New Tirucallane-Type Triterpenoids from Guarea guidonia

Abstract

The aerial parts of Guarea guidonia afforded three new tirucallane-type triterpenoids: 3,4-seco-tirucalla-4(28),8(9),24(25)-trien-7α,11α-dihydroxy-21,23-epoxy-3,11-olide, named guareolide (1), 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21-hydroxy-21,23-epoxy-3-oic acid, named guareoic acid A (2), and 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21,23-epoxy-3-oic acid, named guareoic acid B (3), of which 1 possessed an unusual seven-membered lactone ring. Seven known terpenes were also isolated and characterized as flindissone, 7-acetyldihydronomilin, picroquassin E, boscartol C, and cneorubins A, B, and X. Their structures were determined by spectroscopic methods including one-dimensional and two-dimensional nuclear magnetic resonance analysis and high-resolution mass spectrometry. The isolates were investigated for their potential cytotoxic activity on Jurkat, HeLa, and MCF7 cancer cell lines. Flindissone and compound 2 showed an antiproliferative activity in all cell lines. Further studies revealed that flindissone, the most active compound, induced in Jurkat and HeLa cells both cytostatic and cytotoxic responses.

Key words

Guarea guidonia - Meliaceae - tirucallane triterpenes - cytotoxicity - antiproliferative activity

Introduction

Guarea is a genus of the Meliaceae family, which is among the richest and most diverse source of secondary metabolites in the Angiospermae, with 50 genera and more than 1400 species. The Guarea genus comprised both trees or small shrubs with about 80 species widespread in African and American countries; eight species of this genus were classified in the wet forest of Venezuela [1]. Guarea guidonia (L.) Sleumer is a tree typical of Brazil, but it is also found in Venezuela, particularly in the Sierra Parima of the Estado Bolivar, where the wood bark is traditionally used as a febrifugal agent and an abortive, while the leaves and the fruits are known to be toxic for the cattle [2], [3].

Limonoids, triterpenes, steroids, sesquiterpenes, diterpenes, and coumarins are typical constituents of this species [3]. The aerial parts of this plant were already investigated and different compositions have been found for specimens collected in different regions [4]. In our continuing investigations on Venezuelan Meliaceae plants [5], [6], the phytochemical study of G. guidonia aerial parts was carried out, and three new tirucallane-type triterpenoids (1–3) ([Fig. 1]) were isolated and characterized, together with seven known compounds. In the framework of a research project aimed to investigate the cytotoxic activity of plant small molecules, all the isolates were assayed.

Fig. 1 Chemical structures of compounds 1 – 3 isolated from G. guidonia aerial parts.

Results and Discussion

The n-hexane and chloroform aerial parts extract of G. guidonia were repeatedly subjected to different chromatographies to afford three new tirucallane-type triterpenes (1–3) ([Fig. 1]), along with seven known compounds. NMR spectra of compounds 1–3 and data for the EC₅₀ determination of cytotoxic activity are available as Supporting Information.

