Regular Articles
Design and Synthesis of New Anticancer Glycyrrhetinic Acids and Oleanolic Acids
2017 Volume 40 Issue 5 Pages 703-710
Details
2017 Volume 40 Issue 5 Pages 703-710
A series of new glycyrrhetinic acids and oleanolic acids has been designed and synthesized based on the principles of combinatorial chemical synthesis. Their anticancer activities were further studied by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with hepatocellular carcinoma (Hep-G2), breast cancer (MCF-7) cell lines and a normal hepatic cell (LO2). Cytotoxicity tests (in vitro) indicated that compound 6a showed the highest cytotoxicity with the lowest IC50 values of 23.34 µM on Hep-G2 cells, 12.23 µM on MCF-7 cells, and 44.47 µM on LO2, which would widen the structural diversity of these anticancer targets and confirm the perspectives of further investigations.
Cancer is a main reason of death all over the world. As shown in GLOBOCAN, about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide, and it is estimated that in 2030 new cases of cancer will increase from 12.7 to 21.4.1,2) In the U.S.A., 1685210 new cancer cases and 595690 cancer deaths are projected to occur in 2016.3) Therefore, more and more researchers are working to develop new therapeutic agents with improved selectivity and higher anticancer activity, most of which are chemically modified natural products.4) It is a very effective way for people to find the drugs by looking for the lead compounds in natural products and structural modifications of it, and now most of the drugs we use are direct or indirect sources in natural products.5) Glycyrrhiza uralensis is a traditional natural product, people have used it as food and medicine to prevent various diseases as early as two thousand years ago.6) Glycyrrhetinic acid (GA), as one of active component of Glycyrrhiza uralensis, plays a more important role in the pharmacological and shows a variety of pharmacological effects, such as antiviral,7,8) anti-allergic,9) anti-inflammatory,10,11) anti-ulcer12) and anti-tumor activities.13,14) Our group has done some research on the synthesis and anticancer activity of glycyrrhetinic acid derivatives, and found that some of them have good anticancer activities.15) Oleanolic acid (OA) and glycyrrhetinic acid (GA) are both pentacyclic triterpenoid compounds (Fig. 1). They have similar structure and distribute widely in nature. Oleanolic acid also has anti-inflammatory,16) anti-tumor,17,18) anti-virus19) and other biological activities.
However, due to the smaller polarity of GA and OA, their bioavailability is very limited. It is reported that the introduction of ester-joined groups at 30-COOH of GA could enhance the anti-tumor effect,20) and the IC50 values of GA and OA were much higher than the introduction of carboxyl groups into the C-3 position of GA and OA.21,22) In these contexts, we envisioned that the lipophilicity of GA and OA should be improved by installing ester to them, and their polarity could be obtained by using suitable additional functional groups. Accordingly, herein we synthesized 12 derivatives of GA and OA—esterified at C(30) or C(28) and varied by coupling with organic acids (indoleacetic acid, indole butyric acid, lipoic acid, salicylic acid,23) cinnamic acid24) and acetylated 4-hydroxy cinnamic acid25)) at C(3). We also designed six additional compounds, the –COOH of GA (and OA) and 4-hydroxycoumarin, sesamol, eugenol to form esters. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to test the anticancer activities of these new-synthesized compounds with the hepatocellular carcinoma (Hep-G2), breast cancer (MCF-7) cell lines and a normal hepatic cell (LO2).
The syntheses of glycyrrhetinic acids and oleanolic acids, viz., 3a–l, are illustrated in Chart 1. In the first series, GA (or OA) esters 2a and b were obtained by the treatment of them with bromoethane in the presence of K2CO3,26) then their 3-OH moieties were subsequently coupled with organic acids (R1OH) in 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI)/N,N-dimethyl-4-aminopyridine (DMAP) system to afford esters 3a–l. Interestingly, treatment of 2a and b with acetylsalicylic acid under the above reaction conditions, esters 5a and b were obtained instead of expected esters 4a and b. The result was further confirmed by reaction of 2a and b with acetic anhydride to prepare authentic esters 5a and b and then by comparing their spectra. A possible reason to understand the above phenomenon is that the carboxyl group of acetylsalicylic acid has a large steric hindrance and cannot be preferentially activated by EDCI, and thus cannot be esterified with 2a and b. The ester exchange reaction between the hydroxyl moiety of 2a and b and the ester moiety of acetylsalicylic acid was preferentially occurred in the presence of DMAP, and thus to give esters 5a and b. The possible reaction mechanism was shown in Fig. 2.27) The high nucleophilicity of DMAP facilitated its nucleophilic addition to the ester moiety of acetylsalicylic acid to form intermediate I, which was converted to a N-acylpyridinium by elimination of a phenoxide anion. Subsequently, the N-acylpyridinium undertook a nucleophilic attack by the hydroxyl moiety of 2a and b to generate intermediate II, which was neutralized by the in-situ generated phenoxide anion, and was then converted to the desired ester 5a or b by elimination of DMAP.
Reagents and conditions: a) EtBr, K2CO3, DMF, room temperature (r.t.); b) EDCI, DMAP, reflux, r.t.; c) acetylsalicylate, EDCI, DMAP, reflux, r.t.
As shown in Chart 2, the treatment of 1a and b with 4-hydroxycoumarin to afford esters 6a and b, and 7a and b. However, the reaction of 1a and b with sesamol or eugenol was failed, which might be resulted from the steric hindrance of the C(28) of OA.
Reagents and conditions: d) 4-Hydroxycoumarin, sesamol and eugenol, EDCI, DMAP, reflux, r.t.
Inhibitory activities of compounds 1a and b, 2a and b, 3a–l, 5a and b, 6a and b and 7a and b were evaluated by MTT method against Hep-G2, MCF-7 cell lines and LO2 cells. The bioassay results are summarized in Table 1. The parent compounds GA and OA exhibited strong inhibitory effect on LO2 cells, but they exhibited no inhibitory effect on Hep-G2 cells and MCF-7 cells. All the synthesized glycyrrhetinic acid and oleanolic acid derivatives, surprisingly, were found to exhibit relatively lower inhibitory activity against LO2 cells. Compounds 2a and b exhibited relatively low inhibitory activities against MCF-7 cells as compared with GA and OA, respectively, suggesting that the introduction of ethyl group at C-30 results in a gain of potency. In contrast to compound 2a, compound 2b showed enhanced inhibitory effect but poor selectivity toward Hep-G2 cells. Further modifications to the 30-position (GA) or 28-position (OA) afforded esters. Compounds 3a, c, f, h, i and l, prepared by esterification with organic acids such as indoleacetic acid, indole butyric acid, lipoic acid, salicylic acid, are potent selective inhibitors of MCF-7 cells instead of Hep-G2 cells. Compounds 3b, d, e, g, j and k did not display inhibitory effect on Hep-G2 cells and MCF-7 cells (IC50>100 µM), indicating that the introduction of organic acid moieties to such a position is not beneficial to improve their anticancer activities. Esterification at the C-3 of compound 5a led to an increase of the inhibitory effects on MCF-7 cells, as compared with compound 2a. On the contrary, compound 5b exhibited no inhibitory activities against Hep-G2 cells and MCF-7 cells as compared with OA and compound 2b, respectively, suggesting that esterification at the C-3 of compound 5b lead to a loss of potency. Compound 6a showed the highest cytotoxicity with the lowest IC50 values of 23.34 µM on Hep-G2 cells and 12.23 µM on MCF-7 cells. Its anticancer activity is similar to that of the positive control, Gefitinib.28) Compounds 6b and 7a exhibited poor inhibitory effect on MCF-7 cells with the relatively high IC50 (73.76 µM, 78.84 µM) and strict selectivity toward Hep-G2 cells. Compound 7b had a similar range of activity as compound 6a, it exhibited potent inhibitory effect on MCF-7 cells with the relatively low IC50 (22.74 µM) and poor selectivity toward Hep-G2 cells (IC50=46.99 µM).
