Introduction

The family Apocynaceae have been known to produce various type of alkaloids with medicinal properties [1,2,3]. In the course of our continuing search for bioactive natural products from tropical plants, we have reported alkaloids from the Apocynaceae family and their bioactivity, including antimalarial activity [4,5,6,7,8,9,10,11,12,13,14,15,16]. Tabernaemontana is a large genus which belongs to the Apocynaceae family and is well known for its alkaloid content. This genus, comprising approximately 100 species, is distributed in the tropical and some subtropical regions [17,18,19]. In Borneo, Indonesia, the exudate from the bark of Tabernaemontana macrocarpa Jack has been used traditionally to cure dental disorders, herpes, and eczema. Phytochemical analysis of the stem of this plant revealed the presence of alkaloids, flavonoids, terpenoids, and tannins [20]. In this paper, we reported the isolation and structure elucidation of two new monoterpene indole alkaloids (1 and 2) from the bark of T. macrocarpa Jack, together with five known alkaloid compounds and their antimalarial activities.

Results and discussions

Structure elucidation of 1 and 2

Compound 1 was obtained as an optically active brownish amorphous solid, \( \left[ \alpha \right]_{\text{D}}^{ 2 5} \) −45 (c 1.0, MeOH). The IR spectrum showed two important absorptions at 3382 and 1678 cm−1 for hydroxyl and carbonyl groups, respectively, while the UV absorption bands at λmax 225 and 271 nm indicated an indole chromophore. The electrospray ionisation mass spectrometry (ESIMS, pos.) of 1 showed a molecular ion peak at m/z 397, and the molecular formula of 1 was established as C23H29N2O4 from high-resolution ESIMS (HRESIMS). Analysis of the 1H NMR data (Table 1) suggested the presence of one substituted indole moiety from three aromatic resonances at δH 6.77 (1H, d, J = 8.0 Hz), 7.02 (1H, t, J = 8.0 Hz), and 7.11 (1H, d, J = 8.0 Hz), three N-methyl or methoxy (δH 3.97, 3.19 and, 3.94), and one olefinic proton (δH 5.50 1H, q, J = 6.0 Hz). The 13C NMR data revealed 23 resonances, comprising 7 sp2 quarternary carbons, 4 sp2 methines, 4 methyls, 4 sp3 methylenes, 3 sp3 methines, and 1 sp3 quarternary carbon (Fig. 1).

Table 1 1H and 13C NMR data of 1 and 2 in CD3OD
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Fig. 1
figure 1

Structures of 1–7

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The structure of 1 was deduced from extensive analysis of the two-dimensional (2D) NMR data, including the 1H–1H correlation spectroscopy (COSY), heteronuclear single–quantum correlation (HSQC), and heteronuclear multiple–bond correlation (HMBC). In particular, the HMBC cross-peaks of H2-17 (δH 3.72 and δH 3.58) to C-22 (Fig. 2) provides additional support for the presence of a carboxyl moiety at C-22. Furthermore, the HMBC correlations of methyl signals at δH 3.94 to C-12, δH 3.97 to C-2 and C-13, and δH 3.19 to C-3, C-5, and C-21 revealed the presence of a methoxy group at C-12 and two N-methyl groups. Moreover, an ethylidene side-chain at C-20 was confirmed by an HMBC cross-peak from H3-18 (δH 1.72) to C-20, and H-19 (δH 5.50) to C-15 and C-21 (Fig. 2).

Fig. 2
figure 2

Selected 2D NMR correlations for 1

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The relative configuration of 1 was elucidated by nuclear Overhauser effect spectroscopy (NOESY) correlation to be similar to that of 12-methoxy-4-methylvoachalotine (MMV) (3) [21]. First, H-3 and H-5/N4-CH3 were assigned to be α-axially oriented from the NOESY correlations of H-3 and H-5/N4-CH3 while CH2-17 were deduced to possess β-orientation from the NOESY correlations of H-17a/H-14a. Furthermore, the α configuration of H-15 and the E configuration of the ethylidene group were confirmed by the NOE correlation of H-15 to H3-18 and H-14b (Fig. 3).

