Synthesis of acyl oleanolic acid-uracil conjugates and their anti-tumor activity
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Oleanolic acid, which can be isolated from many foods and medicinal plants, has been reported to possess diverse biological activities. It has been found that the acylation of the hydroxyl groups of the A-ring in the triterpene skeleton of oleanolic acid could be favorable for biological activities. The pyrimidinyl group has been constructed in many new compounds in various anti-tumor studies.
Five acyl oleanolic acid-uracil conjugates were synthesized. Most of the IC50 values of these conjugates were lower than 10.0 μM, and some of them were even under 0.1 μM. Cytotoxicity selectivity detection revealed that conjugate 4c exhibited low cytotoxicity towards the normal human liver cell line HL-7702. Further studies revealed that 4c clearly possessed apoptosis inducing effects, could arrest the Hep-G2 cell line in the G1 phase, induce late-stage apoptosis, and activate effector caspase-3/9 to trigger apoptosis.
KeywordsAcyl oleanolic acid Uracil Anti-tumor activity Cytotoxicity Apoptosis
human alveolar adenocarcinoma cell line
- Annexin V-FITC
apoptosis detection kit
acridine orange/ethidium bromide
human grastic cancer cell line
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester
dulbecco’s modified eagle’s medium
fetal calf serum
caspase-3 detection kit
caspase-9 detection kit
- G1 phase
gap1, pre-synthetic period
gap2, post-synthetic period/mitosis
human hepatoma cell line
- HL-7702 (L-O2)
hepatic immortal cell line
high resolution mass spectrometry
half maximal inhibitory concentration
human breast adenocarcinoma cell line
3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
nuclear magnetic resonance
phosphate buffered saline
human prostatic carcinoma cell line
roswell park memorial institute
traditional Chinese medicines
the mitochondrial membrane potential
The importance of C-3 in the oleanolic acid skeleton was elucidated (Fig. 1). The SAR analysis of oleanolic acid derivatives modified at C-3 and C-28 indicated that hydrogen-bond acceptor substitution at the C-3 position of oleanolic acid may be advantageous for the improvement of cytotoxicity against PC-3, A549 and MCF-7 cell lines . Gnoatto found that the derivative with an acetylation at C-3 of the oleanolic acid backbone had much better activity against the L. amazonensis strain . 3-Oxo oleanolic acid (3-oxo-olea-12-en-28-oic acid), a derivative of oleanolic acid modified at C-3, was found to significantly inhibit the growth of cancer cells derived from different tissues, particularly on melanoma in vivo . Some other acyl compounds, generated from the modification of the hydroxyl groups of the A-ring in the triterpene skeleton of oleanolic acid and maslinic acid (MA, Fig. 1) with 10 different acyl groups, displayed cytotoxic effects against b16f10 murine melanoma cells and showed apoptotic effects with high levels of early and total apoptosis (up to 90%). These acyl compounds also exhibited better inhibition effects to anti-HIV-1-protease, with IC50 values between 0.31 and 15.6 μM, which are 4–186 times lower than their non-acylated precursors . Compound 2 (Fig. 1), un benzyl (2α,3β) 2,3-diacetoxy-olean-12-en-28-amide, exhibited much better cytotoxicity against human tumor cell lines compared with its deacylation product, while it showed a rather low cytotoxicity for human fibroblasts (WW030272) .
On the other hand, pyrimidine has been widely used as an anti-tumor pharmacophore in medicinal chemical research . For instance, some new pyridines and pyrazolo [1,5-α] pyrimidines exhibited potent anti-tumor cytotoxic activity in vitro against different human cell lines . The evaluation of several ring-A fused hybrids of oleanolic acid against seven human cancer cell lines showed that the fused pyrimidine moiety seemed important to enhance the antiproliferative activity of oleanolic acid . Thus, the pyrimidinyl group has been constructed in many new compounds in various anti-tumor studies .
