Acylated Lignans Isolated from Brachanthemum gobicum and Their

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Acylated Lignans Isolated from Brachanthemum gobicum and Their Trypanocidal Activity Batsukh Odonbayar,† Toshihiro Murata,*,† Keisuke Suganuma,‡,§ Yoshinobu Ishikawa,∥ Buyanmandakh Buyankhishig,† Javzan Batkhuu,⊥ and Kenroh Sasaki† †

Department of Pharmacognosy, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan National Research Center for Protozoan Diseases and §Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan ∥ School of Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan ⊥ School of Engineering and Applied Sciences, National University of Mongolia, POB-617, Ulaanbaatar-46A, 14201, Mongolia

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‡

S Supporting Information *

ABSTRACT: Eight isovaleryllignans (1−4 and 8−11), three isovalerylphenylpropanoids (5−7), three known lignans (12− 14), and four known compounds were isolated from an extract of the aerial part of Brachanthemum gobicum. The structures of the isolated compounds were elucidated based on NMR and MS data analyses. The enantiomers of compounds 1−3, 5, 8, and 9 were isolated using chiral-phase HPLC, and the absolute configurations of 1a/1b−3a/3b, 5a/5b, 8a/8b, and 9a/9b were elucidated from their optical rotations and ECD spectra; the other lignans were assumed to be racemic or scalemic by chiralphase HPLC analyses and optical rotation data. Some of the acylated lignans (racemic mixtures) (1−4, 8, 9, and 12−14) exhibited moderate inhibitory activities against Trypanosoma congolense, the causative agent of nagana disease in animals.

Brachanthemum gobicum Krasch belongs to the family Asteraceae and is known as “Umkhii tulee-awful firewood” among Mongolians. This shrub is distributed in the Gobi Desert and has adapted to extreme climatic conditions, such as dry winds, irregular water availability, high UV irradiation, and dramatic changes in temperature, and it is valuable to the local population,1 who use it as livestock fodder. Its leaves, stems, and aqueous extracts are burned, and the smoke is used to remove external parasites, such as the parasitic louse Linognathus, from domesticated sheep.2 Phytochemical studies of B. gobicum have led to the identification of sesquiterpenoids from its essential oils.3 In this study, eight isovaleryllignans (1−4 and 8−11), three isovalerylphenylpropanoids (5−7), three known lignans (12−14), and four known compounds were isolated from the aerial parts of B. gobicum. Racemic mixtures of lignans, such as those of neolignans and oxyneolignans, have been discussed in recent reports, including racemic mixtures isolated from Phyllanthus glaucus (Euphorbiaceae),4 Gardenia ternifolia (Rubiaceae),5 and Picrasma quassioides (Simaroubaceae).6 Compounds 1−3, 5, 8, and 9 were isolated as pairs of enantiomers; these compounds were separated using chiral-phase HPLC, and their absolute © XXXX American Chemical Society and American Society of Pharmacognosy

configurations were determined from their optical rotations and electronic circular dichroism (ECD) spectra. Trypanosoma congolense is the most pathogenic trypanosome affecting vertebrates. It causes nagana disease in various animal species in the sub-Saharan region. An estimated 55 million cattle are at risk of contracting this disease, and it kills over 3 million animals every year. Few drugs are available for the treatment of this disease, and drug-resistant trypanosomosis has already been reported. New treatments are expected to be highly effective for controlling nagana. Non-tsetse-transmitted animal trypanosomoses (NTTAT) also occur worldwide, and these include dourine and surra, which are caused by T. equiperdum and T. evansi, respectively, and have been reported in Mongolia.7 Given that trypanocidal compounds may be effective in treating NTTAT, in the present study, we sought to identify the components of traditional Mongolian medicinal plants with inhibitory activity against T. congolense, with a specific focus on compounds isolated from B. gobicum. Received: August 8, 2018

A

DOI: 10.1021/acs.jnatprod.8b00670 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

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RESULTS AND DISCUSSION An acetone−H2O extract of the aerial parts of B. gobicum was dissolved in H2O and partitioned into Et2O. The Et2O fraction was subjected to silica gel column chromatography. Forty fractions were separated, and some were purified by reversedphase HPLC. Eight isovaleryllignans (1−4, 8−11), three isovalerylphenylpropanoids (5−7), and seven known compounds were obtained, and their chemical structures were determined spectroscopically. The known compounds were identified as dimeric coniferyl acetate (12),8 3-acetoxymethyl5-[(E)-2-formylethen-1-yl]-2-(4-hydroxy-3-methoxyphenyl)-7methoxy-2,3-dihydrobenzofuran (13),9 balanophonin (14),10 coniferyl isovalerate,11 trans-ferulic acid,12 4-hydroxy-3-methoxycinnamaldehyde,13 and vanillin14 by comparing their NMR and MS data with published data. The molecular formula of compound 1 was determined as C30H38O8 based on high-resolution (HR) FABMS (m/z 549.2458 [M + Na]+; calcd for C30H38O8Na, 549.2464) and 13 C NMR data. In the 1H NMR data of 1 (Table 1), an AMX2 spin system [δH 6.59 (1H, brd, J = 16.0 Hz, H-7), 6.15 (1H, dt, J = 16.0, 6.5 Hz, H-8), and 4.72 (2H, dd, J = 6.5, 1.0 Hz, H-9)] and an AMXY spin system [δH 5.47 (1H, d, J = 7.5 Hz, H-7′), 4.42 (1H, dd, J = 11.5, 5.5 Hz, H-9′), 4.33 (1H, dd, J = 11.5, 7.5 Hz, H-9′), and 3.76 (1H, dt, J = 7.5, 5.5 Hz, H-8′)] were observed. Their corresponding six carbons were identified based on the HMQC spectrum [δC 134.2, C-7; 121.3, C-8; 88.9, C-7′; 65.0, C-9′; 65.0, C-9; 50.4, C-8′]. The 13C NMR spectrum of 1 (Table 1) showed 12 aromatic carbons [δC 148.2, C-4; 146.7, C-3′; 145.9, C-4′; 144.4, C-3; 132.2, C-1′; 130.5, C-1; 127.8, C-5; 119.6, C-6′; 115.3, C-6; 114.3, C-5′; 110.6, C-2; and 108.6, C-2′]. These features indicated the presence of two phenylpropanoid moieties. A dihydrobenzofuranlignan fragment was constructed from the two- and three-

