Preparatory materials for the research work were obtained from commercial sources i.e. Loba Chemie, Pvt Ltd. Mumbai, India; Central Drug House (CDH) Pvt. Ltd., New Delhi, India and HiMedia Laboratory Pvt. Ltd., Delhi, India, used without further purification. All reactions were monitored by thin-layer chromatography on 0.25 mm silica gel (Merck) plates, using benzene as mobile phase and spots were observed by exposure to iodine vapours or visualized with UV light. Melting points of synthesized compounds was determined in open capillary tube. An infrared spectrum was recorded (KBr-pellets) in Bruker 12060280, Software: OPUS 7.2.139.1294 spectrometer. 1H-NMR and 13C-NMR were recorded at 600 and 150 MHz, respectively on Bruker Avance III 600 NMR spectrometer by appropriate deuterated solvents. The results are conveyed in parts per million (δ, ppm) downfield from tetramethyl silane (internal standard). 1H-NMR spectral details of the synthesized derivatives are represented with multiplicity like singlet (s); doublet (d); triplet (t); multiplet (m) and the number hydrogen ion. Elemental analysis of the new synthesized compounds was obtained by Perkin–Elmer 2400 C, H and N analyzer. All the compounds gave C, H and N analysis within ±0.4% of the theoretical results. Mass spectra were taken on Waters Micromass Q-ToF Micro instrument.
General procedure of the synthesized compounds
Step a: synthesis of 3,3′-(1,4-phenylene)bis(1-(4-hydroxyphenyl)prop-2-en-1-one (intermediate-I)
The reaction mixture of 1-(4-hydroxyphenyl)ethanone (0.02 mol) and terephthalaldehyde (0.01 mol) were stirred for 2–3 h in methanol (5–10 mL) followed by drop wise addition of sodium hydroxide solution (10 mL 40%) with constant stirring at room temperature till a dark yellow mass was obtained. Then reaction mixture was allowed to stand overnight at room temperature and then poured into icecold water and acidified with hydrochloric acid and the precipitated 3,3′-(1,4-phenylene)bis(1-(4-hydroxyphenyl)prop-2-en-1-one was filtered, dried and recrystallized from methanol [23].
Step b: synthesis of 4,4′-(6,6′-(1,4-phenylene)bis(2-aminopyrimidine-6,4-diyl))diphenol (intermediate-II)
The solution of 3,3′-(1,4-phenylene)bis(1-(4-hydroxyphenyl)prop-2-en-1-one (0.01 mol) (synthesized in previous step-a) in methanol (80 mL) was added with 0.01 mol of potassium hydroxide and 40 mL of 0.50 M solution of guanidine hydrochloride and refluxed for 5–6 h. The reaction mixture was then cooled and acidified with few drops of hydrochloric acid (20 mL of 0.5 M solution) and the resultant precipitate of 4,4′-(6,6′-(1,4-phenylene)bis(2-amino pyrimidine-6,4-diyl))diphenol was separated out dried and recrystallized from methanol [24].
Step c: synthesis of bis-pyrimidine Schiff bases (q1–q20) from intermediate-II (4,4′-(6,6′-(1,4-phenylene)bis(2-aminopyrimidine-6,4-diyl))diphenol)
A mixture of 4,4′-(6,6′-(1,4-phenylene)bis(2-aminopyrimidine-6,4-diyl))diphenol (0.01 mol) (synthesized in previous step-b) and 0.02 mol of substituted aldehyde was refluxed for 3–4 h in methanol with few drops of glacial acetic acid. The reaction mixture was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was poured into ice cold water and the precipitated title compound was filtered, dried and recrystallized from methanol.
Spectral characteristic of the synthesized bis-pyrimidine compounds (q1–q20) and intermediate (I and II)
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(4-hydroxyphenyl)prop-2-en-1-one) (I)
FT-IR (K–Br, cm−1): 3088 (C–H str.), 1427 (C=C str.), 1693 (C=O str.), 1497 (C=C st., alkyl chain), 2864 (C–H sym. str., alkyl chain), 3363 (O–H str., Ar–OH); 1H-NMR (DMSO-d
6): 6.91–7.63 (m, 12H, Ar–H), 5.23 (s, 2H, Ar–OH), 7.59 (d, 2H, CH), 8.06 (d, 2H, CH); 13C-NMR (δ, DMSO-d
6): 192.92, 164.30, 145.1, 139.69, 134.78, 131.72, 130.21, 129.91, 116.21.