Results and discussion
Evaluation of 4a–4e against different tumor cell lines
7.83 ± 0.69
4.01 ± 0.37
2.81 ± 0.22
15.5 ± 1.34
6.53 ± 0.35
5.24 ± 0.05
3.01 ± 0.28
0.22 ± 0.02
6.99 ± 0.57
0.28 ± 0.05
8.49 ± 0.68
0.27 ± 0.03
0.11 ± 0.01
3.51 ± 0.22
10.61 ± 1.13
4.19 ± 0.35
8.76 ± 0.07
5.26 ± 0.41
2.51 ± 0.05
15.90 ± 1.13
16.29 ± 1.36
23.74 ± 1.53
12.60 ± 1.09
72.74 ± 6.88
55.74 ± 5.09
22.62 ± 2.19
8.82 ± 0.78
21.47 ± 1.99
12.23 ± 1.18
The results showed that these compounds exhibited excellent antiproliferative activities against the tested cells, with the IC50 values mainly under 10.0 μM, except for compounds 4a and 4b which showed no inhibition against the PC-3 cell line. In the Hep-G2, A549, BGC-823 and MCF-7 cell line assays, all the synthesized compounds displayed much better inhibition than that of 1 and 5-FU. With a propionyloxy group at C-3, compound 4b possessed the best inhibition activity against the Hep-G2 cell line, almost 5.5-fold and 20-fold stronger than 1 and 5-FU, respectively. With a dodecanoyloxy group at C-3, compound 4d showed the best inhibition activity against the A549 cell line, almost 60-fold and 84-fold stronger than 1 and 5-FU, respectively. Meanwhile, compound 4a, with an acetoxy group at C-3, exhibited the best inhibition activity against the MCF-7 cell line, more than 126-fold and 215-fold more effective than 1 and 5-FU respectively. Compounds 4a (acetoxy), 4b (propionyloxy) and 4e (palmitoxy) exhibited excellent antiproliferative activities against the BGC-823 cell line (IC50 < 0.1 μM). Although compounds 4a (acetoxy) and 4b (propionyloxy) possessed good antiproliferative activities against the Hep-G2, A549, BGC-823 and MCF-7 cell lines, they showed no inhibition against the PC-3 cell line. In the PC-3 assay, the butyryloxy compound 4c exhibited the best antiproliferative activity, being 260-fold and 44-fold stronger than 1 and 5-FU, respectively. The results above reveal that in general, the acyl groups at the C-3 position of these uracil conjugates have primarily made a great contribution to the antiproliferative activities against the tested cell lines.
For further analysis, conjugate 4c was selected to determine its cytotoxicity selectivity and mechanism of growth inhibition on an adherent Hep-G2 cell line. The controls of the figures were reused from our previous work .
Cell cycle analysis
AnnexinV/propidium iodide assay
Mitochondrial membrane potential detection
Caspase-3/9 activity assay
Five acyl oleanolic acid-uracil conjugates were synthesized and their anti-tumor activities were evaluated. These conjugates exhibited excellent antiproliferative activities against the tested cells (Hep-G2, A549, BGC-823, MCF-7 and PC-3) except compounds 4a and 4b, which showed no inhibition against the PC-3 cell line. Most of the IC50 values were under 10.0 μM, with some of them even under 0.1 μM.
Conjugate 4c was selected for further analysis including for its cytotoxicity selectivity and its mechanism of growth inhibition on the Hep-G2 cell line. The inhibition rate of 4c against the HL-7702 cell line was only approximately 15% (90% against Hep-G2) at the concentration of 10 μM, indicating that it had strong cytotoxicity selectivity to human hepatocellular carcinoma cells in vitro. The treatment of Hep-G2 cells with this compound, could induce changes in the permeability of the mitochondrial membrane, and thus cause a decline in the mitochondrial membrane potential. With the disruption of the mitochondrial membrane potential, changes in cellular morphology appeared as a result of significant apoptosis induction. Then, cell proliferation in the G1 phase was arrested and apoptotic signaling activated caspase-9. Caspase-9, as a protease, can activate the apoptotic effector caspase-3, eventually causing nuclear apoptosis. Further studies of the specific mechanisms of these compounds in human malignant tumors are currently underway.
All commercially available solvents and reagents used were of analytical grade and were used without further purification. All commercial reagents were purchased from Aladdin (Shanghai) Industrial Corporation. Melting points were measured on a RY-1 melting point apparatus. 1H and 13C-NMR spectra were recorded on Bruker AV-500 (500/125 MHz for 1H/13C) spectrometers. Chemical shifts are reported as values relative to an internal tetramethylsilane standard. The low-resolution mass spectra were obtained on a Bruker Esquire HCT spectrometer, and HRMS were recorded on a Thermo Scientific Accela—Exactive High Resolution Accurate Mass spectrometer.
General procedures for synthesis of oleanolic acid-uracil conjugates 4a–4e
To a solution of different acyl oleanolic acid compound (3a–3e, 0.2 mmol) in anhydrous CH2Cl2 (3 mL) at 0 °C, oxalyl chloride (0.34 mL, 3.6 mmol) was added. After stirring at room temperature for 12 h, the mixture was evaporated, and co-evaporated with CH2Cl2 (3 × 1 mL). The residue was dissolved in dry THF (3 mL), and then Et3N (1 mL, 0.7 mmol) and uracil (0.067 g, 0.6 mmol) were added at 0 °C. After stirring at r.t. for 24 h, the solvent was evaporated. The residue was then taken up in H2O (35 mL) and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with H2O and brine, dried with MgSO4, filtered and concentrated to give a crude product. The crude product was purified by flash column chromatography to afford the corresponding product (4a–4e) respectively (Additional file 1).
