Synthesis and biological evaluation of novel asiatic acid derivatives with anticancer activity

Abstract

Asiatic acid (AA) is a pentacyclic triterpenoid with recognized anticancer properties. Structural modification of AA may afford derivatives with improved anticancer potency. Hence, in this paper, a series of new lactol and A-nor AA derivatives were prepared, and their antiproliferative activities against several human cancer cell lines and a non-tumoral fibroblast cell line (BJ) were tested. Among all the derivatives tested, compound 24 proved to be the most active compound, with IC₅₀ values ranging from 0.11 μM to 0.65 μM for cancer cell lines. The molecular mechanisms underlying its antiproliferative activity were further investigated using the HeLa cell line. Our results showed that treatment of HeLa cells with compound 24 led to cell-cycle arrest at the G0/G1 phase, which was associated with an upregulation of p21^cip1/waf1 and p27^kip1 and a downregulation of cyclin D₃. Moreover, compound 24 induced apoptotic HeLa cell death via the activation of caspase-9, caspase-8, and caspase-3. The downregulation of the Bcl-2 and Bid proteins and the upregulation of the Bax protein suggest that the mitochondrial pathway is activated during the apoptotic process.

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1. Introduction

Despite recent advances in cancer therapy, this complex disease remains one of the leading causes of death worldwide.^1,2 To solve this public health problem, research efforts toward the development of new therapeutic strategies and more effective anticancer drugs have been made.

Pentacyclic triterpenoids (PTs) are a large class of natural products that are widely distributed in nature and have a vast range of unique biological activities.^3–5 In the last decades, the anticancer and anti-inflammatory activities of PTs and their semi-synthetic derivatives have been extensively studied, and some papers support the contention that these compounds are ideal candidates for the development of new anticancer therapies.^4,6–11 Asiatic acid (AA) (1) (Fig. 1) is a PT that is mainly extracted from Centella asiatica that, in addition to other important pharmacological activities, shows promising anticancer effects. The mechanisms underlying the anticancer effect of AA (1) include cell-cycle arrest,¹² strong antiangiogenic activity,¹³ inhibition of cancer cell proliferation, and induction of apoptosis in several cancer cell lines.^13–16

Fig. 1 Chemical structure of asiatic acid 1.

Apoptotic cell death induced by AA (1) has been reported to be mediated by alterations in calcium homeostasis in the HepG2, PCC-1, and U87-MG cancer cell lines;^14,16,17 activation of the mitochondrial pathway in SW480 human colon cancer cells;¹⁸ and generation of reactive oxygen species (ROS), upregulation of BAX, and activation of caspase-3 in SK-MEL-2 human melanoma cells.¹⁵

Because of its promising anticancer activity, low toxicity, and commercial availability, AA (1) has been receiving increased attention from scientists who aim for the development of new anticancer drugs. Several groups performed structural modifications of the AA (1) backbone; some of the semi-synthetic derivatives obtained displayed improved antiproliferative activity in several cancer cell lines compared with AA (1).^19–22 However, further investigation is needed to develop and synthesize new AA (1) derivatives as anticancer agents.

Bhagirath Sing and coworkers reported the conversion of the hexameric ring A of AA (1) into a pentameric ring containing an α,β-unsaturated carbonyl group.²³ It has been well established in several studies that the presence of an α,β-unsaturated carbonyl group in the A-ring of PTs significantly enhances their biological activities.^4,24–26 However, the transformations performed in the A ring of AA (1) were not thoroughly explored, which encouraged us to develop and synthesize a series of new AA (1) derivatives containing a 5-carbon ring A with an α,β-unsaturated carbonyl moiety, combined with additional modifications at C-23, C-11, and C-28, to obtain AA (1) derivatives with improved anticancer activity.

In addition, the nitrile group has been gaining great importance in the design and development of new drugs^27,28, because its presence in organic molecules plays several biologically important roles.^27–32 This functional group could work as a hydroxyl and carboxyl surrogate, as nitrile is a strong hydrogen acceptor.²⁷ Taking this into account, we also designed and prepared a series of pentameric A-ring AA (1) derivatives containing a nitrile group.

The in vitro antiproliferative activities of the newly synthesized derivatives against the MCF-7, HT-29, and HeLa cell lines were tested, and a structure–activity relationship (SAR) was established. Most of the new derivatives showed improved cytotoxic activities compared with AA (1). Compound 24 exhibited the best antiproliferative profile among all new derivatives and was selected for further experiments aimed at exploring its mechanism of action in HeLa cells.

2. Results and discussion

2.1 Chemistry

A series of new AA (1) derivatives containing a pentameric ring A were synthesized and their structures were fully elucidated. The synthesis started by the treatment of AA (1) with anhydrous potassium carbonate (K₂CO₃) and methyl iodide or ethyl iodide in DMF, to give the methyl derivative 2 and the ethyl derivative 3, respectively, in almost quantitative yields (Scheme 1). The lactol derivatives 4, 5, and 6 were obtained in good yield via the reaction of compounds 1, 2, and 3, respectively, with sodium periodate (NaIO₄) in methanol/water.²³ Further treatment of compounds 4, 5, and 6 with catalytic amounts of piperidine and acetic acid in benzene at 60 °C afforded the pentameric A-ring derivatives 7, 8, and 9, respectively.²³

Scheme 1 Synthesis of asiatic acid derivatives 1–9. Reagents and conditions: (a) CH₃I, K₂CO₃, DMF, r.t.; (b) CH₃CH₂I, K₂CO₃, DMF, r.t.; (c) NaIO₄, MeOH/H₂O, r.t.; (d) i – acetic acid, piperidine, dry benzene, reflux 60 °C, N₂; ii – anhydrous MgSO₄, reflux 60 °C, N₂.

The proton of the aldehyde group of the lactol derivatives 4–6 appeared as a singlet at δ = 9.94 ppm on ¹H NMR spectra, and the ¹³C NMR signal for the CHO carbon was observed around δ = 206 ppm. In the ¹H NMR spectra of compounds 7–9, the signals of the aldehyde and olefinic protons in the A ring were consistently observed as two singlet signals at δ = 9.72 ppm and δ = 6.66 ppm, respectively. The ¹³C NMR signal for the CHO carbon was observed at δ = 190.81–190.85 ppm, whereas the signals for the carbons of the A ring double bond appeared at δ = 159.24–159.29 ppm and δ = 158.90–159.95 ppm. The characteristic IR bands for the C=O and C=C stretching vibrations of the α,β-unsaturated aldehydes were observed around 1689 cm⁻¹ and 1581 cm⁻¹, respectively.

To study the impact of the introduction of a carbonyl group at C-11 on the anticancer activity, we decided to synthesize several AA (1) derivatives with an α,β-unsaturated ketone in the C ring. As shown in Scheme 2, the reaction of compound 2 with acetic anhydride in the presence of DMAP in THF at room temperature gave compound 10, which was further oxidized with a mixture of potassium permanganate and iron sulfate, to afford the 11-oxo-12-en derivative 11 in 95% yield.^33,34 Compound 11 was then deacetylated with potassium hydroxide (KOH) in methanol, to afford intermediate 12. The lactol derivative 13 was prepared by reaction of compound 12 with sodium periodate (NaIO₄) in methanol/water at room temperature. The α,β-unsaturated aldehyde 14 was obtained from 13 using the procedure that was described previously for the preparation of compounds 7–9.

Scheme 2 Synthesis of asiatic acid derivatives 10–14. Reagents and conditions: (a) acetic anhydride, DMAP, THF, r.t.; (b) KMnO₄, Fe₂(SO₄)₃·nH₂O, t-BuOH, H₂O, CH₂Cl₂, r.t.; (c) KOH, MeOH, reflux, 30 min; (d) NaIO₄, MeOH/H₂O, r.t.; (e) i – acetic acid, piperidine, dry benzene, reflux 60 °C, N₂; ii – anhydrous MgSO₄, reflux 60 °C, N₂.

Taking into consideration that the nitrile group can act as a carbonyl bioisostere,²⁷ and considering its pharmaceutical importance, we prepared a panel of AA (1) nitrile derivatives. The treatment of compounds 7, 8, and 14 with iodine and 25% aqueous ammonium solution in THF at room temperature³⁵ afforded the derivatives 17, 16, and 18, respectively, which have an α,β-unsaturated nitrile group in the pentameric A ring (Scheme 3). The direct conversion of aldehyde into nitrile was confirmed by the specific IR absorption band for CN stretching vibration observed at 2215.8–2217.7 cm⁻¹ and by the ¹³C NMR signal for the CN carbon observed around δ = 117 ppm. Moreover, no signal corresponding to the proton of the CHO group was observed on the ¹H NMR spectra. These structural data were consistent for all derivatives that had a pentameric A ring with an α,β-unsaturated nitrile (16–18 and 25–31).

Scheme 3 Synthesis of asiatic acid derivatives 15–18. Reagents and conditions: (a) methanesulfonyl chloride, triethylamine, dry CH₂Cl₂, r.t. (b) I₂, aq. NH₃, THF, r.t.

In addition, we decided to investigate the influence of C-23 hydroxyl substitution on the anticancer activity of compounds 14 and 16. Thus, starting from these two compounds, we prepared a panel of C-23-substituted ester, carbamate, and mesylate derivatives (Schemes 4 and 5). The ester derivatives 19, 20, and 25–28 were prepared in moderate-to-good yields via the reaction of compound 14 or compound 16 with the corresponding anhydrides in the presence of DMAP at room temperature. The treatment of 14 or 16 with cinnamoyl chloride and DMAP in dry benzene at 60 °C afforded the 23-cinnamic ester derivatives 21 and 29, respectively. As shown in Scheme 4, compound 14 was treated with CDI or CBMI in THF at reflux, to give the carbamate derivatives 22 and 23 in 12% and 33% yield, respectively, after FCC. Derivative 30 was prepared in 59% isolated yield via the reaction of 16 with CBMI in THF at 70 °C (Scheme 5). The successful preparation of the carbamate derivatives 22, 23, and 30 was confirmed by the IR band observed at 1747–1764 cm⁻¹, corresponding to the C=O stretching vibration, and by the ¹³C NMR signal for the carbamate carbonyl carbon observed at 148.56–149.47 ppm. In addition, in the ¹H NMR spectra of compounds 23 and 30, the two specific hydrogen atoms of the methyl imidazole moiety appeared as two peaks at 7.27–7.28 ppm and 6.84–6.87 ppm. In the case of the derivative 22, the imidazole protons appeared as three singlets at 8.09, 7.30, and 7.06 ppm (ref. 36 and 37) on the ¹H NMR spectra.

Scheme 4 Synthesis of asiatic acid derivatives 19–24. Reagents and conditions: (a) acetic anhydride or butyric anhydride, DMAP, THF, r.t.; (b) cinnamoyl chloride, dry benzene, DMAP, 60 °C, N₂; (c) CDI or CBMI, THF, 70 °C, N₂; (d) methanesulfonyl chloride, triethylamine, dry CH₂Cl₂, r.t.

Scheme 5 Synthesis of asiatic acid derivatives 25–31. Reagents and conditions: (a) acetic or butyric or benzoic anhydride, DMAP, THF, r.t.; (b) succinic anhydride, DMAP, CH₂Cl₂, r.t.; (c) cinnamoyl chloride, dry benzene, DMAP, 60 °C, N₂; (d) CBMI, THF, 70 °C, N₂; (e) methanesulfonyl chloride, triethylamine, dry CH₂Cl₂, r.t.

Finally, the 23-methanesulfonyloxy derivatives 15, 24, and 31 were obtained in moderate yields by treatment of compounds 8, 14, and 16, respectively, with methanesulfonyl chloride and triethylamine in dry dichloromethane at room temperature (Schemes 3–5). The successful preparation of compounds 15, 24, and 31 was confirmed by the characteristic IR absorptions for the asymmetric and symmetric S=O vibrations observed around 1355–1359 cm⁻¹ and 1174–1176 cm⁻¹. In the ¹H NMR spectra, the protons from the mesylate methyl group were observed as a singlet at 3.01–3.03 ppm.

2.2 Biological activities

2.2.1 Evaluation of antiproliferative activity. The antiproliferative activity of AA (1) and all the newly synthesized derivatives against MCF-7 (human breast adenocarcinoma), HT-29 (human colon adenocarcinoma), and HeLa (human cervix adenocarcinoma) cell lines was evaluated by MTT assay. The intermediates (2, 4, 5, 7, 8, 10, 11, 12) and the control drug cisplatin were tested against the HeLa cell line. The cell lines were treated with increasing concentrations of each compound, and the IC₅₀ values (concentration that inhibited 50% of cell growth) were determined after 72 h of incubation.

