Design, synthesis, and biological evaluation of novel asiatic acid derivatives as potential anticancer agents

Abstract

A series of new asiatic acid derivatives modified in the A-ring and at C-28 were synthesized and their antiproliferative activity was evaluated against HT-29 and HeLa cell lines. Most of the derivatives tested here exhibited improved antiproliferative activity compared with asiatic acid. Among them, the best compounds, 7 and 8, were further evaluated against additional cancer cell lines (MCF-7, Jurkat, and PC-3 cells) and a nontumoral cell line (BJ). The most active compound, 7, exhibited IC₅₀ values ranging from 1.62 μM in HeLa cells to 9.93 μM in MCF-7 cells. Further studies revealed that compound 7 arrested the cell cycle at the G0/G1 phase and induced caspase-dependent apoptosis in HeLa cells. Furthermore, this compound showed selectivity toward cancer cells, and a synergistic effect was observed after simultaneous treatment of HeLa cells with compound 7 and cisplatin. Collectively, our results suggest that compound 7 may be useful for the development of new anticancer therapies; thus, additional preclinical studies are warranted.

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1. Introduction

Natural triterpenes represent a structurally diverse group of organic compounds that display several pharmacological activities.^1–5 Because of these activities, these compounds are very interesting leads in the field of drug discovery, particularly regarding the development of innovative anticancer drugs.^4,6–11

Asiatic acid 1 [AA (1), Fig. 1], is a naturally occurring pentacyclic triterpenoid that is found mainly in the traditional medicinal herb Centella asiatica. This compound has long been used in the field of dermatology as a wound-healing agent^12–14 and is the major effective ingredient of the commercial drug Madecassol™.¹⁵ AA (1) also has other clinically useful therapeutic effects, including antioxidant, anti-inflammatory,^16,17 neuro-protective,^18,19 antidiabetic, hepato-protective,²⁰ and, especially, antitumor activities.^21–23 Because its antitumor activity, this compound has attracted a great interest and its anticancer effect has been studied by several research groups. AA (1) inhibits the proliferation of, and induces apoptosis in several cancer cell lines,^21–26 and possesses a strong antiangiogenic potential against malignant gliomas.²⁷ AA (1) also enhances the sensitivity of HT-29 cells to the anticancer drug irinotecan, with a synergistic effect when cells were first exposed to AA (1) and then to irinotecan.²⁸ In addition, this compound proved to be relatively nontoxic in normal cells, which renders it a promising anticancer agent. However, the clinical utility of AA (1) in the treatment of cancer is limited by its modest anticancer activity and poor bioavailability. The chemical modification of the AA (1) backbone had a high impact on its biological activity and may represent the solution to improve not only the antitumor activity of AA (1), but also its pharmacokinetic properties.

Fig. 1 Chemical structure of asiatic acid 1.

Previous studies revealed that the introduction of amino moieties^29–31 at C-28 of AA (1) significantly improved the anticancer activity of the compound against several cancer cell lines. The modification of the A-ring of AA (1) also increased its anticancer activity.¹¹ In addition, in the last decades, the hydroxamic acid group was shown to be a potent moiety in the field of cancer therapy.^32–35 Hydroxamic acid derivatives are well-known inhibitors of metal-containing enzymes, such as matrix metalloproteinases (MMPs)³⁴ and histone deacetylases (HDACs), which are two enzyme families that are important for tumor development. However, the introduction of a hydroxamic acid moiety into the AA (1) structure has not been addressed. Therefore, to develop new AA (1) derivatives as potential anticancer drug candidates, here we designed and prepared a panel of new AA (1) derivatives bearing amino, amino acid, and hydroxamic acid moieties at C-28, combined with modifications at the A ring. The antiproliferative activities of the newly synthesized AA (1) derivatives against the HT-29 and HeLa cancer cell lines were evaluated and a structure–activity relationship (SAR) was established based on the IC₅₀ obtained in HeLa cells. Additional studies were then conducted in HeLa cells to explore the mechanism of action of the most active compound, 7. Finally, the existence of synergism between compound 7 and cisplatin was investigated.

2. Results and discussion

2.1 Chemistry

In this work, a series of novel AA (1) derivatives were successfully synthesized. As shown in Scheme 1, the synthesis started by the preparation of the intermediates 2, 3, 4, 5 and 6. The lactol derivative 2 was obtained by treatment of AA (1) with sodium periodate in methanol/water, by adapting a previously reported procedure.³⁶ The treatment of derivative 2 with acetic acid and piperidine in dry benzene gave the α,β-unsaturated aldehyde 3. This compound was then reduced with sodium borohydride in anhydrous methanol, affording the diol derivative 4 in good yield (92%). The two hydroxyl groups of compound 4 were diacetylated with acetic anhydride and DMAP in THF, affording the intermediate 5. Commercially available asiatic acid was treated with acetic anhydride to give the triacetate derivative 6 in good yield. The intermediates 5 and 6 were then used as a starting point for the synthesis of new derivatives of AA (1).

Scheme 1 Reagents and conditions: (a) NaIO₄, MeOH/H₂O, rt; (b) (i) acetic acid, piperidine, dry benzene, reflux 60 °C, N₂, 1 h; (ii) anhydrous MgSO₄, reflux 60 °C, N₂; (c) NaBH₄, MeOH, rt, 2 h. (d) Acetic anhydride, DMAP, THF, rt. (e) Acetic anhydride, THF, DMAP, rt; (f) (i) THF, CDI, reflux, N₂, 28 h; (ii) hydroxylamine hydrochloride or methylhydroxylamine hydrochloride, rt.

In the last decades, medicinal chemists have shown a renewed interest in hydroxamic acids and their derivatives; in the particular case of triterpenoid compounds, previous studies reported hydroxamic acid derivatives of glycyrrhetinic acid as being selective inhibitors of 11β-hydroxysteroid dehydrogenase 2.^37,38 In this study, we prepared AA (1) hydroxamic acid derivatives from 1,1′-carbonyldiimidazole (CDI)-activated carboxylic acids and hydroxylamine hydrochloride. As CDI promoted the deprotonation of hydroxylamine hydrochloride, no additional base was necessary.³⁹ As depicted in Scheme 1, the carboxylic acid derivative 6 was treated with CDI in THF to afford the respective N-acylimidazole intermediate, which was reacted with hydroxylamine hydrochloride or methylhydroxylamine hydrochloride to give the corresponding hydroxamic derivatives 7 and 8, in good yields. The hydroxamic acid derivative 9 was prepared from 5, according to the procedure described for compound 7 and using hydroxylamine hydrochloride. In the ¹H NMR spectra of hydroxamic acid derivatives 7 and 9, the characteristic peak of the proton attached to the nitrogen atom was observed at around 6.25–6.27 ppm. In addition, on the ¹³C NMR spectrum, the signal of the hydroxamic acid carbonyl group was found at 178.4 ppm, a lower value than that observed for the corresponding carbonyl group of carboxylic acid (at around 183.1–183.6 ppm). The ester carbonyl carbons of compound 7 appeared in ¹³C NMR spectrum as signals at 170.8, 170.5 and 170.4 ppm. The signal at 125.5 ppm corresponded to the tertiary carbon C12, as this signal was present on DEPT 135.

Previous studies performed by our group revealed that the introduction of an imidazole moiety at the C-28 position of triterpenoid compounds improved their cytotoxic activities.^40–42 Thus, the N-acylimidazole derivative 10 was prepared in 63% yield after FCC via the reaction of compound 5 with CDI in THF at reflux (Scheme 2). The successful preparation of compound 10 was confirmed by the presence of three signals at 8.23, 7.52 and 7.03 ppm in the ¹H NMR spectrum, which are typical of imidazole protons. A signal was also observed at δ 174.7 ppm in the ¹³C NMR spectrum corresponding to the C-28 carbon, which was different from the signal observed for the C-28 carboxylic acid carbonyl, at δ 183.6 ppm. The signals at 151.1 and 137.1 ppm were attributed to the quaternary carbons C2 and C13. The signals at 132.0 and 126.1 ppm were attributed to the tertiary carbons C3 and C12, respectively, as these signals appeared in the DEPT 135. The signals for the three tertiary carbons of the imidazole ring were found at 137.1, 129.7 and 117.4 ppm.

Scheme 2 Reagents and conditions: (a) (i) THF, CDI, reflux, N₂, 24 h; (ii) hydroxylamine hydrochloride, rt, 48 h. (b) CDI, THF, 70 °C, N₂; (c) SOCl₂, benzene, reflux temperature; (d) methylamine or ethylamine or propylamine or 4-methylbenzylamine or 4-fluorobenzylamine, Et₃N, dichloromethane, rt; (e) glycine or L-alanine methyl ester, Et₃N, THF, rt.

As depicted in Scheme 2, the treatment of compound 5 with thionyl chloride in dry benzene, gave the acyl chloride derivative, followed by treatment with the respective amines in dry dichloromethane afforded the amide derivatives 11–15 in good yields. The formation of an amide bond at C-28 was confirmed by the presence of a strong N–H bending band at 1509–1527 cm⁻¹ on the IR spectra. On the ¹³C NMR spectrum, the signal of the carbonyl group of the amide was observed at 177.6–178.7 ppm, which was a lower value than that observed for the carbonyl group of carboxylic acid (at 183.6 ppm).

