Chemistry
General
Melting points were determined on Electrothermal IA 9100 capillary melting point apparatus and are uncorrected. UV spectra were recorded on UV–Vis spectrophotometer model type of Genesys 10 s and expressed in nm. Thermo Scientific. Glass cuvettes were used. All the samples were dissolve in chloroform or methanol. FT-IR spectroscopic studies were carried out on FTIR spectrophotometer 1000 model Perkin Elmer at room temperature 25 °C. KBr pellets were dried in oven and scanned for calibration purpose. 1H NMR spectra of compounds were recorded on a Bruker Ascend TM 600 MHz machine, while the spectra of compounds 12, 16, 17 were recorded on 500 MHz NMR spectrometers. The chemical shifts (δ) are presented with references to CDCl3 (δ: 7.25) and TMS (δ: 0.00) as the internal reference. Electron-spray ionization mass spectra in positive mode (ESI–MS) were recorded on a Bruker Esquire 3000 spectrometer. Column chromatography purifications were carried out on Silica Gel 60 (Merck, 70–230 mesh, ASTM) and flash silica gel (230–400 mesh, ASTM, Merck). The purity of all compounds were checked by thin-layer chromatography (TLC) and 1H-NMR spectra. All reagents used were of analytical grade. All the chemicals were purchased from Aldrich, U.S.A. Other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd., China.
Synthetic procedures
Method A (acid-catalyzed)
A typical Claisen-Schmidt condensation reaction procedure was used to prepare all curuminoids. Appropriate mono ketone (cyclohexanone, acetone and cyclopentanone) 10 mol (1 equiv) was dissolved in absolute ethanol (15–20 mL). Substituted benzaldehydes 20 mol, (2 equiv) was added slowly. About 1–2 mL concentrated HCl was added drop wise over 5–10 min in a stirred mixture of ketone. The reaction mixture was stirred overnight (12–24 h). The product was monitored by comparing the Co-TLC with the starting material. The products were extracted with ethyl acetate by dissolving the compounds in distilled water (100 mL). Curcuminoids were purified by silica gel column chromatography (ethyl acetate/hexane) and re-crystallized with hot solution of ethyl acetate and ethanol.
Method B (base-catalyzed)
The general procedure Claisen–Schmidt condensation reaction was used to synthesize curcuminoids by using this method involved in addition of certain amount of mono ketone (cyclohexanone, acetone and cyclopentanone) to a solution of substituted aldehydes in MeOH or C2H5OH by adding KOH or NaOH. The reaction mixture is stirred at room temperature and monitored by TLC. The products are extracted and purified as described as in acid catalysed [43, 44].
(2E,6E)-2,6-bis(2-Methoxybenzylidene)cyclohexanone (1)
Yellow liquid; yield (86%); UV–Vis (CHCl3) λmax: 302, 339 nm; IR (KBr,) v 3023 (Ar C–H stretch), 1636 (C=O), 1527 (Ar C=C<) cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.75 (m, 2H, 4-H), 2.84 (m, 4H, 3, 5-H), 3.86 (s, 6H, OCH3, C-2′ & C-2″), 6.92 (m, 2H, 3′, 3″-H), 6.96 (m, 2H, 5′, 5″-H), 7.32 (m, 2H, 4′, 4″-H), 7.33–7.30 (m, 4H, 4′, 4″, 6′, 6″-H), 7.98 (brs, 2H, –C=C–H). 13C NMR (CDCl3, 150 MHz) δ 23.5 (C-4), 28.6 (C-3, C-5), 55.5 (OCH3), 110.6 (C-3′, C-3″), 119.9 (C-5′, C-5″), 125.2 (C-4′, C-4″, C-6′, C-6″), 130.3 (C-1′, C-1″), 132.5 (C-2, C-6), 136.6 (–C=C–H), 158.4 (C-2′, C-2″), 190.6 (C=O); EI-MS m/z 334.0 (10), 303.1 (20), 240.3 (14), 161.2 (19), 107.4 (23), 77.0 (64); HREI-MS for C22H22O3 M+, calcd.: m/z 334.1575, found: m/z 334.1589.
