Design, synthesis and cytotoxic evaluation of novel betulonic acid-diazine derivatives as potential antitumor agents

With the purpose to improve antiproliferative activity, 26 new betulonic acid-diazine derivatives were designed and synthesized from betulinic acid. The anticancer activity of these semi-synthetic compounds was evaluated by MTT assay in both tumor cell lines and normal cell line. The results indicated that majority of new compounds exhibited improved antitumor activity compared with the parent compound betulonic acid. Compound BoA2C, in particular, had the most significant action with IC50 value of 3.39 μM against MCF-7 cells, while it showed lower cytotoxicity on MDCK cell line than cisplatin. Furthermore, we discovered that BoA2C strongly increased MCF-7 cell damage mostly by influencing arginine and fatty acid metabolism. In addition, the structure-activity relationships were briefly discussed. The results of this study suggested that the introduction of different diazines at C-28 could selectively inhibit different kinds of cancer cells and might be an effective way to synthesize potent anticancer lead compound from betulonic acid.

. It has been reported that oxidation of the hydroxyl group at position C-3 markedly enhanced the cytotoxic potency (Baratto et al., 2013;Zhang et al., 2014). Thus, betulonic acid (BoA), which had a ketone instead of a hydroxyl with βconfiguration at the C-3 of BA, was used as a starting molecule in the synthetic transformations described in this research for the identification of novel anticancer drugs.
BoA has two major functional groups, 3-=O and 28-COOH, which are susceptible to functionalization by transformations such as amination, esterification, sulfonation, hydroxylation, and alkylation. And many of these derivatives ( Figure 1) are known to possess various types of biological activities (Saxena et al., 2006;Dangroo et al., 2017;Eignerova et al., 2017;Gupta et al., 2017;Khlebnicova et al., 2017;Mzhelskaya et al., 2017;Khlebnicova et al., 2019;Popov et al., 2019;Sousa et al., 2019;Hoenke et al., 2020;Yang et al., 2020). A previous study demonstrated that BoA derivatives with fused azole and triazine displayed impressive cytotoxic activity against several cancer cell lines (Dinh Ngoc et al., 2014;Grishko et al., 2017). And BoA derivatives of nitrogen-containing heterocycles at the C-28 position exhibited excellent anticancer activities (Yang et al., 2015). There are also studies that boc-lysinated-betulonic acid (100 μM) had been shown 95.7% inhibition of LNCaP prostate cancer cells whereas little effect on normally proliferating fibroblast cells (Saxena et al., 2006). Besides, a series of C-28 derived 1,2,3-triazolyl derivatives of BoA possessed promising cytotoxicity activity (IC 50 value in the range of 4-6 μM) (Dangroo et al., 2017). The preceding investigations piqued our curiosity in whether BoA with nitrogen-containing heterocycles at C-28 might be beneficial for anticancer activities. In this study, we successfully synthesized 26 new BoA derivatives by introducing pyrazines including TMP, pyrimidines and pyridazines at the C-28 ( Figure 2), in order to improve its anticancer activity. The cytotoxicity of all these derivatives (BoAA-BoAE, BoAnA-BoAnGn (n = 2, 3, 4)) was evaluated against four human cancer cell lines (including HepG2, Bel-7402, HeLa, MCF-7) and normal cell line Figure 2 (MDCK). And the ability of the most potent active compound to trigger cell injury was preliminary analyzed in vitro.

General aspects
Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz. Reagents were bought from commercial suppliers without any further purification. NMR spectra were recorded on a Bruker-500 or -400 spectrometer (Bruker, Dresden, Germany) with tetramethylsilane (TMS; TCI, Tokyo, Japan) as an internal standard; high-resolution mass spectra (HRMS) were acquired using a Thermo Scientific TMLTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY, United States); Melting points were measured at a rate of 5°C/ min using an X-5 micro melting point apparatus (Beijing, China) and were not corrected.

DAPI staining
Morphological observation of nuclear changes was performed by DAPI staining in this assay. MCF-7 cells in the logarithmic growth phase were seeded in 12-well plates at a density of 5 × 10 3 cells/well and were allowed to grow for 24 h. Then the cells were treated with different concentration ranges of BoA2C (0, 2, 4 8 μM) for 72 h. After the treatment period, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Then the liquid was discarded, and the cells were stained with DAPI (1 mg/ml, Molecular Probes/Invitrogen Life Technologies, Carlsbad, CA, United States) for 1 min in dark. After staining, cells were visualized under a fluorescence microscope (Olympus IX71, Tokyo, Japan).

