Isolation and Antimalarial Activity of Alkaloids from Pseudoxandra cuspidata

• Abstract
• Introduction
• Materials and Methods
• Instrumentation
• Plant material
• Extraction and isolation of compounds
• Assays on Plasmodium falciparum in vitro
• Cytotoxicity evaluation
• 1-Aza-7,8,9,10-tetramethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (1)
• 1S,1′R-Rodiasine (2)
• Results and Discussion
• Acknowledgements
• References

Abstract

A novel and very unusual azaanthracene alkaloid, 1-aza-7,8,9,10-tetramethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (1) and a new diastereoisomer of the bis-benzylisoquinoline alkaloid rodiasine, 1S,1′R-rodiasine (2), as well as the alkaloids O-methylpunjabine (3) and O-methylmoschatoline (4) have been isolated from Pseudoxandra cuspidata bark, used in French Guiana as an antimalarial. Their structures were elucidated by spectroscopic analyses, especially 2D-NMR techniques (ADEQUATE and NOESY). We found that the antimalarial activity of this bark was mostly due to bis-benzylisoquinoline 1S,1′R-rodiasine (2) (IC₅₀= 1 μM) also displaying a low cytotoxicity.

Introduction

Malaria remains one of the most important diseases in tropical developing countries. Over 3 billion people live under the threat of malaria and over a million are killed every year - mostly children [1]. Despite international commitment to control this plague and the effort put into research, reality for many people is much as it was years ago: patients still die because they are unable to get access to treatment for economic reasons and, often, drug effectiveness is impaired due to parasite resistance. In this context, safe, affordable and effective new drugs against resistant strains of Plasmodium are desperately needed. Discovery of new antimalarial drugs is partly based on natural product analysis from traditional medicines, which may lead to new compounds, such as, for example, artemisinin in Artemisia annua [2]. Many plant preparations are used all over the world as antimalarials or febrifuges, but have been poorly studied.

In 2002, an ethnopharmacological study, centered on local antimalarial treatments, was performed in French Guiana. Among the remedies used by the population, the Pseudoxandra cuspidata Maas (Annonaceae) bark of the tree, in the form of a decoction, was mentioned as a good antimalarial. This traditional preparation was evaluated in vitro for blood schizonticidal activity on Plasmodium falciparum (concentration inhibiting 50 % of parasite growth: IC₅₀ = 7 mg/mL) and in vivo against Plasmodium yoelii in rodents [3].

P. cuspidata bark was chosen for bio-assay-guided fractionation because of its good antimalarial potential and because no previous work had been undertaken on this particular species. We report here the antimalarial bioassay-guided fractionation of P. cuspidata bark, leading to the isolation and identification of a bis-benzylisoquinoline (2) as an active principle. Three other alkaloids were also isolated and evaluated (see Fig. [1]), particularly a novel and unusual 1-aza-7,8,9,10-tetramethoxy-4-methyl-2-oxo-1,2-dihydroanthracene alkaloid (1).