(2E,6E)-2,6-bis(4-Methoxybenzylidene)cyclohexanone (2)
Yellow crystals; yield (74%); m.p. 152–153 °C (lit. [29] 148–149 °C); UV–Vis (CHCl3) λmax: 362 nm; IR (KBr) v 3010 (Ar C–H stretch), 1660 (C=O), 1508–1594 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.80 (m, 2H, 4-H), 2.92 (m, 4H, 3, 5-H), 3.84 (s, 6H, OCH3, C-4′, 4″), 6.93 (d, 4H, 3′, 3″, 5′, 5″-H, J = 6.78 Hz), 7.45 (d, 4H, 2′, 2″, 6′, 6″-H, J = 6.78 Hz), 7.76 (brs, 2H, –C=C–H); EI-MS m/z 334.0 (100), 303.45 (36), 240.1 (23), 161.2 (10), 107.0 (28); HREI-MS for C22H22O3 M+, calcd.: m/z 334.1568, found: m/z 334.1573.
(2E,6E)-2,6-bis(2,3-Dimethoxybenzylidene)cyclohexanone (3)
Yellow crystals; yield (92%); m.p. 105–106 °C (lit. [36] 107–109 °C); UV–Vis (CHCl3) λmax: 324 nm; IR (KBr) v 3023 (Ar C–H stretch), 1622 (C=O), 1536–1536 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.75 (m, 2H, 4-H), 2.80 (m, 4H, 3, 5-H), 3.82 (s, 6H, OCH3, C3′, 3″), 3.88 (s, 6H, OCH3, C-2′, 2″), 6.93 (m, 4H, 4′, 4″, 6′, 6″-H), 7.06 (brt, 2H, 5′, 5″-H, J = 7.98 Hz), 7.94 (brs, 2H, –C=C–H); 13C NMR, (150 MHz, CDCI3) δ 23.3 (C-4), 28.78 (C-3, C-5), 55.9 (OCH3), 61.2 (OCH3), 112.8 (C-5′, C-5″), 122.2 (C-4′, C-4″), 123.5 (C-6′, C-6″), 130.5 (C-1′, C-1″), 132.5 (C-2, C-6), 137.5 (C=C–H), 152.9 (C-2′, C-2″, C-3′, C-3″), 190.4 (C=O); EI-MS m/z 394 (5), 363.0 (100), 331.2 (68), 161.23 (86), 227.33 (24), 136.18 (29); HREI-MS for C24H26O5 M+, calcd.: m/z 394.1783, found: m/z 394.1778.
(2E,6E)-2,6-bis(4-Hydroxy-3-methoxybenzylidene)cyclohexanone (5)
Synthesis, purification and experimental data of compound 5 was recently published by us [31].
(2E,6E)-2,6-bis(2-Chlorobenzylidene)cyclohexanone (6)
Yellow crystals; yield (68%); m.p. 109–110 °C (lit. [36] 94–95 °C); UV–Vis (CHCl3) λmax: 320 nm; IR (KBr) v 3073 (Ar C–H stretch), 1663 (C=O), 1574–1433 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.76 (m, 2H, 4-H), 2.78 (m, 4H, 3, 5-H), 7.33 (m, 2H, 3′, 3″-H), 7.28 (m, 4H, 4′, 4″, 5′, 5″-H), 7.44 (m, 2H, 6′, 6″-H), 7.91 (brs, 2H, –C=C–H); EI-MS m/z 343.0 (5), 307 (100), 272 (8), 166 (4), 138 (6), 112 (17); HREI-MS for C20H16Cl2O M+, calcd.: m/z 342.0578, found: m/z 342.0572.
(2E,6E)-2,6-bis(4-Chlorobenzylidene)cyclohexanone (7)
Yellow crystals; yield (86%); m.p. 149–153 °C (lit. [29] 147–149 °C); UV–Vis (CHCl3) λmax: 335 nm; IR (KBr) v 3063 (Ar C–H stretch), 1604 (C=O), 1576–1487 (Ar C=C) cm−1; 1H NMR (CDCI3, 500 MHz) δ 1.80 (m, 2H, 4-H), 2.89 (m, 4H, 3, 5-H), 7.34 (m, 2H, 2′, 2″-H), 7.34 (m, 2H, 3′, 3″-H), 7.34 (m, 2H, 5′, 5″-H), 7.34 (m, 2H, 6′, 6″-H), 7.73 (brs, 2H, –C=C–H); EI-MS m/z 343 (76), 307 (87), 272 (71), 244 (31), 166 (14), 138 (22), 112 (9); HREI-MS for C20H16Cl2O M+, calcd.: m/z 342.0678, found: m/z 342.0672.
