Isolation and Structural Characterization of Two New Furanoditerpenes from Pterodon emarginatus (Fabaceae)

Abstract

A furanoditerpene-enriched fraction was obtained from the fruits of Pterodon emarginatus and submitted to semipreparative high performance liquid chromatography (HPLC). Two new furanoditerpenes, 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan and 6α-acetoxy-7β,14β-dihydroxyvouacapan, in addition to the known compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate were obtained. Compound structures were determined by 1D and 2D nuclear magnetic resonance (NMR) experiments and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The major compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate was evaluated against promastigote forms of Leishmania amazonensis and L. braziliensis, presenting the concentration which causes lysis on 50% of parasites IC₅₀ < 30 µg mL^-1.

Keywords:
Pterodon; furanoditerpenes; vouacapan; Leishmania

Introduction

According to the website The Plant List,¹Pterodon (Fabaceae) comprises two native Brazilian species, popularly known as "sucupira-branca" or "faveiro": Pterodon abruptus (Moric.) Benth. (synonym: P. apparicioi) and P. emarginatus Vogel (synonyms: P. polygalaeflorus, P. polygaliflorus and P. pubescens). These species are found in the Brazilian Cerrado and their fruits have often been used in popular medicine for their anti-rheumatic, analgesic and anti-inflammatory properties.² Vouacapan furanoditerpenes are constituents of these fruits, and present some pharmacological properties, such as anti-inflammatory,³ antiproliferative against human cancer cells,⁴ antiedematogenic⁵ and antinociceptive activities.⁶

In the present study, two new furanoditerpenes, as well as one that has been previously described in the literature, were obtained from P. emarginatus through semi-preparative high performance liquid chromatography (HPLC). Their structures were determined via one and two-dimensional nuclear magnetic resonance (1D and 2D NMR) experiments, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS), and comparison with data from literature. In addition to the chemical study, compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate was evaluated against promastigote forms of Leishmania amazonensis and L. braziliensis.

Experimental

General procedures

NMR experiments were performed on a Bruker Avance III 11.75 Tesla spectrometer at 25 °C, using a 5 mm broadband probehead with a z-gradient. Spectra were obtained at 500.13 MHz for ¹H, using CDCl₃ as the solvent and tetramethylsilane (TMS) as the internal standard. Complete signal assignment was also obtained by heteronuclear multiple bond correlation (HMBC) and heteronuclear single quantum correlation (HSQC) experiments, and eventually by ¹³C analysis. A Model 9.4 T Solarix mass spectrometer (Bruker Daltonics, Bremen, Germany) was set to operate over a mass range of m/z 150-2000. The ESI source conditions were as follows: nebulizer gas pressure: 3 bar; capillary voltage: 3.5 kV; transfer capillary temperature: 250 °C. Ions accumulated in the hexapole collision cell in 5 × 10^-3 s were then transported to the analyzer cell (ICR) through the multipole ion guide system (another hexapole). The time-of-flight in the hexapole was 0.5 ms. Each spectrum was acquired by accumulating 200 scans of time-domain transient signals in 4 mega-point time-domain data sets. All mass spectra were externally calibrated using arginine (for ESI+) and sodium salt of trifluoroacetic acid (NaTFA) (for ESI-) solutions (m/z 200-2000). A resolving power, m/Dm_50% ca. 730 000, in which Dm_50% is the full width at half maximum height, with m/z ca. 400 and mass accuracy of < 1 ppm, provided the unambiguous molecular formula assignments for singly charged molecular ions. Mass spectra were acquired and processed using data analysis software (Bruker Daltonics, Bremen, Germany). The MS data was processed and compounds' elemental compositions were determined by measuring m/z values. Semi-preparative HPLC was performed using a Shimadzu LC-8A model (Kyoto, Japan), equipped with an SPD-20A UV detector and a Shimadzu C18 column (250 × 20 mm, 5 µm). Data processing was performed by LC Solution. All solvents used in chromatographic separations were of HPLC grade (Merck KGaA, Darmstadt, Germany), and analytical grade solvents were used for extraction (Qhemis, Brazil).

Plant materials

Fruits of P. emarginatus were collected in the Cerrado region of Bela Vista, Goiás State, Brazil. A voucher specimen was authenticated by Prof José Realino de Paula and deposited in the herbarium of Universidade Federal de Goiás (UFG), Brazil, under No. 27,155.