Wound healing assay
MCF-7 cells were seeded in six-well plates at a density of 2 105 cells/ml and incubated at 37°C to achieve 100 percent confluence. After starving the cells for 24 h, a wound (cell-free) was drawn across the center of the well using the tip of a 1 ml plastic pipette. Wells were cleaned twice with PBS before being treated for 48 h with BoA2C (8 μM). At 0 h, 24 h, and 48 h, cells were viewed and photographed in randomly selected fields using an inverted light microscope (Nikon).

Cellular metabolomics study
To eliminate excess medium, cells were washed twice with 1 ml of PBS solution. The wells were then filled with 2 ml of ACN: MeOH: H 2 O 50:30:20 (v/v) and placed in the freezer for 20 min. The extracts obtained in an Eppendorf tube after the cells were scraped and vortexed. The mixture was then centrifuged for 10 min at 10,000 rpm and 4°C, and the supernatant was collected and dried with an evaporator. 300 μl MeOH was used for reconstitution. 15 μL of the reconstituted extract was mixed with 15 μl H 2 O and 70 L ACN for the metabolomics analysis of the intracellular material. After centrifugation (10 min, 4°C, 11,800 g), the supernatant was transferred to an LC-MS vial for analysis.
An Acquity UPLC HSS T3 column (1.8 μm 100 × 2.1 mm) using an UltiMate 3000 high performance LC system coupled to Q Exactive MS was used for metabolic profiling (Shu et al., 2020). At a flow rate of 0.3 ml/min, mobile phase A (water containing 0.1% acid) and mobile phase B (methanol) were used. A was reduced from 80 % to 70% from 0 to 2 min, A was reduced to 55% from 2 to 5 min, A was reduced to 40% from 5 to 6.5 min, A was reduced to 35% from 6.5 to 12 min, A was last reduced to 0 from 12 to 18 min. The gradient of the mobile phase was as follows: The column oven temperature was set to 40°C, and the autosampler temperature was set to 4°C. The injection volume was 1 μl. MS used electron spray ionization to operate in both positive and negative ionization modes (ESI). To assess the stability and repeatability of the LC-MS-based metabolomics method, quality control (QC) samples were used. Each QC
Frontiers in Chemistry frontiersin.org sample was made by combining five samples of the same volume (100 μL) in the order of analysis. To ensure the system method's stable operation, all combined QC samples for running all QC samples made by every five samples before analysis. After five samples were tested during the analysis run, the combined QC samples of the five samples were taken.  Frontiers in Chemistry frontiersin.org 13

Statistical analysis
The data were expressed as mean ± SD. Statistical testing using GraphPad Prism 8.0.2. The observed data were compared and analyzed by one-way ANOVA. The levels of statistical significance were set at pp < 0.05, ppp < 0.01, pppp < 0.001.

Chemistry
The designed derivatives were prepared following the procedures in Scheme 1. The compound BoA was synthesized from botulin according to the route depicted in Scheme 1, as in detail shown previously (Krasutsky, 2006). In Scheme 1, BoAA and BoAB were synthesized through a combination of BoA and chloromethyl pyrazine in N, N-dimethylformamide (DMF) containing K 2 CO 3 at room temperature, and the yield was 49%-62%. And BoAE was obtained by the same synthetic route at 85°C instead of at room temperature with 82% yield. In addition, methyl-2-pyrazinylmethanol in dry dichloromethane (DCM) containing dichlorosulfoxide (SOCl 2 ) was allowed to stir at room temperature, which was further reacted with BoA in dry DMF in the presence of K 2 CO 3 to yield compounds BoAC and BoAD.

