Introduction

Chukrasia velutina is a deciduous tree of the genus Chukrasia, a monotypic genus in the family Meliaceae. It is native to Indochina through Myanmar to Indonesia [1]. The plant is widely used in Ayurveda as an important medicinal plant and the extract has been reported to exhibit considerable antimalarial activity as well as antibacterial and antifungal activities. The plants of this genus have been reported to produce tetranor-triterpenoids, such as chukrasins A–E [2] from the woods of C. tabularis. Furthermore, tetranor-triterpenoids from Chukrasia plants have been reported to show various activity. Tabulalins B, C, and E, D‐ring‐opened phragmalin‐type limonoids, chukvelutins E and F, C-15-isobutyryl 16-norphragmalin-type limonoids, and velutabularins B, D, E, and I have been reported to show inhibitory activities against LPS‐induced NO production in a macrophage cell line [3,4,5]. On the other hand, tabulalide G exhibited moderate cytotoxic activity against MCF-7 [6].

In our search for new bioactive compounds from medicinal plants [7,8,9,10,11,12,13,14,15,16,17,18,19], we investigated the MeOH extract of Chukrasia velutina leaves which showed antimalarial activity. Bioactivity guided separation of the extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A–I (1–9) (Fig. 1). Structure elucidation of 1–9 and the antimalarial activity of the isolated compounds are reported herein.

Fig. 1
figure 1

Structures of 1–9

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Results and discussion

Compound 1 was obtained as an optically active white amorphous solid. Its molecular formula of C20H32O2 was determined by HRESIMS. IR absorptions implied the presence of ketone (1697 cm−1) and hydroxy (3449 cm−1) groups. The 1H and 13C NMR spectra (Table 1) as well as HSQC spectra implied the presence of four sp3 methines, six sp3 methylenes, four sp3 methyl groups, three sp3 quaternary carbons, one vinyl group (δC 147.1; δH 5.99, δC 111.7; δH 5.02 and 5.06), and a carbonyl group (δC 214.7). Among them, one sp3 methine (δC 71.6; δH 3.98) was ascribed to that attached to an oxygen atom.

Table 1 1H and 13C NMR data of 1–5 in CDCl3
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Analyses of the HSQC and 1H-1H COSY spectra (Fig. 2) revealed the presence of five partial structures; a (C-1 ~ C-3), b (C-5 ~ C-6), c (C-8, C-9, and C-14), d (C-11 ~ C-12), and e (C-15 ~ C-16). The connections between partial structures a–e were deduced mainly from the HMBC correlations of H3-17, H3-19, H3-20 (Fig. 2). In addition, the presence of a carbonyl at C-7 and the connectivity of C-9 and C-11 were deduced from the HMBC correlations of H-6 and H-14 to C-7, and H2-12 to C-9. Thus, 1 was revealed as a pimarane or isopimarane type diterpenoid.

Fig. 2
figure 2

Selected 2D-NMR correlations of 1

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The relative configuration of 1 was assigned by analyses of the 1H-1H coupling constant data and the NOESY correlations (Fig. 2). The NOESY correlations of H3-20/H-8, H-11β, and H3-19, H-5/H-9 and H3-18, and H3-17/H-8 and H-11β indicated H-8, CH3-17, CH3-19, and CH3-20 as β-oriented, whereas H-5 and H-9 are α-oriented. Finally, H-14 was inferred to be β-oriented from the coupling constant data of H-8 (d, 12.9 Hz) and H-14 (s). Therefore, 1 (chukranoid A) was deduced to be a new isopimarane type diterpenoid.

Compound 2 was revealed to have the molecular formula C20H26O2 by HRESIMS [m/z 321.1852 (M + Na)+, ∆ + 2.1 mmu]. The presence of two carbonyls was indicated by IR absorption bands at 1714 and 1701 cm−1 and conjugated system was suggested by UV absorption bands at 204 (ε 8513), 271 (ε 3725), and 311 (ε 4659) nm.

