Design, synthesis, and biological evaluation of new arjunolic acid derivatives as anticancer agents

Bruno M F Gonçalves; Vanessa I S Mendes; Samuel M Silvestre; Jorge A R Salvador

doi:10.1039/d2md00275b

. 2022 Dec 19;14(2):313–331. doi: 10.1039/d2md00275b

Design, synthesis, and biological evaluation of new arjunolic acid derivatives as anticancer agents^†

Bruno M F Gonçalves ^a,^b,^‡, Vanessa I S Mendes ^a,^b,^‡, Samuel M Silvestre ^b,^c, Jorge A R Salvador ^b,^d,^✉

PMCID: PMC9945870 PMID: 36846362

Abstract

Arjunolic acid (AA) is a pentacyclic triterpenoid with promising anticancer properties. A series of novel AA derivatives containing a pentameric A-ring with an enal moiety, combined with additional modifications at C-28, were designed and prepared. The biological activity on the viability of human cancer and non-tumor cell lines was evaluated in order to identify the most promising derivatives. Additionally, a preliminary study of the structure–activity relationship was carried out. The most active derivative, derivative 26, also showed the best selectivity between malignant cells and non-malignant fibroblasts. For compound 26, the anticancer molecular mechanism of action in PANC-1 cells was further studied and the results showed that this derivative induced a cell-cycle arrest at G0/G1 phase and significantly inhibited the wound closure rate of PANC-1 cancer cells in a concentration-dependent manner. Additionally, compound 26 synergistically increased the cytotoxicity of Gemcitabine, especially at a concentration of 0.24 μM. Moreover, a preliminary pharmacological study indicated that at lower doses this compound did not demonstrate toxicity in vivo. Taken together, these findings suggest that compound 26 may be a valuable compound for the development of new pancreatic anticancer treatment, and further studies are needed to explore its full potential.

Arjunolic acid derivatives containing a pentameric A-ring were prepared. The biological activity on the viability of human cancer and non-tumor cell lines was evaluated as well as the molecular mechanism of action in PANC-1 cells for compound 26.

Introduction

Cancer is a significant cause of morbidity and mortality worldwide, in countries of all income levels.¹ Additionally, the number of cancer cases and deaths is expected to rise as populations grow, age, and adopt lifestyles more exposed to cancer risk factors. It is noteworthy to mention that, depending on the degree of economic and social development, as well as lifestyle factors, the most common cancers diagnosed and the leading cause of cancer death substantially vary across countries and even within each country.^2–4 These numbers show that despite the significant advances made over the last decade in research, early detection methods and treatment, we are still far from curing cancer.^5,6 In addition, cancer is viewed as a highly heterogeneous disease, consisting of different types of cells characterized by different molecular features and diverse therapeutic responses.^7–9 For this reason, there is a demand to find new and efficient therapies that treat cancer as a global disease, considering its complexity and heterogeneity in order to boost cancer survival rate and alleviate critical side effects.

Natural products (NPs) still represent a prime source of clinical candidates for the development of new drugs, including oncological agents, mainly due to their unique wide range of structures, biological activities, drug-like properties, and chemical diversity.^10–13 Among the myriad of NPs, pentacyclic triterpenoids (PTs) have gained enormous attention in the last two decades due to their multifunctional anticancer properties. Their reduced toxicity coupled with inexpensive availability from several natural sources have also contributed to the continued success of PTs in anticancer drug discovery, which is widely documented in the literature.^14–21 Arjunolic acid (AA1, Fig. 1) is an oleanane-type triterpenoid and a main constituent of the bark of Terminalia arjuna, a well-known herb in Ayurvedic medicine system.²²

This compound has been widely reported for its protective effects, including against the toxicity-induced by various drugs and chemicals, towards cells, tissues and organs, such as liver,^23–27 kidney²⁸ and heart.^29,30 The cellular and molecular mechanisms underlying the protective effects of this triterpenoid are related, at least in part, with its potent antioxidant activity, which is demonstrated by its free radical scavenging effects.³¹ In addition, AA1 has been shown to exhibit a wide variety of pharmacological activities, including antibacterial,³² antifungal,³³ insect growth inhibition,³⁴ wound healing,³⁵ antiallergic and anti-asthmatic³⁶ and anticholinesterase inhibition.³⁷ This metabolite was also identified as promising antitumor agent against mouse³⁸ and human³⁹ cancer cell lines and in in vivo cancer models.^40,41 Moreover, AA1 has been shown to possess a relative safe profile even at higher doses, not originating any alterations to the hematological and biochemical parameters or any signs of physical or behavioral anomalies in orally treated rats up to 2 g kg⁻¹ concentration.⁴²

Chemical modifications of the triterpenoid scaffold to produce novel derivatives have proved to be a fairly useful strategy in improving not only their anticancer activity but also their pharmacokinetic properties.^43,44 Nevertheless, despite its promising anticancer properties and low toxicity, the chemical modification of AA1 remains practically unexplored with a very limited number of semisynthetic derivatives prepared and evaluated for antitumor activity.⁴⁵

As part of our ongoing research program towards the development of anticancer drug candidates from terpenoid natural products,^46–50 the AA1 chemical derivatization was undertaken to obtain novel compounds with improved antitumor activity. Herein, the rationale behind the synthesis focused on the introduction of amide, ester and α,β-unsaturated carbonyl group moieties into the AA1 backbone, since these moieties have been reported as important structural features not only to increase the cytotoxic activity against human cancer cell lines, but also to promote apoptosis-inducing properties.^48,50–52 Thus, chemical modifications were designed and performed to obtain a series of AA1 derivatives with modifications at the A-ring coupled with additional modifications at C-28. All new compounds were evaluated for their in vitro antitumor activity in human colon (HT-29) and pancreatic (PANC-1) cancer cell lines. This screening allowed a preliminary structure–activity relationship (SAR) study that allows correlating the observed biological activity with a given set of structural features. Four highly cytotoxic derivatives (25, 26, 27, 28) were identified and further tested in additional human cancer cell lines (A375, SK-Mel-28 (melanoma), A549, and H2170 (lung) and in a non-tumor cell line (BJ cells)). Further experiments were conducted with the most active compound (26), aiming to shed some light on its mechanism of action in the PANC-1 cell line and to evaluate its toxicity.

Results and discussion

Chemistry

As outlined in Schemes 1 and 2, a series of novel AA1 derivatives were prepared in an attempt to improve its cytotoxicity and selectivity profiles and establish a meaningful structure–activity relationship. The structures of new synthesized compounds were fully confirmed by comprehensive spectroscopic analyses.

Amide bonds are frequently present in relevant bioactive molecules, including both natural products and synthetic or semi-synthetic compounds. The biological importance of this functional group is tightly related with its high stability and ability to adopt different three-dimensional structures. The amide bound formation is among the most frequently used reactions in medicinal chemistry,⁵³ and as a result, amides are present in 25% of known pharmaceuticals.⁵⁴ In addition, it has been frequently reported that C-28 amide oleanane and ursane-type triterpene derivatives have enhanced anticancer activity with respect to the natural triterpenoid compounds.^16,55–57 Thus, to explore the effect of the introduction of an amide substituent on AA1 anticancer properties, we prepared the C28-amide derivatives 3–8. As shown in Scheme 1, to avoid the formation of multiple derivatives, the highly reactive A-ring hydroxyl groups of AA1 were protected by preparing the triacetate derivative 2 using acetic anhydride, in the presence of 4-dimethylaminopyridine (DMAP). Afterwards, compound 2 was activated with thionyl chloride followed by the addition of either aqueous ammonia solution, methylamine or ethylamine to yield amides 3, 4 and 5, respectively. The presence of the amide moiety was confirmed by the proton (NH) signals at 5.71 (3), 5.89 (4) and 5.81 ppm (5) in the ¹H NMR spectra. Moreover, when comparing the 13C NMR spectra of the new derivatives to that of compound 2, C-28 carbonyl carbon signals were shifted upfield (from δC 184.19 in 2 to δC 181.31 in 3, δC 178.84 in 4 and δC 178.2 in 5) which was indicative of an amide moiety. Deacetylation of compounds 3–5 was achieved with potassium hydroxide (KOH) in methanol (MeOH) to afford the triol derivatives 6–8.

The C-28 ester derivatives 9–12 were synthesized to better understand the influence of the structural diversification on this position on the anticancer activity. AA1 was treated with the corresponding alkyl and allyl halides in dry DMF in the presence of anhydrous potassium carbonate to generate the methyl (9), ethyl (10), butyl (11) and allyl (12) ester derivatives. As expected, when compared with AA1, in addition to the new ¹H NMR signals assigned to the new aliphatic and allylic moieties, the main difference in the ¹³C-NMR spectra was a mild deshielded shift of the C-28 signal from δC 181.82 in AA1 to δC 177.36–178.30 in ester derivatives 9–12.

Several publications support that electrophilic natural products, designed by nature to establish specific interactions with certain classes of proteins eliciting biological responses, are attractive molecules for the development of new drugs.⁵⁸ In addition, Michael acceptor systems, namely α,β-unsaturated carbonyl groups, have been reported as key structural features for the therapeutic activities of natural terpenoid compounds, including with anti-inflammatory and anticancer effects.⁵⁹ Thus, the preparation of new triterpenoid derivatives bearing an α,β-unsaturated carbonyl group was explored. To obtain these structures, we started by oxidizing the 6-membered A-ring derivatives 6–8 and 9–12 with sodium periodate (NaIO₄) in methanol/water at room temperature to afford the intermediate A-ring lactol products 13–20.^45,60 The reaction of these intermediates with piperidine and acetic acid (catalytic amounts) in dry benzene at 60 °C under nitrogen afforded the desired derivatives 21–28 bearing a pentameric A-ring containing an α,β-unsaturated carbonyl moiety (Scheme 2). The chemical structures of all these derivatives were assigned by comparison with the data of precursor compounds and based on their NMR data including 2D-NMR experiments COSY, NOESY, HMBC and HSQC (see NMR spectra data at ESI†).

The absolute configuration of the lactol A-ring in derivatives 13–20 was established based on NOESY correlations (ESI,† page 63). The aldehydic proton H-3 was found to present an α configuration as it correlates with the α-methyl group at C25. These results reveals that the conversion of the hexameric A-ring compounds 1–12 into the lactol ring of the corresponding derivatives 13–20 occurred with inversion of configuration of C-24 methyl group from β to α, according to the proposed mechanism at ESI,† page 65.

In the ¹H NMR spectra of compounds 21–28, it was possible to observe two downfield resonances at δH 9.70–9.71 and δH 6.65–6.70 assigned to the aldehydic proton and olefinic proton at C-3, respectively. Regarding the ¹³C NMR spectrum, the aldehyde carbonyl signals were displayed at δC 190.75–190.94 and two olefinic carbon signals at δC 158.66–158.84 and δC 159.02–160.39 were assigned to C-2 and C-3, respectively, confirming the presence of the α,β-unsaturated carbonyl moiety. Additionally, the characteristic IR carbonyl stretch (C O) vibration band of the α,β-unsaturated aldehyde appeared at 1680.21–1726.47 cm⁻¹. NOESY correlations (ESI,† page. 64), allowed the establishment of the A-nor ring configuration. The substituents at C4 position presented again an inversion of configuration. A mechanistic proposal for the conversion of the lactol A-ring into A-nor ring bearing an α,β-unsaturated carbonyl moiety could be found in ESI† (page 65).

Biological studies

In vitro cytotoxic activity

The in vitro cytotoxic activity of AA1 and semisynthetic derivatives was initially determined in two human cancer cell lines of pancreas ductal adenocarcinoma (PANC-1) and colorectal adenocarcinoma (HT-29) using a MTT (dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) colorimetric assay. These results, expressed as IC₅₀ values (the concentration needed to inhibit 50% of cell viability compared with the control condition) are summarized in Table 1. Acetylation of the three hydroxyl groups of AA1 to afford compound 2 significantly improved the cytotoxic activity against both cell lines relative to the parent compound AA1. The further introduction of the amide moieties at C-28 to obtain derivatives 3–5 led to a significant increase in the cytotoxic activity. In PANC-1 cells, it was observed a 15 to 89-fold increase in cytotoxicity relative to that of the parent compound AA1. The best cytotoxic activity of this series was exhibited by the ethylamide derivative 5 with IC₅₀ values of 0.63 μM (PANC-1). Furthermore, the removal of acetyl groups to generate 6–8 derivatives dramatically lowered cytotoxicity against both cell lines. Considering these results, it can be deduced that the increased potency might be related to the improvement in membrane permeability caused by the triacetate moieties and amide side chain.

Cytotoxic activity for arjunolic acid 1 and its semisynthetic derivatives against HT-29 and PANC-1 cancer cell lines^a.

Compound	Cell line^a/IC₅₀^b (μM ± SD)
Compound	PANC-1	HT-29
AA1	56.31 ± 8.61	36.42 ± 1.88
2	14.90 ± 0.71	12.43 ± 1.78
3	3.63 ± 0.44	19.26 ± 0.50
4	0.93 ± 0.23	16.53 ± 4.50
5	0.63 ± 0.06	1.17 ± 0.18
6	>30	>30
7	>30	>30
8	27.57 ± 2.62	25.48 ± 2.32
9	19.65 ± 1.33	16.73 ± 1.06
10	20.20 ± 1.19	11.60 ± 0.26
11	20.25 ± 1.56	13.70 ± 0.42
12	18.95 ± 1.35	12.83 ± 0.87
13	>30	>30
14	29.32 ± 1.14	N.D.
15	16.65 ± 0.17	25.92 ± 1.46
16	22.03 ± 1.69	N.D.
17	6.70 ± 0.38	5.59 ± 0.33
18	7.14 ± 0.60	6.70 ± 0.65
19	4.58 ± 0.32	4.46 ± 0.37
20	4.92 ± 0.32	4.38 ± 0.34
21	5.27 ± 0.27	5.73 ± 0.98
22	3.74 ± 0.25	5.81 ± 0.19
23	2.38 ± 0.42	7.41 ± 0.70
24	4.15 ± 0.38	3.43 ± 0.12
25	0.82 ± 0.04	0.87 ± 0.03
26	0.47 ± 0.04	0.51 ± 0.02
27	0.43 ± 0.02	0.55 ± 0.04
28	0.84 ± 0.05	0.55 ± 0.04

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HT-29 and PANC-1 cells were treated with crescent concentrations of each compound for 72 h. IC₅₀ values were determined by MTT assay and are represented as the mean ± SD of three independent experiments. N.D.: not determined.

IC₅₀ is the concentration of compound that inhibits 50% of cell growth.

To explore the potential of the C-28 substituted derivatives, the cytotoxic activity of ester analogues 9–12 was also assessed and compared with that of compound AA1. The findings showed that the introduction of the ester moieties led to an increase in potency of the compounds, making them, in PANC-1 and HT-1 cancer cells, respectively, 2.7–3 and 2.2–3.1 times more active than the parent AA1.These results are in agreement with previous results that showed that the presence of short chain alkyl esters at C-28 have a positive impact on the cytotoxicity of triterpenoids towards cancer cell lines.⁶¹

Regarding the A-ring lactol derivatives, the conversion of the hexameric A-ring into the heptameric lactol ring did not alter significantly the activity for derivatives bearing carboxylic acid (e.g. compare AA1vs.13) or amide (e.g. compare 8vs.16) moieties at C-28. Differently, C-28 ester derivatives with a 7-membered lactol ring exhibited improved cytotoxicity compared to the corresponding parent molecules (compare 9vs.17) on both cell lines. The conversion of the lactol derivatives 13–20 into pentameric A-ring derivatives 21–28 led to a profound increase in potency for all derivatives. Within this series of derivatives, the C-28 ester analogues displayed the highest activities with IC₅₀ values in the low micromolar range, from 0.43–0.84 μM on PANC-1 and 0.51–0.87 μM on HT29 cells. According to these results, derivatives 25–28 were selected to further explore their effect on different malignant cells, namely A375 and SK-MEL-28 (melanoma), A549 and H2170 (lung cancer) cells. As it can be observed in Table 2, the four compounds proved to be very potent against all these tumor cell lines.

