Synthesis of ring-A modified lupeol derivatives and their anti-proliferative activity: identification of potent lead active against MCF-7 breast cancer cells

Abstract In continuation of our research program aimed at the development of new natural product-based anticancer agents, a series of lupeol derivatives (5a-5k and 6a-6i) were prepared with the introduction of aryl functionalities and amino acids at C-3 position. All the synthesised derivatives were assessed for in vitro anticancer activity against four human cancer cell lines using MTT assay. Interestingly, the compounds 5j, 5k, and 6 g showed potent activity against MCF7 cells as compared with the parent compound. Further, the flowcytometry analysis revealed that the 5j,5k, and 6 g arrest the cells at the G2/M phase and induce the early apoptosis in MCF7 cells. In addition, the selected compounds inhibit the BcL2 expression and increase the Bax protein expression in MCF7 cells. Overall, these results indicated that the lupeol derivatives could serve as a promising launch point for the development of anticancer agents. Graphical Abstract


Introduction
Natural products have proven to be a rich source of inspiration for anticancer drugs as three-quarters of the anticancer drugs currently in the global market are either derived from natural products or natural product-related compounds (Newman and Cragg 2020).Triterpenoids constitute a large number of naturally occurring substances with diverse structures endowed with various biological activities (Bacho r ık and Urban 2021).Lupeol (1) is a natural pentacyclic triterpenoid and has been found widely in many traditional medicinal plants (Xu et al. 2018).Lupeol and its derivatives have been reported to exhibit a wide range of biological activities such as antifungal (Javed et al. 2021), antiviral (Xu et al. 2020), anticancer (Min et al. 2019), anti-inflammatory (Ahmad et al. 2010), anti-osteoporosis (Nguyen et al. 2021) and anti-neurodegenerative activities (Oliveira-Junior et al. 2019).In view of its high abundance from plants, nontoxic nature and profound biological activities, lupeol has attracted medicinal chemists for chemical modification studies.Structurally, this lead compound possesses various functional sites such as hydroxyl group at C-3 and exo-double bond (Saleem 2009), which are amenable for chemical transformations.Consequently, several lupeol analogues have been reported in the literature by modification of these functionalities (Machado et al. 2018;Saini et al. 2019;Liu et al. 2021).However, ring-A of lupeol is still the attractive site to perform chemical modifications to generate diverse hybrid structures as ring-A modified analogue have been reported to be much more potent than the other analogues.The 3b-hydroxyl functionality, through its oxidation product, is most amenable to form oximes, which plays an important role in the functional group conversions and also as intermediates for synthesis of many hybrid structures are expected to exhibit potent biological activities.With this background and in continuation to our research program focused on the development of new natural productbased anticancer agents (Amujuri et al. 2021;Dileep Kumar et al. 2022;Gaja et al. 2022), we have synthesised derivatives using Sonogashira coupling reaction and also various amino acids are also attached in C-3 position of Ring-A of lupeol.The prepared derivatives were assessed for anticancer activity against panel of cancer cells and the flowcytometry analysis.In this communication, we report synthesis, anticancer activity and mode of action of the synthesised compounds.

Chemistry
As part of our phytochemical-pharmacological integrated studies of Indian flora, lupeol (1) was isolated as a major bioactive compound from Strychnos wallachina.In the drug discovery process, incorporation of an active pharmacophoric moiety into core bioactive natural product is a key step to obtain a wide range of New Chemical Entities (NCEs) with enhanced biological activities.With this objective in mind and in continuation of our research interest, we have designed analogues based on intuitive modification of lupeol template.As shown in Scheme 1, lupeol (1) was subjected to oxidation reaction using Pyridiniumchlorochromate (PCC) in DCM to furnish the corresponding ketone.Thus, to verify the influence of the carbonyl functionality, we converted the carbonyl group into oximes through the reaction of hydroxyl amine hydrochloride and CH 3 COONa in ethanol at room temperature to afford corresponding oxime (3).This ketoxime (3) was further transformed to 4 through standard alkylation procedures using propargyl bromide and NaH in dry THF.The resultantintermediate4 is treated with different aryl iodides in the presence of TEA, copper iodide and palladium catalyst under Sonogashira cross-coupling reaction conditions to afford the final desired products (5a-5k) in good yields (60-80%).Previously it has been reported that the palladium complex stimulated the cross-coupling carbon-carbon bond of a wide range of aryl iodides with the terminal alkyne-containing compounds in good to excellent yields (Su et al. 2019).
Next, we investigated the synthesis of oxime esters by the condensation of amino acids and the resultant derivatives are expected to have unique properties like Scheme 1. Reagents and conditions: (i) PCC, Dry DCM, 0 C, rt, 1 h, 80% (ii) NH 2 OH.HCl, EtOH, CH 3 COONa, 0 C, rt, overnight, 85% (iii) Propargyl Bromide, THF, NaH, 0 C, rt, 2 h,70% (iv) R-I, Et 3 N, CuI,(Pd(PPh 3 ) 3 Cl), 12-24 h, 60-80%.chirality, hydrophilicity/hydrophobicity, enhanced bioavailability and optical properties .As outlined in Scheme 2, different amino acids were reacted with ketoxime3 in the presence of DCC and DMAP in DCM to afford the target compounds (6a-6i).All the reactions proceeded smoothly to give the desired products in excellent yields.The prepared lupeol derivatives were purified using silica gel column chromatography and characterised using NMR and HRMS analysis.

