Spiranthenones A and B, Tetraprenylated Phloroglucinol Derivatives from the Leaves of Spiranthera odoratissima

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• In vitro test against Plasmodium falciparum
• In vitro tests against Leishmania chagasi and Trypanosoma cruzi
• Cytotoxicity test
• Results and Discussion
• Acknowledgments
• References

Abstract

Two new polyprenylated acylphloroglucinols, spiranthenones A (1) and B (2), a sesquiterpenoid, 6α-acetoxy,1β-hydroxyeudesm-4(15)-ene (3), along with sesamin and β-sitosterol, were isolated from the EtOAc extract of the leaves of Spiranthera odoratissima, and shown to display antiprotozoal activity. Their structures and relative stereochemistry were elucidated by NMR and mass spectrometry. These compounds exhibited moderate antiprotozoal activity, but without significant cytotoxicity against fibroblasts cell line NIH-3T3. Compound 3 was the most selective towards parasites.

Introduction

Spiranthera odoratissima (Rutaceae) is a small shrub known popularly as manacá and found in the cerrado region, Central Brazil. It is used in traditional medicine for the treatment of rheumatism, gout, kidney infection, urinary retention, abdominal pain, acne and furuncle, and for its analgesic and anti-inflammatory properties [1]. Previous phytochemical investigations of S. odoratissima led to the isolation of monoterpenes, sesquiterpenes, furoquinoline alkaloids with fungicidal activity, limonoids with insecticidal activity, and coumarins [2], [3], [4].

In our search for new antiprotozoal agents, S. odoratissima was selected from a preliminary screening of cerrado plant extracts [5], in which the EtOAc extract of its leaves displayed in vitro activity against chloroquine-resistant P. falciparum and T. cruzi with IC₅₀ values of 9.2 and 56.3 µg/mL, respectively. It was less active against L. chagasi and showed no significant cytotoxicity against NIH-3T3 cells. Herein, we report the isolation and structure elucidation of compounds 1–3, and their antiprotozoal and cytotoxic activities.

Materials and Methods

General experimental procedures

Optical rotations were measured on a Perkin Elmer model 341 polarimeter at 20 °C. IR spectra were taken on a Shimadzu FTIR-8400S spectrophotometer. Mass spectra data were recorded using an electrospray time of flight mass spectrometer (ESI-TOF-MS) operating in the positive mode (QSTAR Pulsar I of Applied Biosystems). ¹³C NMR spectra were recorded on an AC 300 BRUKER spectrometer operating at 75.47 MHz for ¹³C. ¹H and 2D-NMR spectra were recorded on an Avance 400 BRUKER spectrometer operating at 400.13 MHz. The ¹H and ¹³C NMR chemical shifts are given in ppm relative to TMS, with coupling constants (J) reported in Hz. For the HMBC experiments, the delay (1/2 J) was 70 ms and for the NOESY experiments the mixing time was 150 ms. Analytical and preparative TLC were carried out on precoated Si gel 60 F254 plates (Merck). Spots were detected under UV (254 and 366 nm) before spraying with vanillin/H₂SO₄ solution in EtOH, followed by heating the plate at 110 °C. Column chromatographies (CC) were performed on 200–400 mesh silica gel 60 (Merck). Preparative medium-pressure liquid chromatographies (MPLC) were performed with a pump K-120 (Knauer) and Flashsmart cartridges (SiO₂ gel 20–40 µm; AIT).

Plant material

The leaves of Spiranthera odoratissima A. Saint-Hilaire, a plant of the Rutaceae family, were collected in the cerrado biome, on the outskirts of Brasilia, Brazil in 2007. The plant was identified by Prof. José Elias de Paula, and a voucher specimen (UB 3768) was deposited in the Herbarium of the Institute of Biology, University of Brasilia.

