Lupeol-3-carbamate Derivatives: Synthesis and Biological Evaluation as Potential Antitumor Agents

In the following study, a series of new lupeol-3-carbamate derivatives were synthesized, and the structures of all the newly derived compounds were characterized. The new compounds were screened to determine their anti-proliferative activity against human lung cancer cell line A549, human liver cancer cell line HepG2, and human breast cancer cell line MCF-7. Most of the compounds were found to show better anti-proliferative activity in vitro than lupeol. Among them, obvious anti-proliferation activity (IC50 = 5.39~9.43 μM) was exhibited by compound 3i against all three tumor cell lines. In addition, a salt reaction was performed on compound 3k (IC50 = 13.98 μM) and it was observed that the anti-proliferative activity and water solubility of compound 3k·CH3I (IC50 = 3.13 μM), were significantly enhanced subsequent to the salt formation process. The preliminary mechanistic studies demonstrated that apoptosis in HepG2 cells was induced by compound 3k·CH3I through the inhibition of the PI3K/AKT/mTOR pathway. In conclusion, a series of new lupeol-3-carbamate derivatives were synthesized via the structural modification of the C-3 site of lupeol, thus laying a theoretical foundation for the design of this new anticancer drug.


Introduction
Cancer is recognized as one of the most lethal diseases affecting humans, with its annual incidence and mortality rates continuing to be high [1].At present, a variety of anti-tumor drugs are being developed and applied in clinical practice.Despite clinical use, many anti-tumor drugs are known to have serious side effects, such as those caused by traditional platinum-based anticancer drugs [2,3].Their broader use is limited due to toxicity and potential drug resistance.Consequently, it is imperative to further develop more effective and low-toxicity anticancer drugs [4].
Natural products have been utilized in medicine for an extensive historical period and continue to be a principal source from which new drugs are derived.Approximately 65% of all new anti-tumor drugs used in clinical practice are natural or nature-based products.Therefore, the search for natural products with novel structures and significant efficacy is one of the most important research directions at present [5][6][7][8].Pentacyclic triterpenes comprise a wide range of natural products that can be extracted from different fungi, plants, and marine invertebrates.Ursolic acid (UA), glycyrrhizic acid (GA), and oleanolic acid (OA), among other pentacyclic triterpenoids, have garnered substantial interest for their diverse pharmacological profiles.Specifically, potent anticancer, anti-inflammatory, antioxidant, virucidal, and bactericidal properties have been demonstrated by these compounds [9][10][11].
Lupeol (Lup, 1, Figure 1), a common natural triterpenoid compound, is derived from medicinal plants, as well as edible fruits and vegetables [12].Recent studies have demonstrated that lupeol possesses anti-inflammatory, anti-tumor, anti-oxidative, and demonstrated that lupeol possesses anti-inflammatory, anti-tumor, anti-oxidati wound healing-promoting properties, with therapeutic effects being exerted on tions such as arthritis, diabetes, heart disease, nephrotoxic injury, and liver toxi [13].With respect to its anti-neoplastic properties, lupeol has been consistently by a wealth of research to exert inhibitory effects on a range of malignant neo including hepatocellular carcinoma, lung carcinoma, colorectal cancer, prostate osteosarcoma, and melanoma.The PI3K/AKT/mTOR pathway has been reported in previous studies to pla otal role in the regulation of the cell cycle, autophagy, apoptosis, and other cellu cesses across a spectrum of different cell types [14].The inhibitory effect of silenc ST3GalⅢ gene on the proliferation, invasion, and metastasis of human breast can has been enhanced by lupeol through the suppression of the activation PI3K/AKT/mTOR signaling pathway, as reported in recent studies [15].Lupeol c induce and enhance the autophagy of cervical cancer cells by inhibiting the phosp tion of PI3K/AKT/mTOR pathway proteins, reducing their proliferation and inva tivities, and promoting their apoptosis [16].In addition, the results of other studi shown that lupeol can inhibit the proliferation of retinoblastoma and promote aut and apoptosis, which may also be related to the PI3K/AKT/mTOR pathway [17].
Carbamate is an organic compound whose amino and carboxyl terminuses placed by a variety of alkyl, aryl, or alkyl aryl substituents with different structure appearing in the form of -O-CO-NH-linkage, such as docetaxel, mitomycin C, irin etc. (Figure 2) [18,19].In recent years, significant interest has been garnered by car derivatives, highlighting their pivotal role in the realm of contemporary drug di and medicinal chemistry.The results of recent studies have shown that compoun taining a carbamate group in their molecules can enhance the biological activit active pharmacophore of structurally different natural or synthetic compounds [2 nificant anti-proliferative activities against the human astrocytoma T67 and H lines, with IC50 values ranging from 0.153 to 0.828 µM, were observed for the p derivatives 8 and 11, which incorporate carbamate group, in studies conducted dolfi F et al. [21].The activity of butyrylcholinesterase (BuChE) was found to be m tently inhibited by the phenylpiperazine carbamate derivative 16, as reported by B et al., with an IC50 ranging from 1.04 to 3.83 µM [22].The PI3K/AKT/mTOR pathway has been reported in previous studies to play a pivotal role in the regulation of the cell cycle, autophagy, apoptosis, and other cellular processes across a spectrum of different cell types [14].The inhibitory effect of silencing the ST3GalIII gene on the proliferation, invasion, and metastasis of human breast cancer cells has been enhanced by lupeol through the suppression of the activation of the PI3K/AKT/mTOR signaling pathway, as reported in recent studies [15].Lupeol can also induce and enhance the autophagy of cervical cancer cells by inhibiting the phosphorylation of PI3K/AKT/mTOR pathway proteins, reducing their proliferation and invasion activities, and promoting their apoptosis [16].In addition, the results of other studies have shown that lupeol can inhibit the proliferation of retinoblastoma and promote autophagy and apoptosis, which may also be related to the PI3K/AKT/mTOR pathway [17].