Cytotoxic activities for arjunolic acid derivatives 25–28 against several cancer cell lines and non-tumor human fibroblast cell line (BJ)^a.

Compound	Cell lines^a/IC₅₀^b (μM)
	Melanoma		Lung		Non-tumoral
	A375	SK-MEL-28	A-549	H-2170	BJ
25	0.85 ± 0.04	0.85 ± 0.09	1.22 ± 0.03	2.11 ± 0.09	—
26	0.58 ± 0.04	0.58 ± 0.02	0.83 ± 0.01	1.33 ± 0.03	3.06 ± 0.23
27	0.55 ± 0.02	0.45 ± 0.02	0.58 ± 0.04	1.30 ± 0.03	2.79 ± 0.08
28	0.59 ± 0.06	0.53 ± 0.02	0.82 ± 0.02	1.51 ± 0.14	—

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The different cell lines were treated with crescent concentrations of each compound for 72 h. IC₅₀ values were determined by MTT assay in all cancer cell lines. IC₅₀ values are expressed as the mean ± SD of three independent experiments. N.D.: not determined.

IC₅₀ is the concentration of compound that inhibits 50% of cell growth.

Derivatives 26 and 27, which bear a C-28 ethyl and butyl ester moieties, respectively, displayed the most potent growth inhibitory activities, thus, they were selected to study the selectivity of their effect by performing a viability assay on non-malignant human cells (BJ fibroblasts). Excluding H2170 cells, both compounds 26 and 27 exhibited an interesting selectivity between tumor cells and non-tumor human BJ cells. This selectivity was more evident in PANC-1 cells, with both compounds showing a 6.5-fold increase in tumor-to-non-tumor sensitivity.

Based on the data obtained from the anticancer studies on PANC-1 and HT-29, preliminary SAR correlations were determined and are summarized in Fig. 2. The conversion of the hexameric A-ring into a lactol ring had a moderate impact on the anticancer activity of the new derivatives. On the other hand, the further conversion into a 5-carbon A-ring bearing an α,β-unsaturated aldehyde moiety significantly improved the anticancer activity. The acetylation of the 3-A-ring hydroxyl groups also exhibited a positive effect on the antitumor activity (Fig. 2). The introduction of amide and specially ester moieties at C28 also positively impacts the activity and selectivity of the derivatives. The SAR analysis showed that the two most active derivatives of this study, 26 and 27, presented similar structural features namely an α,β-unsaturated carbonyl moiety inserted in a pentameric A-ring, and an ester group at C-28. These moieties may represent key structural requirements for the activity and selectivity of AA 1 derivatives as anticancer agents.

Cell cycle assay

To better understand the mechanism behind the inhibitory growth activity of compound 26, the cell-cycle distribution was evaluated. The cell cycle is a tightly regulated process where a cell is divided into two daughter cells, and comprises two distinct phases, the mitosis (M), in which a cell undergoes cell division, and the interphase, which includes the G1 (pre-DNA synthesis), S (DNA synthesis), and G2 (pre-division) phases. Following interphase, the cell returns to the G0 phase (quiescence).⁶² The propidium iodide (PI) fluorescence dye can be used to assess the cell cycle state using flow cytometry after staining the cellular DNA with this dye. This method allows differentiating the several cell cycle phases. Thus, this method was used to evaluate the potential effect of compound 26 in PANC-1 cells, which were treated with increasing concentrations of this derivative for 24 h. As it can be observed in Fig. 3A and B, derivative 26 induced a dose-dependent accumulation of cells in the G0/G1 phase (from 51.8% in control to 70.2% in cells treated with 1.88 μM of compound 26), which was accompanied by a decrease in the percentage of cells in the S phase (from 19.8% in control cells to 3.7% in cells treated with 1.88 μM of 26). Therefore, the analysis of the cell-cycle progression suggests that the mechanism of action of this compound is related with cell-cycle arrest at the G1 phase.

Annexin V-FITC/PI double-staining assay

Annexin V-FITC/PI double-staining assay can be used to evaluate if a compound is able to induce apoptosis. The loss of membrane symmetry observed in the earliest apoptosis stages, led to the translocation of phosphatidylserine (PS) molecules from the inner to outer surface of the plasma membrane bilayer. Annexin V-FITC binds with nanomolar affinity to the exposed apoptotic cell surface PS, thus allowing the quantitative assessment of apoptosis. On the other hand, in the late stages of apoptosis membrane loses its integrity allowing PI to enter the cell.⁶³ As it can be observed in Fig. 3C and D, the number of apoptotic cells did not increase upon the treatment of PANC-1 cells with increasing concentrations of compound 26 for 24 h, which suggests that this compound does not induce apoptosis in PANC-1 cells. Considering that the sub-G1 phase in cell cycle analysis has been suggested as a possible indicator for the presence of apoptotic cells,⁶⁴ these results are in good agreement with the cell cycle analysis, in which the sub-G1 phase was not altered by treating cells with this AA derivative.

C Hoechst 33342 staining assay

Hoechst 33342 is a fluorescent stain that labels DNA, staining the condensed chromatin of apoptotic cells more brightly when compared to non-apoptotic cells. Thus, PANC-1 cells were treated with different concentration of compound 26 for 24 h, then stained with Hoechst and observed by fluorescence microscopy. As expected, there were no significant morphological changes associated with apoptosis, such as chromatin condensation and formation of apoptotic bodies, when comparing control cells with treated cells (Fig. 4A). However, there is an evident reduction in the number of cells in the wells treated with increasing concentrations of the drug. These results may be explained by a possible cytostatic effect induced by compound 26 as a result of its effect on the cell-cycle.

Scratch assay

Cell migration is a key procedure involved in many biological processes including cancer progression. The wound healing assay can be used to evaluate cell motility and migration by monitoring the evolution of a wounded monolayer of cells, which can be achieved by growing a confluent monolayer of cells, then creating a wound (or scratch) on each well and observing the wound process over a period of time.⁵⁶ Thus, the wound-healing assay was used to evaluate the effect of compound 26 on PANC-1 cell migration. As it can be observed in Fig. 4B, this compound significantly inhibited the wound closure rate of PANC-1 cancer cells in a concentration-dependent manner, which suggests that derivative 26 exhibits anti-migration activity by modifying the cell motility.

Drug combination assay

Gemcitabine is indicated for the treatment of patients with locally advanced or metastatic pancreas adenocarcinoma. However, the growing resistance to gemcitabine in pancreatic patients is an important issue,⁶⁵ and exploring possible combination regimens that could improve cells sensitivity to Gemcitabine is desirable. Therefore, aiming at evaluating the type of in vitro interactions between compound 26 and the anticancer drug gemcitabine, their effects were assessed in a combination chemotherapy model using PANC-1 cancer cells. The extent of interaction was calculated by the combination index (CI) as suggested by Chou and Talalay,^66,67 with a CI ≈ 1 indicating additivity, CI < 1 defining synergism, and CI > 1 being related to antagonism. For this, PANC-1 cells were treated with different combinations of gemcitabine and compound 26 at different concentrations for 72 h. As shown in Table 3, compound 26 synergistically enhanced the cytotoxicity of gemcitabine, especially at a concentration of 0.24 μM which produced the lowest CI (0.75).

Study of the interaction type between gemcitabine and compound 26 on PANC-1 cells. Combination index (CI) values were calculated based on a constant ratio of 50 : 1 ([gemcitabine] : [26]) using the Chou–Talalay method and the CompuSyn software.⁶⁷ CI values <1 indicate the existence of synergism.

26 (μM)	Gemcitabine (μM)	1 – viability	CI
0.12	6	0.36	0.93
0.24	12	0.42	0.75
0.47	23.5	0.49	0.98
0.94	47	0.76	0.86
1.88	94	0.92	0.79

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Pharmacology safety study

After an in silico prediction of relevant physicochemical parameters as well as drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties (ESI,† pages 66 and 67), a preliminary pharmacology safety study with compound 26 was performed using the primary observation (Irwin) test to evaluate the behavioral effects of this AA derivative in female Balb/c mice. This primary observation (Irwin) test is a method used to detect the first toxic dose, the active dose-range and the main effects of a test substance on the behavior and physiological function, according to the work described by Irwin.⁶⁸ For this, female Balb/c mice were administered with increasing concentrations of compound 26 (i.p. route) and were observed in simultaneous comparison with a control group given vehicle (non-blind conditions). All animals within a treatment group were observed simultaneously. At the lower dose range (0.01, 0.1, 1, 5, 10 mg kg⁻¹) compound 26 did not demonstrate toxicity since the sole sign observed was transient and occasional writhing following administration. At the higher doses of 50 to 100 mg kg⁻¹, dose-dependent and late effects were observed (tremor, decreased activity, abnormal gait with rolling, decrease of abdominal muscle tone, loss of traction indicative a muscle weakness, piloerection, decreased of respiration, decreased fear/startle, decreased reactivity to touch, ptosis, and important hypothermia). In addition, one mouse was found dead at the higher dose of 100 mg kg⁻¹ after 48 hours. Thus, these data suggest that compound 26 at lower doses did not demonstrate toxicity and, consequently, the lower dose range should be selected to further evaluate the effect of derivative 26 on in vivo tumor progression.

Conclusions

This work focused on the generation of a set of new semisynthetic derivatives of the underexplored AA1 scaffold. For that, different modifications were explored at A-ring and C-28 moieties, aiming to optimize the anticancer properties of this natural product. In a general overview, an increase in cytotoxic activity towards human pancreatic (PANC-1) and colon (HT-29) cancer cell lines was observed for most derivatives when compared with AA1. Regarding these two cell lines, the most active compounds were those bearing a 5 carbon A-ring containing a α,β-unsaturated carbonyl group and an ester moiety at C-28 position (25, 26, 27 and 28). Furthermore, these compounds revealed potent cytotoxic activities in a set of additional cancer cell lines. Within this group of AA derivatives, compound 26 has a significantly increased antitumor effect when compared with the parent compound, while retaining selectivity towards tumor cells against non-tumor cells (BJ cells). Therefore, this compound was selected for further studies regarding its mechanism of action using PANC-1 cells. While compound 26 did not display apoptosis-induction effects, it originated a cell-cycle arrest at G0/G1 phase and significantly inhibited the wound closure rate of PANC-1 cancer cells in a concentration-dependent manner. Additionally, compound 26 enhanced synergistically the cytotoxicity of gemcitabine, especially at the concentration of 0.24 μM. Moreover, a preliminary in vivo pharmacology study indicated that compound 26 at lower doses did not demonstrate toxicity. These findings suggest that derivative 26 may be a valuable compound for the development of a new pancreatic anticancer treatment, and further studies are needed to explore its full potential.

Experimental section

Chemistry

General remarks

Arjunolic acid (AA1) was purchased from Sigma Aldrich, in over 90% purity. All other reagents were purchased from commercial suppliers and unless otherwise stated used as received, while the solvents were distilled and dried according to usual procedures. Thin layer chromatography (TLC) performed in Kieselgel 60H F254 coated plates was used to monitor the reaction progress. Flash column chromatography (FCC) using Kieselgel 60 (230–400 mesh, Merck) was employed for purification of the compounds. IR spectra were acquired on a Fourier transform IR spectrometer [VM1]. NMR spectra were recorded on a Bruker Digital NMR-Avance 400 apparatus spectrometer (1H 400 MHz, 13C 100 MHz), using CDCl3 or CH3OD as internal standards. The chemical shifts (δ) were expressed in parts per million (ppm) and coupling constants (J) in hertz (Hz). Mass spectra were obtained on a Linear Ion Trap (LIT-MS) mass spectrometer (LTQ XL, Thermo Scientific) and elemental analysis was performed by chromatographic combustion using an Analyzer Elemental Carlo Erba 1108.

Synthesis and structural characterization of compounds 2–28 is described below

2α,3β,23-Triacetoxyolean-12-en-28-oic acid (2)

To 175.8 mg of compound AA1 (0.36 mmol), dissolved in 5.3 mL of dry THF (5.3 mL), were added 0.22 mL of acetic anhydride (2.35 mmol) and 17.6 mg of DMAP (catalytic). The reaction mixture was then allowed to stir at room temperature in anhydrous conditions for 5 hours. After evaporation of the solvent under reduced pressure, the obtained crude was dispersed in water (25 mL) and extracted with ethyl acetate (2 × 25 mL). The resulting organic phase was washed with 5% aqueous HCl (70 mL), 10% aqueous NaHCO₃ (70 mL), water (2 × 70 mL) and brine (70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 1 : 1) to yield compound 2 as a white solid (86.54 mg, 39.10%). IR (ATR) ν_max: 2945.07, 2869.68, 1778.70, 1739.74, 1463.54, 1366.98, 1228.93, 1041.44 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ = 5.26 (t, J = 3.34 Hz, 1H, H12), 5.14 (td, J = 10.8, 4.4 Hz, 1H, H2), 5.06 (d, J = 10.3 Hz, 1H, H3), 3.83 (d, J = 11.8 Hz, 1H, H23), 3.57 (d, J = 11.8 Hz, 1H, H23), 2.81 (dd, J = 13.9, 4.6 Hz, 1H, H18), 2.08 (s, 3H, OCOCH̲_3̲), 2.01 (s, 3H, OCOCH̲_3̲), 1.97 (s, 3H, OCOCH̲_3̲), 1.10 (s, 3H), 1.07 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.73 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ = 184.19 (C28), 170.99 (OCO), 170.63 (OCO), 170.56 (OCO), 143.78 (C13), 122.21 (C12), 74.95 (C3), 70.01 (C2), 65.37 (C23), 47.77, 47.65, 46.61, 45.87, 43.62, 42.03, 41.66, 41.00, 39.41, 38.01, 33.88, 33.17, 32.49, 32.24, 30.79, 27.65, 25.89, 23.67, 23.55, 22.89, 21.22, 21.02, 20.91, 17.99, 17.10, 17.00, 13.98. DI-ESI-MS m/z: 613.48 (100%) [M–H]. Calcd. for C₃₆H₅₄O₈·0.75H₂O: C, 68.82; H, 8.90; found: C, 68.71; H, 8.96%.

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-amine (3)

To 1050.6 mg of compound 2 (1.71 mmol), dissolved in 38 mL of dry THF (38 mL), were added 0.26 mL of thionyl chloride (3.61 mmol). The resultant solution was heat-refluxed at 65 °C for 14 hours. The solvent was then removed under reduced pressure, and the acyl chloride intermediate was dissolved in 70 mL of THF. Then, 80 mL of cold 25% aqueous ammonia solution was added, and the resultant mixture was allowed to stir vigorously in an ice-cold bath for 3 hours. After evaporation of the solvent under reduced pressure, the obtained crude was dispersed in water (30 mL) and extracted with ethyl acetate (3 × 100 mL). The resulting organic phase was sequentially washed with 5% aqueous HCl (120 mL), 10% aqueous NaHCO₃ (120 mL), water (2 × 120 mL) and brine (120 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to afford 3 as a white solid (136.25 mg, 12.98%). IR (ATR) ν_max: 3474.2, 3362.43, 2944.82, 2870, 1738.99, 1658.18, 1598.32, 1463.89, 1433.03, 1366.93, 1030.28 cm⁻¹.1H NMR (400 MHz, CDCl₃) δ = 5.85 (s, 1H, NH), 5.71 (s, 1H, NH), 5.35–5.34 (m, 1H, H12) 5.17–5.11 (m, 1H, H2), 5.06 (d, J = 10.4 Hz, 1H, H3), 3.83 (d, J = 11.7 Hz, 1H, H23), 3.57 (d, J = 11.7 Hz, 1H), 2.53 (dd, J = 13.2, 4.4 Hz, 1H, H18), 2.07 (s, 3H, OCOCH̲_3̲), 2.01 (s, 3H, OCOCH̲_3̲), 1.97 (s, 3H, OCOCH̲_3̲), 1.14 (s, 3H), 1.08 (s, 3H), 0.90 (6H), 0.88 (s, 3H), 0.81 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ = 181.31 (CON), 170.95 (OCO), 170.60 (OCO), 170.53 (OCO), 145.03 (C13), 122.42 (C12), 74.88 (C3), 69.98 (C2), 65.36 (C23), 47.71, 47.61, 46.72, 46.49, 43.68, 42.59, 42.13, 42.03, 39.37, 37.93, 34.17, 33.10, 32.61, 32.05, 30.84, 27.34, 25.72, 23.81, 23.68, 23.64, 21.22, 21.00, 20.90, 18.00, 17.11, 17.06, 13.99. DI-ESI-MS m/z: 614.45 (100%) [M + H]⁺. Calcd. for C₃₆H₅₅NO₇·1.5H₂O: C, 67.47; H, 9.12; N, 2.19. Found: C, 67.48; H, 8.90; N, 2.54%.

