Picrajavanicins A–G, Quassinoids from Picrasma javanica Collected in

Nov 25, 2015 - Faculty of Pharmacy, Hasanuddin University, Makassar 90245, South ... Faculty of Pharmaceutical Sciences, Tokushima Bunri University, ...
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Picrajavanicins A−G, Quassinoids from Picrasma javanica Collected in Myanmar Nwet Nwet Win,*,†,‡ Takuya Ito,† Ismail,§ Takeshi Kodama,† Yi Yi Win,‡ Masami Tanaka,⊥ Hla Ngwe,‡ Yoshinori Asakawa,⊥ Ikuro Abe,∥ and Hiroyuki Morita*,† †

Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan Department of Chemistry, University of Yangon, Yangon 11041, Myanmar § Faculty of Pharmacy, Hasanuddin University, Makassar 90245, South Sulawesi, Indonesia ⊥ Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan ∥ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡

S Supporting Information *

ABSTRACT: Seven new tetracyclic quassinoids, picrajavanicins A−G (1−7), along with three known analogues, were isolated from a CHCl3-soluble extract of the bark of Picrasma javanica collected in Myanmar. The structures of these compounds were elucidated using spectroscopic techniques, including 1D and 2D NMR. The absolute configuration at C-2 of 2 was determined to be S by the modified Mosher method. All the isolates were tested for their antiproliferative activities against a small panel of five human cancer cell lines. However, none of the isolated compounds exhibited inhibitory activity against any of the cancer cells used (IC50 values >10 μM).

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atural products have played a very important role as established cancer chemotherapeutic agents in both unmodified (naturally occurring) and synthetically modified forms. The bisindole (vinca) alkaloids, camptothecins, epipodophyllotoxins, and taxanes are plant-derived compounds that have been widely used as antitumor agents.1 In an ongoing search for new anticancer agents from Myanmar medicinal plants,2−8 the crude extracts were screened against a panel of five human cancer cell lines (A549, human lung cancer; HeLa, human cervix cancer; PANC-1 and PSN-1, human pancreatic cancer; and MDA-MB-231, human breast cancer). Since the CHCl3-soluble fraction of the Picrasma javanica bark exhibited antiproliferative activity against the cancer cell lines utilized, we continued to isolate its secondary metabolites. P. javanica Blume (Simaroubaceae) is a medium-sized tree that is distributed wildly in the tropical regions of Asia, including Myanmar, Indonesia, and India. It is known as “Nann-pawkyawt” in Myanmar and has been used extensively for selfmedication by malaria, cancer, and AIDS patients. Decoctions of its bark are used in folk medicine as a febrifuge and as a substitute for quinine. A number of quassinoids and alkaloids have been reported as phytoconstituents of P. javanica.9−26 In this investigation, seven new tetracyclic quassinoids were isolated, named picrajavanicins A−G (1−7), along with three known analogues, javanicins B,18 F,20 and I,17 from the bark of P. javanica collected in Myanmar. Herein, the structure elucidation of 1−7 and the evaluation of the antiproliferative activity of all isolated quassinoids are reported. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The chloroform extract of P. javanica bark exhibited antiproliferative activity against a panel of five human cancer cell lines (A549, human lung cancer; HeLa, human cervix cancer; PANC-1 and PSN-1, human pancreatic cancer; and MDA-MB-231, human breast cancer), with IC50 values ranging from 1.6 to 22.1 μg/mL. Thus, it was subjected to a series of chromatographic separations, which led to the characterization Received: September 13, 2015

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DOI: 10.1021/acs.jnatprod.5b00824 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data (600 MHz, CDCl3) of Picrajavanicins A−D (1−4) (δ in ppm and J Values in (Hz) in Parentheses) position 2 3/3α 3β 4α 4β 5α 6α 6β 7β 9α 14β 15α 15β 18 19 MeO-2 MeO-12 Me-13 CHO-13 HO-14

1

2 4.78, 1.38, 2.48, 1.46, 1.83, 1.81, 1.78, 1.97, 4.25, 3.23, 2.42, 2.61, 3.00, 1.43, 1.20,