The analysis of IC₅₀ values (Table 1) revealed that the great majority of the new derivatives showed better antiproliferative activities than AA (1) against the tested cell lines. These new derivatives were particularly active against the HeLa cell line, with the exception of compound 29; therefore, their antiproliferative activities against HeLa cells were used to establish a SAR (Fig. 2).

Table 1 Cytotoxic activities (expressed as IC₅₀) of AA, its derivatives and cisplatin against breast (MCF-7), colon (HT-29) and cervix (HeLa) cancer cell lines

Compound	Cell line^a/IC50 (μM)
	MCF-7	HT-29	HeLa
a The cell lines were treated with increasing concentrations of each compound for 72 h. IC50 values were determined by MTT assay and are expressed as means ± SD (standard deviation) of three independent experiments. N.D. not determined. IC50 is the concentration of compound that inhibits 50% of cell growth.
Asiatic acid (1)	68.50 ± 2.12	64.33 ± 3.21	52.47 ± 0.06
2	N.D.	N.D.	27.50 ± 2.50
3	N.D.	N.D.	20.67 ± 1.53
4	N.D.	N.D.	21.67 ± 1.04
5	N.D.	N.D.	4.70 ± 0.40
6	4.00 ± 0.02	5.70 ± 0.46	4.75 ± 0.21
7	N.D.	N.D.	5.30 ± 0.2
8	N.D.	N.D.	0.60 ± 0.07
9	0.70 ± 0.05	0.64 ± 0.05	0.51 ± 0.03
10	N.D.	N.D.	0.60 ± 0.04
11	N.D.	N.D.	3.13 ± 0.32
12	N.D.	N.D.	37.17 ± 2.57
13	12.03 ± 0.40	12.20 ± 0.26	8.48 ± 1.31
14	0.74 ± 0.05	0.59 ± 0.04	0.30 ± 0.02
15	0.47 ± 0.02	0.45 ± 0.05	0.30 ± 0.02
16	14.33 ± 1.53	10.27 ± 1.55	8.90 ± 0.53
17	>60	>60	43.17 ± 3.55
18	16.10 ± 1.40	17.00 ± 1.32	14.60 ± 1.65
19	1.02 ± 0.11	0.97 ± 0.09	0.60 ± 0.05
20	0.88 ± 0.07	0.77 ± 0.03	0.49 ± 0.00
21	1.03 ± 0.04	0.84 ± 0.05	0.53 ± 0.01
22	1.20 ± 0.05	0.73 ± 0.02	0.53 ± 0.03
23	0.98 ± 0.02	0.66 ± 0.05	0.54 ± 0.02
24	0.65 ± 0.04	0.59 ± 0.02	0.24 ± 0.02
25	11.50 ± 1.32	12.20 ± 1.04	10.75 ± 0.35
26	>30	>60	12.00 ± 1.41
27	>60	>60	15.50 ± 0.71
28	30.00 ± 1.41	>30	26.80 ± 2.34
29	>60	>60	>60
30	7.00 ± 0.00	6.13 ± 0.32	6.33 ± 0.15
31	19.50 ± 2.12	9.77 ± 0.23	7.27 ± 0.91
Cisplatin	19.10 ± 4.50	6.11 (ref. 41)	2.28 ± 0.26

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Fig. 2 Schematic representation of the SAR for the antiproliferative activity against HeLa cell line. The SAR was established based on IC₅₀ values. The derivatives with pentameric ring A containing an α,β-unsaturated carbonyl moiety showed the higher antiproliferative activities, while the compounds containing an hexameric ring A showed the lower antiproliferative activities. The introduction of the methanesulfonyloxy group at C-23 significantly increases the antiproliferative activity. Derivative 2, is 2-fold more potent than asiatic acid 1. Derivatives 8 and 14 are 46-fold and 92-fold more potent than derivative 2, respectively. The most active compound 24 is 115-fold more potent than derivative 2.

According to previous studies, the small carbon chain ester derivatives at C-28 of ursane-type triterpenoids present increased cytotoxic activity.^38,39 Similar results were obtained in our study, the methyl ester derivatives 2, 5, and 8 and the ethyl ester derivatives 3, 6, and 9 exhibited increased cytotoxic activity compared with the parent compounds that had a free carboxyl group (AA (1), 4, and 7, respectively). The antiproliferative activity of C-28 ester derivatives was significantly increased with the increase in the length of the carbon chain.

The direct comparison of the antiproliferative activities (against the HeLa cell line) of compounds 15 and 24, as well as compounds 8 and 14, indicated that the introduction of a keto group at position C-11 improves antiproliferative activity. However, an opposite effect was observed when we compared the activity of the following pairs of compounds: 2 and 12, 10 and 11, and 5 and 13. These results suggest that there is no direct relationship between the introduction of a keto group at C-11 and the antiproliferative activity of the compound, which is in accordance with the results of a previous study.⁴⁰

The conversion of hexameric ring A into the corresponding heptameric lactol ring substantially improved the growth inhibitory activity in all tested cell lines. The lactol derivatives (4, 5, 6, and 13) showed IC₅₀ values ranging from 4.70 μM (5) to 8.48 μM (13), which was 6–11 times lower than the IC₅₀ of AA (1) (52.47 μM) against the HeLa cell line.

The group of derivatives that had a pentameric ring A with an α,β-unsaturated carbonyl moiety proved to be the most active among all tested derivatives in all tested cancer cell lines. These compounds presented IC₅₀ values ranging from 0.24 μM (compound 24) to 5.30 μM (compound 7) against the HeLa cell line. Moreover, with the exception of compound 7, all the derivatives of this group were more active than cisplatin, which indicated that the α,β-unsaturated carbonyl moiety in ring A is important for the antiproliferative activity of these compounds against cancer cell lines.

Compounds 17, 16, and 18, with a pentameric ring A with an α,β-unsaturated nitrile, were 1.2-, 5.9-, and 3.6-fold more potent than was AA (1), respectively, against the HeLa cell line.

The effect of different substituents at the C-23 position of compounds 14 and 16 on their antiproliferative activities was also investigated. We found that, with the exception of compound 30, only the introduction of the methanesulfonyloxy group at C-23 (compounds 15, 24, and 31) resulted in a marked increase in antiproliferative activity. In fact, compounds 15 and 24 were the most active compounds among all the synthesized derivatives, as they were approximately 175- and 218-fold more active than AA (1), respectively, against the HeLa cell line.

The most active compounds, 9, 14, 15, and 24, were selected and their antiproliferative activity was further evaluated against a panel of other four cancer cell lines (Jurkat, PC-3, MIA PaCa-2, and A-375) and against a nontumoral fibroblast cell line (BJ), to evaluate selectivity. As depicted in Table 2, the selected compounds markedly inhibited the proliferation of all tested cancer cell lines. Compounds 14, 15, and 24 exhibited a decreased toxicity for the normal fibroblast cell line BJ compared with cancer cell lines. Compound 24 presented the best antiproliferative profile, and was especially active against the HeLa and Jurkat cell lines, with IC₅₀ values of 0.24 μM and 0.11 μM, respectively. Thus, this compound was selected for further studies aimed at exploring the mechanism underlying its antiproliferative effect against the HeLa cell line.

Table 2 Cytotoxic activities of asiatic acid, derivatives 9, 14, 15, 24 and cisplatin against leukemia (Jurkat), prostate (PC-3), pancreas (MIA PaCa-2), melanoma (A375) cancer cell lines and non-tumoral fibroblast cell line (BJ)

Compound	Cell lines^a/IC50 (μM)
	Jurkat	PC-3	MiaPaca-2	A-375	BJ
a The cell lines were treated with increasing concentrations of each compound for 72 h. IC50 values were determined by MTT assay in PC-3, MIA PaCa-2, A375 and BJ cell lines and by XTT assay in Jurkat cell line. The results shown are expressed as means ± SD (standard deviation) of three independent experiments. N.D. not determined. IC50 is the concentration of compound that inhibits 50% of cell growth.
Asiatic acid 1	37.18 ± 3.75	67.25 ± 0.35	50.67 ± 1.15	50.33 ± 2.57	88.70 ± 0.58
9	0.45 ± 0.04	0.57 ± 0.04	0.83 ± 0.04	0.63 ± 0.05	N.D.
14	0.18 ± 0.02	0.60 ± 0.05	0.60 ± 0.06	0.38 ± 0.01	3.33 ± 0.25
15	0.27 ± 0.01	0.41 ± 0.02	0.60 ± 0.06	0.36 ± 0.03	1.94 ± 0.08
24	0.11 ± 0.01	0.42 ± 0.02	0.46 ± 0.04	0.25 ± 0.01	2.43 ± 0.11
Cisplatin	1.94 (ref. 44)	4.60 (ref. 45)	5.00 ± 1.00 (ref. 46)	3.11 ± 0.98 (ref. 47)	10.10 ± 2.00

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2.2.2 Effects of compound 24 on cell-cycle distribution. First, we investigated the effect of compound 24 on the cell-cycle distribution of HeLa cells. Cells were treated with increasing concentrations of 24 (0 μM, 0.24 μM, 0.48 μM, 0.96 μM, and 1.44 μM) for 24 h and the distribution of the cell cycle was assessed by FACS analysis after staining the cells with propidium iodide (PI). As observed in Fig. 3A and B, treatment of HeLa cells with 0.96 μM of compound 24 increased the percentage of cells in the G0/G1 phase, from 50.53% in control cells to 68.65% in treated cells. Concomitantly, the percentage of cells in the S phase decreased from 36.24% in control cells to 12.89% in treated cells. These results suggest that compound 24 inhibits the proliferation of HeLa cells via cell-cycle arrest at the G0/G1 phase.

Fig. 3 Effects of compound 24 on cell cycle distribution. (A) Representative histograms of cell cycle analysis of HeLa cells untreated (control) or treated with the indicated concentrations of compound 24 during 24 h. After treatment, cells were stained with PI and analysed by flow cytometry. (B) Cell cycle analysis of HeLa cells after treatment with compound 24 at specified concentrations for 24 h. The results are presented as means ± SD of a minimum three independent experiments (C) effect of compound 24 on the levels of cell-cycle-related proteins. HeLa cells were treated with compound 24 at the indicated concentrations during 24 h. Protein levels were analysed by western blot. α-Actin was used as loading control.

We also observed an increase of around 17% in the percentage of cells in the sub-G0/G1 phase (cells with hypodiploid DNA) after treatment of HeLa cells with 1.44 μM of compound 24, which suggests that this compound has the ability to induce cell death in a dose-dependent manner.

2.2.3 Effects of compound 24 on the levels of cell-cycle-related proteins. As the experiment described above indicated that compound 24 arrested the cell cycle at the G0/G1 phase, we explored the effects of this compound on the levels of some cell-cycle-regulatory proteins using western blot analysis. As shown in Fig. 3C, treatment of HeLa cells with compound 24 at 0.96 μM significantly increased the levels of p27^kip1, which was in agreement with the observation of cell-cycle arrest at the G0/G1 phase.

Upregulation of p21^waf1/cip1 was also observed after treatment with 1.44 μM compound 24. These data suggest that 24 preferably targets p27^kip1. Compound 24 also decreased the levels of cyclin D₃ in a concentration-dependent manner, but did not affect the levels of CDK4.

Considering that activated cyclin D/CDK4 complexes promote the progression of the G1 phase of the cell cycle and that the CDKIs p21^waf1/cip1 and p27^kip1 inhibit the kinase activity of such complexes, our results suggest that the upregulation of p27^kip1 and p21^waf1/cip1 and the downregulation of cyclin D₃ induced by compound 24 lead to cell-cycle arrest at the G0/G1 phase, with the consequent inhibition of cell proliferation.

2.2.4 Annexin V-FITC/PI flow cytometric assay. We also explored the possibility that compound 24 induces apoptosis in HeLa cells. In the early stages of the apoptotic process, the membrane loses its symmetry and externalization of phosphatidylserine (PS) occurs. Annexin V-FITC specifically binds to externalized PS, thus allowing the quantitative assessment of apoptosis.⁴² In the late stages of apoptosis, the membrane loses its integrity and PI can enter the cell.⁴² Untreated (control) or treated HeLa cells with compound 24 at concentrations of 0.24 μM, 0.48 μM, 0.96 μM and 1.44 μM for 24 h, were double stained with annexin V-FITC/PI and analyzed by flow cytometry. This assay permits the differentiation between live cells (annexin V −/PI −), early apoptotic cells (annexin V +/PI −), late apoptotic cells (annexin V +/PI +), and necrotic cells (annexin V −/PI +).

