Res Chem Intermed DOI 10.1007/s11164-015-2086-2
Synthesis of a new series of pyrimidine derivatives: exploration of anti-proliferative activity on EAT cells and molecular docking N. Senthilkumar1 • Y. Dominic Ravichandran1,3 K. M. Kumar2 • Sudha Ramaiah2
•
Received: 11 April 2015 / Accepted: 30 April 2015 Springer Science+Business Media Dordrecht 2015
Abstract A new series of pyrimidine derivatives was designed and synthesized from 2-thiouracil via multicomponent, Biginelli-type reactions and structurally characterized by all spectral means. Synthesized compounds were evaluated for antiproliferative activity against Ehrlich ascites tumour (EAT) cells. A molecular docking study was carried out to establish the binding mode of these compounds into human casein kinase-2 inhibitor (CK2). The established binding modes of these compounds into human CK2 were in very good agreement with the in vitro antiproliferative activity. Compound 4-(2-(1H-indol-2-yl)ethylamino)-2-(2-(diethylamino)ethylthio)-6-(4-fluorophenyl)pyrimidine-5-carbonitrile 4h exhibited stronger cytotoxic activity against EAT cells with an IC50 value of 5.2 lM which was the nearest cytotoxic activity compared with the standard drug methotrexate (MTX) that showed an IC50 value of 3.6 lM. Compound 4h has the maximum cytotoxicity against EAT cell, the lowest binding energy (-8.7 kcal/mol) and good ligand efficiency with CK2 compared to all other compounds. Keywords 2-Thiouracil Ehrlich ascites tumour cell Antiproliferative activity Casein kinase-2 inhibitor-CK2 Binding energy
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2086-2) contains supplementary material, which is available to authorized users. & Y. Dominic Ravichandran
[email protected] 1
Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India
2
Bioinformatic Division, School of Bioscience and Technology, VIT University, Vellore 632 014, Tamil Nadu, India
3
Department of Science and Humanities, Karpagam College of Engineering, Coimbatore 641032, Tamil Nadu, India
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Introduction The exploration of new chemotherapeutic drugs for cancer continues to be a dynamic area of research in recent times [1]. Casein kinase-2 (CK2) is the major therapeutic target enzyme involved in many cellular processes, such as cell cycle progression, apoptosis, cell differentiation and transcriptions [2]. CK2 is also a highly pleiotropic and conserved serine/threonine kinase that plays key roles in cell growth, proliferation, and survival. It has been well demonstrated that overexpressed CK2 is closely related to a number of cancers, including head and neck, breast, colorectal, renal, lung, leukemias, and prostate cancer [3]. CK2 has a number of physiological targets and participates in a complex series of cellular functions, including the maintenance of cell viability. The level of CK2 in normal cells is tightly regulated, and it has long been considered to play a role in cell growth and proliferation [4]. Further, an induction of apoptosis in cancer cells by the down-regulation of CK2 activity, emphasizes the importance of the development of CK2 inhibitors in the treatment of cancers [5]. CK2, as a protein kinase target, is considered receptive to drug administration and could be used in the development of antitumor, antiviral and antiinflammatory drugs [6]. Polyhydroxylated aromatic compounds, halogenated benzimidazole, benzimidazole [7] and pyrimidine compounds [6] act as CK2 inhibitors. Highly selective and potent CK2 inhibitors have a broad spectrum of antiproliferative activity in cell lines, including inflammatory breast cancer [6, 8]. Currently, fluorine has become an essential motif in drug discovery. Incorporating fluorine in prospective medicines can have amazing effects on the molecules due to special properties such as high electro negativity, a small atomic radius and low polarizability of C–F bonds. These properties make fluorine molecules more selective with increased efficacy [9]. Pyrimidine derivatives are extensively explored in medicinal chemistry due to their importance in a wide range of biological activity [10–22]. Moreover, it is also used as a central core for kinase inhibitors such as anaplastic lymphoma kinase [23], axl kinase [24], phosphoinositide-3-kinase [25], tie kinase [26], Janus tyrosine [27] and aurora-A kinase [28]. The N,N-diethylamino)ethyl group acts as a radioactive probe for the targeting of muscarinic receptor antagonist [29] and melanotic melanoma [30] due to its ability to accept hydrogen bonds and its aliphatic hydrophobic nature. The ability of a bioactive molecule to recognize the receptors or active sites of enzymes results from a combination of steric and electronic properties. Further, 2,4,6-trisubstituted 5-cyano-pyrimidine derivatives have shown immunosuppressive activity [31]. Methotrexate (MTX) is a pyrimidine-based chemotherapy drug necessary for DNA synthesis and has a therapeutic effect on many types of cancer cells. It is also widely used in the treatment of malignancies, including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin’s lymphoma, Hodgkin’s disease, head and neck cancer, lung cancer, breast cancer, psoriasis, choriocarcinoma and trophoblastic tumors [32]. Hence, a new series of pyrimidine derivatives with thio-(N,N-diethylamino)ethyl at the 2-position, a fluorinated aromatic moiety at the 4-position, cyano groups at the 5-position and various aromatic, aliphatic and heterocyclic amines at the 6-position of pyrimidine
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were synthesized and evaluated for their anti-proliferative activity against Ehrlich ascites tumour (EAT; spontaneous mammary carcinoma tumor) cells with MTX as the standard drug. Molecular docking and the structure activity relationship (SAR) were studied to establish the binding mode of these compounds into CK2.