5.58, dd (5.7, 2.3) 2.21, 2.31, 2.30, 1.80, 2.02, 4.33, 3.06, 2.97, 2.46, 3.05, 1.56, 1.16, 3.61, 4.00,

m m m dt (14.6, 3.0) td (14.6, 2.3) t (3.0) s dd (11.1, 7.3) dd (18.7, 11.1) dd (18.7, 7.3) s s s s

3

dd (11.4, m m m m m m m t (2.6) s dd (12.2, dd (18.7, dd (18.7, s s

7.7)

4.44, 1.56, 2.37, 1.51, 1.82, 1.80, 1.78, 1.94, 4.24, 3.28, 2.41, 2.65, 2.99, 1.44, 1.21, 3.41, 3.66, 1.91,

6.8) 12.2) 6.8)

3.65, s 1.91, s

dd (11.6, m m m m m m m t (2.8) s dd (12.3, dd (18.6, dd (18.6, s s s s s

4 7.0)

6.7) 12.3) 6.7)

4.82, 1.40, 2.50, 1.48, 1.82, 1.84, 1.86, 1.95, 4.62, 3.18,

t (7.6) m m m m m m m t (2.8) s

3.05, 2.99, 1.50, 1.16,

d (18.4) d (18.4) s s

3.69, s 1.94, s

10.27, s 2.29, s

Table 2. 1H NMR Spectroscopic Data (600 MHz, CDCl3) of Picrajavanicins E−G (5−7) (δ in ppm and J Values in (Hz) in Parentheses) position 3/3α 3β 4α 4β 5α 6α 6β 7β 9α 14β 15/15α 15β 18 19 MeO-2 MeO-3 MeO-12 Me-13 HOCH2-13

5

6

5.56, t (2.4) 2.28, 2.42, 2.18, 1.76, 1.98, 4.26, 2.92, 2.38, 2.59, 2.99, 1.50, 1.19, 3.61, 3.91, 3.68, 1.87,

dd (17.7, 4.7) dd (17.7, 11.3) m dt (14.5, 3.2) td (14.5, 2.2) t (2.8) s dd (11.8, 7.0) dd (18.7, 11.8) dd (18.7, 7.0) s s s s s s

2.32, 2.44, 2.18, 1.81, 1.94, 4.65, 2.86,

dd (17.7, 4.7) dd (17.7, 11.2) m dt (14.6, 3.3) td (14.6, 2.3) t (2.9) s

2.30,a m 2.30,a m 2.29, m 1.90, m 2.06, m 4.31, t (2.8) 3.14, s

3.04, 2.99, 1.55, 1.15, 3.61, 3.93, 3.70, 1.90,

d (18.5) d (18.5) s s s s s s

6.13, s 1.50, s 1.30, s 3.60, s 3.90, s 4.49, d (12.9) 4.67, d (12.9)

HO-14 a

7

2.69, s

Overlapping resonances within the same column.

5.7, 2.3 Hz, H-3)], an oxymethine [δH 4.33, t (J = 3.0 Hz, H7β)], three methines [δH 2.30, m (H-5α), 2.97, dd (J = 11.1, 7.3 Hz, H-14β), 3.06, s (H-9α)], three methylenes, two methoxy groups [δH 3.61, s (MeO-2), 4.00, s (MeO-12)], two tertiary methyls [δH 1.16, s (H3-19), 1.56, s (H3-18)], and an aldehyde group [δH 10.27, s (CHO-13)]. The 13C NMR data (Table 3) included 21 signals for four olefinic carbons [δC 110.4 (C-3), 129.8 (C-13), 148.9 (C-2), 158.5 (C-12)], an oxymethine [δC 81.7 (C-7)], three methines [δC 35.8 (C-5), 37.9 (C-14), 48.0 (C-9)], three methylene carbons [δC 26.8 (C-4), 29.0 (C-6), 32.1 (C-15)], two methoxy groups [δC 55.0 (MeO-2), 60.8

of seven new quassinoids, named picrajavanicins A−G (1−7), and three known quassinoids, javanicins B,18 F,20 and I.17 Picrajavanicin A (1) was obtained as a pale yellow, amorphous solid, and its molecular formula was determined as C21H24O7 from the HREIMS and 13C NMR data. Spectroscopic analysis revealed UV absorption band at 264 nm due to a conjugated enone system. The IR spectrum displayed absorption bands at 1730, 1702, and 1666 cm−1, indicative of the presence of δ-lactone and α,β-unsaturated carbonyl functionalities. The 1H NMR data (Table 1) displayed signals corresponding to an olefinic proton [δH 5.58, dd (J = B