| Compd. | IC50 (µM)a) | ||
|---|---|---|---|
| LO2 | Hep-G2 | MCF-7 | |
| GA(1a) | 23.84 | >100 | >100 |
| OA(1b) | 26.62 | >100 | >100 |
| 2a | >100 | >100 | 89.24 |
| 2b | 80.65 | 40.71 | 54.83 |
| 3a | >100 | >100 | 84.48 |
| 3b | >100 | >100 | >100 |
| 3c | >100 | >100 | 76.69 |
| 3d | >100 | >100 | >100 |
| 3e | >100 | >100 | >100 |
| 3f | >100 | >100 | 88.96 |
| 3g | >100 | >100 | >100 |
| 3h | >100 | >100 | 44.78 |
| 3i | >100 | >100 | 72.87 |
| 3j | >100 | >100 | >100 |
| 3k | >100 | >100 | >100 |
| 3l | >100 | >100 | 97.68 |
| 5a | 97.84 | >100 | 74.83 |
| 5b | >100 | >100 | >100 |
| 6a | 44.47 | 23.34 | 12.23 |
| 6b | >100 | >100 | 73.76 |
| 7a | >100 | >100 | 78.84 |
| Gefitinib | — | 20.73±16.36 | 17.83±7.85 |
| Doxorubicin | 0.85±0.29 | 0.89±0.12 | 0.66±0.08 |
a) IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by MTT method. If the IC50 of test compound is over 100 µM, it is regarded as inactive. ‘—’: The IC50 value was not determined.
In summary, a series of new glycyrrhetinic acids and oleanolic acids (3a–l, 6a, b and 7a, b) has been designed and synthesized, and their effects on the inhibition of Hep-G2, MCF-7 cell lines and LO2 cells have been evaluated by MTT assay. The results showed that all of the synthesized glycyrrhetinic acids and oleanolic acids exhibited relatively lower inhibitory activity against LO2 cells, as compared with GA and OA. This series of compounds on the inhibition of MCF-7 cells are better than the inhibitory effect of Hep-G2 cells which exhibited certain selectivity toward Hep-G2 cells. Among these new-synthesized compounds, compound 6a showed the highest cytotoxicity on Hep-G2 cells and MCF-7 cells, which would widen the structural diversity of these anticancer targets and confirm the perspectives of further investigations. Furthermore, SAR analysis revealed that the introduction of ester-joined groups at –COOH of GA and OA could increase the anticancer activity, as compared with GA and OA. Work on this area is in progress in our laboratory and will be reported later.
Reagents and solvents were bought from local reagent company (Wehai Xinyue Chemical and Glass Co., Ltd., Weihai, China) and used without further purification. Column chromatography and TLC were performed on silica gel and silica gel plates (both from Qingdao Ocean Chemical Co., Ltd., Qingdao, China). 1H- and 13C-NMR spectra were recorded on Bruker-400 instrument (Bruker; Fallanden, Switzerland) at 25°C with TMS as an internal standard and CDCl3 as solvents. Mass spectra were measured on a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, U.S.A.). Melting points (mp) were measured on SGW X-4 micro-melting point of the instrument (Hangzhou Kexiao Chemical Equipment Co., Ltd., Hangzhou, China) and were uncorrected. The anhydrous solvent required for the experiment was re-evaporated before use.
General Experimental Method for the Synthesis of 2a and bTo a solution of 1a and b (1 mmol) in dry N, N-dimethylformamide (DMF) (10 mL), anhydrous potassium carbonate (500 mg, 3.62 mmol) was added. After 30 min of stirring at room temperatures, bromoethane (0.4 mL, 4.4 mmol) was added and the resulting mixture was stirred for an additional 12 h. When the reaction was completed, 10 mL of saturated sodium chloride was added and the mixture was extracted with ethyl acetate (25 mL). The combined organic phases were washed with water until pH=7, dried over anhydrous sodium sulfate, filtered and concentrated without further treatment. The next reaction can be carried out to give compounds 2a and b.
General Experimental Method for the Synthesis of 3a–l and 5a and bTo a solution of R1OH (1 mmol) in dry dichloromethane (15 mL), EDCI (230 mg, 1.2 mmol) and DMAP (24 mg, 0.2 mmol) were added. The resulting mixture was stirred at 0°C for 1 h. Simultaneously, a solution of 2a and b (1 mmol) in dry dichloromethane (15 mL) was stirred at 25°C for 1 h. Mixing them up after 1 h and stirring them at room temperature. The reaction was indicated by TLC and when it completed, the organic layer was washed with 1 M HCl solution and concentrated in vacuo, the residue was purified by column chromatography on silica gel with ethyl acetate/petroleum as eluent to yield pure compounds 3a–l. Compounds 2a and b reacting with acetylsalicylic acid in the presence of EDCI/DMAP follow the procedure given for 3a–l that we obtained 5a and b.
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-indole-3-acetic Acid (3a)Compound 3a was obtained from 2a and indole-3-acetic acid as a white solid (292 mg, 45%); mp 117–119°C; 1H-NMR (CDCl3) δ: 8.21 (s, 1H, NH), 7.65 (d, J=7.2 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.19 (dt, J=18.8, 7.2 Hz, 2H), 7.13 (s, 1H), 5.67 (s, 1H, H-12), 4.57 (dd, 1H, H-3, J=11.6, 4.6 Hz), 4.18 (q, 2H, COOCH2CH3), 3.80 (s, 2H, CH2CO), 2.81 (d, 1H, H-1, J=13.5 Hz), 2.38 (s, 1H, H-9), 2.08 (dd, 1H, H-18, J=13.7, 3.77 Hz), 2.01 (ddd, 1H, H-15, J=13.3, 13.3, 4.27 Hz), 1.96 (m, 1H, H-21), 1.98 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J=13.6, 3.8, 2.97 Hz), 1.84 (ddd, 1H, J=13.7, 13.7, 4.77 Hz), 1.70 (m, 1H, H-2), 1.67 (m, 1H, H-7), 1.60 (dd, 1H, J=13.6, 13.67 Hz), 1.56 (m, 1H, H-6), 1.49 (m, 1H, H-6′), 1.46 (m, 1H, H-7′), 1.43 (m, 1H, H-22), 1.38 (s, 3H, H-27), 1.34 (m, 1H, H-22′), 1.33 (m, 1H, H-21′), 1.29 (t, 3H, COOCH2CH3, J=7.17 Hz), 1.21 (m, 1H, H-16′), 1.17 (s, 6H, H-25 and H-28), 1.14 (s, 3H, H-26), 1.04 (ddd, 1H, H-1′, J=13.5, 13.5, 3.57 Hz), 0.91 (m, 1H, H-15′), 0.85 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H); 13C-NMR (CDCl3) δ: 200.1, 176.4, 171.9, 169.3, 136.1, 128.5, 127.3, 122.9, 122.1, 119.5, 118.9, 111.1, 108.9, 81.0, 61.7, 60.2, 55.0, 48.4, 45.4, 42.9, 42.2, 41.1, 38.8, 38.1, 37.7, 36.9, 32.7, 31.8, 31.7, 31.2, 28.6, 28.2, 27.9, 26.5, 26.5, 23.6, 23.3, 18.7, 17.3, 16.7, 16.3, 14.3; high resolution (HR)-MS electrospray ionization (ESI): m/z 678.4147 [M+Na]+ (Calculated for C42H57O5NNa, 678.4129).