Materials and Methods

Instrumentation

Optical rotation was measured on a Perkin-Elmer 241 polarimeter with a sodium lamp (λ = 589 nm) in a 10 cm microcell. IR spectroscopy (in KBr) was performed on a Perkin-Elmer Paragon 1000 FT-IR spectrophotometer. NMR spectra were recorded on a Bruker Avance II 500 spectrometer equipped with a TBI z-gradient 5 mm probe. 1D (¹H, ¹³C) and 2D (COSY, HSQC, HMBC, NOESY) NMR experiments were performed at 298 K using standard pulse sequences. A 2D echo-antiecho ADEQUATE spectrum (¹³C DQ, ¹H) was acquired on a Bruker Avance II 600 spectrometer equipped with a TXI cryogenic cooled probe. 100 % deuterated chloroform (Eurisotop; Gif-sur-Yvette, France) was used as solvent. Chemical shifts (δ) are given in ppm and coupling constants (J) are reported in Hz. ESI-MS (positive ion mode) spectra, 3.5 kV (MeOH-CH₃CN 1 : 1), were recorded on a Perkin-Elmer Sciex API 365 mass spectrometer. HR-ESI-MS spectra (positive ion mode) were recorded on a Q-ToF Ultima (Waters; Milford, MA, USA) apparatus. Column chromatography: silica gel 60 SDS 70 - 200 μm (SDS; Peypin, France). Medium-pressure column chromatography: silica gel 60 SDS 6 - 35 μm (SDS). Reversed-phase chromatography: Mega Bond Elut C₁₈ cartridge (Varian Chromatography; Walnut Creek, California, USA). Preparative and analytic HPLC were performed using X-terra RP18 columns (Varian Chromatography): 10 μm 1.9 × 25 cm i. d. and 5 μm 0.46 × 25 cm i. d., respectively, with a UV DAD Hitachi L 7455 as detector. Fractionations were monitored by TLC (silica gel 60 F-254; Merck; Darmstadt, Germany, treated with sodium acetate) with visualization under UV (254 and 365 nm) and Dragendorf reagent. All solvents were spectral grade or distilled from glass prior to use.

Plant material

Pseudoxandra cuspidata Maas (Annonaceae) was collected in French Guiana, in the vicinity of Saint Georges de l’Oyapock (51°48’W 3°53’N) in November 2002. The herbarium voucher (GB2983) was deposited in the French Guiana Herbarium (CAY) and determined by specialists. The trunk bark was dried in an air drier in the shade. Traditionally, a decoction of the bark of this species is used among the Palikour Amerindians to treat fever, malaria and diarrhoea.

Extraction and isolation of compounds

Ground bark from P. cuspidata (1.2 kg) was first defatted with petroleum ether (5 L × 2) at room tempertaure. After removal of petroleum ether under reduced pressure, the dried powder obtained was impregnated with an ammonia solution and percolated with Et₂O (15 L). After removal of Et₂O, 35 g of dry extract was obtained. This extract was subjected to column chromatography on silica gel (70 - 200 μm, 10 × 45 cm), treated with sodium acetate (20 : 1, SiOH-NaOAc), and eluted with hexane-CH₂Cl₂-MeOH [from hexane-CH₂Cl₂ 1 : 1 to CH₂Cl₂-MeOH 1 : 1 (20 L)] to give five fractions, A to E. After biological evaluation, the active fraction C (6 g) was chromatographed on a silica gel medium pressure column (6 - 35 μm, 4.5 × 20 cm) with a gradient of CH₂Cl₂ (100 %) to CH₂Cl₂-MeOH 1 : 1 (15 L). Three fractions C₁, C₂, C₃ were obtained. Fraction C₁ (1.57 g) was chromatographed under medium pressure as previously described, and a final purification was performed by crystallization in CH₂Cl₂-MeOH-H₂O 1 : 4:0.5 (50 mg in 1 mL). Compound 3 was obtained as white needles (45 mg). Compounds 1 (16 mg), 2 (12 mg) and 4 (13 mg) were obtained from fraction A by three successive medium pressure chromatographies on silica gel treated with sodium acetate (6 - 35 μm, 1.5 × 20 cm) and final isocratic preparative RP18-HPLC (Varian Chromatograph) was done using CH₃CN-NaOAc (pH = 9) 25 : 75 for 1 (t_R = 30 min), 58 : 42 for 2 (t_R = 32 min) and 30 : 70 for 4 (t_R = 40 min) at 10 mL/min.

Assays on Plasmodium falciparum in vitro

The FcB1-Columbia and FcM29-Cameroon strains of P. falciparum, both being chloroquine-resistant [IC₅₀ for chloroquine (Sigma; Saint Quentin Fallavier, France): 186 nM and 540 nM, respectively] were cultured continuously according to Trager and Jensen et al. [4], with modifications described by Van Huyssen and Rieckmann et al. [5]. The IC₅₀ values for chloroquine were checked every 2 months, and we observed no significant variations. The parasites were maintained in vitro in human red blood cells (O±; EFS; Toulouse, France), diluted to 1 % hematocrit in RPMI 1640 medium (Cambrex; Emerainville, France) supplemented with 25 mM Hepes and 30 mM NaHCO₃ (Sigma) and complemented with 5 % human AB+ serum (EFS).