Experimental For all the reactions, analytical grade solvents were used and dried. Tetrahydrofuran (THF) was prepared in house from laboratory reagent grade materials. All the moisture-sensitive reactions were carried out under a nitrogen atmosphere. Analytical thin-layer chromatography (TLC) was performed on pre-coated silica gel 60 F254 plates (Merck). Visualization was performed by ultraviolet (UV) light and staining with iodine, ninhydrin and potassium permanganate. Organic solutions were concentrated by rotary evaporation at ambient temperature. Purification techniques were performed by column chromatography using 60–120-mesh silica gel. Proton nuclear magnetic resonance (1H NMR) and 13C NMR spectra were acquired using a Bruker 400 MHz spectrometers. Chemical shifts were reported in parts per million (ppm) and are calibrated to residual solvent peaks: proton (CDCl3 7.26 ppm & DMSO-d6 2.50 ppm) for 1H NMR and carbon (CDCl3 77.0 ppm & DMSO-d6 39.52 ppm) for 13C NMR. For 1H NMR, coupling constants (J) were reported in Hz. Multiplicities were reported using the following abbreviations: s singlet, br s broad singlet, d doublet, dd doublet of doublet, t triplet, q quartet, m multiplet. Infrared spectroscopic data was recorded with KBr pellet plates using a Shimadzu infrared (IR) spectrophotometer. High-resolution mass spectra were recorded on a Mass-EI-Quadrupole spectrometer. 4-(4-Fluorophenyl)-6-hydroxy-2-mercaptopyrimidine-5-carbonitrile (1) [1, 15, 16] Yellow solid (3.18 g, yield 80 %). 1H NMR (DMSO-d6, 400 MHz) d: 7.31 (s, 2H, ArH), 7.82 (s, 2H, ArH), 11.68 (s, 1H, OH); 13C NMR (DMSO-d6, 100 MHz) d: 84.95, 114.87, 115.08, 118.87, 130.62, 130.71, 134.03, 134.06, 161.88 (d, J = 246.9 Hz, C–F), 162.56, 166.14, 182.95. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-hydroxypyrimidine-5carbonitrile (2) Compound 1 (3.18 g, 12.8 mmol) was dissolved in 10 mL of DMSO followed by addition of triethylamine (3.9 g, 38 mmol) and stirred for 15 min. To the reaction mixture, 2-bromo-N,N-diethylethanamine hydrobromide (3.36 g, 12.8 mmol) was added and stirred for 10 h at room temperature. Reaction completion was monitored by TLC. After addition of cold water to the reaction mixture, a pale yellow solid was precipitated, filtered and dried through vacuum. Pale yellow solid (4 g, yield 90 %), mp: 170–175 C; IR (KBr) mmax/cm-1: 3481.51. 3419.79, 2993.52, 2196.92, 1598.99, 1550.77, 1450.47, 1396.46, 1280.73, 1232.51, 1157.29, 1006.84, 866.04,
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798.53, 569.00; 1H NMR (DMSO-d6, 400 MHz) d: 1.26 (6H, s, (CH3)2), 3.21 (4H, s, N(CH2)2), 3.35 (4H, s, N(CH2)2), 7.35 (2H, s), 7.86 (2H, s), 10.90 (br, 1H, OH, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz) d: 8.63, 24.75, 46.64, 52.63, 89.85, 115.03, 115.24, 119.21, 130.55, 130.63, 133.55, 133.58, 161.83 (d, J = 247.9 Hz, C–F), 166.41, 169.11, 170.83; 19F NMR (DMSO-d6, 376 MHz) d: -110.79; HRMS (EI) (m/z) calcd for C17H19FN4OS 346.1264, found 346.1265. 4-Chloro-2-(2-(diethylamino)ethylthio)-6-(4-fluorophenyl)pyrimidine-5carbonitrile hydrochloride (3) Phosphorous oxychloride (1.3 g, 8.6 mmol) was added to compound 2 (0.3 g, 0.86 mmol) in a nitrogen atmosphere and heated at 80 C for 5 h. Reaction completion was monitored by TLC. The reaction mixture was cooled to room temperature and added to crushed ice in a beaker with constant stirring. White solid was precipitated and filtered through vacuum in a nitrogen atmosphere. Filtered product was taken to the next step immediately. General procedure for title compounds (4a–m) Compound 3 (0.14 g, 0.349 mmol) was dissolved in dried THF (10 mL) followed by addition of triethylamine (0.105 g, 1.0 mmol) and amines (0.418 mmol); the reaction mixture was stirred at reflux for 4 h. Reaction completion was monitored by TLC. The reaction mixture was evaporated to dryness and diluted with water and extracted with ethyl acetate (3 9 10 ml). The organic layer was evaporated to dryness and the crude product was purified using a silicagel column with chloroform and methanol (9:1) as the eluent. Pure product was obtained with a yield of 50–80 %. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(4-methylpiperazin-1yl)pyrimidine-5-carbonitrile (4a) Reddish brown semi-solid (0.11 g, yield 70 %), mp: 155–160 C; IR (KBr) mmax/cm-1: 3414.00, 2935.66, 2856.58, 2206.57, 1598.99, 1535.34, 1512.19, 1479.40, 1313.52, 1278.81, 1226.73, 1159.22, 1134.14, 1111.00, 1068.56, 1043.49, 979.84, 954.76, 844.82; 1H NMR (CDCl3, 400 MHz) d: 1.01(6H, t, J = 7.1 Hz), 2.32 (3H, s), 2.52 (4H, t, J = 4.5 Hz), 2.59 (4H, q, J = 7.0 Hz), 2.80 (2H, t, J = 8.0 Hz), 3.23 (2H, t, J = 7.2 Hz), 3.99 (4H, t, J = 4.2 Hz), 7.13 (2H, t, J = 8.4 Hz), 7.86 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 11.78, 22.78, 28.88, 46.00, 47.09, 47.14, 52.37, 54.86, 83.50, 115.58, 115.79, 118.44, 131.65, 131.74, 132.44, 132.47, 173.77, 162.35, 163.41 (d, J = 251.7 Hz, C–F), 169.79; 19F NMR (CDCl3, 376 MHz) d: -108.39; HRMS (EI) (m/z) calcd for C22H29FN6S 428.2158, found 428.2152. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(piperidin-1yl)pyrimidine-5-carbonitrile (4b) Pale brown solid (0.10 g, yield 70 %), mp: 170–175 C; IR (KBr) mmax/cm-1: 3414.00, 2935.66, 2856.58, 2206.57, 1598.99, 1535.34, 1512.19, 1479.40, 1313.52,
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1278.81, 1226.73, 1159.22, 1134.14, 1111.00, 1068.56, 1043.49, 979.84, 954.76, 844.82; 1H NMR (CDCl3, 400 MHz) d: 1.01 (6H, t, J = 7.1 Hz), 1.73 (6H, s), 2.60 (4H, q, J = 7.0 Hz), 2.81 (2H, t, J = 8.0 Hz), 3.23 (2H, t, J = 7.2 Hz), 3.91 (4H, t, J = 4.2 Hz), 7.14 (2H, t, J = 8.5 Hz), 7.89 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 24.34, 25.99, 47.04, 48.55, 52.48, 83.03, 115.39, 115.61, 118.49, 131.59, 131.68, 132.58, 132.61, 162.10, 163.25(d, J = 251.8 Hz, C–F), 169.62, 173.51; 19F NMR (CDCl3, 376 MHz) d: -108.68; HRMS (EI) (m/z) calcd for C22H28FN5S 413.2049, found 413.2050. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-morpholinopyrimidine5-carbonitrile (4c) Pale brown solid (0.10 g, yield 70 %), mp: 180–190 C; IR (KBr) mmax/cm-1: 3130.47, 2970.38, 2860.43, 2804.50, 2580.76, 2455.38, 2208.49, 1604.77, 1535.34, 1512.19, 1402.25, 1384.89, 1313.52, 1226.73, 1159.22, 1128.36, 987.55, 844.82, 567.07; 1H NMR (CDCl3, 400 MHz) d: 1.01 (6H, t, J = 7.1 Hz), 2.59 (4H, q, J = 7.0 Hz), 2.80 (2H, t, J = 8.0 Hz), 3.23 (2H, t, J = 7.2 Hz), 3.79 (4H, q, J = 4.6 Hz), 3.96 (4H, t, J = 4.2 Hz), 7.14 (2H, t, J = 8.5 Hz), 7.87 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 11.76, 28.69, 47.06, 47.54, 52.28, 66.66, 83.64, 115.79, 118.25, 132.30, 132.33, 162.59, 163.42 (d, J = 250.4 Hz, C– F), 169.72, 173.97; 19F NMR (CDCl3, 376 MHz) d: -108.16; HRMS (EI) (m/z) calcd for C21H26FN5OS 415.1842, found 415.1843. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(4methoxyphenylamino)pyrimidine-5-carbonitrile (4d) Pale yellow solid (0.11 g, yield 70 %), mp: 145–150 C; IR (KBr) mmax/cm-1: 3298.28, 2968.45, 2808.36, 1604.77, 1570.06, 1543.05, 1510.26, 1485.19, 1398.39, 1311.59, 1236.37, 1149.57, 844.82, 786.96, 646.15; 1H NMR (CDCl3, 400 MHz) d: 0.95 (6H, t, J = 7.2 Hz), 2.47 (4H, q, J = 6.8 Hz), 2.71 (2H, t, J = 7.9 Hz), 3.18 (2H, t, J = 7.2 Hz), 3.83 (3H, m), 6.92 (2H, d, J = 8.8 Hz), 7.19 (2H, t, J = 8.5 Hz), 7.24 (1H, s), 7.45 (2H, d, J = 8.8 Hz), 8.02 (2H, q, J = 5.2 Hz); 13C NMR (CDCl3, 100 MHz) d: 11.57, 23.30, 29.98, 30.31, 30.79, 32.04, 32.54, 47.22, 52.63, 56.12, 84.42, 114.89, 116.37, 116.58, 117.13, 125.38, 130.01, 131.59, 131.68, 132.49, 158.23, 161.07, 166.97; 19F NMR (CDCl3, 376 MHz) d: -107.92; HRMS (EI) (m/z) calcd for C24H26FN5OS 451.1842, found 451.1836. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(3(trifluoromethyl)phenylamino)pyrimidine-5-carbonitrile (4e) Pale yellow solid (0.10 g, yield 60 %), mp: 168–170 C; IR (KBr) mmax/cm-1: 3315.63, 2970.38, 2802.57, 2208.49, 1604.77, 1577.77, 1541.12, 1475.54, 1332.81, 1234.44, 1112.93, 786.93, 692.44; 1H NMR (CDCl3, 400 MHz) d: 0.96 (6H, t, J = 7.2 Hz), 2.53 (4H, q, J = 6.8 Hz), 2.76 (2H, t, J = 7.8 Hz), 3.27 (2H, t, J = 7.2 Hz), 7.21 (2H, t, J = 8.5 Hz), 7.44 (1H, d, J = 6.0 Hz), 7.47 (1H, s), 7.53 (1H, t, J = 7.6 Hz), 7.74 (1H, d, J = 8.0 Hz), 8.05 (2H, q, J = 5.2 Hz); 13C NMR