The methyl ester amino acid derivatives 16 and 17 were obtained by treating 5 with thionyl chloride, to give the acyl chloride intermediate, which was further reacted with the corresponding amino acid methyl ester hydrochloride.

2.2 Biology

2.2.1 Antiproliferative activity and selectivity of the asiatic acid derivatives. The in vitro antiproliferative activity of the newly synthesized derivatives was evaluated against colon cancer (HT-29) and cervix cancer (HeLa) cell lines using the MTT assay. The clinically used antineoplastic drug cisplatin was used as the reference drug. The results, which are expressed as the concentration of compound required for inhibiting 50% of cell growth (IC₅₀), are summarized in Table 1 and Fig. 2. The IC₅₀ values determined in HeLa cells were used to establish a structure–activity relationship (SAR) (Fig. 3). As depicted in Table 1, all new derivatives exhibited improved antiproliferative activity against HeLa cells compared with AA (1), as they showed IC₅₀ values ranging from 1.62 to 45 μM.

Table 1 Cytotoxic activities (IC₅₀) of AA, its derivatives and cisplatin against human colon adenocarcinoma (HT-29) and human cervical cancer (HeLa) cell lines^a

Compound	Cell line/IC50^b (μM)
	HT-29	HeLa
a HT-29 and HeLa cells were treated with increasing concentrations of each compound for 72 h. Viable cells were determined by MTT assay and IC50 values are expressed as the mean ± SD of three independent experiments. N.D. not determined.b IC50 is the concentration of compound that inhibits 50% of cell growth.c IC50 value obtained from literature, determined using the same experimental methodology and included here for comparison.
AA 1	64.33 ± 3.21	52.47 ± 0.06
2	N.D.	21.67 ± 1.04
3	N.D.	5.30 ± 0.20
4	N.D.	45.00 ± 1.55
5	N.D.	22.50 ± 0.75
6	N.D.	6.10 ± 0.28
7	4.12 ± 0.30	1.62 ± 0.10
8	7.03 ± 0.06	3.77 ± 0.23
9	19.00 ± 0.71	4.80 ± 0.28
10	13.15 ± 0.78	6.25 ± 0.07
11	16.15 ± 0.92	7.60 ± 0.42
12	14.53 ± 1.45	4.54 ± 0.84
13	14.10 ± 1.27	6.50 ± 0.5
14	>60	6.25 ± 0.35
15	>60	5.38 ± 0.530
16	22.00 ± 1.41	10.65 ± 0.35
17	13.75 ± 0.35	7.23 ± 0.39
Cisplatin	6.11^c (ref. 43)	2.28 ± 0.26

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Fig. 2 Dose-dependent effect of compounds 7 and 8 on HeLa and HT-29 cell viability. Results are presented as mean ± SD of three independent experiments.

Fig. 3 Schematic representation of the SAR for the antiproliferative activity of the several synthetic derivatives of AA (1) against HeLa cell line. The SAR was established based on IC₅₀ values. The esterification of C-2, C-3 and C-23 hydroxyl groups improved the antiproliferative activity: compound 6 was 8.6-fold more potent than AA (1). The introduction of the hydroxamic moiety at C-28 led to an increase of the activity: compound 7 was 32.4- and 3.8-fold more active than AA (1) and compound 6, respectively. The conversion of the hexameric A-ring of AA (1) into the pentameric A-ring of compound 5, led to an increase of the antiproliferative activity: compound 5 was 2.3-fold more active than AA (1). The introduction of amide moieties at C-28 of compound 5 increased the activity: compound 12 was 4.9-fold more active than precursor compound 5 and 11.6-fold more active than AA (1).

As shown in Table 1 and Fig. 2, the hydroxamic (7) and the methyl hydroxamic (8) derivatives, exhibited remarkable antiproliferative activities against HT-29 and HeLa cells, compared with AA (1). Compound 7 showed IC₅₀ values of 4.12 and 1.62 μM against the HT-29 and HeLa cell lines, respectively. This compound was 32.4-fold more potent than AA (1) and 3.8-fold more potent than 6 against HeLa cells. Compound 7 also exhibited a stronger antiproliferative activity against HeLa cells than did the clinically used drug cisplatin. Derivative 8 was 14- and 1.6-fold more active against HeLa cells than AA (1) and compound 6, respectively. The conversion of the carboxylic acid group of compound 5 into a hydroxamic acid group afforded derivative 9, with an improvement of 4.7- and 10.9-fold in antiproliferative activity against HeLa cells compared with 5 and AA (1), respectively. These combined results suggest that the introduction of a hydroxamic acid group at C-28 has a positive impact on the anticancer activity of the compound.

The introduction of an imidazole ring at C-28 of compound 5, to afford compound 10, led to an increase in cell-growth-inhibition activity. The imidazole derivative 10 was 3.5- and 8.4-fold more active than 5 and AA (1), respectively, in HeLa cells (Table 1). These results are in good agreement with previous reports.⁴²

Finally, the comparison of the IC₅₀ values of compounds 11–17 revealed that the introduction of the amide functionality at C-28 of compound 5 resulted in a significant improvement of antiproliferative activity against HeLa cells. Compound 12 was 11.6-fold more potent than AA (1) and 5-fold more potent than compound 5. Derivatives 16 and 17, bearing a methyl ester amino acid residue at C-28, exhibited better antiproliferative activity in HeLa cells than did compound 5.

As compounds 7 and 8 were the most active compounds, they were selected, and their antiproliferative activity was further tested in breast (MCF-7), leukemia (Jurkat), and prostate (PC-3) cancer cell lines. As depicted in Table 2, both compounds exhibited a much higher antiproliferative activity against the tested cancer cell lines than did AA (1). Moreover, the selectivity of compound 7 was assessed on the nontumoral fibroblast cell line BJ. The analysis of the selectivity index (IC₅₀ in the BJ cell line/IC₅₀ in the cancer cell line) (Table 3) values revealed that compound 7 was 6 to 36 times more active in cancer cell lines than on nontumoral BJ cells. In contrast, this index ranged from 1.3 to 2.4 for AA (1). These results clearly suggest that 7 is more selective for cancer cells than AA (1). As compound 7 displayed the strongest antiproliferative effect, it was chosen for further studies in HeLa cells aimed at exploring its anticancer mechanism.

Table 2 Cytotoxic activities (IC₅₀) of compounds 7 and 8 against several cancer cell lines (MCF-7, Jurkat, PC-3) and the non-tumoral human fibroblast cell line BJ^a

Compounds	Cell line/IC50^b (μM)
	MCF-7	Jurkat	PC-3	BJ
a The cell lines were treated with increasing concentrations of each compound for 72 h. Viable cells were determined by MTT assay and IC50 values are expressed as the mean ± SD of three independent experiments. N.D. not determined.b IC50 is the concentration of compound that inhibits 50% of cell growth.c IC50 values obtained from literature determined using the same experimental methodology and included here for comparison.
AA (1)	68.5 ± 2.5	37.17 ± 3.75	67.25 ± 0.35	88.7 ± 0.58
7	9.93 ± 1.01	2.47 ± 0.12	3.73 ± 0.46	59.0 ± 0.27
8	9.17 ± 0.58	2.43 ± 0.21	3.8 ± 0.8	N.D.
Cisplatin	19.0 ± 4.5	1.94^c (ref. 44)	4.6^c (ref. 45)	10.10 ± 2.00

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Table 3 Selectivity index of AA (1) and compound 7 towards cancer cell lines

Compounds	Selectivity index (IC50 BJ cell line/IC50 cancer cell line)
	HT-29	HeLa	MCF-7	Jurkat	PC-3
AA (1)	1.36	1.69	1.29	2.39	1.32
7	14.32	36.42	5.95	23.90	15.81

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2.2.2 Cell cycle and Annexin V/PI flow cytometry assays of derivative 7. To gain a deeper insight into the mechanism underlying the growth inhibition afforded by compound 7, we first evaluated its effects on cell-cycle distribution. HeLa cells were treated with different concentrations of 7 (2, 4 and 6 μM) for 24 h, and the cell-cycle distribution was analyzed by flow cytometry after staining the cellular DNA with PI. We found that treatment of HeLa cells with 4 and 6 μM 7 led to a dose-dependent accumulation of cells in the G0/G1 phase (Fig. 4A).

Fig. 4 (A) Cell cycle distribution of HeLa cells untreated (control) or treated with the indicated concentrations of compound 7 for 24 h. Representative histograms for cell cycle analysis and graph bar summarizing the variation of the percentage of cells in each phase of cell cycle. Results are presented as mean ± SD of three independent experiments. (B) Analysis of apoptosis in HeLa cells untreated (control) or treated with the indicated concentrations of compound 7 during 24 h. Representative flow cytometry histograms that depict the variation of the percentage of cells which are alive (lower left quadrant), in early apoptosis (lower right quadrant) and in late apoptosis/necrosis (upper left and right quadrants). The graph bar summarizes the variation of the percentage of cells which are in early apoptosis and in late apoptosis/necrosis. Results are presented as mean ± SD of three independent experiments. Differences between treated and control groups were considered statistically significant at p < 0.05 (*).

