, NCOCH3); 6.70 (s, 1H, C2-thiadiazoline-H); 7.50-7.56 (m, 5H, Ar- H); 7.77-7.79 (d, 1H, C8-chromone-H); 7.90-7.92 (dd, 1H, C7-chromone-H); 8.05-8.06 (d, 1H, C2- chromone-H); 8.17 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 21.4 (N3- thiadiazoline-acetyl-CH3); 23.4 (N-acetyl-CH3); 55.3 (thiadiazoline-C2); 118.5 (chromone-C3);
120.8 (chromone-C8); 123.4 (chromone-C4a); 125.6 (chromone-C5); 126.0 (chromone-C6); 126.5 (phenyl-C2, C6); 128.1 (phenyl-C4); 130.6 (phenyl-C3, C5); 136.3 (chromone-C7); 137.7 (phenyl- C1); 153.7 (thiadiazoline-C5); 155.1 (chromone-C8a); 157.2 (chromone-C2); 161.8 (N-C=O); 168.0 (N3-thiadiazoline-C=O); 177.4 (chromone-C4). Anal. Calcd. (%) for C21H16ClN3O4S (441.89): C, 57.08; H, 3.65; N, 9.51; S, 7.26. Found: C, 57.21; H, 3.47; N, 9.12; S, 7.12. MS (EI, 70 eV): m/z 442 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N- allylacetamide (3f)
Yield 68%. White powder, mp: 220 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.23 (s, 3H, N3- thiadiazoline-COCH3); 2.26 (s, 3H, NCOCH3); 4.52-4.62 (m, 2H, N-CH2); 5.21-5.27 (m, 2H, CH=CH2); 6.01 (m, 1H, CH); 6.65 (s, 1H, C2-thiadiazoline-H); 7.76-7.78 (d, 1H, C8-chromone-H):
7.89-7.91 (dd, 1H, C7-chromone-H): 8.02-8.03 (s, 1H, C2-chromone-H); 8.19 (s, 1H, C5- chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 22.0 (N3-thiadiazoline-acetyl-CH3); 22.8 (N-acetyl-CH3); 48.6 (allyl-CH2); 56.3 (thiadiazoline-C2); 118.5 (chromone-C3); 119.7 (CH2=);
120.8 (chromone-C8); 123.4 (chromone-C4a); 125.6 (chromone-C5); 126.1 (chromone-C6); 126.5 (CH=); 136.3 (chromone-C7); 155.1 (chromone-C8a); 155.0 (chromone-C2); 155.6 (thiadiazoline- C5); 164.3 (N-C=O); 168.4 (N3-thiadiazoline-C=O); 178.9 (chromone-C4). Anal. Calcd. (%) for C18H16ClN3O4S (405.86): C, 53.27; H, 3.97; N, 10.35; S, 7.90. Found: C, 53.14; H, 4.03; N, 9.98;
S, 7.64. MS (EI, 70 eV): m/z 406 [M+].
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N-(4- methoxyphenyl)acetamide (3g)
Yield 64%. Grey powder, mp: 171 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.89 (s, 3H, N3- thiadiazoline-COCH3); 1.93 (s, 3H, NCOCH3); 2.45 (s, 3H, chromone-CH3); 3.83 (s, 3H, OCH3);
6.68 (s, 1H, C2-thiadiazoline-H); 7.06-7.08 (d, 2H, J = 8.5, Ar-H); 7.43-7.45 (d, 2H, J = 8.5, Ar- H); 7.59-7.61 (d, 1H, C8-chromone-H), 7.67-7.69 (dd, 1H, C7-chromone-H), 7.89 (d, 1H, C2- chromone-H), 8.05 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.9 (chromone-CH3); 21.2 (N3-thiadiazoline-acetyl-CH3); 23.9 (N-acetyl-CH3); 53.4 (thiadiazoline-
C2); 55.8 (OCH3); 114.3 (phenyl-C3, C5); 115.6 (chromone-C8); 119.1 (chromone-C3); 121.8 (chromone-C4a); 126.7 (phenyl-C2, C6); 129.4 (chromone-C5); 133.5 (phenyl-C1); 133.7 (chromone-C6); 137.2 (chromone-C7); 153.4 (chromone-C8a); 154.6 (thiadiazoline-C5); 156.0 (chromone-C2); 160.9 (phenyl-C4); 161.2 (N-C=O); 166.9 (N3-thiadiazoline-C=O); 176.9 (chromone-C4). Anal. Calcd. (%) for C23H21N3O5S (451.49): C, 61.18; H, 4.69; N, 9.31; S, 7.10.
