key: cord-0075937-mi4fnxoy
authors: Ogasawara, Ai; Noguchi, Ryo; Shigi, Takuya; Nugroho, Alfarius Eko; Hirasawa, Yusuke; Kaneda, Toshio; Tougan, Takahiro; Horii, Toshihiro; Hadi, A. Hamid A.; Morita, Hiroshi
title: Caloforines A–G, coumarins from the bark of Calophyllum scriblitifolium
date: 2022-03-22
journal: J Nat Med
DOI: 10.1007/s11418-022-01613-6
sha: 33f2ebe3260822591b0303b6e64e4842fb337828
doc_id: 75937
cord_uid: mi4fnxoy

Bioactivity-guided separation of the methanol extract of Calophyllum scriblitifolium bark led to the isolation of five new pyranocoumarins, caloforines A–E (1–5) and two new coumarins, caloforines F and G (6 and 7). Their structures were elucidated by 1D and 2D NMR spectroscopy, and their absolute configurations were investigated by a combination of CD spectroscopy and DFT calculation. Caloforines A–F (1–6) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11418-022-01613-6.

Malaria is the largest parasitic protozoan infection in humankind in which malaria infects humans through the anopheles mosquito. Malaria is not only widespread throughout the tropics but also occurs in many temperate regions. In light of this problem, scientists have turned to naturally occurring compounds obtained from plants recurrently used in traditional medicine [1] . As part of our ongoing effort to isolate and identify novel antimalarial natural products from a variety of natural sources, our laboratory reported the discovery of various types of skeletal natural products showing antimalarial activity [2] [3] [4] [5] [6] [7] [8] .

Calophyllum, the largest genus in the Calophyllaceae family, is composed of over 200 species distributed mainly in the tropical region [9] . Plants of this genus have been reported for several ethnomedicinal uses in the traditional systems of medicine [10] . In addition, plants of this genus have been reported to contain xanthones, flavonoids, acylphloroglucinols, terpenoids and chromanones [11, 12] . The scientific study of the genus Calophyllum revealed that it is a rich source of bioactive secondary metabolites showing a wide range of biological activities. Some coumarins from C. flavoranulum showed activity against Plasmodium berghei parasite [13] .

We have reported Calophyllum chromanones (calofolic acids A-F) showing dose-dependent vasorelaxation activity on isolated rat aorta, have been isolated from the tropical tree C. scriblitifolium [14] . In our search for new bioactive compounds, we investigated the MeOH extract of C. scriblitifolium which showed antimalarial activity. Bioactivityguided separation of the extract led to the isolation of five new pyranocoumarins, caloforines A-E (1-5) and two new coumarins, caloforines F and G (6 and 7) (Fig. 1) . Structure elucidation of 1-7 and the antimalarial activity of the isolated coumarins, caloforines A-F (1-6) are reported herein.

Caloforine A (1) was obtained as an optically active colorless amorphous solid, − 40 (c 1.0, MeOH) and revealed to have the molecular formula C 17 H 20 O 5 , requiring eight degrees of unsaturation, by HRESIMS. The IR spectrum showed an important absorption at 1717 cm −1 for the unsaturated ester carbonyl group. The UV spectrum (λ max 221, 232, 259, and 321 nm) indicated the presence of a coumarin chromophore. The 1 H and 13 C NMR data (Tables 1 and 3) and HSQC spectrum of 1 revealed the presence of seventeen carbon signals due to three sp 3 methines, one sp 3 methylene, four methyls, seven sp 2 quaternary carbons, and two sp 2 methines. Among them, three sp 2 quaternary carbons (δ C 153.5, 156.7, and 160.4) and one methyl carbon (δ C 56.0) were attributed to those attached to an oxygen atom. The gross structure of 1 was elucidated by analysis of 2D NMR data including the 1 H-1 H COSY, HSQC, and HMBC spectra. Analyses of the HSQC and 1 H-1 H COSY spectra revealed the presence of two partial structures; a (C-9 ~ C-13) and b (C-14 ~ C-15). The connections between two partial structures and coumarin skeleton were deduced mainly from the HMBC correlations of H-9, H 2 -14, and H 3 -15 (Fig. 2 ). In addition, the presence of a methoxy group at C-7 was deduced from the HMBC correlations of OCH 3 -7, H-8 and H-9 to C-7. Thus, 1 was revealed as a tetrahydropyranoyl coumarin as shown in Fig. 1 , which may be derived from tiglyl moiety at C-6, with an ethyl group at C-4 and a methoxy group at C-7.