The percentage of cells in the G0/G1 phase rose from 49.45% in control cells to 65.97% in cells treated with 6 μM 7. Concomitantly, the percentage of cells in the S and G2/M phases was gradually reduced. An analysis of cell-cycle progression indicated that compound 7 induced cell-cycle arrest at the G1 phase.

We also investigated if the growth-inhibitory activity of compound 7 was related to the induction of apoptosis. Apoptosis is a tightly regulated cell death program that plays a critical role in cell homeostasis by controlling cell proliferation.⁴⁶ The dysregulation of the apoptotic process is related to the development of many types of cancer.⁴⁷ In fact, the ability of cancer cells to evade apoptotic cell death is one of the hallmarks of cancer.⁴⁸ Therefore, the induction of apoptosis in cancer cells is one of the recognized strategies that have been used for the development of more effective anticancer drugs.^47,49,50

In the early stages of apoptosis, the cell membrane loses it asymmetry and phosphatidylserine (PS) translocates from the inner to the outer side of the cell membrane. The externalized PS can be detected using Annexin V-FITC, a fluorescent active dye that selectively binds to PS.⁵¹

Therefore, to explore whether compound 7 has the ability to induce apoptosis in HeLa cells, we performed an Annexin V-FITC/PI flow cytometry analysis. HeLa cells treated with different concentrations of compound 7 (2, 4, and 6 μM) for 24 h were analyzed by FACS after double staining with Annexin V/PI. The combined results of three independent experiments are depicted in Fig. 4B. HeLa cells treated with 7 at 2 μM for 24 h showed an increase in the percentage of Annexin-V-positive cells, from 4.94% in control cells to 10.53% in treated cells (5.70% of early apoptotic cells and 4.83% of late apoptotic cells). After increasing the concentration of the drug to 4 and 6 μM, the percentage of Annexin-V-positive cells rose to 19.55% and 29.18%, respectively. Our results suggest that compound 7 induces apoptosis in HeLa cells in a concentration-dependent manner.

2.2.3 Morphological analysis by fluorescence microscopy after Hoechst 333258 staining. Taking into consideration the fact that cells in apoptosis acquired a series of typical morphological features, we also assessed the morphological changes in HeLa cells after treatment with 7. To observe in greater detail chromatin configuration, we used the Hoechst staining technique. Hoechst is a membrane-permeable blue fluorescent dye that intercalates within cellular DNA. HeLa cells treated with compound 7 for 24 h were stained with Hoechst and analyzed by fluorescence microscopy. Non treated cells were used as controls. As depicted in Fig. 5, control cells were uniformly stained and presented a normal round morphology; thus, no obvious morphological changes were observed in these cells. Conversely, typical apoptotic features, such as chromatin condensation and formation of apoptotic bodies, were observed in cells treated with 4 and 6 μM compound 7. Moreover, an evident reduction in the number of cells with increasing concentrations of the drug was also observed. These results clearly confirmed that compound 7 has the ability to trigger apoptosis in HeLa cells.

Fig. 5 Representative fluorescence microscopic images of HeLa cells untreated or treated with the indicated concentrations of compound 7 for 24 h. Before the fluorescence microscopic observation, HeLa cells were stained with Hoechst 33258. White arrows represent apoptotic cells.

2.2.4 Caspase inhibitor assay. One of the most important mechanisms involved in apoptotic cell death is the activation of caspases. To explore if caspase activation was involved in the apoptotic cell death induced by compound 7, we used the general caspase inhibitor z-VAD-fmk. HeLa cells pretreated or not for 45 min with 50 μM z-VAD-fmk were treated for 24 h with compound 7 at 6 μM.

The effects of z-VAD-fmk on compound 7-induced apoptosis were evaluated by flow cytometry after staining with Annexin V-FITC/PI. Pretreatment with z-VAD-fmk did not affect the apoptosis levels in control cells (Fig. 6). However, the percentage of apoptotic cells among HeLa cells treated with compound 7 was significantly reduced when cells were pretreated with the caspase inhibitor (29.18% of total apoptotic cells in nontreated cells vs. 6.31% of apoptotic cells in cells pretreated with the caspase inhibitor), as shown in Fig. 6. These results suggest that compound 7 induces apoptosis via a caspase-dependent mechanism.

Fig. 6 Flow cytometry quantification of apoptosis in HeLa cells. HeLa cells untreated (control) or treated with compound 7 at 6 μM for 24 h, pretreated or not for 45 min with z-VAD-fmk at 50 μM, were analyzed by flow cytometry after staining with Annexin V-FITC/PI. (A) Representative flow cytometry histograms that depict the variation of the percentage of cells which are alive (lower left quadrant), in early apoptosis (lower right quadrant) and in late apoptosis/necrosis (upper left and right quadrants). (B) Graph bar that summarizes the variation of the percentage of cells which are in early apoptosis (dark gray bars) and in late apoptosis/necrosis (light gray bars). Results are presented as mean ± SD of three independent experiments. Differences between treated and control groups were considered statistically significant at p < 0.05 (*).

2.2.5 Evaluation of synergism between cisplatin and compound 7. Drug side effects and the development of resistance in some types of cancer are a limitation to the use of cisplatin,^52,53 which is one of the most powerful chemotherapeutic agents that are in current clinical use for the treatment of several types of cancer.⁵² Drug-combination therapies have been widely employed to achieve a synergistic therapeutic effect that allows the minimization of toxicity and resistance effects.⁵⁴ Keeping that in mind, we hypothesized that the combined use of the derivative 7 and cisplatin exerts a synergistic inhibitory effect on the proliferation of HeLa cells. The proliferation assays revealed that compound 7 alone exhibited an IC₅₀ of 1.69 μM in HeLa cells, which is about 1.5 times lower than the IC₅₀ of cisplatin in the same cell line (2.28 μM).

To confirm our hypothesis, HeLa cells were treated simultaneously with cisplatin and compound 7 at different concentration combinations (maintaining the ratio of 2 : 1 between the concentrations of cisplatin and compound 7, respectively) for 72 h, and the combination index (CI) values were calculated based on the Chou–Talalay method⁵⁵ using the CompuSyn software. As shown in Table 4, the CI values obtained were lower than 1 for all the combinations tested. These data suggest the existence of a synergistic effect between cisplatin and compound 7 in the inhibition of HeLa cells viability.

Table 4 Combination index (CI) for combinations of cisplatin and compound 7 at a constant ratio of 2 : 1, in HeLa cell line. CI values were calculated using the Chou–Talalay method and the CompuSyn software. CI values <1 indicate the existence of synergism^a

Cisplatin (μM)	7 (μM)	1 – Viability	CI
a HeLa cells were treated with compound 7 and cisplatin simultaneously.
0.42	0.21	0.30	0.57
1.02	0.51	0.46	0.84
1.80	0.90	0.71	0.69
3.00	1.50	0.77	0.91
4.20	2.10	0.88	0.73
6.00	3.00	0.89	0.92
10.20	5.10	0.97	0.64
18.00	9.00	0.99	0.46

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3. Conclusions

In the present study, we developed and synthesized a panel of new derivatives of AA (1) modified in the A-ring and at C-28. The new derivatives showed improved antiproliferative activities against several cancer cell lines compared with AA (1). Compound 7, bearing a hydroxamic acid moiety at C-28, displayed the best antiproliferative activity among all the derivatives tested and was more selective toward cancer cell lines than AA (1). Further studies revealed that compound 7 led to cell-cycle arrest at the G0/G1 phase and induced apoptosis mediated by caspases in the HeLa cell line. Moreover, we observed a synergistic inhibitory effect on the proliferation of HeLa cells between compound 7 and cisplatin. These findings led us to conclude that compound 7 may be a valuable candidate for further studies aimed at the development of effective anticancer drugs.

4. Experimental

4.1 Chemistry

4.1.1 General. Asiatic acid and all reagents were purchased from Sigma-Aldrich, whereas the solvents (analytical grade) were bought from Merck Co. Thin layer chromatography (TLC) analysis was performed using Kieselgel 60HF254/Kieselgel 60G. For flash column chromatography (FCC), Kieselgel 60 (230–400 mesh, Merck) was used. Melting points were determined on a BUCHI Melting point B-540 apparatus and are uncorrected. IR spectra were obtained in a Fourier transform spectrometer. NMR spectra were recorded using a Brucker Digital NMR—Avance 400 apparatus spectrometer, with CDCl₃ or DMSO-d6 as internal standards. The chemical shifts (δ) are given in parts per million (ppm), and coupling constants (J) are given in hertz (Hz). Mass spectra were recorded on a Quadrupole/Ion Trap Mass Spectrometer (QIT-MS) (LCQ Advantage MAX, THERMO FINNIGAN). HRMS was performed on a Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer (Bruker Apex Ultra with a 7 Tesla actively shielded magnet). Elemental analysis was performed in an Analyzer Elemental Carlo Erba 1108 by chromatographic combustion.