4,4′-(6,6′-(1,4-Phenylene)bis(2-aminopyrimidine-6,4-diyl))diphenol (II)
FT-IR (K–Br, cm−1): 3058 (C–H str.), 1537 (C=C str.), 1604 (C=N str. pyrimidine), 3331 (C–N str. –NH2), 1388 (C–O st. and OH in pbv); 1H-NMR (DMSO-d6): 7.37–7.53 (m, 12H, Ar–H), 7.26 (s, 2H, CH of pyrimidine), 3.86 (s, 2H, C–NH2); 13C-NMR (δ, DMSO-d
6): 193.00, 192.67, 167.12, 139.34, 139.71, 129.71, 129.94, 129.77, 129.30, 128.16, 126.61, 116.16.
1,1′-((1E,1′E)-((6,6′-(1,4-Phenylene)bis(4-(4-hydroxyphenyl)pyrimidinediyl))bis (azanylylidene))bis(methanylylidene))bis(naphthalen-2-ol) (q1)
Light greenish crystals; Yield: 70.45%; mp: 90–92 °C; R
f
value: 0.55; IR (KBr, cm−1): 2927 (C–H str.), 1594 (C=C str.), 1698 (N=CH str.), 1313 (C–N str.), 3359 (O–H str.); 1H-NMR (δ, DMSO-d
6): 6.96–7.87 (m, 24H, Ar–H), 8.11 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 163.9, 118.7, 128.7, 124.1, 131.6, 129.2, 118.7, 122.1, 127.5, 112.4; CHN: Calc. C48H32N6O4: C, 76.18; H, 4.26; N, 11.10; Found: C, 76.13; H, 4.24; N, 11.12; MS ES + (ToF): m/z 758 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(3,4,5-trimethoxybenzylidene)amino)pyrimidine-6,4-diyl))diphenol (q2)
Dark yellow crystals; Yield: 78.32%; mp: 250–252 °C; R
f
value: 0.15; IR (KBr, cm−1): 2830 (C–H str.), 1604 (C=C str.), 1697 (N=CH str.), 1363 (C–N str.), 3352 (O–H str.), 2928 (C–H str., Ar–OCH3); 1H-NMR (δ, DMSO-d
6): 7.48–7.55 (m, 16H, Ar–H), 8.10 (s, 2H, N=CH), 7.71 (s, 2H, (CH)2 of pyrimidine), 3.41{s, 18H, (OCH3)3}; 13C-NMR (δ, DMSO-d
6): 153.2, 129.9, 106.7, 142.8, 131.6, 139.7, 162.6, 167.8,106.7, 150.9, 56.5, 56.2; CHN: Calc. C46H40N6O8: C, 68.65; H, 5.01; N, 10.44; Found: C, 68.63; H, 5.04; N, 10.43; MS ES + (ToF): m/z 806 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-nitrobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q3)
Dark yellow crystals; Yield: 65.34%; mp: 275–277 °C; R
f
value: 0.52; IR (KBr, cm−1): 2931 (C–H str.), 1605 (C=C str.), 1700 (N=CH str.), 1301 (C–N str.), 3335 (O–H str.), 1347 (C–NO2 sym. str., NO2), 1534 (C–NO2 asym. str., NO2); 1H-NMR (δ, DMSO-d
6): 7.04–8.17 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d6): 150.5, 129.9, 124.2, 140.0, 130.6, 139.7, 158.5, 116.4, 104.1, 167.8, 160.2, 121.1; CHN: Calc. C40H26N8O6: C, 67.22; H, 3.67; N, 15.68; Found: C, 67.24; H, 3.60; N, 15.70; MS ES + (ToF): m/z 716 [M+ +1].