The relative stereochemistry of C-9, C-10, and C-11 was elucidated from 3 J coupling constants as in the case of those of calanolides from Calophyllum lanigerum [15] . The 3 J H10-H11 constant (10.8 Hz) indicated anti relation between H-10 and H-11, whereas that of 3 J H9-H10 (3.5 Hz) syn relation between H-9 and H-10.

Caloforine B (2) was determined to have the molecular formula, C 18 H 22 O 5 which was larger than that of 1 by a CH 2 unit. The 1 H and 13 C NMR data are highly similar and the presence of a methoxy group (δ H 3.49) at C-9, indicating the structure of 2 as 9-methoxy caloforine A. Analysis of the 2D NMR data further supported the proposed structure (Fig. 3 ). In particular, the HMBC correlation of the methoxymethyl to C-9 confirmed the position of the methoxy group at C-9. The same relative stereochemistry of C-9, C-10, and C-11 as 1 was elucidated from 3 J coupling constants ( 3 J H10-H11 12.3 Hz and 3 J H9-H10 2.8 Hz) as in the case of those of 1.

The molecular formulae of caloforines C (3) and D (4) were also determined to be C 17 H 20 O 5 and C 18 H 22 O 5 , respectively, as those of 1 and 2. Furthermore, their NMR data are also highly similar to 1 and 2, respectively. However, 3 J coupling constants of the signals associated with H-9 -H-11 were different from those of 1 and 2. The 3 J H10-H11 constant (1.6 Hz) indicated syn relation between H-10 and H-11, whereas that of 3 J H9-H10 (1.2 Hz) syn relation between H-9 and H-10 [15] . Analysis of the 2D NMR data (Fig. 4 ) supported the structures of 3 and 4 to be as shown in Fig. 1 . Each stable conformer of 1 and 3 was shown in Fig. 5 to clarify the relationship of 3 J H-H coupling constants associated with H-9 -H-11 (syn and anti relation).

Caloforine E (5) are observed instead. The proposed structure as shown in Fig. 1 was confirmed through analysis of the 2D NMR data (Fig. 4) .

Electronic circular dichroism (ECD) may provide a powerful approach to the determination of the absolute configuration of natural products [16] . The absolute configuration of caloforines A-E (1-5) was assigned by comparing the experimental CD spectra shown in Fig. 6 and the calculated CD spectra. CD calculation was performed by Turbomole 7.1 [17] using RI-TD-DFT-B3LYP/def2-SVPD level of theory on RI-DFT-B3LYP/ def2-SVP optimized geometries. The experimental CD spectra show a similar CD pattern compared to calculated CD spectra (Fig. 6) . Therefore, the absolute configuration of caloforines A and B (1 and 2) was proposed to be 9S, 10R, 11S and that of caloforines C-E (3-5) 9R, 10R, 11R as shown in Fig. 1 .

Caloforine F (6) was revealed to have the molecular formula C 19 H 22 O 5 by HRESIMS. The IR spectrum showed two absorptions at 1735 and 1660 cm −1 for unsaturated ester carbonyl and unsaturated ketone groups, respectively. The UV spectrum (λ max 226, 242, and 322 nm) indicated the presence of a coumarin chromophore.

The 1 H and 13 C NMR data (Tables 1 and 3 ) and HSQC spectrum of 6 revealed the presence of nineteen carbon signals due to one ketone, one ester ketone, two methoxyls, two sp 3 methylenes, three methyls, three sp 2 methines, and seven sp 2 quaternary carbons. Among them, three sp 2 quaternary carbons (δ C 155.8 156.7, and 159.4) and two methyl carbons (δ C 56.3 and 63.7) were attributed to those attached to an oxygen atom.