4.1.2 2α,23-Lactol-3-formyl-urs-12-en-28-oic acid (2). To a stirred solution of AA (1) (200 mg, 0.41 mmol) in methanol/water [5 mL/0.25 mL (20 : 1)], NaIO₄ (131.30 mg, 0.61 mmol, 1.5 eq.) was added. The reaction mixture was stirred at room temperature. After 2 h the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (40 mL) and extracted with ethyl acetate (3 × 40 mL). The resulting organic phase was washed with water (4 × 40 mL) and brine (40 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 2 as a white powder (quantitative). Mp: 198.5–201.4 °C. ν_max/cm⁻¹ (KBr): 3421.1, 2948.6, 2927.4, 2871.5, 2732.6, 2630.4, 1716.3, 1695.1, 1457.0, 1378.9, 1037.5. ¹H NMR (400 MHz, CDCl₃): δ = 9.94 (s, 1H, CHO), 5.29 (t, J = 3.3 Hz, 1H, H-12), 5.14–5.11 (m, 1H, H-2), 3.94 (d, J = 13.4 Hz, 1H), 3.75 (d, J = 13.2 Hz, 1H), 1.08 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.95 (d, J = 6.0 Hz, 3H), 0.86 (s, 3H), 0.85 (d, J = 5.4 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.1 (CHO), 182.9 (C28), 138.1 (C13), 126.0 (C12), 93.7, 65.4, 61.2, 53.4, 62.7, 48.1, 45.2, 43.7, 42.6, 40.1, 40.0, 38.9, 38.8, 38.6, 33.6, 30.6, 27.8, 24.6, 24.1, 23.2, 21.1, 20.6, 20.4, 17.9, 16.9, 14.6 ppm. DI-ESI-MS m/z: 487.15 ([M + H]⁺). Calcd for C₃₀H₄₆O₅·H₂O: C, 71.39; H, 9.59. Found: C, 71.49; H, 9.85%.

4.1.3 2-Formyl-23-hydroxy-A(1)-norursa-2,12-dien-28-oic acid (3). To a stirred solution of compound 2 (500 mg, 1.03 mmol) in dry benzene (50 mL), piperidine (3 mL) and acetic acid (3 mL) were added. The resultant solution was heat-refluxed at 60 °C. After 1 hour, anhydrous magnesium sulfate (500 mg) was added and the reaction mixture was heat-refluxed under nitrogen atmosphere for 4 h 20 min. The reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (4 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a yellow powder. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 3 : 1 → 1 : 1) to afford 3 as a white solid (353.77 mg, 73%). Mp: 183.5–186.1 °C. ν_max/cm⁻¹ (KBr): 3428.8, 2946.7, 2925.5, 2869.6, 2726.9, 2632.4, 1689.3, 1581.3, 1454.1, 1380.8, 1041.4. ¹H NMR (400 MHz, CDCl₃): δ = 9.72 (s, 1H, CHO), 6.66 (s, 1H, H-3), 5.28 (t, J = 3.1 Hz, 1H, H-12), 3.62 (d, J = 10.7 Hz, 1H, H-23), 3.45 (d, J = 10.7 Hz, 1H, H-23), 1.25 (s, 3H), 1.10 (s, 3H), 1.01 (s, 3H), 0.93 (d, J = 6.2 Hz, 3H), 0.88 (s, 3H), 0.84 (d, J = 6.3 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.9 (CHO), 183.3 (C28), 159.3 (C3), 158.9 (C2), 137.5 (C13), 126.6 (C12), 69.4, 56.3, 52.6, 50.9, 49.4, 47.9, 44.1, 42.4, 41.4, 38.8 (2C), 36.6, 33.5, 30.6, 28.2, 27.1, 24.0 (2C), 21.2, 19.0, 18.7, 17.4, 17.0, 15.9 ppm; DI-ESI-MS m/z: 469.03 ([M + H]⁺). Calcd for C₃₀H₄₄O₄·H₂O: C, 74.04; H, 9.53. Found: C, 73.68; H, 9.75%.

4.1.4 2-Hydroxymethyl-23-hydroxy-A(1)-norursa-2,12-dien-28-oic acid (4). To a stirred solution of 3 (500 mg, 1.07 mmol) in anhydrous methanol (15 mL), sodium borohydride (101.20 mg, 2.67 mmol, 2.5 eq.) was added. The resultant solution was stirred at room temperature. After 2 h, some drops of acetone were added to consume the excess of sodium borohydride and the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 4 as a white solid (465 mg, 92%). ν_max/cm⁻¹ (ATR): 3369.0, 2925.5, 2871.5, 1695.0, 1455.5, 1380.0. ¹H NMR (400 MHz, DMSO-d6): δ = 5.34 (s, 1H), 5.12 (s, 1H), 4.58 (t, J = 4.8 Hz, 1H), 4.46 (t, J = 5.3 Hz, 1H), 4.04–3.99 (m, 1H), 3.95–3.90 (m, 1H), 3.24–3.20 (m, 1H), 3.13–3.09 (m, 1H), 1.09 (s, 3H), 1.07 (s, 3H), 0.92 (s, 3H), 0.87 (s, 3H), 0.82 (d, J = 6.3 Hz, 3H), 0.80 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO-d6): δ = 178.3 (C28), 156.2 (C2), 138.6 (C13), 129.8 (C3), 124.6 (C12), 70.1, 59.3, 57.0, 52.5, 49.8, 47.2, 46.8, 42.7, 42.0, 40.6, 38.5, 38.1, 36.2, 33.4, 30.1, 27.8, 25.8, 23.7, 23.5, 21.0, 19.1, 18.5, 17.6, 17.0, 16.7 ppm. DI-ESI-MS m/z: 471.02 ([M + H]⁺).

4.1.5 2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oic acid (5). To a stirred solution of 4 (500 mg, 1.06 mmol) in dry THF (15 mL), acetic anhydride (250.5 μL; 2.66 mmol, 2.5 eq.) and a catalytic amount of DMAP (50 mg) were added. The mixture was stirred at room temperature, in anhydrous conditions. After 2 hours, the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with 5% aqueous. HCl (2 × 50 mL), 10% aqueous NaHCO₃ (2 × 50 mL), 10% aqueous Na₂SO₃ (50 mL), water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 5 as a white powder (574 mg, 97%). Mp: 93.5–95.6 °C. ν_max/cm⁻¹ (ATR): 2926.0, 2870.0, 1739.0, 1694.5, 1455.5, 1381.0, 1234.5, 1031.5. ¹H NMR (400 MHz, CDCl₃): δ = 5.44 (s, 1H, H-3), 5.22 (t, J = 2.9 Hz, 1H, H-12), 4.70 (d, J = 14.3 Hz, 1H), 4.57 (d, J = 14.1 Hz, 1H), 3.93 (d, J = 10.7 Hz, 1H, H-23), 3.84 (d, J = 10.7 Hz, 1H, H-23), 2.08 (s, 3H, OCOCH̲₃), 2.06 (s, 3H, OCOCH̲₃), 1.17 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 5.6 Hz), 0.85 (d, J = 6.6 Hz), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 183.6 (C28), 171.3 (OCO), 170.8 (OCO), 151.3 (C2), 138.7 (C13), 131.8 (C3), 125.3 (C12), 72.3, 62.6, 58.0, 52.6, 50.7, 47.9, 46.3, 43.1, 42.3, 41.1, 38.8, 38.8, 36.6, 33.7, 30.5, 28.2, 26.2, 24.0, 23.8, 21.2, 21.0 (2C), 19.1, 18.6, 17.8, 17.0, 16.6 ppm. DI-ESI-MS m/z: 555.04 ([M + H]⁺).

4.1.6 2α,3β,23-Triacetoxyurs-12-en-28-oic acid (6). To a stirred solution of AA (1) (1000 mg, 2.05 mmol) in dry THF (30 mL), acetic anhydride (1.15 mL, 12.28 mmol, 6 eq.) and a catalytic amount of DMAP (100 mg) were added. The mixture was stirred at room temperature in anhydrous conditions. After 4 h, the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (150 mL) and aqueous phase was extracted with EtOAc (3 × 150 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 100 mL), 10% aqueous NaHCO₃ (2 × 100 mL), 10% aqueous Na₂SO₃ (100 mL), water (100 mL) and brine (100 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford compound 6 as a white powder (quantitative). Mp: 150–152 °C. ν_max/cm⁻¹ (KBr): 3463.5, 2948.6, 2871.5, 1816.6, 1747.2, 1456.0, 1369.2, 1236.2, 1045.2. ¹H NMR (400 MHz, CDCl₃): δ = 5.23 (t, J = 3.2 Hz, 1H, H-12), 5.19–5.13 (m, 1H, H-2), 5.08 (d, J = 10.3 Hz, 1H, H-3), 3.85 (d, J = 11.9 Hz, 1H, H-23), 3.58 (d, J = 12.0 Hz, 1H, H-23), 2.08 (s, 3H, CH₃CO), 2.02 (s, 3H, CH₃CO), 1.97 (s, 3H, CH₃CO), 1.10 (s, 3H), 1.07 (s, 3H), 0.94 (d, J = 5.3 Hz, 3H), 0.87 (s, 3H), 0.85 (d, J = 5.4 Hz, 3H), 0.76 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 183.1 (C28), 170.9 (OCO), 170.5 (OCO), 170.4 (OCO), 138.0, 125.3, 74.8, 69.9, 65.3, 52.5, 47.9, 47.6, 47.8, 43.7, 41.9, 41.9, 39.5, 39.0, 38.8, 37.8, 36.6, 32.4, 30.5, 27.9, 24.0, 23.4, 23.3, 21.1, 21.1, 20.9, 20.8, 17.9, 17.0, 16.9, 16.9, 13.9 ppm. DI-ESI-MS m/z: 615.3 ([M + H]⁺). ESI-HRMS m/z calcd for C₃₆H₅₄O₈ [M + Na]⁺: 637.3716, found: 637.3711.

