No author has any associations that may represent a potential conflict of interest.
Cancer is the leading cause of death worldwide, and there is a constant need for new treatment strategies. Sesquiterpene lactones containing a 3-methylenedihydrofuran-2(3
The incidence of cancer is increasing, and different strategies for controlling the disease are developed. The pool of secondary plant metabolites has always been an important provider of low-molecular anti-cancer drugs, and is expected to be so also in the future. As our understanding about the molecular mechanisms of cancer development and progression, as well as the development of treatment resistance, has increased, our ability to design new anti-cancer drugs has improved.
In general, electrophilic compounds are considered potentially toxic and have been rejected by the pharmaceutical industry in their search for novel drug candidates.
Several studies of structure-activity relationships (SARs) of natural terpenoid Michael acceptors have shown that guaianolide and pseudoguaianolide sesquiterpenes with a α-methylene-γ-lactone moiety possess the most potent anti-cancer and cytotoxic activities.
In the present study we compare the cytotoxicity of 23 α-methylene-γ-lactones based on the natural product damsin (1) (see
Chemicals (analytical grade) were purchased from different commercial suppliers and used without further purification. Damsin (1) and coronopilin (2) (Figure 1) were isolated from A. arborescens as previously described.13 IR spectra were recorded with a Brucker Alpha FT-IR Spectrometer, and the optical rotations was measured with a Perkin-Elmer 141 polarimeter. HRMS spectra were recorded with a Waters XEVO-G2 QTOF instrument, while NMR spectra were recorded in CDCl3 using a Bruker DRX spectrometer operating at 400 MHz for 1H and at 100 MHz for 13C. Chemical shifts are given in ppm relative to the solvent signals (7.26 ppm for 1H and 77.00 ppm for 13C). All compounds described here were completely analysed by 2D NMR (COSY, HMQC, HMBC and NOESY), and as the syntheses start with the pure enantiomer damsin (1) all compounds are given with the absolute configuration in Figures 1 and 2. Chromatography was performed with 60 Å 30-75 μm silica gel, while TLC analyses were made on Silica Gel 60 F254 (Merck) plates.
A mixture of 1 (1 eq., 0.4027 mmol), aldehyde (1.3 eq., 0.5235 mmol), and p-TsOH (1.5 eq., 0.6040 mmol) in benzene (10 ml) was prepared in a sealed tube, and stirred at 80 °C for 19 to 66 h until the reaction was completed (monitored by TLC). The reaction was quenched by adding 2 ml of 5 % NaHCO3 followed by 15 ml brine, and the mixture was extracted with DCM (3 x 15 ml). The combined organic layers were dried with MgSO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (EtOAc:petroleum ether 1:1).
A mixture of aldehyde (3 eq., 1.2081 mmol) and 1 (1 eq., 0.4027 mmol) in 50 % PhMe:H2O 1:1 (3 ml) was cooled in an ice-bath, and TBAH (1.2 eq., 0.4832 mmol, 1.5 M in H2O) was added dropwise. The ice-bath was removed after 10 min and the reaction was left to reach room temperature. The reaction was monitored by TLC, and after 3 to 24 h the reaction was quenched by addition of 2 ml of 5 % HCl and stirred for 15 min at r.t. The reaction mixture was diluted with 15 ml brine, and extracted with DCM (3 x 10 ml), and the combined organic layers were dried with MgSO4 and concentrated in vacuo. The crude condensation product was purified by silica gel chromatography (EtOAc:petroleum ether 1:1).
To a solution of hydroxybenzaldehydes 4l – 4n (284.9 mg, 2.3333 mmol) in 6 ml dry DCM was added EtN(i-Pr)2 (2 ml, 11.6665 mmol), followed by MOMBr (400 μl, 4.8999 mmol) dropwise under N2 at 0 °C, whereafter the mixture was stirred at room temperature for 6 h. The reaction was quenched with 10 ml of a saturated solution of NaHCO3 by stirring for 15 min at r.t., and the mixture was extracted with DCM (2 x 15 ml). The combined organic layers were dried with MgSO4 and concentrated in vacuo. The product was purified by chromatography on silica gel (n-heptane:DCM 1:1).
To a solution of a MOM protected Claisen-Schmidt adduct (50 mg, 0.1261 mmol) in MeOH (2.6 ml), HCl conc. (22.5 μl, 0.2695 mmol, 37 %) was added slowly. The reaction mixture was heated to 40 °C for 5 h and then cooled to room temperature, whereafter the organic solvent was evaporated in vacuo. The residue was dissolved in DCM (15 ml), washed with brine (10 ml), dried with MgSO4, and concentrated in vacuo. The product was purified by chromatography on silica gel (EtOAc:petroleum ether 1:1).