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(CDCl3, 100 MHz) d: 12.06, 47.38, 52.17, 84.85, 116.43, 116.65, 119.62, 122.44, 122.47, 123.00, 125.76, 130.28, 131.66, 131.75, 131.95, 132.25, 132.58, 138.09, 160.63, 164.18 (d, J = 253.2 Hz, C–F), 167.05, 176.21; 19F NMR (CDCl3, 376 MHz) d: -62.72, 107.34; HRMS (EI) (m/z) calcd for C24H23F4N5S 489.1610, found 489.1612. 2-(2-(Diethylamino)ethylthio)-4-(dimethylamino)-6-(4fluorophenyl)pyrimidine-5-carbonitrile (4f) Pale yellow solid (0.07 g, yield 60 %), mp: 190–195 C; IR (KBr) mmax/cm-1: 3446.79, 3421.72, 2972.31, 2202.71, 1560.41, 1517.98, 1485.19, 1425.40, 1404.18, 1313.52, 1273.02, 1226.73, 1134.14, 1004.91, 825.54, 792.74, 731.02, 572.86; 1H NMR (CDCl3, 400 MHz) d: 1.02 (6H, t, J = 7.1 Hz), 2.60 (4H, q, J = 7.0 Hz), 2.80 (2H, t, J = 8.0 Hz), 3.23 (2H, t, J = 7.2 Hz), 3.39 (6H, s), 7.15 (2H, t, J = 8.4 Hz), 7.87 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.03, 28.85, 40.39, 47.17, 52.53, 82.35, 115.52, 115.73, 118.81, 131.65, 131.74, 132.70, 162.17, 163.34 (d, J = 250 Hz, C–F), 169.78, 173.43; 19F NMR (CDCl3, 376 MHz) d: -108.79; HRMS (EI) (m/z) calcd for C19H24FN5S 373.1736, found 373.1732. 4-(3-Chlorobenzylamino)-2-(2-(diethylamino)ethylthio)-6-(4fluorophenyl)pyrimidine-5-carbonitrile (4g) Pale yellow solid (0.09 g, yield 60 %), mp: 250–280 C; IR (KBr) mmax/cm-1: 3446.79, 3340.71, 2974.23, 2210.42, 1595.13, 1546.91, 1489.05, 1433.11, 1396.46, 1315.45, 1242.16, 1161.15, 1138.00, 1066.64, 937.40, 844.82, 788.89, 700.16; 1H NMR (CDCl3, 400 MHz) d: 0.99 (6H, t, J = 6.3 Hz), 2.56 (4H, q, J = 7.0 Hz), 2.78 (2H, t, J = 8.0 Hz), 3.26 (2H, t, J = 7.2 Hz), 4.76 (2H, d, J = 4.6 Hz), 6.07 (1H, br s), 7.17 (2H, t, J = 8.4 Hz), 7.21 (1H, s), 7.28 (2H, d, J = 15.8 Hz), 7.99 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.09, 29.12, 44.71, 47.11, 52.66, 83.32, 115.77, 115.99, 116.72, 125.86, 127.92, 128.19, 130.25, 131.04, 131.13, 132.02, 132.05, 134.84, 139.45, 161.84, 163.50 (d, J = 243.6 Hz, C–F), 166.01, 175.4; 19F NMR (CDCl3, 376 MHz) d: -107.97; HRMS (EI) (m/z) calcd for C24H25ClFN5S 469.1503, found 469.1500. 4-(2-(1H-indol-2-yl)ethylamino)-2-(2-(diethylamino)ethylthio)-6-(4fluorophenyl)pyrimidine-5-carbonitrile (4h) Yellow solid (0.10 g, yield 60 %), mp: 185–190 C; IR (KBr) mmax/cm-1: 3340.71, 2968.45, 2210.42, 1587.42, 1544.98, 1517.98, 1489.05, 1433.11, 1396.46, 1313.52, 1236.37, 1159.22, 1072.42, 954.76, 844.82, 788.89, 738.74, 640.37; 1H NMR (CDCl3, 400 MHz) d: 1.01 (6H, t, J = 7.1 Hz), 2.60 (4H, q, J = 7.1 Hz), 2.82 (2H, q, J = 5.6 Hz), 3.12 (2H, t, J = 6.7 Hz), 3.26 (2H, q, J = 7.4 Hz), 3.89 (2H, q, J = 6.6 Hz), 5.75 (1H, t, J = 5.2 Hz), 7.13 (4H, m), 7.23 (1H, d, J = 7.2 Hz), 7.40 (1H, d, J = 8.04 Hz), 7.65 (1H, d, J = 7.8 Hz), 7.95 (2H, m), 8.12 (1H, s); 13C NMR (CDCl3, 100 MHz) d: 12.02, 25.16, 28.81, 41.51, 47.14, 52.51, 83.33, 111.51, 112.20, 115.67, 116.73, 118.69, 119.72, 122.38, 122.50, 127.11, 130.96, 131.05,
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132.17, 132.20, 136.64, 161.75, 163.37 (d, J = 253.2 Hz, C–F), 165.64, 175.11; 19F NMR (CDCl3, 376 MHz) d: -108.31; HRMS (EI) (m/z) calcd for C27H29FN6S 488.2158, found 488.2155. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(2-oxoazepan-3ylamino)pyrimidine-5-carbonitrile (4i) Pale yellow solid (0.07 g, yield 50 %), mp: 250–280 C; IR (KBr) mmax/cm-1: 2968.45, 2931.80, 2204.64, 1672.28, 1598.99, 1564.27, 1541.12, 1490.97, 1390.68, 1313.52, 1238.30, 1159.22, 1068.56, 844.82, 788.89, 572.66; 1H NMR (CDCl3, 400 MHz) d: 1.02 (6H, t, J = 7.1 Hz), 1.54 (2H, m), 1.88 (2H, m), 2.09 (1H, d, J = 3.3 Hz), 2.19 (1H, d, J = 12.8), 2.61 (4H, q, J = 6.8 Hz), 2.80 (2H, t, J = 6.6 Hz), 3.23 (1H, m), 3.34 (3H, m), 4.78 (1H, q, J = 4.6 Hz), 6.29 (1H, t, J = 6.2 Hz), 7.17 (2H, t, J = 8.4 Hz), 7.24 (2H, t, J = 5.5 Hz), 7.99 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 11.39, 27.93, 28.95, 29.81, 30.99, 42.22, 47.05, 52.21, 53.73, 84.41, 115.73, 115.95, 116.18, 131.06, 131.15, 132.16, 132.19,160.15, 163.42 (d, J = 249.2 Hz, C–F), 165.93, 174.42, 174.84; 19F NMR (CDCl3, 376 MHz) d: -108.32; HRMS (EI) (m/z) calcd for C23H29FN6OS 456.2108, found 456.2106. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(pyridin-2ylmethylamino)pyrimidine-5-carbonitrile (4j) Pale yellow solid (0.07 g, yield 50 %), mp: 195–200 C; IR (KBr) mmax/cm-1: 3354.21, 3120.82, 2972.31, 2818.00, 2206.57, 1585.49, 1564.27, 1541.12, 1517.98, 1490.97, 1479.40, 1396.46, 1313.52, 1232.51, 1159.22, 1138.22, 999.13, 864.11; 1H NMR (CDCl3, 400 MHz) d: 1.01 (6H, s), 2.62 (4H, s), 2.84 (2H, s), 3.29 (2H, s), 4.85 (2H, s), 7.14 (2H, s), 7.25 (2H, d, J = 15.2), 7.70 (1H, s), 8.00 (2H, s), 8.61 (1H, s); 13C NMR (CDCl3, 100 MHz) d: 11.75, 45.76, 47.08, 52.36, 83.77, 115.64, 115.85, 116.48, 121.81, 122.68, 130.97, 131.05, 132.09, 136.87, 149.27, 154.90, 161.42, 163.33 (d, J = 253.2 Hz, C–F), 165.84; 19F NMR (CDCl3, 376 MHz) d: -108.28; HRMS (EI) (m/z) calcd for C23H25FN6S 436.1845, found 436.1845. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6(methylamino)pyrimidine-5-carbonitrile (4k) Pale yellow solid (0.10 g, yield 80 %), mp: 155–160 C; IR (KBr) mmax/cm-1: 3363.86, 2968.45, 2798.71, 2210.42, 1600.92, 1550.77, 1492.90, 1435.04, 1394.53, 1321.24, 1280.73, 1238.30, 1161.15, 1124.50, 954.76, 837.11, 786.99, 570.93; 1H NMR (CDCl3, 400 MHz) d: 1.03 (6H, t, J = 7.1 Hz), 1.28 (3H, t, J = 7.2 Hz), 2.62 (4H, q, J = 7.0 Hz), 2.82 (2H, t, J = 7.6 Hz), 3.12 (3H, d, J = 4.5 Hz), 3.27 (2H, t, J = 7.2 Hz), 5.75 (1H, s), 7.15 (2H, t, J = 8.4 Hz), 7.97 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.02, 29.78, 47.18, 52.51, 83.32, 115.72, 115.94, 116.93, 131.00, 131.09, 132.15, 132.18, 162.44, 163.42 (d, J = 251.7 Hz, C–F),
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165.58, 175.13; 19F NMR (CDCl3, 376 MHz) d: -108.26; HRMS (EI) (m/z) calcd for C18H22FN5S 359.1580, found 359.1580. 2-(2-(Diethylamino)ethylthio)-4-(ethylamino)-6-(4-fluorophenyl)pyrimidine5-carbonitrile (4l) Pale yellow solid (0.07 g, yield 60 %), mp: 180–185 C; IR (KBr) mmax/cm-1: 3446.79, 3332.99, 2968.45, 2929.87, 2810.28, 2214.28, 1593.20, 1546.91, 1517.98, 1489.05, 1427.32, 1309.67, 1236.37, 1155.36, 1105.21, 1066.64, 999.13, 842.89, 788.89, 646.15, 565.14; 1H NMR (CDCl3, 400 MHz) d: 1.01 (6H, t, J = 7.1 Hz), 1.28 (3H, t, J = 7.2 Hz), 2.61 (4H, q, J = 7.0 Hz), 2.81 (2H, t, J = 8.0 Hz), 3.25 (2H, t, J = 7.2 Hz), 3.62 (2H, m), 5.6 (1H, s), 7.15 (2H, t, J = 8.4 Hz), 7.96 (2H, q, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.08, 28.82, 36.59, 47.19, 52.58, 83.19, 115.72, 115.94, 116.96, 130.08, 131.08, 132.22, 132.25, 161.78, 163.42 (d, J = 251.7 Hz, C–F), 165.73, 175.16; 19F NMR (CDCl3, 376 MHz) d: -108.34; HRMS (EI) (m/z) calcd for C19H24FN5S 373.1736, found 373.1736. 2-(2-(Diethylamino)ethylthio)-4-(4-fluorophenyl)-6-(octylamino)pyrimidine5-carbonitrile (4m) Pale yellow solid (0.09 g, yield 60 %), mp: 195–200 C; IR (KBr) mmax/cm-1: 3334.92, 2920.23, 2852.72, 2214.28, 1587.42, 1544.98, 1517.98, 1485.19, 1315.45, 1155.33, 842.89, 788.89, 648.08; 1H NMR (CDCl3, 400 MHz) d: 0.87 (3H, t, J = 6.2 Hz), 1.03 (6H, t, J = 7.1 Hz), 1.33 (8H, m), 1.65 (2H, m), 2.61 (4H, q, J = 7.0 Hz), 2.82 (2H, t, J = 7.8 Hz), 3.26 (2H, t, J = 7.2 Hz), 3.58 (2H, q, J = 6.7 Hz), 5.62 (1H, s), 7.16 (2H, t, J = 8.5 Hz), 7.98 (2H, q, J = 5.5 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.11, 22.83, 27.01, 32.07, 41.71, 47.20, 52.60, 83.18, 115.73, 115.95, 116.98, 131.00, 131.09, 132.24, 132.28, 161.90, 163.44 (d, J = 251.6 Hz, C–F), 165.71, 175.16; 19F NMR (CDCl3, 376 MHz) d: -108.34; HRMS (EI) (m/z) calcd for C25H36FN5S 457.2675, found 457.2670. Biological experiments Cytotoxicity assay The cytotoxicity of all the synthesized compounds was screened against EAT cells. EAT cells were seeded in six well plates and incubated with 100 lM of test samples (4a–m) for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), and 1 % penicillin–streptomycin in a humidified incubator with 5 % CO2 at 37 C. Cell viability was checked using a trypan blue dye exclusion method. MTX was also screened against EAT cells. After the treatment of compounds with EAT cells, cells were analyzed for their proliferation and survival. The % cell viability was determined using the following formula and a graph was plotted between % cell viability and concentration. From