Extraction and isolation

Oleoresin extraction was performed by mechanical pressing using a continuous mini-press (MPE-40 ECIRTEC, Brazil). The extraction yield was 30% in weight. Regarding prior purification, 10.06 g of P. emarginatus fruit oil were extracted with 100 mL hexane. The insoluble fraction was solubilized in 100 mL methanol:water (9:1, v/v) and submitted to partitioning with 105 mL of a dichloromethane:hexane (6.3:3.7, v/v) mixture. Following agitation, distilled water was added until phase separation was complete (45 mL). The dichloromethane:hexane (DHF) fraction was submitted to rotaevaporation at 40 °C and used to isolate furanoditerpenes.

Approximately 170 mg of DHF was purified by semi-preparative reversed phase (RP)-HPLC under the following column chromatography conditions: Shimadzu C18 column (250 × 20 mm, 5 µm), detection wavelength of 190 and 220 nm, injection volume of 1.5 mL, mobile phase of acetonitrile:water (65:35, v/v) mixture acidified with 0.05% acetic acid (v/v), at a flow rate of 8 mL min^-1. Three samples were injected and fractions related to the three main chromatographic peaks (retention time ca. 17, 26 and 30 min) were collected, concentrated in a rotary evaporator at 40 °C, and freeze-dried (MicroModulyo115 freeze-dryer-ThermoFisher Scientific). The major constituents of the three collected fractions had their structures established by NMR and corroborated by mass spectrometry as 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan (1) ([M - H]^- of m/z 433.22309), 6α-acetoxy-7β,14β-dihydroxyvouacapan (2) ([M + Na]⁺ of m/z 399.21415), 6α-acetoxy-7β-hydroxyvouacapan-17β-oate (3) ([M - H]^- of m/z 403.21248).

Antileishmanial assay

Promastigote forms of L. amazonensis and L. braziliensis were cultivated in M199 (Sigma-Aldrich) supplemented with 10% bovine fetal serum (Gibco), 0.22% NaHCO₃, and 0.5% penicillin-streptomycin solution (Sigma-Aldrich) at 23 °C for 6 days. Promastigote forms of L. amazonensis and L. braziliensis (10⁶ parasites mL^-1) were deposited in 96-well microplates and treated with different concentrations of compound 3 (8, 32 and 128 µg mL^-1) for 24 and 72 h.

After treatments, 50 µL of tetrazolium dye MTT (2.5 mg mL^-1) were added to each well in the plate, which was incubated for 4 h at 23 °C. Formazan crystals produced by viable cells were solubilized with 50 µL per well of SDS (10% m/v) and plates were incubated at 37 °C for 60 min. Following incubation, absorbance was measured at 570 nm using a microplate reader (BioTek, Synergy H1 Hybrid Reader). Bioassays were performed in triplicate, with dimethylsulfoxide (DMSO) solution being used in the same concentration that was applied for sample solubilization, as negative control and amphotericin B as reference drug.

The leishmanicidal activity of compound 3 was expressed as IC₅₀ values, corresponding to the concentration which causes lysis on 50% of parasites. These values were calculated by the statistical method of sigmoid concentration-response curve, via GraphPad Prism 5.0.

Results and Discussion

Vouacapan furanoditerpenes were obtained from an enriched sample (DHF) by semipreparative RP-HPLC. Fractions amounting to shaded peaks 1, 2, and 3-4 (Figure 1) were collected from three injections, which produced white solids following concentration and freeze-drying steps. Fractions from shaded areas 3 and 4 (Figure 1) were grouped, hence yielding 30.0 mg of a pure compound. Fractions 1 and 2 yielded 7.5 mg each. As may be observed in the chromatogram and confirmed by ¹H NMR spectra, fractions 1 and 2 contain impurities due to the coelution of substances whose retention times are close to those of the major compounds (Figure 1, particularly at 190 nm).

Figure 1
Chromatographic profile of the enriched fraction of the P. emarginatus oleoresin, obtained via semi-preparative HPLC at 190 and 220 nm. Shaded areas 1, 2, and 3-4 correspond to the collected fractions. The chromatogram at 220 nm is displaced on the y-axis for a clearer presentation.

The structures of the new compounds 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan (1) and 6α-acetoxy-7β,14β-dihydroxyvouacapan (2) were elucidated by NMR experiments and confirmed by MS and data from literature (Figure 2).^7,8 These experiments were also used to identify compound 3 as methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate, previously identified in P. apparicioi and P. emarginatus.^9-11

Figure 2
Structure of vouacapan diterpenes 1, 2 and 3.