The normal-like epithelial MCF-10A cell line was purchased from American Type Culture Collection (Manassas, VA, USA) (CRL-10781) and was used as a representative for normal breast epithelial cells. The cell line retains many normal traits, including lack of tumorigenicity in nude mice, anchorage-dependent growth, and dependence on growth factors and hormones for proliferation and survival.
The JIMT-1 human breast carcinoma cell line (ACC589) was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). It carries an amplified HER-2 oncogene and is insensitive to HER-2 inhibiting drugs and belongs to the HER2 sub-type of breast cancer.
Both cell lines were kept at 37 °C in a humidified incubator with 5 % CO2 in air. For the experiments, cells were seeded at the following densities: MCF-10A: 104 cells/cm2, and JIMT-1: 1.5×104 cells/cm2, in tissue culture vessels of the appropriate size to obtain the desired cell number for the different assays. The volume of medium used was 0.2–0.3 ml per cm2. The cells were allowed to attach for 24 hours before the addition of the compounds.
The sample compounds were dissolved in DMSO to 100 mM stock solutions that were kept at -20 °C. Working solutions were diluted in PBS and all had a DMSO concentration of 0.2 %. In the assay, the cells were exposed to PBS with 0.02 % DMSO, or with the respective compounds at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, and 20 µM concentrations.
The dose response to treatment with the compounds was evaluated using an MTT assay, which is based on reduction of MTT in the mitochondria of live cells. The amount of formazan produced is proportional to the number of living cells.
For the assay, cells were trypsinized and counted in a hemocytometer. Aliquots of 180 µl cell suspension containing 3000 cells (MCF-10A) or 5000 cells (JIMT-1) were seeded in 96-well plates. Compounds were added 24 h later to the final concentrations described above. At 72 h of drug treatment, 20 µl of MTT solution (5 mg/ml in PBS) was added to each well and the 96-well plates were returned to the CO2 incubator for 1 h. The medium was then removed and the blue formazan crystals were dissolved by adding of 100 µl of 100 % DMSO per well. The plates were swirled gently at room temperature for 10 minutes to dissolve the crystals in the cells. Absorbance was monitored at 540 nm in a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific, Lund, Sweden) using the software SkanIt 3.1. For each compound, three dose response experiments were performed with six replicates for each one of them. GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla, CA, USA), was used for drawing dose response curves and calculating the IC50 values, i.e. the dose giving 50 % reduction in cell number.
3a was obtained as a colourless oil in 72 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4a with 1 under acidic conditions as described in Experimental Procedure (EP) section 1a. (α)D20 +8.1 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 3366, 3059, 2926, 2870, 1755, 1714, 1623, 1573, 1449, 1271, 1232, 1186, 1158, 1111, 983, 768, 733, 714, 693, 646; TOFMS: [M+H+], found 337.1832, C22H25O3 requires 337.1804. See
3b was obtained as a colourless oil in 70 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4b with 1 under acidic conditions as described in EP section 1a. (α)D20 +29 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2921, 2862, 1752, 1712, 1625, 1603, 1271, 1253, 1179, 1159, 1117, 119, 814, 743, 521; TOFMS: [M+H+], found 351.1922, C23H27O3 requires 351.1915. See
3c was obtained as a colourless oil in 90 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4c with 1 under acidic conditions as described in EP section 1a. (α)D20 +3.0 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2922, 1758, 1714, 1625, 1271, 1157, 1118, 993, 693; TOFMS: [M+H+], found 351.1933, C23H27O3 requires 351.1915. See
3d was obtained as a colourless oil in 90 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4d with 1 under acidic conditions as described in EP section 1a. (α)D20 +62.6 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2922, 2862, 1757, 1714, 1623, 1597, 1270, 1251, 1161, 1119, 982, 949, 759, 735; TOFMS: [M+H+], found 351.1934, C23H27O3 requires 351.1915. See