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Synthesis of a new series of pyrimidine derivatives…
Scheme 1 Synthetic route for synthesis of compound 4 (a–m)
this plot, an IC50 value was calculated. % cell viability = (viable cells (unstained)/total number of cells) 9 100.
Results and discussion Synthesis The Biginelli-type multicomponent reaction of 4-fluorobenzaldehyde with thiourea and ethylcyanoacetate in the presence of potassium carbonate as a base in ethanol [11, 33, 34] gave 4-(4-fluorophenyl)-6-hydroxy-2-mercaptopyrimidine-5-carbonitrile (1) (Scheme 1). The selective S-alkylation of compound (1) could not be achieved using 2-bromo (N,N-diethylamino)ethyl hydrobromide in solvents like DMF and THF with a weak base like K2CO3 and triethylamine (Table 1), but when the same reaction was performed by changing the solvent to DMSO at room temperature for 8 h it gave compound 2 at a yield of 90 %. Then, the conversion of the hydroxyl group in compound (2) to chlorination by treating 2 with phosphorous oxy chloride at 70 C for 5 h yielded compound (3), which was used in the ensuing step without purification. When compound (3) was then refluxed for 4 h with various aliphatic, substituted aromatic and heterocyclic amines in the presence of triethanolamine (TEA) as a base in dried THF as solvent under nitrogen atmosphere afforded compounds 4 (a–m) at a yield of 50–80 % (Table 2). All the newly synthesized 4 compounds (a–m) were confirmed by IR, 1H NMR, 13 C NMR, 19F NMR, distortionless enhancement by polarization transfer (DEPT) and HRMS spectral data, which were in full agreement with their structures. In the IR spectrum, absorption bands at 2210 and 3330 cm-1 confirmed the presence of cyano and N–H functional groups, respectively [35]. The 1H NMR spectrum of
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N. Senthilkumar et al. Table 1 Reaction conditions and solvent screening for synthesis of compound 2
Entry
Base
Solvent
Temperature (C)
Time
Yield (%)
1
K2CO3
DMF
RT
24 h
25
2
K2CO3
DMF
Reflux
24 h
40
3
TEA
DMF
Reflux
24 h
30
4
TEA
THF
RT
24 h
20
5
TEA
THF
Reflux
10 h
35
6
TEA
THF
Reflux
24 h
35
7
K2CO3
DMSO
RT
24 h
40
8
TEA
DMSO
RT
8h
90
Reaction conditions: compound 1 (12.8 mmol) in 10 mL of DMSO, stirring in triethylamine (38 mmol) for 15 min. 2-bromo-N,N-diethyl-ethanamine hydrobromide (12.8 mmol), 8 h at RT
compound 4f was taken as an example of the prepared series 4 (a–m). A methylene proton of ethylthio (–SCH2 CH2–) in position 2 of pyrimidine resonated in the region of d 3.2–2.8 (t) [36], methyl and methylene protons of diethyl amino (–NCH2CH3) resonated at d 1.0 (t) and 2.6 (q), respectively [37]. In addition, phenyl protons showed two signals in position 4 of pyrimidine, and a signal at d 7.9 (q) indicated the coupling of two aromatic equivalent protons with fluorine in the p position of the phenyl ring. The signal at d 7.1 (t) confirmed two decoupled protons [34], and a singlet at d 3.3 confirmed two methyl protons of the N,N- dimethyl group in position 6 of pyrimidine [38]. 13C spectrum confirmed the presence of 19 carbons. In the DEPT spectrum of compound 4f, the signals at d 47.0, 52.4 and 28.7 on the negative axis confirmed the carbons of ethyl thio (–SCH2 CH2–) and –NCH2 as methylene, respectively. Further, the signals on the positive axis at d 12 and 40.2 confirmed the carbons to be methyls of –NCH2CH3 and N,N-dimethyl, respectively. The signals at d 115 and 131 on the positive axis also confirmed the carbons in the phenyl ring to be CH. The absence of signals at d 162–173 confirmed the carbons in pyrimidine and fluorine-substituted carbon in the phenyl ring and the carbon connecting the phenyl with the pyrimidine as quaternary carbons. The number of protons, carbons and fluorines corresponding to compound 4f are shown below (Fig. 1). Above all, HRMS authenticated the structure of 4f.