Carbamate is an organic compound whose amino and carboxyl terminuses are replaced by a variety of alkyl, aryl, or alkyl aryl substituents with different structures, often appearing in the form of -O-CO-NH-linkage, such as docetaxel, mitomycin C, irinotecan, etc. (Figure 2) [18,19].In recent years, significant interest has been garnered by carbamate derivatives, highlighting their pivotal role in the realm of contemporary drug discovery and medicinal chemistry.The results of recent studies have shown that compounds containing a carbamate group in their molecules can enhance the biological activity of the active pharmacophore of structurally different natural or synthetic compounds [20].Significant anti-proliferative activities against the human astrocytoma T67 and HeLa cell lines, with IC 50 values ranging from 0.153 to 0.828 µM, were observed for the pyridine derivatives 8 and 11, which incorporate carbamate group, in studies conducted by Pandolfi F et al. [21].The activity of butyrylcholinesterase (BuChE) was found to be most potently inhibited by the phenylpiperazine carbamate derivative 16, as reported by Bajda M et al., with an IC 50 ranging from 1.04 to 3.83 µM [22].
In the past decade, the synthesis of a multitude of carbamate-based compounds has been pursued with vigor, with these compounds emerging as promising candidates in the realm of oncology, either as therapeutic drugs or prodrugs [23].Certain carbamates have been demonstrated to effectively suppress the proliferation of endothelial cells in vitro and exert a significant inhibitory effect on tumor-induced angiogenesis and tumor growth in murine models in vivo [24].The results of previous studies show that carbamate derivatives have a good inhibitory effect on tumor cells and show good water solubility [25,26].Based on the outcomes of the aforementioned studies, a series of lup-3-carbamate derivatives were designed utilizing the biological isostructural substitution method.The biological activities (IC 50 value) of all derivatives against three human cancer cell lines were evaluated using the MTT method to provide an important reference for the development of new anti-tumor drugs.In the past decade, the synthesis of a multitude of carbamate-based compou been pursued with vigor, with these compounds emerging as promising candidate realm of oncology, either as therapeutic drugs or prodrugs [23].Certain carbama been demonstrated to effectively suppress the proliferation of endothelial cells and exert a significant inhibitory effect on tumor-induced angiogenesis and tumor in murine models in vivo [24].The results of previous studies show that carbamat atives have a good inhibitory effect on tumor cells and show good water solubility Based on the outcomes of the aforementioned studies, a series of lup-3-carbamate tives were designed utilizing the biological isostructural substitution method.The ical activities (IC50 value) of all derivatives against three human cancer cell lines we uated using the MTT method to provide an important reference for the develop new anti-tumor drugs.
However, it should be noted that lupeol, characterized as a fat-soluble com with inherently low water solubility, has attributes that may restrict the investig its anti-proliferative activity [27][28][29].Therefore, increasing water solubility and en activity may be important strategies for the structural modification of this compo address this issue, in our previous study we found that attaching a structurally polar substituent at the C-3 position of 1 can increase its anti-proliferative activit also increasing solubility.N-heterocyclic rings were found in a variety of bioacti pounds and readily form water-soluble salts with organic or inorganic acids [30]. of the above, N-heterocyclic rings at the C-3 position of 1 (Figure 3) were introd improve the water solubility and biological activity of the compound.By stru modifying C-3, potentially important derivatives can be derived, thus promoting ther development as an anti-tumor agent.However, it should be noted that lupeol, characterized as a fat-soluble compound with inherently low water solubility, has attributes that may restrict the investigation of its anti-proliferative activity [27][28][29].Therefore, increasing water solubility and enhancing activity may be important strategies for the structural modification of this compound.To address this issue, in our previous study we found that attaching a structurally simple polar substituent at the C-3 position of 1 can increase its anti-proliferative activity while also increasing solubility.N-heterocyclic rings were found in a variety of bioactive compounds and readily form water-soluble salts with organic or inorganic acids [30].In light of the above, N-heterocyclic rings at the C-3 position of 1 (Figure 3) were introduced to improve the water solubility and biological activity of the compound.By structurally modifying C-3, potentially important derivatives can be derived, thus promoting its further development as an anti-tumor agent.In the past decade, the synthesis of a multitude of carbamate-based compounds has been pursued with vigor, with these compounds emerging as promising candidates in the realm of oncology, either as therapeutic drugs or prodrugs [23].Certain carbamates have been demonstrated to effectively suppress the proliferation of endothelial cells in vitro and exert a significant inhibitory effect on tumor-induced angiogenesis and tumor growth in murine models in vivo [24].The results of previous studies show that carbamate derivatives have a good inhibitory effect on tumor cells and show good water solubility [25,26].Based on the outcomes of the aforementioned studies, a series of lup-3-carbamate derivatives were designed utilizing the biological isostructural substitution method.The biological activities (IC50 value) of all derivatives against three human cancer cell lines were evaluated using the MTT method to provide an important reference for the development of new anti-tumor drugs.
However, it should be noted that lupeol, characterized as a fat-soluble compound with inherently low water solubility, has attributes that may restrict the investigation of its anti-proliferative activity [27][28][29].Therefore, increasing water solubility and enhancing activity may be important strategies for the structural modification of this compound.To address this issue, in our previous study we found that attaching a structurally simple polar substituent at the C-3 position of 1 can increase its anti-proliferative activity while also increasing solubility.N-heterocyclic rings were found in a variety of bioactive compounds and readily form water-soluble salts with organic or inorganic acids [30].In light of the above, N-heterocyclic rings at the C-3 position of 1 (Figure 3) were introduced to improve the water solubility and biological activity of the compound.By structurally modifying C-3, potentially important derivatives can be derived, thus promoting its further development as an anti-tumor agent.