3e was obtained as a colourless oil in 46 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4e with 1 under acidic conditions as described in EP section 1a. (α)D20 +12.2 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1); 2922, 2857, 1758, 1713, 1625, 1606, 1448, 1384, 1336, 1271, 1241, 1190, 1162, 1119, 1068, 984, 816. TOFMS: [M+H+], found 365.2109, C24H29O3 requires 365.2117. See
3f was obtained as a colourless oil in 88 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4-(trifluoromethyl)benzaldehyde with 1 under acidic conditions as described in EP section 1a. (α)D20 +0.9 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1): 2925, 2863, 1758, 1717, 1630, 1322, 1163, 1116, 1068, 1014, 984; TOFMS: [M+H+], found 405.1657, C23H24F3O3 requires 405.1633. See
3g was obtained as a colourless oil in 56 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4g with 1 under basic conditions as described in EP section 1b. (α)D20 -6.7 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1): 2924, 2864, 1758, 1716, 1630, 1327, 1271, 1160, 1073, 1119, 1000, 986, 808, 696, 736; TOFMS: [M+H+], found 405.1655 C23H24F3O3 requires 405.1633. See
3h was obtained as a colourless oil in 88 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4h with 1 under acidic conditions as described in EP section 1a. (α)D20 +36.8 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1): 2925, 1758, 1719, 1631, 1486, 1452, 1387, 1335, 1313, 1286, 1271, 1252, 1154, 1116, 1059, 1034, 985, 814, 799, 769, 735, 666; TOFMS: (M+H+), found 405.1634 C23H24F3O3 requires 405.1633. See
3i was obtained as a colourless oil in 87 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4i with 1 under acidic conditions as described in EP section 1a. (α)D20 +54.4 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2942, 2902, 2869, 2846, 1752, 1709, 1623, 1592, 1510, 1272, 1254, 1179, 1148, 1122, 1027, 979, 940, 835, 814; TOFMS: [M+H+], found 367.1891 C23H27O4 requires 367.1865. See
3j was obtained as a colourless oil in 73 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4j with 1 under acidic conditions as described in EP section 1a. (α)D20+2.4 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2958, 2862, 1752, 1709, 1623, 1592, 1510, 1272, 1254, 1179, 1148, 1122, 1027, 979, 940, 835; TOFMS: [M+H+], found 367.1856 C23H27O4 requires 367.1865. See
3k was obtained as a colourless oil as the single product obtained after the Claisen-Schmidt condensation of 4k with 1 under acidic conditions as described in EP section 1a. (α)D20 +58.2 (c 1.00, CH2Cl2);IR spectrum ( film, γ, cm-1): 2928, 2859, 1757, 1712, 1619, 1596, 1486, 1463, 1437, 1270, 1247, 1188, 1161, 1119, 984, 814, 754, 743; TOFMS: [M+H+], found 367.1849 C23H27O4 requires 367.1865. See
4l was protected to 4x according to EP section 1c, 4x was condensed with 1 under basic conditions as described in EP section 1b to form 3x. 3x was subsequently deprotected to 3l according to EP section 1d, 3l was obtained as a colourless oil in 23 % overall yield (from 1) after chromatographic purification of the crude product. (α)D20: +33.0 (c 1.00, CH2Cl2); IR spectrum; 3319, 2923, 2853, 1755, 1737, 1706, 1595, 1578, 1511, 1469, 1443, 1272, 1254, 1168, 1157, 1120,1104, 984, 833, 816; TOFMS: [M+H+], found 353.1722 C22H25O4 requires 353.1708. See
4m was protected to 4y according to EP section 1c, 4y was condensed with 1 under basic conditions as described in EP section 1b to form 3y. 3y was subsequently deprotected to 3m according to EP section 1d, 3l was obtained as a colourless oil in 43 % overall yield (from 1) after chromatographic purification of the crude product. (α)D20 -11.7 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1); 3353, 2927, 2864, 1735, 1712, 1621, 1591, 1578, 1490, 1471, 1449, 1272, 1228, 1158, 1112, 1119, 992, 785, 687; TOFMS: [M+H+], found 353.1733 C22H25O4 requires 353.1708. See
4n was protected to 4z according to EP section 1c, 4z was condensed with 1 under basic conditions as described in EP section 1b to form 3z. 3z was subsequently deprotected to 3n according to EP section 1d, 3n was obtained as a colourless oil in 27 % overall yield (from 1) after chromatographic purification of the crude product. (α)D20 +29.5 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 3353, 2927, 2864, 1735, 1712, 1621, 1591, 1578, 1490, 1471, 1449, 1272, 1228, 1158, 1112, 1119, 992, 785, 687; TOFMS: [M+H+], found 353.1704 C22H25O4 requires 353.1708. See