(2E,6E)-2,6-bis(3,4-Dimethoxybenzylidene)cyclohexanone (10)
Yellow crystals; yield (74%); m.p. 146–149 °C (lit. [37] 148–150 °C); UV–Vis (CHCl3) λmax: 373 nm; IR (KBr) v 3036 (Ar C–H stretch), 1614 (C=O), 1489–1462 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.83 (m, 2H, 4-H), 2.95 (m, 4H, 3, 5-H), 3.90 (s, 6H, OCH3, C-3′, 3″), 3.92 (s, 6H, OCH3, C-4′, 4″), 6.91 (d, 2H, 5′, 5″-H, J = 8.34 Hz), 7.02 (d, 2H, 2′, 2″-H, J = 1.92 Hz), 7.12 (dd, 2H, 6′, 6″-H, J = 8.34, 1.92 Hz), 7.76 (brs, 2H, –C=C–H); EI-MS m/z 394 (3), 363 (100), 331 (9), 161 (4), 227 (23), 136 (3), 77 (31); HREI-MS for C24H26O5 M+, calcd.: m/z 394.1784, found: m/z 394.1787.
(1E,4E)-1,5-bis(2-Methoxyphenyl)-penta-1,4-dien-3-one (11)
Yellow crystals; yield (66%); m.p. 111–114 °C (lit. [45] 118–120 °C); UV–Vis (CHCl3)λmax: 312, 360 nm; IR (KBr) v 3023 (Ar C–H stretch), 1614 (C=O), 1489–1462 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 3.90 (s, 6H, OCH3, C-2′, 2″), 6.93 (d, 2H, 3′, 3″-H, J = 8.34 Hz), 6.99 (t, 2H, 4′, 4″-H, J = 8.46, 7.4 Hz), 7.18 (d, 2H, 2, 4-H, J = 16.08 Hz), 7.36 (td, 2H, 5′, 5″-H, J = 7.4, 1.6 Hz), 7.62 (dd, 2H, 6′, 6″-H, J = 7.6, 1.6 Hz), 8.08 (d, 2H, 1, 5-H, J = 16.08 Hz); EI-MS m/z 294 (100), 263 (8), 234 (15), 186 (50), 161 (36), 133 (33), 77 (16); HREI-MS for C19H18O3 M+, calcd.: m/z 294.1255, found: m/z 294.1251.
(1E,4E)-1,5-bis(4-Methoxyphenyl)-penta-1,4-dien-3-one (12)
Yellow crystals; yield (79%); m.p. 121–122 °C (lit. [46] 119–120 °C); UV–Vis (CHCl3) λmax: 354 nm; IR (KBr) v 3033 (Ar C–H stretch), 1624 (C=O), 1590–1488 (Ar C=C) cm−1. 1H NMR (CDCl3, 500 MHz) δ 3.87 (s, 6H, OCH3, C-4′, 4″), 6.94 (d, 4H, 3′, 3″, 5′, 5″-H, J = 8.75 Hz), 6.99 (d, 2H, 2, 4-H, J = 16.0 Hz), 7.60 (d, 4H, 2′, 2″, 6′, 6″-H, J = 8.75 Hz), 7.74 (d, 2H, 1, 5-H, J = 16.0 Hz); EI-MS m/z 294.14 (100), 263 (15), 234 (20), 186 (54), 161 (38), 133 (36), 77 (21); HREI-MS for C19H18O3 M+, calcd.: m/z 294.1264, found: m/z 294.1257.
(1E,4E)-1,5-bis(2,3-Dimethoxyphenyl)-penta-1,4-dien-3-one (13)
Yellow solid; yield (68%); m.p. 103–104 °C (lit. [36] 106–108 °C); UV–Vis (CHCl3) λmax: 330 nm; IR (KBr) v 3011–2943 (Ar C–H stretch), 1619 (C=O), 1577–1479 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 3.87 (s, 12H, OCH3, C-2′, 2″, 3′, 3″), 6.97 (dd, 2H, 4′, 4″-H, J = 8.16, 1.44 Hz), 7.10 (t, 2H, 5′, 5″-H, J = 8.04, 8.00 Hz), 7.16 (d, 2H, 2, 4-H, J = 16.1 Hz), 7.26 (dd, 2H, 6′, 6″-H, J = 8.00, 1.44 Hz), 8.04 (d, 2H, 1, 5-H, J = 16.1 Hz); EI-MS m/z 354 (5), 323 (3), 230 (4), 186 (9), 132 (13), 191 (4), 163 (7), 77 (52); HREI-MS for C21H22O5 M+, calcd.: m/z 354.1467, found: m/z 394.1462.