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Table 3. 13C NMR Spectroscopic Data (150 MHz, CDCl3) of Picrajavanicins A−G (1−7) (δ in ppm) position

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 MeO-2 MeO-3 MeO-12 Me-13 CHO-13 HOCH2-13

196.9 148.9 110.4 26.8 35.8 29.0 81.7 37.3 48.0 45.8 194.5 158.5 129.8 37.9 32.1 168.5 11.4 22.1 55.0

212.9 70.1 37.8 24.4 41.6 28.9 82.0 36.8 47.3 48.4 190.6 148.1 140.1 47.1 31.4 168.7 13.9 22.9

209.6 79.2 34.5 24.8 41.4 29.0 82.2 36.7 47.2 49.2 190.7 148.2 139.7 47.3 31.4 168.7 13.8 23.1 57.8

212.9 70.1 37.8 24.5 41.8 28.8 79.3 42.7 50.1 48.8 190.2 148.2 140.8 75.1 39.6 168.0 13.9 16.1

59.6 15.7

59.7 15.7

59.7 10.6

197.9 133.3 158.9 29.8 33.1 28.5 79.1 43.3 49.4 44.9 191.0 148.4 138.5 75.1 39.4 168.4 12.3 15.9 60.1 57.7 59.4 10.3

196.6 149.0 110.1 27.1 36.1 28.4 77.5 42.4 53.7 45.7 192.0 152.0 131.9 159.9 111.7 164.0 10.2 23.0 55.0

60.8

197.7 133.4 158.4 29.9 33.1 28.7 82.0 37.4 46.4 44.6 191.2 148.4 137.2 46.5 31.5 168.8 12.3 22.3 60.1 57.7 59.4 15.2

60.5

191.7 56.4

Figure 1. COSY (bold lines) and key HMBC (1H → 13C) (arrows) correlations in compounds 1−7.

Figure 2. Key NOESY correlations (double-headed arrows) in picrajavanicins A (1) and B (2).