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-methyl amine (4)

To 114.6 mg of compound 2 (0.19 mmol), dissolved in 5 mL of dry THF (5 mL), were added 0.03 mL of thionyl chloride (0.40 mmol). The resultant solution was heat-refluxed at 65 °C for 16 hours. After evaporation of the solvent under reduced pressure, the obtained acyl chloride derivative was dissolved without purification in 5 mL of dry CH₂Cl₂. The obtained solution was basified to pH 8–9 with approx. 0.40 mL of triethylamine and methylamine solution (33 wt% in absolute ethanol) was added. After stirring in an ice-cold bath for 4 hours, the resulting mixture was concentrated under reduced pressured. The obtained crude was dispersed in water (20 mL), acidified with 10% aqueous HCl until pH 5–7 and extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with water (2 × 50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to yield 4 as a white solid (53.7 mg, 45.0%). IR (ATR) ν_max: 3429.93, 2944.42, 1740.39, 1643.83, 1366.94, 1229.60, 1042.87 cm⁻¹.¹H NMR (400 MHz, CDCl₃) δ = 5.89 (q, J = 4.7 Hz, 1H, NH), 5.38–5.36 (m, 1H, H12), 5.15 (td, J = 10.9, 4.5 Hz, 1H, H2), 5.07 (d, J = 10.4 Hz, 1H, H3), 3.84 (d, J = 11.8 Hz, 1H, H23), 3.58 (d, J = 11.9 Hz, 1H, H23), 2.73 (d, J = 4.7 Hz, 3H, CH₃N), 2.49 (dd, J = 13.1, 4.3 Hz, 1H, H-18), 2.08 (s, 3H, OCOCH̲_3̲), 2.02 (s, 3H, OCOCH̲_3̲), 1.98 (s, 3H, OCOCH̲_3̲), 1.14 (s, 3H), 1.09 (s, 3H), 0.90 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ = 178.84 (CON), 170.95 (OCO), 170.64 (OCO), 170.53 (OCO), 145.53 (C13), 122.36 (C12), 74.88 (C3), 70.00 (C2), 65.36 (C23) 47.73, 47.61, 46.92, 46.36, 43.68, 42.30, 42.09, 42.06, 39.46, 37.93, 34.22, 33.13, 32.31, 31.84, 30.89, 27.29, 26.33, 25.85, 23.89, 23.72, 23.70, 21.25, 21.02, 20.93, 18.01, 17.04, 16.72, 14.00. DI-ESI-MS m/z: 628.51 (100%) [M + H]⁺; 650.50 (44.33%) [M + Na]⁺. Calcd. for C₃₇H₅₇NO₇·0.5H₂O: C, 69.78; H, 9.18; N, 2.20. Found: C, 69.49; H, 9.32; N, 2.54%.

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-ethyl amine (5)

To 368.6 mg of compound 2 (0.60 mmol), dissolved in 13 mL of dry THF (13 mL), were added 0.09 mL of thionyl chloride (1.27 mmol). The resultant solution was heat-refluxed at 65 °C for 15 hours. After evaporation of the solvent under reduced pressure, the obtained acyl chloride intermediate was dissolved without purification in 13 mL of dry CH₂Cl₂ (13 mL). The obtained solution was basified to pH 8–9 with approx. 0.55 mL of triethylamine and ethylamine solution (2.0 M in THF) was added. After stirring in an ice-cold bath for 3.5 hours, the resulting mixture was concentrated under reduced pressured. The obtained crude was dispersed in water (20 mL), acidified with 10% aqueous HCl until pH 5–7 and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (2 × 65 mL) and brine (65 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 2 : 1) to yield 5 as a white solid (70.79 mg, 18.38%). IR (ATR) ν_max: 3417.78, 2943.03, 2875.70, 1742.40, 1639.82, 1520.00, 1463.58, 1367.28, 1230.28, 1042.18 cm⁻¹. 1H NMR (400 MHz, CDCl3) δ = 5.81 (t, J = 5.4 Hz, 1H, NH), 5.36 (t, J = 3.6 Hz, 1H, H12), 5.15 (td, J = 10.9, 4.6 Hz, 1H, H2), 5.07 (d, J = 10.3 Hz, 1H, H3), 3.84 (d, J = 11.8 Hz, 1H, H23), 3.58 (d, J = 11.8 Hz, 1H, H23), 3.36–3.27 (m, 1H, NCH̲), 3.16–3.08 (m, 1H, NCH̲), 2.08 (s, 3H, OCOCH̲_3̲), 2.02 (s, 3H, OCOCH̲_3̲), 1.98 (s, 3H, OCOCH̲_3̲), 1.14 (s, 3H), 1.09–1.08 (6H), 0.90 (6H), 0.88 (s, 3H), 0.77 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ = 178.02 (CON), 170.96 (OCO), 170.63 (OCO), 170.54 (OCO), 145.38 (C13), 122.21 (C12), 74.90 (C3), 70.00 (C2), 65.38 (C23), 47.73, 47.61, 46.89, 46.21, 43.71, 42.33, 42.18, 42.05, 39.53, 37.94, 34.51, 34.24, 33.14, 32.61, 32.00, 30.88, 27.32, 25.76, 23.81, 23.72 (2C), 21.25, 21.02, 20.93, 18.01, 17.06, 17.00, 14.76, 14.01. DI-ESI-MS m/z: 642.46 (100%) [M + H]⁺. Calcd. for C₃₈H₅₉NO₇·0.75H₂O: C, 69.64; H, 9.30; N, 2.14. Found: C, 69.83; H, 8.90; N, 2.24.

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-amine (6)

To a stirred solution of methanolic potassium hydroxide (8 mL, 5% w/v), were added 89.1 mg of compound 3. The resulting solution was allowed to stir at room temperature for 10.5 hours. After evaporation of the solvent under reduced pressure, the obtained crude was dispersed in water (20 mL), acidified with 10% aqueous HCl until pH 5–7 and extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with water (2 × 50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 1 : 2 → 1 : 8) to yield compound 6 as a white solid (46.3 mg, 63.29%). IR (ATR) ν_max: 3350.86, 2941.65, 1640.73, 1586.33, 1462.76, 1384.59, 1047.19 cm⁻¹. 1H NMR (400 MHz, CH₃OD) δ = 5.39–5.37 (m, 1H, H12), 3.74–3.68 (m, 1H, H2), 3.52 (d, J = 11.1 Hz, 1H, H23), 3.37 (d, J = 9.6 Hz, 1H, H3), 3.29 (d, J = 11.1 Hz, H23), 2.79–2.75 (m, 1H, H18), 1.22 (s, 3H), 1.06 (s, 3H), 0.97 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.72 (s, 3H). ¹³C NMR (101 MHz, CH₃OD) δ 183.64 (CON), 145.48 (C13), 123.81 (C12), 78.13 (C3), 69.64 (C2), 66.24 (C23), 49.00, 48.13, 47.87, 47.69, 47.52, 44.13, 43.09, 42.83, 40.54, 39.01, 35.10, 34.25, 33.54, 33.19, 31.62, 28.50, 26.49, 24.66, 24.10, 23.99, 19.08, 17.82, 17.54, 13.86. MS: DI-ESI-MS m/z: 488.42 (100%) [M + H]⁺. Calcd. for C₃₀H₄₉NO₄·1H₂O: C, 70.00; H, 10.18; N, 2.72; O, 13.12. Found: C, 69.62; H, 9.97; N, 2.93.

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-methyl amine (7)

To a stirred solution of methanolic potassium hydroxide (42 mL, 5% w/v), were added 419.5 mg of compound 4 (0.84 mmol). The resulting solution was allowed to stir at room temperature for 16 h. After evaporation of the solvent under reduced pressure, the obtained crude was dispersed in water (30 mL), acidified with 10% aqueous HCl until pH 5–7 and extracted with ethyl acetate (3 × 70 mL). The combined organic phases were washed with water (2 × 90 mL) and brine (90 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 1 : 2 → 1 : 5) to yield compound 7 as a white solid (131.25 mg, 31.14%). IR (ATR) ν_max: 3382.82, 2942.74, 2875.30, 1634.81, 1532.78, 1463.67, 1454.99, 1386.64, 1364.33, 1047.81 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ = 5.98–5.95 (m, 1H, NH), 5.38 (t, J = 3.6 Hz, 1H, H12), 3.79–3.72 (m, 1H, H2), 3.64 (d, J = 10.7 Hz, 1H, H23), 3.43–3.39 (2H, H3 and H23), 2.73 (d, J = 4.6 Hz, 3H, NCH̲_3̲), 2.47 (dd, J = 13.2, 4.3 Hz, 1H, H-18), 1.15 (s, 3H), 1.02 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.73 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ = 179.27 (CON), 145.34 (C13), 122.79 (C12), 80.38 (C3), 70.34 (C23), 68.64 (C2), 49.04, 47.59, 46.93, 46.37, 45.99, 42.61, 42.33, 42.19, 39.43, 38.20, 34.22, 33.12, 32.26, 31.93, 30.88, 27.34, 26.38, 26.05, 23.96, 23.71, 23.69, 18.43, 17.20, 16.81, 12.93. MS: DI-ESI-MS m/z: 502.43 (100%) [M + H]⁺. Calcd. for C₃₁H₅₁NO₄·1H₂O: C, 71.64; H, 10.28; N, 2.69. Found: C, 71.38; H, 10.18; N, 2.84.

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-ethyl amine (8)

To a stirred solution of methanolic potassium hydroxide (40 mL, 5% w/v), were added 500.7 mg of compound 5 (0.78 mmol). The resulting solution was allowed to stir at room temperature for 16.5 hours. After evaporation of the solvent under reduced pressure, the obtained crude was dispersed in water (30 mL), acidified with 10% aqueous HCl until pH 5–7 and extracted with ethyl acetate (3 × 60 mL). The combined organic phases were washed with water (2 × 80 mL) and brine (80 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 1 : 2 → 1 : 5) to yield compound 8 as a white solid (76.44 mg, 19.00%). IR (ATR) ν_max: 3366.98, 2939.67, 2875.90, 1723.14, 1634.85, 1527.06, 1452.00, 1386.16, 1364.01, 1047.45 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ = 5.88 (t, J = 5.3 Hz, 1H, NH), 5.37 (t, J = 3.6 Hz, 1H, H12), 3.78–3.72 (m, 1H, H2), 3.64 (d, J = 10.6 Hz, 1H, H23), 3.43–3.99 (2H, H3 and H23), 3.35–3.28 (m, 1H, NCH̲_2̲CH₃), 3.13–3.07 (m, 1H, NCH̲_2̲CH₃), 2.49 (dd, J = 13.0, 4.4 Hz, 1H, H18), 1.15 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H, NCH₂CH̲_3̲), 1.02 (s, 3H), 0.90 (6H), 0.86 (s, 3H), 0.76 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ = 178.46 (CON), 145.20 (C13), 122.64 (C12), 80.36 (C3), 70.31 (C23), 68.64 (C2), 49.03, 47.58, 46.91, 46.24, 46.03, 42.61, 42.39, 42.28, 39.51, 38.21, 34.59, 34.25, 33.12, 32.56, 32.10, 30.87, 27.36, 25.96, 23.87, 23.71 (2C), 18.43, 17.23, 17.08, 14.66, 12.94. DI-ESI-MS m/z: 516.48 (100%) [M + H]⁺. Calcd. for C₃₂H₅₃NO₄·1.5H₂O: C, 70.81; H, 10.40; N, 2.58. Found: C, 70.75; H, 9.95; N, 2.61.

Methyl 2α,3β,23-trihydroxyolean-12-en-28-oate (9)

To 1000 mg of compound AA1 (2.05 mmol) and 700 mg of anhydrous potassium carbonate (5.07 mmol), dissolved in 10 mL of dry DMF, were added 0.255 mL of methyl Iodide (255 μL, 4 mmol). The reaction mixture obtained was allowed to stir at room temperature in anhydrous conditions for 2 hours. The reaction mixture was then diluted with water (70 mL) and extracted with ethyl acetate (3 × 70 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 70 mL), 10% aqueous NaHCO₃ (2 × 70 mL), 10% aqueous Na₂SO₃ (70 mL), water (100 mL) and brine (100 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 1 : 1 → 1 : 5) to yield compound 9 as a white solid (1010 mg, quantitative). IR (ATR) ν_max: 3380.20, 2945.71, 2929.40, 2864.20, 1725.00, 1456.22, 1162.42, 1035.49 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ = 5.27 (s, 1H, H12), 3.80–3.67 (m, 2H, H2 and H3), 3.61 (s, 3H, COOCH̲_3̲), 3.40–3.39 (m, 2H, H23), 2.87–2.84 (m, 1H, H18), 1.12 (s, 3H), 1.00 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.83 (s, 3H), 0.71 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 178.30 (C28), 143.82 (C13), 122.08 (C12), 80.43 (C3), 70.48 (C23), 68.70 (C2), 51.54, 49.08, 47.53, 46.67, 45.97, 45.81, 42.42, 41.67, 41.21, 39.26, 38.19, 33.82, 33.09, 32.35, 32.26, 30.68, 27.62, 25.99, 23.61, 23.43, 23.00, 18.32, 16.99, 16.87, 12.74.ppm. DI-ESI-MS m/z: 503.26 (100%) [M + H]⁺, 525.38 (38%) [M + Na]⁺. Anal. calcd. for C₃₁H₅₀O₅·1H₂O: C, 71.5; H, 10.07. Found: C, 71.47; H, 9.95%.

Ethyl 2α,3β,23-trihydroxyolean-12-en-28-oate (10)

To 300 mg of compound AA1 (0.61 mmol) and 211.8 mg of anhydrous potassium carbonate (1.53 mmol), dissolved in 5.3 mL of dry DMF, were added 0.125 mL of ethyl Iodide (1.55 mmol). The resulting reaction mixture was allowed to stir at room temperature in anhydrous conditions for 3 hours. The reaction mixture was then diluted with 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. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 2 : 1 →1 : 5) to yield compound 10 as a white solid (45.19 mg, 14.25%). IR (ATR) ν_max: 3387.97, 2942.56, 2866.50, 1722.28, 1462.28, 1035.11 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 5.28 (t, J = 3.31, 3.34 Hz, 1H, H12), 4.13–4.03 (m, 2H, COOCH̲_2̲CH₃), 3.77–3.71 (m, 1H, H2), 3.63 (d, J = 11.5 Hz, 1H, H3), 3.40 (d, J = 9.70 Hz, 2H, H23), 2.86 (dd, J = 13.85, 3.55 Hz, 1H, H18), 1.22 (t, J = 7.15, 7.15 Hz, 3H, COOCH₂CH̲_3̲), 1.13 (s, 3H), 1.01 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.73 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.73 (C28), 143.84 (C13), 122.01 (C12), 80.25 (C3), 69.95 (C23), 68.70 (C2), 60.09 (COOC̲H₂CH₃), 48.94, 47.52, 46.44, 46.04, 45.85, 42.47, 41.74, 41.22, 39.34, 38.18, 33.88, 33.10, 32.38, 32.34, 30.69, 27.57, 25.93, 23.60, 23.45, 22.93, 22.60, 18.31, 17.01, 14.24, 12.79 ppm. DI-ESI-MS m/z: 503.26 (100%) [M + H]⁺, 525.38 [M + Na]⁺. Anal. calcd. for C₃₂H₅₂O₅·H₂O: C, 71.87; H, 10.18. Found: C, 72.08; H, 10.28.