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-indolebutyric Acid (3b)Compound 3b was obtained from 2a and indolebutyric acid as a white solid (315 mg, 46%); mp 159–161°C; 1H-NMR (CDCl3) δ: 8.09 (s, 1H, NH), 7.64 (d, 1H, J=7.9 Hz), 7.38 (d, 1H, J=8.0 Hz), 7.17 (dt, 2H, J=30.3, 7.4 Hz), 7.01 (s, 1H), 5.68 (s, 1H, H-12), 4.58 (dd, 1H, H-3, J=11.7, 4.8 Hz), 4.26–4.10 (q, 2H, COOCH2CH3), 2.84 (t, 2H), 2.42 (t, 2H), 2.36 (s, 1H, H-9), 2.09 (dd, 1H, H-18, J=13.5, 3.67 Hz), 2.02 (ddd, 1H, H-15, J=13.5, 13.5, 4.47 Hz), 1.98 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J=13.6, 3.8, 2.97 Hz), 1.82 (ddd, 1H, J=13.7, 13.7, 4.77 Hz), 1.71 (m, 1H, H-2), 1.68 (m, 2H), 1.67 (m, 1H, H-7), 1.64 (m, 2H), 1.60 (dd, 1H, J=13.6, 13.67 Hz), 1.60 (m, 1H), 1.52 (m, 1H, H-6), 1.49 (m, 1H, H-6′), 1.45 (m, 1H, H-7′), 1.42 (m, 1H, H-22), 1.40 (s, 3H, H-27), 1.37 (m, 1H, H-22′), 1.35 (m, 1H, H-21′), 1.29 (t, 3H, COOCH2CH3, J=7.17 Hz), 1.23 (m, 1H, H-16′), 1.19 (s, 3H, H-25), 1.18 (s, 3H, H-28), 1.16 (s, 3H, H-26), 1.05 (ddd, 1H, H-1′, J=13.5, 13.5, 3.57 Hz), 1.03 (m, 1H, H-15′), 0.91 (s, 6H, H-23 and H-24), 0.88 (m, 1H, H-5), 0.83 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.1, 176.4, 173.6, 169.4, 136.4, 128.5, 127.5, 121.9, 121.5, 119.8, 118.9, 115.7, 111.1, 80.4, 61.8, 60.2, 55.1, 48.4, 45.4, 42.9, 42.2, 41.1, 39.8, 39.1, 37.7, 36.9, 34.4, 32.7, 31.8, 31.1, 28.6, 28.3, 28.1, 26.4, 25.6, 24.6, 23.6, 23.3, 18.70, 17.4, 16.8, 16.4, 14.3; HR-MS (ESI): m/z 706.4444 [M+Na]+ (Calculated for C44H61O5NNa, 706.4442).
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-dl-thioctic Acid (3c)Compound 3c was obtained from 2a and dl-thioctic acid as a white solid (388 mg, 56%); mp 141–143°C; 1H-NMR (CDCl3) δ: 5.67 (s, 1H, H-12), 4.55 (dd, 1H, H-3, J=11.6, 4.67 Hz), 4.25–4.10 (q, 2H, COOCH2CH3), 2.80 (ddd, 1H, H-1, J=13.7, 3.7, 3.77 Hz), 2.39–2.34 (m, 1H), 2.35 (s, 1H, H-9), 2.08 (dd, 1H, H-18, J=13.7, 3.77 Hz), 2.06–2.02 (m, 2H), 2.01 (ddd, 1H, H-15, J=13.3, 13.3, 4.27 Hz), 1.97 (m, 1H, H-21), 1.90 (ddd, 1H, H-19, J=13.7, 4.2, 2.97 Hz), 1.86–1.78 (m, 1H), 1.81 (ddd, 1H, H-16, J=13.7, 13.7, 4.67 Hz), 1.70 (m, 1H, H-2), 1.64 (m, 1H, H-7), 1.60 (m, 1H, H-2′), 1.59 (dd, 1H, H-19′, J=13.3, 13.37 Hz), 1.58 (m, 1H), 1.57 (m, 1H, H-6), 1.51–1.46 (m, 3H), 1.45 (m, 1H, H-6′), 1.40 (m, 1H, H-7′), 1.56 (m, 3H, H-27), 1.39 (s, 3H, H-27), 1.31 (m, 1H, H-22′), 1.29 (m, 1H, H-21′), 1.26 (t, 3H, COOCH2CH3, J=7.17 Hz), 1.25–1.23 (m, 2H), 1.22 (m, 1H, H-16′), 1.19 (s, 3H, H-25), 1.17 (s, 3H, H-28), 1.15 (s, 3H, H-26), 1.05 (ddd, 1H, H-1′, J=13.7, 13.7, 3.77 Hz), 0.98 (m, 1H, H-15′), 0.90 (s, 6H, H-23 and H-24), 0.86 (m, 1H, H-5), 0.83 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.1, 176.4, 172.3, 169.4, 128.4, 80.5, 61.7, 60.2, 59.5, 56.3, 55.0, 52.4, 48.4, 45.4, 43.9, 43.2, 41.1, 40.2, 38.8, 38.5, 38.1, 37.7, 36.9, 34.5, 32.7, 31.8, 31.1, 28.8, 28.6, 28.3, 26.5, 24.9, 23.6, 23.4, 18.7, 18.4, 17.4, 16.8, 16.4, 14.3; HR-MS (ESI): m/z 709.3933 [M+Na]+ (Calculated for C40H62O5S2Na, 709.3931).
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-cinnamic Acid (3d)Compound 3d was obtained from 2a and cinnamic acid as a white solid (356 mg, 57%); mp 217–219°C; 1H-NMR (CDCl3) δ: 7.67 (d, 1H, J=16.0 Hz), 7.53 (dd, 1H, J=7.6, 3.6 Hz), 7.38 (m, 3H), 6.45 (d, 1H, J=16.0 Hz), 5.65 (s, 1H, H-12), 4.66 (dd, 1H, H-3, J=7.6, 4.8 Hz), 4.16 (q, 2H, COOCH2CH3), 2.84 (ddd, 1H, H-1, J=12, 6.8, 4 Hz), 2.40 (s, 1H, H-9), 2.09 (ddd, 1H, H-18, J=11.4, 4.0, 0.87 Hz), 2.02 (ddd, 1H, H-15, J=12.4, 12.4, 4.47 Hz), 1.98 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J=13.6, 4.1, 2.87 Hz), 1.82 (ddd, 1H, H-16, J=13.6, 13.6, 4.57 Hz), 1.71 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.66 (m, 1H, H-2′), 1.60 (dd, 1H, H-19′, J=13.7, 13.77 Hz), 1.57 (m, 1H, H-6), 1.49 (m, 1H, H-6′), 1.44 m, 1H, H-7′), 1.41 (m, 1H, H-22), 1.39 (s, 3H, H-27), 1.35 (m, 1H, H-22′), 1.32 (m, 1H, H-21′), 1.26 (t, 3H, COOCH2CH3, J=7.17 Hz), 1.24 (m, 1H, H-16′), 1.21 (s, 3H, H-25), 1.15 (s, 6H, H-28 and H-26), 1.08 (ddd, 1H, H-1′, J=13.7, 13.7, 3.67 Hz), 1.01 (m, 1H, H-15′), 0.97 (s, 3H, H-23), 0.93 (s, 3H, H-24), 0.89 (m, 1H, H-5), 0.80 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.3, 169.3, 166.8, 144.3, 134.6, 130.1, 128.8, 128.4, 128.0, 118.8, 80.7, 61.7, 60.3, 58.3, 55.1, 48.4, 45.4, 43.8, 43.3, 41.1, 38.8, 38.3, 37.7, 37.0, 32.8, 31.8, 31.1, 28.5, 28.3, 28.1, 26.5, 26.4, 23.6, 23.4, 23.3, 18.7, 18.4, 17.4, 16.8, 16.4, 14.3; HR-MS (ESI): m/z 629.4210 [M+H]+ (Calculated for C41H57O5, 629.4201).