C

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(MeO-12)], two tertiary methyl groups [δC 11.4 (C-18), 22.1 (C-19)], two quaternary carbons [δC 37.3 (C-8), 45.8 (C-10)], an aldehyde carbon [δC 191.7 (CHO-13)], two carbonyls [δC 194.5 (C-11), 196.9 (C-1)], and one lactone carbonyl carbon [δC 168.5 (C-16)]. These data suggested that 1 is a tetracyclic quassinoid similar to javanicin F, which was reported by Koike et al.20 However, there were significant differences found, including the absence of a tertiary methyl group and the presence of an aldehyde group in 1. The 1H−13C HMBC correlations (Figure 1) of an aldehyde proton (δH 10.27, s) to C-13 (δC 129.8) and of H-14 [δH 2.97, dd (J = 11.1, 7.3 Hz)] to an aldehyde carbonyl carbon (δC 191.7) was used to locate an aldehyde group at C-13. The relative configuration of 1 was assigned on the basis of a 2D NOESY NMR experiment. The NOESY correlations (Figure 2) between H-5 and H-9 suggested α orientations of these protons, whereas those between H-7 and H-14/H3-19 and between H3-18 and H3-19 suggested that they are β-oriented. Hence, the structure of 1 was assigned as shown and was assigned the trivial name picrajavanicin A. To the best of our knowledge, this is the first report of the substitution by an aldehyde group at C-13 in a compound bearing the des-4-methylated picrasane skeleton. Picrajavanicin B (2) was obtained as an amorphous solid with a molecular formula of C20H26O6. The IR spectrum showed absorption bands of a hydroxy group (3471 cm−1), a δlactone (1733 cm−1), and an α,β-unsaturated carbonyl group (1685 cm−1). The UV spectrum displayed an absorption maximum at 252 nm. The 1H and 13C NMR spectroscopic data (Tables 1 and 3) of 2 indicated the presence of five methines, including two oxygenated signals, four methylenes, two tertiary methyls, a vinyl methyl, a methoxy, two quaternary carbons, two tetrasubstituted olefins, two carbonyls, and a δ-lactone carbonyl carbon. These data were similar to those of picrasin B.27 Compound 2 was found to differ from picrasin B in that it lacks a secondary methyl group at C-4. This suggested that compound 2 is a des-4-methylpicrasin B derivative, as was confirmed by the COSY correlations between H2-3 (δH 1.38, 2.48, both m) and H2-4 (δH 1.46, 1.83, both m) and the HMBC correlations shown in Figure 1. The NOESY correlations of H318 to H-2/H3-19 and H3-19 to H-7/H-14/H3-18 revealed that these groups are β-oriented. In contrast, α-orientations of H-5 and H-9 were established based on their NOESY correlations (Figure 2). The absolute configuration at C-2 of 2 was established by the modified Mosher method.28,29 The (S)- and (R)-MTPA esters of 2 (2a and 2b) were obtained by treating 2 with (R)-(−)- and (S)-(+)-MTPA chloride, and their 1H NMR resonances were assigned based on their COSY correlations. The chemical shift differences (ΔSR = δS − δR) of the individual protons of 2a and 2b are shown in Figure 3. In the 1H NMR spectrum of the (S)-MTPA ester (2a), H2-3 and H2-4 appeared shielded, whereas H-9α was deshielded compared with the corresponding signals of the (R)-MTPA ester (2b). Thus, the H2-3 and H2-4 resonances of 2a were affected by the phenyl ring of the MTPA part, indicating that the absolute