4.1.7 N-Hydroxy-[2α,3β,23-triacetoxyurs-12-en-28-oyl]amine (7). To a stirred solution of 6 (153 mg, 0.25 mmol) in anhydrous THF (6.5 mL), carbonyldiimidazole (CDI) (201.35 mg, 1.24 mmol, 5 eq.) was added. The mixture was stirred at reflux temperature and under nitrogen atmosphere. After 28 h, the heating was stopped and powdered hydroxylamine hydrochloride (103.50 mg, 1.49 mmol, 6 eq.) was added. The resulting mixture was stirred at room temperature. After 23 h 35 min the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was diluted with 5% aqueous KHSO₄ (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated under vacuum to give 7 as a white powder (140.4 mg, 90%). Mp: 152.1–154.8 °C. ν_max/cm⁻¹ (ATR): 3302.2, 2947.0, 2921.6, 2870.0, 1739.0, 1455.5, 1368.5, 1228.5, 1042.5, 1028.5. ¹H NMR (400 MHz, CDCl₃): δ = 6.25 (s, 1H, NH̲–OH), 5.28 (t, J = 3.0 Hz, 1H, H-12), 5.19–5.13 (m, 1H, H-2), 5.08 (d, J = 10.3 Hz, 1H, H3), 3.85 (d, J = 11.9 Hz, 1H, H-23), 3.57 (d, J = 11.8 Hz, 1H, H-23), 2.08 (s, 3H, OCOCH₃), 2.02 (s, 3H, OCOCH₃), 1.97 (s, 3H, OCOCH₃), 1.10 (s, 3H), 1.07 (s, 3H), 0.94 (d, J = 5.8 Hz, 3H), 0.88 (s, 3H), 0.85 (d, J = 6.2 Hz, 3H), 0.75 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.4 (C28), 170.8 (OCO), 170.5 (OCO), 170.4 (OCO), 138.0 (C13), 125.5 (C12), 74.8, 69.9, 65.3, 52.6, 48.0, 47.6, 47.5, 43.7, 41.9, 41.9, 39.5, 38.9, 38.7, 37.8, 36.6, 32.4, 30.5, 27.9, 24.0, 23.5, 23.3, 21.1 (2C), 20.9, 20.8, 17.9, 17.0, 16.9 (2C), 13.9 ppm. DI-ESI-MS m/z: 630.12 ([M + H]⁺), 652.46 ([M + Na]⁺). Calcd for C₃₆H₅₅NO₈: C, 68.65; H, 8.80; N, 2.22. Found: C, 69.01; H, 9.20; N, 1.84.

4.1.8 N-Hydroxy-N-methyl-[2α,3β,23-triacetoxyurs-12-en-28-oyl]amine (8). To stirred a solution of 6 (300 mg, 0.49 mmol) in anhydrous THF (12.9 mL) was added carbonyldiimidazole (CDI) (276.90 mg, 1.71 mmol, 3.5 eq.). The mixture was stirred under reflux and nitrogen atmosphere. After 28 h the heating was stopped and powdered N-methylhydroxylamine hydrochloride (203.80 mg, 2.44 mmol 5 eq.) was added. The resulting mixture was stirred at room temperature. After 21 h the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was diluted with 5% aq. KHSO₄ (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with brine (50 mL), dried over Na₂SO₄ filtered and concentrated under vacuum to afford 8 as a white powder (308.6 mg, 98%). Mp: 119.5–121.9 °C. ν_max/cm⁻¹ (ATR): 3244.0, 2947.0, 2926.5, 2871.5, 1740.0, 1455.5, 1368.5, 1228.0, 1043.0, 1028.5. ¹H NMR (400 MHz, CDCl₃): δ = 5.29 (t, J = 3.0 Hz, 1H, H-12), 5.19–5.12 (m, 1H, H-2), 5.08 (d, J = 10.3 Hz, 1H, H-3), 3.85 (d, J = 11.9 Hz, 1H, H-23), 3.57 (d, J = 11.9 Hz, 1H, H-23), 2.76 (s, 3H, N–CH̲₃), 2.08 (s, 3H, OCOCH₃), 2.02 (s, 3H, OCOCH₃), 1.97 (s, 3H, OCOCH₃), 1.09 (s, 3H), 1.08 (s, 3H), 0.94 (d, J = 6.1 Hz, 3H), 0.88 (s, 3H), 0.85 (d, J = 6.3 Hz, 3H), 0.78 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.6 (C28), 170.9 (OCO), 170.4 (OCO), 170.4 (OCO), 137.9 (C13), 125.5 (C12), 74.8, 69.9, 65.3, 52.6, 47.7, 47.6, 47.5, 43.8, 42.1, 41.9, 39.7, 39.6, 39.0, 38.7, 37.8, 36.7, 32.5, 30.5, 27.8, 23.9, 23.4, 23.3, 21.1, 21.1, 20.9, 20.8, 17.9, 17.2, 17.0, 16.9, 13.9 ppm. DI-ESI-MS m/z: 644.21 ([M + H]⁺), 666.45 ([M + Na]⁺). Calcd for C₃₇H₅₇NO₈: C, 69.02; H, 8.92; N, 2.18. Found: C, 68.62; H, 9.30; N, 2.21.

4.1.9 N-Hydroxy-[2-acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]amine (9). To a stirred solution of compound 5 (150 mg, 0.27 mmol) in anhydrous THF (6 mL), carbonyldiimidazole (CDI) (219.22 mg, 1.35 mmol, 5 eq.) was added. The reaction mixture was stirred at reflux temperature and under nitrogen atmosphere. After 24 hours the heating was stopped and powdered hydroxylamine hydrochloride (112.75 mg, 1.62 mmol, 6 eq.) was added. The resulting mixture was stirred at room temperature. After 48 hours the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was diluted with 5% aqueous KHSO₄ (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated under vacuum to give a light yellow oil, which was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 2 : 1) to afford 9 as a white solid (46.6 mg, 30%). Mp: 86.1–88.8 °C. ν_max/cm⁻¹ (ATR): 3250.5, 2926.0, 2.871.5, 1741.0, 1380.5, 1233.0, 1029.5. ¹H NMR (400 MHz, CDCl₃): δ = 6.27 (s, 1H, NH̲OH), 5.44 (s, 1H, H-3), 5.26 (s, 1H), 4.70 (d, J = 14.2 Hz, 1H), 4.57 (d, J = 14.2 Hz, 1H), 3.93 (d, J = 10.5 Hz, 1H, H-23), 3.84 (d, J = 10.5 Hz, 1H, H-23), 2.08 (s, 3H, OCOCH₃), 2.06 (s, 3H, OCOCH₃), 1.17 (s, 3H), 1.11 (s, 3H), 1.00 (s, 3H), 0.95 (s, 3H), 0.86–0.81 (3, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.4 (C28), 171.3 (OCO), 170.8 (OCO), 151.2 (C2), 138.6 (C13), 131.8 (C3), 125.5 (C12), 72.3, 62.6, 58.1, 52.7, 50.6, 48.1, 46.3, 43.1, 42.3, 41.1, 38.7, 38.7, 36.6, 33.7, 30.5, 28.2, 26.1, 24.0, 23.8, 21.1, 21.0 (2C), 19.1, 18.5, 17.8, 17.0, 16.6 ppm. DI-ESI-MS m/z: 570.09 ([M + H]⁺).

4.1.10 2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-yl-1H-imidazole-1-carboxylate (10). To a stirred solution of compound 5 (200 mg, 0.36 mmol) in anhydrous THF (8 mL) at 60 °C, carbonyldiimidazole (CDI) (263.0 mg, 1.63 mmol, 4.5 eq.) was added. The reaction mixture was stirred overnight at reflux temperature and N₂ atmosphere. The reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with water (4 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a yellowish powder. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 3 : 1 → 2 : 1) to afford 10 as a white solid (136.3 mg, 63%). Mp: 85.4–88.0 °C. ν_max/cm⁻¹ (ATR): 3135.0, 2927.0, 2872.5, 1737.5, 1458.5, 1367.5, 1226.5, 1034.0. ¹H NMR (400 MHz, CDCl₃): δ = 8.23 (s, 1H, H2′), 7.52 (s, 1H, H5′), 7.03 (s, 1H, H4′), 5.43 (s, 1H, H-3), 5.19 (t, J = 3.0 Hz, 1H, H-12), 4.67 (d, J = 14.2 Hz, 1H), 4.54 (d, J = 14.2 Hz, 1H), 3.91 (d, J = 10.6 Hz, 1H, H-23), 3.82 (d, J = 10.6 Hz, 1H, H-23), 2.07 (s, 3H, OCOCH₃), 2.05 (s, 3H, OCOCH₃), 1.14 (s, 3H), 1.12 (s, 3H), 1.00–0.98 (m, 6H), 0.90 (d, J = 6.1 Hz, 3H), 0.74 (s, 3H) ppm. ¹³C (100 MHz, CDCl₃): δ = 174.7 (C28), 171.2 (OCO), 170.7 (OCO), 151.1 (C2), 137.7, 137.1, 132.0 (C3), 129.7, 126.1 (C12), 117.4, 72.2, 62.5, 58.1, 54.2, 50.9, 50.4, 46.3, 43.1, 42.4, 41.0, 39.0, 38.7, 35.5, 33.5, 30.3, 27.9, 26.1, 25.0, 23.8, 21.0, 20.9 (2C), 19.1, 18.3, 17.7, 17.1, 16.5 ppm. DI-ESI-MS m/z: 604.89 ([M + H]⁺). Calcd for C₃₇H₅₂N₂O₅·0.5H₂O: C, 72.40; H, 8.70; N, 4.56. Found: C, 72.66; H, 8.77; N, 4.48.