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-acetylated trans-4-Hydroxylcinnamic Acid (3e)Compound 3e was obtained from 2a and acetylated trans-4-hydroxylcinnamic acid as a white solid (378 mg, 55%); mp 237–239°C; 1H-NMR (CDCl3) δ: 7.65 (d, 1H, J=15.9 Hz), 7.55 (d, 2H, J=8.5 Hz), 7.13 (d, 2H, J=8.5 Hz), 6.41 (d, 1H, J=16.0 Hz), 5.67 (s, 1H, H-12), 4.67 (dd, 1H, H-3, J=11.8, 4.87 Hz), 4.25–4.09 (m, 2H, COOCH2CH3), 2.85 (dt, 1H, H-1, J=13.5, 3.6 Hz), 2.40 (s, 1H, H-9), 2.32 (s, 3H), 2.09 (ddd, 1H, H-18, J=13.4, 4.0, 0.87 Hz), 2.03 (ddd, 1H, H-15, J=12.5, 12.5, 4.47 Hz), 1.99 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J=12.5, 4.1, 2.87 Hz), 1.82 (ddd, 1H, H-16, J=11.2, 11.2, 4.57 Hz), 1.71 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.66 (m, 1H, H-2′), 1.60 (dd, 1H, H-19′, J=13.7, 13.77 Hz), 1.57 (m, 1H, H-6), 1.44 (m, 1H, H-6′), 1.43 (m, 1H, H-7′), 1.42 (m, 1H, H-22), 1.39 (s, 3H, H-27), 1.35 (m, 1H, H-22′), 1.32 (m, 1H, H-21′), 1.26 (t, 3H, COOCH2CH3, J=5.60 Hz), 1.22 (m, 1H, H-16′), 1.20 (s, 3H, H-25), 1.16 (s, 3H, H-28), 1.15 (s, 3H, H-26), 1.09 (ddd, 1H, H-1′, J=13.7, 13.7, 3.67 Hz), 1.04 (m, 1H, H-15′), 0.97 (s, 3H, H-23), 0.93 (s, 3H, H-24), 0.88 (m, 1H, H-5), 0.82 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.1, 176.4, 169.4, 169.1, 166.7, 151.9, 143.2, 132.3, 129.1, 128.4, 122.0, 118.9, 80.7, 61.7, 60.3, 58.4, 55.6, 53.4, 48.4, 45.4, 43.8, 43.2, 41.1, 38.8, 38.3, 37.7, 36.9, 32.7, 31.8, 31.1, 28.6, 28.3, 28.1, 26.5, 26.4, 23.7, 23.4, 21.1, 18.7, 18.4, 17.4, 16.8, 16.4, 14.3; HR-MS (ESI): m/z 687.4263 [M+H]+ (Calculated for C43H59O7, 687.4255).
Ethyl 3β-Hydroxy-11-oxo-olean-12-en-30-oate-salicylic Acid (3f)Compound 3f was obtained from 2a and salicylic acid as a white solid (201 mg, 32%); mp 238–240°C; 1H-NMR (CDCl3) δ: 10.93 (s, 1H, OH), 7.85 (d, 1H, J=8.0 Hz), 7.46 (t, 1H, J=7.6 Hz), 6.99 (d, 1H, J=8.4 Hz), 6.90 (t, 1H, J=3.2 Hz), 5.68 (s, 1H, H-12), 4.81 (dd, 1H, H-3, J=11.7, 4.8 Hz), 4.26–4.08 (m, 2H, COOCH2CH3), 2.90 (ddd, 1H, H-1, J=13.6, 3.5, 3.57 Hz), 2.42 (s, 1H, H-9), 2.10 (ddd, 1H, H-18, J=12.4, 4.0, 0.87 Hz), 2.02 (ddd, 1H, H-15, J=13.6, 13.6, 4.47 Hz), 1.98 (m, 1H, H-21), 1.92 (ddd, 1H, H-19, J=13.6, 4.1, 2.87 Hz), 1.82 (ddd, 1H, H-16, J=13.6, 13.6, 4.57 Hz), 1.71 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.66 (m, 1H, H-2′), 1.60 (dd, 1H, H-19′, J=13.7, 13.77 Hz), 1.57 (m, 1H, H-6), 1.50 (m, 1H, H-6′), 1.47 (m, 1H, H-7′), 1.44 (m, 1H, H-22), 1.40 (s, 3H, H-27), 1.36 (m, 1H, H-22′), 1.33 (m, 1H, H-21′), 1.29 (t, 3H, COOCH2CH3, J=7.20 Hz), 1.26 (m, 1H, H-16′), 1.23 (s, 3H, H-25), 1.17 (s, 3H, H-26 and H-28), 1.14 (ddd, 1H, H-1′, J=13.6, 13.6, 3.67 Hz), 1.12 (m, 1H, H-15′), 1.06 (s, 3H, H-23), 0.98 (s, 3H, H-24), 0.89 (m, 1H, H-5), 0.83 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.4, 169.9, 169.5, 161.7, 135.5, 129.7, 128.4, 119.1, 117.6, 112.0, 82.1, 61.7, 60.3, 55.1, 53.4, 48.4, 45.4, 43.8, 43.3, 41.1, 38.7, 38.4, 37.7, 36.9, 32.7, 31.8, 31.1, 28.6, 28.3, 28.2, 26.5, 26.4, 23.6, 23.4, 18.7, 17.4, 16.9, 16.4, 14.3; HR-MS (ESI): m/z 619.3999 [M+H]+ (Calculated for C39H55O6, 619.3993).