bond HMBC correlations of H-7′ with C-4, C-2′, and C-6′; H8′ with C-4, C-5, and C-1′; and H-7 with C-2 and C-6 (Table 1).5 The coupling constant between H-7 and H-8 (16.0 Hz) indicates that the C-7/C-8 double bond is in the trans configuration.5 The five overlapping aromatic ring proton resonances from δH 6.80 to δH 6.95 (5H, overlapping, H-2, 6, 2′, 5′, 6′) and two methoxy proton resonances at δH 3.90 (3H, s, 3-OMe) and 3.86 (3H, s, 3′-OMe) indicate the presence of a dimeric coniferyl moiety, as these fragments are similar to those found for dimeric coniferyl acetate (12).8 Furthermore, unlike the two acetyl groups present in 12, the presence of two isovaleric acid moieties [(δH 2.16, 2H, brd, J = 6.5 Hz, H-2″; 2.03, 1H, m, H-3′′; 0.90, 6H, d, J = 6.5 Hz, H-4″ and 5′′) and (δH 2.23, 2H, brd, J = 7.0 Hz, H-2‴; 2.14, 1H, m, H-3″′; 0.98, 6H, d, J = 6.5 Hz, H-4‴ and 5″′)] were indicated by the 1H NMR data of 1.8 Although no spectroscopic data were reported, an isomer of 1 with the cis-oriented dihydrobenzofuran moiety has previously been reported from Pyrethrum santolinoides.15 The trans configuration of the dihydrobenzofuran moiety of 1 was established based on the NOE correlations from H-7′ to H-9′ and from H-2′ and H-6′ to H-8′ (Figure 1). A lignan with a molecular structure identical to that of 1 has previously been reported;16 however, its absolute configuration was not determined. The optical rotation of 1 was 0, and there were no Cotton effects in its ECD spectrum, suggesting that 1 is a racemic mixture. Because the enantiomers of 1 could not be separated on an ODS column, 1 was subjected to chiral-phase HPLC (Daicel Chiralpak AS-H), and the enantiomers of 1 (1a and 1b) were obtained (Figure 2). The specific rotation of 1a ([α]25D −17) was the inverse of that of 1b ([α]25D +15). The ECD curve of 1a at ∼200−350 nm was the mirror image of that of 1b (Figure 3). The positive Cotton effect observed at 202 nm B



Article

Table 1. NMR Spectroscopic Data for Compounds 1 and 8−10 1b position

δC

δH (J in Hz)

8a HMBC

δC

1 2

130.5 110.6

3 4 5 6

144.4 148.2 127.8 115.3

7

134.2

8

121.3

9

65.0

1′ 2′

132.2 108.6

3′ 4′ 5′

146.7 145.9 114.3

6.80−6.95c

6′

119.6

6.80−6.95c

7′

120.2

7′

88.9

5.47 d (7.5)

90.7

8′

50.4

9′

65.0

3.76, dt (7.5, 5.5) 4.42, dd (11.5, 5.5) 4.33, dd (11.5, 7.5)

4, 1′, 2′, 6′, 8′, 9′ 4, 5, 1′, 7′, 9′

1″ 2″

172.9 43.3

3″ 4″

3, 6, 7

129.9 114.4

6.89c

2, 7

146.1 152.8 130.1 119.7

6.59, brd (16.0) 6.15, dt (16.0, 6.5)

2, 6, 9

155.8

1, 9

127.3

4.72, dd (6.5, 1.0)

7, 8, 1″

196.1

6.80−6.95c

7′

133.1 110.8

6.87c

149.2 148.2 116.3

9a

δH (J in Hz)

δC

HMBC 3, 4, 6, 7

133.0 114.2

7.27, brs

2, 4, 7

146.4 155.5 130.1 122.0

7.62, d (16.0) 6.69, dd (16.0, 8.0) 9.59, d (8.0)

2, 6, 9

192.6

7.25, brs

6.95, d (2.0)

δH (J in Hz)

10a δH (J in Hz)

HMBC

δC

1, 3, 4, 6, 7

missing 115.6

7.58, brs

4, 6, 7

7.51, d (1.0) 9.79, s

1, 2, 4, 7

145.4 153.7 129.4 120.6

7.62, brs

2, 4, 7

1, 2, 6

170.0

6.95, d (2.0)

1′, 4′, 6′, 7′

6.95, d (2.0)

4′, 6′, 7′

6.79, d (8.0) 6.84, dd (8.0, 2.0) 5.56, d (7.0) 3.79c

1′, 3′

7.50, d (1.0)

HMBC

1 8

4′, 6′, 7′

132.8 110.9

1′, 3′

149.3 148.3 116.4

4′, 6′, 7′

120.3

4, 1′, 2′, 6′, 8′, 9′ 5, 1′, 9′

91.2

51.4

6.79, d (8.0) 6.83, dd (8.0, 2.0) 5.57, d (7.0) 3.83c

66.0

4.44, m

5, 7′, 8′, 1‴

2.14, d (8.5) 1.95, m 0.86, d (7.0) 0.85, d (7.0)