The procedure for preparing 3o is shown in
3p was obtained as a colourless oil in 42 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4p with 1 under acidic conditions as described in EP section 1a. (α)D20 -33.6 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1): 2923, 2851, 1757, 1720, 1649, 1448, 1384, 1346, 1270, 1250, 1190, 1156, 1101, 993, 953, 802. TOFMS [M+H+], found 343. 2261, C22H31O3 requires 343. 2273. See
3q was obtained as a colourless oil in 20 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4q with 1 under acidic conditions as described in EP section 1a. (α)D20 +34.4 (c 1.00, CH2Cl2);IR spectrum (film, γ, cm-1): 2924, 2870, 1738, 1660, 1449, 1384, 1341, 1271, 1162, 1119, 1002, 977, 949. TOFMS [M+H+], found 343. 2279 C22H31O3 requires 343. 2273. See
3r was obtained as a colourless oil in 20 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4r with 1 under acidic conditions as described in EP section 1a. (α)D20 -53.7 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2927, 1757, 1649, 1271, 979; TOFMS: [M+H+], found 289.1786, C18H25O3 requires 289.1759. See
3s was obtained as a colourless oil in 42 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4s with 1 under basic conditions as described in EP section 1b. (α)D20 -44.0 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2929, 1758, 1719, 1649, 1269, 1111, 954, 814, 731; TOFMS: [M+H+], found 303.1864, C19H27O3 requires302.1882. See
3t was obtained as a colourless oil in 42 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4t with 1 under basic conditions as described in EP section 1b. (α)D20 -63.0 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2955, 1757, 1649, 1271, 967; TOFMS: [M+H+], found 317.2100, C20H29O3 requires317.2072. See
3u was obtained as a colourless oil in 36 % yield after chromatographic purification of the product obtained after the Claisen-Schmidt condensation of 4u with 1 under basic conditions as described in EP section 1b. (α)D20 -50.6 (c 1.00, CH2Cl2); IR spectrum (film, γ, cm-1): 2921, 1757, 1719, 1850, 1271, 1112, 994; TOFMS: [M+H+], found 315.1926 C20H27O3 requires315.1915. See
Damsin (1) and coronopilin (2) were isolated from the plant Ambrosia arborescens as previously described.
Compound | MCF-10A (µM) | JIMT-1 (µM) | Ratio MCF-10A:JIMT-1 |
1 | 8.1 ± 0.42 | 3.3 ± 0.62 | 2.5 |
2 | 15.3 ± 0.92 | 5.6 ± 0.82 | 2.7 |
3a | 8.2 ± 1.62 | 1.7 ± 0.41 | 4.8 |
3b | 3.7 ± 0.42 | 2.1 ± 0.32 | 1.8 |
3c | 12.6 ± 1.62 | 4.8 ± 0.32 | 2.6 |
3d | 11.1 ± 1.82 | 4.7 ± 0.11 | 2.4 |
3e | 5.2 ± 1.52 | 3.5 ± 0.72 | 1.5 |
3f | 3.1 ± 0.32 | 1.8 ± 0.22 | 1.7 |
3g | 11.9 ± 0.41 | 4.4 ± 0.71 | 2.7 |
3h | 13.0 ± 0.81 | 8.1 ± 0.6 |
1.6 |
3i | 7.9 ± 1.22 | 1.6 ± 0.12 | 4.9 |
3j | > 201 | 9.0 ± 1.01 | na |
3k | > 201 | 7.1 ± 0.21 | na |
3l | 13.6 ± 0.62 | 2.9 ± 0.21 | 4.7 |
3m | 10.6 ± 1.32 | 2.4 ± 0.11 | 4.4 |
3n | 6.7 ± 0.92 | 2.1 ± 0.21 | 3.2 |
3o | 7.1 ± 0.62 | 2.0 ± 0.62 | 3.6 |
3p | 11.7 ± 1.92 | 8.1 ± 3.12 | 1.4 |
3q | > 202 | 12.3 ± 1.32 | na |
3r | 5.5 ± 1.11 | 1.4 ± 0.11 | 3.9 |
3s | 12.6 ± 0.81 | 3.7 ± 0.11 | 3.4 |
3t | 20.3 ± 0.31 | 8.1 ± 0.11 | 2.5 |
3u | 17.5 ± 5.11 | 1.7 ± 0.01 | 10.3 |
The structures of all compounds prepared in this investigation was carefully determined by 1- and 2D NMR experiments (including COSY, NOESY, HMQC and HMBC experiments), in combination with the IR and HRMS data reported in the Experimental section. The configuration of the C-3/C-1' double bond was determined by NOESY NMR experiments. For the E isomers correlations were observed between 2-H2 and 2''-H/6''-H (for 3a - 3n) or 1''-H2 (for 3p - 3u), and between 1'-H and 2-H2 for the Z-isomer 3q. The absolute configuration of C-3 in derivative 3o was suggested by the observed NOESY correlation between 1-H and 3-H, as well as the lack of NOESY correlations between both 14-H3 and 15-H3, and 3-H. This configuration is also supported by the 1H-1H coupling constants observed, 8.4 and 12.1 Hz between 3-H and 2-H2 as well as 3.9 and 8.8 Hz between 3-H and 1'-H2.