Butyl 2α,3β,23-trihydroxyolean-12-en-28-oate (11)

To 300 mg of compound AA1 (0.61 mmol) and 223.0 mg of anhydrous potassium carbonate (1.61 mmol), dissolved in 6 mL of dry DMF, were added 0.167 mL of n-butyl bromide (155 mmol). The reaction mixture was allowed to stir at room temperature in anhydrous conditions for 4.5 hours. The reaction mixture was then diluted with water (70 mL) and extracted with ethyl acetate (3 × 70 mL). The resulting organic phase was washed with 5% aqueous HCl (2 × 70 mL), 10% aqueous NaHCO₃ (2 × 70 mL), 10% aqueous Na₂SO₃ (70 mL), water (70 mL) and brine (70 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 2 : 1→ 1 : 5) to yield compound 11 as a white solid (33.9 mg, 10.20%). IR (ATR): 3401.54, 2932.11, 2872.88, 1780.53, 1720.98, 1462.28, 1042.91 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 5.28 (t, J = 3.25, 3.25 Hz, 1H, H12), 4.03–3.99 (m, 1H, COOCH̲_2̲CH₂CH₂CH₃), 3.79–3.73 (m, 1H, H2), 3.68–3.66 (d, J = 10.23 Hz, 1H, H3), 3.50 (s, 1H), 3.44–3.42 (d, J = 8.27 Hz, 1H, H23), 3.42–3.40 (d, J = 7.89 Hz, 1H, H23), 3.06 (s, 1H), 2.87 (dd, J = 13.9, 4.5 Hz, 1H, H18), 1.12 (s, 3H), 1.02 (s, 3H), 0.92 (m, 6H), 0.90 (s, 3H), 0.89 (s, 3H), 0.73 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.79 (C28), 143.85 (C13), 122.06 (C12), 80.77 (C3), 71.29 (C23), 68.70 (C2), 64.00, 49.31, 47.54, 46.61, 45.96, 45.83, 42.35, 41.74, 41.25, 39.32, 38.21, 33.87, 33.09, 32.44, 32.38, 30.70, 30.66, 27.57, 25.91, 23.58, 23.44, 22.94, 19.23, 18.37, 17.04, 16.99, 13.71, 12.68. ppm. DI-ESI-MS m/z: 567.43 (100%) [M + Na]⁺. Anal. calcd. for C₃₄H₅₆O₅·1.25H₂O: C, 71.98; H, 10.39. Found: C, 71.75; H, 9.96.

Allyl 2α,3β,23-trihydroxyolean-12-en-28-oate (12)

To 361.4 mg of compound AA1 (0.74 mmol) and 254.7 mg of anhydrous potassium carbonate (1.84 mmol), dissolved in 6.5 mL of dry DMF were added 0.130 mL of allyl iodine (1.42 mmol). The reaction mixture was allowed to stir at room temperature in anhydrous conditions for 4.5 hours. The reaction mixture was then diluted with 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 NaHCO₃ (2 × 60 mL), 10% aqueous Na₂SO₃ (60 mL), water (60 mL) and brine (60 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 1 : 1 → 1 : 5) to yield compound 12 as a white solid (74.31 mg, 19.00%). IR (ATR) ν_max: 3388.32, 2940.28, 2866.90, 1723.37, 1648.54, 1046.66, 1033.17 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 5.94–5.84 (m, 1H, COOCH₂CH̲CH₂), 5.33–5.28 (m, 2H, H12 and COOCH₂CHCH_{_2}), 5.20 (dd, J = 10.5, 1.2 Hz, 1H, COOCH₂CHCH_{_2}), 4.53–4.50 (m, 2H, –COOCH̲_2̲CHCH₂), 3.78–3.70 (m, 1H, H2), 3.65 (d, J = 10.60 Hz, 1H, H3), 3.43–3.39 (m, 2H, H23), 2.88 (dd, J = 13.85, 4.10 Hz, 1H, H18), 1.13 (s, 3H), 1.01 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H), 0.72 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.36 (C28), 143.75 (C13), 132.50 (COOCH₂C̲HCH₂), 122.14 (C12), 117.72 (COOCH₂CHC̲H₂), 80.43 (C3), 70.46 (C23), 68.70 (C2), 64.81 (COOC̲H₂CHCH₂), 49.08, 47.52, 46.70, 46.00, 45.83, 42.42, 41.73, 41.26, 39.35, 38.19, 33.85, 33.08, 32.41, 32.33, 30.68, 27.59, 25.94, 23.60, 23.43, 22.99, 18.33, 17.00 (2C), 12.74. ppm. DI-ESI-MS m/z: 551.45 (100%) [M + Na]⁺. Anal. calcd. for C₃₃H₅₂O₅·0.75H₂O: C, 73.09; H, 9.94. Found: C, 73.11; H, 9.80.

2α,23-Lactol-3-formyl-olean-12-ene-28-oic acid (13)

To 450.0 mg of compound AA1 (0.92 mmol), dissolved in 11.3 mL methanol/0.55 mL water 20 : 1), were added 290.6 mg of NaIO₄ (1.36 mmol).The reaction mixture was allowed to stir at room temperature for 2 hours and 10 minutes. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (50 mL) and extracted with dichloromethane (3 × 50 mL). The resulting organic phase was washed with water (3 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 1 : 1) to yield compound 13 as a white solid (62.16 mg, 13.87%). IR (ATR) ν_max: 3421.15, 2948.73, 2927.31, 2872.49, 2732.44, 2630.40, 1716.54, 1696.12, 1456.99, 1378.85 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.95 (s, 1H, H̲CO), 5.32 (t, J = 3.08, 3.08 Hz, 1H, H12), 5.14–5.10 (m, 1H, H2), 3.94 (d, J = 13.0 Hz, 1H, H23), 3.72 (d, J = 13.2 Hz, 1H, H23), 2.83 (m, 1H, H18), 1.13 (s, 3H), 1.05 (s, 3H), 0.99 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.25 (C̲HO), 183.19 (C28), 143.45 (C13), 122.73 (C12), 93.71 (C2), 65.37, 61.03, 53.49, 46.61, 45.53, 44.67, 43.71, 42.15, 41.16, 40.18, 39.85, 33.80, 33.04, 32.31, 30.65, 27.57, 25.50, 24.69, 23.51, 22.89, 22.60, 22.53, 20.53, 20.38, 17.72, 17.47, 14.41. ppm. DI-ESI-MS m/z: 487.15 (100%) [M + H]⁺. Anal. calcd. for C₃₀H₄₆O₅·0.30CH₂Cl₂: C, 71.06; H, 9.17. Found: C, 71.07; H, 8.76%.

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-amine (14)

To 502.4 mg of compound 6 (1.03 mmol), dissolved in 12 mL methanol/0.6 mL water (20 : 1), were added 327.3 mg of NaIO₄ (1.53 mmol). The reaction mixture was allowed to stir at room temperature for 5 hours. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (30 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with water (2 × 80 mL) and brine (80 mL), dried over Na₂SO₄, altered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to yield compound 14 as a white solid (62.38 mg, 12.47%). IR (ATR) ν_max: 3474.20, 3348.80, 2943.43, 2873.50, 2755.60, 1714.04, 1654.21, 1594.08, 1462.93, 1378.83, 1363.27, 1035.38 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ = 9.94 (s, 1H, CH̲O), 5.88 (br s, 1H, NH), 5.78 (br s, 1H, NH), 5.40 (t, J = 3.7 Hz, 1H, H12), 5.11 (dd, J = 9.6; 5.0 Hz, 1H, H2), 3.93 (d, J = 13.4 Hz, 1H), 3.72 (d, J = 13.3 Hz, 1H), 2.54 (dd, J = 13.1, 4.4 Hz, 1H, H18), 1.16 (s, 3H), 1.05 (s, 3H), 0.99 (s, 3H), 0.91 (9H). ¹³C NMR (100 MHz, CDCl₃) δ = 206.00 (CHO), 181.49 (CON), 144.90 (C13), 123.02 (C12), 93.72 (C2), 65.49, 61.02, 53.66, 46.67, 46.48, 44.93, 43.82, 42.80, 42.74, 40.24, 39.96, 34.23, 33.11 (2C), 32.62, 30.84, 27.37, 25.49, 24.92, 23.89, 23.62, 20.72, 20.53, 17.84, 14.60. DI-ESI-MS m/z: 486.37 (100%) [M + H]⁺. Calcd. for C₃₀H₄₇NO₄·1.5H₂O: C, 70.28; H, 9.83; N, 2.73. Found: C, 70.19; H, 9.63; N, 2.69.

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-methyl amine (15)

To 541.9 mg of compound 7 (1.08 mmol), dissolved in 13 mL methanol/0.65 mL water (20 : 1), were added 344.4 mg of NaIO₄ (1.61 mmol). The reaction mixture was allowed to stir at room temperature for 3 hours. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (30 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with water (2 × 80 mL) and brine (80 mL), dried over Na₂SO₄, altered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to yield compound 15 as a white solid (155.93 mg, 28.89%). IR (ATR) ν_max: 3374.51, 2943.22, 2877.60, 2740.00, 1714.00, 1634.76, 1463.72, 1379.00, 1362.97, 1036.94 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.95 (s, 1H, CHO), 5.92 (q, J = 4.8 Hz, 1H, NH), 5.41 (t, J = 3.6 Hz, 1H, H12), 5.12 (dd, J = 9.6, 5.0 Hz, 1H, H2), 3.93 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 2.74 (d, J = 4.7 Hz, 3H, NNCH̲_3̲), 1.16 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.91–0.90 (6H), 0.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ = 205.94 (CHO), 178.90 (CON), 145.42 (C13), 122.88 (C12), 93.79 (C2), 65.57, 60.98, 53.68, 46.66, 46.50, 44.84, 43.84, 42.68, 42.49, 40.25, 40.01, 34.26, 33.13, 32.85, 32.31, 30.88, 27.32, 26.35, 25.62, 24.95, 23.95, 23.66, 20.70, 20.53, 17.34, 14.59. DI-ESI-MS m/z: 500.44 (100%) [M + H]⁺. Calcd. for C₃₁H₄₉NO₄·1.25H₂O: C, 71.29; H, 9.94; N, 2.68. Found: C, 71.00; H, 9.99; N, 2.84%.

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-ethyl amine (16)

To 531.3 mg of compound 8 (1.03 mmol), dissolved in 12 mL methanol/0.6 mL water (20 : 1), were added 327.3 mg of NaIO₄ (1.53 mmol). The reaction mixture was allowed to stir at room temperature for 2 hours. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (30 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with water (2 × 80 mL) and brine (80 mL), dried over Na₂SO₄, altered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (petroleum ether/ethyl acetate 5 : 1 → 1 : 1) to yield compound 16 as a white solid (132.03 mg, 24.95%). IR (ATR) ν_max: 3374.90, 2946.22, 2872.72, 2736.10, 1714.14, 1634.84, 1527.03, 1462.16, 1379.02, 1363.08, 1036.32 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.94 (s, 1H, CHO), 5.83 (t, J = 5.5 Hz, 1H, NH), 5.41 (t, J = 3.7 Hz, 1H, H12), 5.11 (dd, J = 9.6, 5.0 Hz, 1H, H2), 3.93 (d, J = 13.2 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 3.34–3.27 (m, 1H, NCH̲_2̲CH₃), 3.17–3.10 (m, 1H, NCH̲_2̲CH₃), 1.16 (s, 3H), 1.11 (s, 3H), 1.09 (t, J = 7.3 Hz, 3H, NCH₂CH̲_3̲), 1.06 (s, 3H), 0.99 (s, 3H), 0.90 (6H), 0.86 (s, 3H).¹³C NMR (100 MHz, CDCl₃): δ = 205.94 (CHO), 178.06 (CON), 145.25 (C13), 122.78 (C12), 93.77 (C2), 65.55, 61.01, 53.66, 46.62, 46.35, 44.92, 43.83, 42.77, 42.55, 40.23, 40.10, 34.53, 34.29, 33.14, 33.06, 32.60, 30.86, 27.33, 25.51, 24.96, 23.87, 23.69, 22.76, 20.71, 20.53, 17.72, 14.73, 14.62. DI-ESI-MS m/z: 514.43 (100%) [M + H]⁺. Calcd. for C₃₂H₅₁NO₄·H₂O: C, 72.28; H, 10.05; N, 2.63. Found: C, 72.62; H, 9.66; N. 2.49%.

Methyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (17)

To 700.0 mg of compound 9 (1.39 mmol), dissolved in 18.5 mL methanol/0.93 mL water (20 : 1), were added 454.0 mg of NaIO₄ (2.12 mmol). The reaction mixture was allowed to stir at room temperature for 3 hours and 20 minutes. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (75 mL) and extracted with ethyl acetate (3 × 75 mL). The resulting organic phase was washed with water (3 × 75 mL) and brine (75 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 2 : 1) to yield compound 17 as a white solid (322.63 mg, 46.27%). IR (ATR) ν_max: 3535.86, 2933.04, 2870.41, 2723.70, 1724.12, 1466.38, 1033.63 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.96 (s, 1H, CH̲O), 5.33 (t, J = 3.32 Hz, 1H, H12), 5.13–5.09 (m, 1H, H2), 3.93 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 3.62 (s, 3H, COOCH̲_3̲), 2.88 (dd, J = 13.8, 3.7 Hz, 1H, H18), 1.13 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.81 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.94 (C̲HO), 178.21 (C28), 143.68 (C13), 122.52 (C12), 93.74 (C2), 65.44, 61.02, 53.52, 51.57, 46.82, 45.53, 44.73, 43.72, 42.18, 41.49, 40.16, 39.86, 33.86, 33.21, 33.10, 32.28, 30.68, 27.59, 25.51, 24.70, 23.57, 23.02, 20.58, 20.42, 17.60, 14.37 ppm. DI-ESI-MS m/z: 523.40 (100%) [M + Na]⁺. Anal. calcd. for C₃₁H₄₈O₅: C, 74.36; H, 9.66. Found: C, 74.25; H, 9.62%.

Ethyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (18)

To 540 mg of compound 10 (1.04 mmol), dissolved in 13.5 mL methanol/0.67 mL water (20 : 1), were added 348.5 mg of NaIO₄ (1.63 mmol). The reaction mixture was allowed to stir at room temperature for 2 hours and 50 minutes. After evaporation of the solvent under reduced pressure 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 (3 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 2 : 1) to afford 18 as a white solid (152.1 mg, 28.29%). IR (ATR) ν_max: 3306.03, 2929.07, 2870.71, 2731.8, 1727.23, 1464.18, 1386.57, 1380.04, 1363.23, 1160.84, 1034.21 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.95 (s, 1H, CHO), 5.33 (t, J = 3.40, 3.40 Hz, 1H, H12), 5.13–5.09 (m, 1H, H2), 4.12–4.04 (m, 2H, COOCH̲_2̲CH₃), 3.93 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 2.88 (dd, J = 13.7, 3.4 Hz, 1H, H18), 1.22 (t, J = 7.04 Hz, 1H, COOCH₂CH̲_3̲), 1.13 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.90 (s, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.94 (C̲HO), 177.64 (C28), 143.70 (C13), 122.47 (C12), 93.74 (C2), 65.43, 61.03, 60.13, 53.51, 46.60, 45.56, 44.77, 43.72, 42.25, 41.50, 40.14, 39.94, 33.91, 33.31, 33.11, 32.32, 30.69, 27.54, 25.43, 24.72, 23.56, 22.95, 20.59, 20.47, 17.78, 14.39, 14.26. ppm. DI-ESI-MS m/z: 537.38 (100%) [M + Na]⁺. Anal. calcd. for C₃₂H₅₀O₅·1.5H₂O: C, 70.94; H, 9.86. Found: C, 70.88; H, 9.60%.

Butyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (19)

To 437.8 mg of compound 11 (0.80 mmol), dissolved in 11 mL methanol/0.55 mL water (20 : 1), were added 282.7 mg of NaIO₄ (1.32 mmol). The reaction mixture was allowed to stir at room temperature for 1 hour and 40 minutes. After evaporation of the solvent under reduced pressure 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 (3 × 50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 3 : 1) to yield compound 19 as a white solid (110.6 mg, 25.36%). IR (ATR) ν_max: 3416.94, 2949.85, 2873.11, 2740.00, 1717.29, 1462.17, 1379.47, 1161.64, 1033.42 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.95 (s, 1H, CH̲O), 5.33 (t, J = 3.5 Hz, 1H, H12), 5.13–5.09 (m, 1H, H2), 4.03–4.00 (m, 2H, COOCH̲_2̲CH₂CH₂CH₃), 3.93 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 2.88 (dd, J = 13.8, 3.9 Hz, 1H, H18), 1.13 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.94–0.90 (m, 6H, COOCH₂CH₂CH₂CH̲_3̲ and CH₃), 0.90 (s, 3H), 0.80 (s, 3H). ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.94 (CHO), 177.71 (C28), 143.71 (C13), 122.51 (C12), 93.73 (C2), 65.43, 64.03, 61.03, 53.51, 46.76, 45.55, 44.77, 43.71, 42.24, 41.52, 40.14, 39.92, 33.91, 33.31, 33.11, 32.38, 30.69, 30.67, 27.53, 25.44, 24.71, 23.55, 22.96, 20.58, 20.43, 19.23, 17.80 (2C), 14.38, 13.71. ppm. DI-ESI-MS m/z: 565.45 (100%) [M + Na]⁺. Anal. calcd. for C₃₄H₅₄O₅·1.5H₂O: C, 71.67; H, 10.08. Found: C, 71.75; H, 10.05%.

Allyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (20)

To 497.3 mg of compound 12 (0.94 mmol), dissolved in 12.5 mL methanol/0.63 mL water (20 : 1), were added 321.2 mg of NaIO₄ (1.50 mmol).The reaction mixture was allowed to stir at room temperature for 5 hours and 30 minutes. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (60 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with water (3 × 60 mL) and brine (60 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 6 : 1 → 3 : 1) to yield compound 20 as a white solid (185.26 mg, 37.40%). IR (ATR) ν_max: 3289.29, 2949.20, 2927.66, 2907.00, 2873.10, 2840.50, 1726.47, 1466.47, 1158.65, 1033.56 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.96 (s, 1H, CH̲O), 5.94–5.84 (m, 1H, COOCH₂CH̲CH₂), 5.35–5.29 (m, 2H, COOCH₂CHCH̲_2̲ and H12), 5.20 (d, J = 10.4 Hz, 1H, COOCH₂CHCH̲_2̲), 5.13–5.09 (m, 1H, H2), 4.52 (t, J = 5.3 Hz, 2H, COOCH̲_2̲CHCH₂), 3.93 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 2.90 (dd, J = 13.8, 3.8 Hz, 1H, H18), 1.13 (s, 3H, CH̲_3̲-27), 1.06 (s, 3H, CH̲_3̲-25), 0.99 (s, 3H, CH̲_3̲-24), 0.93 (s, 3H, CH̲_3̲-29), 0.90 (s, 3H, CH̲_3̲-30), 0.82 (s, 3H, CH̲_3̲-26) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 205.92 (CH̲O), 177.27 (C28), 143.61 (C13), 132.49 (COOCH₂C̲HCH₂), 122.60 (C12), 117.81 (COOCH₂CHC̲H₂), 93.74 (C2), 65.44 (C23), 64.86 (COOC̲H₂CHCH₂), 61.03 (C5), 53.51 (C4), 46.85 (C17), 45.55 (C19), 44.77 (C1), 43.72 (C9), 42.24 (C14), 41.53 (C18), 40.14 (C10), 39.95 (C8), 33.90 (C15), 33.29 (C21), 33.09 (C30), 32.35 (C22), 30.68 (C20), 27.56 (C7), 25.45 (C27), 24.70 (C11), 23.56 (C29), 23.01 (C16), 20.59 (C24), 20.42 (C6), 17.77 (C26), 14.39 (C25) ppm. DI-ESI-MS m/z: 527.46 (10%) [M + H]⁺, 527.46 (10%). Anal. calcd. for C₃₃H₅₀O₅·0.25H₂O: C, 74.61; H, 9.58. Found: C, 74.21; H, 9.49%.

2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oic acid (21)

To 186.0 mg of compound 13 (0.38 mmol), dissolved in 18 mL of dry benzene, were added 0.95 mL of piperidine and 0.95 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then 180 mg of anhydrous magnesium sulfate was added and the reaction mixture was heat-refluxed under nitrogen atmosphere for 2 hours and 50 min. After evaporation of the solvent under reduced pressure, 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, altered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 5 : 1 → 2 : 1) to yield compound 21 as a white solid (85.5 mg, 47.73%). IR (ATR) ν_max: 3432.1, 2931.26, 2865.72, 2728.40 (falta aqui um pico, ver no espectro), 1688.06, 1581.84, 1462.02, 1383.71, 1364.08, 1036.39 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.70 (s, 1H, CH̲O), 6.66 (s, 1H, H3), 5.31 (t, J = 3.28, 3.28 Hz, 1H, H12), 3.61 (d, J = 10.6 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 2.83–2.80 (m, 1H, H18), 1.23 (s, 3H), 1.14 (s, 3H), 1.01 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.81 (C̲HO), 183.82 (C28), 159.16 (C3), 158.70 (C2), 143.03 (C13), 123.30 (C12), 69.28, 56.31, 50.92, 49.38, 46.47, 45.53, 44.14, 41.99, 41.12, 33.75, 33.34, 33.04, 32.38, 30.62, 27.89, 27.19, 26.47, 23.55, 22.77, 18.92, 18.79, 17.33, 15.91 ppm. DI-ESI-MS m/z: 491.33 (100%) [M + Na]⁺. Anal. calcd. for C₃₀H₄₄O₄·0.5H₂O: C, 75.43; H, 9.50. Found: C, 75.38; H, 9.34%.

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-amine (22)

To 267.1 mg of compound 14 (0.55 mmol), dissolved in 25 mL of dry benzene, were added 1.6 mL of piperidine and 1.6 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then 267.0 mg of anhydrous magnesium sulfate was added, and the reaction mixture was once again heat-refluxed under nitrogen atmosphere and allowed to stir for 4 hours. After evaporation of the solvent under reduced pressure the obtained crude was dispersed in water (25 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (2 × 70 mL) and brine (70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to yield compound 22 as a white solid (63.95 mg, 24.86%). IR (ATR) ν_max: 3443.60, 3340.60, 3191.20, 2943.00, 2864.08, 2751.40, 1680.21, 1652.97, 1585.02, 1364.23, 1382.93, 1048.39 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.70 (s, 1H, CHO), 6.70 (s, 1H, 3H), 5.97 (br s, 1H, NH̲_2̲), 5.58 (br s, 1H, NH̲_2̲), 5.40 (t, J = 3.6 Hz, 1H, H12), 3.62 (d, J = 10.7 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 1.23 (s, 3H), 1.18 (s, 3H), 1.03 (s, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ = 190.90 (CHO), 181.69 (CON), 160.29 (C3), 158.73 (C2), 144.63 (C13), 123.82 (C12), 69.38, 56.37, 50.96, 49.63, 46.56 (2C), 44.23, 42.92, 42.60, 41.24, 34.23, 33.32, 33.12, 32.44, 30.83, 27.75, 27.48, 26.44, 23.88, 23.67, 19.08, 18.93, 17.46, 16.11. DI-ESI-MS m/z: 468.37 (100%) [M + H]⁺. Calcd. for C₃₀H₄₅NO₃·1.1H₂O: C, 73.91; H, 9.76; N, 2.87. Found: C, 73.73; H, 9.72; N, 3.03%.

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-methyl amine (23)

To 269.9 mg of compound 15 (0.54 mmol), dissolved in 25 mL of dry benzene, were added 1.6 mL of piperidine and 1.6 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then were added 267.0 mg of anhydrous magnesium sulfate (2.22 mmol) and the reaction mixture was heat-refluxed and allowed to stir under nitrogen atmosphere for 4 hours. After evaporation of the solvent under reduced pressure the obtained crude was dispersed in water (25 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (2 × 70 mL) and brine (70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 4 : 1 → 1 : 1) to afford 23 as a white solid (90.73 mg, 34.88%). IR (ATR) ν_max: 3406.05, 2940.64, 2869.58, 2732.30, 1683.70, 1631.27, 1532.80, 1463.42, 1383.64, 1363.75, 1045.37, cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.70 (s, 1H, CHO), 6.70 (s, 1H, H3), 6.02 (d, J = 4.8 Hz, 1H, NH̲), 5.41 (t, J = 3.6 Hz, 1H, H12), 3.62 (d, J = 10.6 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 2.74 (d, J = 4.7 Hz, 3H, NHCH̲_3̲), 1.23 (s, 3H), 1.17 (s, 3H), 1.03 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ = 190.90 (CHO), 179.19 (CON), 160.34 (C3), 158.68 (C2), 145.01 (C13), 123.74 (C12), 69.37, 56.35, 50.95, 49.64, 46.70, 46.37, 44.24, 42.53, 41.31, 34.25, 33.13, 32.14, 30.85, 27.69, 27.51, 26.55, 26.31, 23.99, 23.96, 23.70, 19.06, 18.51, 17.45, 16.12. DI-ESI-MS m/z: 482.38 (100%) [M + H]⁺. Calcd. for C₃₁H₄₇NO₃·1.5H₂O: C, 73.19; H, 9.91; N, 2.75. Found: C, 72.94; H, 9.71; N, 2.84%.

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-ethyl amine (24)

To 213.5 mg of compound 16 (0.42 mmol), dissolved in 20 mL of dry benzene, were added 1.2 mL of piperidine and 1.2 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then were added 204 mg of anhydrous magnesium sulfate and the reaction mixture was once again heat-refluxed. The reaction mixture was allowed to stir under nitrogen atmosphere for 4 h. After evaporation of the solvent under reduced pressure the obtained crude was dispersed in water (20 mL) and extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (2 × 70 mL) and brine (70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue obtained was purified by preparative TLC (dichloromethane : diethyl ether 2 : 1) to yield compound 24 as a white solid (51.69 mg, 22.33%). IR (ATR) ν_max: 3397.93, 2928.05, 2866.73, 1682.90, 1632.80, 1525.23, 1461.23, 1047.10 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.70 (s, 1H, CHO), 6.70 (s, 1H, H3), 5.93 (t, J = 5.3 Hz, 1H, NH), 5.40 (t, J = 3.6 Hz, 1H, H12), 3.62 (d, J = 10.6 Hz, 1H), 3.46 (d, J = 10.5 Hz, 1H), 3.35–3.28 (m, 1H, NCHH), 3.15–3.08 (m, 1H, NCHH), 1.23 (s, 3H), 1.17 (s, 3H), 1.10 (t, J = 7.3 Hz, 3H, CH₃CH₂N), 0.90–0.89 (s, 6H), 0.87 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ = 190.94 (CHO), 178.41 (CON), 160.39 (C3), 158.66 (C2), 144.91 (C13), 123.59 (C12), 76.84, 69.36, 56.32, 50.95, 49.66, 46.67, 46.22, 44.26, 42.64, 42.62, 41.39, 34.53, 34.27, 33.25, 33.13, 32.40, 30.84, 27.72, 27.51, 26.46, 23.83, 23.73, 19.07, 18.79, 17.44, 16.12, 14.58. DI-ESI-MS m/z: 496.39 (100%) [M + H]⁺. Calcd. for C₃₂H₄₉NO₃·0.5H₂O: C, 76.15; H, 9.99; N, 2.78. Found: C, 75.97; H, 9.59; N, 2.47%.

Methyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (25)

To 385.0 mg of compound 17 (0.77 mmol), dissolved in 38 mL of dry benzene, were added 1.92 mL of piperidine and 1.92 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then, were added 385.0 mg of anhydrous magnesium sulfate and the reaction mixture was heat-refluxed and allowed to stir under nitrogen atmosphere for 4 h 10 min. After evaporation of the solvent under reduced pressure 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, altered, and concentrated under vacuum. The residue obtained was purified by preparative TLC (dichloromethane/methanol 15 : 1) to yield compound 25 as a white solid (80.56 mg, 21.68%). IR (ATR) ν_max: 3456.91, 2946.30, 2930.70, 2866.00, 2808.50, 2718.50, 1722.20, 1686.76, 1582.16, 1461.29, 1262.48, 1162.12, 1037.89 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.71 (s, 1H, CH̲O), 6.65 (s, 1H, H3), 5.31 (t, J = 3.05 H, 1H, H12), 3.63–3.60 (m, 4H, –COOCH̲_3̲ and H23), 3.46 (d, J = 10.5 Hz, 1H, H23), 2.85 (dd, J = 13.4, 3.4 Hz, 1H, H18), 1.24 (s, 3H), 1.14 (s, 3H), 1.02 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.82 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.36 (CHO), 178.34 (C28), 158.95 (C3), 158.84 (C2), 143.28 (C13), 123.08 (C12), 69.42, 56.38, 51.53, 50.97, 49.42, 46.71, 45.57, 44.18, 42.04, 41.39, 41.14, 33.83, 33.38, 33.10, 32.35, 30.60, 27.96, 27.21, 26.49, 23.64, 23.00, 18.92, 18.64, 17.42, 15.95 ppm. DI-ESI-MS m/z: 483.32 (100%) [M + H]⁺. Anal. calcd. for C₃₁H₄₆O₄·0.5H₂O: C, 75.72; H, 9.63. Found: C, 75.60; H, 9.50%.

Ethyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (26)

To 396.6 mg of compound 18 (0.77 mmol), dissolved in 39 mL of dry benzene, were added 1.97 mL of piperidine and 1.97 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then were added 396.6 mg of anhydrous magnesium sulfate and the reaction mixture was heat-refluxed and allowed to stir under nitrogen atmosphere for 2 hours and 50 min. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (50 mL) and extracted with dichloromethane (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. The residue obtained was purified by flash column chromatography (petroleum ether/ethyl acetate 6 : 1 → 3 : 1) to yield compound 26 as a white solid (187.4 mg, 48.97%). IR (ATR) ν_max: 3444.91, 2942.63, 2866.32, 2727.80, 1719.33, 1687.70, 1582.39, 1462.30, 1383.55, 1159.82, 1036.10 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.71 (s, 1H, CH̲O), 6.65 (s, 1H, H3), 5.31 (t, J = 3.5 Hz, 1H, H12), 4.08 (q, J = 7.1 Hz, 2H, COOCH̲_2̲CH₃), 3.61 (d, J = 10.5 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 2.85 (dd, J = 13.8, 4.1 Hz, 1H, H18), 1.24–1.21 (m, 6H, COOCH₂CH̲_3̲ and CH₃), 1.14 (s, 3H), 1.02 (s, 3H), 0.92 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.77 (C̲HO), 177.78 (C28), 159.02 (C3), 158.82 (C2), 143.32 (C13), 123.00 (C12), 69.41, 60.08, 56.35, 46.47, 45.58, 44.19, 42.08, 41.40, 41.21, 33.86, 33.44, 33.11, 32.37, 30.67, 27.89, 27.22, 26.41, 23.63, 22.91, 18.92, 18.76, 17.40, 15.95, 14.22. ppm. DI-ESI-MS m/z: 497.36 (100%) [M + H]⁺. Anal. calcd. for C₃₂H₄₈O₄·0.5CH₂Cl₂: C, 72.40; H, 9.16. Found: C, 72.42; H, 9.26%.

Butyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (27)

To 195.4 mg of compound 19 (0.36 mmol), dissolved in 19.5 mL of dry benzene, were added 0.97 mL of piperidine and 0.97 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then were added 195.4 mg of anhydrous magnesium sulfate and the reaction mixture was heat-refluxed and allowed to stir under nitrogen atmosphere for 4 hours and 45 min. After evaporation of the solvent under reduced pressure 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 (3 × 50 mL) and brine (50 mL), dried over Na₂SO₄, altered, and concentrated under vacuum. The crude solid was purified by flash column chromatography (petroleum ether/ethyl acetate 6 : 1 → 2 : 1) to yield compound 27 as a white solid (50.3 mg, 26.62%). IR (ATR) ν_max: 3441.93, 2932.24, 2866.34, 2728.50, 1719.15, 1688.16, 1582.09, 1461.98, 1382.97, 1160.84, 1033.27 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.71 (s, 1H, CH̲O), 6.66 (s, 1H, H3), 5.30 (t, J = 3.2 Hz, 1H, H12), 4.02 (t, J = 6.5 Hz, 2H, COOCH̲_2̲CH₂CH₂CH₃), 3.62 (d, J = 10.6 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 2.86 (dd, J = 13.8, 3.9 Hz, 1H, H18), 1.24 (s, 3H), 1.14 (s, 3H), 1.02 (s, 3H), 0.95–0.92 (m, 6H, COOCH₂CH₂CH₂CH̲_3̲ and CH₃), 0.89 (s, 3H), 0.83 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.77 (C̲HO), 177.85 (C28), 159.07 (C3), 158.84 (C2), 143.35 (C13), 123.07 (C12), 69.42, 63.99, 56.36, 50.97, 49.43, 46.65, 45.58, 44.19, 42.09, 41.42, 41.20, 33.86, 33.45, 33.11, 32.44, 30.68, 30.67, 27.89, 27.23, 26.43, 23.62, 22.91, 19.24, 18.91, 18.79, 17.41, 15.95, 13.72 ppm. DI-ESI-MS m/z: 547.48 (100%) [M + Na]⁺. Anal. calcd. for C₃₄H₅₂O₄·0.2H₂O: C, 77.29; H, 10.00. Found: C, 77.12; H, 9.92%.

Allyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (28)

To 230.0 mg of compound 20 (0.44 mmol), dissolved in 23 mL of dry benzene (23 mL), were added 1.15 mL of piperidine and 1.15 mL of acetic acid. The resultant solution was heat-refluxed at 60 °C and allowed to stir for 1 hour. Then were added 230.0 mg of anhydrous magnesium sulfate and the reaction mixture was once again heat-refluxed at 60 °C and allowed to stir under nitrogen atmosphere for 2 hours and 20 min. After evaporation of the solvent under reduced pressure the obtained crude was dispersed by water (60 mL) and extracted with ethyl acetate (3 × 60 mL). The resulting organic phase was washed with water (3 × 60 mL) and brine (60 mL), dried over Na₂SO₄, altered, and concentrated under vacuum. The residue obtained was purified by preparative TLC (dichloromethane/methanol 30 : 1) to yield compound 28 as a white solid (40.26 mg, 18.12%). IR (ATR) ν_max: 3289.24, 2949.2, 2927.66, 2907.00, 2873.10, 2840.50, 1726.47, 1466.47, 1453.00, 1158.65, 1033.56 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ = 9.71 (s, 1H, CH̲O), 6.65 (s, 1H, H3), 5.94–5.84 (m, 1H, COOCH₂CH̲CH₂), 5.34–5.30 (m, 2H, H12 and COOCH₂CHCH̲_2̲), 5.20 (dd, J = 10.5, 1.0 Hz, 1H, COOCH₂CHCH̲_2̲), 4.53 (d, J = 5.5 Hz, 2H, COOCH̲_2̲CHCH₂), 3.61 (d, J = 10.6 Hz, 1H, H23), 3.46 (d, J = 10.6 Hz, 1H, H23), 2.88 (dd, J = 13.7, 4.0 Hz, 1H, H18), 1.23 (s, 3H, CH̲_3̲-25), 1.14 (s, 3H, CH̲_3̲-27), 1.02 (s, 3H, CH̲_3̲-24), 0.92 (s, 3H, CH̲_3̲-29), 0.89 (s, 3H, CH̲_3̲-30), 0.82 (s, 3H, CH̲_3̲-26) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 190.75 (C̲HO), 177.41 (C28), 159.03 (C3), 158.83 (C2), 143.23 (C13), 132.49 (COOCH₂C̲HCH₂), 123.16 (C12), 117.65 (COOCH₂CHC̲H₂), 69.41 (C23), 64.79 (COOC̲H₂CHCH₂), 56.36 (C5), 50.97 (C10), 49.43 (C4), 46.74 (C17), 45.58 (C19), 44.19 (C9), 42.08 (C14), 41.42 (C18), 41.23 (C8), 33.85 (C15), 33.43 (C21), 33.10 (C30), 32.41 (C22), 30.66 (C20), 27.91 (C7), 27.21 (C11), 26.44 (C27), 23.63 (C29), 22.98 (C16), 18.93 (C25), 18.76 (C26), 17.41 (C6), 15.95 (C24) ppm. DI-ESI-MS m/z: 509.49 (100%) [M + H]⁺. Anal. calcd. for C₃₃H₄₈O₄·0.6H₂O: C, 76.29; H, 9.55. Found: C, 76.05; H, 9.47%.

Biology

Cells and reagents

Human cancer cell lines (HT-29, PANC-1, SK-MEL-28, A375, A549, H2170) and human fibroblast BJ cell line, were obtained from American Type Culture Collection (USA). Cell culture media, supplements and reagents were obtained from commercial sources: Dulbecco's modified Eagle medium (DMEM), minimum essential medium (MEM), Roswell Park Memorial Institute (RPMI), and Dulbecco's phosphate buffered saline (DPBS) and l-glutamine acquired from Biowest; penicillin/streptomycin solution and fetal bovine serum (FBS) obtained from Gibco; sodium pyruvate solution 100 mM and trypsin/EDTA obtained from Biological Industries; sodium bicarbonate solution 7.5% and glucose solution 45%, annexin V-FITC, propidium iodide (PI), Hoechst 33342 and gemcitabine were purchased from Sigma Aldrich. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder was purchased from Applichem Panreac.

Compounds

All compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare 20 mM stock solutions that were stored at −80 °C. Further dilutions of the compounds were performed with culture media. The final concentration of DMSO was equal or lower than 0.5% in working solutions.

Cell culture

PANC-1, A549, A375 and HT-29 cells were cultured in DMEM high glucose, with l-glutamine, supplemented with 10% (V/V) FBS and P/S solution (1×). H2170 cells were cultured in RPMI supplemented with 10% (V/V) FBS and P/S solution (1×). BJ cells were cultured in DMEM high glucose, supplemented with 10% (V/V) FBS and P/S solution (1×), 2 mM, l-glutamine, 1 mM sodium pyruvate and sodium bicarbonate (7.5%). SK-MEL-28 were grown in MEM supplemented with 10% (V/V) FBS and P/S solution (1×). HT29. Cells were maintained at 37 °C with 5% CO₂ in a humidified incubator.

MTT assay

Cell viability was determined by using MTT assay. HT-29 (4500 cells per well), PANC-1 (4000 cells per well), SK-MEL-28 (3200 cells per well), A375 (1200 cells per well), A549 (2500 cells per well), H2170 (10 000 cells per well), and BJ cell lines (10 000 cells per well) were plated in 96-well plates. After 24 h culture medium was removed, and cells were treated with increasing concentration of each compound in culture medium and incubated for additional 72 h. The supernatant was then removed and replaced by 100 mL of filtered MTT solution (0.5 mg mL⁻¹). After 1 h of incubation, MTT solution was removed and the formazan crystals were dissolved with 100 μL of DMSO added to each well. The absorbance of the resulting solutions was measure using a ELISA plate reader (Synergy™ H1, Biotek) at 550 nm. Three independent experiments were performed to calculate the IC₅₀ values which are expressed as mean ± standard error of mean (SEM).

Cell cycle assay

PANC-1 cells were seeded at a density of 2 × 10⁵ cells per well onto 6-well plates with 2 mL medium and incubated for 24 h. Then cells were treated with compound 26 at indicated concentrations. After treatment cells were harvested, washed twice with PBS and stained in Tris-buffered saline containing 50 mg mL⁻¹ PI, 10 mg mL⁻¹, DNase-free RNase and 0.1% Igepal CA-360, for 1 h, at 4 °C in darkness. The DNA contents were assessed by flow cytometry using a fluorescence-activated cell sorter (FACS) (BD Accuri™ C6 flow cytometer, BD Biosciences).

Annexin V-FITC/PI flow cytometry assay

Annexin V-FITC/PI flow cytometric assay was employed to detect apoptosis in PANC-1 cells. Briefly, cells were seeded at a density of 2 × 10⁵ cells per well onto 6-well plates and incubated for 24 hours. After treatment with compound 26 at the indicated concentrations, cells were incubated for additional 24 hours. Afterwards, cells were harvested collected by centrifugation, suspended in 95 mL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 10 mM NaCl, 2.5 mM CaCl₂) and stained with Annexin V-FITC conjugate for 30 min at room temperature, protected from the light. Then, 500 mL of binding buffer and 20 mL of 1 mg mL⁻¹ PI solution were added to each vial of cells immediately before flow cytometry analysis (BD Accuri™ C6, BD Biosciences).

Hoechst 33342 staining assay

PANC-1 cells were seeded into 96-well plates to a density of 6500 cells per well and incubated for 24 h. After treatment with compound 26 at the indicated concentrations cells were incubated for additional 24 h. Cells were then washed with PBS and stained with Hoechst 33342 solution (1 μg mL⁻¹ in PBS) for 10 min at 37 °C protected from the light. The morphological changes of PANC-1 cells were analyzed using the InCell Analyzer 2000 (GE Healthcare) with a DAPI filter.

Migration assay

Migration assay of PANC-1 was performed using the wound healing method. PANC-1 cells at a density of 4000 cells per well were seeded in 96-well plates and incubated during 24 h. Afterwards, the confluent monolayers were scrap away vertically using a P200 pipette. After washing each well with PBS, cells were supplied with 200 μL of culture medium (0.5% DMSO, control group) or treated with compound 26 at different concentrations. Wounded areas were observed during 72 h using high-content imaging microscope (In Cell Analyzer 2200, GE Healthcare). A reduction in the size of the scraped region is indicative of cell migration.

Synergy study

PANC-1 cells were seeded at a density of 4000 cells per well in 96-well plates with 200 μL of medium and incubated for 24 h. Following culture media was replaced with new media containing increasing concentrations of compound 26, gemcitabine, or the combination of both with a constant ratio of concentrations and cells were incubated for 72 h. Next, the relative cell viability was analyzed as described above for the MTT assay. The combination index (CI) values were determined using CompuSyn software following the Chou and Talalay method for drug combination^66,67 CI values below 1, equal to 1, and above 1 indicate synergy, additivity, and antagonism, respectively.

Primary observation (Irwin) test in the mouse

Experiments were conducted in strict accordance with Council Directive No. 2010/63/UE of 22 September 2010, and the French decree no. 2013-118 of 1 February 2013, on the protection of animals for use and care of laboratory animals. The study was performed following in an accredited lab from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All experiments were also approved by the ethics committee for animal experimentation of Porsolt's laboratory (Porsolt's agreement n° F 53 1031).The method, which detects the first toxic dose, the active dose-range and the principal effects of a test substance on behavior and physiological function, follows that described by Irwin⁶⁸ and was conducted by Porsolt S.A.S. Female Balb/c mice, 7–8 weeks old at the beginning of the experiment, were administered with compound 26 and were observed in simultaneous comparison with a control group given vehicle (non-blind conditions). Between 1 and 3 treated groups were compared with the same control at any one time. All animals within a treatment group were observed simultaneously. The order of testing doses depends on effects observed with previous doses. Behavioral modifications, physiological and neurotoxicity symptoms, rectal temperature and pupil diameter were recorded according to a standardized observation grid derived from that of Irwin. The grid contains the following items: death*, convulsions*, tremor*, Straub tail*, altered activity, jumping*, abnormal gait* (rolling, tiptoe), motor incoordination*, altered abdominal muscle tone, loss of grasping, akinesia, catalepsy, loss of traction, writhing*, piloerection*, stereotypies* (sniffing, chewing, head movements), head-twitches*, scratching*, altered respiration*, aggression*, altered fear/startle, altered reactivity to touch, ptosis, exophthalmia, loss of righting reflex, loss of corneal reflex, analgesia, defecation/diarrhea, salivation, lacrimation, rectal temperature (hypothermia/hyperthermia) and pupil diameter (myosis/mydriasis). Observations were performed 15, 30, 60, 120 and 180 minutes after administration of the test substance and 24 hours later. The symptoms marked (*) were observed continuously from 0 to 15 minutes after administration. Three mice were studied per group. The compound 26 was evaluated at a maximum of 7 doses, administered i.p. immediately before the test. The starting doses were 0.01, 1, and 100 mg kg⁻¹. Based on the effects observed (potential high toxicity at low doses), the remaining doses were tested or adapted, which led to the following testing concentrations of compound 26: 0.01, 0.1, 1, 5, 10, 50 and 100 mg kg⁻¹. A 0.2% hydroxypropylmethylcellulose (HPMC) in physiological saline solution was used to prepare the compound for administration, and thus control group was treated with this solution.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-014-D2MD00275B-s001

MD-014-D2MD00275B-s001.pdf^{(2.8MB, pdf)}

Acknowledgments

This article has been developed in the framework of “Drugs2CAD” project CENTRO-01-0247-FEDER-003269, led Chem4Pharma, LDA in partnership with University of Coimbra. Project supported by CENTRO 2020 of PT2020 through the European Regional Development Fund (ERDF).