Ethyl 3β-Hydroxyolean-12-en-28-oate-indole-3-acetic Acid (3g)Compound 3g was obtained from 2b and indole-3-acetic acid as a white solid (312 mg, 49%); mp 138–139°C; 1H-NMR (CDCl3) δ: 8.59 (s, 1H, NH), 7.62 (d, 1H, J=7.6 Hz), 7.32 (d, 1H, J=8.0 Hz), 7.19 (dt, 2H, J=22.8, 6.8 Hz), 7.08(s, 1H), 5.31 (1H, dd, H-12, J=3.1, 3.1 Hz), 4.56 (t, 1H, H-3, J=8.8 Hz), 4.16–4.10 (m, 2H, COOCH2CH3), 3.80 (s, 2H, CH2CO), 2.92 (dd, 1H, H-18, J=13.9, 4.2 Hz), 1.96 (1H, m, H-11), 1.95 (ddd, 1H, H-16, J=13.6, 13.6, 4.8 Hz), 1.87 (m, 1H, H-16′), 1.68 (m, 1H, H-7), 1.64 (m, 1H, H-19), 1.61 (m, 3H, H-11′ and H-15), 1.60 (m, 1H, H-1), 1.52 (m, 3H, H-9 and H-6), 1.51 (m, 1H, H-7′), 1.34 (m, 1H, H-21), 1.26 (t, 3H, COOCH2CH3, J=6.8 Hz), 1.20 (m, 1H, H-21′), 1.10 (m, 1H, H-19′), 0.96 (s, 3H, H-27), 0.95 (s, 6H, H-23 and H-30), 0.88 (m, H-1′), 0.85 (s, 3H, H-29), 0.82 (s, 3H, H-25 and H-24), 0.78 (s, 3H, H-26), 0.74 (m, 1H, H-5); 13C-NMR (CDCl3) δ: 177.8, 172.1, 143.8, 136.2, 127.3, 123.2, 121.8, 119.3, 118.8, 111.3, 108.4, 81.4, 60.1, 55.3, 47.7, 47.5, 46.5, 45.9, 41.7, 41.4, 39.4, 38.1, 37.7, 36.9, 33.9, 33.2, 32.7, 32.5, 31.7, 30.7, 28.2, 28.0, 27.7, 27.7, 27.2, 25.9, 25.8, 23.8, 23.6, 23.5, 23.5, 23.1, 18.2, 17.0, 16.7, 15.3, 14.31; HR-MS (ESI): m/z 664.4332 [M+Na]+ (Calculated for C42H59O4NNa, 664.4336).
Ethyl 3β-Hydroxyolean-12-en-28-oate-Indolebutyric Acid (3h)Compound 3h was obtained from 2b and indolebutyric acid as a white solid (345 mg, 52%); mp 79–82°C; 1H-NMR (CDCl3) δ: 8.09 (s, 1H, NH), 7.64 (d, 1H, J=7.7 Hz), 7.37 (d, 1H, J=8.0 Hz), 7.21 (t, 1H, J=8.0 Hz), 7.13 (t, 1H, J=7.2 Hz), 7.00 (s, 1H), 5.31 (dd, 1H, H-12, J=3.8, 3.8 Hz), 4.56(t, 1H, H-3, J=10 Hz), 4.15–4.09 (m, 2H, COOCH2CH3), 2.91 (dd, 1H, H-3, J=15, 4.8 Hz), 2.87 (dt, 1H, H-18, J=24.6, 8.3 Hz), 2.42 (t, 2H, J=7.4 Hz), 1.96 (m, 1H, H-11), 1.95 (ddd, 1H, H-16, J=13.6, 13.6, 4.8 Hz), 1.87 (m, 1H, H-16′), 1.68 (m, 1H, H-7), 1.64 (m, 1H, H-19), 1.61 (m, 3H, H-11′ and H-15), 1.60 (m, 1H, H-1), 1.52 (m, 3H, H-9, H-6), 1.51 (m, 1H, H-7′), 1.30 (m, 1H, H-21), 1.26 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.17 (s, 3H, H-27), 1.10 (m, 1H, H-21′), 1.04 (m, 1H, H-19′), 0.97 (s, 6H, H-23 and H-30), 0.95 (m, 1H, H-1′), 0.94 (s, 3H, H-29), 0.91 (s, 3H, H-25), 0.89 (s, 3H, H-24), 0.82 (m, 1H, H-5), 0.79 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.7, 173.5, 143.8, 136.4, 127.5, 122.3, 121.8, 121.5, 119.2, 118.9, 111.1, 80.7, 60.0, 55.4, 47.6, 46.6, 45.9, 41.8, 41.4, 39.4, 38.2, 37.8, 36.9, 34.5, 33.9, 33.1, 32.7, 32.5, 30.7, 28.2, 27.7, 25.8, 25.7, 24.6, 23.6, 23.6, 23.4, 23.0, 18.3, 17.0, 16.8, 15.4, 14.3; HR-MS (ESI): m/z 670.4853 [M+H]+ (Calculated for C44H64O4N, 670.4830).
Ethyl 3β-Hydroxyolean-12-en-28-oate-dl-thioctic Acid (3i)Compound 3i was obtained from 2b and dl-thioctic acid as a white solid (381 mg, 45%); mp 65–67°C; 1H-NMR (CDCl3) δ: 5.30 (dd, 1H, H-12, J=4.0, 4.0 Hz), 4.52 (t, 1H, H-3, J=8 Hz), 4.10 (q, 2H, COOCH2CH3, J=7.0 Hz), 2.89 (dd, 1H, H-18, J=13.8, 4.5 Hz), 2.47 (m, 1H), 2.33 (t, 2H, J=7.4 Hz), 1.96 (1H, m, H-11), 1.97 (ddd, 1H, H-16, J=23.2, 13.6, 4.6 Hz), 1.90 (q, 2H, J=6. 4 Hz), 1.87 (1H, m, H-16′), 1.68 (1H, m, H-7), 1.64 (1H, m, H-19), 1.63 (m, 4H), 1.61 (m, 3H, H-11′ and H-15), 1.60 (m, 1H, H-1), 1.53 (q, 2H, J=9.4 Hz), 1.52 (m, 3H, H-9 and H-6), 1.51 (m, 1H, H-7′), 1.34 (m, 1H, H-21), 1.25 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.19 (m, 1H, H-21′), 1.15 (m, 1H, H-19′), 1.15 (s, 3H, H-27), 1.06 (m, 1H, H-1′), 0.96 (s, 3H, H-23), 0.95 (s, 3H, H-30), 0.92 (s, 3H, H-29), 0.88 (s, 3H, H-25 and H-24), 0.82 (m, 1H, H-5), 0.77 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.7, 173.2, 143.8, 122.2, 80.8, 60.0, 56.4, 55.3, 47.6, 46.5, 45.9, 41.7, 41.3, 40.2, 39.4, 38.5, 38.2, 37.8, 36.9, 34.6, 34.6, 33.9, 33.1, 32.7, 32.4, 30.7, 28.8, 28.1, 27.6, 25.8, 24.9, 23.6, 23.6, 23.4, 23.0, 18.2, 17.0, 16.8, 15.6, 14.3; HR-MS (ESI): m/z 695.4144 [M+Na]+ (Calculated for C40H64O4S2Na, 695.4138).