The gross structure of 6 was elucidated by analysis of 2D NMR data including the 1 H-1 H COSY, HSQC, and HMBC spectra (Fig. 7) . Analyses of the HSQC and 1 H-1 H COSY spectra revealed the presence of two partial structures; a (C-11 ~ C-12) and b (C-14 ~ C-16). The connections between the partial structure a and a coumarin skeleton were deduced mainly from the HMBC correlations of H-14 and H 2 -15. In addition, the presence of a methoxy group at C-5 and C-7 was deduced from the NOESY correlation between OCH 3 -5 and H 2 -14, and the HMBC correlations of OCH 3 -7 and H-8 to C-7. Thus, 6 was revealed as a coumarin with a propyl group at C-4, an angelyl moiety at C-6, and two methoxy groups at C-5 and C-7.

Caloforine G (7) was revealed to have the molecular formula C 18 H 20 O 6 by HRESIMS. The IR spectrum showed two absorptions at 1731 and 1662 cm −1 for unsaturated ester carbonyl and unsaturated ketone groups, respectively. Furthermore, the NMR data of 6 and 7 are highly similar to each other. However, the signals associated with the angelyl moiety in 6 is not observed in 7, and sp 2 methylene and an oxygen-bearing doublet methyl signals (δ C 128.3, δ H 5.79 and 6.18, and δ C 22.2, δ H 1.48 and δ C 66.5, δ H 4.87 for 7) are observed instead. Furthermore, an ethyl signal instead of a propyl at C-4 is also observed. Therefore, 7 should have a 3-hydroxy-2-methylenebutanoyl moiety instead of an angelyl moiety. Analysis of the 2D NMR data (Fig. 7) supported the structure of 6 and 7 to be as shown in Fig. 1 . Caloforine G (7), which was considered to be racemate, showed no optical rotation.

Coumarins, which exhibited various biological properties such as antimicrobial, anti-inflammatory, enzyme inhibitory properties, and so on, also displayed potential in vitro anti-plasmodial and in vivo antimalarial activities [18, 19] . Moreover, many of coumarin derivatives. have already been used in clinical practice for the treatment of several diseases.

Caloforines A-G (1-7) were tested for the antimalarial activity against P. falciparum 3D7 strain. The result showed that most of them showed moderate in vitro antimalarial activity [the half-maximal (50%) inhibitory concentration (IC 50 ) = 9.4 ~ 35.5 µM, respectively.] (Table 2 ), whereas 7 did not (> 50 µM). The activity for pyranocoumarins 1-5 

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. CD spectra were recorded on a JASCO J-820 polarimeter. High-resolution ESI MS was 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 

The barks of C. scriblitifolium (1.0 kg) were extracted with MeOH to obtain 130.8 g of extract. A part of the MeOH extract (50 g) was successively partitioned with n-hexane, EtOAc, n-BuOH and water. The EtOAc and n-BuOH-soluble materials were combined (25.5 g), and further separated by HP-20 column chromatography (H 2 O/ MeOH 1:0 → 4:1 → 3:2 → 1:4 → 0:1, 100% Acetone) to obtain 3 fractions (eb-1-3). The third fraction (eb-3, 1.1 g) was further separated with a silica gel column (Hexane/EtOAc 1:0 → 9:1 → 4:1 → 7:3 → 6:4, CHCl 3 /MeOH 0:1 → 50:1 → 30:1 → 10:1 → 0:1) to obtain 8 fractions 

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 using RI-TD-DFT-B3LYP/def2-SVPD level of theory on RI-DFT-B3LYP/def2-SVP optimized geometries [17] .

Parasite strain 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% heat-inactivated 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 [20] . Ring-form parasites were collected using the sorbitol synchronization technique [21] . 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).

Ring-form-synchronized parasites were cultured with compounds 1-7 at sequentially decreasing concentrations (50, 15, 5, 1.5, 0.5, and 0.15 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) [22, 23] . 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 [22] . 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:

The IC 50 was calculated from GI (%) using GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, CA, USA) [24] .

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The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11418-022-01613-6.

Ai Ogasawara 1 · Ryo Noguchi 1 · Takuya Shigi 1 · Alfarius Eko Nugroho 1 · Yusuke Hirasawa 1 · Toshio Kaneda 1 · Takahiro Tougan 2 · Toshihiro Horii 3