The ¹H NMR spectrum for compound 1 showed hydrogen furans at δ_H 6.42 (1H, d, J 1.9 Hz, H₁₅) and δ_H 7.26 (1H, d, J 1.9 Hz, H₁₆). Two acetyl groups were assigned by the signals at δ_H 5.36 (1H, dd, J 11.8 and 9.2 Hz, H₆), δ_H 4.12 (1H, d, J 11.3 Hz, H-19a), d_H 4.22 (1H, d, J 11.3 Hz, H-19b), δ_H 2.19 (3H, s, H_6"), and δ_H 2.05 (3H, s, H_19"). Three methyl groups were observed at δ_H 1.46 (3H, s, H₁₇), 1.14 (3H, s, H₁₈) and 1.05 (3H, s, H₂₀).

The ¹³C NMR associated with cross peaks observed in HSQC and HMBC experiments corroborated typical signals for vouacapan's furan ring at δ_C 148.2, 141.9, 123.9 and 107.2. Two acetoxy and three carbinolic groups were also observed at δ_C 172.6, 171.2, 77.5, 75.8 and 72.6.

These data are very similar to that presented for known compound 3 (Table 1), except for the replacement of carbomethoxy and hydrogen groups at C₁₄ for hydroxyl and methyl groups. Replacement of hydrogen by one acetoxy group at C₁₉ was also observed. The hydroxyl group at C₁₄ was confirmed by cross peaks between hydrogens H₇, H₈ and H₁₅ (δ_H 3.81, 2.02 and 6.42) with C₁₄ at δ_C 72.6 in the HMBC experiment. The doublets at δ_H 4.12 and 4.22 (J 11.3 Hz) as well as the cross peak between H₁₉ and carbonyl at δ_C 171.2 confirmed the position of this new acetoxy group. Cross peaks between these signals and carbons C₄, C₅ and C₁₈ (δ_C 38.7, 55.0 and 30.6), observed in the HMBC experiment, suggested that the acetoxy group is attached to C₁₉. The C₄ and C₁₄ stereochemistry were evaluated by nuclear Overhauser effect (NOE) experiments (Figures S6 and S7). The observed NOE between H₅, H₇ and Me-18 corroborate acetoxy group on C₁₉. On the other hand, the NOE between Me-17, H₇ and H₁₅ confirmed hydroxyl group on β position. No NOE was observed between H₈ and Me-17. The C₁₄ stereochemistry in is agreement with that proposed for two furanoditerpenes voucapanes containing methyl and hydroxyl groups attached on C₁₄.¹² The proposed structure is in agreement with 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan (1).

Table 1
¹H and ¹³C NMR spectra data for diterpenes 1, 2 and 3, isolated from the fruit oil of P. emarginatus (500 MHz, CDCl₃)

H/C	1		2		3
	δ 1H / ppm (mult., J / Hz)	δ 13C / ppm	δ 1H / ppm (mult., J / Hz)	δ 13C / ppm	δ 1H / ppm (mult., J / Hz)	δ 13C / ppm
1	1.7-1.6 (m); 1.2-1.1 (m)	38.4	1.7-1.6 (m); 1.0-0.9 (m)	39.4	1.7-1.6 (m); 1.0-0.9 (m)	39.5
2	1.6-1.4 (m)	18.0	1.6-1.4 (m)	18.1	1.6-1.4 (m)	18.5
3	1.8-1.7 (m); 1.1-1.0 (m)	39.5	1.2-1.1 (m); 1.4-1.3 (m)	43.3	1.2-1.1 (m); 1.4-1.3 (m)	43.7
4	-	38.7	-	32.9	-	33.2
5	1.46 (d, 11.8)	55.0	1.29 (d, 11.6)	54.3	1.28 (d, 11.5)	55.0
6	5.36 (dd, 11.8, 9.2)	75.8	5.33 (dd, 11.6, 9.2)	75.8	5.21 (dd, 11.5, 9.4)	75.7
7	3.81 (dd, 10.6, 9.2)	77.5	3.85 (dd, 10.6, 9.2)	76.7	3.31 (dd, 10.6, 9.4)	81.4
8	2.02 (dd, 12.5, 10.6)	49.5	2.04 (dd, 12.5, 10.6)	49.6	2.37 (ddd, 11.7, 10.6, 9.1)	42.7
9	1.5-1.4 (m)	46.9	1.5-1.4 (m)	46.7	1.45 (ddd, 11.7, 5.6, 4.9)	48.2
10	-	37.0	-	38.6	-	38.6
11	2.62 (dd, 16.6, 6.6) 2.45 (dd, 16.6, 10.5)	22.5	2.61 (dd, 16.6, 6.5) 2.44 (dd, 16.6, 10.7)	22.2	2.64 (dd, 16.1, 5.6) 2.41 (dd, 16.1, 4.9)	22.2
12	-	148.2	-	148.4	-	150.5
13	-	123.9	-	123.7	-	113.6
14	-	72.6	-	72.6	3.38 (d, 9.1)	46.6
15	6.42 (d, 1.9)	107.2	6.42 (d, 1.9)	107.0	6.14 (d, 1.7)	108.6
16	7.26 (d, 1.9)	141.9	7.26 (d, 1.9)	141.6	7.21 (d, 1.7)	141.6
17	1.46 (s)	25.9	1.47 (s)	25.6	-	175.6
18	1.14 (s)	30.6	1.06 (s)	36.1	1.00 (s)	36.5
19	4.22 (d, 11.3) 4.12 (d, 11.3)	67.0	0.96 (s)	22.4	0.92 (s)	22.7
20	1.05 (s)	16.3	1.07 (s)	15.6	1.05 (s)	15.7
6'	-	172.6	-	172.0	-	172.1
6"	2.19 (s)	21.9	2.16 (s)	21.7	2.09 (s)	22.6
19'	-	171.2	-	-	-	-
19"	2.05 (s)	20.9	-	-	-	-
OMe-17	-	-	-	-	3.70 (s)	52.5