1″, 3″, 4″, 5″ 2″, 4″, 5″ 2″, 3″, 5″

26.8 22.7

2″, 3″, 4″

22.7

3 3′

56.7 56.5

51.1 66.0

6.79, d (8.0) 6.84, dd (8.0, 2.0) 5.62, d (7.0) 3.87, m 4.45, d (6.0)

133.1 110.8

1′, 3′

149.3 148.2 116.3

2′, 4′, 7′

120.3

4, 1′, 2′, 6′, 8′, 9′ 4, 5, 1′, 7′, 9′ 5, 7′, 8′,1‴

90.8 51.3 66.2

2′, 4′, 7′ 1′, 2′, 6′, 9′ 5, 7′, 9′

4.42, d (6.5)

5, 7′, 8′, 1‴

2.15, m

1″, 3″, 4″, 5″ 4″, 5″ 2″, 3″, 5″

5, 7′, 8′,1‴ 174.4 44.1

25.6 22.4

2.16, brd (6.5) 2.03, m 0.90, d (6.5)

1″, 3″, 4″, 5″ 2″, 4″, 5″ 2″, 3″, 5″

26.8 22.6

5″

22.4

0.90, d (6.5)

2″, 3″, 4″

22.6

1‴ 2‴

173.0 43.5

3‴ 4‴ 5‴ 3-OMe 3′-OMe

25.7 22.5 22.5 56.0 56.0

2.23, brd (7.0) 2.14, m 0.98, d (6.5) 0.98, d (6.5) 3.90, s 3.86, s

1‴, 3‴, 4‴, 5‴ 2‴ 2‴, 3‴, 5‴ 2‴, 3‴, 4‴ 3 3′

56.8 56.4

3.91, s 3.83, s

174.4 44.1

2.15, m 1.95, m 0.86, d (7.0) 0.85, d (7.0)

3.92, s 3.82, s

174.5 44.1

1″, 3″, 4″, 5″ 4″, 5″ 2″, 3″, 5″

26.8 22.7

2″, 3″, 4″

22.7

3 3′

56.7 56.5

1.96, m 0.87, d (6.5) 0.86, d (6.5)

3.90, s 3.83, s

2″, 3″, 4″

3 3′

a

In methanol-d4 solution. bIn CDCl3 solution. cUnclear signal pattern due to overlapping.

Hz, H-5; 3.77, 3H, s, 3-OMe) and (δH 6.95, 1H, d, J = 2.0 Hz, H-2′; 6.84, 1H, dd, J = 8.0, 2.0 Hz, H-6′; 6.73, 1H, d, J = 8.0 Hz, H-5′; 3.85, 3H, s, 3′-OMe)]. In addition, resonances of two methines (δH 4.14, 1H, d, J = 12.0 Hz, H-7; 3.90, 1H, ddd, J = 12.0, 8.0, 3.5 Hz, H-8), a methylene (δH 4.07, 1H, dd, J = 10.5, 3.5 Hz, H-9a; 3.94, 1H, dd, J = 10.5, 8.0 Hz, H-9b), an olefinic methylene (δH 6.49, 1H, s, H-7′a and 6.14, 1H, s, H7′b), and a formyl group (δH 9.36, 1H, s, H-9′) were observed in the spectra of this compound. In the 1H−1H COSY spectrum, H-8 was coupled with H-7, H-9a, and H-9b, indicating the presence of a C-7-C-9 propanyl moiety. The three-bond HMBC correlations of H-2, H-6, H-2′, and H-6′ with C-7 (δC 52.8) and the NOE correlations from H-7 to H-2,

and the negative Cotton effect at 289 nm indicated a (7′R,8′S)-absolute configuration for 1a.4 Similarly, the negative and positive Cotton effects at 202 and 288 nm, respectively, indicated a (7′S, 8′R)-absolute configuration for 1b. On the basis of these data, the structures of 1a and 1b were assigned as (7′R,8′S)-brachangobinan A and (7′S,8′R)-brachangobinan A, respectively (Figure 2). Compound 2 was assigned the molecular formula C25H30O7 based on its HRFABMS (m/z 465.1901 [M + Na]+; calcd for C25H30O7Na, 465.1889) and 13C NMR data. The 1H NMR data of 2 (Table 2) showed the presence of two sets of 4hydroxy-3-methoxyphenyl moieties [(δH 6.77, 1H, d, J = 2.0 Hz, H-2; 6.62, 1H, dd, J = 8.0, 2.0 Hz, H-6; 6.59, 1H, d, J = 8.0 C



Article

Figure 1. Structures and key HMBC and NOE correlations of 1, 2, 4, 5, 8, and 11.