The three partial structures; a (C-1 ~ C-2), b (C-9, C-11 and C-12), and c (C-15 ~ C-16), which indicated by the HSQC and 1H-1H COSY spectra was connected by the HMBC correlations as shown in Fig. 3. The structure of 2 with three double bonds at C-5, C-8 ~ C-14, and C-15 was concluded as chukranoid B with isopimarane skeleton. The NOESY correlations between CH3-20 and H-11β, CH3-17 and H-11β, and CH3-19 and CH3-20 showed β-orientations for these protons. The similar ECD spectra at 259 (Δ ε -0.83), 309 (Δ ε -1.13) nm to 1 indicated that 2 had the same absolute structure with isopimarane skeleton as 1.

Fig. 3
figure 3

Selected 2D-NMR correlations of 2

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Compound 3 was obtained as an optically active white amorphous solid. Its molecular formula of C20H26O2 was determined by HRESIMS. The four partial structures; a (C-1 ~ C-2), b (C-5 ~ C-7), c (C-9, C-11 and C-12), and d (C-15 ~ C-16), which indicated by the HSQC and 1H-1H COSY spectra was connected by the HMBC correlations as shown in Fig. 4. The structure of 3 with two α,β-unsaturated ketone system at C-1 ~ C-3, and C-7, C-8 and C-14 was concluded as chukranoid C with isopimarane skeleton.

Fig. 4
figure 4

Selected 2D-NMR correlations of 3 and 5

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The 1H and 13C NMR data (Table 1) of 4 with molecular formula of C20H28O2 were similar to 3, suggesting their structural similarity of each other. Furthermore, the observed differences were mainly for the signals ascribed to the two adjacent methylenes (C-1 ~ C-2) in 4. The proposed structure was further supported by the 2D NMR correlations. Based on the observed differences, the structure of 4 (chukranoid D) was concluded as shown in Fig. 1.

The molecular formula, C20H30O2 of chukranoid E (5) was determined by HRESIMS. The four partial structures; a (C-1 ~ C-3), b (C-5 ~ C-7) c (C-9, C-11 and C-12), and d (C-15 ~ C-16), which indicated by the HSQC and 1H-1H COSY spectra was connected by the HMBC correlations as shown in Fig. 4. The configuration of an oxymethine at C-6 was assigned to be β by the 3JH-5/H-6 (10.3 Hz).

Compound 6 was revealed to have the molecular formula C20H28O2 by HRESIMS. The 1H and 13C NMR data (Table 2) suggested the presence of a ketone (δC 210.0), an α,β-unsaturated ketone (δH 7.08 and δH 5.98; δC 203.4, 154.1 and 126.7), and vinyl group (δC 149.7; δH 5.81, δC 109.8; δH 4.91 and 4.99). Analyses of the 2D NMR data (Fig. 5) further supported the structure of 6. In particular, the HMBC correlations among each partial structure a ~ e clarified the structure of the isopimarane skeleton with the above functions. The NOESY correlations also indicated the relative structure of 6 as shown in Fig. 5.

Table 2 1H and 13C NMR data of 6–9 in CDCl3
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Fig. 5
figure 5

Selected 2D-NMR correlations of 6

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Compound 7 was obtained as an optically active white amorphous solid. Its molecular formula of C20H26O3 was determined by HRESIMS. IR absorptions implied the presence of two ketone groups (1708 and 1675 cm−1). The 1H and 13C NMR data (Table 2) implied the presence of an epoxy (δH 3.84; δC 59.3 and δC 57.8) as well as a ketone (δC 208.7), an α,β-unsaturated ketone (δH 6.81 and δH 5.93; δC 203.6, 152.1 and 126.4), and a vinyl group (δC 142.0; δH 5.98, δC 113.8; δH 5.07 and 5.12).

Analyses of the HSQC and 1H-1H COSY spectra (Fig. 6) revealed the presence of four partial structures; a ~ d. The HMBC correlations in Fig. 6, from which the connections between partial structures a ~ d were deduced, suggested the structure of 7 as isopimarane skeleton with the epoxy moiety at C-7 and C-8. Finally, H-7 was inferred to be β-oriented from the broad singlet peak of H-7 (δH 3.84). The relative configuration of 7 was assigned by analyses of the NOESY correlations (Fig. 6). In addition, the 13C chemical shifts of C-5, C-7, and C-8 for both 7,8-α-epoxy-7 and 7,8-β -epoxy-7 were computed from DFT calculations using the ωB97X-D/6-31G(d) functional and basis set combination supplied with Spartan’20 software for Windows [20]. Experimental data of δC5 41.7, δC7 59.3, and δC8 57.8 were well coincident with those of the calculated values (δC5 41.7, δC7 58.1, and δC8 58.3) of 7,8-β-epoxy-7, when compared with those (δC5 47.7, δC7 55.7, and δC8 63.2) of 7,8-β-epoxy-7 (Supporting information).

