key: cord-0030977-zzp18cpo
authors: Nugroho, Alfarius Eko; Tange, Masaki; Kusakabe, Sumi; Hirasawa, Yusuke; Shirota, Osamu; Matsuno, Michiyo; Mizukami, Hajime; Tougan, Takahiro; Horii, Toshihiro; Morita, Hiroshi
title: Chukranoids A–I, isopimarane diterpenoids from Chukrasia velutina
date: 2022-05-05
journal: J Nat Med
DOI: 10.1007/s11418-022-01623-4
sha: d0afd4be92bb143a6642fa4faf230fe6b1b8a927
doc_id: 30977
cord_uid: zzp18cpo

Bioactivity guided separation of Chukrasia velutina root methanolic extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A–I (1–9). The absolute configuration was then assigned by comparing the experimental CD spectra and the calculated CD spectra. Chukranoids A–I (1–9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. It seems that conjugate system in the isopimarane skeleton may influence their antimalarial activity. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11418-022-01623-4.

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, tetranortriterpenoids from Chukrasia plants have been reported to show various activity. Tabulalins B, C, and E, D-ringopened 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.

Compound 1 was obtained as an optically active white amorphous solid. Its molecular formula of C 20 H 32 O 2 was determined by HRESIMS. IR absorptions implied the presence of ketone (1697 cm −1 ) and hydroxy (3449 cm −1 ) groups. The 1 H and 13 C NMR spectra (Table 1 ) as well as HSQC spectra implied the presence of four sp 3 methines, six sp 3 methylenes, four sp 3 methyl groups, three sp 3 quaternary carbons, one vinyl group (δ C 147.1; δ H 5.99, δ C 111.7; δ H 5.02 and 5.06) , and a carbonyl group (δ C 214.7). Among them, one sp 3 methine (δ C 71.6; δ H 3.98) was ascribed to that attached to an oxygen atom.

Analyses of the HSQC and 1 H-1 H 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 H [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . 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 H 2 -12 to C-9. Thus, 1 was revealed as a pimarane or isopimarane type diterpenoid.

The relative configuration of 1 was assigned by analyses of the 1 H-1 H coupling constant data and the NOESY correlations (Fig. 2) . The NOESY correlations of H are α-oriented. Finally, H-14 was inferred to be β-oriented from the coupling constant data of 12.9 Hz) and H-14 (s). Therefore, 1 (chukranoid A) was deduced to be a new isopimarane type diterpenoid.

Compound 2 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 1 H-1 H 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 CH 3 -20 and H-11β, CH [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] and CH 3 -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.

Compound 3 was obtained as an optically active white amorphous solid. Its molecular formula of C 20 H 26 O 2 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 1 H-1 H COSY spectra was connected by the HMBC correlations as shown in Fig. 4 . The structure of 3 with two α,βunsaturated ketone system at C-1 ~ C-3, and C-7, C-8 and C-14 was concluded as chukranoid C with isopimarane skeleton.

The 1 H and 13 C NMR data (Table 1 ) of 4 with molecular formula of C 20 H 28 O 2 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 15 5.99 (dd, 17.5, 11.0) 147. 1 5.83 (dd, 17.5, 10.7) 145. 6 6.16 (dd, 17.5, 10.8) 143. 2 6.17 (dd, 17.5, 11.1) 143. 4 6.16 (dd, 17.7, 10.7) 143 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 1 H-1 H 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 3 J H-5/H-6 (10.3 Hz).

Compound 6 was revealed to have the molecular formula C 20 H 28 O 2 by HRESIMS. The 1 H and 13 C NMR data ( Table 2) suggested the presence of a ketone (δ C 210.0), an α,β-unsaturated ketone (δ H 7.08 and δ H 5.98; δ C 203.4, 154.1 and 126.7) , and vinyl group (δ C 149.7; δ H 5.81, δ C 109. 8; δ H 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 .

Compound 7 was obtained as an optically active white amorphous solid. Its molecular formula of C 20 H 26 O 3 was determined by HRESIMS. IR absorptions implied the presence of two ketone groups (1708 and 1675 cm −1 ). The 1 H and 13 C NMR data (Table 2) implied the presence of an epoxy (δ H 3.84; δ C 59.3 and δ C 57.8) as well as a ketone (δ C 208.7), an α,β-unsaturated ketone (δ H 6.81 and δ H 5.93; δ C 203. 6, 152.1 and 126.4) , and a vinyl group (δ C 142.0; δ H 5.98, δ C 113. 8; δ H 5.07 and 5.12) .

Analyses of the HSQC and 1 H-1 H 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 (δ H 3.84). The relative configuration of 7 was assigned by analyses of the NOESY correlations ( Fig. 6 ). In addition, the 13 C 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] . Table 2 ). The conjugated positions were revealed to be C-1 ~ C-3 by the HMBC correlations of H-1 (δ H 7.18) to C-3 (δ C 202.9) and C-7 ~ C-9 by those of H-6 (δ H 2.58) to C-8 (δ C 130.5) and H-12 (δ H 1.43) to C-9 (δ C 160.5). Compound 9 with the molecular formula C 20 H 30 O 2 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 1 H and 13 C NMR data (Table 2) suggested the presence of a hydroxy methyl group [δ H 3.85 and 3.55 (each d, 10.9 Hz) δ C 64.9]. The HMBC correlations of H-7 (δ H 6.89) and H 3 -17 to C-14 (δ C 203.2) and H-7 to C-9 (δ C 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.