Figure 3. ECD spectra of compounds 1a/1b, 2a/2b, 5a/5b, 8a/8b, and 9a/9b.

secolignan (−)-lucidenal.17 The presence of an isovaleric acid moiety of 2, instead of the acetyl group of (−)-lucidenal, was indicated by the 1H NMR resonances at δH 2.09 (2H, brd, J = 8.0 Hz, H-2″), 1.97 (1H, m, H-3′′), and 0.90 (3H, d, J = 6.5 Hz, H-4″ and 5′′). The small specific rotation of 2 ([α]D −4) and the apparent lack of a Cotton effect in its ECD spectrum indicate that 2 is a scalemic mixture. Similar to 1, compounds 2a and 2b were isolated as a pair of enantiomers by chiralphase HPLC separation of 2. The ECD curve of 2a at ∼200− 400 nm is the mirror image of that of 2b. The specific rotation of 2a ([α]25D −54) was the inverse of that of 2b ([α]25D +32). Based on its structural similarity to (−)-lucidenal,17 2a was determined to have an 8R configuration. Therefore, 2a and 2b were identified as (8R)-brachangobinan B and (8S)-brachangobinan B, respectively, and their structures are shown in Figure 2. The 1H and 13C NMR and HREIMS data (m/z 544.2660 [M+]; calcd for C30H40O9, 544.2673) of 3 showed that it was 1-(4-hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-isovaleroyloxy-1propenyl]-2-methoxyphenoxy}-propan-3-isovaleroyloxy-1-ol, which has previously been isolated from Nannoglottis carpesioides.18 The coupling constant between H-7 and H-8 of 3 (J = 16.0 Hz) indicated a trans-coniferyl alcohol moiety. The large vicinal coupling constant between H-7′ and H-8′ indicated a threo configuration (J = 8.0 Hz) rather than an erythro configuration (J = 3.2 Hz),19,20 which is consistent with the reported compound.18 The specific rotation of 3 ([α]23D 0) suggests that 3 is a (7′RS,8′RS) racemic mixture. Similar to the racemate of 1, chiral-phase HPLC separation of 3 provided the two enantiomers 3a and 3b. The (7′R,8′R) absolute

Figure 2. Structures of 1a/1b-3a/3b, 5a/5b, 8a/8b, and 9a/9b.

H-6, H-2′, and H-6′ constructed bonds between the two 4hydroxy-3-methoxyphenyl moieties and the double benzylic carbon (C-7) (Figure 1). The HMBC correlations of the formyl (H-9′) and olefinic (H-7′a and H-7′b) protons with C8 (δC 41.8) and the NOE correlations from H-7′a and H-7′b to the formyl proton suggested a structure of a secolignan, as shown in Figure 2. These data are similar to those of the D



Article

Table 2. NMR Spectroscopic Data for Compounds 2−4 and 11 2a position

δC

1 2 3 4 5 6

136.2 112.6 148.8 145.9 116.0 121.6

7

52.8

8

41.8

9

66.8

1′ 2′

136.3 112.4

3′ 4′ 5′ 6′

149.2 146.3 116.4 121.3

7′

138.6

8′

151.4

9′

196.2

3b

δH (J in Hz)

HMBC

6.77, d (2.0)

4, 6, 7

6.59, d (8.0) 6.62, dd (8.0, 2.0) 4.14, d (12.0)

3, 4 4, 7

3.90, ddd (12.0, 8.0, 3.5) 4.07, dd (10.5, 3.5) 3.94, dd (10.5, 8.0)

6.95, d (2.0)

δC 132.3 109.9 150.8 148.0 120.1 120.4

1, 2, 6, 8, 9, 1′, 2′, 6′ 7

133.6 122.8

7’

64.7

4b

δH (J in Hz)

HMBC

δC

6.97, d (2.0)

4, 7

7.08, d (8.0) 6.94, dd (8.0, 2.0) 6.60, brd (16.0) 6.21, dt (16.0, 6.5) 4.73, dd (6.5, 1.0)

1, 3, 4 4, 7

131.5 110.2 150.1 146.9 117.3 119.8

1, 2, 6, 9

133.7

1, 9

122.2

7, 8, 1″

64.8

4′, 6′, 7′

127.7 110.8 146.6 151.1 114.1 124.4

δH (J in Hz)

HMBC

δC

δH (J in Hz)

6.88, s

3, 4

6.82c 6.82c

3 4

6.54, d (16.0) 6.14, m

2, 6, 9

190.7

9.74, s

1, 2

4.69, brd (8.0)

7, 8, 1″

7.67, d (2.0)

1′, 3′, 4′, 6′, 7′

128.7 110.4

7.62, d (2.0)

3′, 4′, 7′

6.86, d (9.0) 7.65, dd (9.0, 2.0)

1′, 3′, 4′ 2′, 4′, 7′

5.44, dd (8.5, 6.0) 4.80, dd (11.0, 8.5) 4.40, dd (11.0, 6.0)

4, 5, 7′, 9′

172.9 43.2

2.12, d (5.5)

25.6

2.02, m

1″, 3″, 4″, 5″ 2″, 4″, 5″ 2″, 3″, 5″ 2″, 3″, 4″

7.33, d (1.5)

6, 7

7.35, d (1.5)

2, 4, 7

1, 9

7, 3′, 4′, 6′

131.1 109.2

6.87, d (8.0) 6.87, brd (8.0)

3′ 2′, 4′, 7′

6.91, brs

6.73, d (8.0) 6.84, dd (8.0, 2.0) 6.49, s

1′, 3′ 7, 2′, 4′ 8

74.3

4.85, d (8.0)

1′, 2′, 6′, 8′, 9′

193.8

6.14, s

8, 8′, 9′ 86.0

4.22, ddd (8.0, 5.5, 3.5) 4.29, dd (12.5, 3.5) 4.00, dd (12.5, 5.5)

7′

80.2

7′

64.3

8, 7′, 8′

62.8

6.89, d (8.0) 7.79, dd (8.0, 2.0)

1′, 3′ 2′, 4′, 7′

195.7

5.62, dd (7.0, 4.0) 4.66, dd (11.5, 4.0) 4.51, dd (11.5, 7.0)

9′

172.9 43.1

2.22, d (7.0)