Extraction and isolation

The dried and powdered leaves (3 kg) of S. odoratissima were subjected to repeated maceration with EtOAc (3 L, 3 × 24 h), and the combined solutions were concentrated to dryness under reduced pressure at 40 °C yielding a crude extract (30 g). A portion (21 g) of this extract was suspended in H₂O (100 mL) into a separating funnel and partitioned successively with cyclohexane, EtOAc, and n-butanol (v/v). The EtOAc soluble extract (12 g) was active against P. falciparum (inhibition > 75 % of the parasite at 10 µg/mL) and chromatographed through an SiO₂ gel open column (800 g, 7 × 65 cm) eluted with a cyclohexane/EtOAc/MeOH gradient (95 : 5 : 0, 2 L; 90 : 10 : 0, 1.5 L; 80 : 20 : 0, 1 L; 70 : 30 : 0, 1 L; 50 : 50 : 0, 1 L; 0 : 100 : 0, 1 L; 0 : 50 : 50, 1 L; 0 : 0 : 100, 1 L) and yielded 22 fractions (F1–F22) grouped according to their TLC behavior. All fractions were tested against P. falciparum and F6, F10, F12 were active with inhibition > 90 % at 10 µg/mL. F6 (450 mg) was purified by SiO₂ gel CC (45 g, 2 × 55 cm) eluted with cyclohexane/EtOAc gradient (90 : 10, 400 mL; 80 : 20, 200 mL; 70 : 30, 200 mL) to yield 10 subfractions. Purification of subfraction F6-5 (22 mg) by MPLC (Flashsmart cartridge, AIT, 2 g) eluted with cyclohexane/EtOAc (95 : 5) at a 1 mL/min constant flow rate gave compound 3 (4 mg). Fraction F10 (435 mg) was purified by SiO₂ gel CC (44 g, 2 × 55 cm) eluted with cyclohexane/EtOAc (95 : 5, 200 mL; 90 : 10, 200 mL; 80 : 20, 200 mL) and afforded β sitosterol (20 mg) and 9 subfractions. Subfraction F10–5 was subjected successively to SiO₂ gel CC (6 g, 1.5 × 22 cm) eluted with cyclohexane/EtOAc (90 : 10, 100 mL) and MPLC (Flashsmart cartridge, AIT, 2 g) eluted with cyclohexane/EtOAc (90 : 10, 60 mL, flow rate 1 mL/min) to give sesamin (4 mg). Fraction F12 (537 mg) was subjected to Sephadex LH-20 CC (200 g, 3 × 120 cm) eluted with MeOH (1 L). Subfraction F12–7 (8 mg) was purified by preparative TLC eluted with cyclohexane/EtOAc (99 : 1, 20 mL) to give 1 (3 mg) and 2 (2 mg).

Spiranthenone A (1): Viscous oil; [α]_D ²⁰ + 11 (c 0.19, CHCl₃); IR (CHCl₃) ν _max: 1774, 1685, 1740, 1458, 1215, 756 cm⁻¹; ¹H-NMR and ¹³C-NMR data in CDCl₃, see [Table 1]; HRESI-MS m/z: 513.3217 [M + H]⁺, C₃₁H₄₅O₆ (calcd.: 513.3216).

Spiranthenone B (2): Viscous yellow oil; [α]_D ²⁰ + 13 (c 0.17, CHCl₃); IR (CHCl₃) ν _max: 1732, 1662, 1543, 1446, 840 cm⁻¹; ¹H-NMR and ¹³C-NMR data in acetone-d ₆, see [Table 1]; HRESI-MS m/z: [M + H]⁺ 483.3485, C₃₁H₄₇O₄ (calcd.: 483.3474).

6α-Acetoxy-1β-hydroxyeudesm-4(15)-ene (3): Amorphous powder; [α]_D ²⁰ = − 24 (c 0.10, CHCl₃); IR (CHCl₃) ν _max: 3448, 1732, 1651, 1458, 1250, 1020 cm⁻¹; ¹H-NMR and ¹³C-NMR data in CD₃OD, see [Table 1]; HRESI-MS m/z: [M + H]⁺ 281.2118 C₁₇H₂₉O₃ (calcd.: 281.2109).

In vitro test against Plasmodium falciparum

It was based on the inhibition of [³H]-hypoxanthine uptake by P. falciparum cultured in human blood and was performed as previously described [5]. Chloroquine diphosphate (purity > 98 %; Sigma Aldrich®) was used as a positive control.