Fig. 6
figure 6

Selected 2D-NMR correlations of 7

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Compound 8 was revealed to have the molecular formula C20H26O2 by HRESIMS. The presence of two α,β-unsaturated ketones (δH 7.18 and δH 6.00, and δC 202.9, 151.9 and 127.3; δC 198.1, 160.5 and 130.5) was indicated like as in 3 by 1H and 13C NMR data (Table 2). The conjugated positions were revealed to be C-1 ~ C-3 by the HMBC correlations of H-1 (δH 7.18) to C-3 (δC 202.9) and C-7 ~ C-9 by those of H-6 (δH 2.58) to C-8 (δC 130.5) and H-12 (δH 1.43) to C-9 (δC 160.5).

Compound 9 with the molecular formula C20H30O2 was obtained as optically active white amorphous solids. IR absorption implied the presence of conjugated ketone (1698 cm−1) and hydroxy (3421 cm−1) groups. Their 1H and 13C NMR data (Table 2) suggested the presence of a hydroxy methyl group [δH 3.85 and 3.55 (each d, 10.9 Hz) δC 64.9]. The HMBC correlations of H-7 (δH 6.89) and H3-17 to C-14 (δC 203.2) and H-7 to C-9 (δC 52.7) verified the presence of a carbonyl at C-14 and a double bond at C-7 in 9 (Fig. 7). Further structure elucidation and chemical shift assignments were done through analyses of the 2D NMR data.

Fig. 7
figure 7

Selected 2D-NMR correlations of 9

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The relative configuration of 9 was confirmed through analyses of the NOESY correlations as shown in Fig. 7, NMR chemical shifts, and 1H-1H coupling constant data. Particularly, C-17, C-19 and C-20 were deduced to be β-oriented from the NOESY correlations of H3-20/H-19, H-11β, and H3-17/H-11β. On the other hand, H-5 and H-9 were deduced to be α-oriented.

The absolute configuration of chukranoid C (3) was assigned by comparing the experimental CD spectra and the calculated CD spectra as shown in Fig. 8. CD calculation was performed by Turbomole 7.1 [21] using RI-DFT-BP86/def-TZVP level of theory on RI-DFT-BP86/def-TZVP optimized geometries. The experimental CD spectra showed similar CD pattern compared to calculated CD spectra. Therefore, the absolute configuration of 3 was proposed as shown in Fig. 1. Based on biogenetic considerations, the absolute configurations of the other chukranoids, showing the negative optical rotation around 250–300 nm, should be considered to be the same as that of 3.

Fig. 8
figure 8

Calculated and experimental CD spectra of chukranoid E (3)

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Antimalarial activity

Malaria still remains one of the leading deadliest diseases throughout the world. The emergence and spread of growing resistance to the first-line antimalarials are an alarmingly serious problem in malaria control, demanding the need for new drugs. So far, some isopimarane diterpenoids from Platycladus orientalis have been reported to show in vitro anti-plasmodial activity [22]. Chukranoids A–I (1–9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain (Table 3). It seems that conjugate system in the isopimarane diterpenoid may influence their antimalarial activity.

Table 3 Antimalarial activity of 1–9 against P. falciparum 3D7 strain
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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. High-resolution ESI MS were obtained on a JMS-T100LP (JEOL). 1H and 2D NMR spectra were measured on a 400 MHz or 600 MHz spectrometer at 300 K, while 13C NMR spectra were on a 100 MHz or 150 MHz spectrometer. The residual solvent peaks were used as internal standards (δH 7.26 and δC 77.0 for CDCl3). Merck silica gel 60 (40–63 μm) was used for the column chromatography, and the separations were monitored by Merck silica gel 60 F254, or Merck silica gel RP C-18 F254 TLC plates.