25.7

2.13, m

1″, 3″, 4″, 5″ 2″, 4″, 5″

7′, 8′, 1‴

146.6 150.8 114.0 124.1

43.5 64.2

7′, 8′, 1‴

1″ 2″

174.5 44.2

2.09, brd (8.0)

1″, 3″,4″, 5″

173.0 43.4

2.24, d (7.0)

3″

26.8

1.97, m

2″, 4″, 5″

25.6

2.15, m

1″, 3″, 4″, 5″ 2″, 4″, 5″

4″

22.8

0.90, d (6.5)

2″, 3″, 5″

22.4

0.93, d (7.0)

2″, 3″, 5″

22.3

0.96, d (7.0)

2″, 3″, 5″

22.3

0.87, d (6.5)

5″

22.8

0.90, d (6.5)

2″, 3″, 4″

22.4

0.93, d (7.0)

2″, 3″, 4″

22.3

0.96, d (7.0)

2″, 3″, 4″

22.3

0.87, d (6.5)

1‴ 2‴

172.7 43.2

2.17, d (7.0)

43.4

2.17 d (7.0)

3‴

25.7

2.06, m

1‴, 3‴, 4‴, 5‴ 2‴, 4‴, 5‴

25.5

2.06, m

4‴

22.4

0.98, d (7.0)

2‴, 3‴, 5‴

22.4

0.90, d (7.0)

5‴

22.4

0.98, d (7.0)

2‴, 3‴, 4‴

22.4

0.90, d (7.0)

1‴, 3‴, 4‴, 5‴ 2‴, 4‴, 5‴ 2‴, 3‴, 5‴ 2‴, 3‴, 4‴

55.9 55.8

3.92, s 3.87, s

3 3′

55.8 56.1

3.78, s 3.95, s

56.4 56.0

3.97, s 3.93, s

3-OMe 3′-OMe

56.4 56.5

3.77, s 3.85, s

HMBC

129.7 107.5 147.3 148.7 122.0 126.5

7′, 1″

146.7 145.7 114.3 120.1

9.36, s

11b

3 3′

3 3′

7′, 1‴

3 3′

a

In methanol-d4 solution. bIn CDCl3 solution. cUnclear signal pattern due to overlapping.

optical rotation, similar to that of 3a.18 Therefore, 3a was identified as (7′R,8′R)-threo-(E)-4′,7′-dihydroxy-9,9′-diisovaleroyloxy-3,3′-dimethoxy-7-en-4-O-8′-neolignan, and 3b was defined as (7′S,8′S)-brachangobinan C. The molecular formula of 4 was established as C30H38O9 based on its HREIMS (m/z 542.2521 [M+]; calcd for C30H38O9, 542.2516), which corresponds to two fewer H

configuration of the reported compound was determined from its negative ECD Cotton effect at 226 nm.18 However, no clear Cotton effects were observed in the ECD spectra of 3a and 3b. Compounds 3a and 3b have specific rotations of [α]24D +7 and [α]24D −7, respectively. (7′R,8′R)-threo-(E)-4′,7′-Dihydroxy9,9′-diisovaleroyloxy-3,3′-dimethoxy-7-en-4-O-8′-neolignan has been reported previously and was found to have a positive E



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Table 3. NMR Spectroscopic Data for Compounds 5−7 5a position

δC

1 2 3 4 5 6 7 8 9

130.9 111.9 149.2 147.8 116.1 121.7 86.0 74.2 66.4

1′ 2′ 3′ 4 5′ 3-OMe 7-OMe

174.7 44.1 26.8 22.7 22.7 56.4 56.9

δH (J in Hz)

6a HMBC (H to C)

6.89, d (1.5)

3, 4, 6, 7

6.78, d (8.0) 6.75, dd (8.0, 1.5) 4.07, d (7.0) 3.85b 3.99, dd (11.5, 3.5) 3.78, dd (11.5, 6.0)

1, 3 2, 4, 7 1, 2, 6, 8, 7-OMe

2.18, 2.04, 0.94, 0.94, 3.85, 3.22,

brd (7.0) m d (6.5) d (6.5) s s

8, 1′ 8, 1′ 1′, 2′, 2′, 2′, 3 7

3′, 4′, 3′, 3′,

4′, 5′ 5′ 5′ 4′

δC 127.9 112.5 149.2 154.0 116.0 125.1 198.2 72.4 67.7 174.5 44.0 26.8 22.6 22.6 56.5

δH (J in Hz)

7a HMBC (H to C)

7.61, d (2.0)

3, 4, 6, 7

6.89, d (8.0) 7.63, dd (8.0, 2.0)

1, 3 2, 4, 7

5.29, dd (6.0, 3.5) 4.47, dd (11.5, 3.5) 4.18, dd (11.5, 6.5)

7, 9 7, 1′ 7, 8, 1′

2.03, 1.99, 0.90, 0.89, 3.92,

d (7.0) m d (7.0) d (7.0) s

1′, 2′, 2′, 2′, 3

3′, 4′, 3′, 3′,

4′, 5′ 5′ 5′ 4′

δC 134.3 111.5 149.0 147.3 116.0 120.7 75.9 74.9 66.6 174.8 44.1 26.8 22.8 22.8 56.4

δH (J in Hz)

HMBC (H to C)

6.97, d (2.0)

4, 6, 7

6.75, d (8.0) 6.78, dd (8.0, 2.0) 4.51, m 3.84b 4.02, d (6.5) 3.83b

1, 2, 1, 7,

2.18, 2.05, 0.95, 0.95, 3.85,

1′, 1′, 2′, 2′, 3

d (7.0) m d (7.0) d (7.0) s

3 4, 7 2, 6, 8, 9 9

7, 8, 1′ 3′, 2′, 3′, 3′,

4′, 5′ 4′, 5′ 5′ 4′

a

In methanol-d4 solution. bUnclear signal pattern due to overlapping.