4,4′-((1E,1′E)-((6,6′-(1,4-Phenylene)bis(4-(4-hydroxyphenyl)pyrimidine-6,2-diyl))bis (azanylylidene))bis(methanylylidene))bis(2-methoxyphenol) (q4)
Dark yellow crystals; Yield: 72.25%; mp: 280–282 °C; R
f
value: 0.54; IR (KBr, cm−1): 2933 (C–H str.), 1603 (C=C str.), 1698 (N=CH str.), 1365 (C-N str.), 3337 (O–H str.), 3064 (C–H str., Ar–OCH3); 1H-NMR (δ, DMSO-d
6): 6.96–7.55 (m, 18H, Ar–H), 8.12 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine), 3.75 (s, 6H, (OCH3)2); 13C-NMR (δ, DMSO-d
6): 153.1, 148.1, 128.6, 126.0, 115.3, 110.6, 55.5, 158.5, 116.4, 162.5; CHN: Calc. C42H32N6O6: C, 70.38; H, 4.45; N, 11.73; Found: C, 70.34; H, 4.40; N, 11.75; MS ES + (ToF): m/z 718 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-chlorobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q5)
Dark yellow crystals; Yield: 70.25%; mp: 123–125 °C; R
f
value: 0.58; IR (KBr, cm−1): 3060 (C–H str.), 1604 (C=C str.), 1700 (N=CH str.), 1384 (C–N str.), 3333 (O–H str.), 776 (C–Cl str. phenyl nucleus); 1H-NMR (δ, DMSO-d
6): 6.91–7.61 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 162.6, 134.7, 131.3, 136.7, 130.1, 129.9, 128.9, 125.6, 115.4; CHN: Calc. C40H26Cl2N6O2: C, 69.27; H, 3.78; N, 12.12; Found: C, 69.25; H, 3.77; N, 12.09; MS ES + (ToF): m/z 695 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-(dimethylamino)benzylidene)amino)pyrimidine-6,4-diyl))diphenol (q6)
Dark yellow crystals; Yield: 72.27%; mp: 250–252 °C; R
f
value: 0.15; IR (KBr, cm−1): 2926 (C–H str.), 1595 (C=C str.), 1697 (N=CH str.), 1353 (C–N str.), 3405 (O–H str.), 2830 (N–CH3 str.); 1H-NMR (δ, DMSO-d
6): 6.78-7.54 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 2.89 (s, 12H, (CH3)2), 7.72 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 131.5, 129.9, 124.4, 111.0, 39.9, 158.7, 116.5, 167.1, 144.5, 130.1, 133.1; CHN: Calc. C44H38N8O2: C,74.35; H, 5.39; N, 15.76; Found: C, 74.30; H, 5.35; N, 15.72; MS ES + (ToF): m/z 712 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(3-nitrobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q7)
Yellow crystals; Yield: 70.82%; mp: 251–253 °C; R
f
value: 0.54; IR (KBr, cm−1): 2927 (C–H str.), 1629 (C=C str.), 1698 (N=CH str.), 1352 (C–N str.), 3386 (O–H str.), 1602 (NO2 str.), 814 (C–N str., –NO2); 1H-NMR (δ, DMSO-d
6): 7.50-8.69 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 139.7, 137.1, 134.8, 130.9, 129.9, 128.5, 126.5, 124.0; CHN: Calc. C40H26N8O6: C, 67.22; H, 3.67; N, 15.68; Found: C, 67.20; H, 3.70; N, 15.63; MS ES + (ToF): m/z 716 [M+ +1].