Found: C, 61.41; H, 4.47; N, 8.92; S, 6.83. MS (EI, 70 eV): m/z 452 [M+].
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N-(3- trifluoromethylphenyl)acetamide (3h)
Yield 70%. Light brown powder, mp: 88 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.89 (s, 3H, N3-thiadiazoline-COCH3); 1.93 (s, 3H, NCOCH3); 2.45 (s, 3H, chromone-CH3); 6.71 (s, 1H, C2- thiadiazoline-H); 7.59-7.61 (d, 1H, C8-chromone-H); 7.67-7.69 (dd, 1H, C7-chromone-H); 7.78- 7.81 (m, 1H, Ar-H); 7.87-7.92 (m, 3H, Ar-H); 8.08 (d, 1H, C2-chromone-H); 8.10 (s, 1H, C5- chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 20.9 (chromone-CH3); 21.7 (N3- thiadiazoline-acetyl-CH3); 24.1 (N-acetyl-CH3); 56.5 (thiadiazoline-C2); 118.8 (chromone-C8);
121.7 (phenyl-C2); 123.2 (chromone-C3); 124.5 (chromone-C4a); 126.4 (CF3); 126.6 (phenyl-C4);
130.6 (phenyl-C5); 130.8 (phenyl-C3); 131.3 (chromone-C5); 133.6 (phenyl-C6);136.0 (chromone- C6); 136.3 (chromone-C7); 140.5 (phenyl-C1); 151.0 (chromone-C8a); 153.6 (thiadiazoline-C5);
154.7 (chromone-C2); 168.8 (N-C=O); 170.6 (N3-thiadiazoline-C=O); 175.6 (chromone-C4). Anal.
Calcd. (%) for C23H18F3N3O4S (489.47): C, 56.44; H, 3.71; N, 8.58; S, 6.55. Found: C, 56.62; H, 3.83; N, 8.24; S, 6.33. MS (EI, 70 eV): m/z 490 [M+].
N-(4-acetyl-5-(6-methyl-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)acetamide (3i) Yield, 65%. Grey crystals, mp: 280 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.04 (s, 3H, N3- thiadiazoline-COCH3); 2.25 (s, 3H, NCOCH3); 2.43 (s, 3H, chromone-CH3); 6.64 (s, 1H, C2- thiadiazoline-H); 7.55-7.57 (d, 1H, C8-chromone-H); 7.63-7.66 (dd, 1H, C7-chromone-H); 7.85 (s, 1H, C2-chromone-H); 8.12 (s, 1H, C5-chromone-H); 11.75 (s, 1H, NH). ). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.6 (chromone-CH3); 22.3 (N3-thiadiazoline-acetyl-CH3); 23.7 (N-acetyl- CH3); 55.2 (thiadiazoline-C2); 115.7 (chromone-C8); 117.4 (chromone-C3); 120.6 (chromone-C4a);
128.6 (chromone-C5); 134.4 (chromone-C6); 136.2 (chromone-C7); 155.2 (chromone-C8a); 157.1 (chromone-C2); 157.4 (thiadiazoline-C5); 167.8 (N-C=O); 168.0 (N3-thiadiazoline-C=O); 176.8 (chromone-C4). Anal. Calcd. (%) for C16H15N3O4S (345.37): C, 55.64; H, 4.38; N, 12.17; S, 9.28.