Ethyl 3β-Hydroxyolean-12-en-28-oate-cinnamic Acid (3j)Compound 3j was obtained from 2b and cinnamic acid as a white solid (266 mg, 43%); mp 153–155°C; 1H-NMR (CDCl3) δ: 7.68 (d, 1H, J=15.9 Hz), 7.53 (t, 2H, J=6.8 Hz), 7.39 (m, 3H), 6.45 (d, 1H, J=16.0 Hz), 5.31 (dd, 1H, H-12, J=4.1, 4.1 Hz), 4.65 (t, 1H, H-3, J=9.2 Hz), 4.10 (m, 2H, COOCH2CH3), 2.90 (dd, 1H, H-18, J=16, 6.0 Hz), 2.02 (m, 1H, H-16), 1.94 (m, 1H, H-16′), 1.89 (m, 1H, H-11), 1.77 (m, 1H, H-22), 1.71 (m, 1H, H-11′), 1.65 (m, 1H, H-1), 1.64 (m, 1H, H-19), 1.60 (m, 1H, H-9), 1.57 (m, 1H, H-22), 1.55 (m, 1H, H-6), 1.40 (m, 1H, H-6′), 1.38 (m, 1H, H-21), 1.35 (m, 1H, H-21′), 1.25 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.19 (m, 1H, H-21′), 1.17 (s, 3H, H-27), 1.10 (m, 1H, H-19′), 1.07 (m, 1H, H-1′), 0.99 (m, 3H, H-23), 0.96 (m, 3H, H-30), 0.95 (m, 3H, H-29), 0.93 (m, 6H, H-25 and H-24), 0.78 (s, 3H, H-26), 0.72 (m, 1H, H-5); 13C-NMR (CDCl3) δ: 177.7, 166.8, 144.3, 143.8, 134.6, 130.1, 128.8, 128.7, 128.0, 127.9, 122.2, 118.8, 81.0, 60.0, 55.4, 47.6, 46.5, 45.9, 41.7, 41.3, 39.4, 38.2, 37.9, 36.9, 33.9, 33.1, 32.7, 32.4, 30.7, 29.1, 28.1, 27.6, 25.8, 23.6, 23.4, 23.0, 18.3, 17.0, 16.8, 15.4, 14.3; HR-MS (ESI): m/z 637.4233 [M+Na]+ (Calculated for C41H58O4Na, 637.4227).
Ethyl 3β-Hydroxyolean-12-en-28-oate-acetylated trans-4-Hydroxylcinnamic Acid (3k)Compound 3k was obtained from 2b and acetylated trans-4-hydroxylcinnamic acid as a white solid (292 mg, 45%); mp 129–131°C; 1H-NMR (CDCl3) δ: 7.64 (d, 1H, J=16.0 Hz), 7.53 (d, 2H, J=11.6 Hz), 7.13 (d, 2H, J=7.2 Hz), 6.40 (d, 1H, J=16.0 Hz), 5.21 (dd, 1H, H-12, J=8.0, 8.0 Hz), 4.65 (t, 1H, H-3, J=8.4 Hz), 4.10 (m, 2H, COOCH2CH3), 2.89 (dd, 1H, H-18, J=13.6, 4.0 Hz), 2.35 (s, 3H, COCH3), 1.98 (m, 1H, H-16), 1.93 (m, 1H, H-16′), 1.91 (m, 1H, H-11), 1.73 (m, 1H, H-22), 1.71 (m, 1H, H-11′), 1.66 (m, 1H, H-1), 1.62 (m, 1H, H-19), 1.58 (m, 1H, H-9), 1.55 (m, 1H, H-22), 1.52 (m, 1H, H-6), 1.38 (m, 1H, H-6′), 1.38 (m, 1H, H-21), 1.35 (m, 1H, H-21′), 1.25 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.16 (s, 3H, H-27), 1.10 (m, 1H, H-19′), 1.06 (m, 1H, H-1′), 0.98 (m, 3H, H-25), 0.94 (m, 3H, H-24), 0.92 (m, 3H, H-30), 0.90 (m, 3H, H-23), 0.87 (m, 3H, H-29), 0.85 (m, 1H, H-5), 0.77 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.7, 169.0, 166.6, 151.9, 143.8, 143.1, 132.3, 129.1, 129.1, 122.2, 122.1, 122.1, 119.0, 81.1, 60.0, 55.4, 47.6, 46.5, 45.9, 41.7, 41.3, 39.4, 38.2, 37.9, 36.9, 33.9, 33.1, 32.7, 32.4, 30.7, 29.0, 28.1, 27.6, 25.8, 23.6, 23.6, 23.4, 23.0, 18.2, 16.9, 16.8, 15.4, 14.2; HR-MS (ESI): m/z 695.4288 [M+Na]+ (Calculated for C43H60O6Na, 695.4282).
Ethyl 3β-Hydroxyolean-12-en-28-oate-salicylic Acid (3l)Compound 3l was obtained from 2b and salicylic acid as a white solid (285 mg, 42%); mp 174–177°C; 1H-NMR (CDCl3) δ: 10.93 (s, 1H, OH), 7.85 (d, 1H, J=7.6 Hz), 7.47 (t, 1H, J=4 Hz), 7.00 (dd, 1H, J=8.5, 4.1 Hz), 6.90 (t, 1H, J=3.6 Hz), 5.32 (d, 1H, H-12, J=4.0 Hz), 4.80 (t, 1H, H-3, J=5.2 Hz), 4.10 (m, 2H, COOCH2CH3), 2.90 (d, 1H, H-18, J=13.6 Hz), 1.99 (m, 1H, H-11), 1.95 (m, 1H, H-16), 1.80 (m, 1H, H-16′), 1.72 (m, 1H, H-7), 1.67 (m, 1H, H-19), 1.58 (m, 3H, H-11′, H-15), 1.55 (m, 1H, H-1), 1.53 (m, 3H, H-9 and H-6), 1.51 (m, 1H, H-7′), 1.34 (m, 1H, H-21), 1.32 (t, 3H, COOCH2CH3, J=15.2 Hz), 1.25 (m, 1H, H-21′), 1.18 (s, 3H, H-27), 1.13 (m, 1H, H-19′), 1.08 (m, 1H, H-1′), 1.05 (s, 3H, H-23), 1.02 (s, 3H, H-30), 0.97 (s, 3H, H-29), 0.93 (s, 6H, H-25 and H-24), 0.85 (m, 1H, H-5), 0.79 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.7, 169.9, 161.7, 135.4, 129.7, 122.1, 119.0, 117.6, 82.4, 60.1, 55.4, 47.6, 46.5, 45.9, 41.8, 41.3, 39.4, 38.1, 38.1, 36.9, 33.9, 33.1, 32.7, 32.4, 30.7, 29.1, 28.2, 27.6, 25.8, 23.6, 23.6, 23.4, 23.0, 18.2, 17.0, 16.9, 15.4, 14.3; HR-MS (ESI): m/z 627.4031 [M+Na]+ (Calculated for C39H56O5Na, 637.4020).
Ethyl 3β-Acetyloxy-11-oxo-olean-12-en-30-oate (5a)Compound 5a was obtained from 2a and acetylsalicylic acid as a white solid (352 mg, 65%); mp 260–261°C; 1H-NMR (CDCl3): δ: 5.61 (s, 1H, H-12), 4.49 (dd, 1H, H-3, J=11.6, 4.8 Hz), 4.22–4.05 (m, 2H, COOCH2CH3), 2.77 (dt, 1H, H-1, J=13. 6, 3.7 Hz), 2.34 (s, 1H, H-9), 2.10 (m, 1H, H-18), 2.06 (m, 1H, H-15), 2.02 (s, 3H, COCH3), 1.96 (m, 1H, H-21), 1.88 (m, 1H, H-19), 1.80 (dd, 1H, H-16, J=13.6, 3.6 Hz), 1.64 (m, 1H, H-2), 1.61 (m, 1H, H-7), 1.58 (m, 1H, H-2′), 1.57 (m, 1H, H-19′), 1.56 (m, 1H, H-6), 1.45 (dd, 1H, H-6′, J=12, 2.4, 3.17 Hz), 1.38 (m, 1H, H-7′), 1.35 (m, 1H, H-22), 1.34 (s, 3H, H-27), 1.30 (m, 1H, H-22′), 1.28 (m, 1H, H-21′), 1.23 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.18 (m, 1H, H-16′), 1.13 (s, 3H, H-28), 1.11 (s, 3H, H-25), 1.10 (s, 3H, H-26), 1.03 (m, 1H, H-15′), 0.95 (m, 1H, H-1′), 0.85 (s, 6H, H-23 and H-24), 0.79 (m, 1H, H-5), 0.78 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.3, 170.9, 169.3, 128.4, 80.6, 61.7, 60.2, 54.9, 48.3, 45.3, 43.8, 43.2, 41.0, 38.7, 38.0, 37.7, 36.9, 32.7, 31.8, 31.1, 28.5, 28.2, 28.0, 26.4, 26.4, 23.5, 23.3, 21.2, 18.6, 17.3, 16.6, 16.4, 14.3; HR-MS (ESI): m/z 541.3888 [M+H]+ (Calculated for C34H53O5, 541.3888).