4,4′-((1E,1′E)-((6,6′-(1,4-Phenylene)bis(4-(4-hydroxyphenyl)pyrimidine-6,2-diyl))bis(azanyl ylidene))bis(methanylylidene))bis(2-ethoxyphenol) (q8)
Yellow crystals; Yield: 64.33%; mp: 260–262 °C; R
f
value: 0.25; IR (KBr, cm−1): 2928 (C–H str.), 1597 (C=C str.), 1629 (N=CH str.), 1353 (C–N str.), 3408 (O–H str.), 2830 (C–H str.,–OC2H5); 1H-NMR (δ, DMSO-d
6): 6.90–7.53 (m, 18H, Ar–H), 8.10 (s, 2H, N=CH), 7.72 (s, 2H, (CH)2 of pyrimidine), 4.08 {q, 4H, (CH2)2, 1.34 (t, 6H, (CH3)2}; 13C-NMR (δ, DMSO-d
6): 147.4, 131.3, 129.9, 128.0, 126.0, 115.5, 111.7, 63.7, 14.5; CHN: Calc. C44H36N6O6: C, 70.96; H, 4.87; N, 11.28; Found: C, 70.91; H, 4.83; N, 11.23; MS ES + (ToF): m/z 746 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-hydroxybenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q9)
Yellow crystals; Yield: 73.45%; mp: 200–202 °C; R
f
value: 0.16; IR (KBr, cm−1): 2927 (C–H str.), 1509 (C=C str.), 1697 (N=CH str.), 1354 (C–N str.), 3387 (O–H str.); 1H-NMR (δ, DMSO-d
6): 6.91–7.53 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.72 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 139.7, 132.0, 131.3, 129.9, 115.0, 158.1, 116.4, 162.4, 104.1, 133.1, 167.5, 160.1; CHN: Calc. C40H28N6O4: C,73.16; H, 4.30; N, 12.80; Found: C, 73.13; H, 4.34; N, 12.84; MS ES + (ToF): m/z 658 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(2-methoxybenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q10)
Yellow crystals; Yield: 77.34%; mp: 244–246 °C; R
f
value: 0.33; IR (KBr, cm−1): 2830 (C–H str.), 1540 (C=C str.), 1604 (N=CH str.), 1353 (C–N str.), 3357 (O–H str.), 2931(C–H str., Ar–OCH3); 1H-NMR (δ, DMSO-d
6): 7.00–7.55 (m, 20H, Ar–H), 8.12 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine), 3.58 {s, 6H, (OCH3)2}; 13C-NMR (δ, DMSO-d
6): 129.9, 127.6, 120.5, 55.8, 158.5, 116.4, 125.7, 167.8, 130.2, 132.1, 55.9; CHN: Calc. C42H32N6O4: C, 73.67; H, 4.71; N, 12.27; Found: C, 73.63; H, 4.74; N, 12.22; MS ES + (ToF): m/z 686 [M+ +1].
4,4′-(6,6′-(1,4-Phenylene)bis(2-((E)-((E)-3-phenylallylidene)amino)pyrimidine-6,4-diyl)) diphenol (q11)
Yellow crystals; Yield: 62.33%; mp: 250–252 °C; R
f
value: 0.45; IR (KBr, cm−1): 3059 (C–H str.), 1538 (C=C str.), 1699 (N=CH str.), 1386 (C–N str.), 3330 (O–H str.), 2932 (C–H str., aliphatic), 1606 (C=C str., alkyl chain); 1H-NMR (δ, DMSO-d
6): 6.85–7.53 (m, 22H, Ar–H), 7.75 (s, 2H, N=CH), 7.7 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 131.1, 129.9, 128.6, 128.4, 158.5, 116.4, 125.7, 162.5, 163.6, 138.2, 126.4, 135.4; CHN: Calc. C44H32N6O2: C, 78.09; H, 4.77; N, 12.42; Found: C, 78.05; H, 4.72; N, 12.39; MS ES + (ToF): m/z 678 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(2-hydroxybenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q12)
Yellow crystals; Yield: 81.84%; mp: 195–197 °C; R
f
value: 0.80; IR (KBr, cm−1): 2930 (C–H str.), 1540 (C=C str.), 1699 (N=CH str.), 1354 (C–N str.), 3372 (O–H str.); 1H-NMR (δ, DMSO-d
6): 6.96–7.54 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.72 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 129.9, 158.5, 116.4, 128.9, 125.7,162.5, 167.8, 118.5, 130.6, 121.5, 132.1, 161.4; CHN: Calc. C40H28N6O4: C, 73.16; H, 4.30; N, 12.80; Found: C, 73.13; H, 4.34; N, 12.78; MS ES + (ToF): m/z 658 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-methoxybenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q13)
