Modification at A-Ring and Cytotoxic Activity of Betulonic Acid and its N-Methylpyperazinyl Amide


 Since cancer remains one of the most prevalent diseases today, there is an urgent need for the development of new agents. Triterpenoids may act in multiple pathways displaying antiproliferative, antiangiogenic, anti-inflammatory, and pro-apoptotic activities that place them as promising multifunctional agents in treating cancer. In this paper a series of betulonic acid and its N-methylpyperazinyl amide derivatives, especially holding C2-nicotinoylidene/furfurylidene/fluorobenzylidene fragments, have been synthesized and evaluated for their cytotoxic activity against the NCI-60 cancer cell line panel. N-Methylpiperazinyl amides of betulinic acid 11 and 4-pyridinoylidene-betulinic acid 9 as well as betulonic acid C2-4-pyridinoylidene- 14 or furfurylidene 16 derivatives were found to be the leading compounds with GI50 values of 0.49 μM for leukemia CCRF-CEM, 1.60 μM and 1.36 μM for colon cancer HCT-116 and 1.66 μM for melanoma LOX IMVI cell lines, respectively. The activity displayed for these compounds was higher than for the standard drug doxorubicin against colon cancer HCT-15 and ovarian cancer NCI/ADR-RES cell lines. Cell cycle analysis indicates that compound 11 promotes cytotoxic activity through the apoptosis induction both in conditionally normal (HEK293) and in cancer (A549, MCF-7) cells, whereas compound 14 exhibits both cytostatic and cytotoxic activity, dependently on cell line evaluated. In particular, in HEK293 cells the compound 14 induces mainly apoptotic cell death, while in A549 and MCF-7 cells cytostatic effect is dependent on cell cycle arrest in G2/M phase.Our results suggest that betulinic acid N-methylpyperazinyl amide 11 is the promising compound for the future drug development antitumor studies.


Introduction
Natural products are a rich source of bioactive compounds that can be employed in obtaining substances with great medicinal potential [1]. Attempts to successfully treat cancer are more necessary than ever, as 9.6 million people worldwide died in 2018, and globally about 1 in 6 deaths is due to cancer [2], there is an urgent need for the development of new agents. In cancer treatment, the in uence of natural products is quite marked. With more than 50% of all new anticancer drugs , natural products and derivatives thereof still play a key role in treating cancer From the 1940s to the end of 2014 nearly 80% of the 246 anticancer worldwide approved drugs are related with natural sources [3]. Among the naturally occurring compounds, triterpenoids display a wide spectrum of biological activities, including anti-in ammatory, antiviral, anticancer, antimicrobial effects [4][5][6][7]. Pentacyclic triterpenoids of lupane, oleanane, ursane, friedelane and dammarane types (among others) have been reported in the literature as possible antitumor agents [8][9][10][11]. These substances may act in multiple pathways displaying antiproliferative, antiangiogenic, anti-in ammatory, and pro-apoptotic activities that place them as promising multifunctional agents in treating cancer [12].
The reaction of compound 11 with diethyl chlorophosphate in pyridine in the presence of 4dimethylaminopyridine (DMAP) led to diethoxyphosphate 12. In the 1 H NMR spectra of compound 12 proton signals of two СН 3 СН 2 O-groups were contained at δ 1.28-1.42 ppm and δ 4.01-4.14 ppm, while four signals of the carbon СН 3 and СН 2 groups appeared at δ 16.1, 16.3 and 61.5, 63.4 ppm in the 13 C NMR spectra.