Synthesis of Lupeol Derivatives
In the present study, a two-step reaction method was designed for the synthesis of lupeol-3-carbamate derivatives (3a-k), as shown in Scheme 1.In the first step, lupeol (1) was selected as the starting material and was reacted with 4-nitrophenyl chloroformate (PNPCF) in dichloromethane (CH 2 Cl 2 ) in the presence of pyridine (Py) as a catalyst, leading to the efficient synthesis of the key intermediate 2. Subsequently, in the presence of triethylamine (Et 3 N), intermediate 2 was reacted with a series of different amines, and through optimized reaction conditions, we successfully synthesized the target products 3a-k.Based on Scheme 1, a salt-forming reaction was further designed, employing compound 3k as the substrate.The reaction with iodomethane (CH 3 I) in acetonitrile (CH 3 CN), conducted under precisely controlled conditions, facilitated the successful synthesis of the target product 3k•CH 3 I.This synthetic route is detailed in Scheme 2. Unless otherwise specified, all reagents were commercially available and were directly employed in the experiments without further purification.The progress of the reactions was monitored using thin-layer chromatography (TLC) techniques, with observation under ultraviolet (UV) light at 254 nm or staining performed using a 5% sulfuric acid (H 2 SO 4 )/ethanol solution (EtOH) to ensure the smooth progression of the reactions.The chemical structures of all the synthesized compounds were confirmed using analytical techniques such as 1 H NMR, 13 C NMR, and HRMS.The experimental section meticulously recorded the physical properties and analytical data of all the synthesized compounds.

Synthesis of Lupeol Derivatives
In the present study, a two-step reaction method was designed for the synthesis of lupeol-3-carbamate derivatives (3a-k), as shown in Scheme 1.In the first step, lupeol (1) was selected as the starting material and was reacted with 4-nitrophenyl chloroformate (PNPCF) in dichloromethane (CH2Cl2) in the presence of pyridine (Py) as a catalyst, leading to the efficient synthesis of the key intermediate 2. Subsequently, in the presence of triethylamine (Et3N), intermediate 2 was reacted with a series of different amines, and through optimized reaction conditions, we successfully synthesized the target products 3a-k.Based on Scheme 1, a salt-forming reaction was further designed, employing compound 3k as the substrate.The reaction with iodomethane (CH3I) in acetonitrile (CH3CN), conducted under precisely controlled conditions, facilitated the successful synthesis of the target product 3k•CH3I.This synthetic route is detailed in Scheme 2. Unless otherwise specified, all reagents were commercially available and were directly employed in the experiments without further purification.The progress of the reactions was monitored using thin-layer chromatography (TLC) techniques, with observation under ultraviolet (UV) light at 254 nm or staining performed using a 5% sulfuric acid (H2SO4)/ethanol solution (EtOH) to ensure the smooth progression of the reactions.The chemical structures of all the synthesized compounds were confirmed using analytical techniques such as 1 H NMR, 13 C NMR, and HRMS.The experimental section meticulously recorded the physical properties and analytical data of all the synthesized compounds.