Material. The root of C. velutina were collected in Popa Mountain Park, Mandalay Region, Myanmar under the Memorandum of Understanding between the Kochi Prefectural Makino Botanical Garden (MBK), Japan and the Forestry Department, the Ministry of Natural Resources and Environmental Conservation, Myanmar. The botanical identification was made by Dr. Nobuyuki TanakaMBK, currently National Museum of Nature and Science. Voucher specimen (Herbarium No. MBK 0,113,430) is deposited in the Herbarium of MBK.

Extraction and isolation. The dried ground root of Chukrasia velutina (100 g) was extracted with MeOH, and the extract (0.96 g) was successively partitioned with n-hexane, EtOAc, n-BuOH, and water. The n-hexane-soluble fraction (664 mg) were separated further by a silica gel column (n-hexane/EtOAc, 4:1) to afford 13 fractions.

Fraction 2 was separated by a silica gel column chromatography (toluene/EtOAc, 1:0 → 9:1) to afford 7 fractions (fractions 2–1–2–7). Fractions 2–4 were separated further by an ODS column (MeOH/H2O, 1:0 → 9:1) to afford chukranoid A (1, 0.6 mg, 0.0006%).

Fraction 3 was separated by a silica gel column chromatography (toluene/EtOAc, 1:0 → 9:1) to afford 13 fractions (fractions 3–1–3–13). Fractions 3–4 were separated further by an ODS column (MeOH/H2O), and then an ODS HPLC column (COSMOSIL 5C18MSII 10 × 250 mm, 68.0% MeOH(aq) at 3.0 mL/min, UV detection at 210 nm) to afford chukranoids B (2, 1.7 mg, 0.0017%), C (3, 0.1 mg, 0.0001%), D (4, 0.3 mg, 0.0003%), and E (5, 1.3 mg 0.0013%).

Fraction 4 was separated by a silica gel column chromatography (toluene/EtOAc, 1:0 → 9:1) to afford 10 fractions (fractions 4–1–4–10). Fraction 4–4 was separated further by an ODS HPLC column (COSMOSIL 5C18MSII 10 × 250 mm, 68.0% MeOH(aq) at 3.0 mL/min, UV detection at 210 nm) to afford chukranoid F (6, 1.6 mg, 0.0016%, tR 24.0 min). Fraction 4–7 was separated further by an ODS HPLC column (COSMOSIL 5C18MSII 10 × 250 mm, 68.0% MeOH(aq) at 3.0 mL/min, UV detection at 210 nm) to afford chukranoid G (7, 1.2 mg, 0.0012%, tR 24.4 min).

Fraction 5 was separated by a silica gel column chromatography (toluene/EtOAc, 1:0 → 9:1) to afford 11 fractions (fractions 5–1–5–11). Fraction 5–6 was separated further by an ODS HPLC column (COSMOSIL 5C18MSII 10 × 250 mm, 67.0% MeOH(aq) at 3.0 mL/min, UV detection at 210 nm) to afford orizalexin C, and fraction 5–7 was separated by the same condition to afford chukranoid H (8, 0.6 mg, 0.0006%, tR 36.0 min).

Fraction 7 was separated by a silica gel column chromatography (n-hexane:EtOAc, 8:2 → 1:1) to afford chukranoid I (9, 1.8 mg, 0.0018%).

Chukranoid A (1): white amorphous solid. [α]D27 -40 (c 0.3, CHCl3). IR (Zn-Se) νmax 3449 and 1697 cm−1; UV (MeOH) λmax 204 (ε 8755), 268 (ε 3841), 310 (ε 4823) nm; CD (MeOH) λmax 289 (Δε -1.82) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 327 (M + Na)+. HRESIMS m/z 327.2324 [calcd. for C20H32O2Na (M + Na)+: 327.2300].

Chukranoid B (2): white amorphous solid. [α]D27 -65 (c 0.9, CHCl3). IR (Zn-Se) νmax 1714 and 1701 cm−1; UV (MeOH) λmax 204 (ε 8513), 271 (ε 4659) nm; CD (MeOH) λmax 259 (Δε -0.83), 309 (Δε -1.13) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 321 (M + Na)+. HRESIMS m/z 321.1852 [calcd. for C20H26O2Na (M + Na)+: 321.1831].