Found: C, 56.01; H, 4.58; N, 12.51; S, 8.92. MS (EI, 70 eV): m/z 346 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)acetamide (3j) Yield 58%. Grey crystals, mp: 300 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 2.04 (s, 3H, N3- thiadiazoline-COCH3); 2.24 (s, 3H, NCOCH3); 6.64 (s, 1H, C2-thiadiazoline-H); 7.76-7.77 (d, 1H, C8-chromone-H); 7.88-7.90 (dd, 1H, C7-chromone-H); 8.02 (d, 1H, C2-chromone-H); 8.23 (s, 1H, C5-chromone-H); 11.77 (s, 1H, NH). 13C NMR (DMSO-d6, 500 MHz, ppm): 21.6 (N3- thiadiazoline-acetyl-CH3); 24.2 (N-acetyl-CH3); 56.4 (thiadiazoline-C2); 118.3 (chromone-C3);
120.1 (chromone-C8); 122.3 (chromone-C4a); 125.5 (chromone-C5); 126.1 (chromone-C6); 135.4 (chromone-C7); 156.3 (chromone-C8a); 156.7 (chromone-C2); 157.0 (thiadiazoline-C5); 168.3 (N- C=O); 169.6 (N3-thiadiazoline-C=O); 178.5 (chromone-C4). Anal. Calcd. (%) for C15H12ClN3O4S (365.79): C, 49.25; H, 3.31; N, 11.49; S, 8.77. Found: C, 49.61; H, 3.17; N, 11.17; S, 8.91. MS (EI, 70 eV): m/z 366 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N-(4- methoxyphenyl)acetamide (3k)
Yield 69%. Grey powder, mp: 181 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.89 (s, 3H, N3- thiadiazoline-COCH3); 1.93 (s, 3H, NCOCH3); 3.83 (s, 3H, OCH3); 6.69 (s, 1H, C2-thiadiazoline- H); 7.06-7.08 (d, 2H, J = 9.0, Ar-H); 7.43-7.45 (d, 2H, J = 9.0, Ar-H); 7.76-7.78 (d, 1H, C8- chromone-H); 7.89-7.91 (dd, 1H, C7-chromone-H); 8.04 (d, 1H, J = 2.5, C2-chromone-H); 8.15 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 19.6 (N3-thiadiazoline-acetyl- CH3); 24.3 (N-acetyl-CH3); 56.1 (thiadiazoline-C2); 57.8 (OCH3); 115.6 (phenyl-C3, C5); 118.8 (chromone-C3); 121.2 (chromone-C8); 124.0 (chromone-C4a); 126.4 (chromone-C5); 126.9 (chromone-C6); 127.8 (phenyl-C2, C6); 134.3 (phenyl-C1); 137.5 (chromone-C7); 155.2 (thiadiazoline-C5); 155.9 (chromone-C8a) ; 156.6 (chromone-C2); 161.8 (phenyl-C4); 163.6 (N- C=O); 169.2 (N3-thiadiazoline-C=O); 177.3 (chromone-C4). Anal. Calcd. (%) for C22H18ClN3O5S
(471.91): C, 55.99; H, 3.84; N, 8.90; S, 6.79. Found: C, 56.26; H, 3.66; N, 8.54; S, 7.04. MS (EI, 70 eV): m/z 472 [M+].
N-(4-acetyl-5-(6-chloro-4-oxo-4H-chromen-3-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N-(3- trifluoromethylphenyl)acetamide (3l)
Yield 72%. Light brown powder, mp: 117 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 1.89 (s, 3H, N3-thiadiazoline-COCH3); 1.93 (s, 3H, NCOCH3); 6.72 (s, 1H, C2-thiadiazoline-H); 7.77-7.81 (m, 2H, chromone-H); 7.87-7.92 (m, 3H, Ar-H); 8.04-8.05 (d, 1H, J = 3.0, Ar-H); 8.08 (d, 1H, C2- chromone-H); 8.20 (s, 1H, C5-chromone-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 21.4 (N3- thiadiazoline-acetyl-CH3); 23.7 (N-acetyl-CH3); 55.9 (thiadiazoline-C2); 117.1 (phenyl-C2); 118.6 (chromone-C3); 121.6 (chromone-C8); 123.9 (chromone-C4a); 125.6 (chromone-C5); 126.3 (chromone-C6); 126.7 (CF3); 127.5 (phenyl-C4); 128.7 (phenyl-C5); 129.1 (phenyl-C3); 130.5 (phenyl-C6); 137.2 (chromone-C7); 139.7 (phenyl-C1); 154.6 (thiadiazoline-C5); 155.3 (chromone- C8a); 157.2 (chromone-C2); 164.3 (N-C=O); 169.1 (N3-thiadiazoline-C=O); 176.9 (chromone-C4).
Anal. Calcd. (%) for C22H15ClF3N3O4S (509.89): C, 51.82; H, 2.97; N, 8.24; S, 6.29. Found: C, 51.76; H, 2.87; N, 7.91; S, 6.12. MS (EI, 70 eV): m/z 510 [M+].