• ¹H and ¹³C NMR assignments are based on ¹H and ¹³C spectra and on ¹H-¹³C HSQC and HMBC contour maps; s: singlet; m: multiplet; d: doublet; dd: double doublets; ddd: double double doublets.

Compounds 1 and 2 show very similar NMR spectra (Table 1). The most significant difference was due to the absence of the acetoxy group attached to C₁₉. One methyl group is attached in this position, according to the signal at δ_H 1.06 (s). Basing on this data, the 6α-acetoxy-7β,14β-dihydroxyvouacapan structure was proposed for compound 2.

Figure 3 shows the ESI(-)-FTICR mass spectrum of these three diterpenes (1, 2 and 3). Diterpene 1 was detected in deprotonated form [M - H]^- of m/z 433.22309 with an error of 0.20 ppm. Diterpene 2 was detected as sodium adduct [M + Na]⁺ of m/z 399.21415 with an error of 0.11 ppm. Diterpene 3 was detected in the negative ion mode in deprotonated form [M - H]^- of m/z 403.21248 with an error of 0.13 ppm. In all cases, the ultra-high resolution mass spectrum provides the exact mass with an error lower than 1 ppm.

Figure 3
ESI-FTICR mass spectrum of vouacapan diterpenes 1, 2 and 3.

The antiparasitic activity of furanoditerpene 3 was assessed in vitro against promastigotes of L. amazonensis and L. braziliensis, resulting in significant leishmanicidal activity (Table 2).¹³

Table 2
Antiparasitic activities of compound 3 and amphotericin B

Compound	IC50 / (µg mL-1)
	Leishmania amazonensis		Leishmania braziliensis
	24 h	72 h	24 h	72 h
3	25.47 ± 1.01	20.12 ± 0.76	26.12 ± 0.18	17.94 ± 1.94
Amphotericin B	3.09 ± 0.08	3.22 ± 0.03	2.68 ± 0.23	4.84 ± 0.12

• IC₅₀: half-maximal inhibitory concentration; data expressed as mean of triplicates ± standard deviation.

Diterpenes rank among the classes of secondary metabolites with promising leishmanicidal activity.¹⁴ Leishmaniasis is regarded as a neglected disease, and this is the first study to report on assays carried out with furanoditerpenes vouacapan isolated from fruits of the Pterodon genus. Further studies on the pharmacological potential of Pterodon furanoditerpenes should be encouraged, as they contribute to the development of drugs for neglected diseases and favor the preservation of Cerrado.

Conclusions

The phytochemical study of P. emarginatus fruits has led to the isolation and elucidation of two new furanoditerpenes, 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan (1) and 6α-acetoxy-7β,14β-dihydroxyvouacapan (2), in addition to the known compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate (3). Compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate (3) was found as a promising leishmanicidal agent.

Acknowledgments

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), and Financiadora de Estudos e Projetos (FINEP) for their financial support.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

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Publication Dates

• Publication in this collection
  Oct 2017

History

• Received
  24 Oct 2016
• Accepted
  15 Feb 2017