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Synthesis of a new series of pyrimidine derivatives…
Fig. 1 Selected 1H,
13
C and
19
F NMR chemical shifts of compound 4f
Fig. 2 Effect of compound 4 (a–m) on EAT cell growth and viability
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N. Senthilkumar et al. Table 2 Synthesized target molecules of compounds 4 (a–m)a with yield and melting points R-H Product
Yield (%)b
(° C)
(R)
123
m.p
4a
70
155-160
4b
70
170-175
4c
70
180-190
4d
70
145-150
4e
60
168-170
4f
60
190-195
4g
60
250-280
4h
60
185-190
Synthesis of a new series of pyrimidine derivatives… Table 2 continued
R-H Product
Yield (%)b
m.p (° C)
(R)
4i
50
250-280
4j
50
195-200
4k
80
155-160
4l
60
180-185
4m
60
195-200
a
Reaction conditions: compound 3 (0.349 mmol) was dissolved in dried THF (10 mL) followed by addition of triethylamine (1.0 mmol) and amines (0.418 mmol) heated at reflux for 4 h
b
Isolated Yield
Biological activity In vitro cytoxicity on EAT cell lines All the synthesized compounds were evaluated for their cytotoxic activity against EAT cells and compared with the standard drug MTX. Representation of percentage cell viability of all the compounds against EAT cells with MTX at a concentration of 100 lM is illustrated in Fig. 2. Based on IC50, compound 4h exhibited stronger cytotoxic activity against EAT cells with an IC50 value of 5.2 lM, which was almost comparable with the standard drug MTX with an IC50 value of 3.6 lM. Further, compounds 4c, 4d, 4g, 4k, and 4m showed substantial activity with IC50 values of 18.7 lM, 17.6 lM, 15.8 lM, 19.7 lM, and 16.8 lM against EAT cells, respectively. Compounds 4a, 4b, 4e, 4f, 4i, 4j, and 4l showed moderate cytotoxicity against EAT cells with IC50 values of 32.8 lM, 25.2 lM, 28.3 lM, 30.8 lM, 32.6 lM, 25.8 lM, and 22.8 lM, respectively (Table 3).
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Fig. 3 Role of substituents in increasing cytotoxicity
Table 3 Percentage cell viability with IC50 of compounds 4 (a–m) along with ClogP and MR Entry
Compounds
% cell viability (IC50 lM)
1
4a
2
4b
3
Clog P
MR
70 (32.8)
4.27
125
58 (25.2)
5.21
121
4c
48 (18.7)
3.82
118
4
4d
47 (17.6)
6.38
129
5
4e
57 (28.3)
7.36
128
6
4f
60 (30.8)
4.53
109
7
4g
45 (15.8)
6.64
132
8
4h
18 (5.2)
6.56
142
9
4i
65 (32.6)
4.45
128
10
4j
57 (25.8)
4.43
125
11
4k
47 (19.7)
4.48
103
12
4l
50 (22.8)
5.01
108
13
4m
45 (16.8)
8.18
135
14
Methotrexate
12 (3.6)
-0.52
118
This study revealed that compounds 4h, 4d, 4g, 4k, and 4m exhibited significant cytotoxicity against EAT cells due to the presence of a cyano group and hydrogen bond donating ability of aliphatic amines present in these compounds (Fig. 3). This may be due to the stronger electron withdrawing ability of the cyano group [39] and other parameters, including the hydrogen bond donating ability of aliphatic and aromatic hydrophobic sites that are necessary for the optimal interaction with specific receptors [12, 27]. To correlate the effect of substitution with cytotoxicity, the lipophilicity (ClogP) of all the synthesized compounds was examined. Compounds 4h, 4d, 4g, and 4m showed higher ClogP values (Table 3, calculated using ChemDraw Ultra 11.0 software). To explain the activity behavior of synthesized compounds, the molar refractivity (MR), which in turn represents size and polarizability of a molecule describing steric effects, were calculated (ChemDraw Ultra 11.0 software). The MR values are shown in Table 3. The higher the lipophilicity and molar refractivity of compounds 4d (ClogP = 6.38, MR = 129), 4g (ClogP = 6.64, MR = 132), 4h (ClogP = 6.56, MR = 142), and 4m (ClogP = 8.18, MR = 135), the higher the inhibitory activity against EAT cells
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Synthesis of a new series of pyrimidine derivatives… Table 4 Interaction of ligands with CK2 Ligand
Binding energy (kcal/mol)
Ligand efficiency (kcal/mol)
H-bond formed
H-bond residues
H-bond distance
4a
-7.47
-0.25
2
(Lys68) Nz-N30
3.6
4b
-7.07
-0.24
1
(Asp175) NH-OD1
1.8
(Val116) N-N15
2.9
4c
-7.27
-0.21
1
(Val116) N-N21
3.3
4d
-7.88
-0.25
1
(Asp175) OD1-NH
1.8
4e
-6.68
-0.2
2
(Val116) O-H39
2.7
4f
-6.07
-0.23
1
(Val116) N-N27
3.1
(Leu45) O-NH
1.9
4g
-7.09
-0.23
1
(His160) NE2-N21
3.5
4h
-8.7
-0.3
2
(Lys64) N-N36
3.2
(Lys64) O-N21
2.6
4i
-7.98
-0.25
1
(Asn189) OD1-N27
3.3
4j
-8.2
-0.26
1
(Asp175) OD1-N21
3.2
4k
-6.32
-0.25
2
(Val116) N-N25
2.9
(Val116) O-H44
2.6
4l
-6.32
-0.25
2
(Val116) O-H45
2.3
(Val116) N-N26
3.0
4m
-5.41
-0.17
1
(Leu45) O-NH
2.1