those of alatusols A and B.21 In contrast, the presence of an isovaleryl moiety (δH 2.18, 2H, brd, J = 7.0 Hz, H-2′; 2.04, 1H, m, H-3′; 0.94, 2H, d, J = 6.5 Hz, H-5′ and 6′; δC 174.7, C-1′) instead of the acetyl group in alatusols A and B was indicated by the NMR data. The isovaleryl moiety was connected to C-9, via the three-bond HMBC correlations of H-9a and H-9b with C-1′ (δC 174.7). The coupling constant between H-7 and H-8 (J = 7.0 Hz) suggests that 5 is in the threo configuration, as these data are analogous to the data published for the threo (J = 7.0 Hz) and erythro (J = 5.4 Hz) configurations.21,22 The enantiomers of 5 (5a and 5b) were obtained by the chiralphase HPLC separation of 5. The specific rotation of 5a ([α]25D −14) was the inverse of that of 5b ([α]25D +14). By comparison with the specific rotation of alatusol A,21 5a also has a (7R,8R) configuration. The ECD curve of 5b at ∼200− 260 nm was the mirror image of that of 5a (Figure 3). On the basis of these data, these structures were identified as (7R,8R)brachangobinan E (5a) and (7S,8S)-brachangobinan E (5b) (Figure 2). Compound 6 has an [M+] ion in its HREIMS data at m/z 296.1271 (calcd for C15H20O6, 296.1260), which corresponds to the molecular formula C15H20O6. The 1H and 13C NMR spectra of 6 were similar to those of 5, except for the presence of a carbonyl carbon (δC 198.2, C-7) in 6 instead of a methoxy methine group (δH 3.92, 3H, s; δC 56.5) in 5. In its HMBC spectrum, H-2 (δH 7.61, 1H, d, J = 2.0 Hz), H-6 (δH 7.63, 1H, dd, J = 8.0, 2.0 Hz), H-8 (δH 5.29, 1H, dd, J = 6.0, 3.5 Hz), and H-9 (δH 4.47 1H, dd, J = 11.5, 3.5 Hz; 4.18, 1H, dd, J = 11.5, 6.5 Hz) showed long-range coupling with the benzylic carbonyl carbon (C-7). Compound 6 was assigned the trivial name brachangobinan F. The small specific rotation of 6 ([α]D +2) and the apparent absence of Cotton effects in its ECD spectrum suggest that 6 may also be a scalemic mixture. The molecular formula of 7 was determined to be C15H22O6 on the basis of its HREIMS data (m/z 298.1421 [M+], calcd for C15H22O6, 298.1417). The 1H and 13C NMR spectra were similar to those for 5, except for the absence of the resonances of a methoxy group. The methoxy protons at δH 3.85 (3H, s, 3OMe) showed a long-range HMBC coupling with C-3 (δC

atoms than in 3. The 1H and 13C NMR data of 4 were similar to those of 3.5 In the 13C NMR spectrum of 4, a signal for a carbonyl carbon at δC 193.8 (C-7′) was observed instead of the oxygenated sp3 carbon at δC 74.3 (C-7′) in 3. In the HMBC spectrum of 4, H-2′ (δH 7.67, 1H, d, J = 2.0 Hz), H-6′ (δH 7.79, 1H, dd, J = 8.0, 2.0 Hz), and H-9′b (δH 4.51, 1H, dd, J = 11.5, 7.0 Hz) showed long-range coupling with C-7′, indicating a benzylic carbonyl group in place of the hydroxymethine moiety of 3. The NOE (Figure 1) between H-5 and H-8′ indicated that 4 is an 4-O-8′-type phenylpropanoid dimer. Therefore, the structure of 4 was identified as shown in Figure 1, and it was named brachangobinan D. The small specific rotation of 4 ([α]D +1) and the apparent lack of Cotton effects in its ECD spectrum indicate that 4 may be a scalemic mixture. The 1H and 13C NMR spectra of 5−7 indicate that they are phenylpropanoids with an isovaleryl group (Table 3). The molecular formula of 5 was determined to be C16H24O6 based on its HREIMS data (m/z 312.1577 [M+], calcd for C16H24O6, 312.1573). Its 13C NMR spectrum showed 16 carbon resonances, including six aromatic (δC 149.2, C-3; 147.8, C4; 130.9, C-1; 121.7, C-6; 116.1, C-5; 111.9, C-2), two sp3 oxymethine (δC 86.0, C-7; 74.2, C-8), an sp3 oxymethylene (δC 66.4, C-9), and two methoxy (δC 56.9, 7-OMe; 56.4, 3-OMe) carbons. The aromatic region of the 1H NMR spectrum showed an ABX spin system of a trisubstituted aromatic ring [δC 6.89 (1H, d, J = 1.5 Hz, H-2), 6.78 (1H, d, J = 8.0 Hz, H5), and 6.75 (1H, dd, J = 8.0, 1.5 Hz, H-6)]. In the 1H−1H COSY spectrum, H-8 (δH 3.85, 1H, overlapping) was correlated with H-7 (δH 4.07, 1H, d, J = 7.0 Hz) and H-9a and H-9b (δH 3.99, 1H, dd, J = 11.5, 3.5 Hz; 3.78, 1H, dd, J = 11.5, 6.0 Hz), which suggested the presence of a 1,2,3trioxygenated propyl moiety. In the HMBC spectrum, the benzylic hydrogen (H-7) showed long-range coupling with C1, C-2, and C-6, indicating the presence of a phenylpropanoid moiety. The three-bond HMBC cross-peaks of the two methoxy resonances (δH 3.85, 3H, s; 3.22, 3H, s) with C-3 and C-7 indicated the methoxy groups were located at these positions (3-OMe and 7-OMe), respectively (Figure 1). The expected substructures constructed from these data resemble F