Compound 4a was prepared from 3a [15, 19, 20] (1.000 g, 2.00 mmol) and uracil (0.673 g, 6.00 mmol) according to the general procedure. The residue was purified by flash column chromatography (Petroleum ether:EtOAc = 4: 1). Yield: 0.349 g, 29%, white solid, mp 209–211 °C. 1H NMR (500 MHz, CDCl3) δ 0.69, 0.85, 0.86, 0.99 and 1.17 (5 s, each 3H, 5 × CH3), 0.92 (s, 6H, 2 × CH3), 0.63–2.15 (m, 22H), 2.05 (s, 3H, CH3COO), 2.99 (dd, 1H, J = 3.2, 13.2 Hz, H-18), 4.48 (dd, 1H, J = 6.6, 9.3 Hz, H-3), 5.28 (t, 1H, J = 3.2 Hz, H-12), 5.74 (d, 1H, J = 8.3 Hz, H-5 Ura ), 7.51 (d, 1H, J = 8.3 Hz, H-6 Ura ), 8.20 (brs, 1H, NH). 13C NMR (125 MHz, CDCl3) δ 15.6, 16.8, 18.2, 18.3, 21.4, 23.0, 23.7, 24.0, 26.1, 27.7, 28.2, 30.0, 30.7, 32.7, 33.2, 33.9, 37.0, 37.9, 38.4, 39.8, 42.0, 43.2, 46.5, 47.5, 53.3, 55.3, 81.0, 103.0, 123.6, 141.7, 143.3, 148.8, 162.7, 171.2, 182.0. HRMS (ESI) m/z: [M−H]+ calcd for C36H51N2O5, 591.3798; found 591.3808.
Compound 4b was prepared from 3b [19, 25] (0.300 g, 0.59 mmol) and uracil (0.196 g, 1.75 mmol) according to the general procedure. The residue was purified by flash column chromatography (Petroleum ether:EtOAc = 5: 1). Yield: 0.213 g, 60%, white solid, mp 119–121 °C. 1H NMR (500 MHz, CDCl3) δ 0.69, 0.84, 0.85, 0.98 and 1.17 (5 s, each 3H, 5 × CH3), 0.92 (s, 6H, 2 × CH3), 1.14 (t, J = 7.6 Hz, CH3), 0.63–2.13 (m, 22H), 2.32 (q, 2H, J = 7.3 Hz, CH2COO), 2.99 (dd, 1H, J = 2.5, 12.8 Hz, H-18), 4.48 (dd, 1H, J = 6.5, 9.3 Hz, H-3), 5.28 (s, 1H, H-12), 5.74 (d, 1H, J = 8.3 Hz, H-5 Ura ), 7.50 (d, 1H, J = 8.3 Hz, H-6 Ura ), 8.97 (brs, 1H, NH). 13C NMR (125 MHz, CDCl3) δ 9.4, 15.6, 16.9, 18.1, 18.2, 22.9, 23.6, 24.0, 26.1, 27.7, 28.1, 28.2, 30.0, 30.6, 32.7, 33.1, 33.9, 37.0, 37.9, 38.4, 39.8, 42.0, 43.2, 46.5, 47.5, 53.2, 55.3, 80.6, 103.0, 123.6, 141.7, 143.3, 149.0, 163.4, 174.4, 182.0. HRMS (FTMS + pESI) m/z: [M−H]+ calcd for C37H53N2O5, 605.3955; found 605.3979.