^†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2md00275b

Notes and references

World Health Organization, Global Health Observatory, World Health Organization, Geneva, 2020, https://www.who.int/health-topics/cancer, (accessed 15 December 2020) [Google Scholar]
Torre L. A. Siegel R. L. Ward E. M. Jemal A. Cancer Epidemiol., Biomarkers Prev. 2016;25:16–27. doi: 10.1158/1055-9965.EPI-15-0578. [DOI] [PubMed] [Google Scholar]
Bray F. Ferlay J. Soerjomataram I. Siegel R. L. Torre L. A. Jemal A. Ca-Cancer J. Clin. 2018;68:394–424. doi: 10.3322/caac.21492. [DOI] [PubMed] [Google Scholar]
Siegel R. L. Miller K. D. Fuchs H. E. Jemal A. Ca-Cancer J. Clin. 2021;71:7–33. doi: 10.3322/caac.21654. [DOI] [PubMed] [Google Scholar]
Falzone L. Salomone S. Libra M. Front. Pharmacol. 2018;9:1–26. doi: 10.3389/fphar.2018.01300. [DOI] [PMC free article] [PubMed] [Google Scholar]
Li K. Zhang Z. Mei Y. Li M. Yang Q. Wu Q. Yang H. He L. Liu S. J. Mater. Chem. B. 2022;10:1709–1733. doi: 10.1039/D1TB02818A. [DOI] [PubMed] [Google Scholar]
Hanahan D. Weinberg R. a. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
Bhatia A. Thakur A. Mol. Aspects Med. 2001;21:167–223. [Google Scholar]
Vasan N. Baselga J. Hyman D. M. Nature. 2019;575:299–309. doi: 10.1038/s41586-019-1730-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
Newman D. J. Cragg G. M. J. Nat. Prod. 2020;83:770–803. doi: 10.1021/acs.jnatprod.9b01285. [DOI] [PubMed] [Google Scholar]
Ghirga F. Quaglio D. Mori M. Cammarone S. Iazzetti A. Goggiamani A. Ingallina C. Botta B. Calcaterra A. Org. Chem. Front. 2021;8:996–1025. doi: 10.1039/D0QO01210F. [DOI] [Google Scholar]
Zhong Z. Vong C. T. Chen F. Tan H. Zhang C. Wang N. Cui L. Wang Y. Feng Y. Med. Res. Rev. 2022;42:1246–1279. doi: 10.1002/med.21876. [DOI] [PMC free article] [PubMed] [Google Scholar]
Cui J. Qian J. Chow L. M.-C. Jia J. Curr. Med. Chem. 2021;28:6773–6804. doi: 10.2174/0929867328666210405111913. [DOI] [PubMed] [Google Scholar]
Salvador J. A. R. Leal A. S. Valdeira A. S. Gonçalves B. M. F. Alho D. P. S. Figueiredo S. A. C. Silvestre S. M. Mendes V. I. S. Eur. J. Med. Chem. 2017;142:95–130. doi: 10.1016/j.ejmech.2017.07.013. [DOI] [PubMed] [Google Scholar]
Valdeira A. S. C. Darvishi E. Woldemichael G. M. Beutler J. A. Gustafson K. R. Salvador J. A. R. J. Nat. Prod. 2019;82:2094–2105. doi: 10.1021/acs.jnatprod.8b00864. [DOI] [PMC free article] [PubMed] [Google Scholar]
Hodon J. Borkova L. Pokorny J. Kazakova A. Urban M. Eur. J. Med. Chem. 2019;182:111653. doi: 10.1016/j.ejmech.2019.111653. [DOI] [PubMed] [Google Scholar]
Brandes B. Hoenke S. Fischer L. Csuk R. Eur. J. Med. Chem. 2020;185:111858. doi: 10.1016/j.ejmech.2019.111858. [DOI] [PubMed] [Google Scholar]
Kamble S. M. Goyal S. Pati C. R. RSC Adv. 2014;4:33370–33382. doi: 10.1039/C4RA02784A. [DOI] [Google Scholar]
Wang Y. Luo Z. Zhou D. Wang X. Chen J. Gong S. Yu Z. Biomater. Sci. 2021;9:4110–4119. doi: 10.1039/D1BM00087J. [DOI] [PubMed] [Google Scholar]
Khan M. W. Zou C. Hassan S. Din F. U. Abdoul Razak M. Y. Nawaz A. Zeb A. Wahab A. Bangash S. A. RSC Adv. 2022;12:14808–14818. doi: 10.1039/D2RA00742H. [DOI] [PMC free article] [PubMed] [Google Scholar]
Wang W.-Y. Yang Z.-H. Li A.-L. Liu Q.-S. Sun Y. Gu W. New J. Chem. 2022;46:2335–2350. doi: 10.1039/D1NJ05294B. [DOI] [Google Scholar]
Ghosh J. Sil P. C. Biochimie. 2013;95:1098–1109. doi: 10.1016/j.biochi.2013.01.016. [DOI] [PubMed] [Google Scholar]
Manna P. Sinha M. Pal P. Sil P. C. Chem.-Biol. Interact. 2007;170:187–200. doi: 10.1016/j.cbi.2007.08.001. [DOI] [PubMed] [Google Scholar]
Ghosh J. Das J. Manna P. Sil P. C. Toxicol. In Vitro. 2008;22:1918–1926. doi: 10.1016/j.tiv.2008.09.010. [DOI] [PubMed] [Google Scholar]
Manna P. Das J. Ghosh J. Sil P. C. Free Radical Biol. Med. 2010;48:1465–1484. doi: 10.1016/j.freeradbiomed.2010.02.025. [DOI] [PubMed] [Google Scholar]
Pal S. Pal P. B. Das J. Sil P. C. Toxicology. 2011;283:129–139. doi: 10.1016/j.tox.2011.03.006. [DOI] [PubMed] [Google Scholar]
Toppo E. Sylvester Darvin S. Esakkimuthu S. Buvanesvaragurunathan K. Ajeesh Krishna T. P. Antony Caesar S. Stalin A. Balakrishna K. Pandikumar P. Ignacimuthu S. Al-Dhabi N. A. Biomed. Pharmacother. 2018;107:979–988. doi: 10.1016/j.biopha.2018.08.019. [DOI] [PubMed] [Google Scholar]
Elsherbiny N. M. Eladl M. A. Al-Gayyar M. M. H. Cytokine+ 2016;77:26–34. doi: 10.1016/j.cyto.2015.10.010. [DOI] [PubMed] [Google Scholar]
Manna P. Sinha M. Sil P. C. Arch. Toxicol. 2008;82:137–149. doi: 10.1007/s00204-007-0272-8. [DOI] [PubMed] [Google Scholar]
Al-Gayyar M. M. H. Al Youssef A. Sherif I. O. Shams M. E. E. Abbas A. Life Sci. 2014;111:18–26. doi: 10.1016/j.lfs.2014.07.002. [DOI] [PubMed] [Google Scholar]
Manna P. Sil P. C. Free Radical Res. 2012;46:815–830. doi: 10.3109/10715762.2012.683431. [DOI] [PubMed] [Google Scholar]
Djoukeng J. D. Abou-Mansour E. Tabacchi R. Tapondjou A. L. Bouda H. Lontsi D. J. Ethnopharmacol. 2005;101:283–286. doi: 10.1016/j.jep.2005.05.008. [DOI] [PubMed] [Google Scholar]
Masoko P. Mdee L. K. Mampuru L. J. Eloff J. N. Nat. Prod. Res. 2008;22:1074–1084. doi: 10.1080/14786410802267494. [DOI] [PubMed] [Google Scholar]
Bhakuni R. S. Shukla Y. N. Tripathi A. K. Prajapati V. Kumar S. Phytother. Res. 2002;16:2000–2002. doi: 10.1002/ptr.748. [DOI] [PubMed] [Google Scholar]
Chaudhari M. Mengi S. Phytother. Res. 2006;20:799–805. doi: 10.1002/ptr.1857. [DOI] [PubMed] [Google Scholar]
Prasad M. V. V. Anbalagan N. Patra A. Veluchamy G. Balakrishna K. Nat. Prod. Sci. 2004;10:240–243. [Google Scholar]
Kim D. H. Han K. M. Chung I. S. Kim D. K. Kim S. H. Kwon B. M. Jeong T. S. Park M. H. Ahn E. M. Baek N. I. Arch. Pharmacal Res. 2005;28:550–556. doi: 10.1007/BF02977757. [DOI] [PubMed] [Google Scholar]
Ramesh A. S. Christopher J. G. Radhika R. Setty C. R. Thankamani V. Nat. Prod. Res. 2012;26:1549–1552. doi: 10.1080/14786419.2011.566870. [DOI] [PubMed] [Google Scholar]
Manna S. Dey A. Majumdar R. Bag B. G. Ghosh C. Roy S. Heliyon. 2020;6:e03456. doi: 10.1016/j.heliyon.2020.e03456. [DOI] [PMC free article] [PubMed] [Google Scholar]
Diallo B. Vanhaelen-Fastré R. Vanhaelen M. Konoshima T. Takasaki M. Tokuda H. Phytother. Res. 1995;9:444–447. doi: 10.1002/ptr.2650090612. [DOI] [Google Scholar]
Elsherbiny N. M. Al-Gayyar M. M. H. Biomed. Pharmacother. 2016;82:28–34. doi: 10.1016/j.biopha.2016.04.046. [DOI] [PubMed] [Google Scholar]
Aamir K. Khan H. U. Hossain C. F. RejinaAfrin M. Shaik I. Salleh N. Giribabu N. Arya A. PeerJ. 2019;2019:1–24. doi: 10.7717/peerj.8045. [DOI] [PMC free article] [PubMed] [Google Scholar]
Chen H. Gao Y. Wang A. Zhou X. Zheng Y. Zhou J. Eur. J. Med. Chem. 2015;92:648–655. doi: 10.1016/j.ejmech.2015.01.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
Ren Y. Kinghorn A. D. Planta Med. 2019;85:802–814. doi: 10.1055/a-0832-2383. [DOI] [PMC free article] [PubMed] [Google Scholar]
Sandjo L. P. Djoumessi A. V. B. S. Rincheval V. Poumale H. M. P. Abegaz B. M. Ngadjui B. T. Nat. Prod. Res. 2013;27:711–718. doi: 10.1080/14786419.2012.691494. [DOI] [PubMed] [Google Scholar]
Salvador J. A. R. Moreira V. M. Gonçalves B. M. F. Leal A. S. Jing Y. Nat. Prod. Rep. 2012;29:1463–1479. doi: 10.1039/C2NP20060K. [DOI] [PubMed] [Google Scholar]
Goncalves B. M. F. Salvador J. A. R. Marín S. Cascante M. Eur. J. Med. Chem. 2016;114:101–110. doi: 10.1016/j.ejmech.2016.02.057. [DOI] [PubMed] [Google Scholar]
Mendes V. I. S. Bartholomeusz G. A. Ayres M. Gandhi V. Salvador J. A. R. Eur. J. Med. Chem. 2016;123:317–331. doi: 10.1016/j.ejmech.2016.07.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
Valdeira A. S. C. Darvishi E. Woldemichael G. M. Beutler J. A. Gustafson K. R. Salvador J. A. R. J. Nat. Prod. 2019;82:2094–2105. doi: 10.1021/acs.jnatprod.8b00864. [DOI] [PMC free article] [PubMed] [Google Scholar]
Figueiredo S. A. C. Salvador J. A. R. Cortés R. Cascante M. Eur. J. Med. Chem. 2017;139:836–848. doi: 10.1016/j.ejmech.2017.08.058. [DOI] [PubMed] [Google Scholar]
Alho D. P. S. Salvador J. A. R. Cascante M. Marin S. Molecules. 2019;24:2938. doi: 10.3390/molecules24162938. [DOI] [PMC free article] [PubMed] [Google Scholar]
Alho D. P. S. Salvador J. A. R. Cascante M. Marin S. Molecules. 2019;24:1–21. doi: 10.3390/molecules24040766. [DOI] [PMC free article] [PubMed] [Google Scholar]
Brown D. G. Boström J. J. Med. Chem. 2016;59:4443–4458. doi: 10.1021/acs.jmedchem.5b01409. [DOI] [PubMed] [Google Scholar]
Ghose A. K. Viswanadhan V. N. Wendoloski J. J. J. Comb. Chem. 1999;1:55–68. doi: 10.1021/cc9800071. [DOI] [PubMed] [Google Scholar]
Dan L. I. U. Yan-qiu M. Juan Z. Li-gong C. Chem. Res. Chin. Univ. 2008;24:42–46. doi: 10.1016/S1005-9040(08)60010-0. [DOI] [Google Scholar]
Meng Y. Q. Liu D. Cai L. L. Chen H. Cao B. Wang Y. Z. Bioorg. Med. Chem. 2009;17:848–854. doi: 10.1016/j.bmc.2008.11.036. [DOI] [PubMed] [Google Scholar]
Medina-O'Donnell M. Rivas F. Reyes-Zurita F. J. Martinez A. Lupiañez J. A. Parra A. Eur. J. Med. Chem. 2018;148:325–336. doi: 10.1016/j.ejmech.2018.02.044. [DOI] [PubMed] [Google Scholar]
Gersch M. Kreuzer J. Sieber S. A. Nat. Prod. Rep. 2012;29:659–682. doi: 10.1039/C2NP20012K. [DOI] [PubMed] [Google Scholar]
Sporn M. B. Liby K. T. Yore M. M. Fu L. Lopchuk J. M. Gribble G. W. J. Nat. Prod. 2011;74:537–545. doi: 10.1021/np100826q. [DOI] [PMC free article] [PubMed] [Google Scholar]
Singh B. Rastogi R. P. Phytochemistry. 1969;8:917–921. doi: 10.1016/S0031-9422(00)85884-7. [DOI] [Google Scholar]
Tu H.-Y. Huang A.-M. Wei B.-L. Gan K.-H. Hour T.-C. Yang S.-C. Pu Y.-S. Lin C.-N. Bioorg. Med. Chem. 2009;17:7265–7274. doi: 10.1016/j.bmc.2009.08.046. [DOI] [PubMed] [Google Scholar]
Jingwen B. Yaochen L. Guojun Z. Cancer Biol. Med. 2017;14:348. doi: 10.20892/j.issn.2095-3941.2017.0033. [DOI] [PMC free article] [PubMed] [Google Scholar]
Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995;184:39–51. doi: 10.1016/0022-1759(95)00072-I. [DOI] [PubMed] [Google Scholar]
Kajstura M. Halicka H. D. Pryjma J. Darzynkiewicz Z. Cytometry, Part A. 2007;71:125–131. doi: 10.1002/cyto.a.20357. [DOI] [PubMed] [Google Scholar]
Fryer R. A. Barlett B. Galustian C. Dalgleish A. G. Anticancer Res. 2011;31:3747–3756. [PubMed] [Google Scholar]
Chou T. C. Talalay P. Adv. Enzyme Regul. 1984;22:27–55. doi: 10.1016/0065-2571(84)90007-4. [DOI] [PubMed] [Google Scholar]
Chou T. C. Cancer Res. 2010;70:440–446. doi: 10.1158/0008-5472.CAN-09-1947. [DOI] [PubMed] [Google Scholar]
Irwin S. Psychopharmacologia. 1968;13:222–257. doi: 10.1007/BF00401402. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

MD-014-D2MD00275B-s001

MD-014-D2MD00275B-s001.pdf^{(2.8MB, pdf)}

[cit1] World Health Organization, Global Health Observatory, World Health Organization, Geneva, 2020, https://www.who.int/health-topics/cancer, (accessed 15 December 2020) [Google Scholar]

[cit2] Torre L. A. Siegel R. L. Ward E. M. Jemal A. Cancer Epidemiol., Biomarkers Prev. 2016;25:16–27. doi: 10.1158/1055-9965.EPI-15-0578. [DOI] [PubMed] [Google Scholar]

[cit3] Bray F. Ferlay J. Soerjomataram I. Siegel R. L. Torre L. A. Jemal A. Ca-Cancer J. Clin. 2018;68:394–424. doi: 10.3322/caac.21492. [DOI] [PubMed] [Google Scholar]

[cit4] Siegel R. L. Miller K. D. Fuchs H. E. Jemal A. Ca-Cancer J. Clin. 2021;71:7–33. doi: 10.3322/caac.21654. [DOI] [PubMed] [Google Scholar]

[cit5] Falzone L. Salomone S. Libra M. Front. Pharmacol. 2018;9:1–26. doi: 10.3389/fphar.2018.01300. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit6] Li K. Zhang Z. Mei Y. Li M. Yang Q. Wu Q. Yang H. He L. Liu S. J. Mater. Chem. B. 2022;10:1709–1733. doi: 10.1039/D1TB02818A. [DOI] [PubMed] [Google Scholar]

[cit7] Hanahan D. Weinberg R. a. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]

[cit8] Bhatia A. Thakur A. Mol. Aspects Med. 2001;21:167–223. [Google Scholar]

[cit9] Vasan N. Baselga J. Hyman D. M. Nature. 2019;575:299–309. doi: 10.1038/s41586-019-1730-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit10] Newman D. J. Cragg G. M. J. Nat. Prod. 2020;83:770–803. doi: 10.1021/acs.jnatprod.9b01285. [DOI] [PubMed] [Google Scholar]

[cit11] Ghirga F. Quaglio D. Mori M. Cammarone S. Iazzetti A. Goggiamani A. Ingallina C. Botta B. Calcaterra A. Org. Chem. Front. 2021;8:996–1025. doi: 10.1039/D0QO01210F. [DOI] [Google Scholar]

[cit12] Zhong Z. Vong C. T. Chen F. Tan H. Zhang C. Wang N. Cui L. Wang Y. Feng Y. Med. Res. Rev. 2022;42:1246–1279. doi: 10.1002/med.21876. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit13] Cui J. Qian J. Chow L. M.-C. Jia J. Curr. Med. Chem. 2021;28:6773–6804. doi: 10.2174/0929867328666210405111913. [DOI] [PubMed] [Google Scholar]

[cit14] Salvador J. A. R. Leal A. S. Valdeira A. S. Gonçalves B. M. F. Alho D. P. S. Figueiredo S. A. C. Silvestre S. M. Mendes V. I. S. Eur. J. Med. Chem. 2017;142:95–130. doi: 10.1016/j.ejmech.2017.07.013. [DOI] [PubMed] [Google Scholar]

[cit15] Valdeira A. S. C. Darvishi E. Woldemichael G. M. Beutler J. A. Gustafson K. R. Salvador J. A. R. J. Nat. Prod. 2019;82:2094–2105. doi: 10.1021/acs.jnatprod.8b00864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit16] Hodon J. Borkova L. Pokorny J. Kazakova A. Urban M. Eur. J. Med. Chem. 2019;182:111653. doi: 10.1016/j.ejmech.2019.111653. [DOI] [PubMed] [Google Scholar]