Lupeol Derivatives Inhibited the Proliferation of Various Human Cancer Cell Lines
The anti-proliferative activities of 3a-k against human lung cancer cell line (A549), human hepatoma cell line (HepG2), and human breast cancer cell line (MCF-7) were examined.With lupeol and cisplatin as positive controls, the MTT assay was used to evaluate the anti-proliferative activity of all of the synthesized compounds in vitro.
As shown in Table 1, most of the carbamate derivatives (3a-k) showed strong inhibitory effects on all three tumor cell lines, and their anti-proliferative activity was higher than that of lupeol.The preliminary structure-activity relationship (SAR) analysis indicated that the presence of nitrogen-containing carbocycle, specifically piperazine, piperidine, and pyridine moieties, significantly enhanced the anti-proliferative activity, as observed with compounds 3e, 3h, 3i, and 3k, which displayed IC50 values less than 20 µM.Notably, the introduction of a methyl group at the para position of the piperazine group (3i) resulted in the most pronounced anti-proliferative activity, with an IC50 ranging from 5.74 to 9.43 µM.In contrast, compounds substituted with N-methyl, N-ethyl, and fivemembered heterocyclic groups (3a, 3b, 3d) showed relatively lower anti-proliferative activities.

Anti-Proliferative Activity of Selected Compounds in Tumor Cells
Due to the unique n-heterocyclic structure of piperazine and pyridinyl, compound 3k was further reacted with salt, using lupeol as a positive control, and compounds 3c, 3e,

Biological Evaluation of Lupeol Derivatives 2.2.1. Lupeol Derivatives Inhibited the Proliferation of Various Human Cancer Cell Lines
The anti-proliferative activities of 3a-k against human lung cancer cell line (A549), human hepatoma cell line (HepG2), and human breast cancer cell line (MCF-7) were examined.With lupeol and cisplatin as positive controls, the MTT assay was used to evaluate the anti-proliferative activity of all of the synthesized compounds in vitro.
As shown in Table 1, most of the carbamate derivatives (3a-k) showed strong inhibitory effects on all three tumor cell lines, and their anti-proliferative activity was higher than that of lupeol.The preliminary structure-activity relationship (SAR) analysis indicated that the presence of nitrogen-containing carbocycle, specifically piperazine, piperidine, and pyridine moieties, significantly enhanced the anti-proliferative activity, as observed with compounds 3e, 3h, 3i, and 3k, which displayed IC 50 values less than 20 µM.Notably, the introduction of a methyl group at the para position of the piperazine group (3i) resulted in the most pronounced anti-proliferative activity, with an IC 50 ranging from 5.74 to 9.43 µM.In contrast, compounds substituted with N-methyl, N-ethyl, and five-membered heterocyclic groups (3a, 3b, 3d) showed relatively lower anti-proliferative activities.a IC 50 was calculated from three independent experimental measurements using an MTT assay after 48 h of treatment.

Anti-Proliferative Activity of Selected Compounds in Tumor Cells
Due to the unique n-heterocyclic structure of piperazine and pyridinyl, compound 3k was further reacted with salt, using lupeol as a positive control, and compounds 3c, 3e, 3k, and 3k•CH 3 I were further tested to determine their anti-tumor activity, as shown in Figure 4. Significant inhibitory effects were observed for the four synthesized compounds on the three tested cell lines, with their anti-proliferative activity being more potent than that of lupeol.Compound 3k•CH 3 I showed the strongest inhibitory effect on the HepG2 cell line (IC 50 = 3.13 ± 0.54 µM), with it being about 10 times more active than lupeol.The inhibition of these four compounds on the HepG2 cell line appears to have obvious anti-proliferative activity compared with the MCF-7 and A549 cell lines.The results of our study clearly demonstrate that the anti-proliferative activity of compound 3k is enhanced subsequent to the salting process.This enhancement suggests that the methyl group is an electron-donating group, leading to an increased electron cloud density on the nitrogen atom after salting.Such an increase in electron density may confer the property of targeting mitochondria, thereby indicating the compound's potential to be developed as an anticancer agent.
3k, and 3k•CH3I were further tested to determine their anti-tumor activity, as shown in Figure 4. Significant inhibitory effects were observed for the four synthesized compounds on the three tested cell lines, with their anti-proliferative activity being more potent than that of lupeol.Compound 3k•CH3I showed the strongest inhibitory effect on the HepG2 cell line (IC50 = 3.13 ± 0.54 µM), with it being about 10 times more active than lupeol.The inhibition of these four compounds on the HepG2 cell line appears to have obvious antiproliferative activity compared with the MCF-7 and A549 cell lines.The results of our study clearly demonstrate that the anti-proliferative activity of compound 3k is enhanced subsequent to the salting process.This enhancement suggests that the methyl group is an electron-donating group, leading to an increased electron cloud density on the nitrogen atom after salting.Such an increase in electron density may confer the property of targeting mitochondria, thereby indicating the compound's potential to be developed as an anticancer agent.