4.1.11 N-[2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]methylamine (11). To a stirred solution of 5 (200 mg, 0.36 mmol), in dry benzene (8 mL), thionyl chloride (54.90 μL, 0.76 mmol, 2.1 eq.) was slowly added. The resultant solution was heat-refluxed at 80 °C. After 3 h, the solvent was removed by evaporation under reduced pressure, and petroleum ether (approx. 2 mL) was added to the residue and concentrated to dryness to give the acyl chloride derivative. Without purification, the obtained acyl chloride was dissolved in dry dichloromethane (8 mL), basified to pH 8–9 with triethylamine, and a solution of methylamine (sol. 33% w/v in ethanol) (135.70 μL, 1.44 mmol, 4 eq.) was added. The resultant mixture was stirred at room temperature. After 45 min, the solvent was removed by evaporation under reduced pressure. The obtained crude was dispersed with water (50 mL) acidified with 5% aqueous HCl (pH 3–4) and extracted with ethyl acetate (3 × 50 mL). The combined organic phase was washed with 10% aqueous NaHCO₃ (2 × 50 mL), water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a light yellow solid. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 2 : 1) to afford 11 as a white solid (187.2 mg, 91%). Mp: 94.8–97.0 °C. ν_max/cm⁻¹ (ATR): 3418.5, 2927.0, 2870.5, 1739.5, 1635.0, 1527.0, 1454.5, 1376.5, 1235.5, 1033.5. ¹H NMR (400 MHz, CDCl₃): δ = 5.91 (d, J = 4.4 Hz, 1H, NH̲CH₃), 5.45 (s, 1H, H-3), 5.28 (t, J = 3.0 Hz, 1H, H-12), 4.69 (d, J = 14.2 Hz, 1H), 4.57 (d, J = 14.2 Hz, 1H), 3.93 (d, J = 10.6 Hz, 1H, H-23), 3.84 (d, J = 10.6 Hz, 1H, H-23), 2.72 (d, J = 4.6 Hz, 3H, NHCH̲₃), 2.08 (s, 3H, OCOCH₃), 2.06 (s, 3H, OCOCH₃), 1.17 (s, 3H), 1.11 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.86 (d, J = 6.4 Hz, 3H), 0.82 (s, 3H) ppm. ¹³C (100 MHz, CDCl₃): δ = 178.7 (C28), 171.2 (OCO), 170.8 (OCO), 150.8 (C2), 141.1 (C13), 132.1 (C3), 124.9 (C12), 72.3, 62.5, 58.0, 53.9, 50.5, 47.6, 46.3, 43.1, 42.7, 41.0, 39.4, 39.1, 36.8, 33.3, 30.8, 28.0, 26.2, 26.2, 24.9, 23.6, 21.2, 21.0, 20.9, 19.0, 18.2, 17.2, 17.3, 16.6 ppm. DI-ESI-MS m/z: 568.52 ([M + H]⁺), 590.49 ([M + Na]⁺).

4.1.12 N-[2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]ethylamine (12). Accordingly to the method described for 11, using compound 5 (150 mg, 0.27 mmol), dry benzene (6 mL), thionyl chloride (41.20 μL, 0.57 mmol, 2.1 eq.) and ethylamine (70.75 μL, 1.08 mmol, 4 eq.). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 2 : 1) to afford 12 as a white solid (113 mg, 72%). Mp: 83.5–86.0 °C. ν_max/cm⁻¹ (ATR): 3404.5, 2929.5, 2871.5, 1739.0, 1635.5, 1522.5, 1450.5, 1360.0, 1234.0, 1028.0. ¹H NMR (400 MHz, CDCl₃): δ = 5.81 (t, J = 4.4 Hz, 1H, NH̲CH₂CH₃), 5.45 (s, 1H, H-3), 5.27 (t, J = 3.0 Hz, 1H, H-12), 4.69 (d, J = 14.3 Hz, 1H), 4.56 (d, J = 14.3 Hz, 1H), 3.93 (d, J = 10.6 Hz, 1H, H-23), 3.83 (d, J = 10.6 Hz, 1H, H-23), 3.34–3.24 (m, 1H, NHCH̲₂CH₃), 3.15–3.05 (m, 1H, NHCH̲₂CH₃), 2.08 (s, 3H, OCOCH₃), 2.05 (s, 3H, OCOCH₃), 1.17 (s, 3H), 1.11 (s, 3H), 1.08 (t, J = 7.4 Hz, 3H, NHCH₂CH̲₃), 0.99 (s, 3H), 0.94 (s, 3H), 0.86–0.85 (m, 6H) ppm. ¹³C (100 MHz, CDCl₃): δ = 177.8 (C28), 171.2 (OCO), 170.1 (OCO), 150.9 (C2), 140.9 (C13), 132.1 (C3), 124.9 (C12), 72.8, 62.5, 58.0, 54.0, 50.5, 47.4, 46.3, 43.1, 42.2, 41.1, 39.4, 39.0, 37.0, 34.3, 33.5, 30.8, 28.0, 26.2, 24.7, 23.4, 21.2, 21.0, 20.9, 19.0, 18.5, 17.8, 17.3, 16.6, 14.5 ppm. DI-ESI-MS m/z: 582.50 ([M + H]⁺), 604.51 ([M + Na]⁺). Calcd for C₃₆H₅₅NO₅·0.25H₂O: C, 73.74; H, 9.54; N, 2.39. Found: C, 73.65; H, 9.93; N, 2.43.

4.1.13 N-[2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]propylamine (13). Accordingly to the method described for 11, using compound 5 (200 mg, 0.36 mmol), dry benzene (8 mL), thionyl chloride (54.90 μL, 0.76 mmol, 2.1 eq.), dichloromethane (8 mL) and propylamine (118.50 μL, 1.44 mmol, 4 eq.). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 3 : 1) to afford 13 as a white solid (122 mg, 57%). Mp: 77.0–79.5 °C. ν_max/cm⁻¹ (ATR): 3368.5, 2959.0, 2921.5, 2872.5, 1739.5, 1635.0, 1525.0, 1455.5, 1369.0, 1234.5, 1033.5. ¹H NMR (400 MHz, CDCl₃): δ = 5.87 (t, J = 4.3 Hz, 1H, NH̲CH₂CH₂CH₃), 5.44 (s, 1H, H-3), 5.27 (t, J = 3.0 Hz, 1H, H-12), 4.68 (d, J = 14.2 Hz, 1H), 4.56 (d, J = 14.2 Hz, 1H), 3.92 (d, J = 10.6 Hz, 1H, H-23), 3.83 (d, J = 10.6 Hz, 1H, H-23), 3.32–3.24 (m, 1H, NHCH̲₂CH₂CH₃), 2.99–2.92 (m, 1H, NHCH̲₂CH₂CH₃), 2.07 (s, 3H, OCOCH₃), 2.05 (s, 3H, OCOCH₃), 1.16 (s, 3H), 1.11 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.89 (t, J = 7.5 Hz, 7.49 Hz, 3H, NHCH₂CH₂CH̲₃), 0.86 (d, J = 6.6 Hz, 3H), 0.84 (s, 3H) ppm. ¹³C (100 MHz, CDCl₃): δ = 177.8 (C28), 171.2 (OCO), 170.7 (OCO), 150.9 (C2), 140.9 (C13), 132.1 (C3), 124.9 (C12), 72.3, 62.5, 58.0, 54.0, 50.5, 47.6, 46.3, 43.1, 42.8, 41.1, 41.1, 39.4, 39.0, 37.1, 33.5, 30.8, 28.0, 26.2, 24.7, 23.5, 22.5, 21.2, 20.9, 20.9, 19.0, 18.5, 17.8, 17.3, 16.6, 11.5 ppm. DI-ESI-MS m/z: 596.54 ([M + H]⁺), 618.53 ([M + Na]⁺).