Parasite cultures were synchronized by D-sorbitol lysis (5 % of D-sorbitol in sterile water; D-sorbitol; Merck) as reported by Lambros and Vanderberg [6]. The antimalarial activities of plant extracts and purified compounds were evaluated by the radioactive micromethod described by Desjardins et al. [7] with the modifications reported by Benoit et al. [8]. Extract testing was performed in triplicate in 96-well culture plates (TPP) with cultures mostly at ring stages (synchronisation interval, 16 h) at 0.5 - 1 % parasitemia (hematocrit, 1 %). Parasite culture was incubated with each extract for 48 h. Parasite growth was estimated by [³ H]-hypoxanthine (Perkin-Elmer; Courtaboeuf, France) incorporation, [³ H]-hypoxanthine being added to the plates 24 h before freezing. After the 48 h incubation, plates were frozen-defrosted and each well was harvested on a glass fiber filter. Incorporated [³ H]-hypoxanthine was then determined with a beta-counter (1450-Microbeta Trilux; Wallac-Perkin Elmer). IC₅₀ values were determined by linear least square regression analysis.

The control parasite cultures free from any extracts were referred to as 100 % growth. IC₅₀ values were determined graphically in concentration versus percent inhibition curves. Chloroquine diphosphate (Sigma) was used as positive control.

Cytotoxicity evaluation

Cytotoxicity of the pure compounds was estimated on human breast cancer cells (MCF7). The cells were cultured in the same conditions as those used for P. falciparum, except for the 5 % human serum, which was replaced by 5 % fetal calf serum (Cambrex). For the determination of pure compound cytotoxicity, cells were distributed in 96-well plates at 2 × 10⁴ cells/well in 100 μL, then 100 μL of culture medium containing extracts at various concentrations were added. Cell growth was estimated by [³ H]-hypoxanthine incorporation after 24 and 72 h incubation exactly as for the P. falciparum assay. The [³ H]-hypoxanthine incorporation in the presence of extracts or pure compounds was compared with that of control cultures without extract (positive control being doxorubicin (Sigma)) [9].

1-Aza-7,8,9,10-tetramethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (1)

Yellow amorphous powder; R_f: 0.38, silica gel 60 F₂₅₄ CHCl₃/MeOH/Et₂NH (95 : 4:1); [α]_D ²⁵: + 3° (c 1.7, CHCl₃); IR (KBr): ν_max = 1657, 3400 cm^-1; UV (CHCl₃): λ_max (log ε) = 254 (5.13), 287 (5.18), 296 (5.16), 351 (4.25), 362 (4.30) nm; ¹H-NMR (CDCl₃, 500 MHz): δ = 2.78 (3H, s, H-11), 3.92 (3H, s, H-14), 3.96 (3H, s, H-12), 4.06 (3H, s, H-13), 6.43 (1H, s, H-3), 7.31 (1H, d, J = 9.5 Hz, H-6), 8.01 (1H, d, J = 9,5 Hz, H-5), 9.17 (1H, s, H-1); ¹³C-NMR (CDCl₃, 125 MHz): δ = 23.2 (C-11), 56.6 (C-13), 62.1 (C-14), 62.7 (C-15), 64.1 (C-12), 112.4 (C-4a), 113.4 (C-6), 120.7 (C-5), 121.3 (C-10a), 122.5 (C-3), 124.3 (C-8a), 129.7 (C-9a), 134.5 (C-9), 141.2 (C-8), 148.5 (C-4), 151.3 (C-7), 151.7 (C-10), 161.9 (C-2); HR-ESI-MS (positive ion mode): m/z = 330.1214 [M + H]⁺ (calcd. for C₁₈H₂₀NO₅: 330.1341).