Biological screening 3.2.1 Cytotoxicity assay
The in vitro cytotoxicity of BoA derivatives was evaluated on four tumor cell lines (HepG2, Bel-7402, Hela, MCF-7) and MDCK cell line using the standard MTT assay. The IC 50 values of these compounds were summarized in Table 2. The results revealed that the majority of the compounds considerably  increased cytotoxicity in all tumor cell lines examined compared to the BoA, while they showed lower cytotoxicity than DDP on MDCK cell line (13.69 ± 1.45 μM). Among the compounds, BoA3G possessed pronounced cytotoxicity against the cell lines Hela at the IC 50 levels of 4.00 ± 0.32 μM (Figure 3). It's worth noting that the most active compound was BoA2C, which revealed more severe cytotoxicity against Bel-7402 and MCF-7 cells at the IC 50 levels of 3.65 ± 0.43 μM and 3.39 ± 0.07 μM, comparing with the cisplatin (DDP) against those cells at the IC 50 levels of 8.86 ± 1.32 μM and 6.98 ± 0.32 μM, respectively. Meanwhile, BoA2C exhibited lower cytotoxic against MDCK cell (IC 50 > 30 μM) than DDP (Figure 4). Structure-activity relationships (SAR) investigations revealed that BoA derivatives including diazines have varying selectivity for four distinct human cancer cells, with pyrazine derivatives showing the best selectivity. The amount of methyl groups in pyrazine derivatives is critical, since the anticancer activity drops dramatically as the number of methyl groups increases. Despite their improved anticancer activity, pyrimidine and pyridazine compounds exhibited no selectivity. Besides, it was observed that the compounds with pyridazine had stronger activity on Hela cells, and those with 2, 5-dimethylpyrazine seemed to be more active on Bel-7402 and MCF-7 cells. However, there was no obvious rule that the relationship among the differing length of carbon chain between BoA and diazines and cytotoxic activity against a variety of cancer cell lines. In this study, the most promising compound BoA2C was selected for further analysis to reveal its preliminary mechanism of growth inhibition on MCF-7 cells and MDCK cells.

Analyses of cell staining
To characterize the effects of apoptosis inducted by BoA2C on MCF-7 cell and MDCK cell, the nuclear morphological changes in BoA2C-treated MCF-7 and MDCK were observed by DAPI staining. As shown in Figure 5, the morphology of MCF-7 cells in the negative control was normal, after treated with BoA2C (2, 4, 8 μM) for 72 h, MCF-7 cells showed nuclear morphological changes typical of apoptosis in a dosedependent manner. Even at low concentration of BoA2C (2 μM), In terms of morphological characteristics, the cells had displayed typical death, such as distinctive condensation, margination of chromatin and nuclear fragmentation. With the increase of the concentration of BoA2C, the characteristic of proliferation inhibition was more and more obvious. When the concentration of BoA2C was raised up to 8 μM, the number of cells decreased drastically, the shape of the cells became irregular, and nuclear fragmentation occurred. In contrast, as seen in Figure 6, MDCK cells did not show a significant nuclear damage with increasing concentration of BoA2C. And these results indicated that BoA2C significantly induced apoptosis in MCF-7 cells but less damaged to MDCK cells.