Compound 1 showed a sodiated molecular ion peak at m/z 491.3122 for [M + Na]⁺ in the HRESIMS, allowing the assignment of molecular formula C₃₀H₄₄O₄. The ¹H NMR of 1 ([Table 1]) showed the presence of six methyl singlets, three of which on double bonds (δ 1.72, 1.73, and 1.82), an exomethylene group (δ 4.80, 5.01), one resonance for olefinic proton (δ 5.24), three hydroxymethines (δ 4.12, 4.67, 5.18), and one hydroxymethylene (δ 3.24, 4.15). The ¹³C NMR ([Table 1]) displayed 30 carbon signals that were assigned as six methyls (δ 18.3, 19.7, 20.2, 24.0, 25.9 double signal), seven methylenes (δ 28.3, 30.5, 30.8, 35.0, 35.3, 39.1, 39.3), three methines (δ 43.4, 46.9, 49.7), three quaternary carbons (δ 41.5, 43.0, 52.4), one methylene double bond (δ 115.8, 148.0), one trisubstituted double bond (δ 127.6, 138.0), one tetrasubstituted double bond (δ 133.2, 145.8), three oxygenated methyne carbons (δ 64.0, 75.1, 76.0), one oxygenated methylene carbon (δ 72.8), and one carboxyl group (δ 178.0). Results obtained from 1D TOCSY and COSY experiments established the correlations of all protons in 1, showing the sequences H-1–H-2, H-5–H-7, H-11–H-12, H-15–H-21, H-15–H-23. Full assignments of all protonated carbon signals were secured by HSQC and HMBC NMR interpretation. The HMBC correlations between δ 5.18 (H-11) and δ 133.2 (C-9), 145.8 (C-8), and 178.0 (C-3), and δ 1.96 (H-1a) and δ 20.2 (Me-19), 41.5 (C-10), 133.2 (C-9), and 178.0 (C-3) suggested that the hydromethine group at C-11 was involved in an unusual lactone ring with C-3. Other key cross-peaks were observed between δ 0.93 (Me-30) and δ 145.8 (C-8), confirming the presence of a C-8/C-9 double bond. The chemical shifts of H₂-21 and H-23 suggested the presence of an epoxy function at these positions, forming a tetrahydrofuran ring in the side chain, while C-24–C-27 formed an isobutenoyl group [7]. The relative stereochemistry of 1 was determined by 1D ROESY (rotating frame Overhauser effect spectroscopy) experiments and comparison with literature data. The signal characteristic of H-7 (br s) and its 1D ROESY correlation with δ 0.93 (Me-30) demonstrated an α-orientation for the 7-OH group. Selective irradiation of H-11 (δ 5.18) showed significant enhancement of the methyl signal at δ 1.09 (Me-19) and of H-2β; similarly, irradiation of δ 1.09 (Me-19) produced enhancement of H-11 and H-7 signals. In light of these data, the structure of 1 was elucidated as 3,4-seco-tirucalla-4(28),8(9),24(25)-trien-7α,11α-dihydroxy-21,23-epoxy-3,11-olide and was named guareolide. To the best of our knowledge, this is the first report of a tirucallane-type triterpene with an unusual seven-membered lactone ring.

Table 1  ¹H and ¹³C-NMR data of compounds 1–3 (CD₃OD, 600 MHz, J in Hz).