Compound 4c was prepared from 3c [19, 25] (0.200 g, 0.38 mmol) and uracil (0.127 g, 1.14 mmol) according to the general procedure. The residue was purified by flash column chromatography (Petroleum ether:EtOAc = 3: 1). Yield: 0.131 g, 56%, white solid, mp 285–287 °C. 1H NMR (500 MHz, CDCl3) δ 0.69, 0.85, 0.86, 0.95, 0.99 and 1.18 (6 s, each 3H, 6 × CH3), 0.92 (s, 6H, 2 × CH3), 0.63–2.15 (m, 24H), 2.28 (t, 2H, J = 7.1 Hz, CH2COO), 2.99 (dd, 1H, J = 3.3, 13.2 Hz, H-18), 4.49 (dd, J = 5.7, 10.1 Hz, 1H, H-3), 5.28 (s, 1H, H-12), 5.74 (d, 1H, J = 8.3 Hz, H-5 Ura ), 7.50 (d, 1H, J = 8.3 Hz, H-6 Ura ), 8.30 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3) 13.7, 15.4, 16.8, 17.2, 18.2, 18.6, 23.4, 23.6, 25.7, 25.8, 27.8, 28.0, 30.7, 32.0, 32.7, 33.0, 33.8, 36.8, 36.9, 37.7, 38.1, 40.0, 41.3, 41.9, 45.8, 47.4, 47.5, 55.3, 80.5, 104.5, 112.3, 123.0, 139.6, 142.9, 149.0, 161.8, 173.5, 175.2. APCI-MS m/z: 619.4 [M−H]+. HRMS (ESI) m/z: [M−H]+ calcd for C38H55N2O5, 619.4111; found 619.4113.
Compound 4d was prepared from 3d  (0.280 g, 0.44 mmol) and uracil (0.148 g, 1.32 mmol) according to the general procedure. The residue was purified by flash column chromatography (Petroleum ether:EtOAc = 5: 1). Yield: 0.072 g, 22%, white solid. 1H NMR (500 MHz, CDCl3) δ 0.70, 1.00 and 1.19 (3 s, each 3H, 3 × CH3), 0.87 and 0.93 (2 s, each 6H, 4× CH3), 0.63–2.10 (m, 43H), 2.30 (s, 2H, CH2COO), 3.01 (d, 1H, J = 11.8 Hz, H-18), 4.50 (s, 1H, H-3), 5.30 (s, 1H, H-12), 5.74 (d, 1H, J = 7.5 Hz, H-5 Ura ), 7.52 (d, 1H, J = 7.7 Hz, H-6 Ura ), 8.72 (brs, 1H, NH). 13C NMR (125 MHz, CDCl3) δ 14.3, 15.6, 16.9, 18.1, 18.2, 22.8, 23.0, 23.6, 24.0, 25.3, 26.1, 27.7, 28.2, 29.3, 29.4, 29.5, 29.6, 29.7, 30.0, 30.7, 32.1, 32.6, 33.2, 33.9, 35.0, 37.0, 37.9, 38.4, 39.8, 42.0, 43.2, 46.5, 47.5, 53.2, 55.3, 80.5. 103.0, 123.6, 141.7, 143.3, 148.9, 163.1, 173.9, 182.0. HRMS (ESI) m/z: [M−H]+ calcd for C46H71N2O5, 731.5363; found 731.5387.
Compound 4e was prepared from 3e  (0.347 g, 0.50 mmol) and uracil (0.168 g, 1.50 mmol) according to the general procedure. The residue was purified by flash column chromatography (Petroleum ether:EtOAc = 5: 1). Yield: 0.044 g, 11%, white solid, mp 89–91 °C. 1H NMR (500 MHz, CDCl3) δ 0.69, 0.85, 0.86, 0.99 and 1.18 (5 s, each 3H, 5× CH3), 0.92 (s, 6H, 2× CH3), 0.63–2.10 (m, 51H), 2.29 (t, 2H, J = 7.5 Hz, CH2COO), 2.99 (dd, 1H, J = 2.7, 12.9 Hz, H-18), 4.48 (dd, 1H, J = 5.9, 9.8 Hz, H-3), 5.28 (s, 1H, H-12), 5.74 (d, 1H, J = 8.3 Hz, H-5 Ura ), 7.50 (d, 1H, J = 8.3 Hz, H-6 Ura ), 8.17 (brs, 1H, NH). APCI-MS m/z: 787.7 [M−H]+. HRMS (ESI) m/z: [M−H]+ calcd for C50H79N2O5, 787.5989; found 787.6003.
Cell lines and culture
The human hepatocellular cell line Hep-G2, human lung cancer cell line A549, human gastric tumor cell line BGC-823, human breast tumor cell line MCF-7, human prostate cancer cell line PC-3 and human hepatocyte cell line HL-7702, these adherent cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai) and cultured in DMEM medium supplemented with 10% FCS (Fetal Calf Serum). The cells were incubated in an atmosphere of 5% CO2 and 95% air at 37 °C.