configuration of C-2 in 2 is S. Accordingly, the absolute structure of 2 (picrajavanicin B) was elucidated as shown. Picrajavanicin C (3) was obtained as an amorphous solid, and its molecular formula was determined as C21H28O6 from the HREIMS and 13C NMR data. The 1H and 13C NMR spectroscopic data (Tables 1 and 3) were very similar to those of 2 except for signals suggesting the presence of an additional methoxy group (δH 3.41, s; δC 57.8) in 3. The HMBC correlations from the methoxy group to an oxymethine carbon

Figure 3. Difference in the ΔSR (δS − δR) values for the (S)- and (R)MTPA esters of 2 in CDCl3.

(δC 79.2) and from an oxymethine proton [δH 4.44, dd (J = 11.6, 7.0 Hz, H-2)] and H2-4 to C-3 (δC 34.5) (Figure 2) suggested that this methoxy group is located at C-2. According to the observed NOESY correlations [H3-18 with H-2/H3-19; H-14 with H-7/H3-19; H-5 with H-9] and from a biogenetic perspective, the absolute configuration at C-2 of 3 was also assigned as S. Hence, the structure of 3 (picrajavanicin C) was established as shown. Picrajavanicin D (4) was isolated as an amorphous solid. The HREIMS of 4 revealed a molecular ion at m/z 378.1686, which, in combination with the 13C NMR data, corresponded to a molecular formula of C20H26O7. The 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 3) closely resembled those of 2 apart from the presence of a signal for an oxygenated quaternary carbon (δC 75.1) instead of a methine group [δH 2.42, dd (J = 12.2, 6.8 Hz); δC 47.1] in 2. The HMBC correlations from H-9 (δH 3.18, s), Me-13 (δH 1.94, s), and H215 to this oxygenated quaternary carbon suggested the presence of a tertiary hydroxy group at C-14. On the basis of the observed NOESY correlations, the configurations of all the stereocenters of 4 were concluded to be identical to those of 2. Therefore, the structure of 4 (picrajavanicin D) was proposed as shown. Picrajavanicin E (5) was obtained as an amorphous solid, and its molecular formula was determined as C22H28O7 from the HREIMS and 13C NMR data. The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 5 were similar to those of javanicin F20 except for the presence of an additional methoxy group (δH 3.91, s; δC 57.7) and a tetrasubstituted olefinic carbon (δC 158.4) in 5 and the lack of an olefinic methine proton at C-3 as in javanicin F.20 The HMBC correlations (Figure 1) from a methoxy signal (δH 3.91) and H2-4 [δH 2.28, dd (J = 17.7, 4.7 Hz, H-4α), 2.42, dd (J = 17.7, 11.3 Hz, H-4β)] to C-3 (δC 158.4) confirmed the methoxy group to be located at C-3. The NOESY correlations between H3-19 and H-7/H-14/H3-18 and between H-5 and H-9 indicated that the relative configurations of all the stereocenters were identical to those of javanicin F.20 Thus, the structure of 5 (picrajavanicin E) was characterized as depicted. To the best of our knowledge, this is the first report of a compound bearing a methoxy group at C-3 with the des-4-methylated picrasane skeleton. Picrajavanicin F (6) was obtained as an amorphous solid, and its molecular formula was determined as C22H28O8 from the HREIMS and 13C NMR data. The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 6 were similar to those of 5. The main differences included the absence of a D