The relative configuration of 9 was confirmed through analyses of the NOESY correlations as shown in Fig. 7 , NMR chemical shifts, and 1 H-1 H coupling constant data. Particularly, C-17, C-19 and C-20 were deduced to be β-oriented from the NOESY correlations of H 3 -20/H-19, H-11β, and H 3 -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.

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. 

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). 1 H and 2D NMR spectra were measured on a 400 MHz or 600 MHz spectrometer at 300 K, while 13 (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/H 2 O, 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/H 2 O), and then an ODS HPLC column (COSMOSIL 5C 18 MSII 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 5C 18 MSII 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%, t R 24.0 min). Fraction 4-7 was separated further by an ODS HPLC column (COSMOSIL 5C 18 MSII 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%, t R 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 5C 18 MSII 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%, t R 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 

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 (NaHCO 3 ), 50 mg/L hypoxanthine, 10 mg/L gentamicin, 10% heatinactivated human serum, and red blood cells (RBCs) at a 3% hematocrit in an atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2 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: .

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 K 2 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:

CHCl 3 ). IR (Zn-Se) ν max 1714 and 1701 cm −1 ; UV (MeOH) λ max 204

Δε -1.13) nm; 1 H and 13 C NMR, see Table 1

CHCl 3 ). IR (Zn-Se) ν max 1725 and 1677 cm −1 ; UV (MeOH) λ max 204

Δε -4.08) nm; 1 H and 13 C NMR, see Table 1

CHCl 3 ). IR (Zn-Se) ν max 1709 and 1685 cm −1 ; UV (MeOH) λ max 203

CD (MeOH) λ max 247 (Δε -2.73) nm; 1 H and 13 C NMR, see Table 1. ESIMS m/z 301 (M + Na) + . HRESIMS m/z 323.1999 [calcd. for C 20 H 28 O 2 Na (M + Na

white amorphous solid. IR

UV (MeOH) λ max 202 (ε 7595), 239 (ε5002) nm

CD (MeOH) λ max 199 (Δε -0.17) nm; 1 H and 13 C NMR, see Table 1. ESIMS m/z 325

HRESIMS m/z 325.2163 [calcd. for C 20 H 30 O 2 Na

white amorphous solid. IR

UV (MeOH) λ max 203 (ε 7879), 227 (ε 9040) nm

CD (MeOH) λ max 227 (Δ ε 2.04) nm; 1 H and 13 C NMR, see Table 2. ESIMS m/z 323

HRESIMS m/z 323.2004 [calcd. for C 20 H 28 O 2 Na (M + Na

white amorphous solid. IR

UV (MeOH) λ max 207 (ε 7860), 224 (ε 8028) nm

CD (MeOH) λ max 228 (Δ ε 1.36) nm

see Table 1. ESIMS m/z 337 (M + Na) +

HRESIMS m/z 337.1790 [calcd. for C 20 H 26 O 3 Na (M + Na

white amorphous solid. IR

UV (MeOH) λ max 226 (ε 8010), 243 (ε 7873) nm

245 (Δ ε -1.71), 330 (Δ ε 0.66) nm; 1 H and 13 C NMR, see Table 1

white amorphous solid. IR (Zn-Se) ν max 3421 and 1698 cm −1

UV (MeOH) λ max 204 (ε 8480) and 244 (ε 8154) nm

241 (Δ ε -0.43) and 334 (Δ ε 0.44) nm; 1 H and 13 C NMR, see Table 1. ESIMS m/z 325 (M + Na) + . HRESIMS m/z 325.2167 [calcd. for C 20 H 30 O 2 Na

The half-maximal (50%) inhibitory concentration (IC 50 ) was calculated from GI (%) using GraphPad Prism version 9.0 (GraphPad Prism Software

References 1. Mabberley DJ (2011) Meliaceae. Flowering Plants Eudicots: Sapindales

The Chukrasines A, B, C, D and E, five new tetranortriterpenes from Chukrasia tabularis A

D-Ring-opened phragmalin-type limonoids from Chukrasia tabularis var. velutina

Three new C-15-isobutyryl 16-norphragmalin-type limonoids from Chukrasia tabularis var. velutina

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GI (% ) = 100 -(test sample -positive control)/ (negativecontrol − positivecontrol

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TURBOMOLE GmbH (2019) TURBOMOLE V7.0, a development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH

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Human malaria parasites in continuous culture

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We express thanks to Prof. Masatsugu Kimura (Osaka City University, Osaka, Japan) for the kind gift of the 3D7 strain, Mr. Yuji Toya and Dr. Kinya Uchihashi (Sysmex) for the setting of the XN-30 analyzer, and Ms. Toshie Ishisaka and Ms. Sawako Itagaki for their technical assistance. We appreciate for supporting of Dr. Nyi Nyi Kyaw, Director General of the Forest Department, Myanmar to correct the plant materials in Myanmar. This work was partly supported by JSPS KAKENHI Grant Number JP19K07152 and JP 16K08309, Japan.

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11418-022-01623-4.