Ethyl 3β-Acetyloxyolean-12-en-28-oate (5b)Compound 5b was obtained from 2b and acetylsalicylic acid as a white solid (335 mg, 64%); mp 216–218°C; 1H-NMR (CDCl3) δ: 5.28 (m, 1H, H-12), 4.49 (q, 1H, H-3, J=8.1 Hz), 4.10 (q, 2H, COOCH2CH3, J=7.2 Hz), 2.86 (1H, dd, H-18, J=11.5, 6.2 Hz), 2.04 (s, 3H, OCH3), 1.96 (m, 1H, H-11), 1.87 (t, 2H, H-16 and H-16′, J=4.8 Hz), 1.68 (m, 1H, H-7), 1.64 (m, 1H, H-19), 1.62 (m, 3H, H-11′, H-15), 1.60 (m, 1H, H-1), 1.52 (m, 3H, H-9 and H-6), 1.51 (m, 1H, H-7′), 1.34 (m, 1H, H-21), 1.23 (t, 3H, COOCH2CH3, J=7.2 Hz), 1.18 (m, 1H, H-21′), 1.17 (m, 1H, H-19′), 1.14 (s, 3H, H-27), 1.12 (s, 3H, H-23), 0.96 (m, 1H, H-1′), 0.91 (s, 6H, H-30 and H-29), 0.85 (s, 6H, H-25 and H-24), 0.75 (m, 1H, H-5), 0.74 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.6, 170.9, 143.8, 122.2, 80.9, 60.0, 55.3, 47.6, 46.5, 45.8, 41.7, 41.3, 39.4, 38.1, 37.6, 36.9, 33.9, 33.1, 32.7, 32.4, 30.7, 28.0, 27.6, 25.8, 23.6, 23.5, 23.4, 22.9, 21.2, 18.2, 16.9, 16.7, 15.3, 14.2; HR-MS (ESI): m/z 6549.3915 [M+H]+ (Calculated for C34H54O4Na, 549.3914).
Compounds 2a and b Reacting with Acetylsalicylic Acid/Pyridine to Obtain 5a and bAccording to the procedure previously reported to obtain 5a and b.29,30) Firstly, to a solution of 2a and b (1 mmol) in pyridine (5 mL), dry acetic anhydride (10 mL) was added. After 24 h of stirring at room temperatures, the reaction solution was poured into ice-water-dilute hydrochloric acid and allowed to stand overnight. Then it was filtered, washed with 2 mol/L hydrochloric acid to pyridine-free taste, washed with water to pH=7, and then recrystallized from methanol, to obtain white granular crystals. Their structures were characterized by 1H-, 13C-NMR and HR-MS, and we found their structures was same with that compounds 2a and b reacting with acetylsalicylic acid in the presence of EDCI/DMAP to afford 5a and b.
General Experimental Method for the Synthesis of 6a and b and 7a and bThe 1a and b (1 mmol) was dissolved in dry dichloromethane (15 mL) and stirred at 0°C for 5 min, added with EDCI (230 mg, 1.2 mmol) and DMAP (24 mg, 0.2 mmol), and the resulting mixture was stirred at 0°C for 1 h. The 4-hydroxycoumarin (162 mg, 1 mmol) was added to the dry dichloromethane (15 mL), stirred at room temperature for 1 h, and added with the above mixture at room temperature. After completion of the reaction (as monitored by TLC), the solvent was washed with aqueous hydrochloric acid, dried with anhydrous Na2SO4, evaporated to dryness under vacuum, purified by chromatography on silica (ethyl acetate/petroleum, gradient elution) to give 6a and b and 7a and b.
4-Hydroxycoumarin 3β-Hydroxy-11-oxo-olean-12-en-30-oate (6a)Compound 6a was obtained from GA and 4-hydroxycoumarin as a white solid (360 mg, 59%); mp 261–263°C; 1H-NMR (CDCl3) δ: 7.60 (t, 2H, J=7.7 Hz), 7.38 (d, 1H, J=7.6 Hz), 7.38 (d, 1H, J=8.4 Hz), 6.48 (s, 1H), 5.68 (s, 1H, H-12), 3.23 (dd, 1H, H-3, J=10.8, 5.4 Hz), 2.79 (dt, 1H, H-1, J=13.4, 3.6 Hz), 2.35 (s, 1H, H-9), 2.23 (dd, 1H, H-18, J=3.6, 1.6 Hz), 2.20 (m, 1H, H-15), 2.12 (m, 1H, H-21), 2.08 (dd, 1H, H-19, J=3.6, 1.2 Hz), 1.86 (m, 1H, H-16), 1.64 (m, 1H, H-2), 1.61 (m, 1H, H-7), 1.58 (m, 1H, H-2′), 1.57 (m, 1H, H-19′), 1.56 (m, 1H, H-6), 1.55 (m, 1H, H-6′), 1.52 (m, 1H, H-7′), 1.49 (m, 1H, H-22), 1.44 (s, 3H, H-27), 1.28 (m, 1H, H-22′), 1.42 (s, 3H, H-28), 1.26 (m, 1H, H-21′), 1.22 (m, 1H, H-16′), 1.13 (s, 6H, H-25 and H-26), 1.08 (m, 1H, H-15′), 1.01 (s, 3H, H-23), 0.95 (m, 1H, H-1′), 0.87 (s, 3H, H-24), 0.81 (s, 3H, H-29), 0.71 (dd, 1H, H-5, J=11.8, 1.9 Hz); 13C-NMR (CDCl3) δ: 199.9, 168.0, 153.6, 132.9, 128.9, 124.5, 122.4, 117.2, 115.7, 104.9, 78.7, 61.8, 54.9, 53.4, 48.4, 45.4, 45.3, 43.2, 41.1, 39.2, 39.1, 37.7, 37.1, 32.7, 32.0, 31.1, 28.5, 28.1, 27.3, 26.4, 26.4, 23.5, 18.7, 17.5, 16.3, 15.6; HR-MS (ESI): m/z 637.3505 [M+Na]+ (Calculated for C39H50O6Na, 637.3500).