Fig. 3
figure 3

Selected NOESY correlations for 1

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Further analysis of 1H and 13C NMR showed that 1 is very similar to MMV, except for the signal of the methyl ester at C-22 in MMV which was not observed in 1. Thus, 1 was assumed to be a 22-demethyl derivative of MMV. Finally, stereochemistry of 1 was confirmed by a methyl esterification reaction of 1 using trimethylsilyldiazomethane (TMSCHN2) to form a product with identical spectroscopic data to that of MMV.

Compound 2 was isolated as an optically active brownish amorphous solid, \( \left[ \alpha \right]_{\text{D}}^{ 2 5} \) −5 (c 1.0, MeOH). The UV spectrum showed two absorption bands at λmax 225 and 280 nm, characteristic of an indole chromophore. The IR absorptions bands at 3383 and 1685 cm−1 resulted from the hydroxyl and carbonyl groups, respectively. The ESIMS spectrum displayed a molecular ion peak at m/z 367, and the molecular formula of 2 was established as C22H27N2O3 from HRESIMS.

The NMR spectra of 2 and 1 exhibited similar resonances, differing by the presence of a doublet signal of methine (δH 2.48) in 2 instead of a methyl alcohol signal at δH 3.58 and δH 3.72 in 1 linkage at C-22, indicating the structure of 2 as a 22-deethyl fuchsiaefoline [22] (Fig. 4). The relative configuration of 2 was assigned by analyses of the 1H–1H coupling constant data and the NOESY correlations similar to 1. In particular, the observed H-16/H-6b NOE indicated that the configuration of C-16 is R*.

Fig. 4
figure 4

Selected 2D NMR correlations for 2

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Antimalarial activity

The two new compounds 1 and 2, together with five known compounds; 12-methoxy-4-methylvoachalotine (3), 16-demethoxycarbonylvoacamine (4) [23], isositsirikine (5) [24], affinisine (6), and affinine (7) [25], were tested for their antimalarial activity against Plasmodium falciparum 3D7 strain. The results showed only the dimeric alkaloid, 16-demethoxycarbonylvoacamine (4), possessed moderate in vitro antimalarial activity [the half-maximal (50%) inhibitory concentration (IC50) = 28.8 μM], while the others did not show activity even at 50 µM. Furthermore, the cytotoxic activity was evaluated using a human cell line, HepG2 cells. The cytotoxic activity of 4 was low [the half-maximal (50%) cytotoxic concentration (CC50) = > 50 µM for HepG2 cells].

Experimental section

General experimental procedures Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer, and IR spectra on a JASCO FT/IR-4100 spectrophotometer. HRESIMS was conducted on a LTQ Orbitrap XL (Thermo Scientific). 1H and 2D NMR spectra were measured on 600-MHz spectrometer at 300 K, while 13C NMR spectra were measured on a 150-MHz spectrometer. The residual solvent peaks were used as internal standards (δH 3.31 and δC 49.0 for CD3OD). Standard pulse sequences were used for the 2D NMR experiments. Merck silica gel 60 (40–63 μm), amino silica, and HP-20 were used for the column chromatography, and the separations were monitored by Merck silica gel 60 F254, or Merck amino silica gel 60 F254 thin-layer chromatography (TLC) plates.

Plant materials The barks of T. macrocarpa Jack were collected in August 2017 from the Centre for Plant Conservation Botanic Gardens, Bogor, Indonesia. Authentication and identification of plant was carried out at the Centre for Plant Conservation Botanic Gardens, Bogor, Indonesia.