Yellow crystals; Yield: 89.14%; mp: 255–257 °C; R
f
value: 0.32; IR (KBr, cm−1): 2830 (C–H str.), 1540 (C=C str.), 1699 (N=CH str.), 1354 (C–N str.), 3359 (O–H str.), 2931 (C–H str., Ar–OCH3); 1H-NMR (δ, DMSO-d
6): 7.12–7.59 (m, 20H, Ar–H), 8.10 (s, 2H, N=CH), 7.68 (s, 2H, (CH)2 of pyrimidine), 3.74 (s, 6H, OCH3); 13C-NMR (δ, DMSO-d
6): 131.7, 129.9, 114.4, 125.8, 162.6, 104.3, 167.8, 160.1, 128.0, 133.1, 158.1, 116.4, 126.1, 163.0, 55.6; CHN: Calc. C42H32N6O4: C, 73.67; H, 4.71; N, 12.27; Found: C, 73.62; H, 4.75; N, 12.31; MS ES + (ToF): m/z 686 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(3-bromobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q14)
Yellow crystals; Yield: 66.90%; mp: 205–207 °C; R
f
value: 0.27; IR (KBr, cm−1): 3057 (C–H str.), 1538 (C=C str.), 1699 (N=CH str.), 1387 (C–N str.), 3329 (O–H str.), 549 (C–Br str.); 1H-NMR (δ, DMSO-d
6): 7.36–782 (m, 20H, Ar–H), 8.12 (s, 2H, N=CH), 7.72 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 139.7, 137.0, 131.9, 128.1, 122.3, 158.5, 116.3, 167.6, 134.0, 162.3, 104.1; CHN: Calc. C40H26Br2N6O2: C, 61.40; H, 3.35; N, 10.74; Found: C, 61.45; H, 3.30; N, 10.77; MS ES + (ToF): m/z 783 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-bromobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q15)
Yellow crystals; Yield: 72.00%; mp: 217–219 °C; R
f
value: 0.58; IR (KBr, cm−1): 3059 (C–H str.), 1538 (C=C str.), 1699 (N=CH str.), 1386 (C–N str.), 3338 (O–H str.), 1168 (C–O–C str., Ar–OCH3), 2830 (C–H str., –OCH3), 551 (C–Br str.); 1H-NMR (δ, DMSO-d
6): 7.46–7.53 (m, 20H, Ar–H), 8.12 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 139.7, 132.2, 131.1, 128.6, 126.5, 158.5, 116.4, 131.8, 125.3, 167.8, 160.2; CHN: Calc. C40H26Br2N6O2: C, 61.40; H, 3.35; N, 10.74; Found: C, 61.45; H, 3.30; N, 10.77; MS ES + (ToF): m/z 783 [M+ +1].
2,2′-((1E,1′E)-((6,6′-(1,4-Phenylene)bis(4-(4-hydroxyphenyl)pyrimidine-6,2-diyl))bis(azanyl ylidene))bis(methanylylidene))bis(4-bromophenol) (q16)
Yellow crystals; Yield: 83.67%; mp: 296–298 °C; R
f
value: 0.25; IR (KBr, cm−1): 542 (C–Br str., C6H5Br), 2928 (C–H str.), 1604 (C=C str.), 1699 (N=CH str.), 1363 (C–N str.), 3357 (O–H str.); 1H-NMR (δ, DMSO-d
6): 7.39–7.61 (m, 18H, Ar–H), 8.11 (s, 2H, N=CH), 7.71 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 158.4, 163.6, 104.1, 167.8, 160.1, 128.0, 130.0, 120.4, 133.1, 115.8, 116.4, 128.9, 125.7, 135.4, 118.2, 160.1; CHN: Calc. C40H26Br2N6O4: C, 58.99; H, 3.22; N, 10.32; Found: C, 58.95; H, 3.20; N, 10.35; MS ES + (ToF): m/z 815 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(2-chlorobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol) (q17)
Yellow crystals; Yield: 79.12%; mp: 265–267 °C; R
f
value: 0.18; IR (KBr, cm−1): 2925 (C–H str.), 1539 (C=C str.), 1699 (N=CH str.), 1363 (C–N str.), 3355 (O–H str.), 775(C–Cl str.); 1H-NMR (δ, DMSO-d
6): 7.48–7.61 (m, 20H, Ar–H), 8.12 (s, 2H, N=CH), 7.70 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 139.7, 135.7, 130.7, 129.7, 127.8; CHN: Calc. C40H26Cl2N6O2: C, 69.27; H, 3.78; N, 12.12; Found: C, 69.23; H, 3.74; N, 12.10; MS ES + (ToF): m/z 695 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(2-nitrobenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q18)