1S,1′R-Rodiasine (2)

White needles, (CHCl₃/MeOH, 1 : 3, v/v); m. p. 203 - 208 °C; R_f: 0.75, silica gel 60 F₂₅₄ CHCl₃/MeOH/Et₂NH (95 : 4:1); [α]_D ²⁵: + 74° (c 1.7, CHCl₃); UV (MeOH): λ_max (log ε) = 204 (4.5), 212 (4.2), 233 (4.1), 284 (4.1), 284 (4.0) nm; IR (KBr): ν_max = 1624, 1648, 1686 cm^-1; ¹H-NMR (CDCl₃, 500 MHz): δ = 2.35 (3H, s, NMe-2), 2.49 (2H, m, H-4), 2.65 (3H, s, NMe-2′), 2.86 - 2.96 (2H, dd, J = 14.0, 8.0 Hz, H-α), 2.97 (1H, m, H-α′s), 2.56 - 3.05 (2H, m, H-4), 2.56 - 3.05 (2H,  m, H-4′), 2.62 - 3.31 (2H,  m, H-3′), 3.09 (1H, m, H-α′r), 2.93 - 3.47(2H, m, H-3), 3.32 (3H, s, OMe-6′), 3.50 (3H, s, OMe-7), 3.69 (1H, bdd, J = 3.9, 4.1 Hz, H-1′), 3.80 (3H, s, OMe-12), 3.83 (3H, s, OMe-6), 4.03 (1H, bd, J = 7.5, <1 Hz, H-1), 6.40 (2H, bs, H-5 and H-5′), 6.85 (1H,  d, J = 8.1 Hz, H-13′),  6.87 (1H,  d, J = 8.1 Hz, H-13), 7.07 (1H,  bs, H-8′), 7.27 (1H,  dd, J = 8.1, 2.2 Hz, H-14), 7.28 (1H, d, J = 2.1 Hz, H-10); ¹³C-NMR (CDCl₃, 125 MHz): δ = 22.7 (C-4), 27.6 (C-4′), 37.6 (C-α′), 42.2 (C-15), 43.5 (C-15′), 41.5 (C-α), 44.6 (C-3), 49.3 (C-3′), 55.6 (OMe-6′), 56.0 (OMe-6), 56.4 (OMe-12), 60.4 (OMe-7), 62.8 (C-1), 65.1 (C-1′), 106.6 (C-5), 110.7 (C-13), 113.4 (C-5′), 117.0 (C-5), 117.9 (C-11′), 118.8 (C-8′), 124.9 (C-8a), 125.5 (C-4’a), 127.8 (C-8′a), 128.2 (C-4a), 129.5 (C-14), 130.7 (C-14′, C-10′), 131.2 (C-9′), 135.4 (C-10), 137.3 (C-9, C-11), 139.1 (C-7), 143.7 (C-7’′), 147.7 (C-6′), 149.3 (C-8), 151.5 (C-6), 151.8 (C-12′), 153.16 (C-12); HR-ESI-MS (positive ion mode): m/z = 623.3129 [M + H]⁺ (calcd. for C₃₈H₄₃N₂O₆: 623.3121).

The physicochemical data of compounds 3 and 4 are presented in the Supporting Information.

Results and Discussion

Compound 1 (Fig. [1]) was found to possess a molecular formula of C₁₈H₁₉NO₅ as derived from its ESI-Q-TOF-HR-MS at m/z = 330.1214 [M + H]⁺ (calcd. 330.1341).