4.1.14 N-[2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]4-methylbenzylamine (14). Accordingly to the method described for 11, using compound 5 (150 mg, 0.27 mmol), dry benzene (6 mL), thionyl chloride (41.20 μL, 0.57 mmol, 2.1 eq.), dichloromethane (6 mL) and 4-methylbenzilamine (137.70 μL, 1.08 mmol, 4 eq.). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1) to afford 14 as a white solid (140.3 mg, 79%). Mp: 82.1–84.9 °C. ν_max/cm⁻¹ (ATR): 3367.0, 2923.0, 2870.5, 1739.5, 1635.5, 1516.5, 1455.5, 1376.0, 1235.0, 1031.0. ¹H NMR (400 MHz, CDCl₃): δ = 7.13 (s, 4H, Ar-H), 6.07 (t, J = 4.2 Hz, 1H, NH̲CH₂ArCH₃), 5.45 (s, 1H, H-3), 5.18 (t, J = 2.6 Hz, 1H, H-12), 4.67 (d, J = 14.2 Hz, 1H), 4.56 (d, J = 14.3 Hz, 1H), 4.49 (dd, J₁ = 14.4 Hz, J₂ = 5.9 Hz, 1H, NHCH̲₂ArCH₃), 4.13 (dd, J₁ = 14.5 Hz, J₂ = 4.3 Hz, 1H, NHCH̲₂ArCH₃), 3.93 (d, J = 10.6 Hz, 1H, H-23), 3.84 (d, J = 10.6 Hz, 1H, H-23), 2.34 (s, 3H, NHCH₂ArCH̲₃), 2.08 (s, 3H, OCOCH₃), 2.06 (s, 3H, OCOCH₃), 1.14 (s, 3H), 1.11 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.84 (d, J = 6.6 Hz, 3H), 0.75 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.6 (C28), 171.2 (OCO), 170.8 (OCO), 150.9 (C2), 140.7, 137.1, 135.3 (Ar-C), 132.0 (C3), 129.3 (2C, Ar-C), 128.1 (2C, Ar-C), 125.1 (C12), 77.3, 62.5, 58.0, 54.0, 50.3, 47.7, 46.3, 43.4, 43.1, 42.8, 41.1, 39.4, 39.0, 37.1, 33.5, 30.8, 28.0, 26.1, 24.7, 23.5, 21.2, 21.1, 20.9 (2C), 19.0, 18.6, 17.8, 17.2, 16.6 ppm. DI-ESI-MS m/z: 658.29 ([M + H]⁺), 680.40 ([M + Na]⁺). Calcd for C₄₂H₅₉NO₅: C, 76.67; H, 9.04; N, 2.13. Found: C, 76.32; H, 9.14; N, 2.23.

4.1.15 N-[2-Acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]4-fluorobenzylamine (15). According to the method described for 11, using compound 5 (150 mg, 0.27 mmol), dry benzene (6 mL), thionyl chloride (41.20 μL, 0.57 mmol, 2.1 eq.), dichloromethane (6 mL) and 4-fluorobenzylamine (126.60 μL, 1.08 mmol, 4 eq.). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 2 : 1) to afford 15 as a white solid (108.8 mg, 61%). Mp: 82.9–85.3 °C. ν_max/cm⁻¹ (ATR): 3413.0, 2928.0, 2870.5, 1739.0, 1635.5, 1509.5, 1455.0, 1381.0, 1220.5, 1033.5. ¹H NMR (400 MHz, CDCl₃): δ = 7.22–7.19 (m, 2H), 7.00 (t, J = 8.61 Hz, 2H), 6.13 (t, J = 5.1 Hz, 1H, NH̲CH₂ArF), 5.44 (s, 1H, H-3), 5.19 (t, J = 2.5 Hz, 1H, H-12), 4.67 (d, J = 14.3 Hz, 1H), 4.55 (d, J = 14.3 Hz, 1H), 4.49 (dd, J₁ = 14.5 Hz, J₂ = 5.9 Hz, 1H, NHCH̲₂ArF), 4.14 (dd, J₁ = 14.9 Hz, J₂ = 4.7 Hz, 14.6 Hz, 1H, NHCH̲₂ArF), 3.92 (d, J = 10.6 Hz, 1H, H-23), 3.83 (d, J = 10.7 Hz, 1H, H-23), 2.07 (s, 3H, OCOCH₃), 2.05 (s, 3H, OCOCH₃), 1.13 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.84 (d, J = 6.2 Hz, 3H), 0.72 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.8 (C28), 171.2 (OCO), 170.7 (OCO), 162.1 (d, J = 245.2 Hz, C4′′), 150.9 (C2), 140.7 (C13), 134.2 (C1′′), 132.1 (C3), 129.6, 129.5, 125.2 (C12), 115.5, 115.3, 72.2, 62.5, 58.0, 54.0, 50.5, 47.7, 46.3, 43.1, 42.9, 42.7, 41.1, 39.4, 39.0, 37.2, 33.5, 30.8, 28.0, 26.1, 24.7, 23.4, 21.1, 20.9 (2C), 19.0, 18.5, 17.8, 17.2, 16.6 ppm. DI-ESI-MS m/z: 662.40 ([M + H]⁺). Calcd for C₄₁H₅₆FNO₅·0.25H₂O: C, 73.90; H, 8.55; N, 2.10. Found: C, 73.95; H, 8.60; N, 2.23.

4.1.16 Methyl N-[2-acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]gycinate (16). To a stirred solution of compound 5 (200 mg, 0.36 mmol), in dry benzene (8 mL), thionyl chloride (54.90 μL, 0.76 mmol, 2.1 eq.) was slowly added. The resultant solution was heat-refluxed at 80 °C. After 3 h, the solvent was removed by evaporation under reduced pressure, and petroleum ether (approx. 2 mL) was added to the residue, concentrated to dryness to give the acyl chloride. Without purification the acyl chloride was dissolved in dichloromethane (8 mL), basified to pH 8–9 with triethylamine, and glycine methyl ester hydrochloride (72.40 mg, 0.58 mmol, 1.6 eq.) was added. The resultant mixture was stirred at room temperature. After 45 min, the solvent was removed by evaporation under reduced pressure. The obtained crude was dispersed with water (50 mL) acidified with 5% aqueous HCl (pH 3–4) and extracted with ethyl acetate (3 × 60 mL). The combined organic phase was washed with 10% aqueous NaHCO₃ (2 × 50 mL), water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a light yellow solid. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 2 : 1) to afford 16 as a white solid (179 mg, 79%). Mp: 74.5–76.6 °C. ν_max/cm⁻¹ (ATR): 3395.0, 2923.5, 2871.5, 1739.0, 1650.0, 1520.5, 1451.0, 1368.0, 1234.0, 1031.0. ¹H NMR (400 MHz, CDCl₃): δ = 6.50 (t, J = 4.1 Hz, 1H, –NH̲CH₂COOCH₃), 5.44 (s, 1H, H-3), 5.38 (t, J = 3.1 Hz, 1H, H-12), 4.69 (d, J = 14.3 Hz, 1H), 4.57 (d, J = 14.2 Hz, 1H), 4.09 (dd, J₁ = 18.5 Hz, J₂ = 5.3 Hz and, 1H, –NHCH̲₂COOCH₃), 3.93 (d, J = 10.5 Hz, 1H, H-23), 3.84 (dd, J₁ = 18.5 Hz, J₂ = 3.9 Hz, 1H, –NHCH̲₂COOCH₃), 3.83 (d, J = 10.7 Hz, 1H, H-23), 3.75 (s, 3H, –NHCH₂COOCH̲₃), 2.08 (s, 3H, OCOCH₃), 2.06 (s, 3H, OCOCH₃), 1.15 (s, 3H), 1.12 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.88 (d, J = 6.3 Hz, 3H), 0.77 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.1, 171.2, 170.7 (OCO), 170.6 (OCO), 151.0 (C2), 140.0 (C13), 131.9 (C3), 125.7 (C12), 72.3, 62.5, 58.0, 53.7, 52.3, 50.5, 47.7, 46.3, 43.2, 42.6, 41.5, 41.1, 39.4, 39.0, 36.9, 33.5, 30.8, 28.0, 26.3, 24.8, 23.6, 21.2, 21.0, 21.0, 19.0, 18.0, 17.8, 17.2, 16.6 ppm. DI-ESI-MS m/z: 626.27 ([M + H]⁺), 648.42 ([M + Na]⁺).