[cit17] Brandes B. Hoenke S. Fischer L. Csuk R. Eur. J. Med. Chem. 2020;185:111858. doi: 10.1016/j.ejmech.2019.111858. [DOI] [PubMed] [Google Scholar]

[cit18] Kamble S. M. Goyal S. Pati C. R. RSC Adv. 2014;4:33370–33382. doi: 10.1039/C4RA02784A. [DOI] [Google Scholar]

[cit19] Wang Y. Luo Z. Zhou D. Wang X. Chen J. Gong S. Yu Z. Biomater. Sci. 2021;9:4110–4119. doi: 10.1039/D1BM00087J. [DOI] [PubMed] [Google Scholar]

[cit20] Khan M. W. Zou C. Hassan S. Din F. U. Abdoul Razak M. Y. Nawaz A. Zeb A. Wahab A. Bangash S. A. RSC Adv. 2022;12:14808–14818. doi: 10.1039/D2RA00742H. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit21] Wang W.-Y. Yang Z.-H. Li A.-L. Liu Q.-S. Sun Y. Gu W. New J. Chem. 2022;46:2335–2350. doi: 10.1039/D1NJ05294B. [DOI] [Google Scholar]

[cit22] Ghosh J. Sil P. C. Biochimie. 2013;95:1098–1109. doi: 10.1016/j.biochi.2013.01.016. [DOI] [PubMed] [Google Scholar]

[cit23] Manna P. Sinha M. Pal P. Sil P. C. Chem.-Biol. Interact. 2007;170:187–200. doi: 10.1016/j.cbi.2007.08.001. [DOI] [PubMed] [Google Scholar]

[cit24] Ghosh J. Das J. Manna P. Sil P. C. Toxicol. In Vitro. 2008;22:1918–1926. doi: 10.1016/j.tiv.2008.09.010. [DOI] [PubMed] [Google Scholar]

[cit25] Manna P. Das J. Ghosh J. Sil P. C. Free Radical Biol. Med. 2010;48:1465–1484. doi: 10.1016/j.freeradbiomed.2010.02.025. [DOI] [PubMed] [Google Scholar]

[cit26] Pal S. Pal P. B. Das J. Sil P. C. Toxicology. 2011;283:129–139. doi: 10.1016/j.tox.2011.03.006. [DOI] [PubMed] [Google Scholar]

[cit27] Toppo E. Sylvester Darvin S. Esakkimuthu S. Buvanesvaragurunathan K. Ajeesh Krishna T. P. Antony Caesar S. Stalin A. Balakrishna K. Pandikumar P. Ignacimuthu S. Al-Dhabi N. A. Biomed. Pharmacother. 2018;107:979–988. doi: 10.1016/j.biopha.2018.08.019. [DOI] [PubMed] [Google Scholar]

[cit28] Elsherbiny N. M. Eladl M. A. Al-Gayyar M. M. H. Cytokine+ 2016;77:26–34. doi: 10.1016/j.cyto.2015.10.010. [DOI] [PubMed] [Google Scholar]

[cit29] Manna P. Sinha M. Sil P. C. Arch. Toxicol. 2008;82:137–149. doi: 10.1007/s00204-007-0272-8. [DOI] [PubMed] [Google Scholar]

[cit30] Al-Gayyar M. M. H. Al Youssef A. Sherif I. O. Shams M. E. E. Abbas A. Life Sci. 2014;111:18–26. doi: 10.1016/j.lfs.2014.07.002. [DOI] [PubMed] [Google Scholar]

[cit31] Manna P. Sil P. C. Free Radical Res. 2012;46:815–830. doi: 10.3109/10715762.2012.683431. [DOI] [PubMed] [Google Scholar]

[cit32] Djoukeng J. D. Abou-Mansour E. Tabacchi R. Tapondjou A. L. Bouda H. Lontsi D. J. Ethnopharmacol. 2005;101:283–286. doi: 10.1016/j.jep.2005.05.008. [DOI] [PubMed] [Google Scholar]

[cit33] Masoko P. Mdee L. K. Mampuru L. J. Eloff J. N. Nat. Prod. Res. 2008;22:1074–1084. doi: 10.1080/14786410802267494. [DOI] [PubMed] [Google Scholar]

[cit34] Bhakuni R. S. Shukla Y. N. Tripathi A. K. Prajapati V. Kumar S. Phytother. Res. 2002;16:2000–2002. doi: 10.1002/ptr.748. [DOI] [PubMed] [Google Scholar]

[cit35] Chaudhari M. Mengi S. Phytother. Res. 2006;20:799–805. doi: 10.1002/ptr.1857. [DOI] [PubMed] [Google Scholar]

[cit36] Prasad M. V. V. Anbalagan N. Patra A. Veluchamy G. Balakrishna K. Nat. Prod. Sci. 2004;10:240–243. [Google Scholar]

[cit37] Kim D. H. Han K. M. Chung I. S. Kim D. K. Kim S. H. Kwon B. M. Jeong T. S. Park M. H. Ahn E. M. Baek N. I. Arch. Pharmacal Res. 2005;28:550–556. doi: 10.1007/BF02977757. [DOI] [PubMed] [Google Scholar]

[cit38] Ramesh A. S. Christopher J. G. Radhika R. Setty C. R. Thankamani V. Nat. Prod. Res. 2012;26:1549–1552. doi: 10.1080/14786419.2011.566870. [DOI] [PubMed] [Google Scholar]

[cit39] Manna S. Dey A. Majumdar R. Bag B. G. Ghosh C. Roy S. Heliyon. 2020;6:e03456. doi: 10.1016/j.heliyon.2020.e03456. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit40] Diallo B. Vanhaelen-Fastré R. Vanhaelen M. Konoshima T. Takasaki M. Tokuda H. Phytother. Res. 1995;9:444–447. doi: 10.1002/ptr.2650090612. [DOI] [Google Scholar]

[cit41] Elsherbiny N. M. Al-Gayyar M. M. H. Biomed. Pharmacother. 2016;82:28–34. doi: 10.1016/j.biopha.2016.04.046. [DOI] [PubMed] [Google Scholar]

[cit42] Aamir K. Khan H. U. Hossain C. F. RejinaAfrin M. Shaik I. Salleh N. Giribabu N. Arya A. PeerJ. 2019;2019:1–24. doi: 10.7717/peerj.8045. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit43] Chen H. Gao Y. Wang A. Zhou X. Zheng Y. Zhou J. Eur. J. Med. Chem. 2015;92:648–655. doi: 10.1016/j.ejmech.2015.01.031. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit44] Ren Y. Kinghorn A. D. Planta Med. 2019;85:802–814. doi: 10.1055/a-0832-2383. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit45] Sandjo L. P. Djoumessi A. V. B. S. Rincheval V. Poumale H. M. P. Abegaz B. M. Ngadjui B. T. Nat. Prod. Res. 2013;27:711–718. doi: 10.1080/14786419.2012.691494. [DOI] [PubMed] [Google Scholar]

[cit46] Salvador J. A. R. Moreira V. M. Gonçalves B. M. F. Leal A. S. Jing Y. Nat. Prod. Rep. 2012;29:1463–1479. doi: 10.1039/C2NP20060K. [DOI] [PubMed] [Google Scholar]

[cit47] Goncalves B. M. F. Salvador J. A. R. Marín S. Cascante M. Eur. J. Med. Chem. 2016;114:101–110. doi: 10.1016/j.ejmech.2016.02.057. [DOI] [PubMed] [Google Scholar]

[cit48] Mendes V. I. S. Bartholomeusz G. A. Ayres M. Gandhi V. Salvador J. A. R. Eur. J. Med. Chem. 2016;123:317–331. doi: 10.1016/j.ejmech.2016.07.045. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit49] Valdeira A. S. C. Darvishi E. Woldemichael G. M. Beutler J. A. Gustafson K. R. Salvador J. A. R. J. Nat. Prod. 2019;82:2094–2105. doi: 10.1021/acs.jnatprod.8b00864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit50] Figueiredo S. A. C. Salvador J. A. R. Cortés R. Cascante M. Eur. J. Med. Chem. 2017;139:836–848. doi: 10.1016/j.ejmech.2017.08.058. [DOI] [PubMed] [Google Scholar]

[cit51] Alho D. P. S. Salvador J. A. R. Cascante M. Marin S. Molecules. 2019;24:2938. doi: 10.3390/molecules24162938. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit52] Alho D. P. S. Salvador J. A. R. Cascante M. Marin S. Molecules. 2019;24:1–21. doi: 10.3390/molecules24040766. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit53] Brown D. G. Boström J. J. Med. Chem. 2016;59:4443–4458. doi: 10.1021/acs.jmedchem.5b01409. [DOI] [PubMed] [Google Scholar]

[cit54] Ghose A. K. Viswanadhan V. N. Wendoloski J. J. J. Comb. Chem. 1999;1:55–68. doi: 10.1021/cc9800071. [DOI] [PubMed] [Google Scholar]

[cit55] Dan L. I. U. Yan-qiu M. Juan Z. Li-gong C. Chem. Res. Chin. Univ. 2008;24:42–46. doi: 10.1016/S1005-9040(08)60010-0. [DOI] [Google Scholar]

[cit56] Meng Y. Q. Liu D. Cai L. L. Chen H. Cao B. Wang Y. Z. Bioorg. Med. Chem. 2009;17:848–854. doi: 10.1016/j.bmc.2008.11.036. [DOI] [PubMed] [Google Scholar]

[cit57] Medina-O'Donnell M. Rivas F. Reyes-Zurita F. J. Martinez A. Lupiañez J. A. Parra A. Eur. J. Med. Chem. 2018;148:325–336. doi: 10.1016/j.ejmech.2018.02.044. [DOI] [PubMed] [Google Scholar]

[cit58] Gersch M. Kreuzer J. Sieber S. A. Nat. Prod. Rep. 2012;29:659–682. doi: 10.1039/C2NP20012K. [DOI] [PubMed] [Google Scholar]

[cit59] Sporn M. B. Liby K. T. Yore M. M. Fu L. Lopchuk J. M. Gribble G. W. J. Nat. Prod. 2011;74:537–545. doi: 10.1021/np100826q. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit60] Singh B. Rastogi R. P. Phytochemistry. 1969;8:917–921. doi: 10.1016/S0031-9422(00)85884-7. [DOI] [Google Scholar]

[cit61] Tu H.-Y. Huang A.-M. Wei B.-L. Gan K.-H. Hour T.-C. Yang S.-C. Pu Y.-S. Lin C.-N. Bioorg. Med. Chem. 2009;17:7265–7274. doi: 10.1016/j.bmc.2009.08.046. [DOI] [PubMed] [Google Scholar]

[cit62] Jingwen B. Yaochen L. Guojun Z. Cancer Biol. Med. 2017;14:348. doi: 10.20892/j.issn.2095-3941.2017.0033. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit63] Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995;184:39–51. doi: 10.1016/0022-1759(95)00072-I. [DOI] [PubMed] [Google Scholar]

[cit64] Kajstura M. Halicka H. D. Pryjma J. Darzynkiewicz Z. Cytometry, Part A. 2007;71:125–131. doi: 10.1002/cyto.a.20357. [DOI] [PubMed] [Google Scholar]

[cit65] Fryer R. A. Barlett B. Galustian C. Dalgleish A. G. Anticancer Res. 2011;31:3747–3756. [PubMed] [Google Scholar]

[cit66] Chou T. C. Talalay P. Adv. Enzyme Regul. 1984;22:27–55. doi: 10.1016/0065-2571(84)90007-4. [DOI] [PubMed] [Google Scholar]

[cit67] Chou T. C. Cancer Res. 2010;70:440–446. doi: 10.1158/0008-5472.CAN-09-1947. [DOI] [PubMed] [Google Scholar]

[cit68] Irwin S. Psychopharmacologia. 1968;13:222–257. doi: 10.1007/BF00401402. [DOI] [PubMed] [Google Scholar]

PERMALINK

Design, synthesis, and biological evaluation of new arjunolic acid derivatives as anticancer agents†

Bruno M F Gonçalves

Vanessa I S Mendes

Samuel M Silvestre

Jorge A R Salvador

Abstract

Introduction

Fig. 1. Chemical structure of arjunolic acid 1. Carbons are numbered.

Results and discussion

Chemistry

Scheme 2. Reagents and conditions: a) NaIO4, MeOH/H2O, r.t.; b) i – acetic acid, piperidine, dry benzene, reflux 60 °C, N2; ii – anhydrous MgSO4, reflux 60 °C, N2.

Biological studies

In vitro cytotoxic activity

Cytotoxic activity for arjunolic acid 1 and its semisynthetic derivatives against HT-29 and PANC-1 cancer cell linesa.

Cytotoxic activities for arjunolic acid derivatives 25–28 against several cancer cell lines and non-tumor human fibroblast cell line (BJ)a.

Fig. 2. A) IC50 values obtained for AA1 and its derivatives towards PANC-1 cell line using MTT assay. B) SAR proposal based on the antiproliferative activity against PANC-1 and HT-29 cell lines and the several structural modifications performed in the AA 1 backbone.

Cell cycle assay

Annexin V-FITC/PI double-staining assay

C Hoechst 33342 staining assay

Scratch assay

Drug combination assay

Study of the interaction type between gemcitabine and compound 26 on PANC-1 cells. Combination index (CI) values were calculated based on a constant ratio of 50 : 1 ([gemcitabine] : [26]) using the Chou–Talalay method and the CompuSyn software.67 CI values <1 indicate the existence of synergism.

Pharmacology safety study

Conclusions

Experimental section

Chemistry

General remarks

Synthesis and structural characterization of compounds 2–28 is described below

2α,3β,23-Triacetoxyolean-12-en-28-oic acid (2)

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-amine (3)

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-methyl amine (4)

N-(2α,3β,23-Triacetoxyolean-12-en-28-oyl)-ethyl amine (5)

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-amine (6)

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-methyl amine (7)

N-(2α,3β,23-Trihydroxyolean-12-en-28-oyl)-ethyl amine (8)

Methyl 2α,3β,23-trihydroxyolean-12-en-28-oate (9)

Ethyl 2α,3β,23-trihydroxyolean-12-en-28-oate (10)

Butyl 2α,3β,23-trihydroxyolean-12-en-28-oate (11)

Allyl 2α,3β,23-trihydroxyolean-12-en-28-oate (12)

2α,23-Lactol-3-formyl-olean-12-ene-28-oic acid (13)

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-amine (14)

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-methyl amine (15)

N-(2α,23-Lactol-3-formyl-olean-12-ene-28-oyl)-ethyl amine (16)

Methyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (17)

Ethyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (18)

Butyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (19)

Allyl 2α,23-lactol-3-formyl-olean-12-ene-28-oate (20)

2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oic acid (21)

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-amine (22)

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-methyl amine (23)

N-(2-Formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oyl)-ethyl amine (24)

Methyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (25)

Ethyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (26)

Butyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (27)

Allyl 2-formyl-23-hydroxy-A(1)-norolean-2,12-diene-28-oate (28)

Biology

Cells and reagents

Compounds

Cell culture

MTT assay

Cell cycle assay

Annexin V-FITC/PI flow cytometry assay

Hoechst 33342 staining assay

Migration assay

Synergy study

Primary observation (Irwin) test in the mouse

Conflicts of interest

Supplementary Material

Acknowledgments

Notes and references

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Design, synthesis, and biological evaluation of new arjunolic acid derivatives as anticancer agents^†

Scheme 2. Reagents and conditions: a) NaIO₄, MeOH/H₂O, r.t.; b) i – acetic acid, piperidine, dry benzene, reflux 60 °C, N₂; ii – anhydrous MgSO₄, reflux 60 °C, N₂.

Cytotoxic activity for arjunolic acid 1 and its semisynthetic derivatives against HT-29 and PANC-1 cancer cell lines^a.

Cytotoxic activities for arjunolic acid derivatives 25–28 against several cancer cell lines and non-tumor human fibroblast cell line (BJ)^a.

Fig. 2. A) IC₅₀ values obtained for AA1 and its derivatives towards PANC-1 cell line using MTT assay. B) SAR proposal based on the antiproliferative activity against PANC-1 and HT-29 cell lines and the several structural modifications performed in the AA 1 backbone.