Metabolomics study in MCF-7 cells treated with BoA2C
Mass spectrometry data of metabolites in MCF-7 cells was used for metabolomics investigation after co-cultivation with BoA2C (2 μM) for 72 h. The effectiveness of the OPLS-DA model to distinguish between the BoA2C and Control groups was assessed using the success criterion of R 2 ≥ 0.7 and Q 2 ≥ 0.4 (Martin-Blazquez et al., 2019). The current model has an R2 of 0.994 and a Q2 of 0.982, indicating that it is capable of distinguishing between BoA2C and Control samples in Figure 7A. VIP-plots were investigated using the OPLS-DA models to examine important factors that led to the differentiation of the metabolome between the BoA2C and Control groups. Variables having a VIP value greater than 1 were deemed significant contenders. Matching retention time and MS fragmentation patterns from the MetaboAnalyst (version 5.0) database verified the presence of distinct metabolites (Pang et al., 2021). These metabolites were further categorized based on their characteristics, including amino acids, amines, aliphatic acids and bile acids. All differential metabolites were loaded into MetaboAnalyst (version 5.0) for metabolic analysis to further explain the fundamental mechanisms of BoA2C's cytotoxic impact on breast carcinoma cells. In accordance with the described guidelines, the Homo sapiens pathway library and Fisher's exact test were used for route enrichment analysis, while relative-betweeness centrality was used for pathway topology analysis (Pang et al., 2021). The impacted metabolic pathways were defined as those with a pathway effect value more than 0.10 and a p value less than 0.05. According to the p and pathway impact values, 15 metabolic pathways were observed in BoA2C and Control groups ( Figure 7B). Six of them ( Figure 7B, Table 3) were primarily involved: 1) arginine and proline metabolism 2) linoleate metabolism 3) bile acid biosynthesis 4) de novo fatty acid biosynthesis 5) fatty acid activation, and (VI) fatty Acid Metabolism.
Arginine is a type of multi-functional amino acid involved in the synthesis of many metabolites, such as nitric oxide, urea, ornithine, and citrulline, and is involved in protein modification and immunoregulation (Morris, 2007;Rath et al., 2014;Szefel et al., 2019). ASS1, one of the key enzymes involved in arginine metabolism, is highly expressed in normal tissues but heterogeneously expressed in tumors (Dillon et al., 2004). These researchers found that ASS1 was completely deficient in HCC and prostate cancer and partially deficient in breast cancer (Dillon et al., 2004;Morettin et al., 2015). Several recent investigations have shown that arginine deprivation caused tumor cell death via degrading extracellular arginine (Alexandrou et al., 2018;Nasreddine et al., 2020;Chu et al., 2021). This suggested that arginine shortage might occur in MCF-7 cells treated with BoA2C based on the noticeably changed in arginine metabolism. Bile acid composition is controlled via the modulation of bile acid-metabolizing enzymes and bile acid transporters, the majority of which are expressed by farnesoid X receptor (FXR) target genes (Sun et al., 2021). FXR regulates the expression of target genes involved in both bile acid production Frontiers in Chemistry frontiersin.org and fatty acid biosynthesis, which is required for the maintenance of whole-body bile acid homeostasis (Matsubara et al., 2013;Chiang and Ferrell, 2020). Furthermore, FXR stimulation weakened cell proliferation and migration under steroid-free media conditions in MCF-7 and T47D breast cancer cells, and other investigators observed that FXR agonists caused apoptosis and inhibited local estrogen synthesis (Alasmael et al., Giordano et al., 2016;Barone et al., 2018). As shown in Figure 8A, the scratch wounds and migratory cells in the wound were imaged at 0, 24, and 48 h after scratched in this study. The results demonstrated that the treatment of BoA2C obviously reduced the migration of MCF-7 cells in the scratch compared to control group, thus potentially inhibited tumor invasion with enhanced FXR expression. Then, a potent FXR antagonist (DY268) was employed (Yu et al., 2014;Hankir et al., 2020) in a cell proliferation investigation to better evaluate the regulatory link between FXR expression and BoA2C treatment. The results ( Figure 8B) unequivocally demonstrated that DY268 reversed BoA2C inhibitory impact on the growth of MCF-7 breast cancer cells in both 24, 48 and 72 h. Thus, BOA2C could disrupt bile acid and fatty acid metabolism and limit tumor development and invasion probably by activating FXR expression.

Conclusion
In this study, 26 new betulonic acid-diazines were designed and synthesized from BoA and all of them were characterized by 1 H NMR, 13 C NMR, HRMS. Furthermore, all these compounds were tested for cytotoxic activity against HepG2, Bel-7402, Hela, MCF-7 and MDCK cell lines by standard MTT assay. After exploring the structure-activity relationship, we found that the introduction of different groups at C-28 can selectively inhibit different kinds of cancer cells. The introduction of a pyridazine structure at the C-28 of BoA was possessed a stronger cytotoxic activity against Hela cell. It is interesting that the introduction of a 2, 5-dimethylpyrazine structure might be more effective cytotoxicity against Bel-7402 and MCF-7 cells and lower cytotoxicity on MDCK than DDP. From the obtained results, compounds BoA2C exerted potent and selective in vitro antiproliferative activity. As shown in metabolomics results, it was found that BoA2C significantly induced cell injury in MCF-7 cells mostly through the interference of arginine metabolism and fatty acid metabolism. It's presently undertaking intense adjustments at C-28 and investigations in the mechanism of BoA2C's antiproliferative impact in our further studies, and the results will be revealed in due course.

FIGURE.8
The effect of BoA2C on the invasive ability of MCF-7 cells and the role of FXR in the anticancer process. (A) Wound healing assays were performed in 0, 24, 48 h; (B) Changes of antiproliferative activity under DY268. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells.
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