Yellow crystals; Yield: 69.23%; mp: 198–200 °C; R
f
value: 0.16; IR (KBr, cm−1): 2926 (C–H str.), 1539 (C=C str.), 1699 (N=CH str.), 1354 (C–N str.), 3375 (O–H str.), 1604 (NO2 str.), 814 (C–N str., –NO2); 1H-NMR (δ, DMSO-d
6): 7.43–8.16 (m, 20H, Ar–H), 8.12 (s, 2H, N=CH), 7.75 (s, 2H, (CH)2 of pyrimidine); 13C-NMR (δ, DMSO-d
6): 167.4, 139.7, 134.1, 130.5, 128.1, 126.5, 124.2; CHN: Calc. C40H26N8O6: C, 67.22; H, 3.67; N, 15.68; Found: C, 67.18; H, 3.62; N, 15.63; MS ES + (ToF): m/z 716 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(4-(diethylamino)benzylidene)amino)pyrimidine-6,4-diyl))diphenol (q19)
Light yellow crystals; Yield: 65.33%; mp: 249–251 °C; R
f
value: 0.23; IR (KBr, cm−1): 2970 (C–H str.), 1590 (C=C str.), 1699 (N=CH str.), 1356 (C–N str.), 2830 (C–H str., alkyl chain), 3350 (O–H str.); 1H-NMR (δ, DMSO-d
6): 6.75–7.55 (m, 20H, Ar–H), 8.1 (s, 2H, N=CH), 7.77 (s, 2H, (CH)2 of pyrimidine), 3.41 {q, 4H, (CH2)2, 1.13 (t, 6H, (CH3)2}; 13C-NMR (δ, DMSO-d
6): 151.9, 139.7, 129.9, 123.9, 110.5, 116.4, 126.9, 160.1, 128.0, 133.1, 130.1, 125.7, 162.6, 104.1, 167.8, 114.4, 44.5, 12.27; CHN: Calc. C48H46N8O2: C, 75.17; H, 6.05; N, 14.61; Found; C,75.14; H, 6.09; N, 14.64; MS ES + (ToF): m/z 768 [M+ +1].
4,4′-((E)-6,6′-(1,4-Phenylene)bis(2-((E)-(2-methoxybenzylidene)amino)pyrimidine-6,4-diyl)) diphenol (q20)
Light yellow crystals; Yield: 89.45%; mp: 274–276 °C; R
f
value: 0.16; IR (KBr, cm−1): 2931 (C–H str.), 1605 (C=C str.), 1699 (N=CH str.), 1365 (C–N str.), 3350 (O–H str.), 3058 (C–H str.,–OCH3); 1H-NMR (δ, DMSO-d
6): 6.91–7.54 (m, 20H, Ar–H), 8.1 (s, 2H, N=CH), 7.62 (s, 2H, (CH)2 of pyrimidine), 3.75 (s, 2H, OCH3); 13C-NMR (δ, DMSO-d
6): 159.1, 130.3, 129.9, 129.1, 128.1, 122.4, 120.9, 118.7, 113.8, 116.3, 126.9, 125.7, 163.6, 104.1, 134.8, 121.5, 128.8, 160.8, 55.3; CHN: Calc. C42H32N6O4: C, 73.67; H, 4.71; N, 12.27; Found: C, 73.62; H, 4.75; N, 12.22; MS ES + (ToF): m/z 686 [M+ +1].
In vitro antimicrobial assay
The in vitro antimicrobial study of the synthesized bis-pyrimidines was evaluated against Gram +ve bacterial species: S. aureus (MTCC 3160), B. subtilis (MTCC 441), Gram −ve species: E. coli (MTCC 443) and fungus species: A. niger (MTCC 281) and C. albicans (MTCC 227) by tube dilution technique [25]. Dilutions of test and reference drug in double strength nutrient broth media I.P. was used for antibacterial study and Sabouraud dextrose broth media I.P. was used for the antifungal study. The stock solution was prepared for the test compounds (q1–q20) and reference drugs (norfloxacin and fluconazole) in dimethyl sulfoxide (DMSO) to get a concentration of 100 µg/mL and this stock solution was used for further tube dilution with six concentration of 50, 25, 12.5, 6.25, 3.125 and 1.562 µg/mL for the antimicrobial study [26]. The MIC values of synthesized bis-pyrimidine Schiff base derivatives were recorded at different incubation period: 37 ± 1 °C (bacterial species) for 24 h, 37 ± 1 °C (C. albicans) for 48 h and 25 ± 1 °C (A. niger) for 7 days and the antimicrobial results have been recorded in terms of minimum inhibitory concentration values in µmol/mL.
