Introduction

Voacanga is a small genus of the Apocynaceae family consisting of 12 species. Species of this genus are distributed mainly in the tropical Africa and Malaysia, and have been reported to contain vobasine, eburnane, iboga, and aspidosperma type of monoterpene indole alkaloids [1]. Various activities have been reported for monoterpene indole alkaloids, such as cytotoxic activity [2,3,4], anti-melanogenesis [5], anti-plasmodial [6], and vasorelaxant activities [7]. In the search for new bioactive compounds from tropical plants [8,9,10,11,12,13,14,15], alkaloid constituents of V. grandifolia leaves were investigated and two new bisindole alkaloids, 12′-O-demethyl-vobtusine-5-lactam and isovobtusine-N-oxide (1 and 2, Fig. 1), were isolated together with vobtusine-N-oxide [16], and vobtusine-3-lactam [17]. The isolation and structure elucidation of 1 and 2 consisting of bis-aspidosperma-type skeleton, from the leaves of Voacanga grandifolia, and their antimalarial activities are reported herein.

Fig. 1
figure 1

Structures of isolated compounds (1 and 2) from Voacanga grandifolia

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Results and discussions

Compound 1 {[α]D24 − 117.6° (c 1.0, MeOH)} was isolated as an optically active, yellow amorphous solid. The molecular formula was determined by HRESIMS (m/z 719.3453 [M + H]+, ∆ + 0.8 mmu) as C42H46N4O7, and the IR absorption at 3340 cm−1 and 1715 cm−1 indicated the presence of hydroxy and carbonyl groups, respectively. Analysis of the 1H and 13C NMR data (Table 1) and the HSQC spectrum of 1 revealed the presence of six sp3 quaternary carbons, five sp3 methines, fourteen sp3 methylenes, one methyl, seven sp2 methines, and nine sp2 quaternary carbons. Among them, two sp3 methines (δC 63.6; δH 2.60, δC 64.9; δH 3.97) and four sp3 methylenes (δC 44.5; δH 2.85 and 4.16, δC 44.6; δH 3.13 and 4.82, δC 48.8; δH 2.29 and 2.84, and δC 52.0; δH 2.21 and 3.02) ascribed to those attached to a nitrogen atom.

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data of 1 and 2 in CDCl3
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The gross structure of 1 was elucidated by analysis of 2D NMR data including the 1H-1H COSY, TOCSY, HSQC, and HMBC spectra in CDCl3 (Fig. 2). The 1H–1H COSY spectrum revealed the connectivity of seven partial structures a (C-9′ ~ C-11′), b (C-5′ ~ C-6′), c (C-3′, C-14′ ~ C-15′), d (C-16′ ~ C-17′, C-22′), e (C-18′ ~ C-19′), f (C-18 ~ C-19), and g (C-9 ~ C-12) as shown in Fig. 2. These partial structures were classified into two units, a ~ e and f ~ g.

Fig. 2
figure 2

Selected 2D NMR correlations of 1

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The connectivity of partial structures b and c and C-21′ through a nitrogen atom was revealed by the HMBC correlations of H-5′b and H-21′ to C-3′ (δC 48.8). The HMBC correlations from H-18′a and H-19′a to C-15′ (δC 80.5), H-14′a to C-20′ (δC 44.0), and H-19′b to C-17′ (δC 32.6) and C-21′ (δC 63.6) established the connections among C-15′, C-17′, C-19′, and C-21′ through C-20'.

In addition, HMBC cross peaks of H-6′b to C-8′ (δC 134.9), H-9′ to C-21′, H-21′ to C-2′ (δC 93.5) and C-6′ (δC 31.2), and H-16′ to C-7′ (δC 55.9) revealed the connectivity between C-6′, C-16′, C-21′, and an indoline ring. Then, the HMBC correlations of H-23′b and OH-12′ to C-13′ (δC 138.2), OH-12′ to C-12′ (δC 139.8), and H-22′a and H-23′a to C-2′ suggested the presence of hydroxyl groups at C-2′ and C-12′ in the indoline ring. These NMR data were characteristic of an aspidosperma-type skeleton.

On the other hand, the similar aspidosperma-type skeleton with a methoxy carbonyl at C-16 and a double bond between C-2 and C-16 was suggested by the HMBC correlations in the units f and g as shown in Fig. 2. The connection of two indole alkaloids through C-14 was revealed by the HMBC correlations from H-23′a to C-14 (δC 38.1) and C-22′ (δC 35.2) and from H-23′b to C-3 (δC 44.5) and C-15 (δC 87.8). In particular, the HMBC correlations of H-3a, H2-6 and H-21 to C-5 (δC 172.6) confirmed the presence of a carbonyl at C-5. These data suggested the structure of 1 as 12′-O-demethyl-vobtusine-5-lactam.

