Ginkgolide B derivative synthesis and their effects on the viability of SKOV3 cells

The natural product Ginkgolide B was used as a raw material and modified by esterification on C10-OH or C1-OH to obtain 11 derivatives (1–11), which were structurally characterized with nuclear magnetic resonance spectroscopy. An MTT assay-based in vitro tumor proliferation inhibitory activity test showed that compounds 2, 3, 6, 7, 10, and 11 exhibited strong inhibitory activity against the human ovarian cancer cells SKOV3, with IC50 values of 16.05 µmol/L, 15.65 µmol/L, 32.00 µmol/L, 63.30 µmol/L, 23.20 µmol/L, and 31.10 µmol/L, respectively. Annexin V/PI double staining assay showed that compound 2 induced apoptosis in SKOV3 cells to a slightly greater extent than GB and compounds 5 and 9, with an apoptosis rate of 31.68%.

The PAF/PAFR signaling axis has emerged as an important determinant of the aggressive phenotype in several malignancies, including ovarian cancer [16,17]. GB, as a potent PAFR antagonist, may be a key regulator in treating tumor cells. Jiang Wei et al. [7,8] conducted in vivo and in vitro experiments and found that GB could inhibit the proliferation of ovarian cancer cells and growth of nude mice tumors with a tumor suppression rate of 48%, and up to 78.2% in combination with cisplatin (CDDP), which could be used as an adjuvant drug for the treatment of ovarian cancer. The mechanism may be that GB inhibits the expression of platelet-activating factor (PAF), plateletactivating factor receptor (PAFR), and the tyrosine kinase Src and the p38 mitogen-activated protein kinase (p38MAPK), preventing p38MAPK from activating downstream transcription factors [18], including the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), which is closely related to cell invasion. This results in the inability of CREB to bind to the promoter in the upstream of the matrix metalloproteinase 2/9 (MMP2/MMP9) gene and the inability of MMP2 mRNA/MMP9 mMRA to be expressed, which inhibits the migration of ovarian cancer cells. In addition, GB upregulated 25 proteins with antitumor effects (e.g., p53) and downregulated 22 proteins associated with tumor migration (e.g., β-catenin).
Yu Y et al. [19] demonstrated that the PAFR upregulation in CDDP-treated ovarian cancer cells provided additional support for the function of the PAF/PAFR signaling axis as a resistance mechanism, with the possible mechanism being that cisplatin (CDDP) acts on the ovarian cancer cells and causes nuclear factor kappa-B (NF-KB) and hypoxia-inducible factor (HIF-1α) to accumulate in the nucleus, leading to the upregulation of the plateletactivating factor receptor PAFR. In addition, GB inhibited the PAFR activity, which may block the downstream signaling pathways of phosphoinositide 3-kinase (PI3K) and the extracellular regulated protein kinase (ERK), thereby enhancing the sensitivity of ovarian cancer cells to CDDP, intensifying drug efficacy and significantly reducing tumor growth.
Ginkgolide B has a unique rigid caged dodeca-carbon skeleton structure, and it was found that the synthesis of GB ester derivatives by converting the hydroxyl groups at the C-1 and (or) C-10 positions of GB into aromatic-containing groups such as ester groups significantly enhanced the anti-PAF activity [20,21]. To explore the effect of GB parent structure and the introduction of small molecule side chains of benzoic acid series on the hydroxyl groups at C-1 and (or) C-10 positions on pharmacological activity, this study constructed GB ester derivatives by the esterification reaction of GB as the parent nucleus with compounds with aromatic groups such as p-chlorobenzoic acid, p-fluorobenzoic acid, p-nitrobenzoic acid, p-methoxybenzoic acid, 3-methoxybenzoic acid, and 3'5'-dinitrobenzoic acid by reference to the design of nicotinic acid esters, cinnamic acid esters, and benzoic esters [22][23][24][25][26][27][28][29][30]. The GB derivatives were structurally characterized by nuclear magnetic resonance spectroscopy (NMR), tested for in vitro tumor proliferation inhibitory activity by thiazolyl blue (MTT), and detected for ovarian cancer SKOV3 cell apoptosis by the Annexin V/PI double staining assay.