Synthesis of 5-chromenyl-thiazolidine-2-thioxo-4-one 4
For synthesis, 1 mmol (0.332 g) of 6,8-dibromo-3-formyl-chromone 1c was refluxed for 3 h with 1 mmol (0.133 g) of 2-thioxo-4-thiazolidinone in 5 ml of acetic acid in the presence of 4 mmol (0.328 g) of anhydrous sodium acetate. The reaction mixture was cooled, and the crude product was filtered under reduced pressure, washed with water on the filter and purified by recrystallisation from ethanol.
5-((6,8-Dibromo-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (4)
Yield 95%. Yellow powder, mp: 308-312 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 7.63 (s, 1H, C7-chromone-H); 8.17 (s, 1H, C2-chromone-H); 8.40 (s, 1H, C5-chromone-H); 9.05 (s, 1H, C=CH); 12.44 (br, s, NH). 13C NMR (DMSO-d6, 500 MHz, ppm): 113.4 (chromone-C3); 113.5 (chromone-C6); 117.4 (chromone-C8); 119.6 (CH); 121.4 (thioxothiazolidinone-C5); 123.8 (chromone-C4a); 128.1 (chromone-C5); 139.6 (chromone-C7); 153.5 (chromone-C8a); 160.2 (thioxothiazolidinone-C4); 162.6 (chromone-C2); 177.7 (chromone-C4); 191.2 (C=S). Anal. Calc.
(%) for C13H5Br2NO3S2 (447.12): C, 34.92; H, 1.13; N, 3.13; S, 14.34. Found: C, 34.90; H, 1.14;
N, 3,14; S, 14,33. MS (EI, 70 eV): m/z 448 [M+].
Synthesis of 5-chromenyl-2-yl-thioacetamide-thiazolinone 5
For synthesis, 1 mmol of 5-chromenyl-2-thioxo-thiazolidine-4-one 4 was stirred for 3 h at room temperature with 1.1 mmol (0.203 g) of iodoacetamide in the presence of 1.1 mmol (0.062 g) of anhydrous potassium hydroxide in 6 ml of DMF. The crude product was filtered under reduced pressure, washed with water on the filter and purified by recrystallisation from ethanol.
2-[5-(6,8-Dibromo-4-oxo-4H-chromen-3-yl-methylene)-4-oxo-4,5-dihydro-thiazol-2-yl-thio]- acetamide (5)
Yield 40%. Red powder, mp: 230-232 °C. 1H NMR (DMSO-d6, 500 MHz, ppm): 4.21 (s, 2H, CH2); 7.41 and 7.81 (2 br s, 1H each, NH2); 7.57 (s, 1H, C7-chromone-H); 8.16 (s, 1H, C2- chromone-H); 8.45 (s, 1H, C5-chromone-H); 9.10 (s, 1H, C=CH). 13C NMR (DMSO-d6, 500 MHz, ppm): 37.2 (CH2); 113.7 (chromone-C6); 118.6 (chromone-C8); 119.3 (CH); 126 (chromone- C4a); 127.7 (chromone-C3); 127.8 (chromone-C5); 128.9 (thiazolinone-C5); 140.3 (chromone-C7);
151.8 (thiazolinone-C4); 163.1 (chromone-C8a); 167.7 (chromone-C2); 174.1 (thiazolinone-C2);
179.1 (H2N-C=O); 194.5 (chromone-C4). Anal. Calcd. (%) for C15H8Br2N2O4S2 (504.17): C, 35.73;
H, 1.60; N, 5.56; S, 12.72. Found: C, 35.85; H, 1.60; N, 9.88; S, 12.78. MS (EI, 70 eV): m/z 505 [M+].
Biological activity
Determination of In Vitro Growth Inhibition Activity in Human Cancer Cells. Seven human cancer cell lines were obtained from the American Type Culture Collection (ATCC,
Manassas, USA), the European Collection of Cell Culture (ECACC, Salisbury, UK) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
These seven cell lines included the A549 non-small-cell lung cancer (NSCLC; DSMZ code ACC107), the SKMEL-28 melanoma (ATCC code HTB-72), the Hs683 oligodendroglioma (ATCC code HTB-138), the U373 (ECACC code 08061901), the T98G (ATCC; code CRL-1690) and U251 (ECACC code 09063001) glioblastoma, and the MCF-7 breast cancer (DSMZ; code ACC115) cell lines.