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Figure 4. Plausible biosynthetic pathway of the isolated neolignans and related compounds.

effect at 327 nm and a positive Cotton effect at 339 nm indicated the structures of (7′R,8′S)-8a and (7′S,8′R)-8b, respectively (Figure 3);6 thus, the structures of 8a and 8b were determined as shown in Figure 2, and they were named (7′R,8′S)-brachangobinan H and (7′S,8′R)-brachangobinan H. The 1H and 13C NMR spectroscopic data of 9 resembled those of 8. Compound 9 has a molecular formula of C23H26O7 based on its HREIMS data (m/z 414.1669 [M+]; calcd for C23H26O7, 414.1679). A formyl proton singlet at δH 9.79 (1H, H-7) showed long-range HMBC coupling with C-6 (δC 122.0) and C-2 (δC 114.2), indicating the formyl group was located at C-1. The enantiomers of 9 (9a and 9b) were obtained by chiral-phase HPLC separation of 9. The specific rotation of 9a ([α]28D −51) was the inverse of that of 9b ([α]28D +64). A negative ECD Cotton effect at 297 nm indicated a structure of (7′R,8′S)-9a (Figure 3).4 In contrast, a positive Cotton effect at 296 nm indicated the structure of (7′S,8′R)-9b. From these data, 9a and 9b were identified as (7′R,8′S)-brachangobinan I and (7′S,8′R)-brachangobinan I, respectively (Figure 2). Based on its HREIMS data (m/z 430.1623 [M+]; calcd for C23H26O8, 430.1628), the molecular formula of 10 was determined to be C23H26O8. The 1H and 13C NMR spectra of 10 were similar to those of 9, except no formyl resonances were observed. The three-bond HMBC correlations of H-2 (δH 7.58, 1H, brs) and H-6 (δH 7.62, 1H, brs) with the carbon

149.0). These data indicated that 7 is the demethylated derivative of 5. Although 1′,2′,4-trihydroxy-3′-O-isobutyrylconiferyl alcohol has been reported, its relative and absolute configurations were not determined.23 The coupling constant between H-7 and H-8 (J = 6.5 Hz) suggests 7 is in the threo configuration, similar to that described for 5.21,22 Thus, 7 was determined to be the demethyl derivative of 5 and named brachangobinan G. The chiral-phase HPLC chromatogram showed two peaks, which suggested that 7 might be a mixture of enantiomers. Compound 8 has the molecular formula C25H28O7 based on its HREIMS data (m/z 440.1823 [M+]; calcd for C25H28O7, 440.1835). Its 1H and 13C NMR spectra revealed that it was a dihydrobenzofuran-4-O-neolignan and is structurally similar to 1 (Table 1). A formyl proton doublet at (δH 9.59, 1H, d, J = 8.0 Hz, H-9) and a single isovaleryl moiety (δH 2.14, 2H, d, J = 8.5 Hz, H-2″; 1.95, 1H, m, H-3′′; 0.86, 3H, d, J = 7.0 Hz, H4″; 0.85, 3H, d, J = 7.0 Hz, H-5″) are present in 8 instead of the oxymethylene and two isovaleryl moieties in 1. The 7′,8′trans configuration of 8 was established based on the NOE correlations from H-7′ to H-9′ (Figure 1). It has previously been reported that 9-isovaleroxybalanophonin has the same structure as 8;24 however, its absolute configuration has not been determined. Chiral-phase HPLC separation of 8 afforded 8a ([α]25D −16) and 8b ([α]25D +11). A negative ECD Cotton G



Article

Table 4. Inhibitory Activity against Trypanosoma congolense for Compounds from B. gobicum