The chemical shifts for all proton and carbon signals observed in CDCl3 are presented in
1-H | 2-H2 | 6-H | 7-H | 8-H2 | 9-H2 | 10-H | 13-H2 | 14-H3 | 15-H3 | 1’-H | 2’’-H | 3’’-H | 4’’-H | 5’’-H | 6’’-H | |
3a | 2.07 | 2.87/2.97 | 4.65 | 3.28 | 1.80/2.06 | 1.74/1.89 | 2.28 | 5.57/6.28 | 1.17 | 1.16 | 7.44 | 7.41 | 7.54 | 7.37 | 7.54 | 7.41 |
3b |
2.06 | 2.84/2.94 | 4.63 | 3.38 | 1.80/2.07 | 1.74/1.88 | 2.27 | 5.56/6.27 | 1.17 | 1.14 | 7.4 | 7.43 | 7.21 | - | 7.21 | 7.43 |
3c |
2.05 | 2.84/2.94 | 4.63 | 3.28 | 1.80/2.06 | 1.73/1.87 | 2.28 | 5.56/6.26 | 1.17 | 1.14 | 7.38 | 7.32 | - | 7.17 | 7.3 | 7.32 |
3d |
2.03 | 2.74/2.94 | 4.64 | 3.28 | 1.81/2.06 | 1.70/1.86 | 2.24 | 5.57/6.28 | 1.16 | 1.18 | 7.65 | - | 7.23 | 7.26 | 7.24 | 7.46 |
3e |
2.03 | 2.75/2.94 | 4.65 | 3.28 | 1.82/2.09 | 1.70/1.86 | 2.24 | 5.57/6.29 | 1.19 | 1.16 | 7.66 | - | 7.07 | - | 7.07 | 7.38 |
3f | 2.1 | 2.86/2.99 | 4.66 | 3.31 | 1.84/2.07 | 1.74/1.91 | 2.3 | 5.58/6.29 | 1.19 | 1.17 | 7.43 | 7.65 | 7.66 | - | 7.66 | 7.65 |
3g | 2.1 | 2.85/2.98 | 4.65 | 3.3 | 1.84/2.08 | 1.75/1.91 | 2.31 | 5.58/6.29 | 1.19 | 1.17 | 7.43 | 7.76 | - | 7.62 | 7.55 | 7.71 |
3h | 2.05 | 2.65/2.90 | 4.64 | 3.29 | 1.81/2.03 | 1.72/1.85 | 2.22 | 5.56/6.27 | 1.14 | 1.18 | 7.71 | - | 7.58 | 7.58 | 7.45 | 7.71 |
3i |
2.06 | 2.82/2.92 | 4.63 | 3.27 | 1.79/2.04 | 1.74/1.86 | 2.27 | 5.56/6.26 | 1.16 | 1.13 | 7.37 | 7.5 | 9.93 | - | 6.93 | 7.5 |
3j |
2.06 | 2.86/2.95 | 4.64 | 3.28 | 1.80/2.03 | 1.72/1.86 | 2.27 | 5.57/6.27 | 1.16 | 1.15 | 7.38 | 7.05 | - | 6.92 | 7.33 | 7.14 |
3k |
2.01 | 2.73/2.91 | 4.59 | 3.25 | 1.77/2.04 | 1.72/1.84 | 2.22 | 5.55/6.24 | 1.14 | 1.14 | 7.82 | - | 6.89 | 7.32 | 6.96 | 7.47 |
3l | 2.07 | 2.81/2.91 | 4.67 | 3.3 | 1.81/2.04 | 1.72/1.90 | 2.27 | 5.59/6.29 | 1.16 | 1.14 | 7.36 | 7.43 | 6.93 | - | 6.93 | 7.43 |