We observed that treatment of HeLa cells with 1.44 μM of compound 24 led to an increase in the number of apoptotic cells, from 2.9% in control cells to 19.17% in treated cells (i.e., 7.53% of early apoptotic cells and 11.64% of late apoptotic cells) (Fig. 4A). Concomitantly, the percentage of live cells decreased from 96.53% in the control to 78.61% in treated cells. Treatment with 0.24 μM or 0.48 μM of compound 24 did not change the apoptotic rates significantly. These results suggest that compound 24 at 1.44 μM induces apoptosis in HeLa cells.

Fig. 4 (A) Flow cytometric quantification of apoptosis in HeLa cells untreated (control) or treated with compound 24 at specified concentrations for 24 h. After treatment, HeLa cells were stained with annexin V-FICT/PI and analysed by flow cytometry. Black bars represent the percentage of live cells. Dark grey bars represent the percentage of early apoptotic cells and light grey bars represent the percentage of late apoptotic cells. The results shown are means ± SD of a minimum three independent experiments. (B) Morphological analysis. Upper panel: representative phase-contrast images of HeLa cells untreated (control) or treated with compound 24 at specified concentrations for 24 h. Lower panel: representative fluorescence microscopic images of HeLa cells untreated (control) or treated with compound 24 at specified concentrations for 24 h. Hela cells were stained with Hoechst 33258 before analysis by fluorescence microscopy. White arrows represent apoptotic cells.

2.2.5 Morphological analysis of HeLa cells treated with compound 24 using phase-contrast microscopy and fluorescence microscopy after Hoechst 33258 staining. Taking into consideration that apoptosis is characterized by typical morphological features,⁴³ we analyzed the morphology of HeLa cells untreated or treated with compound 24 at 0.96 μM and 1.44 μM for 24 h using microscopic observation, to evaluate further the proapoptotic effect of compound 24.

As shown in the phase-contrast microscopic pictures (Fig. 4B, upper panel), treatment of HeLa cells with compound 24 reduced the cell density and induced remarkable morphological changes. Compared with control cells, treated cells became smaller and nonadherent and acquired a rounded morphology.

To assess the nuclear morphological changes in greater detail, HeLa cells were stained with Hoechst 33258 after treatment with compound 24 and were analyzed by fluorescence microscopy. As shown in Fig. 4B, lower panel, control cells were uniformly stained and presented a normal morphology. Conversely, HeLa cells treated with 0.96 μM of compound 24 exhibited typical apoptotic morphological changes, such as chromatin condensation and cell shrinkage. Nuclear fragmentation and membrane blebbing were evident after treatment with 1.44 μM of compound 24. A decrease in the number of cells with increasing drug concentrations was also observed. The morphological changes induced by compound 24 were consistent with an apoptotic cell death process.

2.2.6 Effects of compound 24 on the levels of apoptosis-related proteins. To gain deeper insight into the mechanism via which compound 24 induces apoptosis in HeLa cells, we investigated the effects of this compound (0–1.44 μM over 24 h) on the levels of some apoptosis-related proteins using western blot analysis (Fig. 5). The activation of caspases is one of the most important mechanisms underlying the execution of apoptosis.⁴⁸ Therefore, we first investigated the effect of compound 24 on the levels of caspase-9, caspase-8, caspase-3, and cleaved PARP. Cleaved PARP is an 89 kDa fragment that is generated during apoptosis from the full length PARP via the proteolytic action of active caspase-3 and is considered a biomarker of apoptosis. As shown in Fig. 5A, treatment of HeLa cells with 1.44 μM of compound 24 led to the cleavage of procaspase-8 and procaspase-3 into their active forms, and to increased levels of cleaved caspase-9 (active form of caspase-9). Compound 24 also induced a significant increase in the levels of cleaved PARP. The activation of caspases 8 and 9 suggest that compound 24 induces apoptosis in HeLa cells via a caspase-driven mechanism with activation of both extrinsic and intrinsic pathways.

Fig. 5 Effect of compound 24 on levels of apoptosis-related proteins. HeLa cells were treated with the indicated concentrations of compound 24 for 24 hours. The levels of apoptosis-related proteins were assessed by western blot analysis. (A) Compound 24 induced the caspase-9, caspase-8 and caspase-3 activation and the cleavage of PARP. (B) Compound 24 downregulated the levels of Bcl-2 and Bid. (C) Compound 24 upregulated the levels of Bax.

Subsequently, we explored the effect of compound 24 on the levels of Bcl-2 protein family members, such as Bcl-2 (antiapoptotic), Bax (proapoptotic), and Bid (proapoptotic). As depicted in Fig. 5B and C, the treatment of HeLa cells with compound 24 caused a downregulation of Bcl-2 in a concentration-dependent manner. These data support that the mitochondrial pathway is involved in compound 24-induced apoptosis. Compound 24 also downregulated the levels of Bid, suggesting the activation of Bid into t-Bid. However, bands corresponding to t-Bid were not detected on western blots, which can be justified by the short half-life and small size of t-Bid. Further studies are needed to confirm and understand better the role of the intrinsic and extrinsic pathways in this apoptotic mechanism.

3. Conclusions

In summary, we successfully synthesized a series of new pentameric A-ring AA derivatives. Our results showed that the conversion of the hexameric ring A of AA into a 5-carbon ring with α,β-unsaturated carbonyl or α,β-unsaturated nitrile moieties significantly improved the antiproliferative activity of compounds. Compound 24 displayed the best antiproliferative profile, with IC₅₀ values ranging from 0.11 μM to 0.65 μM. This compound arrested the cell cycle at the G0/G1 phase and induced apoptosis in HeLa cells via the activation of caspases-9, -8, and -3, cleavage of PARP, and modulation of the ratio of Bax/Bcl-2. In light of our results, this compound might represent a promising lead for the development of new anticancer agents.

4. Experimental

4.1 Chemistry

4.1.1 General. Asiatic acid and all reagents were purchased from Sigma-Aldrich Co. The solvents used in the reactions were obtained from Merck Co and were purified and dried according to usual procedures. The solvents used in the workups were purchased from VWR Portugal. Thin layer chromatography (TLC) analysis was carried out in Kieselgel 60HF254/Kieselgel 60G. Separation and purification of compounds by flash column chromatography (FCC) was performed using Kieselgel 60 (230–400 mesh, Merck). Melting points were measured using a BUCHI melting point B-540 apparatus and are uncorrected. IR spectra were obtained on a Fourier transform spectrometer. ¹H and ¹³C NMR spectra were recorded on Bruker Digital NMR-Avance 400 apparatus spectrometer, using CDCl₃ as internal standard. The chemical shifts (δ) were reported in parts per million (ppm), and coupling constants (J) in hertz (Hz). The mass spectrometry was carried out on Quadrupole/Ion Trap Mass Spectrometer (QIT-MS) (LCQ Advantage MAX, THERMO FINNINGAN). Elemental analysis was performed in an Analyser Elemental Carlo Erba 1108 by chromatographic combustion.

4.1.2 Methyl 2α,3β,23-trihydroxyurs-12-en-28-oate (2). To a stirred solution of AA (1) (1000 mg, 2.05 mmol) and anhydrous potassium carbonate (707.20 mg, 5.12 mmol, 2.5 eq.) in dry DMF (20 mL), methyl iodide (254.7 μL; 4.04 mmol, 2 eq.) was added. The reaction 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 (100 mL) and extracted with ethyl acetate (3 × 100 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 2 as a white powder (1040 mg, quantitative). Mp: 211.6–214.1 °C. v_max/cm⁻¹ (KBr): 3419.17, 2946.70, 2925.48, 2873.42, 1725.98, 1454.06, 1049.09. ¹H NMR (400 MHz, CDCl₃): δ = 5.23 (t, J = 3.09 Hz, 1H, H-12), 3.79–3.71 (m, 1H, H-2), 3.59 (m, 4H), 3.40 (d. J = 9.97 Hz, 1H, H-23), 3.38 (d, J = 10.32 Hz, 1H, H-23), 1.07 (s, 3H), 1.01 (s, 3H), 0.94 (d, J = 5.93 Hz, 3H), 0.85 (d, J = 6.48 Hz, 3H), 0.81 (s, 3H), 0.72 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.06 (C28), 138.25 (C13), 125.22 (C12), 79.83, 69.51, 68.73, 52.80, 51.45, 48.64, 48.04, 47.41, 46.3, 42.58, 42.05, 39.48, 39.02, 38.83, 38.10, 36.59, 32.53, 30.61, 27.95, 24.18, 23.69, 23.33, 21.16, 18.24, 17.15, 17.03, 16.91, 12.89 ppm; DI-ESI-MS m/z: 502.98 ([M + H]⁺); calcd. For C₃₁H₅₀O₅·0.25H₂O: C, 73.41; H, 10.03. Found: C, 73.29; H, 10.46%.

4.1.3 Ethyl 2α,3β,23-trihydroxyurs-12-en-28-oate (3). Accordingly to the method described for 2, using AA (1) (500 mg, 1.02 mmol), anhydrous potassium carbonate (353.50 mg, 2.56 mmol, 2.5 eq.), dry DMF (10 mL) and ethyl iodide (246.77 μL; 3.07 mmol, 3 eq.) for 3 hours at room temperature, to afford 3 as a white powder (507.3 mg, 96%). Mp: 177.5–179.1 °C. v_max/cm⁻¹ (KBr): 3417.24, 2973.70, 2925.48, 2871.49, 1724.05, 1454.06, 1037.52. ¹H NMR (400 MHz, CDCl₃): δ = 5.24 (t, J = 3.10 Hz, 1H, H-12), 4.05 (q, J = 7.20 Hz, 2H, COOC̲H̲₂CH₃), 3.79–3.72 (m, 1H, H-2), 3.66 (d, J = 10.36 Hz, 1H, H-3), 3.44 (d, J = 9.05 Hz, 1H, H-23), 3.41 (d, J = 9.40 Hz, 1H, H-23), 1.21 (t, J = 7.00 Hz, 3H, COOCH₂C̲H̲₃), 1.08 (s, 3H), 1.03 (s, 3H), 0.94 (d, J = 6.05 Hz, 3H), 0.87 (s, 3H), 0.85 (d, J = 6.20 Hz, 3H), 0.76 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.53 (C28), 138.25 (C13), 125.18 (C12), 68.75, 60.02, 52.81, 47.83, 47.47, 46.24, 42.13, 39.57, 39.07, 38.85, 38.15, 38.62, 32.70, 30.58, 27.91, 24.13, 23.58, 23.34, 21.18, 18.32, 17.16, 17.11, 17.01, 14.21, 12.76 ppm; DI-ESI-MS m/z: 516.97 ([M + H]⁺); calcd. For C₃₂H₅₂O₅·0.5H₂O: C, 73.10; H, 10.16. Found: C, 73.31; H, 10.32%.

4.1.4 2α,23-Lactol-urs-12-ene-28-oic acid (4). 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 hours 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 4 as a white powder (quantitative). Mp: 198.5–201.4 °C. v_max/cm⁻¹ (KBr): 3421.1, 2948.63, 2927.41, 2871.49, 2732.64, 2630.43, 1716.34, 1695.12, 1456.99, 1378.85, 1037.52. ¹H NMR (400 MHz, CDCl₃): δ = 9.94 (s, 1H, CHO), 5.29 (t, J = 3.25 Hz, 1H, H-12), 5.14–5.11 (m, 1H, H-2), 3.94 (d, J = 13.41 Hz, 1H), 3.75 (d, J = 13.17 Hz, 1H), 1.08 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.95 (d, J = 5.96 Hz, 3H), 0.86 (s, 3H), 0.85 (d, J = 5.42 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.09 (CHO), 182.94 (C28), 138.05 (C13), 125.98 (C12), 93.68, 65.37, 61.16, 53.41, 62.71, 48.07, 45.15, 43.65, 42.57, 40.05, 39.98, 38.94, 38.77, 38.58, 33.57, 30.59, 27.83, 24.63, 24.06, 23.17, 21.10, 20.58, 20.37, 17.88, 16.93, 14.57 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%.