In vitro tests against Leishmania chagasi and Trypanosoma cruzi

Epimastigote forms of T. cruzi (Berenice strain, Universidade de Brasilia) were grown at 28 °C in liver infusion tryptose (LIT) medium supplemented with 5 % heat-inactivated fetal calf serum (FBS) and antibiotics. L. chagasi (MCER/BR/79/M6445, Instituto Evandro Chagas, Brasil) promastigotes were maintained in NNN, Schneider (Sigma) medium containing 10 % heat-inactivated FBS, at 22 °C. Assays were performed as previously described [5]. Benznidazole (purity > 99 %; Roche®) was used as the reference drug for T. cruzi and miltefosine (purity > 98 %; Sigma Aldrich®) for L. chagasi.

Cytotoxicity test

NIH-3T3 cells (Sigma Aldrich®) were seeded on 96-well plates at a density of 8 × 10³ cells in DMEM containing 10 % FBS overnight at 37 °C in 5 % CO₂. The medium was changed and incubated with or without different concentrations of samples at 37 °C in 5 % CO₂. After 24 h, the cell viability was determined by the MTT assay [6]. Assays were performed as previously described [5].

Results and Discussion

The active EtOAc-soluble material obtained after partition of the crude extract of the leaves of S. odoratissima was fractionated to give five compounds, two new tetraprenylated acylphloroglucinols (1–2) and an eudesmene-type sesquiterpene (3) ([Fig. 1]), along with sesamin [7] and β-sitosterol [8].

Compound 1 was isolated as a viscous oil optically active [α]_D ²⁰ + 11 (c 0.19, CHCl₃). In its HRESI-MS, the protonated molecular ion [M + H]⁺ was depicted at m/z 513.3217 in agreement with the molecular formula C₃₁H₄₄O₆ implying ten degrees of unsaturation. The IR spectrum exhibited bands at 1685 cm⁻¹ characteristic of an α,β unsaturated CO, 1774 and 1740 cm⁻¹ for CO and at 1458, 1215 cm⁻¹. The ¹H NMR spectrum ([Table 1]) displayed two strong singlets at δ 9.38 and 7.48, three olefinic protons at δ 6.25, 5.21, and 4.98, several aliphatic protons between δ 1.28 and 2.33, among which three were methyls. The ¹³C NMR spectrum exhibited four COs, one of them being conjugated, six methyls, five methylenes, five methines, and eight quaternary carbons. The ¹³C spectrum displayed the CO at δ 194.5, one methylene at δ 28.9, one methine at δ 151.5, one quaternary carbon at δ 141.1, and the shielded methyl group at δ 9.3 which had correlations in the HMBC consistent with an α,β-unsaturated aldehyde.

The COSY spectrum allowed determination of the partial structures a–d, which were linked by HMBC long-range connectivities to give the complete structure of 1 ([Fig. 2 a]). Two isoprenyl groups were thus identified and attached to C-1 and C-5. The correlations of the aldehyde proton at δ 9.38 with carbons at δ 9.3 (C-20), 141.0 (C-19), and 151.5 (C-18) defined the 3-al-3-methylbutenyl substituent. HMBC connectivities of H₂-6 to the quaternary carbons at δ 55.0 (C-5), 107.2 (C-9), and the CO at δ 208.1 (C-4) attached the third partial structure (c) at C-5. In addition, the proton at δ 7.48 was attributed to a hydroxyl due to absence of correlation in the HSQC spectrum. The presence of the hemiketalic C-9 was suggested by the downfield shift of the quaternary carbon at δ 107.2 and was confirmed by HMBC connectivities of the hydroxyl with C-9 and the two bridgehead carbons C-1 and C-5. Further interactions of methyls at δ 1.00 (CH₃-10) and 1.23 (CH₃-11) with C-8, C-7, and C-1 evidenced the presence of a gem-dimethyl and joined the substructure (c) to C-1, resulting in the formation of the first six-membered ring. The connectivity of protons H₂-27 with the downfield quaternary carbon at δ 98.2 corroborated that it was engaged in an ether linkage. An additional cross-peak was observed between H-27a and CO at δ 207.5 (C-2) while H-27b correlated with CO at δ 208.1 (C-4). Indeed, no HMBC correlation was observed between H-27b and C-2. This is in agreement with the linkage of the isoprenyl group (a) to this totally substituted six-membered ring at C-3.