Position	1		2		3
	δ H	δ C	δ H	δ C	δ H	δ C
Data assignments were confirmed by DQF-COSY, 1D TOCSY, HSQC, and HMBC experiments. a Overlapped signal.
1a	1.96 br dd (14.7, 8.0)	35.0	1.28 m	32.0	1.40	32.8
1b	1.50a
2a	3.00 br dd (14.0, 10.0)	30.5	2.00a	30.0	2.38 ddd (15.5, 11.0, 3.8)	29.1
2b	2.44 br dd (14.5, 7.6)		1.80a		2.21a
3		178.0		178.0		175.0
4		148.0		149.0		148.3
5	2.74 dd (14.5, 2.5)	46.9	2.47 dd (12.0, 6.0)	50.4	2.49 dd (12.0, 6.0)	50.5
6a	2.03 m	28.3	2.24 m	31.3	2.24a	31.2
6b	1.57a		2.03a		2.03a
7	4.12 br s	64.0	5.26 br d (3.5)	119.1	5.39 br d (3.0)	119.5
8		145.8		147.0		147.9
9		133.2	2.60 m	42.2	2.57 m	42.2
10		41.5		37.9		38.0
11	5.18 br d (5.6)	75.1	1.61a	18.0	1.61a	18.7
12a	2.16a	39.3	1.63a	33.0	1.72a	32.7
12b	1.70a		1.50a		1.63a
13		43.0		45.0		44.8
14		52.4		52.0		52.0
15a	2.06 ddd (17.0, 13.0, 3.5)	35.3	1.99a	36.7	1.65a	35.5
15b	1.69a		1.50a		1.52 m
16a	1.50a	30.8	1.70a	34.4	1.72a	32.5
16b	1.37a		1.63a		1.49a
17	1.78a	49.7	2.11 br dd (15.0, 9.4)	46.0	1.72a	52.9
18	1.07 s	19.7	0.94 s	24.0	0.92 s	23.4
19	1.09 s	20.2	0.86 s	16.0	0.87 s	16.2
20	2.35 m	43.4	2.02a	49.0	2.05a	43.5
21a	4.15 t (8.5)	72.8	4.83a	104.0	4.01 t (8.3)	73.0
21b	3.24 t (8.5)				3.28 br t (9.2)
22a	2.20a	39.1	1.72a	37.0	1.72a	39.6
22b	1.79a		1.50a
23	4.67 ddd (13.5, 8.0, 5.0)	76.0	4.76 ddd (13.0, 8.3, 4.6)	76.4	4.65 ddd (13.3, 8.5, 4.6)	75.5
24	5.24 br d (8.3)	127.6	5.18 br d (9.0)	128.9	5.20 br d (8.4)	127.1
25		138.0		135.0		138.0
26	1.73 s	18.3	1.69 s	18.0	1.70 s	17.9
27	1.72 s	25.9	1.72 s	26.0	1.72 s	25.0
28a	5.01 br s	115.8	4.86a	114.4	4.89a	114.6
28b	4.80 br s		4.84a		4.85a
29	1.82 s	24.0	1.81 s	23.0	1.81 s	22.8
30	0.93 s	25.9	1.06 s	27.0	1.05 s	27.9

The molecular formula of C₃₀H₄₆O₄ was assigned to compound 2, obtained as white amorphous powder, by its negative HRESIMS spectrum (m/z 469.6838 [M – H]⁻). Analysis of ¹H and ¹³C NMR data ([Table 1]) suggested a triterpenoid structure. The ¹³C, HSQC, and HMBC NMR spectra of 2 showed 30 carbon signals, indicative of six methyls, eight methylenes, four methines, three quaternary carbons, an exomethylene group, two trisubstituted double bonds, one hydroxymethine, a carboxyl group, and an emiacetal function. These data led to establish the presence of a seco-ring A tirucallane triterpene [8]. COSY and 1D TOCSY experiments, together with HMBC cross-peaks correlations between H-5–C-1, H-5–C-4, H-5–C-28, Me-29–C-4, Me-29–C-5, further confirmed the 3,4-seco-A-ring form in 2. The ¹H NMR spectrum of 2 revealed the presence of six singlet methyl groups, of which three were linked to double bonds, two resonances for olefinic hydrogens, two protons for an exocyclic double bond, one hydroxymethine, and one emiacetal function ([Table 1]). A spin system, CH(17)−CH(20)−CHOH(21) and CH(17)−CH(20)−CH₂(22)−CH(23)−CH(24), determined from the COSY spectrum, suggested associations of C-21 with C-20 and C-24, which would require a five-membered ring anchored at C-17. This finding was confirmed by the HMBC correlations of H-17 (δ _H 2.11) with C-20 (δ _C 49.0), H-20 (δ _H 2.02) with C-17 (δ _C 46.0) and C-21 (δ _C 104.0), H-21 (δ _H 4.83) with C-22 (δ _C 37.0), C-20 (δ _C 49.0), and C-23 (δ _C 76.4). The HMBC correlations from the proton signals at δ _H 1.69 (Me-26) and 1.72 (Me-27) to the carbon signals at δ _C 128.9 (C-24) and 135.0 (C-25) supported the occurrence of an isobutenyl group at C-23 [7]. The relative stereochemistry of 2 was obtained through 1D ROESY correlations between δ 1.06 (Me-30) and 0.86 (Me-19) and δ 0.94 (Me-18) and 2.02 (H-20). Furthermore, similar ¹H and 1D ROESY NMR data showed the relative configuration of the side chain in 2 to be the same as 3-epi-flindlissol [7]. Therefore, compound 2 was identified as 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21-hydroxy-21,23-epoxy-3-oic acid, named as guareoic acid A.