4.1.5 Methyl 2α,23-lactol-urs-12-ene-28-oate (5). Accordingly to the method described for 4, using compound 2 (1000 mg, 1.99 mmol), methanol/water (25 mL/1.25 mL [20 : 1]) and NaIO₄ (645.60 mg; 3.02 mmol, 1.52 eq.) for 3 hours to afford 5 as a white powder (978.70 mg, 98%). Mp: 144.7–147.1 °C. v_max/cm⁻¹ (KBr): 3444.24, 2948.63, 2927.41, 2871.49, 2730.71, 2626.57, 1722.12, 1454.06, 1378.85, 1037.52. ¹H NMR (400 MHz, CDCl₃): δ = 9.94 (s, 1H, CHO), 5.30 (t, J = 3.26 Hz, H-12), 5.13–5.09 (m, 1H, H-2), 3.93 (d, J = 13.31 Hz, 1H), 3.74 (d, J = 13.31 Hz, 1H), 3.60 (s, 3H, COOC̲H̲₃), 1.08 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 6.03 Hz), 0.85–0.83 (m, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.85 (CHO), 177.99 (C28); 138.30 (C13); 125.75 (C12); 93.67; 65.41, 61.12, 53.45, 53.03, 51.49, 48.23, 45.17, 43.67, 42.59, 40.03, 39.97, 38.94, 38.81, 36.52, 33.62, 30.64, 27.86, 24.63, 24.17, 23.17, 21.10, 20.60, 20.38, 17.86, 16.97, 14.52 ppm; DI-ESI-MS m/z: 500.99 ([M + H]⁺); calcd. For C₃₁H₄₈O₅·0.25H₂O: C, 73.70; H, 9.68. Found: C, 73.75; H, 9.97%.

4.1.6 Ethyl 2α,23-lactol-urs-12-ene-28-oate (6). Accordingly to the method described for 4, using compound 3 (480 mg, 0.93 mmol), methanol/water (12.90 mL/0.65 mL (20 : 1)) and NaIO₄ (299.45 mg; 1.40 mmol, 1.5 eq.) for 1 h 30 min to afford 6 as a white powder (477.90 mg, quantitative). Mp: 138.1–142.0 °C. v_max/cm⁻¹ (KBr): 3444.24, 2950.55, 2927.41, 2971.49, 2732.64, 1720.19, 1454.06, 1378.85, 1230.36, 1141.65, 1037.52. ¹H NMR (400 MHz, CDCl₃): δ = 9.94 (s, 1H, CHO), 5.29 (t, J = 3.10 Hz, 1H, H-12), 5.12–5.09 (m, 1H, H-2), 4.06 (q, J = 7.15 Hz, 2H, COOC̲H̲₂CH₃), 3.93 (d, J = 13.29 Hz, 1H), 3.75 (d, J = 13.29 Hz, 1H), 1.21 (t, J = 7.14 Hz, 3H, COOCH₂C̲H̲₃), 1.08 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 6.10 Hz, 3H), 0.84 (m, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.85 (CHO), 177.45 (C28), 138.31 (C13), 125.70 (C12), 93.65, 65.39, 61.12, 60.04, 53.43, 53.01, 47.99, 45.21, 43.68, 42.65, 40.11, 39.94, 38.97, 38.81, 36.53, 33.71, 30.68, 27.80, 24.63, 24.12, 23.07, 21.10, 20.60, 20.37, 18.05, 16.95, 14.53, 14.21 ppm. DI-ESI-MS m/z: 514.92 ([M + H]⁺); calcd. For C₃₂H₅₀O₅·0.25H₂O: C, 74.02; H, 9.80. Found: C, 73.84; H, 10.05%.

4.1.7 2-Formyl-23-hydroxy-A(1)-norurs-2,12-diene-28-oic acid (7). To a stirred solution of compound 4 (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 7 as a white solid (353.77 mg, 73%). Mp: 183.5–186.1 °C. v_max/cm⁻¹ (KBr): 3428.81, 2946.7, 2925.48, 2869.56, 2726.85, 2632.36, 1689.34, 1581.34, 1454.06, 1380.78, 1041.37. ¹H NMR (400 MHz, CDCl₃): δ = 9.72 (s, 1H, CHO), 6.66 (s, 1H, H-3), 5.28 (t, J = 3.05 Hz, 1H, H-12), 3.62 (d, J = 10.67 Hz, 1H, H-23), 3.45 (d, J = 10.70 Hz, 1H, H-23), 1.25 (s, 3H), 1.10 (s, 3H), 1.01 (s, 3H), 0.93 (d, J = 6.24 Hz, 3H), 0.88 (s, 3H), 0.84 (d, J = 6.32 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.85 (CHO), 183.27 (C28), 159.29 (C3), 158.90 (C2), 137.49 (C13), 126.60 (C12), 69.36, 56.26, 52.57, 50.88, 49.41, 47.87, 44.10, 42.41, 41.36, 38.78 (2C), 36.63, 33.53, 30.55, 28.19, 27.09, 23.99 (2C), 21.16, 19.03, 18.74, 17.35, 16.98, 15.92 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%.

4.1.8 Methyl 2-formyl-23-hydroxy-A(1)-norurs-2,12-diene-28-oate (8). Accordingly to the method described for 7, using compound 5 (500 mg, 0.99 mmol), dry benzene (50 mL), piperidine (2.5 mL) acetic acid (2.5 mL) and anhydrous magnesium sulfate (327 mg), to afford compound 8 as a yellow powder (488.5 mg, quantitative). Mp: 119.7–122.4 °C. v_max/cm⁻¹ (KBr): 3448.10, 2948.63, 2925.48, 2869.56, 2724.92, 2634.29, 1724.05, 1687.41, 1581.34, 1455.99, 1382.71, 1232.29, 1145.51, 1047.16. ¹H NMR (400 MHz, CDCl₃): δ = 9.72 (s, 1H, CHO), 6.66 (s, 1H, H-3), 5.28 (t, J = 3.20 Hz, 1H, H-12), 3.62–3.60 (m, 4H), 3.46 (d, J = 10.72 Hz, 1H, H-23), 1.25 (s, 3H), 1.09 (s, 3H), 1.02 (s, 3H), 0.93 (d, J = 6.15 Hz, 3H), 0.83 (m, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.81 (CHO), 178.09 (C28), 159.24 (C3), 158.93 (C2), 137.74 (C13), 126.34 (C12), 69.39, 56.27, 52.81, 51.45, 50.90, 49.42, 48.03, 44.12, 42.43, 41.34, 38.83, 38.81, 36.57, 33.55, 30.59, 28.25, 27.09, 24.16, 23.99, 21.17, 19.00, 18.67, 17.37, 17.04, 15.99 ppm; DI-ESI-MS m/z: 482.98 ([M + H]⁺); calcd. For C₃₁H₄₆O₄·0.25H₂O: C, 76.42; H, 9.62. Found: C, 76.58; H, 9.78%.

4.1.9 Ethyl 2-formyl-23-hydroxy-A(1)-norurs-2,12-diene-28-oate (9). Accordingly to the method described for 7, using compound 6 (300 mg, 0.58 mmol), dry benzene (30 mL), piperidine (1.5 mL), acetic acid (1.5 mL) and anhydrous magnesium sulfate (300 mg) to afford a yellow crude. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 3 : 1 → 2 : 1) to afford 9 as a white solid (193.2 mg, 67%). Mp: 207.5–209.7 °C. v_max/cm⁻¹ (KBr): 3550.31, 2977.55, 2960.20, 2946.70, 2925.48, 2867.63, 2809.78, 2726.85, 1708.62, 1689.34, 1581.34, 1452.11, 1238.08, 1143.58, 1045.23. ¹H NMR (400 MHz, CDCl₃): δ = 9.72 (s, 1H, CHO), 6.66 (s, 1H, H-3), 5.28 (t, J = 3.00 Hz, 1H, H-12), 4.09–4.02 (m, 2H, COOC̲H̲₂CH₃), 3.62 (d, J = 8.50 Hz, 1H, H-23), 3.46 (d, J = 8.78 Hz, 1H, H-23), 1.25 (s, 3H), 1.22 (t, J = 7.20 Hz, 3H, COOCH₂C̲H̲₃), 1.09 (s, 3H), 1.02 (s, 3H), 0.93 (d, J = 6.07 Hz, 3H), 0.86 (s, 3H), 0.83 (d, J = 6.54 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.84 (CHO), 177.57 (C28), 159.29 (C3), 158.95 (C2), 137.79 (C13), 126.28 (C12), 69.39 (C23), 60.02, 56.25, 52.81, 50.91, 49.43, 47.79, 44.15, 42.49, 41.44, 38.84 (2C), 36.57, 33.62, 30.64, 28.19, 27.10, 24.11, 23.90, 21.17, 19.01, 18.81, 17.37, 17.02, 15.93, 14.18 ppm. DI-ESI-MS m/z: 497.07 ([M + H]⁺); calcd. For C₃₂H₄₈O₄: C, 77.38; H, 9.74. Found: C, 76.94; H, 10.12%.

4.1.10 Methyl 2α,3β,23-triacetoxyurs-12-ene-28-oate (10). To a stirred solution of compound 2 (953 mg, 1.89 mmol) in dry THF (28 mL), acetic anhydride (1163.30 μL; 12.33 mmol, 6.5 eq.) and a catalytic amount of DMAP (95.30 mg) were added. The reaction mixture was stirred at room temperature in anhydrous conditions. After 5 hours the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (80 mL) and extracted with ethyl acetate (3 × 80 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 80 mL), 10% aqueous NaHCO₃ (2 × 80 mL), 10% aqueous Na₂SO₃ (80 mL), water (80 mL) and brine (80 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 10 as a white powder (1182 mg, 99% quantitative). Mp: 118.8–121.1 °C. v_max/cm⁻¹ (KBr): 2948.63, 2927.41, 2871.49, 1745.26, 1455.99, 1369.21, 1234.22, 1043.30. ¹H NMR (400 MHz, CDCl₃): δ = 5.24 (t, J = 3.00 Hz, 1H, H-12), 5.19–5.13 (m, 1H, H-2), 5.08 (d, J = 10.29 Hz, 1H, H-3), 3.85 (d, J = 11.80 Hz, 1H, H-23), 3.57 (d, J = 11.80 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 2.08 (s, 3H, CH₃CO), 2.01 (s, 3H, CH₃CO), 1.97 (s, 3H, CH₃CO), 1.09 (s, 3H), 1.06 (s, 3H), 0.94 (d, J = 6.06 Hz, 3H), 0.88 (s, 3H), 0.84 (d, J = 6.23 Hz, 3H), 0.74 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.95 (C28), 170.85 (OCO), 170.44 (OCO), 170.37 (OCO), 138.30 (C13), 125.01 (C12), 74.85, 69.91, 65.28, 52.81, 51.46, 48.04, 47.59, 47.49, 43.75, 41.98, 41.89, 39.50, 39.00, 38.81, 37.77, 36.54, 32.44, 30.60, 27.90, 24.12, 23.42, 23.34, 21.14, 21.06, 20.86, 20.76, 17.88, 17.00, 16.95, 16.85, 13.90 ppm; DI-ESI-MS m/z: 629.36 ([M + H]⁺); calcd. For C₃₇H₅₆O₈: C, 70.67; H, 8.98. Found: C, 70.55; H, 8.98%.

4.1.11 Methyl 2α,3β,23-triacetoxy-11-oxours-12-ene-28-oate (11). To a stirred solution of compound 10 (100 mg, 0.16 mmol) in dry dichloromethane (2 mL), a mixture of KMnO₄ (440 mg) and Fe₂(SO₄)₃·nH₂O (220 mg), previous reduced to fine powder, water (22 μL) and t-butanol (0.1 mL) were added. The reaction mixture was stirred at room temperature. After 4 h 45 min the reaction mixture was diluted with diethyl ether (20 mL) and filtered thought a Celite pad, with further washing with diethyl ether (100 mL). The resulting organic phase was washed with 10% aqueous NaHCO₃ (2 × 50 mL) and water (2 × 50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 11 as a white powder (97.5 mg, 95%). Mp: 139.2–141.5 °C. v_max/cm⁻¹ (KBr): 2977.55, 2950.55, 2875.34, 1747.19, 1662.34, 1457.92, 1369.21, 1234.22, 1043.30. ¹H NMR (400 MHz, CDCl₃): δ = 5.62 (s, 1H, H-12), 5.31–5.25 (m. 1H, H-2), 5.04 (d, J = 10.30 Hz, 1H, H-3), 3.83 (d, J = 11.76 Hz, 1H, H-23), 3.57 (d, J = 11.76 Hz, 1H, H-23), 3.59 (s, 3H, COOC̲H̲₃), 2.07 (s, 3H, CH₃CO), 2.00 (s, 3H, CH₃CO), 1.94 (s, 3H, CH₃CO), 1.28 (s, 6H), 0.96 (d, J = 6.31 Hz, 3H), 0.90 (s, 6H), 0.85 (d, J = 6.49 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 198.82 (C11), 177.09 (C28), 170.79 (OCO), 170.46 (OCO), 170.10 (OCO), 163.26 (C13), 130.43 (C12), 74.88, 68.96, 65.21, 61.11, 52.71, 51.87 (2C), 47.61, 47.32, 44.55, 44.17, 43.72, 41.88, 38.58, 37.65, 35.88, 32.45, 30.26, 28.29, 23.84, 20.98, 20.95 (2C), 20.88, 20.75, 18.81, 17.70, 17.05, 16.99, 13.85 ppm; DI-ESI-MS m/z: 643.12 ([M + H]⁺); calcd. For C₃₇H₅₄O₉·0.25H₂O: C, 68.65; H, 8.49. Found: C, 68.46; H, 8.38%.