The MTT assay was carried out according to a description in a published study [30, 31]. Cells were seeded in 96-well plates and incubated in a CO2 incubator at 37 °C. The tested compounds were dissolved in fresh culture medium with 2% DMSO to afford various concentrations (100, 50, 10, 5, 1 or 0.1 μmol/L). When the cells adhered, compounds at different concentrations were added to every well. The control wells contained medium supplemented with 2% DMSO. After incubation for another 72 h, 20 μL MTT (5%) was added to each well, and the cells were incubated for an additional 4 h at 37 °C. At last, the medium was removed carefully and dimethyl sulfoxide (100 μL) was added to each well. Then the plate was kept on a shaker for 10 min to mix these solutions properly. The absorbance of each well was scanned with an electrophotometer at 570 nm. Each concentration treatment was performed in triplicate wells. The IC50 values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation.
This assay was carried out according to a description in a published study . Cells were seeded at a concentration of 5 × 104 cell/mL in a volume of 2 mL on a sterile cover slip in six-well tissue culture plates. Following incubation, the RPMI 1640 medium was removed and replaced with fresh medium plus 10% FCS and supplemented with compound 4c at the indicated concentration. After the treatment period (24 h), the cover slip with monolayer cells was inverted on a glass slide with 20 μL of AO/EB stain (100 mg/mL). The fluorescence was read on a Nikon ECLIPSETE2000-S fluorescence microscope (Japan).
Hoechst 33258 staining
This assay was carried out according to a description in a published study . Cells grown on a sterile cover slip in six-well tissue culture plates were treated with compound for a certain range of time. The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. After being washed twice with PBS, the cells were stained with 0.5 mL of Hoechst 33258 (0.5 μg/mL, Beyotime) for 5 min and then again washed twice with PBS. The stained nuclei were observed under a Nikon ECLIPSETE2000-S fluorescence microscope using 350 nm excitation and 460 nm emission.
Mitochondrial membrane potential measurement
This assay was carried out as described in a published study . The depolarization of the mitochondrial membrane potential for cell apoptosis results in the loss of Rhodamine123 from the mitochondria and a decrease in the intracellular fluorescence intensity. Prepared Hep-G2 cells were harvested and washed twice in cold PBS and then resuspended in Rhodamine 123 (2 μM) for 30 min in the dark. The fluorescence was measured by flow cytometry with an excitation wavelength of 485 nm and emission wavelength of 530 nm.
Flow cytometric analysis of cell cycle and apoptosis
This assay was carried out according to a description in a published study . The induced apoptosis was assayed by the Annexin V-FITC Apoptosis Detection kit (Beyotime, China), according to the manufacturer’s instructions. Briefly, the prepared Hep-G2 cells (1 × 106 cells/mL) were washed twice with ice-cold PBS and then resuspended gently in 500 μL of binding buffer. Thereafter, the cells were stained in 5 μL of Annexin V-FITC and shaken well. Finally, the cells were mixed with 5 μL of PI, incubated for 20 min in the dark and subsequently analyzed using an FACS AriaII (Becton–Dickinson).
Determination of caspase-3 and caspase-9 activities by flow cytometric analysis
According to a description in a published study , the measurement of the caspase-3 and caspase-9 activities was performed by a CaspGLOW™ Fluorescein Active Caspase-3 and Caspase-9 Staining Kit. The prepared Hep-G2 cells were harvested at a density of 1 × 106 cells/mL in RPMI 1640 medium supplemented with 10% FCS. A total of 300 μL each from the induced and control cultures were incubated with 1 μL of FITC-DEVD-FMK (caspase-3) or FITC-LEHD-FMK (caspase-9) for 1 h in a 37 °C incubator with 5% CO2. Flow cytometric analysis was performed using a FACS AriaII flow cytometer (Becton–Dickinson) equipped with a 488 nm argon laser.
The experiments were repeated three times, and the results were presented as mean ± standard deviation (SD). Student’s t test was used to process the statistical significance and the differences between groups with P < 0.05 were considered significant.
WBM developed the pharmacology part and co-wrote the manuscript, CHS developed the synthesis and co-wrote the manuscript, JYH participated in the synthesis, JL, ZFC and KGC conceived and designed the experiments. All authors read and approved the final manuscript.
The controls of the figures in this study were reused from our previous work with permission from the Royal Society of Chemistry (Med. Chem. Commun., 2016, 7, 972. doi: 10.1039/c6md00061d). This study was financially supported by Grants from the National Natural Science Foundation of PRC (21562006), the Guangxi Natural Science Foundation of China (2015GXNSFAA139186), the Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), the Ministry of Education of China (CMEMR2012-B03/B04, CMEMR2013-A01/C02), Guangxi’s Medicine Talented Persons Small Highland Foundation (1506), IRT1225 and the Bagui Scholar Program of Guangxi Province of China.
The authors declare that they have no competing interests.
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