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methine proton [δH 2.38, dd (J = 11.8, 7.0 Hz, H-14)] in 5 and the appearance of signals corresponding to the presence of an oxygen-bearing quaternary carbon (δC 75.1) and a tertiary hydroxy proton (δH 2.69, s) in 6. The HMBC correlations (Figure 1) from H-9 (δH 2.86, s), HO-14 (δH 2.69, s), H-15α [δH 3.04, d (J = 18.5 Hz)], H-15β [δH 2.99, d (J = 18.5 Hz)], and H3-19 (δH 1.15, s) to C-14 (δC 75.1) indicated the tertiary hydroxy group is located at C-14. On the basis of the observed NOESY correlations, the relative configurations at C-5, C-7, C8, C-9, C-10, and C-14 were found to be identical to those of 5. Accordingly, the structure of 6 (picrajavanicin F) was established as shown. Picrajavanicin G (7) was obtained as an amorphous solid, and its molecular formula was established as C21H24O7 from the HREIMS and 13C NMR data. The 1H NMR spectrum of 7 exhibited signals attributable to two olefinic protons [δH 5.56, t (J = 2.4 Hz, H-3), 6.13, s (H-14)], an oxymethylene [δH 4.49, 4.67, both d (J = 12.9 Hz, HOCH2-13)], an oxymethine [δH 4.31, t (J = 2.8 Hz, H-7)], two methines [δH 2.29, m (H-5), 3.14, s (H-9)], two methylenes [δH 2.30, m (H2-4), 1.90, 2.06, both m (H2-6)], two methoxy groups [δH 3.60 (MeO-2), 3.90 (MeO-12), both s], and two tertiary methyl groups [δH 1.30 (H3-19), 1.50 (H3-18), both s]. The 13C NMR spectrum showed 21 signals corresponding to two carbonyls [δC 192.0 (C-11), 196.6 (C-1)], a lactone carbonyl [δC 164.0 (C-16)], six olefinic carbons [δC 110.1 (C-3), 111.7 (C-15), 131.9 (C-13), 149.0 (C-2), 152.0 (C-12), 159.9 (C-14)], an oxymethine [δC 77.5 (C-7)], two methines [δC 36.1 (C-5), 53.7 (C-9)], an oxymethylene [δC 56.4 (HOCH2-13)], two methylenes [δC 27.1 (C-4), 28.4 (C-6)], two methoxy groups [δC 55.0 (MeO2), 60.5 (MeO-12)], two tertiary methy groups [δC 10.2 (C18), 23.0 (C-19)], and two quaternary carbons [δC 42.4 (C-8), 45.7 (C-10)]. The 1H and 13C NMR spectroscopic data of 7 (Tables 2 and 3) were similar to those of javanicin I17 except for the replacement of an oxymethylene group with a vinyl methyl group at C-13. The HMBC correlations (Figure 1) from the oxymethylene signals to C-12, C-13, and C-14 confirmed that the oxymethylene group is located at C-13. The NOESY correlations between H-5 and H-9 suggested that these protons have α orientations. Furthermore, the NOESY correlations between H-7 and H3-19 and between H3-18 and H3-19 revealed that they have β orientations. Hence, the structure of 7 (picrajavanicin G) was determined as shown. This is the first report of the presence of a hydroxylated methylene group at C13 in a des-4-methylated picrasane quassinoid. The three known compounds, javanicins B,18 F,20 and I,17 were identified by comparing their observed and reported NMR data. However, it was found that some of the 13C NMR assignments of javanicin I17 should be reassigned, and this was conducted using 1D and 2D NMR spectroscopic analyses. All the isolates, consisting of picrajavanicins A−G (1−7) and javanicins B,18 F,20 and I,17 were tested for their antiproliferative activities against five human cancer cell lines by the aforementioned procedure.8 However, none of the isolated compounds exhibited inhibitory activity against any of the cancer cells used (IC50 values >10 μM). According to the previous reports,9−26 P. javanica contains quassinoids and βcarboline alkaloids. The strong antiproliferative activity of the crude extracts may be due to the actions of other quassinoids and β-carboline alkaloids.