[40–44]. Furthermore, the theoretical results are in good correlation with the in vitro cytotoxic activity in most of the compounds. Molecular docking In order to acquire a consistent and more precise picture of the biologically active molecules at the atomic level and to provide new insights and to establish the strength of association or binding between two molecules, molecular docking and the knowledge of the preferred orientation is essential. Hence, in order to predict their binding modes to CK2 and to get deeper insight into the inhibitory potency of the newly synthesized pyrimidine compounds, molecular docking studies were performed using Autodock 4.0 software. All the synthesized compounds were docked by CK2 (Pdb: 3OWJ). From the docking analysis, the binding mode of compounds based on binding energy was calculated and listed (Table 4). The binding conformation for each ligand molecule into the CK2 target protein was determined and the one having the lowest binding energy with CK2 among the different conformations was generated. The lower energy scores represent better protein–ligand binding affinity compared to higher energy values [45, 46]. Among all the compounds, compound 4h and 4j showed lower binding energy than other compounds. Compound 4h had the least binding energy value with CK2 (binding energy value = -8.7 kcal/mol), and compound 4j had a binding energy value of -8.2 kcal/mol. Docked conformation
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Fig. 4 a Binding mode of compound 4h into the cavity of CK2. b A close-up view of the binding mode. c Close-up view of ribbon model. d Interaction of compound 4h with CK2. Ligand atoms are colored by type. The interacted amino acids residues, hydrogen bond networks in the binding pocket and the distance (in Angstrom units) of bonds are all shown
was analyzed further for finding the binding mode of compound 4h and 4j into CK2 to validate the position obtained likely to represent reasonable binding conformations. Interaction analysis of the binding mode of compound 4h in CK2 revealed that it formed two hydrogen bonds between the Lys64 group in pyrimidine and N ˚ , and another hydrogen bond was atom of Lys64 with the bond distance 3.2 A observed between the nitrogen atom of the NH group in pyrimidine and the oxygen ˚ (Fig. 4). From analysis of the binding atom of Lys64 with the bond distance 2.6 A modes of compound 4j in CK2, it was found to be due to the formation of an H-bond by the interaction of NH of the ligand and oxygen atom of Asp175 with the ˚ ) (Fig. 5). Molecular modeling studies correlated well with bond distance 3.2 A pharmacological activity. In this regards, compound 4h showed the maximum cytotoxicity against EAT cells and the lowest binding energy and good ligand
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Synthesis of a new series of pyrimidine derivatives…
Fig. 5 a Binding mode of compound 4j into the cavity of CK2. b A close-up view of the binding mode. c Close-up view of ribbon model. d Interaction of compound 4j with CK2. Ligand atoms are colored by type. The interacted amino acids residues, hydrogen bond networks in the binding pocket and the distance (in Angstrom units) of bonds are all shown
efficiency with CK2 compared to all other compounds. The results of the docking studies were in good agreement with in vitro anticancer activity.
Conclusion In conclusion, a new series of pyrimidine derivatives was synthesized by introducing various amines at the 6-position of pyrimidine. Compound 4h exhibited stronger cytotoxic activity against EAT cells with an IC50 value of 5.2 lM, which was almost comparable with the positive control MTX with an IC50 value of 3.6 lM. Further, compounds 4c, 4d, 4g, 4k, and 4m showed substantial activity
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with IC50 values of 18.7 lM, 17.6 lM, 15.8 lM, 19.7 lM, and 16.8 lM against EAT cell, respectively. 4h has the maximum cytotoxicity against EAT cells, the lowest binding energy (-8.7 kcal/mol) and good ligand efficiency with CK2 compared to all other compounds. The studies revealed that the cytotoxicity of compounds against EAT cell lines were mainly governed by lipophilicity and molar refractivity. The in vitro results and binding interactions from docking studies exhibited good correlation, which, in turn, indicated compound 4h to be a potential candidate with good anti-proliferative activity. Acknowledgments The authors thank the management of VIT University, Vellore for all the support and encouragement. In addition the support from SAIF, School of Advanced Sciences, VIT University, Vellore and DST-FIST is greatly acknowledged for the spectral analysis.
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