Chukranoid C (3): white amorphous solid. [α]D27 -34 (c 0.2, CHCl3). IR (Zn-Se) νmax 1725 and 1677 cm−1; UV (MeOH) λmax 204 (ε 6450), 231 (ε7637) nm; CD (MeOH) λmax 231 (Δε 2.44), 248 (Δε -4.08) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 321 (M + Na)+. HRESIMS m/z 321.1861 [calcd. for C20H26O2Na (M + Na)+: 321.1831].

Chukranoid D (4): white amorphous solid. [α]D25 -11 (c 0.2, CHCl3). IR (Zn-Se) νmax 1709 and 1685 cm−1; UV (MeOH) λmax 203 (ε 6968), 251 (ε8310) nm; CD (MeOH) λmax 247 (Δε -2.73) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 301 (M + Na)+. HRESIMS m/z 323.1999 [calcd. for C20H28O2Na (M + Na)+: 323.1987].

Chukranoid E (5): white amorphous solid. IR (Zn-Se) νmax 3566 and 1716 cm−1; UV (MeOH) λmax 202 (ε 7595), 239 (ε5002) nm; CD (MeOH) λmax 199 (Δε -0.17) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 325 (M + Na)+. HRESIMS m/z 325.2163 [calcd. for C20H30O2Na (M + Na)+: 325.2143].

Chukranoid F (6): white amorphous solid. IR (Zn-Se) νmax 1701 and 1669 cm−1; UV (MeOH) λmax 203 (ε 7879), 227 (ε 9040) nm; CD (MeOH) λmax 227 (Δ ε 2.04) nm; 1H and 13C NMR, see Table 2. ESIMS m/z 323 (M + Na)+. HRESIMS m/z 323.2004 [calcd. for C20H28O2Na (M + Na)+: 323.1987].

Chukranoid G (7): white amorphous solid. IR (Zn-Se) νmax 1708 and 1675 cm−1; UV (MeOH) λmax 207 (ε 7860), 224 (ε 8028) nm; CD (MeOH) λmax 228 (Δ ε 1.36) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 337 (M + Na)+. HRESIMS m/z 337.1790 [calcd. for C20H26O3Na (M + Na)+: 337.1780].

Chukranoid H (8): white amorphous solid. IR (Zn-Se) νmax 1709 and 1685 cm−1; UV (MeOH) λmax 226 (ε 8010), 243 (ε 7873) nm; CD (MeOH) λmax 211 (Δ ε 3.34), 245 (Δ ε -1.71), 330 (Δ ε 0.66) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 321 (M + Na)+. HRESIMS m/z 321.1847 [calcd. for C20H26O2Na (M + Na)+: 321.1831].

Chukranoid I (9): white amorphous solid. IR (Zn-Se) νmax 3421 and 1698 cm−1; UV (MeOH) λmax 204 (ε 8480) and 244 (ε 8154) nm; CD (MeOH) λmax 217 (Δ ε 1.54), 241 (Δ ε -0.43) and 334 (Δ ε 0.44) nm; 1H and 13C NMR, see Table 1. ESIMS m/z 325 (M + Na)+. HRESIMS m/z 325.2167 [calcd. for C20H30O2Na (M + Na)+: 325.2143].

CD calculation

The conformations were obtained using Monte Carlo analysis with MMFF94 force field and charges on Macromodel 9.1. CD calculations were performed in Turbomole 7.1 [21] using RI-DFT-BP86/def-TZVP level of theory on RI-DFT-BP86/def-TZVP optimized geometries.

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 [23]. Ring-form parasites were collected using the sorbitol synchronization technique [24]. 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 1–9 at sequentially decreasing concentrations (50, 15, 5, 1.5, 0.5, and 0.15 µM) for 48 h. Parasitemia was measured by 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) [25, 26]. 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 [25]. 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:

$${\rm{GI (\% ) = 100 - (test sample - positive control)/(negative control - positive control)}} \times {\rm{100 }}$$

The half-maximal (50%) inhibitory concentration (IC50) was calculated from GI (%) using GraphPad Prism version 9.0 (GraphPad Prism Software, San Diego, CA, USA) [27].