3m | 2.01 | 2.80/2.90 | 4.71 | 3.34 | 1.84/2.04 | 1.74/1.90 | 2.24 | 5.59/6.29 | 1.14 | 1.12 | 7.48 | 7.08 | - | 6.91 | 7.25 | 7.25 |
3n | 2.04 | 2.79/2.94 | 4.64 | 3.29 | 1.82/2.06 | 1.73/1.87 | 2.24 | 5.58/6.29 | 1.16 | 1.17 | 7.92 | - | 6.94 | 7.22 | 6.9 | 7.45 |
3o |
1.99 | 1.71/1.87 | 4.5 | 3.31 | 1.79/2.02 | 1.69/1.81 | 2.16 | 5.53/6.29 | 1 | 0.91 | 2.66/3.19 | 7.17 | 7.28 | 7.21 | 7.28 | 7.17 |
3p |
2 | 2.51/2.59 | 4.59 | 3.26 | 1.78/2.03 | 1.71/1.84 | 2.23 | 5.54/6.26 | 1.1 | 1.08 | 6.47 | 1.23/1.66 | 1.26/1.75 | 1.77 br | 1.26/1.75 | 1.23/1.66 |
3q |
2.01 | 1.82/2.55 | 4.47 | 3.28 | 1.82/2.03 | 1.72/1.80 | 2.21 | 5.52/6.26 | 1.05 | 1.03 | 5.43 | 1.59/2.08 | 1.65/1.97 | 1.71 br | 1.65/1.97 | 1.59/2.08 |
3r |
2 | 2.50/2.58 | 4.6 | 3.26 | 1.79/2.02 | 1.71/1.86 | 2.23 | 5.55/6.26 | 1.11 | 1.09 | 6.61 | 2.16 | 1.06 | - | - | - |
3s |
1.98 | 2.47/2.53 | 4.56 | 3.24 | 1.76/2.00 | 1.68/1.79 | 2.19 | 5.52/6.20 | 1.06 | 1.04 | 6.57 | 2.09 | 1.44 | 0.88 | - | - |
3t |
1.96 | 2.50/2.54 | 4.59 | 3.25 | 1.74/1.86 | 1.70/1.84 | 2.19 | 5.53/6.23 | 1.07 | 1.05 | 6.62 | 2.02 | 1.76 | 0.89 | 0.89 | - |
3u |
2 | 2.47/2.55 | 4.6 | 3.26 | 1.78/2.02 | 1.73/1.85 | 2.24 | 5.51/6.26 | 1.09 | 1.08 | 6.61 | 2.25 | 2.22 | 5.78 | 5 | - |
Aromatic methyl group at 2.37.
Aromatic methyl group at 2.37.
Aromatic methyl group at 2.39.
Aromatic methyl groups at 2.34 and 2.38.
Aromatic methoxy group at 3.83.
Aromatic methoxy group at 3.83.
Aromatic methoxy group at 3.83.
A signal for 3-H at 2.56.
A signal for 1''-H at 2.21.
A signal for 1''-H at 2.54.
Signals for the ethyl group at 2.16 and 1.06.
Signals for the propyl group at 2.09, 1.44 and 0.88.
Signals for the 2-methyl propyl group at 2.02, 1.76 and 0.89.
Signals for the 3-butenyl group at 5.78, 5.00, 2.25 and 2.22.