In vitro cytotoxicity assay
The anticancer screening of synthesized compounds was determined against human colorectal carcinoma [HCT-116 (ATCC (American Type Culture Collection) CCL-247)] cancer cell line using sulforhodamine B (SRB) assay. The optimal cell count (2500 cells/180 μL/well) of HCT-116 was seeded onto the 96 flat-bottom well plates and incubated overnight to allow attachment. 20 μL of pure compounds at tenfold the final concentrations were added in quadruplicates. Both drug-free control and treated cells were then incubated for 72 h. The drug-induced cytotoxicity was assessed using the SRB assay as previously described by Skehan et al. [27], but with minor modifications. Briefly, upon removal of media, cells in each well were fixed with 200 μL of 10% cold TCA [Sigma-Aldrich, St Louis, Missouri, USA] (w/v; in deionised water). After incubation at 4 0 °C for 30 min, the individual wells were rinsed with water for five times. Cells in each well were allowed to stain in 100 µL of 0.4% SRB [Sigma-Aldrich, St Louis, Missouri, USA] (w/v; in 1% acetic acid) for 15 min. Unincorporated dye was rinsed off with 1% acetic acid [Fisher Scientific, Loughborough, Leicestershire, UK] (v/v; in deionised water) and plates were left to air-dry at room temperature overnight. The air-dried plates were placed on a plate shaker and bound SRB was solubilised in 100 µL of 10 mM Tris base solution [Sigma-Aldrich, St Louis, Missouri, USA]. Absorbance was measured by a computer-interfaced 96-well plate spectrophotometer at 570 nm. A dose–response curve (percentage of cell viability vs log concentration) was plotted from which the IC50 value of each molecule against each cell type was graphically determined.
Molecular docking protocol
In order to reveal the binding modes of synthesized twenty bis-pyrimidine Schiff base derivatives, docking simulation was performed targeting the crystal structure of cyclin-dependent kinase 8 (CDK8). Prior to docking, the crystal structure [PDB ID: 5FGK] was retrieved from the protein data bank (PDB) [28]. The CDK8 structure was prepared using protein preparation wizard and optimized by removing the water molecules, hetero atoms and co-factors. Hydrogen, missing atoms, bonds and charges were computed through Maestro [Schrodinger Release 2015-1: Maestro, version 10.1, Schrodinger, LLC, New York, 2015]. The synthesized twenty.
Bis-pyrimidine Schiff base derivatives were used for docking. Meanwhile, the bis-pyrimidine Schiff base derivatives were prepared and optimized using built and LigPrep module implemented in Schrodinger Maestro. Ligands preparation includes generating various tautomers, assigning bond orders, ring conformations and stereo chemistries. All the conformations generated were minimized using OPLS2005 force field prior to docking study.
Molecular docking studies were performed using GOLD (Genetic Optimization for Ligand Docking) program version 5.1. GOLD is an automated docking program that employs the genetic algorithm to search the ligand conformational flexibility with a partial flexibility of protein’s active site [29]. GOLD uses genetic algorithm method for protein–ligand docking and it is well-known for its performance and accuracy specifically for the protein targets with buried active site. The GOLD software has four scoring functions namely ChemPLP, GoldScore, ChemScore, and ASP (the Astex Statistical Potential) which take into account the terms of hydrogen bonding, van der Waal and intramolecular energies. In the present GOLD docking study targeting cyclin-dependent kinase CDK8, the ChemPLP scoring function was used as it outperformed the other scoring function. All the bis-pyrimidine derivatives were docked to cyclin-dependent kinase CDK8 active site, using co-complex 5XG ligand as the reference with 12 A radius. Further, the population size was set to (100); selection-pressure (1.1); number of operations (10,000); number of islands (1); niche size (2); operator weights for migrate (0), mutate (100), and cross-over (100) and with 100 GA run. Results divergent by less than 1.50 A in ligand-all atom RMSDs were clustered together. Best cluster poses and top ranked scores were saved and visually analyzed by Pymol [PyMOL Molecular Graphics System, Schrödinger L, NY, USA, 2010.]. Additional, Qikprop prediction of ADME properties were done for all the synthesized twenty bis-pyrimidine derivatives [Rapid ADME, QikProp, Schrödinger LLC, New York, 2012].