4.1.12 Methyl 2α,3β,23-trihydroxy-11-oxours-12-ene-28-oate (12). To a stirred solution of compound 11 (353 mg, 0.55 mmol) in methanol (23.5 mL), KOH (2350 mg) was added and the reaction mixture was heated under reflux. After 30 min the reaction mixture was evaporated under reduced pressure to remove the methanol, and acidified (pH 5–6) with 6 M aqueous HCl solution. Water (20 mL) was added and the aqueous layer was extracted with ethyl acetate (3 × 80 mL). The resulting organic phase was washed with 10% aqueous NaHCO₃ (3 × 50 mL), water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 12 as a white powder (280.2 mg, quantitative). Mp: 174.9–177.3 °C. v_max/cm⁻¹ (KBr): 3424.96, 2948.63, 2929.34, 2873.42, 1727.91, 1660.41, 1455.99, 1388.50, 1201.43, 1047.16. ¹H NMR (400 MHz, CDCl₃): δ = 5.60 (s, 1H, H-12), 3.87–3.81 (m, 1H, H-2), 3.64 (d, J = 11.09 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 3.42 (d, J = 9.51 Hz, 1H, H3) 3.36 (d, J = 11.09 Hz, 2H, H-23), 1.30 (s, 3H), 1.21 (s, 3H), 0.96 (d, J = 6.10 Hz, 3H), 0.89 (s, 3H), 0.86 (d, J = 6.31 Hz, 3H), 0.82 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 199.70 (C11), 177.19 (C28), 163.51 (C13), 130.43 (C12), 68.50, 61.04, 52.73, 51.85 (2C), 47.64, 46.89, 44.66, 43.86, 42.78, 38.63, 38.56, 38.01, 35.94, 32.54, 30.28, 28.35, 23.90, 21.19, 20.96 (2C), 18.91, 17.88, 17.21, 17.09, 12.99 ppm; DI-ESI-MS m/z: 516.93 ([M + H]⁺); calcd. For C₃₁H₄₈O₆·H₂O: C, 69.63; H, 9.42. Found: C, 69.19; H, 8.92%.

4.1.13 Methyl 2α,23-lactol-11-oxours-12-ene-28-oate (13). Accordingly to the method described for 4, using compound 12 (1950 mg, 3.78 mmol), methanol/water (48.70 mL/2.40 mL (20 : 1)) and NaIO₄ (1227.40 mg; 5.74 mmol, 1.52 eq.) for 1 hour at room temperature. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 4 : 1 → 1 : 1) to afford 13 as a white solid (1280.30 mg, 66%). Mp: 154.0–157.0 °C. v_max/cm⁻¹ (KBr): 3436.53, 2948.63, 2938.98, 2873.42, 2734.57, 1725.98, 1658.48, 1455.99, 1205.29, 1141.65, 1037.52. ¹H NMR (400 MHz, CDCl₃): δ = 9.97 (s, 1H, CHO), 5.64 (s, 1H, H-12), 5.36–5.33 (m, 1H, H-2), 3.98 (d, J = 13.23 Hz, 1H), 3.71 (d, J = 13.59 Hz, 1H), 3.60 (s, 3H, COOC̲H̲₃), 1.34 (s, 3H), 1.32 (s, 3H), 0.97 (m, 6H), 0.93 (s, 3H), 0.86 (d, J = 6.37 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.65 (CHO), 198.89 (C11), 177.12 (C28), 162.74 (C13), 130.85 (C12), 93.49, 65.25, 61.38, 57.40, 53.20, 52.75, 51.89, 47.71, 45.70, 44.65, 44.15, 39.21, 38.60, 38.57, 35.92, 33.16, 30.30, 28.35, 23.88, 20.93, 20.70, 20.47, 19.60, 19.28, 17.03, 14.38 ppm; DI-ESI-MS m/z: 515.42 ([M + H]⁺); calcd. For C₃₁H₄₆O₆·0.75H₂O: C, 70.49; H, 9.06. Found: C, 70.31; H, 9.41%.

4.1.14 Methyl 2-formyl-11-oxo-23-hydroxy-A(1)-norurs-2,12-diene-28-oate (14). Accordingly to the method described for 7, using compound 13 (300 mg, 0.58 mmol), dry benzene (30 mL), piperidine (1.45 mL), acetic acid (1.45 mL) and anhydrous magnesium sulfate (300 mg). The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 2 : 1 → 1 : 1) to afford 14 as a white solid (142.90 mg, 49%). Mp: 144.8–146.6 °C. v_max/cm⁻¹ (KBr): 3442.31, 2977.55, 2948.63, 2929.34, 2871.49, 1727.91, 1662.34, 1614.13, 1581.34, 1455.99, 1226.50. ¹H NMR (400 MHz, CDCl₃): δ = 10.15 (s, 1H, CHO), 6.35 (s, 1H, H-3), 5.67 (s, 1H, H-12), 3.62 (s, 3H, COOC̲H̲₃, H28), 3.56 (d, J = 11.08 Hz, 1H), 3.39 (d, J = 11.08 Hz, 1H), 1.45 (s, 3H), 1.33 (s, 3H), 1.00 (s, 3H), 0.97 (m, 6H), 0.85 (d, J = 6.35 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 199.38 (C11), 195.49 (CHO), 177.17 (C28), 164.67 (C13), 157.47 (C2); 144.75 (C3), 129.47 (C12), 69.24, 58.26, 54.83, 52.99, 51.88, 48.70, 48.23, 47.57, 45.83, 44.44, 38.61, 38.36, 35.87, 33.44, 30.21, 28.77, 23.87, 21.39, 21.06, 20.96, 20.54, 17.11, 16.90, 16.10 ppm. DI-ESI-MS m/z: 497.47 ([M + H]⁺). Anal. calcd. For C₃₁H₄₄O₅·0.5H₂O: C, 73.63; H, 8.97. Found: C, 73.27; H, 8.50%.

4.1.15 Methyl 2-formyl-23-methanesulfonyloxy-A(1)-norurs-2,12-diene-28-oate (15). To a stirred solution of compound 8 (250 mg, 0.52 mmol) in dry dichloromethane (10 mL), triethylamine (144.60 μL; 1.04 mmol, 2 eq.) and methanesulfonyl chloride (80.20 μL, 1.04 mmol, 2 eq.) were added. The reaction mixture was stirred at room temperature. After 4 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 water (4 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a yellow crude. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 4 : 1 → 2 : 1) to afford 15 as a white solid (206.9 mg, 71%). Mp: 105.6–107.2 °C. v_max/cm⁻¹ (KBr): 2948.63, 2925.48, 2871.49, 2728.78, 1722.12, 1689.34, 1585.2, 1455.99, 1357.64, 1176.36, 958.45. ¹H NMR (400 MHz, CDCl₃): δ = 9.73 (s, 1H, CHO), 6.60 (s, 1H, H-3), 5.26 (t, J = 3.01 Hz, 1H, H-12), 4.13 (d, J = 9.59 Hz, 1H, H-23), 4.06 (d, J = 9.59 Hz, 1H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 3.03 (s, 3H), (S–C̲H̲₃), 1.25 (s, 3H), 1.10 (s, 3H), 1.08 (s, 3H), 0.93 (d, J = 5.87 Hz, 3H), 0.84–0.83 (m, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.56 (CHO), 178.03 (C28), 158.95 (C2), 155.79 (C3), 137.85 (C13), 126.10 (C12), 74.70, 56.93, 52.79, 51.45, 50.85, 48.01, 47.69, 44.29, 42.43, 41.30, 38.82, 38.78, 37.49, 36.53, 33.43, 30.56, 28.17, 27.03, 24.11, 23.86, 21.16, 18.78, 18.66, 17.30, 17.05, 15.88 ppm. DI-ESI-MS m/z: 560.91 ([M + H]+); calcd. For C₃₂H₄₈O₆S·0.5H₂O: C, 67.45; H, 8.67; S, 5.63. Found: C, 67.56; H, 8.73; S, 5.27%.

4.1.16 Methyl 2-cyano-23-hydroxy-A(1)-norurs-2,12-diene-28-oate (16). To a stirred solution of compound 8 (295 mg, 0.61 mmol) in THF (4.40 mL), aqueous ammonium solution 25% (13.70 mL) and iodine (340.75 mg, 1.34 mmol, 2.20 eq.) were added. The mixture was stirred at room temperature for 8 hours (the dark solution became colorless). The reaction mixture was charged with 5% aqueous Na₂S₂O₃ (50 mL) and extracted with ethyl acetate (3 × 60 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 a yellow powder. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 3 : 1 → 2 : 1) to afford 16 as a white solid (146.20 mg, 50%). Mp: 115.1–118.1 °C. v_max/cm⁻¹ (KBr): 3504.02 (OH), 2948.63, 2925.48, 2871.49, 2215.81, 1724.05, 1652.70, 1587.13, 1455.99, 1362.71, 1234.22, 1197.58, 1145.51, 1049.09. ¹H NMR (400 MHz, CDCl₃): δ = 6.48 (s, 1H, H-3), 5.26 (t, J = 3 Hz, 1H, H-12), 3.61 (s, 3H, COOC̲H̲₃), 3.55 (d, J = 10.07 Hz, 1H, H-23), 3.39 (d, J = 9.92 Hz, 1H, H-23), 1.27 (s, 3H), 1.13 (s, 3H), 1.02 (s, 3H), 0.94 (d, J = 5.36 Hz, 3H), 0.87 (d, J = 6.13 Hz, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.02 (C28), 153.59 (C3), 138.68 (C13), 127.16 (C2), 125.01 (C12), 117.64 (CN), 69.26, 55.37, 52.90 (2C), 51.48, 50.83, 48.03, 43.55, 42.39, 40.92, 38.83 (2C), 36.54, 33.41, 30.57, 28.20, 24.55, 24.09, 24.01, 21.16, 19.27, 18.44, 17.65, 17.08, 16.01 ppm. DI-ESI-MS m/z: 480.08 ([M + H]⁺); calcd. For C₃₁H₄₅NO₃·0.5H₂O: C, 76.19; H, 9.49; N, 2.87. Found: C, 76.41; H, 9.62; N, 2.97%.

4.1.17 2-Cyano-23-hydroxy-A(1)-norurs-2,12-diene-28-oic acid (17). Accordingly to the method described for 16, using compound 7 (240 mg, 0.51 mmol), THF (2.40 mL), aqueous ammonium solution 25% (9.60 mL) and iodine (142.96 mg, 0.56 mmol, 1.10 eq.) at room temperature for 2 hours. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 4 : 1 → 2 : 1) to afford 17 as a white solid (134.4 mg, 56%). Mp: 147.1–150.1 °C. v_max/cm⁻¹ (KBr): 3423.03, 2948.63, 2925.48, 2871.49, 2217.74, 1697.05, 1455.99, 1382.71, 1043.30. ¹H NMR (400 MHz, CDCl₃): δ = 6.48 (s, 1H, H-3), 5.25 (t, J = 2.99 Hz, 1H, H-12), 3.54 (d, J = 10.58 Hz, 1H, H-23), 3.38 (d, J = 10.58 Hz, 1H, H-23), 1.27 (s, 3H), 1.13 (s, 3H), 1.01 (s, 3H), 0.95 (d, J = 6.04 Hz, 3H), 0.87 (m, 6H) ppm. ₁₃C NMR (100 MHz, CDCl₃): δ = 183.40 (C28), 153.72 (C3), 138.50 (C13), 127.02 (C2), 125.13 (C12), 117.62 (CN), 69.17 (C23), 55.34, 52.85, 52.65, 50.81, 47.89, 43.53, 42.37, 40.92, 38.79 (2C), 36.63, 33.38, 30.54, 28.13, 24.54, 24.00, 23.93, 21.15, 19.29, 18.46, 17.62, 17.03, 16.00 ppm. DI-ESI-MS m/z: 466.03 ([M + H]⁺); calcd. For C₃₀H₄₃NO₃·0.75H₂O: C, 75.20; H, 9.36; N, 2.92. Found: C, 75.10; H, 9.83; N, 2.79%.