C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | C-7 | C-8 | C-9 | C-10 | C-11 | C-12 | C-13 | C-14 | C-15 | C-1’ | C-1’’ | C-2’’ | C-3’’ | C-4’’ | C-5’’ | C-6’’ | |
3a | 43.6 | 31.4 | 133.3 | 207.9 | 54.8 | 81.8 | 44.8 | 26.5 | 34.2 | 34.1 | 140.1 | 170.2 | 121.2 | 15.7 | 14.5 | 133.9 | 129 | 130.6 | 128.8 | 135.4 | 128.8 | 130.6 |
3b |
43.6 | 31.4 | 132.4 | 208.1 | 54.8 | 81.9 | 44.8 | 26.5 | 34.3 | 34.1 | 140.1 | 170.3 | 121.3 | 15.8 | 14.5 | 133.9 | 132.6 | 130.6 | 129.6 | 140 | 129.6 | 130.6 |
3c |
43.6 | 31.4 | 133.2 | 208.1 | 54.8 | 81.9 | 44.8 | 26.5 | 34.2 | 34 | 140.1 | 170.3 | 121.3 | 15.8 | 14.5 | 134 | 135.3 | 131.4 | 138.4 | 130.4 | 128.7 | 127.6 |
3d |
43.9 | 31.3 | 134.1 | 207.9 | 54.9 | 81.9 | 44.8 | 26.4 | 34.1 | 34 | 140.1 | 170.3 | 121.2 | 15.8 | 14.5 | 131.5 | 134.1 | 139 | 130.7 | 129.4 | 125.9 | 128.7 |
3e |
44 | 31.5 | 133.3 | 208 | 55 | 82 | 45 | 26.5 | 34.3 | 34.2 | 140.2 | 170.4 | 121.2 | 15.9 | 14.6 | 131.7 | 131.4 | 139.3 | 131.6 | 139.8 | 126.7 | 128.8 |
3f |
43.6 | 31.4 | 135.8 | 207.6 | 55 | 81.7 | 44.8 | 26.4 | 34.1 | 34 | 139.9 | 170.2 | 121.5 | 15.7 | 14.4 | 132 | 138.9 | 130.6 | 125.7 | 130.8 | 125.7 | 130.6 |
3g |
43.7 | 31.2 | 135.2 | 207.7 | 54.9 | 81.8 | 44.8 | 26.4 | 34.2 | 34 | 140 | 170.3 | 121.4 | 15.7 | 14.5 | 132 | 131.4 | 126.8 | 136.2 | 125.9 | 129.4 | 133.5 |
3h |
43.7 | 31.1 | 137 | 207 | 55 | 81.7 | 44.7 | 26.3 | 34 | 34 | 139.9 | 170.3 | 121.3 | 15.7 | 14.4 | 129.4 | 129.8 | 134.1 | 130.1 | 131.7 | 128.8 | 126.3 |
3i |
43.6 | 31.4 | 130.9 | 208 | 54.7 | 81.9 | 44.9 | 26.6 | 34.3 | 34.1 | 140.2 | 170.3 | 121.2 | 15.8 | 14.6 | 133.7 | 128.1 | 132.4 | 114.3 | 160.7 | 114.3 | 132.4 |
3j |
43.6 | 31.4 | 133.7 | 208 | 54.8 | 81.8 | 44.8 | 26.5 | 34.2 | 34 | 140.1 | 170.3 | 121.3 | 15.7 | 14.5 | 133.8 | 136.7 | 116.1 | 159.7 | 115 | 129.8 | 123.1 |
3k |
43.6 | 31.4 | 133.2 | 207.9 | 54.7 | 81.9 | 44.8 | 25.4 | 34.1 | 34 | 140.1 | 170.4 | 121.1 | 15.8 | 14.5 | 128.5 | 124.3 | 158.8 | 110.8 | 131.1 | 120.2 | 129.6 |
3l | 43.8 | 31.4 | 130.3 | 208.7 | 54.8 | 82.3 | 44.8 | 26.5 | 34.3 | 34.1 | 140.1 | 170.9 | 121.7 | 15.8 | 14.6 | 134.7 | 127.5 | 132.8 | 116.1 | 158.2 | 116.1 | 132.8 |
3m | 43.6 | 31.5 | 133.3 | 208.8 | 54.9 | 82.4 | 44.5 | 26.3 | 34.1 | 34 | 139.9 | 171.1 | 121.9 | 15.7 | 14.5 | 134.8 | 136.8 | 122 | 156.7 | 117.3 | 129.8 | 118.5 |
3n | 43.8 | 31.4 | 132 | 209.1 | 55 | 82.1 | 44.8 | 26.5 | 34.2 | 34.1 | 140.1 | 170.7 | 121.5 | 15.8 | 14.6 | 129.8 | 122.5 | 156.8 | 116.4 | 131.5 | 120 | 129.6 |
3o | 44.1 | 30.6 | 50.3 | 218.6 | 55.3 | 81.9 | 44.5 | 25.6 | 33.1 | 34.2 | 139.6 | 170.3 | 120.8 | 14.6 | 14 | 36.3 | 139.4 | 129.2 | 128.5 | 126.4 | 128.5 | 129.2 |
3p | 43.2 | 28.6 | 132.8 | 207.6 | 55.4 | 81.9 | 44.9 | 26.5 | 34.2 | 34.2 | 140.2 | 170.4 | 121.1 | 15.8 | 14.4 | 143.1 | 39.1 | 31.7 | 25.6 | 25.1 | 25.6 | 31.7 |
3q | 44.1 | 28.3 | 135.4 | 219.9 | 55.3 | 82 | 44.6 | 25.3 | 33.3 | 34.3 | 139.6 | 170.3 | 120.8 | 16.2 | 14.3 | 122.9 | 39.5 | 31.6 | 25.8 | 25.5 | 25.8 | 31.6 |
3r | 43.1 | 28.5 | 134.1 | 207.2 | 55.5 | 81.9 | 44.9 | 26.5 | 34.2 | 34.2 | 140.1 | 170.3 | 121.2 | 15.8 | 14.4 | 139.7 | 23.2 | 12.9 | - | - | - | - |
3s | 43 | 28.6 | 134.8 | 207 | 55.3 | 81.8 | 44.7 | 26.3 | 34 | 34 | 140 | 170.3 | 121.1 | 15.7 | 14.2 | 138.1 | 31.7 | 21.6 | 13.9 | - | - | - |