Water Solubility Testing of Compound 3k•CH3I
The water solubility of compounds 1 and 3k•CH3I was determined, as shown in Figure 5.The concentration gradients of 3k•CH3I were 0.01, 0.03, 0.05, 0.12, 0.20, and 0.35 mg/mL.Compounds 1 and 3k•CH3I were dissolved in 2 mL of distilled water, and distilled water was used as a blank control.The absorbance characteristics of compounds 1 and 3k•CH3I were ascertained utilizing a UV-VIS spectrophotometer at a wavelength of 234 nm.The results showed that compound 3k•CH3I is 60 times more soluble than compound 1, thus indicating that the water solubility of compound 3k•CH3I significantly improved following salt modification by compounds 1 and 3k.

Effect of Compound 3k•CH3I on the Apoptosis Rate of HepG2 Cells
The apoptotic morphological alterations induced by compound 3k•CH3I in HepG2 cells were scrutinized after a 48-h treatment using the acridine orange/ethidium bromide (AO/EB) double staining method, as depicted in Figure 6.Normal green fluorescence and no morphological alterations were observed in the control group following AO/EB staining.HepG2 tumor cells treated with compound 3k•CH3I showed bright red fluorescence

Effect of Compound 3k•CH 3 I on the Apoptosis Rate of HepG2 Cells
The apoptotic morphological alterations induced by compound 3k•CH 3 I in HepG2 cells were scrutinized after a 48-h treatment using the acridine orange/ethidium bromide (AO/EB) double staining method, as depicted in Figure 6

Effect of Compound 3k•CH3I on Reactive Oxygen Species (ROS) in HepG2 Cells
As shown in Figure 7, with the increase in drug concentration, the ROS fluorescence in HepG2 cells treated with compound 3k•CH3I was also gradually enhanced.Flow cytometry was additionally used to determine the effect of compound 3k•CH3I on ROS levels in HepG2 cells.After 48 h of drug administration, ROS levels in HepG2 cells treated with compound 3k•CH3I (0, 2, 4, and 8 µmol/L) increased significantly compared with those of the control group.With the increase in drug concentration, these levels increased significantly in a dose-dependent manner.The results of our experiment showed that Additionally, annexin V/propidium iodide (AV/PI) staining was used to determine the apoptotic rate of HepG2 cells treated with different concentrations of 3k•CH 3 I via flow cytometry.Following treatment with 0, 2, 4, and 8 µmol/L of compound 3k•CH 3 I for 48 h, compared with the control group, the proportion of early and late apoptotic cells increased significantly, and the apoptotic rate of HepG2 cells increased with the increase in drug concentration.At concentrations of 4 µmol/L and 8 µmol/L, the total percentage of apoptotic and necrotic cells increased from 3.03% to 45.10% and 55.6% in the normal cell population.The above data indicated that the proliferation of HepG2 cells was inhibited by compound 3k•CH 3 I through the induction of apoptosis.

Effect of Compound 3k•CH 3 I on Reactive Oxygen Species (ROS) in HepG2 Cells
As shown in Figure 7, with the increase in drug concentration, the ROS fluorescence in HepG2 cells treated with compound 3k•CH 3 I was also gradually enhanced.Flow cytometry was additionally used to determine the effect of compound 3k•CH 3 I on ROS levels in HepG2 cells.After 48 h of drug administration, ROS levels in HepG2 cells treated with compound 3k•CH 3 I (0, 2, 4, and 8 µmol/L) increased significantly compared with those of the control group.With the increase in drug concentration, these levels increased significantly in a dose-dependent manner.The results of our experiment showed that compound 3k•CH 3 I significantly increased the production of ROS, which may also be responsible for its induction of apoptosis.