4.1.18 Methyl 2-cyano-11-oxo-23-hydroxy-A(1)-norurs-2,12-diene-28-oate (18). Accordingly to the method described for 16, using compound 14 (300 mg, 0.60 mmol), THF (3 mL), aqueous ammonium solution 25% (12 mL) and iodine (168.60 mg, 0.66 mmol, 1.10 eq.) at room temperature for 3 hours. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 3 : 1 → 1 : 1) to afford 18 as a white solid (144.6 mg, 48%). Mp: 139.1–142.0 °C. v_max/cm⁻¹ (KBr): 3444.24, 2948.63, 2929.34, 2871.49, 2217.74, 1725.98, 1670.05, 1612.2, 1581.34, 1457.92, 1226.50. ¹H NMR (400 MHz, CDCl₃): δ = 6.57 (s, 1H, H-3), 5.71 (s, 1H, H-12), 3.60 (s, 3H, COOC̲H̲₃), 3.55 (d, J = 10.93 Hz, 1H, H-23), 3.38 (d, J = 10.92 Hz, 1H, H-23), 1.40 (s, 3H), 1.33 (s, 3H), 1.01 (s, 3H), 0.97 (d, J = 6.26 Hz, 3H), 0.91 (s, 3H), 0.87 (d, J = 6.43 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 197.16 (C11), 177.19 (C28), 163.92 (C13), 155.23 (C3), 129.34 (C12), 128.72 (C2), 117.24 (CN), 68.76, 57.20, 54.68, 52.93, 51.88, 50.86, 49.96, 47.56, 45.43, 44.25, 38.61, 38.45, 35.91, 33.14, 30.20, 28.74, 23.81, 21.51, 20.96, 20.76, 20.44, 17.23, 16.86, 15.86 ppm. DI-ESI-MS m/z: 494.37 ([M + H]⁺); calcd. For C₃₁H₄₃NO₄·0.5H₂O: C, 74.07; H, 8.82; N, 2.79. Found: C, 74.10; H, 8.83; N, 2.94%.

4.1.19 Methyl 2-formyl-11-oxo-23-acetoxy-A(1)-norurs-2,12-diene-28-oate (19). To a stirred solution of compound 14 (95 mg, 0.19 mmol) in dry THF (2.85 mL), acetic anhydride (27 μL; 0.29 mmol, 1.50 eq.) and a catalytic amount of DMAP (9.5 mg) were added. The mixture was stirred at room temperature in anhydrous conditions. After 1 h 30 min, the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 30 mL), 10% aqueous NaHCO₃ (2 × 30 mL), 10% aqueous Na₂SO₃ (30 mL), water (30 mL) and brine (30 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 19 as a white powder (73.2 mg, 71%). Mp: 104.2–107.3 °C. v_max/cm⁻¹ (KBr): 2948.63, 2933.20, 2873.42, 1735.62, 1662.34, 1614.13, 1585.2, 1455.99, 1384.84, 1232.29, 1039.44. ¹H NMR (400 MHz, CDCl₃): δ = 10.15 (s, 1H, CHO), 6.39 (s, 1H, H-3), 5.67 (s, 1H, H-12), 3.97 (d, J = 10.78 Hz, 1H, H-23), 3.89 (d, J = 10.76 Hz, 1H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 2.05 (s, 3H, CH₃CO), 1.45 (s, 3H), 1.31 (s, 3H), 1.05 (s, 3H), 0.96 (m, 6H), 0.86 (d, J = 6.51 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 199.12 (C11), 195.25 (CHO), 177.13 (C28), 171.05 (OCO), 164.61 (C13), 156.37 (C2), 143.96 (C3), 129.46 (C12), 70.75, 58.41, 56.75, 52.99, 51.88, 48.01, 47.56, 46.64, 45.77, 44.37, 38.61, 38.36, 35.84, 33.49, 30.19, 28.71, 23.85, 21.21, 21.02, 20.95, 20.85, 20.25, 17.12, 17.00, 16.28 ppm. DI-ESI-MS m/z: 539.88 ([M + H]⁺); calcd. For C₃₃H₄₆O₆·0.75H₂O: C, 71.77; H, 8.67. Found: C, 71.80; H, 8.65%.

4.1.20 Methyl 2-formyl-11-oxo-23-butyroxy-A(1)-norurs-2,12-diene-28-oate (20). Accordingly to the method described for 19, using compound 14 (300 mg, 0.60 mmol), dry THF (9 mL), butyric anhydride (98.80 μL; 0.60 mmol, 1 eq.) and a catalytic amount of DMAP (30 mg) at room temperature for 1 hour. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 7 : 1 → 5 : 1) to afford 20 as a white solid (59.1 mg, 17%). Mp: 77.8–80.3 °C. v_max/cm⁻¹ (KBr): 2950.55, 2933.20, 2873.42, 1731.76, 1662.34, 1614.13, 1585.20, 1457.92, 1224.58, 1197.58, 1174.44. ¹H NMR (400 MHz, CDCl₃): δ = 10.15 (s, 1H, CHO), 6.39 (s, 1H, H-3), 5.68 (s, 1H, H-12), 3.98 (d, J = 10.78 Hz, 1H, H-23), 3.90 (d, J = 10.63 Hz, 1H, H-23), 3.62 (s, 3H, COOCH₃), 1.46 (s, 3H), 1.31 (s, 3H), 1.05 (s, 3H), 0.96–0.92 (m, 6H), 0.86 (d, J = 6.23 Hz, 3H) ppm. ₁₃C NMR (100 MHz, CDCl₃): δ = 199.13 (C11), 195.22 (CHO), 177.14 (C28), 173.61 (OCO), 164.59 (C13), 156.36 (C2), 144.10 (C3), 129.49 (C12), 70.33, 58.41, 56.64, 53.00, 51.89, 47.98, 47.57, 46.73, 45.79, 44.37, 38.62, 38.38, 36.16, 35.86, 33.51, 30.20, 28.72, 23.86, 21.21, 21.01, 20.97, 20.30, 18.47, 17.14, 16.97, 16.31, 13.73 ppm. DI-ESI-MS m/z: 567.40 ([M + H]⁺). Anal. calcd. For C₃₅H₅₀O₆: C, 74.17; H, 8.89. Found: C, 73.79; H, 9.21%.

4.1.21 Methyl 2-formyl-11-oxo-23-cinnamoxy-A(1)-norurs-2,12-diene-28-oate (21). To a stirred solution of compound 14 (300 mg, 0.60 mmol) in dry benzene (18 mL), cinnamoyl chloride (402.50 mg, 2.41 mmol, 4 eq.) and DMAP (295.20 mg, 2.41 mmol, 4 eq.) were added. The mixture was stirred at 60 °C under nitrogen atmosphere. After 3 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 water (4 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a light yellow crude. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 6 : 1 → 4 : 1) to afford 21 as a white solid (268.7 mg, 71%). Mp: 97.0–100.2 °C. v_max/cm⁻¹ (KBr): 3083.62, 3060.48, 3025.76, 2948.63, 2931.27, 2873.42, 1718.26, 1664.27, 1637.27, 1452.14, 1382.71, 1309.43, 1272.79, 1228.43, 1201.43, 1164.79. ¹H NMR (400 MHz, CDCl₃): δ = 10.17 (s, 1H, CHO), 7.69 (d, J = 15.98 Hz, 1H, H-3′), 7.54–7.52 (m, 2H, H-2′′ and H-6′′), 7.40–7.38 (m, 3H, H-3′′, H-4′′, H-5′′), 6.47 (s, 1H, H-3), 6.43 (d, J = 16.02 Hz, 1H, H-2′), 5.68 (s, 1H, H-12), 4.11 (d, J = 11.01 Hz, 1H, H-23), 4.07 (d, J = 11.05 Hz, 1H, H-23), 3.62 (s, 3H, COOC̲H̲₃), 1.48 (s, 3H), 1.29 (s, 3H), 1.11 (s, 3H), 0.96–0.95 (m, 6H), 0.84 (d, J = 6.36 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 199.18 (C11), 195.29 (CHO), 177.14 (C28), 166.94 (OCO), 164.66 (C13), 156.39 (C2), 145.30, 144.13 (C3), 134.22, 130.43, 129.43 (C12), 128.89 (2C), 128.19 (2C), 117.61, 70.67, 58.42, 56.73, 52.98, 51.88, 48.03, 47.55, 46.92, 45.78, 44.37, 38.59, 38.32, 35.84, 33.51, 30.18, 28.69, 23.83, 21.19, 21.02, 20.94, 20.29, 17.10, 17.03, 16.37 ppm. DI-ESI-MS² m/z: 627.36 ([M + H]⁺, 42%), 609.48 (62), 566.37 (72), 479.38 (100), 419.39 (29); calcd. For C₄₀H₅₀O₆·H₂O: C, 74.50; H, 8.13. Found: C, 74.10; H, 7.76%.

4.1.22 Methyl 2-formyl-11-oxo-23-(1H-imidazole-1-carbonyloxy)-A(1)-norurs-2,12-diene-28-oate (22). To a stirred solution of compound 14 (280 mg, 0.56 mmol) in dry THF (11 mL), CDI (184.10 mg, 1.14 mmol, 2 eq.) was added. The reaction mixture was stirred at reflux temperature under nitrogen atmosphere. 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 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, 1 : 1) to afford 22 as a white solid (41.4 mg, 12%). Mp: 111.6–114.1 °C. v_max/cm⁻¹ (KBr): 3129.90, 2948.63, 2873.42, 1764.55, 1725.98, 1662.34, 1614.13, 1587.13, 1457.92, 1400.07, 1288.22, 1240.00, 1004.73. ¹H NMR (400 MHz, CDCl₃): δ = 10.14 (s, 1H, CHO), 8.09 (s, 1H, H-2′′), 7.30 (s, 1H, H-5′′), 7.06 (s, 1H, H-4′′), 6.37 (s, 1H, H-3), 5.67 (s, 1H, H-12), 4.29 (d, J = 10.50, 1H, H-23), 4.24 (d, J = 10.33 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 1.24 (s, 3H), 1.13 (s, 3H), 0.95 (s, 6H), 0.84 (d, J = 5.41 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 198.78 (C11), 194.76 (CHO), 177.06 (C28), 164.76 (C13), 157.09 (C2), 148.56 (OCO), 142.24 (C3), 136.89, 130.90, 129.34 (C12), 116.90, 73.85, 58.35, 56.69, 52.95, 51.85, 48.10, 47.50, 46.95, 45.68, 44.29, 38.56, 38.29, 35.77, 33.46, 30.12, 28.66, 23.77, 21.01, 20.97, 20.90, 20.20, 17.11, 17.05, 16.20 ppm. DI-ESI-MS m/z: 591.52 ([M + H]⁺); calcd. For C₃₅H₄₆N₂O₆·0.25H₂O: C, 70.62; H, 7.87; N, 4.71. Found: C, 70.30; H, 7.71; N, 4.35%.

4.1.23 Methyl 2-formyl-11-oxo-23-(2′-methyl-1H-imidazole-carbonyloxy)-A(1)-norurs-2,12-diene-28-oate (23). Accordingly to the method described for 22, using compound 14 (300 mg, 0.60 mmol), dry THF (12.5 mL), and CBMI (287.20 mg, 1.51 mmol, 2.5 eq.) at reflux temperature under nitrogen atmosphere for 6 hours. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 2 : 1 → 1 : 2) to afford 23 as a white solid (121.4 mg, 33%). Mp: 128.7–131.0 °C. v_max/cm⁻¹ (KBr): 3122.19, 2948.63, 2931.27, 2873.42, 1758.76, 1725.98, 1660.41, 1614.13, 1587.13, 1552.42, 1511.92, 1457.92, 1398.14, 1295.93, 1143.58. ¹H NMR (400 MHz, CDCl₃): δ = 10.15 (s, 1H, CHO), 7.28 (d, J = 1.47 Hz, 1H, H-5′′), 6.84 (d, J = 1.48 Hz, 1H, H-4′′), 6.39 (s, 1H, H-3), 5.67 (s, 1H, H-12), 4.27 (d, J = 10.81 Hz, 1H, H-23), 4.20 (d, J = 10.81 Hz, 1H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 2.62 (s, 3H, CH₃ imidazole), 1.47 (s, 3H), 1.23 (s, 3H), 1.13 (s, 3H), 0.97–0.95 (m, 6H), 0.85 (d, J = 6.30 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 198.80 (C11), 194.81 (CHO), 177.08 (C28), 164.75 (C13), 157.03 (C2), 149.47 (OCO), 147.91, 142.61 (C3), 129.39 (C12), 128.20, 117.78, 73.30, 58.38, 56.42, 52.98, 51.88, 48.07, 47.52, 46.93, 45.72, 44.32, 38.59, 38.33, 35.81, 33.48, 30.15, 28.70, 23.80, 21.06, 20.98, 20.92, 20.26, 17.13, 16.94, 16.85, 16.26 ppm. DI-ESI-MS m/z: 605.40 ([M + H]⁺); calcd. For C₃₆H₄₈N₂O₆: C, 71.50; H, 8.00; N, 4.63. Found: C, 71.16; H, 8.46; N, 4.36%.