The relative configuration of each monoterpene indole unit in 1 was assigned by NOESY correlations as shown in computer-generated 3D drawing (Fig. 3). In 1a (partial structures a ~ e), the NOESY correlations of H-21′/H-18′a and H-9′, and H-15′/H-18′b and H-17′b suggested that both C-21′ and C-15′ should be S* and C-7′ and C-20′ should be R*. The relative configuration of C-2′ (δC 93.5) was deduced to be R* based on the similarity with the chemical shift (δC 93.7) of vobtusine [17]. Stereochemistry of H-16′ was elucidated to be α-oriented by 3JH-16′/H-17′a (13.0 Hz) and 3JH-16′/H-22′b (13.4 Hz), and the NOESY correlations in 1a. In 1b (partial structures f ~ g), H-15 is β-oriented and C-20 should be R* by the NOESY correlations of H-21/H-18 and H-19a. Stereochemistry of a spiro carbon (C-14) in 1c should be assigned as R* by the NOESY correlations, suggesting that the diazaspiro [5.5] undecane ring took a chair-chair conformation, as shown in Fig. 3. Finally, the NOESY correlations and 1H-1H coupling constant values suggested the relative configuration of 1 to be, as shown in Fig. 1, the same as vobtusine-3-lactam.

Fig. 3
figure 3

Selected NOESY correlations of 1

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Compound 2 {[α]D24 − 123.6° (c 1.0, MeOH)} was isolated as an optically active, yellow amorphous solid. The molecular formula was determined by HRESIMS (m/z 735.3765 [M + H]+, ∆ 0.7 mmu) as C43H50N4O7, and the IR absorption at 3340 cm−1 and 1685 cm−1 indicated the presence of hydroxy and carbonyl groups, respectively. In addition, the 13C NMR data of 2 (Table 1) were similar to those of vobtusine-N-oxide and isovobtusine [16,17]. The chemical shift differences between 2 and vobtusine-N-oxide (i.e. C-3, C-14, C-15, C-18, C-16′, C-22′ and C-23′) suggested 2 to be the 14-epi derivative of vobtusine-N-oxide, isovobtusine-N-oxide, and the downfield shifts of signals ascribed to C-3′, C-5′ and C-21′ in 2 relative to isovobtusine further supported this conclusion.

Biological activity of 1 and 2

The antimalarial activity and cytotoxic activity of 1 and 2 were evaluated in vitro. Against P. falciparum 3D7 strain, 1 and 2 showed potent antimalarial activity [Table 2: half-maximal (50%) inhibitory concentration (IC50) = 5.1 and 3.3 μM, respectively]. Chloroquine was used as a positive control (IC50 = 0.026 μM). In contrast, the cytotoxic activity of both 1 and 2 was low [Table 2: half-maximal (50%) cytotoxic concentration (CC50) ≧ 50 µM] against the human cell line, HepG2 cells.

Table 2 Antimalarial and cytotoxic activities of 1 and 2 against Plasmodium falciparum 3D7
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Experimental section

General experimental procedures

Optical rotations were measured on a JASCO DIP-1000 polarimeter, UV spectra on a Shimadzu UVmini-1240 spectrophotometer, and IR spectra on a JASCO FT/IR-4100 spectrophotometer. High-resolution ESI MS were obtained on a JMS-T100LP (JEOL). 1H and 2D NMR spectra were measured on a 600 MHz spectrometer at 300 K, and 13C NMR spectra on a 150 MHz spectrometer. Residual solvent chemical shifts were used as internal standard, δH 7.26 and δC 77.0 for CHCl3. Standard pulse sequences were used for the 2D NMR experiments.

Material

The leaves of V. grandiflora were collected at the Purwodadi Botanical Garden in March 2008. The botanical identification was made by Ms. Sri Wuryanti, Purwodadi Botanical Garden. A voucher specimen has been deposited in the herbarium at Purwodadi Botanical Garden, Pasuruan, Indonesia.

Extraction and isolation

Finely powdered leaves of V. grandiflora (400 g) were extracted with MeOH (1L × 3) at room temperature. The combined extracts were evaporated under reduced pressure to give a residue (30 g). The extract was dissolved in 3% aqueous tartaric acid (pH 2) and then partitioned with EtOAc. The aqueous layer was treated with Na2CO3 to pH 9 and was partitioned successively by CHCl3 and n-BuOH. The CHCl3 soluble materials (3.9 g) was subjected to an LH-20 column (CHCl3/MeOH, 1:1) to obtain ten fractions.

Fraction 7 (360 mg) was fractionated by silica gel column chromatography (CHCl3/MeOH, 0:1 → 1:0), ODS column chromatography (MeOH/H2O) and RP-HPLC (Nacalai tesque Cholester, 10 × 250 mm; 24% MeCN in 0.1% aqueous HCO2H; flow rate 3.5 mL/min; UV detection at 254 nm) to obtain 1 (1.5 mg, 0.0004%), 2 (1.5 mg, 0.0004%), vobtusine-N-oxide (1.9 mg, 0.0005%) [16], and vobtusine-3-lactam (0.8 mg, 0.0002%) [17].