The cells were cultured in RPMI (Lonza, Verviers, Belgium) culture medium supplemented with 10% heat-inactivated foetal calf serum (Lonza), 4 mM glutamine, 100 µg/ml gentamicin, and penicillin-streptomycin (200 U/ml and 200 µg/ml; Lonza).
The overall growth level of each cell line was determined using the colorimetric MTT (3- [4,5-dimethylthiazol-2yl-diphenyl tetrazolium bromide, Sigma, Belgium) assay as detailed previously [23-26]. The data are represented as the mean values from one experiment with six replicates in each experimental condition.
Computer-Assisted Phase-Contrast Microscopy (Videomicroscopy). The direct visualisation of the compound-induced effects on the cell proliferation and morphology was carried out by means of computer-assisted phase contrast microscopy, i.e., quantitative videomicroscopy, as detailed elsewhere [28,29].
3. Results and discussion Chemistry
Our synthetic routes to the target compounds 3a-l, 5 are shown in Schemes 1-2, the synthesis of compound 6 being reported previously [30]. The structure of compound 6 is presented in Fig. 1. The derivatives of chromenyl-4,5-dihydrothiadiazoles 3a-l were prepared by reacting the obtained chromenyl-thiosemicarbazones 2a-l with the cyclisation agent acetic anhydride in the presence of small quantities of pyridine as a catalyst [17,31,32]. The intermediate thiosemicarbazones 2a-l were obtained by a simple condensation between the 3-formyl-chromones 1a,b and N4-substituted thiosemicarbazides in absolute ethanol using concentrated sulphuric acid as catalyst [33,34]. The N4-substituted phenyl, methyl, allyl, 3-trifluoro-methyl-phenyl, and 4- methoxy-phenyl thiosemicarbazides were obtained in good yields (>90%) by the addition of hydrazine hydrate to phenylisothiocyanate, methylisothiocyanate, allylisothiocyanate, 3-trifluoro- methylphenyl-isothiocyanate and 4-methoxyphenyl-isothiocyanate, respectively, with stirring in absolute ethanol at room temperature for 3 h [34]. These thiosemicarbazides are also commercially available.
The 5-chromenyl-methylene-2-thioxo-4-thiazolidinone 4 was obtained by the condensation of 2-thioxo-4-thiazolidinone with the 3-formyl-chromone 1c in glacial acetic acid at reflux and in the presence of anhydrous sodium acetate. After isolation and purification, the obtained derivative 4 was alkylated with 2-iodoacetamide in the presence of anhydrous potassium
hydroxide in dimethylformamide at room temperature to obtain the novel N-substituted compound 5.
The purity of the compounds was confirmed by TLC, and all new compounds were characterized by m.p., elemental analysis and spectroscopic data (1H NMR, 13C NMR, MS).
Scheme 1. Synthesis of chromen-yl-thiadiazoline derivatives 3a-l: (i) H2NNHCSNHZ/abs EtOH, reflux 3 h; (ii) Ac2O/pyridine, reflux 4 h
Scheme 2. Synthesis of chromen-yl-methylene-thiazolin-2-thioacetamide 5; (i) Thiazolidine-2-thioxo-4-one/AcOH, AcONa, reflux 3 h; (ii) ICH2CONH2/KOH, DMF, rt, 3 h
Fig. 1. Chemical structure of compound 6
Characterization of in vitro anticancer activity
The data in Table 1 show that the most potent compounds displayed an in vitro IC50 ranging between 12 (3h) and 35 (3l, 3k, 3j) µM, while the remaining compounds in the study displayed weak or no (> 100 µM for 3i) in vitro growth inhibition activity. In fact, nine of the 14 compounds in the study displayed an antiproliferative activity of < 100 µM against all cancer cell lines that were analysed. Compound 3h displayed the highest in vitro anticancer activity (Table 1).