4.1.24 Methyl 2-formyl-11-oxo-23-methanesulfonyloxy-A(1)-norurs-2,12-diene-28-oate (24). Accordingly to the method described for 15, using compound 14 (300 mg, 0.60 mmol), dry CH₂Cl₂ (12.50 mL), triethylamine (168.50 μL; 1.21 mmol, 2 eq.) and methanesulfonyl chloride (93.50 μL, 1.21 mmol, 2 eq.) at room temperature in anhydrous conditions for 1 h 10 min. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 5 : 1 → 1.5 : 1) to afford 24 as a white solid (183.1 mg, 53%); Mp: 114.0–116.5 °C. v_max/cm⁻¹ (KBr): 2948.63, 2933.20, 2873.42, 1725.98, 1662.34, 1614.13, 1587.13, 1457.92, 1355.71, 1174.44, 958.45 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 10.15 (s, 1H, CHO), 6.30 (s, 1H, H-3), 5.68 (s, 1H, H-12), 4.07 (d, J = 10.05 Hz, 1H, H-23), 4.04 (d, J = 10.05 Hz, 1H, H-23), 3.62 (s, 3H, COOC̲H̲₃), 3.01 (s, 3H, S–C̲H̲₃), 1.46 (s, 3H), 1.33 (s, 3H), 1.09 (s, 3H), 0.97–0.96 (m, 6H), 0.86 (d, J = 6.21 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 199.04 (C11), 194.93 (CHO), 177.14 (C28), 164.93 (C13), 157.18 (C2), 141.96 (C3), 129.35 (C12), 75.09, 58.28, 56.08, 53.01, 51.89, 48.15, 47.56, 46.91, 45.75, 44.44, 38.61, 38.35, 37.55, 35.84, 33.28, 30.18, 29.69, 23.83, 21.25, 21.05, 20.95, 20.27, 17.12, 16.94, 16.06 ppm. DI-ESI-MS m/z: 575.42 ([M + H]⁺), 597.30 ([M + Na]⁺). Anal. calcd. For C₃₂H₄₆O₇S: C, 66.87; H, 8.07; S, 5.58. Found: C, 66.48; H, 8.45; S, 5.20%.

4.1.25 Methyl 2-cyano-23-acetoxy-A(1)-norurs-2,12-diene-28-oate (25). Accordingly to the method described for 19, using compound 16 (200 mg, 0.42 mmol), dry THF (6 mL), acetic anhydride (43.30 μL; 0.46 mmol, 1.10 eq.) and a catalytic amount of DMAP (20 mg) at room temperature for 1 hour. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 6 : 1 → 4 : 1) to afford 25 as a white solid (126.4 mg, 58%). Mp: 86.7–88.6 °C. v_max/cm⁻¹ (KBr): 2948.63, 2925.48, 2871.49, 2215.81, 1745.26, 1724.05, 1650.77, 1589.06, 1455.99, 1382.71, 1236.15, 1039.44. ¹H NMR (400 MHz, CDCl₃): δ = 6.44 (s, 1H, H-3), 5.26 (t, J = 2.95 Hz, 1H, H-12), 3.97 (d, J = 11.05 Hz, 1H, H-23), 3.90 (d, J = 10.95 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 2.06 (s, 3H, COC̲H̲₃), 1.26 (s, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.94 (d, J = 5.92 Hz, 3H), 0.87 (d, J = 6.32 Hz, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.95 (C28), 170.88 (OCO), 152.65 (C3), 138.72 (C13), 127.03 (C2), 124.93 (C12), 117.46 (CN), 69.73, 56.16, 52.89, 52.61, 51.47, 48.99, 48.01, 43.79, 42.33, 40.88, 38.81, 38.81, 36.51, 33.42, 30.54, 28.12, 24.54, 24.06, 23.81, 21.15, 20.80, 19.02, 18.42, 17.49, 17.11, 16.19 ppm. DI-ESI-MS m/z: 522.31 ([M + H]⁺); calcd. For C₃₃H₄₇NO₄: C, 75.97; H, 9.08; N, 2.68. Found: C, 76.22; H, 9.25; N, 2.85%.

4.1.26 Methyl 2-cyano-23-butyroxy-A(1)-norurs-2,12-diene-28-oate (26). Accordingly to the method described for 19, using compound 16 (290 mg, 0.61 mmol), dry THF (11.6 mL), butyric anhydride (108.8 μL, 0.67 mmol, 1.10 eq.) and a catalytic amount of DMAP (30 mg) at room temperature for 1 h 15 min. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 7 : 1 → 6 : 1) to afford 26 as a white solid (101.1 mg, 30%); Mp: 65.5–68.0 °C. v_max/cm⁻¹ (KBr): 2948.63, 2927.41, 2873.42, 2215.81, 1735.55, 1652.70, 1589.06, 1455.99, 1172.51. ¹H NMR (400 MHz, CDCl₃): δ = 6.44 (s, 1H, H-3), 5.27 (t, J = 3.0 Hz, 1H, H-12), 3.98 (d, J = 10.94 Hz, 1H, H-23), 3.90 (d, J = 10.94 Hz, 2H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 1.27 (s, 3H), 1.10 (s, 3H), 1.04 (s, 3H), 0.97–0.93 (m, 6H), 0.87 (d, J = 6.09 Hz, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.96 (C28), 173.45 (OCO), 152.81 (C3), 138.70 (C13), 126.99 (C2), 124.95 (C12), 117.47 (CN), 69.27, 55.99, 52.88, 52.60, 51.48, 49.09, 48.01, 43.76, 42.33, 40.89, 38.82 (2C), 36.52, 36.16, 33.43, 30.55, 28.13, 24.54, 24.06, 23.79, 21.15, 19.06, 18.48, 18.40, 17.44, 17.12, 16.23, 13.71 ppm. DI-ESI-MS m/z: 550.33 ([M + H]⁺); calcd. For C₃₅H₅₁NO₄: C, 76.46; H, 9.35; N, 2.55. Found: C, 76.75; H, 9.62; N, 2.58%.

4.1.27 Methyl 2-cyano-23-benzoxy-A(1)-norurs-2,12-diene-28-oate (27). Accordingly to the method described for 19, using compound 16 250 mg, 0.52 mmol, dry THF (10 mL), benzoic anhydride (141.50 mg, 0.63 mmol, 1.20 eq.) and a catalytic amount of DMAP (25 mg) at room temperature for 1 h 30 min. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 10 : 1 → 7 : 1) to afford 27 a white solid (158.2 mg, 52%). Mp: 100.2–103.1 °C. v_max/cm⁻¹ (KBr): 3089.40, 3060.48, 2948.63, 2925.48, 2871.49, 2215.81, 1722.12, 1602.56, 1585.20, 1452.14, 1270.86, 1112.73. ¹H NMR (400 MHz, CDCl₃): δ = 8.00 (d, J = 7.23 Hz, 2H, H2′′ and H6′′), 7.58 (t, J = 7.34 Hz, 1H, H4′′), 7.45 (t, J = 7.75 Hz, 2H, H3′′ and H5′′), 6.54 (s, 1H, H-3), 5.26 (t, J = 2.80 Hz, 1H, H-12), 4.24 (d, J = 11.00 Hz, 1H, H-23), 4.14 (d, J = 11.00 Hz, 1H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 1.30 (s, 3H), 1.15 (s, 3H), 1.03 (s, 3H), 0.94 (d, J = 5.79 Hz, 3H), 0.86 (d, J = 6.64 Hz, 3H). 0.84 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.95 (C28), 166.27 (OCO), 152.76 (C3), 138.73 (C13), 133.26, 129.75, 129.54, 128.55, 127.18 (C2), 124.92 (C12), 117.45 (CN), 69.85, 56.08, 52.87, 52.67, 51.47, 49.39, 48.00, 43.88, 42.31, 40.90, 38.83, 38.78, 36.52, 33.49, 30.54, 28.09, 24.57, 24.04, 23.63, 21.11, 19.00, 18.41, 17.49, 17.12, 16.36 ppm. DI-ESI-MS m/z: 584.29 ([M + H]⁺); calcd. For C₃₈H₄₉NO₄: C, 78.18; H, 8.46; N, 2.40. Found: C, 77.80; H, 8.58; 2.52%.

4.1.28 Methyl 2-cyano-23-succinoxy-A(1)-norurs-2,12-diene-28-oate (28). To a stirred solution of compound 16 (250 mg, 0.52 mmol) in dry CH₂Cl₂ (15 mL), succinic anhydride (130.38 mg; 1.30 mmol, 2.5 eq.) and DMAP (95.50 mg, 0.78 mmol, 1.50 eq.) were added. The reaction mixture was stirred at room temperature in anhydrous conditions. After 3 hours, the reaction mixture was evaporated under reduced pressure to remove the organic phase. The obtained crude was dispersed by water (60 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 60 mL), 10% aqueous Na₂SO₃ (60 mL), water (60 mL) and brine (60 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 28 as a white solid (213.9 mg, 71%). Mp: 105.8–107.7 °C. v_max/cm⁻¹ (KBr): 2948.63, 2927.41, 2871.49, 2657.43, 2559.08, 2215.81, 1743.33, 1727.91, 1712.48, 1589.06, 1455.99, 1201.43, 1162.87. ¹H NMR (400 MHz, CDCl₃): δ = 6.43 (s, 1H, H-3), 5.26 (t, J = 2.85 Hz, 1H, H-12), 4.01 (d, J = 11.01 Hz, 1H, H-23), 3.92 (d, J = 11.01 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 2.68–2.64 (m, 4H, H-2′ and H-3′), 1.26 (s, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.94 (d, J = 5.96 Hz, 3H), 0.87 (d, J = 6.33 Hz, 3H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ = 178.00 (C28), 177.25, 171.89, 152.49 (C3), 138.71 (C13), 127.10 (C2), 124.91 (C12), 117.41 (CN), 70.06, 56.21, 52.88, 52.62, 51.49, 49.05, 48.02, 43.74, 42.33, 40.88, 38.80 (2C), 36.51, 33.40, 30.54, 28.76, 28.70, 28.10, 24.53, 24.06, 23.82, 21.15, 19.02, 18.41, 17.49, 17.10, 16.17 ppm. DI-ESI-MS m/z: 580.35 ([M + H]⁺); calcd. For C₃₅H₄₉NO₆·H₂O: C, 70.32; H, 8.60; N, 2.34. Found: C, 70.22; H, 8.50; N, 2.38%.

4.1.29 Methyl 2-cyano-23-cinnamoxy-A(1)-norurs-2,12-diene-28-oate (29). Accordingly to the method described for 21, using compound 16 (300 mg, 0.63 mmol), dry benzene (18 mL), cinnamoyl chloride (416.75 mg, 2.50 mmol, 4.0 eq.) and DMAP (305.60 mg, 2.50 mmol, 4 eq.) at 60 °C under nitrogen atmosphere for 1 h 40 min. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 3 : 1) to afford 29 as a white solid (189 mg, 50%) Mp: 102.3–105.0 °C. v_max/cm⁻¹ (KBr): 3083.62, 3060.48, 2946.70, 2925.48, 2871.49, 2215.81, 1718.26, 1637.27, 1579.41, 1496.49, 1452.14, 1162.87. ¹H NMR (400 MHz, CDCl₃): δ = 7.69 (d, J = 16.15 Hz, 1H, H-3′), 7.54–7.52 (m, 2H, H-2′′and H-6′′), 7.41–7.39 (m, 3H, H-3′′, H-4′′ and H-5′′), 6.51 (s, 1H, H-3), 6.43 (d, J = 16.17 Hz, 1H, H-2′), 5.27 (t, J = 2.65 Hz, 1H, H-12), 4.10 (d, J = 10.93 Hz, 1H, H-23), 4.05 (d, J = 10.99 Hz, 1H, H-23), 3.61 (s, 3H, COOC̲H̲₃), 1.29 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H), 0.93 (d, J = 6.04 Hz, 3H), 0.85–0.84 (m, 6H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.95 (C28), 166.75, 152.77 (C3), 145.50, 138.72 (C13), 134.16, 130.50, 128.91 (2C), 128.17 (2C), 127.06 (C2), 124.91 (C12), 117.48 (CN), 117.42, 69.70, 56.22, 52.88, 52.66, 51.46, 49.23, 48.01, 43.81, 42.34, 40.89, 38.81, 38.77, 36.51, 33.44, 30.53, 28.11, 24.54, 24.05, 23.85, 21.13, 19.06, 18.44, 17.54, 17.08, 16.28 ppm. DI-ESI-MS m/z: 610.33 ([M + H]⁺); calcd. For C₄₀H₅₁NO₄: C, 78.78; H, 8.43; N, 2.30. Found: C, 78.82; H, 8.20; N, 2.46%.