Results and discussion
According to the reaction described in Scheme 1, eleven novel GB derivatives 1-11 were synthesized by esterification using GB as a raw material. Two products can be simultaneously obtained in one pot reaction. Due to the higher reaction activity of C-10 hydroxyl in GB, the yields of esterification at the C-10 position were higher than at the C-1 position. Conformational analysis showed that compounds 1, 3, 5, 7, 9, and 11 were hydroxyesterification products at the C-1 position of GB, and compounds 2, 4, 6, 8, and 10 were hydroxyesterification products at the C-10 position of GB. A method for the rapid separation and purification of GB derivatives by preparative chromatography was established. The structure of all the final compounds 1-11 was confirmed by 1 H NMR and 13 C NMR. For the NMR spectra of compounds 1-11, a singlet at δ 1.0-0.9 ppm that corresponded to tert butyl functionality, signals at δ 9.1-6.0 ppm revealed the presence of the benzene ring.
On the basis of previous studies, the final products 1-11 and GB were evaluated for their in vitro antiproliferative activity against ovarian cancer SKOV3 cell lines by MTT assay. The proliferation inhibitory activity of these compounds was measured at different concentrations. The IC 50 values for each sample were obtained by plotting the inhibition rate against the drug concentration. The results are presented in Table 1, and inhibition curves for various compounds were shown in Fig. 1. As we defined that compounds showing <50% inhibitory rate at 100 µM were inactive. Compounds 3, 4, 7, 8, and 11 hardly showed any activity, the IC 50 values against the SKOV3 cell lines are all above 100 µM. The rest of the compounds showed moderate to good activity on SKOV3 cell lines with IC 50 values 15.65-63.30 μM. Obviously, as compared with compounds 1 and 2, the esterification position of C-1 or C-10 has little effect on the activity of the compounds. However, the inhibitory activities were increased by introduce of the electron-donating group into the benzyl group.
The apoptosis of ovarian cancer SKOV3 cells was induced by 100 μmol/L of GB, compounds 2, 5, and 9. The apoptosis rates were found to be 7.98%, 31.68%, 8.82%, and 9.46% (Fig. 2), respectively. Preliminary in vitro antitumor cell proliferation activity test and the Annexin V/PI double staining assay showed that compound 2 had the highest inhibition rate against ovarian cancer SKOV3 cells and could inhibit the proliferation of tumor cells and induce apoptosis. Ginkgolide B derivatives hydroxyesterified at the C-10 position showed significantly higher apoptosis rates IC 50 values are shown in µM against SKOV3 cell line, respectively

Conclusion
In this study, 11 novel GB derivatives 1-11 were synthesized and evaluated for their in vitro antiproliferative activities against human ovarian cancer SKOV3 cell lines. The majority of the compounds showed moderate to good activity on SKOV3 cell lines. Among these derivatives, compound 2 was found to exhibit better cytotoxic activity on against SKOV3 cells with IC 50 values 15.65 µM. Annexin V/PI double staining assay showed that compound 2 induced apoptosis in SKOV3 cells to a slightly greater extent than GB and compounds 5 and 9, with an apoptosis rate of 31.68%. The above findings will be of great significance for the development of GB derivatives as potential antitumor agents.

Chemistry
All reagents and solvents used in this study were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. (P.R. China), Shanghai Macklin Biochemical Technology Co., Ltd. (P.R. China), and Energy Chemical (P.R. China), and used as received without further purification unless otherwise noted.
In addition, some biological reagents were purchased from Sigma or GIBCO, USA. TLC was carried out on Silica Gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd) and spots were visualized by iodine vapors or by irradiation with UV light (254 nm). All water employed was ultrapure (> 18.2 MΩcm-1 at 25°C, Milli-Q, Millipore, Billerica, MA). The NMR spectra were measured by Bruker Avance 600 NMR analyzer (Bruker, Switzerland) in the indicated solvents. Chemical shifts are expressed in ppm (δ units) relative to TMS signal as internal reference, and the coupling constant values (J) are shown in Hertz. Signal multiplicities are reported by the following abbreviations: s (singlet), d (doublet), t (triplet), dd (double doublet), q (quartet), m (multiplet), and brs (broad signal). HB DAC-50 was used for preparative liquid chromatograph (Jiangsu Hanbon Science & Technology Co., Ltd.). The high resolution mass spectrometry (HRMS) was carried out on a Thermo Q Exactive Focus. Agilent 1260 high performance liquid chromatograph (Agilent Technologies Co. Ltd, USA), SCIENTZ-10N vacuum freeze dryer (Ningbo Xinzhi Biotechnology Co. Ltd.), YG-875B ultra clean bench (Suzhou Medical Equipment Factory), inverted phase contrast microscope (Zeiss, Germany), enzyme marker (Biotek, USA), CO 2 cell incubator (Thermo Fisher Scientific), and GB (98.24% purity, lab-made) were used.

Tumor cell growth inhibitory assay
The SKOV3 cells used in the following cell experiments were obtained from the Shanghai EK-Bioscience Co., Ltd. (P.R. China). SKOV3 cells were maintained in DMEM containing 10% fetal bovine serum (FBS). All cells were grown at 37°C in a humidified atmosphere of 5% CO 2 . All the reagents used in this study, unless otherwise indicated, were purchased from Sigma (USA). The compounds with inhibitory activity against human ovarian cancer cells SKOV3 were screened by the MTT assay [31]. Briefly, SKOV3 cells were cultured at a density of General procedure for the synthesis of GB derivatives 1-11 The synthetic route was shown in Fig. 3  solution or acetic acid-iodine. The reaction was considered complete when the spots of GB disappeared. After recovering the dichloromethane solution under reduced pressure, the residue was dissolved with 30.0 mL of ethyl acetate and underwent reverse extraction three times with water, with 15.0 mL/time. The ethyl acetate layer was dried by anhydrous sodium sulfate and concentrated under reduced pressure to dryness. The product of each synthesis was dissolved in acetonitrile to make a sample solution of 30.0 mg/mL. After passing through a 0.45 µm filter membrane, the product was purified by high pressure preparative chromatography eluting with acetonitrile/water to produce the corresponding derivatives.