Compound 3 was purified as a white amorphous powder. Its molecular formula was determined to be C₃₀H₄₆O₃ from the deprotonated molecular ion [M – H]⁻ at m/z 453.3329 in the negative HRESIMS spectrum. Analysis of its NMR spectra ([Table 1]) suggested the same 3,4-seco-tirucallane skeleton described for 1. Comparison of NMR spectra with those of 2 revealed 3 to differ from 2 only by functionalities of ring E. In fact, in compound 3 the chemical shifts of H₂-21 at δ 4.01 (1H, t, J = 8.3 Hz) and 3.28 (1H, br t, J = 9.2 Hz), and H-23 at δ 4.65 (1H, ddd, J = 13.3, 8.5, 4.6 Hz), suggested that the emiacetal function at C-21 was replaced by a hydroxymethylene group [9]. Thus, the structure of 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21,23-epoxy-3-oic acid was determined for compound 3 and was named guareoic acid B.

Flindissone [10], 7-acetyldihydronomilin [11], picroquassin E [12], boscartol C [13], and cneorubins A, B, and X [14] were also purified and identified by comparison with published spectroscopic data.

The antiproliferative activity of the isolates was evaluated in Jurkat (T-cell leukemia), HeLa (cervical carcinoma), and MCF-7 (breast cancer) cancer cell lines by ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) (MTT) assay. Half maximal effective concentration (EC₅₀) values, obtained from dose-response curves, are shown in [Table 2]. Compound 2 and flindissone showed an antiproliferative activity in all cell lines. In particular, Jurkat resulted slight more susceptible than HeLa and MCF7 cells. Furthermore, the cytotoxic potential of active compounds was evaluated in human peripheral blood mononuclear cells (PBMC) from healthy donors, chosen as the normal counterpart of leukemia-derived Jurkat cell line. Compound 2 and flindissone did not causes any significant reduction of the number of freshly isolated non-proliferating PBMC, at least in the range of cytotoxic doses in leukemia cells.

Table 2 EC₅₀ (µM) antiproliferative activity of isolates in different cell lines.

Compound	Jurkata	HeLab	MCF7c	PBMCd
a T-cell leukemia; b cervical carcinoma; c breast carcinoma; d human peripheral blood mononuclear cells
1	> 100	> 100	> 100	–
2	39	55	75	> 100
3	> 100	> 100	> 100	–
Flindissone	25	27	50	> 100
Picroquassin E	88	> 100	> 100	–
Etoposide	2.5	4	12	–

The mechanism(s) underlying the antiproliferative effect of the most active flindissone was further studied in HeLa and Jurkat cells. To investigate whether flindissone reduced cells number by affecting cell cycle progression and/or by inducing cell death, HeLa and Jurkat cells were exposed for 48 h to concentrations (15 and 35 µM) close to the EC₅₀ value of flindissone. DNA content was evaluated by flow cytometry analysis of propidium iodide stained nuclei. As shown in [Fig. 2], flindissone caused in HeLa ([Fig. 2 A]) and in Jurkat ([Fig. 2 B]) an increase of cell population in G₀/G₁ and G₂/M phases. Moreover, an increase of cells with subG₀/G₁ DNA content was also observed, thus indicating the onset of apoptotic events.

Fig. 2 Effect of flindissone on cell proliferation. Flow cytometric evaluation of DNA content in cells exposed for 48 h to flindissone or vehicle alone (controls). A HeLa cells were exposed to 15 µM and 35 µM flindissone; B Jurkat cells exposed to 15 µM and 35 µM flindissone. Data in A and B have been subtracted for the corresponding values in control cells (exposed to vehicle only): HeLa, subG₀/G₁ ≤ 2%; G₀/G₁, 53 ± 1.9%; S, 37 ± 2.2%; G₂/M, 10 ± 0.3%; Jurkat, subG₀/G₁ ≤ 2.2%; G₀/G₁, 56 ± 1.8%; S, 37 ± 2.1%; G₂/M, 6 ± 0.9%. Data are the mean values from three experiments: standard deviation (SD) values never exceeded 12% of the means.