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P2100 polarimeter. UV spectra were measured on a Shimadzu UV-160 A. Infrared spectra were recorded using KBr pellets on a JASCO FT/IR-460 Plus spectrometer. The NMR spectra were recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR) using a Varian Unity 600 spectrometer. Chemical shift values were expressed in δ (ppm) downfield from tetramethylsilane (TMS) as an internal standard. The mass spectra, including HRMS data, were recorded on a JEOL MStation JMS-700 spectrometer. Open-column chromatography was performed with normal-phase silica gel (silica gel 60N, Spherical, neutral, 40−50 μm, Kanto Chemical Co., Inc., Japan) and Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto, Japan). TLC was conducted on precoated silica gel 60F254 and RP-18 F254 plates (Merck, 0.25 or 0.50 mm thickness). The cell lines, human lung A549 adenocarcinoma (RCB0098), human cervix HeLa adenocarcinoma (RCB0007), human pancreatic PANC-1 epithelioid carcinoma (ATCC CRL-1469), human pancreatic PSN-1 adenocarcinoma (ATCC CRM-CRL-3211), and human breast MDA-MB-231 adenocarcinoma (ATCC HTB-26), were available and maintained in our laboratory.30−32 The cell culture flasks and 96-well plates were supplied by Corning Inc. (Corning, NY, USA). An SH-1200 microplate reader (Corona, Hitachinaka, Japan) was used to measure the absorbance of the cells in the antiproliferative activity assay. Plant Material. The dried bark of P. javanica was collected from Kayin State, Myanmar, in May 2014 and identified by Dr. Khin Cho Cho Oo, an authorized botanist of the Department of Botany, University of Yangon. A voucher specimen (TMPW 28303) was deposited at the Museum for Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan. Extraction and Isolation. The dried bark of P. javanica (550 g) was extracted with CHCl3 under sonication (2 L, 1.5 h, × 3) at 30 °C, and the solvent was evaporated under reduced pressure to yield a CHCl3 extract (17 g). The CHCl3 extract (15 g) was chromatographed on silica gel with EtOAc−n-hexane and EtOAC−MeOH solvent systems to give eight fractions [1: EtOAc−n-hexane (1:9) eluate, 2.20 g; 2: EtOAc−nhexane (1:4) eluate, 1.88 g; 3: EtOAc−n-hexane (3:7) eluate, 690 mg; 4: EtOAc−n-hexane (2:3) eluate, 1.50 g; 5: EtOAc−n-hexane (1:1) eluate, 745 mg; 6: EtOAc−n-hexane (2:1) eluate, 635 mg; 7: EtOAc− MeOH (20:1) eluate, 3.7 g; 8: EtOAc−MeOH (10:1) eluate, 1.10 g]. Fraction 7 (3.7 g) was rechromatographed on a silica gel column chromatograph with n-hexane−CHCl3−EtOAc (1:1:1) to give four subfractions [7-1: 131 mg; 7-2: 534 mg; 7-3: 1.80 g; 7-4: 550 mg]. Purification of subfraction 7−-2 using Cosmosil 75C18-OPN with MeCN−acetone−MeOH−H2O (1:1:1:1) followed by a normal-phase preparative TLC with toluene−CHCl3−EtOAc (1:1:1) afforded picrajavanicin A (1, 18 mg) and javanicin I (40 mg). Subfraction 7-3 was purified by Cosmosil 75C18-OPN with MeOH−H2O (3:1) to yield javanicins B (100 mg) and F (1.2 g). Purification of subfraction 7-4 using Cosmosil 75C18-OPN with MeOH−H2O (3:1) followed by a normal-phase preparative TLC with n-hexane−EtOAc− propan-2-ol (15:4:1) (development four times) provided picrajavanicins B (2, 24 mg), C (3, 5 mg), D (4, 2 mg), E (5, 19 mg), F (6, 6 mg), and G (7, 4 mg). Picrajavanicin A (1): pale yellow, amorphous solid; [α]25D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 264 (3.57) nm; IR (KBr) νmax 2938, 2362, 1730, 1702, 1666, 1632, 1461, 1236, 1036, 970 cm−1; 1H and 13C NMR data, see Tables 1 and 3; EIMS m/z 388 [M]+ (100); HREIMS m/z 388.1525 [M]+ (calcd for C21H24O7 388.1522). Picrajavanicin B (2): amorphous solid; [α]25D −20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.78) nm; IR (KBr) νmax 3471, 2935, 2361, 1733, 1685, 1618, 1448, 1378, 1242, 1035, 897 cm−1; 1H and 13 C NMR data, see Tables 1 and 3; EIMS m/z 362 [M]+ (100); HREIMS m/z 362.1736 [M]+ (calcd for C20H26O6 362.1729). Picrajavanicin C (3): amorphous solid; [α]25D −66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (4.04) nm; IR (KBr) νmax 3470, 2836, 1733, 1685, 1638, 1355, 1298, 1122, 964 cm−1; 1H and 13C NMR data, E