12′-demethyl-vobtusine-5-lactam (1): Yellow amorphous solid; [α] -117.6 (c 1.0, MeOH); IR (film) vmax 3340 and 1715 cm−1; UV/Vis λmax (MeOH) nm (log ε): 306 (3.57), 273 (3.84), 227 (4.41) and 206 (4.41) nm; ESIMS (pos.) m/z 719 (M + H)+; HRMS-ESI: m/z [M + H]+ calcd for C42H47N4O7: 719.3445; found 719.3453.

Isovobtusine-N-oxide (2): Yellow amorphous solid; [α] -123.6 (c 1.0, MeOH); IR (film) vmax 3370 and 1685 cm−1; UV/Vis λmax (MeOH) nm (log ε): 328 (4.22), 299 (4.16), 259 (3.93) and 220 (4.47) nm; ESIMS (pos.) m/z 735 (M + H)+; HRMS-ESI: m/z [M + H]+ calcd for C43H51N4O7: 735.3758; found 735.3765.

Parasite culture

P. falciparum laboratory strain 3D7 was obtained from Prof. Masatsugu Kimura (Osaka City University, Osaka, Japan). For the assessment of antimalarial activity of the compounds in vitro, the parasites were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 0.5 g/L l-glutamine, 5.96 g/L HEPES, 2 g/L sodium bicarbonate (NaHCO3), 50 mg/L hypoxanthine, 10 mg/L gentamicin, 10% heat-inactivated human serum, and red blood cells (RBCs) at a 3% hematocrit in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 37 °C as previously described [18]. Ring-form-infected RBCs were collected using the sorbitol synchronization technique [19]. Briefly, the cultured cells were collected by centrifugation at 840 g for 5 min at room temperature, suspended in a fivefold volume of 5% d-sorbitol (Nacalai Tesque, Kyoto, Japan) for 10 min at room temperature, and then they were washed twice with RPMI 1640 medium to remove the d-sorbitol. The utilization of blood samples of healthy Japanese volunteers for the parasite culture was approved by the institutional review committee of the Research Institute for Microbial Diseases (RIMD), Osaka University (approval number: 22–3).

Antimalarial activity

Ring-form-synchronized parasites were cultured with compounds 1 and 2 at sequentially decreasing concentrations (50, 15, 5, 1.5, 0.5, 0.15, 0.05, and 0.015 µM) for 48 h for the flow cytometric analysis using an automated hematology analyzer, XN-30. The XN-30 analyzer was equipped with a prototype algorithm for cultured falciparum parasites [prototype; software version: 01–03, (build 16)] and used specific reagents (CELLPACK DCL, SULFOLYSER, Lysercell M, and Fluorocell M) (Sysmex, Kobe, Japan) [20,21]. Approximately 100 µL of the culture suspension diluted with 100 µL phosphate-buffered saline was added to a BD Microtainer MAP Microtube for Automated Process K2 EDTA 1.0 mg tube (Becton Dickinson and Co., Franklin Lakes, NJ, USA) and loaded onto the XN-30 analyzer with an auto-sampler as described in the instrument manual (Sysmex). The parasitemia shown as MI-RBC% was automatically reported [20]. Then 0.5% DMSO alone or containing 5 µM artemisinin used as the negative and positive controls, respectively. The growth inhibition (GI) rate was calculated from the MI-RBC% according to the following equation:

$$ {\text{GI}}\left( \% \right) = 100 - {{\left( {{\text{test}}\,{\text{sample}} - {\text{positive}}\,{\text{control}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{test}}\,{\text{sample}} - {\text{positive}}\,{\text{control}}} \right)} {\left( {{\text{negative}}\,{\text{control}} - {\text{positive}}\,{\text{control}}} \right)}}} \right. \kern-\nulldelimiterspace} {\left( {{\text{negative}}\,{\text{control}} - {\text{positive}}\,{\text{control}}} \right)}} \times 100 $$

The IC50 was calculated from GI (%) using GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, CA, USA) [22].

Cytotoxic activity

HepG2 (JCRB1054) cell line was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium [DMEM (1.0 g/L glucose) with l-glutamine and sodium pyruvate; Nacalai Tesque, Kyoto, Japan] supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY, USA) in a humidified incubator with 5% CO2 at 37 °C. For the assay, the cells (5 × 103/well) were seeded in a 96-well plate. 1 and 2 at gradually decreasing concentrations (50, 15, 5, 1.5, 0.5, 0.15, 0.05, and 0.015 µM) were added to the cell culture after 24 h and the cells were subsequently cultured for 48 h. Cell viability was measured using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 10 µL CCK-8 reagent was added to each well containing culture medium and incubated for 2 h under standard culture conditions. The absorbance of the sample was measured at 450 nm using a PowerWave HT microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). The cell viability was expressed as a percentage of the absorbance of the untreated control cells after subtracting the appropriate background intensity. The CC50 was calculated from the cell viability using GraphPad Prism version 5.0.