Table 1. Characterization of the in vitro growth inhibitory activity (using the MTT colorimetric assay) of compounds 3 a-l, 5, 6
Compounds
IC50 concentrations (µM) after having cultured the cancer cells for 3 days with the compound of interest
A549 SKMEL
-28 U373 U251 Hs683 MCF-7 Mean
± SEM
3a * * * 79 85 84 **
3b 36 44 69 58 36 21 44 ± 7
3c 94 * * * 99 87 **
3d * 43 70 21 * 22 **
3e 84 * * * 85 43 **
3f 75 72 48 41 42 40 53 ± 7
3g 45 39 63 77 54 34 52 ± 7
3h 19 17 12 17 27 32 21 ± 3
3i * * * * * * **
3j 30 39 33 32 38 31 34 ± 2
3k 32 27 30 44 35 23 32 ± 3
3l 27 35 28 30 32 29 30 ± 1
5 77 67 82 65 73 56 70 ± 4
6 48 68 56 n.d. n.d. 59 58 ± 4
n.d.- not determined
* IC50> 100 µM
** The mean value could not be calculated because at least one cell line displayed an IC50 > 100 µM
Structure Activity Relationship (SAR) analyses indicate that, of the two chemical series that were investigated, the chromenyl-thiadiazolines displayed the highest in vitro anticancer activity. With respect to the thiadiazoline derivatives (3a-l compounds), the substitution of the exocyclic amine with a substituted-phenyl moiety increased the in vitro growth inhibition of cancer cells (Table 1), 3-trifluoromethyl-phenyl-derivatives (3h, 3l) displaying the highest growth inhibition activity (Table 1).
Thiazolinones 5 and 6 showed moderate growth inhibition activity against the cancer cell lines that were analysed in the current study.
So, it seems that the presence of fluorine atoms induces positive influences on the antiproliferative activity of these compounds against cancer cells.
The data in Table 1 further reveal that the most potent compounds, i.e., 3h, 3j, 3k and 3l, display similar growth inhibition effects in cancer cells independently of whether these cells are associated (A549 [21,22], SKMEL-28 [23], U373 [24,25]) or not associated (Hs683 [25,26], MCF-7 [27]) to various levels of resistance to pro-apoptotic stimuli.
Compounds 3h, 3k and 3l were further assayed by means of quantitative videomicroscopy. Compounds 3h and 3l induced morphological cytostatic effects in the human MCF-7 breast cancer (Fig. 2) and T98G glioblastoma (data not shown) cell lines, while the compound 3k induced morphological cytotoxic effects in both the human MCF-7 breast cancer (Fig. 2) and the T98G glioblastoma (data not shown) cell lines.
both h quantit betwee obtaine 3h- an cell gr MCF-7
Fig. 2. (a) Gx200) of h or 29 µM 3 concentratio total of 1,0 was perfor controls in F to the ratio present in th or 3l- (d) tr growth rati MCF-7 can In fact, the human MCF tative determ en 3h- and
ed via morph nd 3l-related
rowth, while 7 breast canc
Morphologic human MCF-7 3l; these conce ons (see Tabl 80 digitised i rmed in tripli Fig. 2b, 2c an of the mean n he first image reated experim io (GGR; Fig.
ncer cells grew ese three com
F-7 breast c mination of c 3l-related cy hological an
cytostatic e e 3k-induced cer (Fig. 2) a
cal illustratio 7 breast canc entrations ref le 1); (b-d) on images during icates; all ex nd 2d are iden number of cell e (at 0 h); we t ment by the r g. 2a) index: a w in the treat observa mpounds indu cancer and cell death (T ytostatic effe nalyses (Fig.
ffects parall d cytotoxic e and in T98G
ons (computer cer cells cultur fer to the MTT ne image has g each 72 h fo xperiments w ntical. Therefo
ls present in t then divided th ratio obtained a GGR value ted experimen ation period uced cell dea
T98G gliob Table 2) did fects versus 2). Indeed, t el a delay in effects are p
glioblastoma
r-assisted pha red for 72 h w T colorimetric been digitised for each exper ere carried o ore, the global the 1,080th ima
his ratio obtai d in the contro
of 0.2 means t compared to
ath rates that blastoma ce d not enable effects from the panels o n MCF-7 (an paralleled by a (data not sh
ase-contrast with 32 µM 3h c test-related d every four m
rimental cond out in parall l growth rates age to the num ined in the 3h ol to calculat for example o the control o
t ranged betw ells (Table
a clear disti m 3k, but tha n the right in nd also T98G complete g hown) cells.
microscopy;
h, 23 µM 3k in vitro IC50
minutes for a dition, which lel, thus the s correspond mber of cells h- (b), 3k- (c) te the global that 20% of over a 72 h
ween 15 and 2). Therefo inction to be at informatio n Fig. 2 sho G, data not growth inhibi
30% in ore, the
e made on was ws that shown) ition in
Table 2. The percentages of cell deaths induced by compounds 3h, 3k and 3l in the human MCF-7 breast cancer and the T98G glioblastoma cell lines.