HMBC correlation of H₂-13 with CO at δ 210.4 allowed identification of an isovaleroyl which was connected to C-1. Finally, the five-membered rings resulting from the oxygen bridge between C-3 and C-9 was suggested by the degrees of unsaturation required for the molecular formula. The planar structure of 1 was then established as a tricyclic tetraprenylated acylphloroglucinol derivative of type A [9].

The relative stereochemistry of 1 was determined from NOESY correlations. The cross-peaks observed between the hydroxyl OH-9 and both CH₃-10 and H-6b indicated that all these protons were axial. They also correlated with H-17a and H-22a which were thus on the same side. The NOE interaction of H-13a with CH₃-10 implied the acyl chain at C-1 to be located on the same α-face of the molecule. The weak ³ J coupling constant (3.5 Hz) of H-6a suggested that it was β-equatorial. Its correlation with H-7 and H-17b implies the prenyl chain at C-7 to be α-axial. Compound 1 has a cage-like structure and the ether bridge constraints the configuration at C-3 and C-9 and prevents any eventuality of a keto-enol equilibrium of the β-dicarbonyl system (C-2 and C-4). Similar results were previously reported for subellinone [10], 9-hydroxyhyperforin 9,3-hemiacetal [11], and its derivative hyperibone J [12], which share a tricyclic structure with a 9,3-hemiacetal. From the above evidence, the structure of this novel compound 1 was elucidated as 9-hydroxy-3,9-hemiacetal-8,8-dimethyl-3,5-di-(3-methyl-2-butenyl)-7-(3-al,3-methyl-2-butenyl)-1(2-methyl-butanoyl) bicyclo[3.3.1]nonane-2,4-dione and named spiranthenone A ([Fig. 1]).

Compound 2 was isolated as a viscous oil optically active [α]_D ²⁰ + 13 (c 0.17; CHCl₃). The molecular formula C₃₁H₄₆O₄ was deduced from the protonated ion [M + H]⁺ at m/z 483.3485 (calcd. for C₃₁H₄₇O₄: 483.3474), observed in its HRESI-TOF compatible with nine degrees of unsaturation. Its IR spectrum exhibited bands at 1732, 1662 cm⁻¹ assignable to saturated and unsaturated CO, and at 1543, 1446, and 840 cm⁻¹ characteristic of olefinic functionalities.

The ¹H NMR and ¹³C NMR spectra ([Table 1]) showed the presence of splitting signals in the 2/1 ratio which suggested 2 to be a mixture of two keto-enol tautomers 2A/2B. The presence of two highly deshielded singlets at δ 18.78 and 18.66 in the ration 2/1 comforted the above hypothesis. These signals were shifted downfield due to their involvement in strong intramolecular hydrogen bonds between a hydroxyl and ketone. Six ethylenic protons were depicted between δ 4.74 and 5.09. The protons at δ 3.06 for the major tautomer (2A) and δ 3.11 for the minor one (2B) were characteristic of a position α to a carbonyl. Several aliphatic protons were present between δ 1.65 and 2.77 whereas methyl groups were observed between δ 0.75 and 1.67. From the HSQC spectrum, a superposition of two protons at δ 4.95 was deduced, which correlated to the carbon at δ 123.5 (C-23) and assigned to the two tautomers.