3t | 43 | 28.8 | 135.3 | 207 | 55.4 | 81.9 | 44.7 | 26.4 | 34.1 | 34.1 | 140.1 | 170.4 | 121.2 | 15.7 | 14.3 | 137.4 | 38.9 | 28.2 | 22.6 | 22.6 | - | - |
3u | 43.1 | 28.7 | 135.1 | 206.9 | 55.5 | 81.8 | 44.8 | 26.4 | 34.2 | 34.2 | 140.1 | 170.3 | 121.2 | 15.7 | 14.4 | 137.3 | 29.2 | 34.3 | 137.3 | 115.6 | - | - |
Aromatic methyl group at 21.5.
Aromatic methyl group at 21.5.
Aromatic methyl group at 20.0.
Aromatic methyl groups at 21.4 and 20.0.
A signal for an aromatic -CF3 group at 122.7.
A signal for an aromatic -CF3 group at 122.5.
A signal for an aromatic -CF3 group at 122.5.
Aromatic methoxy group at 55.4.
Aromatic methoxy group at 55.4.
Aromatic methoxy group at 55.5.
The MCF-10A cell line used in this study is a non-tumorigenic breast epithelial cell line
The cytotoxicity of the reduced derivative 3o is similar to that of 3a, indicating that the double bond created at C-3 by the condensation is unimportant. Replacing the phenyl group of 3a with a cyclohexyl (in 3p) decreases both potency and selectivity, and when comparing 3p with 3q it is obvious that an E configuration of the C-3 double bond is preferable. None of the methylated derivatives 3b – 3e is more potent compared to 3a, although the p-substituted derivative 3b has a similar potency, and they are all less selective. Methyl substituents in the benzene ring are therefore not beneficial. For the trifluoromethyl derivatives 3f – 3h there is a strong tendency that p-substitution is better for the potency than m-substitution, and m is better than o, but all three suffer from lower selectivity. For the methoxylated derivatives 3i – 3k, it is obvious that the p-methoxy derivative 3i not only retains the potency of 3a but also the selectivity. However, the m- and o-methoxy derivatives 3j and 3k are considerably less cytotoxic. Furthermore, the p-hydroxy derivative 3l retains much of the potency and selectivity of 3a, although the m- and o-hydroxyl derivatives 3m and 3n are even more potent than 3l. The differences in the trends observed may be explained by the ability of the hydroxyl group to both give and accept hydrogen bonds, which is not the case for the other substituents used here. Also, the size of a hydroxyl group is smaller than a trifluoromethyl or methoxy group, so there may be a steric component. For the alkenyl derivatives 3r – 3u, the results are quite interesting. The smallest, 3r, is as potent and selective as 3a, indicating that a short alkyl group influences the molecular interaction as much as a phenyl group. An extra methyl group (as in 3s) is less advantageous, while two extra methyl groups (as in 3t) is even worse for both potency and selectivity. Remarkably, by keeping the same number of carbons in the chain but without branching and with a double bond in the end (as in 3u), the potency is the same as that of 3a while the selectivity is high. With a ratio of IC50 MCF-10A cells and IC50 JIMT-1 cells greater than 10, 3u is the most selective derivative prepared in this investigation, and will be a future starting point for the development of this class of compounds.
The authors thank the Swedish International Development Agency (SIDA) for the financial support of this study, which is part of the project “Biomolecules of medicinal and industrial interest” developed between the Universidad Mayor de San Andrés (UMSA La Paz – Bolivia) and Lund University (Sweden).