Compounds*
Human cancer cell lines analysed
IC50-related MTT
concentration*
GGR***
% of cell death****
Global
morphological effects
24
h 48 h 72 h
3h
MCF-7 32 0.5
7.7
± 2.0
14.0 ± 3.8
14.7
± 3.2
Cytostatic
T98G** 30 0.4
9.7
± 1.9
12.7 ± 2.3
18.7
± 3.8
Cytostatic
3k
MCF-7 23 0.2
5.7
± 1.2
16.3 ± 6.4
22.0
± 8.7
Cytotoxic
T98G 26 0.1
23.3
± 1.9
29.0 ± 1.0
28.0
± 2.9
Cytotoxic
3l
MCF-7 29 0.5
7.0
± 1.5
7.7
± 2.4
9.8 ± 2.4
Cytostatic
T98G 62 0.3
10.7
± 4.4
11.3 ± 1.3
22.7
± 2.7
Cytostatic
* Compounds 3h, 3k and 3l have been assayed by means of quantitative videomicroscopy (see Fig.
2) at their IC50-related MTT colorimetric assay concentrations in the human MCF-7 apoptosis- sensitive (Dumont et al. 2007) breast cells (Table 1) and the human T98G apoptosis-resistant**
(Branle et al. 2002) glioblastoma cells (IC50 concentrations: 3h = 30, 3k = 26 and 3l = 62 µM).
*** Based on the phase-contrast microscopic pictures obtained by means of quantitative videomicroscopy (Fig. 2), we calculated the global growth rate (GGR), which corresponds to the ratio of the mean number of cells present in the last image captured in the experiment (conducted at 72 h) to the number of cells present in the first image (at 0 h). We divided this ratio obtained in each treated experiment by the ratio obtained in the control. Thus, for example, a GGR value of 0.4 in the current Table means that 40% of cells grew in the treated condition compared to the control condition over a 72 h observation period.
**** The % of cell deaths were determined by observation of the video films available for each experimental condition. For each experiment lasting for 72 h, one image was digitised every four minutes, yielding a whole set of 1,080 images after 72 h of observation. These 1,080 images were compressed into a one-minute video that clearly indicated whether the compound-induced effects were cytostatic or cytotoxic. Dynamic analyses of these videos allowed for easy discrimination between cytostatic (cell proliferation arrest) and cytotoxic (cell death) effects. The percentages of cell death in each control condition were < 2%.
It thus appears that compounds 3h, 3k and 3l are able to overcome the intrinsic resistance of cancer cells (Table 1) to pro-apoptotic stimuli by inducing in cells either cytostatic (3h, 3l) or cytotoxic (3k) effects (Fig. 2; Table 2).
4. Conclusions
Several types of metastatic cancers and primary cancers that have not yet metastasized by the time of initial diagnosis, including gliomas, melanomas, oesophageal cancers, pancreatic cancers, and NSCLCs, resist conventional chemotherapy and radiotherapy because these treatments induce pro-apoptotic factors in cancer cells, while the above-mentioned cancer types display various levels of resistance to pro-apoptotic stimuli.
The current study demonstrates that thiadiazoline derivatives, such as the 3- trifluoromethyl-phenyl-derivatives (3h, 3l), are able to overcome cancer cell resistance to pro- apoptotic stimuli because these compounds display in vitro growth inhibition activity in both cancer cell lines that are sensitive to pro-apoptotic stimuli and those that are resistant to pro- apoptotic stimuli. Therefore, 3-trifluoromethyl-phenyl-derivatives deserve further investigation as potential anticancer drugs that could be assayed in vivo by the oral route in various mouse tumour models. We are currently pursuing this goal.
Acknowledgements
We warmly thank Thierry Gras for his excellent technical assistance. Robert Kiss is a Director of Research with the Fonds National de la Recherche Scientifique (FRS-FNRS; Belgium) and Hélène Leclercqz is the holder of a Grant Télévie (FRS-FNRS; Belgium).
The current work was partly granted by the POS DRU/107/1.5/S/78702 European project.
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