Synthesis of gypsogenin derivatives with capabilities to arrest cell cycle and induce apoptosis in human cancer cells

Thirty-two gypsogenin derivatives were synthesized and screened for their cytotoxic activities. Their structures were established using IR, 1H NMR, 13C NMR, and LC-MS spectroscopic data. In MTT assays nearly all the compounds displayed good cytotoxicity in the low μM range for several human tumour cell lines (A549, LOVO, SKOV3 and HepG2). Low IC50 values were obtained especially for the carboxamides 7a–7j, for an oxime derivative 3 and a (2,4-dinitrophenyl)hydrazono derivative 4. In particular, the IC50 values of compounds 4 (IC50 = 2.97 ± 1.13 µΜ) and 7 g (IC50 = 3.59 ± 2.04 µΜ) against LOVO cells were found to be much lower than those of the other derivatives and parent compound. These compounds were submitted to an extensive biological testing and proved compounds 4 and 7 g to act mainly by an arrest of the tumour cells in the S phase of the cell cycle. In addition, compounds 4 and 7 g triggered the apoptotic pathway in cancer cells, showing high apoptosis ratios.


Introduction
Cancer is one of the most challenging problems in medicine. One of the several treatments is chemotherapy, and many chemotherapeutics have been developed so far. Many of them were gained from secondary natural products, for example vinca-alkaloids (from poisonous evergreen, Catharanthus roseus), taxeles (diterpenes first derived from the Pacific yew tree, Taxus brevifolia) and camptothecins (lactone alkaloids from Camptotheca acuminata). Moreover, it was reported that a regular consumption of fruits and herbs helps reduce carcinogenic risk [1,2].
Gypsophila oldhamiana, known as 'xia cao' in China, belongs to the Caryophyllaceae family [3]. The roots of Gypsophila species are an especially rich source of triterpenoid saponins [4,5]. Some triterpenoid saponins from Gypsophila have shown a variety of biological activities including anticarcinogenic [6], immunostimulatory [7], cytotoxicity [8][9][10] and α-glucosidase inhibition activities [11]. Gypsogenin (3hydroxy-23-oxoolean-12-en-28-oic acid), a natural pentacyclic triterpenoid, has four active sites such as C-3 hydroxyl, ring-C double bond, C-23 aldehyde group and C-28 carboxylic acid, which are amenable for a wide range of chemical transformations. The structure of gypsogenin is shown in figure 1. This valuable compound has been detected and isolated from Gypsophila oldhamiana. Many biological activities have been credited to gypsogenin, such as inhibitory activity [12,13], cytotoxicity [14,15], antimicrobial and antiproliferative activities [16]. Of special interest are its cytotoxicity and its antiproliferative properties. In previous study, The C-28 carboxylic acid and C-23 aldehyde group were used to prepare nitrile and different types of esters. These compounds are the first pentacyclic triterpenoids described as a potent AChE-selective inhibitor [17]. The C-23 aldehyde group of gypsogenin was treated with hydroxylamine hydrochloride to provide oxime. This compound triggered the apoptotic pathway in cancer cells, showing high apoptosis ratios [16]. Thus, there is strong evidence that gypsogenin has anti-cancer activity.
Oleanolic acid (3-hydroxy-12-en-28-oic acid), a pentacyclic triterpenoic acid, has three active sites such as C-3 hydroxyl, ring-C double bond and C-28 carboxylic acid. The structure of gypsogenin is very similar to the structure of oleanolic acid. The C-3 hydroxyl and C-28 carboxylic acid of oleanolic acid were used to prepare acetoxy and different types of amide and esters [18,19]. In particular, the different 3-O-acetyl oleanolic acid derived amides have been prepared and screened for their cytotoxic activity [20]. The results reveal that most of the carboxamides displayed good cytotoxicity in the low micromolar range for several human tumour cell lines. These compounds were submitted to an extensive biological testing and proved some compounds to act mainly by an arrest of the tumour cells in the S phase of the cell cycle. In addition, some compounds triggered the apoptotic pathway in cancer cells, showing high apoptosis ratios. These findings make oleanolic acid a promising lead compound for developing new cytotoxic/antitumour active compounds. In previous study, we have never found any references about the cytotoxicity of gypsogenin derived amides. Thus, it is valuable to investigate gypsogenin derived amides.
Gypsogenin aglycone is found at high concentrations in Gypsophila oldhamiana [21]; therefore, it can be obtained with ease [22]. In this study, the C-23 aldehyde group has been used to prepare hydrazone and oxime. The different types of esters and amide were designed, and synthesized at C-28 carboxylic acid. In addition, they were evaluated for their cytotoxic activities against four different human cancer cell cultures. More investigations about the mechanism of cell death induced by these gypsogenin derivatives were performed.