Biological activity
Evaluation of cytotoxicity activity against NCI-60 cell line panel Compounds 1-16 were selected by National Cancer Institute and tested at one dose assay (10 -5 M) towards a panel of approximately sixty cancer cell lines according to the NCI protocol as described elsewhere (see e.g. http://dtp.nci.nih.gov) [43][44][45][46][47].
Compounds 6-8 did not show cytotoxic activity against the studied cell lines (Table 1).. Compound 1 inhibited the cell growth of the leukemia SR, NSC lung cancer NCI-H460, and colon cancer HCT-116 cell lines.
Amides with pyridinoylidene fragments 2 and 3 were active against colon cancer HCT-116 and HT29, prostate cancer PC-3, breast cancer MCF-7 cell lines, and all six of leukemia cell lines. Compound 4 inhibited two leukemia MOLT-4 and SR cell lines, whereas furfurylidene derivative 5 showed activity towards leukemia MOLT-4 and SR, colon cancer HT29, CNS cancer SF-295 cell lines and melanoma line LOX IMVI. The oxime 10 and azepanobetulinic acid N-methylpiperazinyl amide 13 inhibited only one leukemia cell line SR, as well as compound 15 was active towards one colon cancer cell line HCC-2998. Compound 12 inhibited two leukemia K-562 and SR cell lines. Compounds 11 and 14 demonstrated a broad spectrum of the cell proliferation inhibition against 38 and 54 human tumor cell lines (the growth percent ranging from -98.30 to 128.95 for compound 11 and from -9.50 to 78.00 for compound 14, respectively). N-Methylpiperazinyl amides 9 and 16 showed the greatest antiproliferative effect towards all 60 cell lines, resulting in 50 cases of cancer cell lethality from -0.44 to -90.75% for compound 9 and in 56 cases of cancer cell lethality from -1.95 to -97.61% for compound 16, respectively (Table 1 and Supp. Material).
Thus, all compounds exhibited signi cant antiproliferative effect towards human cancer cell lines, and among them, the highest cytotoxic activity in 5-dose testing mode screening was observed for the betulinic acid N-methylpiperazinyl amide 11 with growth inhibitory (GI 50 ) against the most sensitive cell lines at submicromolar (0.487 μM) and micromolar concentrations (1-2 μM), respectively. Cytotoxic activity (LC 50 ) of this compound against the most sensitive cancer cell lines was also high (5-9 μM) ( Table 2).
The anticancer activity results showed that the modi cation of the betulinic, betulonic and azepanbetulinic  (Table 1).
A raw comparison of the activities of compounds 9, 11, 14 and 16 with respect to the activity reported for doxorubicin [48], re ects that the activity displayed for these compounds was lower than for the standard drug except colon cancer HCT-15 and ovarian cancer NCI/ADR-RES. Furthermore, at the LC 50 level of cytotoxicity, compound 16 was more e cient against 3 cancer cell lines, compounds 9 and 14 -against 12 cell lines, compounds 11 -against 37 cancer cell lines, respectively (Table 2).  The selectivity index (SI) was calculated by dividing the full panel MG_MID 60 (μM) of the compounds 9, 11, 14 and 16 by their individual subpanel MG_MID of the cell line (μM) and is to be considered as a measure of the compounds' selectivity (Table 3). Ratios between 3 and 6 mean moderate selectivity, ratios greater than 6 indicate high selectivity towards the corresponding cell line, while compounds not meeting either of these criteria are rated nonselective [49]. In this context, the compounds 9, 14, and 16 in the present study were found to be nonselective at all the GI 50    Elemental analysis was performed on a Euro EA-3000 CHNS analyzer (Eurovector, Milan, Italy); the main standard is acetanilide. Thin-layer chromatography analyses were performed on Sorb l plates (Sorbpolimer, Krasnodar, Russian Federation), using the solvent system chloroform-ethyl acetate, 40:1. Substances were detected by 10% H 2 SO 4 with subsequent heating to 100-120 °C for 2-3 min. Betulonic acid (Flekhter et al. 2002), compounds 1, 5 and 11 [28], 13 [42], 14 and 16 [50] were obtained according to the methods described previously.

Cell cycle analysis
The cell cycle was measured by ow cytometry assay with PI (propidium iodide) staining. Human embryonic kidney HEK293 cells (2 × 10 5 cells/well), lung carcinoma A549 and, breast adenocarcinoma MCF-7 cells (1 × The data are expressed as mean ± S.E.M from 3 experiments performed in triplicate. Comparison of cell cycle phases was performed using Wilcoxon t-test (Statistica 6.1 (StatSoft. Inc., USA); * -p 0.05 vs. vehicle for certain cell line.
Declarations Figure 1 The effect of compound 11 on cell cycle distribution in HEK293, A549 and MCF-7 cells. The data are expressed as mean ± S.E.M from 3 experiments, performed in triplicate. Comparison of cell cycle phases was performed using Wilcoxon t-test; * -p 0.05 vs. vehicle for certain cell line.