4-Hydroxycoumarin 3β-3-Hydroxyolean-12-en-28-oate (6b)Compound 6b was obtained from OA and 4-hydroxycoumarin as a white solid (263 mg, 44%); mp 146–148°C; 1H-NMR (CDCl3) δ: 8.12 (s, 1H, OH), 7.68 (d, 1H, J=6.4 Hz), 7.60 (t, 1H, J=6.8 Hz), 7.38 (d, 1H, J=8.0 Hz), 7.32 (t, 1H, J=8.0 Hz), 6.45 (s, 1H), 5.43 (t, 1H, H-12, J=3.7 Hz), 3.24 (dd, 1H, H-3, J=11.3, 4.6 Hz), 3.01 (m, 1H, H-18), 2.20 (td, 1H, H-11, J=13.5, 4.0 Hz), 2.01 (td, 1H, H-16, J=13.6, 4.8 Hz), 1.89 (m, 1H, H-16′), 1.78 (m, 1H, H-7), 1.66 (m, 1H, H-19), 1.63 (m, 3H, H-11′ and H-15), 1.60 (m, 1H, H-1), 1.52 (m, 3H, H-9, H-6), 1.48 (m, 1H, H-7′), 1.38 (m, 1H, H-21), 1.36 (m, 1H, H-21′), 1.31 (m, 1H, H-19′), 1.28 (s, 3H, H-27), 1.23 (s, 3H, H-23), 1.07 (m, 1H, H-1′), 1.02 (s, 3H, H-30), 1.01 (s, 3H, H-29), 0.99 (s, 3H, H-25), 0.92 (s, 6H, H-24 and H-26), 0.75 (m, 1H, H-5); 13C-NMR (CDCl3) δ: 173.5, 161.5, 158.7, 153.6, 142.7, 132.6, 124.2, 123.6, 122.6, 117.1, 115.9, 104.8, 79.0, 55.2, 48.5, 47.5, 45.7, 41.8, 41.6, 39.5, 38.7, 38.5, 37.0, 33.7, 32.9, 32.8, 32.5, 30.7, 29.7, 28.1, 27.8, 27.2, 28.9, 23.6, 23.3, 18.3, 17.4, 15.6, 15.3; HR-MS (ESI): m/z 623.3714 [M+Na]+ (Calculated for C39H52O5Na, 623.3707).
Sesamol 3β-Hydroxy-11-oxo-olean-12-en-30-oate (7a)Compound 7a was obtained from GA and sesamol as a white solid (356 mg, 60%); mp 279–281°C; 1H-NMR (CDCl3) δ: 6.79 (d, 1H, J=8.3 Hz), 6.57 (s, 1H), 6.49 (dd, 1H, J=8.3, 2.3 Hz), 5.99 (s, 1H), 5.68 (s, 1H, H-12), 3.24 (dd, 1H, H-3, J=10.8, 5.4 Hz), 2.80 (dt, 1H, H-1, J=13.5, 3.5 Hz), 2.36 (s, 1H, H-9), 2.24 (ddd, 1H, H-18, J=13.5, 4.3, 1.7 Hz), 2.10 (m, 1H, H-15), 2.06 (m, 1H, H-21), 1.87 (td, 1H, H-19, J=13.7, 4.5 Hz), 1.72 (m, 1H, H-16), 1.68 (m, 1H, H-2), 1.62 (m, 1H, H-7), 1.58 (m, 1H, H-2′), 1.57 (m, 1H, H-19′), 1.56 (m, 1H, H-6), 1.47 (m, 1H, H-6′), 1.45 (m, 1H, H-7′), 1.42 (m, 1H, H-22), 1.40 (s, 3H, H-27), 1.28 (m, 1H, H-22′), 1.26 (m, 1H, H-21′), 1.33 (s, 3H, H-28), 1.22 (m, 1H, H-16′), 1.15 (s, 6H, H-25 and H-26), 1.07 (m, 1H, H-15′), 1.02 (s, 3H, H-23), 0.95 (dd, 1H, H-1′, J=12.8, 4.4 Hz), 0.87 (s, 3H, H-24), 0.82 (s, 3H, H-29), 0.71 (dd, 1H, H-5, J=11.8, 2.0 Hz); 13C-NMR (CDCl3) δ: 200.1, 175.4, 168.8, 148.0, 145.3, 145.1, 128.7, 113.7, 108.0, 103.6, 101.7, 78.7, 61.8, 54.9, 48.5, 45.4, 44.2, 43.2, 41.1, 39.1, 39.1, 37.7, 37.1, 32.8, 31.9, 31.1, 28.6, 28.1, 28.1, 27.3, 26.5, 26.4, 23.4, 18.7, 17.5, 16.4, 15.6; HR-MS (ESI): m/z 617.4204 [M+H]+ (Calculated for C40H57O5, 617.4201).
Eugenol 3β-Hydroxy-11-oxo-olean-12-en-30-oate (7b)Compound 7b was obtained from GA and eugenol as a white solid (348 mg, 56%); mp 114–117°C; 1H-NMR (CDCl3) δ: 6.90 (d, 1H, J=7.6 Hz), 6.78 (m, 2H), 5.97 (m, 1H), 5.72 (s, 1H, H-12), 5.10 (m, 2H), 3.80 (s, 3H), 3.42–3.34 (m, 2H), 3.23 (dd, 1H, H-3, J=10.9, 5.3 Hz), 2.79 (dt, 1H, H-1, J=13.5, 3.6 Hz), 2.36 (s, 1H, H-9), 2.12 (m, 1H, H-18), 2.10 (m, 1H, H-15), 2.08 (m, 1H, H-21), 1.87 (td, 1H, H-19, J=13.7, 5.0 Hz), 1.74 (t, 1H, H-16, J=13.6 Hz), 1.69 (m, 1H, H-2), 1.64 (m, 1H, H-7), 1.58 (m, 1H, H-2′), 1.57 (m, 1H, H-19′), 1.56 (m, 1H, H-6), 1.47 (m, 1H, H-6′), 1.45 (m, 1H, H-7′), 1.42 (m, 1H, H-22), 1.35 (s, 3H, H-27), 1.30 (m, 1H, H-22′), 1.27 (s, 3H, H-28), 1.25 (dd, 1H, H-16′, J=15.5, 13.6 Hz), 1.21 (m, 1H, H-21′), 1.16 (s, 3H, H-25), 1.15 (s, 3H, H-26), 1.08 (m, 1H, H-15′), 1.02 (s, 3H, H-23), 0.98 (m, 1H, H-1′), 0.88 (s, 3H, H-24), 0.82 (s, 3H, H-29), 0.72 (d, 1H, H-5, J=12 Hz); 13C-NMR (CDCl3) δ: 200.2, 174.6, 169.3, 150.9, 138.8, 137.9, 137.1, 128.5, 122.3, 120.6, 116.0, 112.8, 78.7, 61.8, 55.8, 55.6, 54.9, 48.0, 45.4, 44.3, 43.2, 41.4, 39.1, 37.5, 37.1, 32.8, 31.8, 31.3, 28.9, 28.1, 27.3, 26.6, 26.5, 23.4, 21.0, 18.7, 17.5, 16.4, 15.6; HR-MS (ESI): m/z 613.3503 [M+Na]+ (Calculated for C37H50O6Na, 617.3500).
Anticancer AssaysHep-G2 cells, LO2 cells and MCF-7 cells in logarithmic growth phase were inoculated in 96-well plates, with a cell density of 3–4×103/well. The cells were incubated in a 5% CO2, 37°C cell culture. When the cells were adhered, the test compound was added at the specified concentration. The same concentration of dimethyl sulfoxide (DMSO) was used as the negative control. After 48 h of incubation, 20 µL MTT (5 mg/mL) was added to each well, and the culture was continued for 4 h. After aspirating the supernatant with a pump, 150 µL of DMSO was added and the OD of each well was measured at 570 nm using a microplate reader. IC50 values were calculated using IC50 software (Prism 5.0).
This study was supported by the Natural Science Foundation of Shandong Province (No.ZR2010BM021) and the National Natural Science Foundation of China (21202028, 21372054).
The authors declare no conflict of interest.