Cytotoxic activity
The invitro anti-proliferative activity of lupeol (1) and its derivatives (5a-5k and 6a-6i) were determined according to MTT assay against a panel of cancer cell lines (hypopharyngeal carcinoma (FADU) cells, human tongue cancer (CAL-27) cells, human lung adenocarcinoma (A549) cells and human breast cancer (MCF7)) and normal cell (HEK-293).Doxorubicin was used as reference standard and results are summarised in Table S1.The results indicated that the majority of synthesised derivatives displayed higher anticancer activity than parent compound, lupeol (1) against the tested cell lines.Interestingly, the compounds 3, 4, 5j, 5k showed potent cytotoxic activity against MCF7 cells and none of the compounds enhanced the activity against A549 cells in comparison with lupeol (1) (Table S1).It is worth mentioning that the addition of various aliphatic and aromatic amino acids with lupeol did not enhanced activity against FADU, CAL27 and A549 cells, however, the addition of lysine with lupeol (6 g) showed enhanced activity against FADU cells, A549 cells and MCF7 cells than the parent molecule (Table S1).Hence, the compounds 5j, 5k and 6 g were selected for further investigation to assess the molecular mechanism underlying cell cycle arrest and apoptosis.

Cell cycle arrest
Cancer cell growth inhibitors are suggested to trigger a modification in cell cycle distribution, preferably either in G0/G1 or G2/M phase (Ji et al. 2022).Herein, the effects of lupeol derivatives exert their cytotoxic action by inhibiting the cancer cell proliferation at certain checkpoints that are diverse phases in the cell cycle.In the present study, the most active compounds in cytotoxic assays 5j, 5k and 6gwas further treated for cell cycle analysis to study the cell cycle phase where the arrest takes place in MCF7 cells.As shown in Figure S1, the selected compounds 5j (21.3%), 5k (21.1%) and 6 g (21.6%) arrested the cell cycle at G2/M phase compared with control (19.9%)MCF7 cells.The results evidently show that the lupeol derivatives inhibited the cellular proliferation of breast cancer cells by arresting the cell cycle at G2/M phase.

Apoptosis studies
Apoptosis is an important physiological function to indicate the cell death via various signaling pathway in response to the anticancer drugs which induce apoptosis in cancer cells (Ramalingam et al. 2022).In the present study, the different concentration of lupeol derivatives were assessed for their potential in inducing apoptosis using Annexin V-FITC staining assay.Annexin V-FITC is a dual staining assay used to detect and calculate the quantity of apoptotic cells using flow cytometry analysis (Chung et al. 2022).Staining the cells with PI and Annexin-FITC discloses the variation between the live, apoptotic, dead, and late apoptotic or necrotic cells, thus offers the confirmation on the signaling pathway mediated cell death (Hu et al. 2021).As shown in Figure S2, the treatment of MCF7 cells with the concentration of IC 50 the percentage of apoptotic cells was calculated as 10.7%, 13.3%, and 15.2% of early apoptosis was observed and increasing the concentration it was calculated as 15.2% 16%, and 17.8% of early apoptosis was observed for the compounds 5j, 5k and 6 g, respectively.Together, it clearly indicated that the lupeol derivatives induce early apoptosis against breast cancer compared with control cells.

Molecular mechanism of action
The underlying mechanism of action of inducing the apoptosis in MCF7 cells by lupeol derivatives was determined using qRT-PCR and Western blotting analysis.Apoptosis is a programmed cell death characterised by oxidative stress mediated DNA damage and the BcL2 family proteins are playing key role in the regulation of apoptosis in cancer cells (Varunkumar et al. 2020).The upregulation of BcL2 is resulting into the increase the resistance of cancers against various chemotherapeutic drugs and radiation therapy which are known for inducing the apoptosis in tumor cells (Garc ıa-Aranda et al. 2018).In the present study, the compounds 5j, 5k and 6gdown regulate the expression of BcL2 gene and protein in MCF7 cells is indicating the compounds induce apoptosis by targeting the BcL2 proteins (Figures S3 and S4).Along with Bax and BcL2 expression analysis, we have also performed the Bax/BcL2 ratio and analysis showed that the cells treated with 5k compound significantly increased the Bax/BcL2 ratio indicating that, 5k compound induced the apoptosis.Bax is one of the BcL2 family proteins and proapoptotic factors which are involved in the intrinsic apoptosis signaling pathway upon response to the oxidative (Ramalingam and Rajaram 2021).Here the compounds 5j, 5k and 6 g increase the expression of Bax gene and protein in MCF7 cells as compared with control cells.The significant regulation of Bax and BcL2 by lupeol derivatives leads to the apoptosis through the intrinsic signaling pathway, i.e.The activation of Bax leads to the release of Cytochrome C into the cytosol where it forms apoptosome and regulates the caspase cascades (caspase 9 and 3) for apoptosis (Muthulakshmi et al. 2022).Together, the results indicate that the selected lupeol derivatives showed excellent anticancer activity against breast cancer cells by targeting the BcL2/Bax expression.

Conclusion
In conclusion, the series of lupeol derivatives were synthesised by attaching various aryl halides using Sonogashira reaction and various amino acids at C-3 position.As synthesised lupeol derivatives showed good to moderate cytotoxic activity against panel of cancer cell lines and the compounds 5j, 5k and 6gshowed excellent activity against MCF7 breast cancer cells.These compounds induce the early apoptosis in MCF7 cells by arresting the cell cycle at G2/M phase confirmed with flowcytometry analysis.Together, the results revealed that the lupeol derivatives can act as an excellent therapeutic agent against breast cancer.