4.1.17 Methyl N-[2-acetoxymethyl-23-acetoxy-A(1)-norursa-2,12-dien-28-oyl]alaninate (17). Accordingly to the method described for 16, using compound 5 (200 mg, 0.36 mmol), dry benzene (8 mL), thionyl chloride (54.90 μL, 0.76 mmol, 2.1 eq.), dichloromethane (8 mL) and L-alanine methyl ester hydrochloride (80.51 mg, 0.58 mmol, 1.6 eq.). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 3 : 1) to afford 17 as a white solid (140 mg, 61%). Mp: 68.9–71.6 °C. ν_max/cm⁻¹ (ATR): 3405.0, 2925.0, 2872.0, 1738.0, 1655.5, 1507.5, 1448.5, 1371.5, 1234.5, 1028.0. ¹H NMR (400 MHz, CDCl₃): δ = 6.59 (d, J = 5.8 Hz, 1H, NH̲CH(CH₃)COOH), 5.43 (s, 1H, H-3), 5.36 (t, J = 3.0 Hz, 1H, H-12), 4.69 (d, J = 14.4 Hz, 1H), 4.57 (d, J = 14.4 Hz, 1H), 4.50–4.42 (m, 1H, NHCH̲(CH₃)COOCH₃), 3.93 (d, J = 10.7 Hz, 1H, H-23), 3.83 (d, J = 10.6 Hz, 1H, H-23), 3.73 (s, 3H, NHCH(CH₃)COOCH̲₃), 2.08 (s, 3H, OCOCH̲₃), 2.05 (s, 3H, OCOCH̲₃), 1.36 (d, J = 7.0 Hz, 3H, NHCH(CH̲₃)COOCH₃), 1.15 (s, 3H), 1.11 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.75 (s, 3H) ppm. ¹³C (100 MHz, CDCl₃): δ = 177.3, 173.7, 171.2 (OCO), 170.2 (OCO), 151.1 (C2), 139.3 (C13), 131.7 (C3), 125.8 (C12), 77.3, 62.5, 58.0, 53.6, 52.4, 50.6, 48.2, 47.6, 46.3, 43.2, 42.6, 41.2, 39.3, 39.0, 37.2, 33.6, 30.8, 28.0, 26.3, 24.6, 23.5, 21.2, 21.0 (2C), 19.0, 18.7, 18.1, 17.8, 17.2, 16.6 ppm. DI-ESI-MS m/z: 640.20 ([M + H]⁺).

4.2 Biology

4.2.1 Cells and reagents. MCF-7, HT-29, Jurkat, PC-3, HeLa and BJ cell lines were obtained from American Type Culture Collection (USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Phosphate Buffered Saline (DPBS) and L-glutamine were obtained from Biowest. Minimum Essential Medium (MEM), penicillin/streptomycin solution and Fetal Bovine Serum (FBS) were obtained from Gibco. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder and the XTT cell proliferation kit were purchased from Applichem Panreac. Sodium pyruvate solution 100 mM and trypsin/EDTA were obtained from Biological Industries. Sodium bicarbonate solution 7.5% and glucose solution 45% were purchased from Sigma-Aldrich Co. Cisplatin was obtained from Sigma Aldrich.

Asiatic acid and its derivatives were suspended in dimethyl sulfoxide (DMSO) to prepare 20 mM stock solutions that were stored at −80 °C. To obtain final assay concentrations, the stock solutions were diluted in culture medium. The final concentration of DMSO in working solutions was always equal or lower than 0.5%.

4.2.2 Cell culture. HT-29, PC-3, and HeLa cells were grown in DMEM high glucose supplemented with 10% heat-inactivated FBS and 100 units per mL penicillin and 100 μg mL⁻¹ streptomycin. MCF-7 cells were maintained in MEM 1× supplemented with 10% heat-inactivated FBS, 10 units per mL penicillin, 10 μg mL⁻¹ streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.01 mg mL⁻¹ insulin, 10 mM glucose and 1× MEM-EAGLE non essential amino acids. BJ cells were cultured in DMEM high glucose supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 100 units per mL penicillin, 100 μg mL⁻¹ streptomycin and 1.5 g L⁻¹ sodium bicarbonate. Jurkat cells were grown in RPMI 1640 supplemented with 10% FBS, 100 units per mL penicillin, 100 μg mL⁻¹ streptomycin and 2 mM L-glutamine. All cell lines were incubated in a humidified atmosphere of 5.0% CO₂ at 37 °C.

4.2.3 Cell viability assay. For MCF-7, HT-29, PC-3, HeLa and BJ cell lines cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, cells were seeded at a concentration of 8 × 10² to 1 × 10⁴ cells per well in 96-well plates and were left to grow. After 24 h, the culture medium was removed and replaced by new medium containing the tested compounds at different concentrations. After 72 h of incubation, cells were incubated with 100 μL of MTT solution (0.5 mg mL⁻¹) for 1 h. After incubation, the MTT solution was removed and 100 μL of DMSO were added to each well to dissolve the formazan crystals. The relative cell viability, compared to the viability of non treated cells, was analyzed by measuring the absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Salzburg, Austria).

For Jurkat cells, the cell viability was determined by XTT assay. Briefly, 4 × 10³ cells per well were seeded in 96 well plates with 100 μL of medium. After 24 h of incubation, 100 μL of new medium containing the tested compounds at different concentrations was added. After an incubation period of 72 hours, 100 μL of XTT solution were added to each well and the plates were incubated for 4 hours at 37 °C. The relative cell viability, compared with the viability of non treated cells, was analyzed by measuring the absorbance at 450 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Salzburg, Austria).

4.2.4 Cell cycle assay. 2.2 × 10⁴ HeLa cells were seeded per well in 6-well plates with 2 mL of medium. After an incubation period of 24 h, cells were treated with specified concentrations of compound 7. After 24 h of incubation, cells were harvested by mild trypsinization, centrifuged, washed twice with PBS and stained in Tris-buffered saline (TBS) containing 50 mg mL⁻¹ PI, 10 mg mL⁻¹ DNase-free RNase and 0.1% Igepal CA-630, for 1 hour, at 4 °C in darkness. The cell cycle was assessed by flow cytometry, using a fluorescence-activated cell sorter (FACS), carried out at 488 nm in an Epics XL flow cytometer (Coulter Corporation, Hialeah, FL). Data from 1 × 10⁴ cells was collected and analysed using the multicycle software (Phoenix Flow Systems, San Diego, CA).

4.2.5 Annexin V-FITC/PI flow cytometry assay. HeLa cells were plated in 6-well plates at a density of 2.2 × 10⁴ cells per well. After 24 h, the cells with or without pretreatment for 45 min of 50 μM z-VAD-fmk, were treated with compound 7 at the indicated concentrations. After 24 h of incubation, cells were harvested by mild trypsinization, collected by centrifugation and suspended in 95 μL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 10 mM NaCl, 2.5 mM CaCl₂). Cells were stained with Annexin V-FITC conjugate for 30 min at room temperature, protected from light. After incubation period, 500 μL of binding buffer were added to each vial of cells and approximately 2 min before FACS analysis, 20 μL of 1 mg mL⁻¹ PI solution were also added. The samples were analysed by flow cytometry.

4.2.6 Fluorescent microscopic observation after Hoechst 33258 staining. 2.2 × 10⁴ cells per well were seeded in 6-well plates with 2 mL of medium and incubated for 24 h. Following incubation, the cells were treated with indicated concentrations of compound 7. After 24 h of treatment, the culture medium was removed and cells were harvested by mild trypsinization and collected by centrifugation. After being washed twice with PBS, the cells were stained with 500 μL of Hoechst solution (2 μg mL⁻¹ in PBS) for 15 min, in darkness. The Hoechst solution was removed and cells were washed twice with PBS, resuspended in 10 μL of PBS and then mounted in a slide. The morphological modifications were then analysed by fluorescence microscopy using a fluorescence microscope DMRB (LeicaMicrosystems, Wetzlar, Germany) with a DAPI filter.

4.2.7 Synergy study. Briefly, 7.5 × 10² cells per well were seeded in 96-well plates. After 24 h cells were treated with compound 7 and cisplatin at different concentrations (the ratio between the concentrations of cisplatin and compound 7 was constant: the concentration of cisplatin was always 2 times higher than concentration of compound 7). After 72 h of incubation, the medium in each well was replaced by 100 μL of filtered MTT solution (0.5 mg mL⁻¹). Following 1 hour, the MTT solution was replaced by 100 μM of DMSO. The relative cell viability, compared to the viability of untreated cells, was analyzed by measuring the absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Salzburg, Austria). The effect of compound 7 and cisplatin, alone or in combination, over HeLa cells viability was evaluated and the results were analyzed by program CompuSyn, which was used to calculate the CI (Combination Index). A CI value below than 1 indicates the existence of synergism.

Acknowledgements

Jorge A. R. Salvador thanks Universidade de Coimbra for financial support. Bruno M. F. Goncalves thanks Fundação para a Ciência e a Tecnologia for financial support (SFRH/BD/69193/2010). MC and SM thanks MICINN of Spain and FEDER Funds (grant number SAF2011-25726) and Agència Catalana d'Ajuts Universitaris I de Recerca (AGAUR) (2014SGR1017). MC thanks also ICREA Foundation (Icrea Academia Award programme, Generalitat de Catalunya). The authors would like to acknowledge UC-NMR facility, which is supported in part by FEDER – European Regional Development Fund through the COMPETE Programme (Operational Programme for Competitiveness) and by National Funds through FCT – Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) through grants REEQ/481/QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear (RNRMN), for NMR data. The authors are grateful to Laboratory of Mass Spectrometry (LEM) of the Node CEF/UC integrated in the National Mass Spectrometry Network (RNEM) of Portugal, for the MS analyses. We acknowledge Fundação para a Ciência e Tecnologia (FCT) for the project REDE/1501/REM/2005 and Dr Carlos Cordeiro for providing data from the FTICR-MS at the Faculdade de Ciências da Universidade de Lisboa, Portugal. The authors would like to acknowledge Centro de Apoio Científico e Tecnolóxico á Investigación (C.A.C.T.I.), Universidade de Vigo, Campos Lagoas – Marcosende, 15, 36310 Vigo, for elemental analysis and CCIT-scientific and technological centers UB for flow cytometry analysis support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04597a

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