Effect of Compound 3k•CH3I on the Mitochondrial Membrane Potential of HepG2 Cells
In order to explore the effect of compound 3k•CH3I on MMP, a JC-1 staining kit was used to detect the depolarization of the MMP of human hepatocellular carcinoma HepG2 cells treated with compound 3k•CH3I, as shown in Figure 8.Compared with the control group, after HepG2 cells were treated with different concentrations of compound 3k•CH3I for 48 h, the red fluorescence gradually weakened until it eventually disappeared, and the green fluorescence gradually strengthened in HepG2 cells.
Flow cytometry was used to detect the intimal potential of HepG2 cells.It was found that compound 3k•CH3I induced a dose-dependent increase in the MMP depolarization of HepG2 cells at concentrations of 0, 2, 4, and 8 µmol/L.It was found to increase from 7.74% to 17.2%, 27.3%, and 48.9%, respectively.It was concluded that the apoptosis of HepG2 cells was induced by compound 3k•CH3I and was associated with the mitochon-

Effect of Compound 3k•CH3I on the Expression of Related Proteins in HepG2 Cells
In order to better comprehend the effect of compound 3k•CH3I on the expression of proteins related to the mitochondrial apoptosis pathway, HepG2 cells were treated with 3k•CH3I at different concentrations (0, 2, 4, and 8 µmol/L), and Western blotting was used to determine the expression of related proteins, as shown in Figure 9. Relative to the control group, it was demonstrated that the expression of key regulatory proteins in HepG2 cells was significantly modulated by compound 3k•CH3I.Specifically, a dose-dependent upregulation of the basal forms of AKT, PI3K, and mTOR was induced, while a downregulation of their phosphorylated counterparts, P-AKT, P-PI3K, and P-mTOR, was concurrently triggered.The above results suggested that compound 3k•CH3I can induce the apoptosis of HepG2 cells through an endogenous mitochondrial pathway, and AKT, PI3K, and mTOR may be involved in inducing the apoptosis of human hepatocellular carcinoma HepG2 cells.Flow cytometry was used to detect the intimal potential of HepG2 cells.It was found that compound 3k•CH 3 I induced a dose-dependent increase in the MMP depolarization of HepG2 cells at concentrations of 0, 2, 4, and 8 µmol/L.It was found to increase from 7.74% to 17.2%, 27.3%, and 48.9%, respectively.It was concluded that the apoptosis of HepG2 cells was induced by compound 3k•CH 3 I and was associated with the mitochondrial-mediated endogenous pathway.

Effect of Compound 3k•CH 3 I on the Expression of Related Proteins in HepG2 Cells
In order to better comprehend the effect of compound 3k•CH 3 I on the expression of proteins related to the mitochondrial apoptosis pathway, HepG2 cells were treated with 3k•CH 3 I at different concentrations (0, 2, 4, and 8 µmol/L), and Western blotting was used to determine the expression of related proteins, as shown in Figure 9. Relative to the control group, it was demonstrated that the expression of key regulatory proteins in HepG2 cells was significantly modulated by compound 3k•CH 3 I.Specifically, a dosedependent upregulation of the basal forms of AKT, PI3K, and mTOR was induced, while a downregulation of their phosphorylated counterparts, P-AKT, P-PI3K, and P-mTOR, was concurrently triggered.The above results suggested that compound 3k•CH 3 I can induce the apoptosis of HepG2 cells through an endogenous mitochondrial pathway, and AKT, PI3K, and mTOR may be involved in inducing the apoptosis of human hepatocellular carcinoma HepG2 cells.

Chemistry
All the reagents were purchased from commercial suppliers (Aladdin, Sha China) and utilized without the need for additional purification steps.Silica gel (Qingdao Ocean Chemical Co., Ltd., Qingdao, China) was employed as the sta phase for thin-layer chromatography (TLC), with the mobile phase consisting of petroleum ether and ethyl acetate or dichloromethane and methanol mixtures.Th gression of all the chemical reactions was carefully monitored through this techniqu umn chromatography was used to purify the intermediates and target derivative 400 mesh silica gel, Hehui, Hangzhou, China). 1 H NMR and 13 C NMR spectra we orded with an Avance DRX400 spectrometer (Bruker, Beijing, China) with TMS as ternal standard, and all the reported chemical shift values are presented in terms of ical shifts (δ) and expressed in units of parts per million (ppm).Mass spectra we orded on an Esquire 6000 mass spectrometer (Thermo Fisher Scientific, Waltham USA).

Synthesis of Lupeol-3-(4-nitrobenzoate) (2)
To a solution of lupeol (1, 500.0mg, 1.0 mmol) in anhydrous CH2Cl2 (20.0 mL dine (278.0 µL, 3.0 mmol, 3.0 eq) and 4-nitrophenyl chloroformate (472.0 mg, 2.0 3.0 eq) were added under nitrogen gas.The mixture was stirred at room temperat 2 h.Following confirmation of completion by TLC, the reaction mixture was subseq diluted in CH2Cl2 (50.0 mL) and sequentially extracted with aqueous sodium car and a brine solution.The organic layer was then dewatered using anhydrous Na2S suring the thorough removal of moisture.The solvent was removed under reduce sure to afford a yellow solid, which was separated by chromatography on silica g petroleum ether/ethyl acetate (20:1, v/v) to obtain the desired intermediate 2.