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see Tables 1 and 3; EIMS m/z 376 [M]+ (100); HREIMS m/z 376.1890 [M]+ (calcd for C21H28O6 376.1886). Picrajavanicin D (4): amorphous solid; [α]25D −51 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.75) nm; IR (KBr) νmax 3439, 2927, 1735, 1693, 1618, 1447, 1262, 1046, 958 cm−1; 1H and 13C NMR data, see Tables 1 and 3; EIMS m/z 378 [M]+ (60); HREIMS m/z 378.1686 [M]+ (calcd for C20H26O7 378.1679). Picrajavanicin E (5): amorphous solid; [α]25D +41 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 264 (3.94) nm; IR (KBr) νmax 3468, 2935, 2836, 1734, 1685, 1618, 1355, 1273, 1058, 983 cm−1; 1H and 13C NMR data, see Tables 2 and 3; EIMS m/z 404 [M]+ (100); HREIMS m/z 404.1830 [M]+ (calcd for C22H28O7 404.1835). Picrajavanicin F (6): amorphous solid; [α]25D −24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 264 (3.76) nm; IR (KBr) νmax 3441, 2926, 2853, 1737, 1694, 1621, 1295, 1242, 989 cm−1; 1H and 13C NMR data, see Tables 2 and 3; EIMS m/z 420 [M]+ (90); HREIMS m/z 420.1788 [M]+ (calcd for C22H28O8 420.1784). Picrajavanicin G (7): amorphous solid; [α]25D +141 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 303 (3.76) nm; IR (KBr) νmax 3535, 2938, 1718, 1698, 1686, 1636, 1450, 1240, 1097, 959 cm−1; 1H and 13C NMR data, see Tables 2 and 3; EIMS m/z 388 [M]+ (97); HREIMS m/z 388.1531 [M]+ (calcd for C21H24O7 388.1522). Javanicin I: 13C NMR (CDCl3, 150 MHz) δ 196.6 (C-1), 191.2 (C11), 164.2 (C-14), 162.7 (C-16), 152.1 (C-12), 149.0 (C-2), 130.8 (C13), 110.8 (C-15), 109.9 (C-3), 77.5 (C-7), 59.7 (MeO-12), 54.9 (MeO-2), 53.5 (C-9), 45.6 (C-10), 42.5 (C-8), 36.2 (C-5), 28.3 (C-6), 27.1 (C-4), 23.2 (C-19), 11.3 (Me-13), 10.2 (C-18). Preparation of the (S)- and (R)-MTPA Esters of 2. Two equal portions of 2 (each 1.5 mg) were dissolved in dichloromethane (300 μL) to which DMAP (5 mg) and (R)-(−)- or (S)-(+)-MTPA chloride (5 μL) were added. The mixtures were stirred at room temperature for 20 min. A saturated aqueous solution of NH4Cl was added to the solution, and the aqueous layer was extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography with n-hexane−EtOAc (2:1) to generate 1.3 mg each of (S)-MTPA ester (2a) and (R)-MTPA ester (2b). The 1H NMR data of 2a obtained were as follows (CDCl3, 600 MHz): δH 1.226 (s, H3-19), 1.551 (m, H-4α), 1.554 (s, H3-18), 1.738 (m, H-3α), 1.805 (m, H-6α), 1.873 (m, H-5α), 1.908 (s, MeO-13), 1.917 (m, H-4β), 1.977 (m, H-6β), 2.298 (m, H-3β), 2.412 (dd, J = 12.0, 6.9 Hz, H14β), 2.618 (dd, J = 18.9, 12.0 Hz, H-15α), 2.996 (dd, J = 18.9, 6.9 Hz, H-15β), 3.223 (s, H-9α), 3.665 (s, MeO-12), 4.257 (t, J = 2.9 Hz, H7β), 5.958 (dd, J = 12.6, 7.5 Hz, H-2β); ESIMS m/z 579 [M + H]+. The 1H NMR data of 2b obtained were as follows (CDCl3, 600 MHz): δH 1.221 (s, H3-19), 1.554 (s, H3-18), 1.609 (m, H-4α), 1.807 (m, H3α), 1.807 (m, H-6α), 1.859 (m, H-5α), 1.900 (s, MeO-13), 1.942 (m, H-4β), 1.985 (m, H-6β), 2.403 (dd, J = 12.0, 6.9 Hz, H-14β), 2.450 (m, H-3β), 2.596 (dd, J = 18.9, 12.0 Hz, H-15α), 2.980 (dd, J = 18.9, 6.9 Hz, H-15β), 3.202 (s, H-9α), 3.549 (s, MeO-12), 4.254 (t, J = 2.6 Hz, H-7β), 5.970 (dd, J = 12.0, 7.0 Hz, H-2β); ESIMS m/z 579 [M + H]+. In Vitro Antiproliferative Activity. The in vitro antiproliferative activities of the crude extracts and isolated compounds were determined by the aforementioned procedure,8 and the data were expressed as the IC50 value. 5-Fluorouracil was used as a positive control. The IC50 values for the antiproliferative activities of 5fluorouracil against A549, HeLa, PANC-1, PSN-1, and MDA-MB-231 were 9.0, 6.4, 0.4, 8.7, and 1.1 μM, respectively.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +95 9 2502 88756. E-mail: [email protected] (N. N. Win). *Tel: +81 76 434 7625. Fax: +81 76 434 5059. E-mail: [email protected] (H. Morita). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (H.M. and I.A.), grants from the Tokyo Biochemical Research Foundation (H.M. and N.N.W.), the Kobayashi International Scholarship Foundation (H.M.), and a Grant-in-Aid for the 2015 Cooperative Research Project II from the Institute of Natural Medicine, University of Toyama (Ismail and H.M). We also thank Ms. Y. Okamoto (Tokushima Bunri University, Tokushima, Japan) for her technical assistance with the MS measurements.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00824. 1 H and 13C NMR, 1H−1H COSY, HMQC, HMBC, NOESY, and HREIMS spectra of compounds 1−7 (Figures S1−S49) (PDF) F

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