4.1.30 Methyl 2-cyano-23-(2′-methyl-1H-imidazole-carbonyloxy)-A(1)-norurs-2,12-diene-28-oate (30). Accordingly to the method described for 22, using compound 16 (280 mg, 0.58 mmol), dry THF (11 mL) and CBMI (277.55 mg, 1.46 mmol, 2.5 eq.) at reflux temperature under nitrogen atmosphere for 8 hours. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 2 : 1 → 1 : 1) to afford 30 as a white solid (204.1 mg, 59%). Mp: 104.1–107.2 °C. v_max/cm⁻¹ (KBr): 3166.54, 2948.63, 2925.48, 2215.81, 1747.19, 1724.05, 1455.99, 1384.64, 1249.65. ¹H NMR (400 MHz, CDCl₃): δ = 7.27 (s, 1H, H-5′), 6.87 (s, 1H, H-4′), 6.47 (s, 1H, H-3), 5.27 (t, J = 3.0 Hz, 1H, H-12), 4.29 (d, J = 11.04 Hz, 1H, H-23), 4.18 (d, J = 11.01 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 2.63 (s, 3H, CH₃ Imidazole), 1.30 (s, 3H), 1.13 (s, 3H), 1.04 (s, 3H), 0.94 (d, J = 5.68 Hz, 3H), 0.86 (d, J = 6.89 Hz, 3H), 0.84 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.94 (C28), 151.40 (C3), 149.37 (OCO), 147.98, 138.73 (C13), 128.33, 127.99 (C2), 124.76 (C12), 117.68, 117.01 (CN), 72.34, 55.96, 52.84, 52.72, 51.48, 49.22, 47.99, 43.91, 42.30, 40.90, 38.82, 38.76, 36.50, 33.45, 30.51, 28.10, 24.51, 24.02, 23.70, 21.13, 18.95, 18.39, 17.43, 17.13, 16.85, 16.20 ppm. DI-ESI-MS m/z: 588.63 ([M + H]⁺); calcd. For C₃₆H₄₉N₃O₄: C, 73.56; H, 8.40; N, 7.15. Found: C, 73.46; H, 8.79; N, 6.86%.

4.1.31 Methyl 2-cyano-23-methanesulfonyloxy-A(1)-norurs-2,12-diene-28-oate (31). Accordingly to the method described for 15, using compound 16 (250 mg, 0.52 mmol), dry CH₂Cl₂ (10.7 mL), triethylamine (145.40 μL; 1.04 mmol, 2 eq.) and methanesulfonyl chloride (80.65 μL, 1.04 mmol, 2 eq.) at room temperature, in anhydrous conditions for 4 hours. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate, 4 : 1 → 3 : 1) to afford 31 as a white solid (179.2 mg, 62%). Mp: 109.50–112.10 °C. v_max/cm⁻¹ (KBr): 2948.63, 2925.48, 2871.49, 2217.74, 1722.12, 1455.99, 1359.57, 1176.36, 960.38. ¹H NMR (400 MHz, CDCl₃): δ = 6.43 (s, 1H, H-3), 5.27 (t, J = 2.54 Hz, 1H, H-12), 4.05 (d, J = 9.84, 1H, H-23), 4.00 (d, J = 9.98 Hz, 1H, H-23), 3.60 (s, 3H, COOC̲H̲₃), 3.02 (s, 3H, S–CH₃), 1.28 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H), 0.94 (d, J = 5.88 Hz, 3H), 0.87 (d, J = 6.44 Hz, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.94 (C28), 150.86 (C3), 138.79 (C13), 128.01 (C2), 124.76 (C12), 117.08 (CN), 74.09, 55.95, 52.87, 52.79, 51.47, 49.07, 48.00, 43.72, 42.38, 40.88, 38.81, 38.79, 37.56, 36.50, 33.27, 30.53, 28.10, 24.49, 24.04, 23.87, 21.15, 19.03, 18.43, 17.52, 17.20, 15.96. DI-ESI-MS m/z: 558.28 ([M + H]⁺); calcd. For C₃₂H₄₇NO₅S·0.25H₂O: C, 68.35; H, 8.51; N, 2.49; S, 5.70. Found: C, 68.55; H, 8.51; N, 2.67; S, 5.30%.

4.2 Biology

4.2.1 Cells and reagents. MCF-7, HT-29, Jurkat, PC-3, A375, MIA PaCa-2, HeLa and BJ cell lines were obtained from American Type Culture Collection (USA). Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 Medium, 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.

Asiatic acid and its derivatives were suspended in DMSO at 20 mM as 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%. Cisplatin was obtained from Sigma Aldrich.

Primary antibodies against p21^cip1/waf1 (sc-397), Bcl-2 (sc-509), Bax (sc-493), cyclin E (sc-247) and cyclin D₃ (sc-182) were obtained from Santa Cruz Biotechnology, inc. Primary antibodies against for p27^kip1 (#610242), Bid (#550365) and poly-(ADP-ribose)-polymerase (PARP) (#556493) were obtained from BD Biosciences. Primary antibodies against caspase 3 (#9662) and caspase 8 (#9746S) were purchased from Cell Signaling. Primary antibody against α-actin (#69100) was obtained from MP Biomedicals. Secondary antibodies anti-mouse (P0260) and anti-rabbit (NA934) were obtained from Dako and from Amersham Biosciences, respectively.

4.2.2 Cell culture. HT-29, PC-3, A375, MIA PaCa-2 and HeLa cells were routinely maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-7 cells were maintained in MEM supplemented with 10% heat-inactivated FBS, 0.1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.01 mg mL⁻¹ of insulin, 10 mM glucose and 1× MEM-EAGLE non essential amino acids. BJ cells were routinely maintained in DMEM supplemented with 10% heat-inactivated FBS, 110 mg L⁻¹ sodium pyruvate, 1% penicillin/streptomycin and 1.5 g L⁻¹ sodium bicarbonate. Jurkat cells were cultivated in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin and 2 mM L-glutamine. All cell lines were incubated in a 5% CO₂ humidified atmosphere at 37 °C.

4.2.3 Cell viability assay. The antiproliferative activities of compounds against MCF-7, HT-29, PC-3, A375, MIA PaCa-2, HeLa and BJ cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 8 × 10² to 1 × 10⁴ cells per well were plated in 96-well plates in 200 μL of medium and were left to grow. After 24 h, the culture medium was removed and replaced by new medium (200 μL) containing the tested compounds at different concentrations, in triplicate. After 72 h of incubation, 100 μL of MTT solution (0.5 mg mL⁻¹) replace the supernatant in each well. Following 1 h of incubation, the supernatant was removed and 100 μL of DMSO were added to each well. Relative cell viability was measured by absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Salzburg, Austria).

The antiproliferative activities of compounds against Jurkat cells were determined by XTT assay. Briefly, Jurkat cells were seeded at a density of 4 × 10³ cells per well in 96 well plates in 100 μL of medium. After 24 h of incubation, 100 μL of medium containing the tested compounds at different concentration, in triplicate, were added. Following 72 h of incubation, 100 μL of XTT solution were added to each well and the plates were incubated for a further 4 hours at 37 °C. Relative cell viability was measured by absorbance at 450 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Salzburg, Austria).

4.2.4 Cell cycle assay. HeLa cells were seeded at a density of 1 × 10⁵ cells per well in 6-well plates with 2 mL of medium and incubated at 37 °C for 24 h. Cells were then treated with compound 24 at the specified concentrations for 24 h. Cells were harvested by mild trypsinization, centrifuged, washed twice with PBS and then stained with 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. Cell cycle was assessed by flow cytometry using a fluorescent activated cell sorter (FACS). FACS analysis was carried out at 488 nm in an Epics XL flow cytometer (coulter Corporation, Hialeah, FL). Data from 1 × 10⁴ cells were collected and analysed using the multicycle software (phoenix Flow Systems, San Diego, CA).

4.2.5 Annexin V-FITC/PI flow cytometry assay. Apoptosis was assessed by evaluation of the annexin-V binding to phosphatidylserine (PS), which was externalized early in the process of apoptosis. HeLa cells were plated in 6-well plates at a density of 1 × 10⁵ cells per well and incubated at 37 °C for 24 h. Cells were then treated with compound 24, at specified concentrations, for 24 h. 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 the stained with annexin V-FITC conjugate for 30 min at room temperature and protected from the light. Then, 500 μL of binding buffer were added to each vial of cells. Approximately 2 min before FACS analysis, 20 μL of 1 mg mL⁻¹ PI solution were added to each vial and the samples were analysed by flow cytometry. Data from 1 × 10⁴ cells were collected and analyzed.

4.2.6 Morphology analysis by phase-contrast microscopy. In this experiment, 1 × 10⁵ HeLa cells per well were seeded in 6-well plates with 2 mL of medium and incubated at 37 °C for 24 h. Cells were then treated with compound 24 at specified concentrations for 24 h. The morphological changes were observed using a inverted phase-contrast microscope (Olympus IMT-2) with an objective of 40× and a digital camera Fujifilm A2O5S (Fuji Foto Film, CO. LTD.).

4.2.7 Hoechst 33258 staining. HeLa cells were seeded at a density of 1 × 10⁵ cells per well in 6-well plates with 2 mL of medium and incubated at 37 °C for 24 h. Cells were then treated with compound 24 at the specified concentrations for 24 h. The culture medium was removed and cells were harvested by mild trypsinization, collected by centrifugation and washed twice with PBS. The cells were then stained with 500 μL of Hoechst 33258 solution (2 μg mL⁻¹ in PBS) for 15 min at room temperature and protected from the light. Subsequently the Hoechst 33258 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 analysed by fluorescence microscopy using a fluorescence microscope (DMRB Leica Microsystems, Wetzlar, Germany) with a DAPI filter.

4.2.8 Preparation of total protein extract. To prepare the total protein extracts, HeLa cells (5.6 × 10⁵) were seeded in 100 mm plates and incubated at 37 °C for 24 h. Cells were then treated with specified concentrations of compound 24 for 24 h. Subsequently HeLa cells were washed with ice cold PBS and resuspended in ice cold lysis buffer [20 mM tris/acetate, pH 7.5, 270 mM sucrose, 1 mM EDTA, pH 8.8, 1 mM EGTA, pH 8.8, 1% Triton X-100 and 1% protease inhibitor cocktail (Sigma-Aldrich)]. The samples were homogenized by sonication, incubated on ice for 15 min and centrifuged at 12 000 rpm for 5 min at 4 °C. The protein content in supernatants was determined using the bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Rockford).

4.2.9 Western blotting. The protein extracts were separated on 10% or 15% SDS-polyacrylamide gel and then transferred to polyvinyl nitrocellulose transfer membranes (BioRad Laboratories, Richmond). Transferred membranes were blocked with tris buffered saline (TBS) buffer (20 mM tris, pH 7.5, and 132 mM NaCl) containing 0.1% Tween and 5% of BSA or nonfat dry milk during 1 h at room temperature. The membranes were then incubated with a primary specific antibody overnight at 4 °C. Following incubation, membranes were washed five times (five minutes each) with TBS–0.1% Tween and incubated with the appropriate secondary antibody for 1 h at room temperature. After incubation, membranes were washed five times (five minutes each) with TBS–0.1% Tween. The membranes were treated with Immobilon ECL Western Blotting Detection Kit Reagent (Millipore) and developed after exposure to an autoradiography film in a film cassette.

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 thank MINECO of Spain and FEDER Funds (grant numbers SAF2011-25726 and SAF2014-56059-R) 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 NRM data. 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/c5ra19120c

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