The CDCl₃ 500 MHz ¹H-NMR spectrum (Table 1S in Supporting Information) of 1 showed the presence of a methyl group (δ = 2.78, s) linked to an sp ² carbon, an olefinic proton as a broad singlet (δ = 6.43) due to long-range coupling with the methyl group, four methoxy singlets (δ = 3.92, 3.95, 3.96 and 4.04) and two vicinal aromatic protons (δ = 7.31 and δ = 8.01, J = 9.5 Hz). The ¹³C-NMR spectrum (Table 1S in Supporting Information) confirmed the previous deductions by the presence of 18 carbons including one methyl and four methoxy groups (δ = 23.2 and 56.6 to 64.1, respectively), ten quaternary carbons including a conjugated carbonyl (δ = 161.9 ppm) and three olefinic carbons (δ = 113.4, 120.7 and 122.5). Except for the methyl and methoxy groups, all ¹H and ¹³C-NMR signals (14 carbons) resonated in the aromatic region, thus suggesting an anthracene skeleton for 1. This was confirmed by UV bands (254, 287, 296, 351 and 362 nm) characteristic of aromatic conjugated rings. The conjugated carbonyl (δ = 161.9) observed in the ¹³C-NMR spectrum was identified as a lactam group by the interpretation of the IR and ¹H-NMR data that showed a low frequency carbonyl band at 1655 cm^-1 and a broad singlet at 9.17 ppm respectively, and corresponding to a non-exchangeable proton linked to a heteroatom. The positions of the four methoxy groups were determined unambiguously from HMBC and NOESY NMR experiments (see Table [1] S in Supporting Information and Fig. [2]). Total ¹³C-NMR assignments were not so clear and left some ambiguities because of long-range couplings (up to four bonds as illustrated in Table [1] S in Supporting Information for H-5) observed in the HMBC spectrum due to the high level of unsaturation and the presence of numerous quaternary carbons in the structure of 1. So, an ADEQUATE NMR spectrum (see Supporting Information) was recorded. The correlations observed in a 2D-¹H-¹³C ADEQUATE NMR spectrum revealed long-range coupling (two bonds) from a proton to its vicinal carbons. This spectrum was very useful to unambiguously assign all NMR data. Key correlations for NOESY and ADEQUATE experiments are illustrated in Fig. [2]. Thus, the structure of this new azaanthracene alkaloid was elucidated as 1-aza-7,8,9,10-tetramethoxy-4-methyl-2-oxo-1,2-dihydroanthracene. To our knowledge, compound 1 is the fourth representative of this kind of azaanthracene alkaloid known, to date, only in Annonaceae thus broadening this as yet restricted group of alkaloids. Indeed, 9,10-dimethoxyazaanthracene was described to be isolated from Polyalthia suberosa [10], [11] and a mixture of two trimethoxy derivatives isolated from Annona ambotay [12] and Annona dioica [13]. We confirm here the chemotaxonomic interest of this type of compound in the chemosystematics of Annonaceae. Moreover, it is worth mentioning that Tadic et al., in 1986, proposed a biosynthetic pathway, which leads to 1-azaanthraquinones. They suggested that these skeletons are oxo-aporphine-derived (see [14] for details) requiring the reduction of the quinone and the O-methylation of the corresponding hydroquinone giving rise to 1-azaanthracene alkaloids [14].

Compound 2 (Fig. [1]) was found to possess a molecular formula of C₃₈H₄₂N₂O₆ as derived from its ESI-Q-TOF-HR-MS at m/z = 623.3129 [M + H]⁺ (calcd. 623.3121). The CDCl₃ 500 MHz ¹H-NMR spectrum (Table 2S in Supporting Information) recorded at room temperature demonstrates the presence of a bis-benzylisoquinoline structure as described before by Jossang et al. [15]. ¹H- and ¹³C-NMR resonances were assigned by analysis of 2D ¹H-¹H COSY, NOESY, [16], [17] and 2D heteronuclear HMQC [18] and HMBC [19] spectra. This compound is described by two spin systems of three aromatic protons (= 6.85 - 7.65), two spin systems of three aliphatic protons (= 2.51 - 3.01), two spin systems of four aliphatic protons (= 2.3 - 3.5), two singlets of N-methyl groups (= 2.35 - 2.65) and four singlets of O-methyl groups (= 3.32 - 3.87) and finally three isolated aromatic protons (= 6.40 - 7.06). The HSQC spectrum is quite helpful to distinguish between vicinal and geminal proton signals in the CH₂CH₂ parts of the tetrahydroisoquinoline ring. Analysis of all spectroscopic data and comparison with values published for bis-benzylisoquinoline alkaloids led us to the identification of compound 2 as rodiasine. The J-based configuration analyses could be successfully applied to our molecule to determine the absolute stereochemistry at C-1 and C-1′. The dihedral angles CH1-CHs or CH1-CHr and CH1’-CH‘s or CH1’-CH‘r were estimated by a Karplus-type relationship [20], [21] from the vicinal proton coupling constant determined on the ¹H-¹H phase-sensitive COSY [22], [23] spectra: 8 Hz for ³ J _HH corresponds to a vicinal HH dihedral angle of 25 - 40° or 140 - 155°; 10 Hz to 20 - 35° or 145 - 155°; <1 Hz to 65 - 115°. From these data and according to previous products described by Janseng et al., and the optical rotation ([α]_D ²⁵: + 74°), we were able to characterize the 1S,1’R configured 16-membered ring of biphenylic bis-benzylisoquinoline alkaloids that generate two conformers at 25 °C in CDCl₃ (one of which is highly populated, more than 90 %). In view of establishing structure-activity relationships, knowledge of the 3D structure and conformational characteristics are of great interest. It is planned to study the exchange system of 1S,1′R-rodiasine by dynamic NMR spectroscopy.