Chemistry
All the reagents were purchased from commercial suppliers (Aladdin, Shanghai, China) and utilized without the need for additional purification steps.Silica gel GF254 (Qingdao Ocean Chemical Co., Ltd., Qingdao, China) was employed as the stationary phase for thin-layer chromatography (TLC), with the mobile phase consisting of either petroleum ether and ethyl acetate or dichloromethane and methanol mixtures.The progression of all the chemical reactions was carefully monitored through this technique.Column chromatography was used to purify the intermediates and target derivatives (300-400 mesh silica gel, Hehui, Hangzhou, China). 1 H NMR and 13 C NMR spectra were recorded with an Avance DRX400 spectrometer (Bruker, Beijing, China) with TMS as an internal standard, and all the reported chemical shift values are presented in terms of chemical shifts (δ) and expressed in units of parts per million (ppm).Mass spectra were recorded on an Esquire 6000 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Synthesis of Lupeol-3-(4-nitrobenzoate) (2)
To a solution of lupeol (1, 500.0mg, 1.0 mmol) in anhydrous CH 2 Cl 2 (20.0 mL), pyridine (278.0 µL, 3.0 mmol, 3.0 eq) and 4-nitrophenyl chloroformate (472.0 mg, 2.0 mmol, 3.0 eq) were added under nitrogen gas.The mixture was stirred at room temperature for 2 h.Following confirmation of completion by TLC, the reaction mixture was subsequently diluted in CH 2 Cl 2 (50.0 mL) and sequentially extracted with aqueous sodium carbonate and a brine solution.The organic layer was then dewatered using anhydrous Na 2 SO 4 , ensuring the thorough removal of moisture.The solvent was removed under reduced pressure to afford a yellow solid, which was separated by chromatography on silica gel with petroleum ether/ethyl acetate (20:1, v/v) to obtain the desired intermediate 2.
White solid (300.0 mg, 77%).To a solution of compound 3k (80.0 mg, 0.1 mmol) in acetonitrile (CH 3 CN, 10.0 mL), CH 3 I (129.0 mg, 1.0 mmol, 7.0 eq) was added, stirred at room temperature for 12 h and filtered.Then the solvent was removed under reduced pressure to afford the crude product.An analytical sample was obtained by recrystallization from acetonitrile.

In Vitro Cytotoxicity
The MTT (Beyotime, Shanghai, China) colorimetric method was used to test the antitumor activity of the synthetic products.The test compound and the control compound were prepared into the liquid with a concentration of 1, 5, 10, 20, and 40 µmol/L, respectively.The tested tumor cells (A549, HepG2, MCF-7) (Chinese Academy of Sciences, Shanghai, China) were inoculated into 96 well plates with a density of 2 × 10 4 cells/mL, and 90 µL PBS was injected into each well.The 96 well plates were cultured overnight in an incubator, and each well was injected with the same concentration of 10 µL of the test compound.After 48 h, 10 µL of MTT solution was injected into each well.After culture for 4 h, the plates were turned upside down and 100 µL DMSO (Kermel, Tianjin, China) was added.The optical density (OD) values of the compound and the control product were determined at 490 nm wavelength with enzyme-labeled apparatus.

Cell Morphology Detection
In this study, human hepatoma HepG2 cells were treated with compound 3k•CH 3 I, and the tested cells were stained by the AO/EB double staining (KeyGEN BioTECH, Nanjing, China) method to observe the morphological changes of the cells.HepG2 cells of logarithmic growth stage were inoculated into confocal six-well plates, and a volume of 2 mL of the cell suspension, adjusted to a concentration of 1 × 10 6 cells per milliliter, was introduced into each well.The cells were cultured in an aseptic constant temperature incubator for 24 h, and 3k•CH 3 I solution with concentrations of 0, 2, 4, and 8 µmol/L was added successively, and the culture continued for 48 h.The cellular specimens were rinsed with phosphate-buffered saline (PBS) on two occasions, after which 500 µL of staining buffer was introduced to the preparation, followed by storage in a light-protected environment for subsequent analysis.AO (5 µL) and EB (5 µL) staining solutions were added to the six-well plates successively, mixed and incubated at 4 • C for 20 min under light protection, then PBS was added and observed under a fluorescence microscope.