Extraction and isolation The barks of T. macrocarpa Jack (550 g) were extracted with MeOH, and part of the extract (8 g) was partitioned between EtOAc and 3% tartaric acid. The aqueous layer was adjusted at pH 9 with saturated Na2CO3 and extracted with CHCl3 to give CHCl3 fraction (1.5 g), followed by partition with butanol to give butanol fraction (3.7 g). The CHCl3 fraction was subjected to CC over silica gel and eluted with hexane/ethyl acetate (49:1 to 1:1, v/v), followed by CHCl3/methanol (49:1 to 100%) to give 16 fractions. Fraction 9 (149 mg) was further separated using Sephadex LH-20 with CHCl3/MeOH (1:1, v/v) to afford 16-demethoxycarbonylvoacamine (4, 3.4 mg, 0.0425%), isositsirikine (5, 0.6 mg, 0.0075%), affinisine (6, 3.1 mg, 0.0388%), and affinine (7, 13.0 mg, 0.1625%). Fraction 11 (160 mg) was purified by amino silica CC and eluted with CHCl3/MeOH (1:1, v/v), which yielded 12-methoxy-4-methylvoachalotine (3, 151.0 mg, 1.8875%). The butanol fraction was subjected to Diaion HP-20 CC with CHCl3/MeOH (20-100%, v/v) to afford 10 fractions. Fraction 6 was further purified by silica gel CC with CHCl3/MeOH (49:1 to 1:1), affording 20 fractions. Fraction 15 was then successively purified using Sephadex LH-20 with CHCl3/MeOH (1:1, v/v) and high-performance liquid chromatography (HPLC; Cholester 10 × 250 mm, 50% MeOH at 2.4 mL/min, UV detection at 254 nm) to yield 1 (6.3 mg, 0.0788%, tR 11.3 min) and 2 (3.1 mg, 0.0388%, tR 23.2 min).

Methyl esterification reaction The preparation of methyl esters from carboxylic acids can be achieved with TMSCHN2 in methanolic benzene. Compound 1 (0.1 mg) was dissolved in MeOH (100 µl), and TMSCHN2 (50 µl) was added. The reaction was easily monitored by the disappearance of the yellow color of TMSCHN2 to form a product with identical spectroscopic data to that of MMV.

Parasite strain and 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 [26]. Ring-form parasites were collected using the sorbitol synchronization technique [27]. Briefly, the cultured parasites 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 1–7 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) [28]. 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 (MI-RBC %) was automatically reported [28]. Then 0.5% DMSO alone or containing 5 µM artemisinin was used as the negative and positive controls, respectively. The growth inhibition (GI) rate was calculated from the MI-RBC % according to the following equation:

$$ {\text{GI(}}\% ) { } = { 1}00 \, {-}{\text{ (test sample }}{-}{\text{ positive control)}}/({\text{negative control }}{-}{\text{ positive control}}) \, \times { 1}00. $$

The IC50 was calculated from GI (%) using GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, CA, USA) [29].

Compound 1 (22-demethyl MMV): brownish amorphous solid. \( \left[ \alpha \right]_{\text{D}}^{ 2 5} \): −45 (c 1.00, MeOH). IR Vmax (KBr): 3382 and 1678 cm−1. UV/Vis λmax (MeOH) (log ε) 225 (4.30), 271 (3.34) nm. CD (MeOH) λmax (∆ε) 228 (−10.4) and 293 (−1.02) nm. 1H and 13C NMR (CD3OD): Table 1. MS (ESI): m/z: 397 [M]+. HRESIMS m/z: 397.2126 [M]+ (calcd. for C23H29N2O4, 397.2122).

Compound 2 (22-deethyl fuchsiaefoline): brownish amorphous solid. \( \left[ \alpha \right]_{\text{D}}^{ 2 5} \): −5 (c 1.00, MeOH). IR Vmax (KBr): 3383 and 1685 cm−1. UV/Vis λmax (MeOH) (log ε) 225 (3.91), 280 (2.91) nm. CD (MeOH) λmax (∆ε) 227 (−3.78) and 291 (−0.81) nm. 1H and 13C NMR (CD3OD): Table 1. MS (ESI): m/z: 367 [M]+. HRESIMS m/z: 367.2143 [M]+ (calcd. for C22H27N2O3, 367.2152).