The COSY spectrum revealed five fragments: (a–e) of which three belong to isoprenyl chains ([Fig. 2 b]). The downfield proton at δ 18.78 correlated with CO at δ 198.6 (C-2) and 205.5 (C-12), quaternary carbons at δ 117.3 (C-3) and 69.0 (C-1), and the methylene at δ 48.7 (C-13). The hydroxyl enolic proton was thus located at C-2, and its proximity with the CO at δ 205.5 together with the interactions of H₂-13 led the acyl side chain to be linked on C-3. The linkage of the isoprenyl chain at C-5 of the A-ring resulted from the connectivities of H₂-17 with carbons at δ 66.5 (C-5), 195.2 (C-4), and 43.6 (C-6). The correlation of protons at δ 2.60 and 2.71 (H₂-27) with the carbon at δ 207.6 (C-9), C-1, C-2, and C-8 allowed the closing of the A-ring by linking C-9 with both C-1 and C-5 forming then. Correlations of H-6 at δ 1.93 with C-5 and C-7, along with the connections of gem dimethyl protons at δ 0.81 (CH₃-10) and 1.20 (CH₃-11) with C-1, C-7, and C-8, led to the formation of the second six-membered ring with the isoprenyl chain at C-7. In this way, the major tautomer 2A was identified as 2-hydroxy-8,8-dimethyl-1,5,7-tri-(3methyl-2-butenyl)-3(2-methyl-butanoyl) bicyclo[3.3.1]non-3-ene-4,9-dione ([Fig. 1]).

For the minor tautomer, connectivities of the enolic OH proton at δ 18.66 with carbons at δ 199.7 (C-4), 116.8 (C-3), 61.0 (C-5) indicated that it is linked at C-4. Moreover, it also correlated with the CO at δ 205.5 (C-12) and the carbon at δ 48.7 (C-13) confirming the linkage of the acyl chain at C-3. Tautomer 2B corresponded to 4-hydroxy-8,8-dimethyl-1,5,7-tri-(3-methyl-2-butenyl)-3(2-methyl-butanoyl) bicyclo[3.3.1]non-3-en-2,9-dione, and 2 is a polyprenylated acylphloroglucinol of type B ([Fig. 1]).

The relative configuration of 2 was assigned from the NOE and coupling constant (J) data. The proton H-6a was in the β-equatorial position as indicated by the small coupling constant of 3.9 Hz. The observed NOE correlations between H-6 eq, H-23, and H-7 confirmed the β-orientation of H-7. As a consequence, the isoprenyl group at C-7 was α-axial. Furthermore, the isoprenyl side chains at C-1 and C-5 were α-equatorial due to the β-position of the C-9 carbonyl bridge. Spectral data for 2 were similar to those of hyperpapuanone, and differences were an ispoprenyl at C-5 and an isovaleroyl at C-3 instead of a methyl group and isobutanoyl moiety [13]. Compound 2 also differs from laxifloranone, a close derivative substituted at C-1 by a cinnamic acid moiety [14]. This new compound 2 was named spiranthenone B.

The HRESI-TOF of compound 3 exhibited a protonated molecular ion peak at m/z 281.1982 [M + H]⁺ in agreement with the formula C₁₇H₂₈O₃ with four degrees of unsaturation. This was confirmed by the ¹³C NMR data which revealed the presence of an exocyclic methylene and a CO group. Compound 3 was then bicyclic. The IR absorption bands at 3448 and 1732 cm⁻¹ suggested the presence of hydroxyl and carbonyl functionalities, whereas those at 1458, 1250, and 1020 cm⁻¹ were attributable to ethylenic groups. The ¹³C-NMR spectrum ([Table 1]) displayed 17 signals, which were assigned to 3 quaternary carbons, 5 methines, among which 2 were oxymethines, 5 methylenes, and 4 methyls. The ¹H NMR spectrum displayed methyl signals for an isopropyl at δ 0.85 (3H, d, J = 6.9 Hz) and 0.93 (3H, d, J = 6.9 Hz) and two methyls at δ 0.72 and δ 1.95. The protons at δ 4.53 and 4.77 correlating with the carbon at δ 107.5 on the HSQC spectrum were assigned to an exomethylene group. Protons at δ 3.36 and δ 5.06 were correlated with carbons at δ 79.7 and 72.5 and identified as oxymethines. All NMR spectral data for 3 ([Table 1]) were similar to those of voleneol from which it mainly differed by the presence of an acetyl group [15].