Materials and Methods

General experimental procedures

Optical rotations were measured on a Autopol IV Automatic Polarimeter (Rudolph) equipped with a sodium lamp (589 nm) and a 1-dm microcell. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH) equipped with a Bruker 5-mm TCI CryoProbe at 300 K. Chemical shifts were expressed in δ (parts per million) referred to the solvent peaks δ _H 3.34 and δ _C 49.0 for CD₃OD. The NMR data were processed as reported previously [15]. HRESIMS were acquired in the positive and negative ion mode on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Column chromatographies were performed over silica gel (70 – 220 mesh, Merck); RP-HPLC separations were conducted on a Waters 590 series pumping system equipped with a Waters R401 refractive index detector, using a C₁₈ µ-Bondapak column (30 cm × 7.8 mm, 10 µm, Waters-Milford) and a mobile phase consisting of MeOH-H₂O mixtures at a flow rate of 2 mL/min. TLC were carried out on precoated Kieselgel 60 F₂₅₄ plates (Merck); compounds were detected by Ce(SO₄)₂/H₂SO₄ (Sigma-Aldrich) solution.

Plant material

The aerial parts of G. guidonia were collected in December 2008, in the Estación Experimental Caparo (7°28′ 13″ N; 71°03′ 16″ O), Llanos Occidentales, Suroeste del Estado Barinas, Municipio Ezequile Zamora, Venezuela. The plant was identified by Prof. Dr. Pablo Melendez, and a voucher specimen (n. 625) was deposited in the Herbarium MERF of the Facultad de Farmacia y Bioánalisis, Universidad de Los Andes, Mérida, Venezuela.

Extraction and isolation

The aerial parts of G. guidonia (400 g) were powdered and successively extracted with n-hexane, CHCl₃, and MeOH (3 × 2 L), to give 6.0 g, 12.1 g, and 15.0 g of the respective dried residues. Part of the n-hexane extract (5 g) was subjected to column chromatography (5 × 150 cm) over silica gel eluting with n-hexane, followed by increasing concentrations of CHCl₃ in n-hexane (between 1% and 100%) and increasing concentrations of MeOH in CHCl₃ (between 1% and 100%). Fractions of 30 mL were collected, analyzed by TLC, and grouped into nine major fractions (A₁–I₁). Fraction D₁ (241.0 mg) was purified by RP-HPLC using MeOH-H₂O (4 : 1) to give cneorubin X (2.0 mg, t _R 42 min). Fraction F₁ (497.2 mg) was chromatographed by RP-HPLC using MeOH-H₂O (75 : 25) to yield cneorubin A (3.5 mg, t _R 34 min) and cneorubin B (4.0 mg, t _R 38 min). Fraction H₁ (274.1 mg) was subject to RP-HPLC using MeOH-H₂O (7 : 3) to give boscartol C (2.2 mg, t _R 37 min) and cneorubin X (2.3 mg, t _R 47 min). Part of the CHCl₃ extract (5.3 g) was subjected to silica gel column chromatography (5 × 150 cm), eluting with CHCl₃ followed by increasing concentrations of MeOH in CHCl₃ (between 1% and 100%). Fractions of 30 mL were collected, analyzed by TLC, and grouped into 10 major fractions (A₂–J₂). Fractions D₂ (990.0 mg) and E₂ (264.5 mg) were purified by RP-HPLC with MeOH-H₂O (85 : 15) as eluent to obtain flindissone (8.5 mg, t _R 27 min), compounds 3 (10.0 mg, t _R 42 min) and 2 (6.0 mg, t _R 50 min), from fraction D₂, and compounds 1 (1.2 mg, t _R 12 min), 3 (2 mg, t _R 42 min), and 2 (2.2 mg, t _R 50 min), from fraction E₂, respectively. Fraction C₂ (228.3 mg) was purified by RP-HPLC using MeOH-H₂O (3 : 2) to give 7-acetyldihydronomilin (1.2 mg, t _R 22 min). Finally, fraction H₂ (145.0 mg) was subjected to RP-HPLC using MeOH-H₂O (9 : 1) to give picroquassin E (2.3 mg, t _R 45 min).