(1E,4E)-1,5-bis(4-Chlorophenyl)-penta-1,4-dien-3-one (16)
Yellow solid; yield (72%); m.p. 193–195 °C (lit. [36] 192–193 °C); UV–Vis (CHCl3) λmax: 333 nm; IR (KBr) v 3065 (Ar C–H stretch), 1608 (C=O), 1584–1489 (Ar C=C str.) cm−1; 1H-NMR (CDCl3, 500 MHz) δ 7.04 (d, 2H, 2, 4-H, J = 15.9 Hz), 7.40 (dd, 4H, 3′, 3″, 5′, 5″-H, J = 8.60 Hz), 7.56 (d, 4H, 2′, 2″, 6′, 6″-H, J = 8.60 Hz), 7.70 (d, H, 1, 5-H, J = 15.9 Hz); 13C NMR (150 MHz, CDCl3) δ 126.0 (C-2, 4), 128.7 (C-3′, 3″), 128.7 (C-5′, 5″), 129.3 (C-2′, 2″), 129.3 (C-6′, 6″), 133.3 (C-1′, 1″), 136.5 (C-4′, 4″), 142.1 (C-1, 5), 188.3 (C=O); EI-MS m/z 302 (60), 267 (32), 232 (5), 203 (20), 165 (35), 137 (49), 77 (5); HREI-MS for C17H12Cl2O M+, calcd.: m/z 302.0265, found: m/z 302.0259.
(1E,4E)-1,5-bis(2,4,6-Trimethoxyphenyl)-penta-1,4-dien-3-one (17)
Yellow solid; yield (68%); m.p. 213–215 °C (lit. [36] 209–211 °C); UV–Vis (CHCl3) λmax: 381 nm. IR (KBr) 3002 (Ar C–H str.), 1629 (C=O), 1561–1466 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 3.74 (s, 6H, 2 × OCH3, C-4′, 4″), 3.85 (s, 12H, 4 × OCH3, C-2′, 2″, 6′, 6″), 6.13 (brs, 4H, 3′, 3″, 5′, 5″-H), 7.46 (d, 2H, 2, 4-H, J = 16.30 Hz), 8.12 (d, 2H, 1, 5-H, J = 16.30 Hz); EI-MS m/z 414 (5), 131 (6), 105 (10); HREI-MS for C23H26O7 M+, calcd.: m/z 414.1671, found: m/z 414.1679.
(2E,5E)-2,5-bis(4-Methoxybenzylidene)cyclopentanone (19)
Yellow solid; yield (66%); m.p. 150–155.5 °C (lit. [29] 158–161 °C); UV–Vis (CHCl3) λmax: 391 nm; IR (KBr) v 2964 (Ar C–H stretch), 1696 (C=O), 1597–1509 (Ar C=C) cm−1; 1H NMR (CDCI3, 600 MHz) δ 3.09 (brs, 4H, 3, 4-H), 3.86 (s, 6H, 2 × OCH3, C-4′, 4″), 6.98 (brd, 2H, 5′, 5″-H, J = 8.34 Hz), 6.98 (brd, 2H, 3′, 3″-H, J = 8.34 Hz), 7.57 (brt, 2H, 2′, 2″-H, J = 8.52 Hz), 7.57 (brt, 2H, 6′, 6″-H, J = 8.52 Hz), 7.58 (brs, 2H, –C=C–H); EI-MS m/z 320 (11), 213 (8), 183 (5), 131 (12), 77 (16); HREI-MS for C21H20O3 M+, calcd.: m/z 320.1412, found: m/z 320.140.
(2E,5E)-2,5-bis(2,3-Dimethoxybenzylidene)cyclopentanone (20)
Yellow solid; yield (54%); m.p. 156–158 °C (lit. [36] 155–157 °C); UV–Vis (CHCl3) λmax: 346 nm; IR (KBr) v 3032 (Ar C-H stretch), 1694 (C=C), 1622 (C=O), 1584–1489 (Ar C=C) cm−1; 1H NMR (CDCI3, 600 MHz) δ 3.02 (brs, 4H, 3, 4-H), 3.87 (s, 6H, OCH3, C-2′, 2″), 3.88 (s, 6H, OCH3, C-3′, 3″), 6.96 (m, 2H, 4′, 4″-H), 7.10 (t, 2H, 5′, 5″-H, J = 7.9 Hz), 7.16 (dd, 2H, 6′, 6″-H, J = 7.9 Hz), 7.93 (brs, 2H, –C=C–H); EI-MS m/z 380 (3), 349 (4), 163 (10), 137 (10), 98 (18); HREI-MS for C23H24O5 M+, calcd.: m/z 380.1618, found: m/z 380.1623.