The relative configuration was established from NOE and coupling constants data. Proton H-1 was α-oriented according to its large coupling with H-2 β-axial (J = 11.6 Hz) indicating their trans-diaxial relationship and then the hydroxyl group was β-equatorial in agreement with NOESY data. H-1 correlated with H-5, H-2b, H-3b, and H-9b and was thus α-axial oriented. A NOE interaction was observed between H-5 and H-7 confirming the isopropyl to be β-equatorial. The coupling constants (t, 10.4 Hz) of H-6 with H-5 and H-7 indicated an axial orientation of H-6. It correlated with CH₃-14, CH₃-13, and H-15b, whereas CH₃-14 correlated with H-8a and H-9a. The A/B ring junction was trans because of the lack of NOE between CH₃-14 and H-5. Compound 3 has been previously synthesized from epoxygermacrene D without NMR data [16], and it is reported here for the first time from a natural source. Thus, the structure of 3 was established as 6α-acetoxy-1β,-hydroxyeudesm-4(15)-ene ([Fig. 1]).

The biosynthesis of PPAPs (polycyclic polyprenylated acylphloroglucinol) 1 and 2 involves a common precursor, but an opposite mechanism of cyclization [9]. A plausible pathway ([Fig. 3]) is the condensation of isovaleroyl Co-A with 3 malonyl-CoA units leading to a tetraketide, which is cyclized into an acylphloroglucinol (A). Its prenylation furnished the intermediate (B) which undergoes a nucleophilic attack of the geminal prenylated group to form a carbocation (C). The intramolecular condensation of C-5 with this carbocation provides the bicyclic tautomers 2A/2B. On the other hand, the condensation of C-1 with the carbocation (C) leads to the triketone E which is further converted by hydration to a tricyclic hemiketal and then the oxidation of the 7-prenyl group results in the formation of 1.

PPAPS are mostly reported from plants belonging to the Guttiferae of the genus Clusia, Garcinia, and Symphonia. Simple acylphloroglucinols such as sessiliflorene and sessiliflorol are found in the Rutaceae family [17]. They are also present in Myrtaceae and Cannabinaceae families [18]. We report here the first occurrence of PPAPs in Rutaceae.

The isolated compounds (purity > 95 %) were tested for their capacity to inhibit the in vitro growth of the chloroquine-resistant strain FcB1 of P. falciparum, T. cruzi, and L. chagasi and for cytotoxicity against NIH-3T3 cells; the selectivity index (SI; IC₅₀ on mammalian cells to the IC₅₀ value on the parasite ratio) was determined ([Table 2]). Sesamin and β-sitosterol exhibited comparable activity as the crude extract against P. falciparum and 1-3 were less active. On the other hand, 3 was the most selective (SI of 96) and active against T. cruzi and L. chagasi with SIs of 13.8 and 22.8, respectively. All the compounds displayed moderate activities, but they exhibited no cytotoxicity against the NIH-3T3 cells.

Acknowledgments

This research was supported by grant No. E07D401533BR of the ALβAN Programme (Europaid) for Lorena Albernaz. The authors gratefully acknowledge also the CNPq, CAPES, FAPDF, FINATEC.

Conflict of Interest

There are no conflicts of interest among all authors.

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Dr. Lengo Mambu

UMR 7245 CNRS-MNHN
Molécules de Communication et Adaptation des Micro-organismes
Muséum National d'Histoire Naturelle

CP 54, 57 rue Cuvier

75231 Paris Cedex 05

France

Phone: +33 1 40 79 56 07

Fax: +33 1 40 79 31 35

Email: mambu@mnhn.fr

References

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  Search in Google Scholar
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Dr. Lengo Mambu

UMR 7245 CNRS-MNHN
Molécules de Communication et Adaptation des Micro-organismes
Muséum National d'Histoire Naturelle

CP 54, 57 rue Cuvier

75231 Paris Cedex 05

France

Phone: +33 1 40 79 56 07

Fax: +33 1 40 79 31 35

Email: mambu@mnhn.fr