Detection of Apoptosis
HepG2 cells with logarithmic growth stage were selected and inoculated into a designated confocal six-well plate with 2 mL cell suspension per well (1 × 10 6 cells/mL) and cultured in a sterile incubator for 24 h.3k•CH 3 I solution with a concentration of 0, 2, 4, and 8 µmol/L was added, and culture continued for 48 h.The cellular preparations were initially rinsed with phosphate-buffered saline (PBS), followed by enzymatic detachment using a 0.25% trypsin solution.Post-collection, the cells underwent centrifugation at 800 revolutions per minute (rpm) for a duration of 5 min to pelletize.The resulting cell pellet was subsequently re-suspended in 1 mL of culture medium designed for cell propagation.The 100 µL cell suspension was transferred into a flow detection tube, with 5 µL AnnexinV added, cultured at room temperature away from light for 15 min, and centrifuged at 800 revolutions per minute (rpm) for a period of 5 min, after which the supernatant containing the staining solution was aspirated.Then, 100 µL of binding buffer for re-suspension and 5 µL of PI dye were added and the solution was mixed well and incubated at low temperature, avoiding light for 15 min.The results were measured by flow cytometry.

Detection of Intracellular ROS
HepG2 cells were inoculated into a six-well plate with 2 mL cell suspension per well at a concentration of 1 × 10 6 cells/mL overnight in an incubation chamber maintained at 37 • C with a 5% CO 2 atmosphere.The cells were treated with different concentrations of compound 3k•CH 3 I (0, 2, 4 and 8 µmol/L) and continued to be cultured for 48 h.The cells underwent a PBS rinse to cleanse them, subsequent digestion with a 0.25% trypsin solution to facilitate detachment, and subsequent centrifugation at 800 revolutions per minute (rpm) for 5 min to sediment the cells.The ensuing cell pellet was then re-suspended in 1 mL of nutrient-rich cell culture medium for further experimental procedures.Then, 5 µmol/L DCFH-DA solution was added to each well and they were incubated for 30 min away from light.After incubation, the cells were digested, the supernatant was centrifuged, and the PBS cell suspension was taken and placed in a flow cytometry to determine the experimental results.

Detection of MMP Depolarization
HepG2 cells with logarithmic growth stage were selected and inoculated into a designated confocal six-well plate with 2 mL cell suspension per well (1 × 10 6 cells/mL) and cultured in a sterile incubator for 24 h.The cells were treated with different concentrations of 3k•CH 3 I (0, 2, 4, 8 µmol/L) and continued to be cultured for 48 h.PBS cleans the cells, 0.25% trypsin digests the cells, and collects the cells.After the drug action time was over, 1 mL of pre-configured JC-1 staining solution was added and incubated for 30 min.After JC-1 incubation, the cells were observed by fluorescence microscope, the supernatant was separated, and the PBS cell suspension was taken and the experimental results were determined by flow cytometry.

Protein Immunoblot Assay
HepG2 cells with logarithmic growth stage were selected to make a single-cell suspension and inoculated into a culture bottle with a cell density of 1 × 10 6 cells/mL.The cells were treated with different concentrations of compound 3k•CH 3 I (0, 2, 4 and 8 µmol/L) and cultured for 48 h.A mixture of 500 µL of phenylmethylsulfonyl fluoride (PMSF) and radio immunoprecipitation assay (RIPA) lysate was added to each well, and the protein content was determined using the bicinchoninic acid (BCA) assay and denatured at high temperature 30 min later.The protein samples were separated by polyacrylamide gel elec-

Figure 3 .
Figure 3.The structure modification strategy of lupeol.

Figure 3 .
Figure 3.The structure modification strategy of lupeol.

Figure 5 .
Figure 5. Evaluation of the aqueous solubility of compounds 1 and 3k•CH 3 I.
. Normal green fluorescence and no morphological alterations were observed in the control group following AO/EB staining.HepG2 tumor cells treated with compound 3k•CH 3 I showed bright red fluorescence due to the chromatin concentration and nuclear fragmentation.Concurrently, the apoptosis phenomenon became more obvious with the increase in drug concentration, indicating that compound 3k•CH 3 I can induce the apoptosis of HepG2 tumor cells in a drug-dependent manner.Molecules 2024, 29, x FOR PEER REVIEW 8 of 17

2. 6 .
Effect of Compound 3k•CH 3 I on the Mitochondrial Membrane Potential of HepG2 Cells In order to explore the effect of compound 3k•CH 3 I on MMP, a JC-1 staining kit was used to detect the depolarization of the MMP of human hepatocellular carcinoma HepG2 cells treated with compound 3k•CH 3 I, as shown in Figure 8.Compared with the control group, after HepG2 cells were treated with different concentrations of compound 3k•CH 3 I for 48 h, the red fluorescence gradually weakened until it eventually disappeared, and the green fluorescence gradually strengthened in HepG2 cells.