(2E,5E)-2,5-bis(4-Hydroxy-3-methoxybenzylidene)cyclopentanone (22)
Yellow solid; yield (58%); m.p. 212–214 °C (lit. [47] 214 °C); UV–Vis (CHCl3) λmax: 388 nm; IR (KBr) v 3043 (Ar C–H stretch), 1690 (C=C), 1620 (C=O), 1588–1485 (Ar C=C) cm−1; 1H NMR (CDCl3, 500 MHz) δ 3.03 (s, 4H, 3, 4-H), 3.88 (s, 6H, OCH3, C-3′, 3″), 6.92 (d, 2H, 5′, 5″-H, J = 8.30 Hz), 7.04 (brs, 2H, 2′, 2″-H), 7.14 (dd, 2H, 5′, 5″-H, J = 8.30, 1.65 Hz), 7.46 (brs, 2H, –C=C–H); EI-MS m/z 352; HREI-MS for C21H20O5 M+, calcd.: m/z 352.1310, found: m/z 352.1305.
(2E,5E)-2,5-bis(3,4-Dimethoxybenzylidene)cyclopentanone (23)
Yellow solid; yield (54%); m.p. 191–193 °C (lit. [37] 188–190 °C); UV–Vis (CHCl3) λmax: 368 nm; IR (KBr) v 3006 (Ar C–H stretch), 1693 (C=O), 1592–1515 (Ar C=C) cm−1; 1H NMR (CDCl3, 600 MHz) δ 3.12 (brs, 4H, 3, 4-H), 3.94, 3.93 (s, 12H, 4 × OCH3, C-3′, 3″, 4′, 4″), 6.96 (d, 2H, 5′, 5″-H, J = 8.34 Hz), 7.14 (s, 2H, 2′, 2″-H), 7.24 (dd, 2H, 6′, 6″-H, J = 8.34 Hz), 7.55 (brs, 2H, –C=C–H); 13C NMR (150 MHz, CDCl3) δ 26.3 (C-3, 4), 56.0 (C–O), 111.2 (C-2′, 2″), 113.5 (C-5′, 5″), 124.6 (C-6′, 6″), 129.0 (C-1′, 1″), 133.7 (–C=C–H), 148.9 (C-2, 5), 150.3 (C-3′, 3″), 150.3 (C-4′, 4″), 196.0 (C=O); EI-MS m/z 380.1 (5), 191.0 (10), 132.2 (18), 77.2 (55); HREI-MS for C23H24O5 M+, calcd.: m/z 380.1624, found: m/z 380.1619.
Anticancer activity
Sample preparation
Stock samples at 1 mg/mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) were prepared and keep at 4 °C.
MTT cell viability assay
Breast cancer MCF-7 and MDA-MB-231 cells, chronic myelogenous leukemia K562 cells, and cervical cancer HeLa cells lines were purchased from American Type Culture Collection (ATCC, USA) and cultured at 37 °C, 5% CO2 and 90% humidity using RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10% Foetal Bovine Serum (FBS) (Thermo Fisher Scientific, USA). For MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) cell viability assay [48], MCF-7, MDA-MB-231, K562 and HeLa cells were seeded overnight in 96-well plates at 8 × 104 cells/well at 37 °C of CO2 [49]. Then, 100 µL of media was discarded for all well-plates and compounds were serially diluted into the seeded cells at the concentration ranging between 30–0.47 µg/mL with cells treated with 3% DMSO (Sigma-Aldrich, USA) as the negative control. All samples were tested for triplicates. After 72 h of incubation, all well was added with 20 µL of MTT solution (5 mg/mL) and further incubated for 3 h. At that point, 170 µL of solution were discarded and 100 µL of DMSO (Sigma-Aldrich, USA) was added to all wells. Finally, absorbance was recorded by ELISA plate reader (Biotek-Instruments, USA) at the wavelength of 570 nm. Percentage of cell viability was calculated using following formula [38, 39]. The assay was performed in triplicate to calculate the half maximal inhibitory concentration (IC50) values. Doxorubicin was used as a positive control.
Cell viability (%) = [OD sample at 570 nm/OD negative control at 570 nm] × 100%
IC50 value (concentration of compounds inhibited 50% of cell viability) was determined from the graph of cell viability vs absorbance.
X-ray crystallographic analysis
X-ray analysis for all these samples were performed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ = 0.71073 Å) with φ and ω scans, at room temperature. Data reduction and absorption correction were performed using SAINT and SADABS programs [50,51,52,53]. The structures of compound 4 was solved by direct methods and refined by full-matrix least-squares techniques on F2 using SHELXTL software package. Crystallographic data of the reported structures have been deposited at the Cambridge Crystallographic Data Centre with CCDC deposition numbers of 1548735. Copy of available material can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).