In addition to compounds 1 and 2, the alkaloids O-methylpunjabine (3) [24] and O-methylmoschatoline (4) were identified by comparison of spectroscopic data with values reported in the literature [25], [26]. These compounds are here described for the first time from Pseudoxandra cuspidata bark.

Because P. cuspidata is a traditional antimalarial plant, and because the antiplasmodial activity of its crude extracts was confirmed in vitro [3], the antiplasmodial activity and cytotoxicity of the compounds purified from the plant were evaluated in vitro (Table [1]). Among the four compounds described, the most active (mean IC₅₀ on FcB1 strain 0.71 μg/ml i. e., 1.14 μM) and least toxic was rodiasine (2). The other compounds were less active or were inactive against P. falciparum. When compared to other bis-benzylisoquinolines, the toxicity of 2 was rather low [9], [27] while antiplasmodial activity was high. However, 1S,1′R-rodiasine as a major component of the extract of Pseudoxandra cuspidata, could be responsible for the known antimalarial activity of the plant stressing the antimalarial interest of this family [28].

Acknowledgements

The authors are grateful to Dr. Véréna Poinsot from Laboratoire des IMRCP (UMR 5623 UPS/CNRS), Toulouse, for high resolution mass spectrometric analyzes, Dr. F. Benoît-Vical from Service de Parasitologie-Mycologie, CHU Rangueil, Toulouse for radioactivity measurements; Eliane Pélissou from UMR 152, for technical assistance in biological evaluations, Antonio Narciso, for sharing his knowledge and help in the plant collection and Pr. Paul Maas for scientific determination of the plant. Finally, many thanks to Dr. Peter Winterton for his kind English revision.

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• 28 Steele J C, Simmonds M S, Veitch N C, Warhust D C. Evaluation of the anti-plasmodial activity of bis-benzylisoquinoline alkaloids from Abuta grandifolia .  Planta Med. 1999;  65 413-6
  Thieme ConnectPubMedSearch in Google Scholar

Dr. Nicolas Fabre

Laboratoire Pharmacochimie des Substances Naturelles et Pharmacophores Redox

UMR 152 IRD

Université Toulouse 3 Paul Sabatier

Faculté des Sciences Pharmaceutiques

31062 Toulouse Cedex 9

France

Phone: +33-5-62-25-68-48

Fax: +33-5-61-55-43-30

Email: nfabre@cict.fr

References

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  Thieme ConnectPubMedSearch in Google Scholar

Dr. Nicolas Fabre

Laboratoire Pharmacochimie des Substances Naturelles et Pharmacophores Redox

UMR 152 IRD

Université Toulouse 3 Paul Sabatier

Faculté des Sciences Pharmaceutiques

31062 Toulouse Cedex 9

France

Phone: +33-5-62-25-68-48

Fax: +33-5-61-55-43-30

Email: nfabre@cict.fr

• Supplementary Material