Chemistry
Thirty-two gypsogenin derivatives were synthesized by a series of reactions as outlined in schemes 1-3. All compounds were obtained in different yields. The gypsogenin (1) was obtained by the hydrolysis of the gypsogenin saponin mixtures. For compound 1, the signals for H-3, H-12 and H-23 could be seen at δ 3.95 (1H, dd, J = 11.41, 5.15 Hz), 5.29 (1H, s) and 9.50 (1H, s), respectively. These signals were also evident by their 13 C NMR spectra showing C-3, C-12 and C-23 at δ 71.52, 122.17 and 207.20, respectively. Gypsogenin was acetylated to afford compound 2 in 95.7% yields. In the 1 H NMR spectrum for compound 2, the proton signal of H-3 at δ 5.23 (dd, J = 11.44, 5. 16 Hz) was observed instead of at δ 3. 95. Compounds 1 and 2 were mixed with hydroxylamine hydrochloride in pyridine at 105°C to provide compounds 3 and 5 in 85.1% and 96.5% yields, respectively. The structures of compounds 3 and 5 were rsos.royalsocietypublishing.org R. Soc. open   confirmed by their respective 13 C NMR spectra, which showed the characteristic C-23 carbon signals at δ 159.46 and 157.15 respectively. Compounds 1 and 2 were treated with 2,4-dinitrophenylhydrazine in acetic acid at room temperature to obtain compounds 4 and 6 in 88.9% and 86.5% yields, respectively. The 13 C NMR spectra of compounds 4 and 6 showed the characteristic carbon signals of C-23 at δ 163. 13 and 158.58 respectively.
Consecutive reactions of the intermediate compounds 2 and 6 with oxalyl chloride and secondary amine in CH 2 Cl 2 at room temperature gave the corresponding amides 7a-7g and 9a-9g in 82-93% and 85-93% yields respectively. Their 13 C NMR spectra showed the characteristic carbon signals at δ 174.45-175.82 attributed to C-28. Under similar conditions, compounds 2 and 6 were coupled with oxalyl chloride and primary alcohols in CH 2 Cl 2 at room temperature to obtain the corresponding esters 7 h-7j and 9h-9j in 89-94% and 89-92% yields, respectively. For compounds 7h-7j and 9h-9j, these carbon signals for C-28 could be seen at δ 177.42-178.22 respectively.
Compounds 7a-7g were coupled with NaOH in C 2 H 5 OH at room temperature to afford the corresponding amides 8a-8g in 82-89% yields. In the 1 H NMR spectra for compounds 8a-8g, the proton signal of H-3 at δ 5. 24-5.26 was converted to δ 4.06-4. 10. The thirty-two compounds' spectral data are presented in the electronic supplementary material.

Cytotoxic activity
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a mitochondrial function assay that is based on the ability of viable cells to reduce the MTT to insoluble formazan crystals rsos.royalsocietypublishing.org R. Soc. open  by mitochondrial dehydrogenase [23]. The anti-cancer activities of the synthesized compounds were studied by an MTT assay using the following human cancer cell lines in cell culture [24]: LOVO (a colon cancer cell line), SKOV3 (an ovary cancer cell line), A549 (a lung cancer cell line) and HepG2 (a liver cancer cell line). Cisplatin along with gypsogenin were taken as reference standards in this study. The IC 50 values were determined for each compound and cell line. The results of tests are shown in table 1. It is evident that the synthesized compounds showed selectivity in exhibiting significant anti-cancer activity against the four cancer cell lines. Among the tested compounds, 3, 4 and 7a-7g were found to be more toxic than others of this series. The structure-activity relationship studies revealed that the introduction of an amide in the 28 position of the target compounds 7a-7g enhanced the anti-cancer activity. Similarly, the introduction of oxime and 2,4-dinitrophenylhydrazone in the 23 position of the target compounds 3 (IC 50 = 12.35 ± 1.34 µM for LOVO cells) and 4 (IC 50 = 2.97 ± 1.13 µM for LOVO cells) also led to an increase in anti-cancer activity. Our results revealed the superior anti-cancer activity of 4 and 7g (IC 50 =3.59 ± 2.04 µM for LOVO cells) compared to other compounds of the same series.

Cell cycle analysis by propidium iodide staining
To further investigate the differential growth inhibition mechanism mediated by amide and 2,4dinitrophenylhydrazone compounds on cell cycle dynamics, the effects of compounds 4 and 7g on cell

Conclusion
In summary, we synthesized thirty-two gypsogenin derivatives in good yield. These compounds were found to be promising lead compounds against the majority of the studied cancer cell lines, exhibiting IC 50 values much lower than that of the parent compound. The synthesized compounds showed selectivity in exhibiting significant anti-cancer activity against the human cancer cell lines. The structure-activity relationship studies revealed that the introduction of amide in the 28 position and 2,4-dinitrophenylhydrazone in the 23 position of target compounds enhanced the anti-cancer activity. Among all of these compounds, compounds 4 and 7g could be considered as possible anti-cancer agents as they were shown to affect the cell cycle, causing cell cycle arrest. In addition, compounds 4 and 7g triggered the apoptotic pathway in cancer cells, showing strong activity in promoting apoptosis.

General
Melting points of all compounds were recorded on an Optimelt-100 automatic melting point apparatus and are uncorrected. IR (KBr) spectra were recorded with a Thermo Nicolet Nexus 670 FT-IR. The NMR spectra were obtained with a Bruker Avance III 600 spectrometer ( 1 H: 600 MHz, 13 C: 150 MHz) with tetramethylsilane as internal standard. Chemical shifts are given in values of ppm and coupling constants in hertz. LC/MS was recorded with an Agilent 1200 capillary spectrometer. Thin-layer chromatography (TLC) was performed on precoated silica gel plates (Qingdao Marine Chemical Industry Factory, Qingdao, China). Column chromatography was carried out using silica gel (200-300 mesh, Qingdao Marine Chemical Industry Factory, Qingdao, China).