Guareolide ( 1 ): white amorphous powder; [α]_D ²⁵ − 143 (c 0.1, MeOH); ¹H and ¹³C NMR data, see [Table 1]; HRESIMS: m/z 491.3122 [M + Na]⁺ (calcd. for C₃₀H₄₄NaO₄, 491.3137).

Guareoic acid A ( 2 ): white amorphous powder; [α]_D ²⁵ + 11.5 (c 0.09, MeOH); ¹H and ¹³C NMR data, see [Table 1]; HRESIMS: m/z 469.6838 [M – H]⁻ (calcd. for C₃₀H₄₅O₄, m/z 469.3318).

Guareoic acid B ( 3 ): white amorphous powder; [α]_D ²⁵ + 139 (c 0.27, MeOH); ¹H and ¹³C NMR data, see [Table 1]; HRESIMS: m/z 453.3329 [M – H]⁻ (calcd. for C₃₀H₄₅O₃, m/z 453.3369).

Cytotoxicity assay

Cells and treatment

Jurkat, HeLa, and MCF-7 cell lines were obtained from the ATCC. Cells were maintained in DMEM (HeLa and MCF-7) or RPMI 1640 (Jurkat), supplemented with 10% FBS, 100 mg/L streptomycin, and penicillin 100 IU/mL at 37 °C in a humidified atmosphere of 5% CO₂. To ensure logarithmic growth, cells were subcultured every 2 d. PBMC were isolated from buffy coats of healthy donors (kindly provided by the Blood Center of the Hospital of Battipaglia, Salerno, Italy) by using a standard Ficoll-Hypaque gradient. Freshly isolated PBMC contained 90.6 ± 1.2% live cells as assessed by the manual Trypan Blue exclusion method. Stock solutions (50 mM) of purified compounds in DMSO were stored in the dark at 4 °C. Appropriate dilutions were prepared in culture medium immediately prior to use. In all experiments, the final concentration of DMSO did not exceed 0.15% (v/v).

Cell viability and cell cycle

Cells were seeded in 96-well plates and incubated for the established times in the absence (vehicle only) and in the presence of different concentrations of compounds. The day before treatments, cells were seeded at a cell density of 1 × 10⁴ cells/well. The number of viable cells was quantified by MTT assay. Absorption at 550 nm for each well was assessed using a microplate reader (LabSystems). In some experiments, cell viability was also checked by Trypan Blue exclusion assay using a Bürker counting chamber. EC₅₀ values were calculated from cell viability dose-response curves and defined as the concentration resulting in 50% inhibition in cell survival as compared to etoposide (etoposide synthetic, ≥ 98%, Sigma-Aldrich), used as positive control. Each experimental condition was tested once in quadruplicate. EC₅₀ values were performed with GraphPad software.

Cell cycle was evaluated by propidium iodide staining of permeabilized cells according to the available protocol and flow cytometry (BD FACSCalibur flow cytometer, Becton Dickinson) [16]. Data from 5000 events per sample were collected. The percentages of the elements in the hypodiploid region and in G₀/G₁, S and G₂/M phases of the cell cycle were calculated using the CellQuest or MODFIT software, respectively.

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors are grateful to University of Salerno for the grant Project “Caratterizzazione dellʼinterattoma di sostanze naturali bioattive” FARB 2016.

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