of a carboxylic acid (δC 170.0, C-7) indicate that 10 is the carboxylic acid derivative of 9, and it was named brachangobinan J. Similar to what was seen with 9, two peaks were detected by chiral-phase HPLC, indicating that 10 may be a mixture of enantiomers. Compound 11 was assigned the molecular formula C23H26O8 based on its HREIMS data (m/z 430.1623 [M+]; calcd for C23H26O8, 430.1629). The 1H NMR spectrum of 11 revealed the presence of two 4-hydroxy-3-methoxyphenyl moieties [(δH 7.35, 1H, J = 1.5 Hz, H-6; 7.33, 1H, J = 1.5 Hz, H-2; 3.97, 3H, s, 3-OMe) and (δH 7.65, 1H, dd, J = 9.0, 2.0 Hz, H-6′; 7.62, 1H, d, J = 2.0 Hz, H-2′; 6.86, 1H, d, J = 9.0 Hz, H-5′; 3.93, 3H, s, 3′-OMe)], a formyl group (δH 9.74, 1H, H7), and an isovaleryl moiety, which resembled those in 9. The spectroscopic data of 11 also showed a benzylic carbonyl carbon at δC 195.7 (C-7′), similar to that of 4, which was confirmed by the three-bond HMBC correlations of H-2′ and H-6′ with C-7′. Hence, the structure of 11 was identified as the 1,2-seco derivative of 9, and it was named brachangobinan K (Figure 1). The specific rotation of 11 ([α]25D 0) and the apparent absence of any Cotton effects in its ECD spectrum suggest that 11 may be a racemic mixture. The enantiomers of compounds 12 and 14 have previously been reported,25,26 and the absolute configuration of 13 has not been determined.9 The chiral-phase HPLC chromatograms of 12−14 showed two peaks, which suggest that 12−14 may also be racemic or scalemic mixtures (Figures 68−70, Supporting Information). The biosynthetic pathway of neolignans and their natural dimerization reactions have attracted the attention of researchers.27,28 Neolignans and related compounds (1−14) may be formed by oxidative dimerization (Figure 4).22 A plausible lignan biosynthesis pathway could involve the oneelectron oxidation of an (E)-coniferyl alcohol unit to form a dimer with another coniferyl alcohol unit. B. gobicum forms 4O-8′ type coniferyl alcohol ethers (3 and 4), 5−8′ type oxyneolignans with dihydrobenzofuran moieties (1, 8−10, and 12−14), and an 8−8′ type dimer (2). The characteristic diphenyl derivative (2) might be biosynthesized from the formed 8−8′ lignan (Figure 4).17 Similar to lucidenal, after the formation of the 8−8′ bond between the two phenylpropanoid units, it is assumed that a key intermediate with a p-quinone methine moiety is produced. This compound could give rise to a cyclized spiroquinone derivative, and the homolytic cleavage might generate a diphenyl derivative (2).17 An examination of the trypanocidal activities of the compounds isolated herein as racemic mixtures revealed that T. congolense was moderately inhibited by compounds 1−4, 8, 9, and 12−14 (IC50 = 2.4−19.9 μM) relative to diminazene (IC50 = 0.1 μM) and pentamidine (IC50 = 0.2 μM) as positive controls. Compounds 1−4, 8, 9, and 11 were either lignans or diphenyl compounds with isovaleric acid moieties (Table 4). Each of the compounds with comparatively higher activities (2, 4, 13, and 14) had a formyl group in its structure. In contrast, monomeric phenylpropanoids showed no activity. Therefore, the diphenyl moieties are assumed to play an important role in the trypanocidal activity of these compounds. Lignans such as licarin A have been reported to show trypanocidal activities against T. cruzi, which is the causative agent of Chagas disease.29,30 Taken together, our findings indicate that the isovaleryl lignans isolated from B. gobicum may be effective in inhibiting or killing trypanosomes.

compound

T. congolensea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 coniferyl isovalerate trans-ferulic acid 4-hydroxy-3-methoxycinnamaldehyde vanillin diminazene16 pentamidine16

13.4 2.8 17.4 9.6 −b −b −b 2.4 19.9 −b −b 16.2 7.6 9.5 −b −b −b −b 0.1 0.2

a IC50 (μM), treatment was replicated three times for each concentration. bIC50 value >25 μM.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2300 polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded with a Shimadzu MPS-2450 (Shimadzu, Kyoto, Japan). ECD spectra were recorded on a JASCO J700 spectropolarimeter (JASCO). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded using a JEOL JNM-AL400 FT-NMR spectrometer (JEOL, Tokyo, Japan), and the chemical shifts are reported as δ values relative to tetramethylsilane as an internal standard (measured in methanol-d4 and CDCl3). Inversedetected heteronuclear correlations were measured using HMQC (optimized for1JC−H = 145 Hz) and HMBC (optimized for nJC−H = 8 Hz) pulse sequences with a pulsed-field gradient. HRFABMS data were obtained using a JEOL JMS700 mass spectrometer (JEOL), with a glycerol matrix. Preparative HPLC separations were performed using a JASCO 2089 instrument with UV detection at 210 nm (JASCO) using the following columns: TSKgel ODS-120T (Tosoh, Tokyo, Japan, 21.5 × 300 mm), Capcell Pak C8 (Shiseido, Japan, 20 × 250 mm), Develosil C30-UG-5 (Nomura Chemical, Aichi, Japan, 20 × 250 mm), Mightysil RP-18 GP (Kanto Chemical, Tokyo, Japan, 10 × 250 mm), and Daicel Chiralpak AS-H (Daicel, Osaka, Japan, 4.6 × 250 mm). Plant Material. The aerial parts of B. gobicum were collected near Khamriin Khiid (N 47°37.349′; E 110°11.096′; H 779 m), Sainshand soum, Dornogobi Province, Mongolia, in September 2012. Dr. Ts. Jamsran, Department of Biology, School of Arts and Sciences, National University of Mongolia, identified the plant species. A voucher specimen (103.29.2.12A) was deposited at the herbarium of the Laboratory of Bioorganic Chemistry and Pharmacognosy, National University of Mongolia. Extraction and Isolation. The aerial parts of B. gobicum (469 g) were dried, sectioned, and extracted by maceration in acetone−water (4:1) (3 × 5 L) at 60 °C. The concentrated extract (62.8 g) was suspended in H2O (1 L), and liquid−liquid partitioning with Et2O (2 × 1 L) was performed; the aqueous (35.1 g), Et2O-soluble (17.4 g), and insoluble fractions (13.3 g) were obtained as brown gums. The Et2O fraction was passed through a Wako gel C-200 (35 × 160 mm) open column eluted with 1 L of n-hexane (fractions 1−3), 2 L of nhexane−acetone (99:1) (fractions 4−10), 2 L of n-hexane−acetone (19:1) (fractions 11−16), 2 L of n-hexane−acetone (4:1) (fractions H



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17−20), n-hexane−acetone (1:1) (fractions 21−26), 2 L of acetone (fractions 27−32), 2 L of acetone−MeOH (1:1) (fractions 33−36), and 2 L of MeOH (fractions 37−40). Fraction 18 was subjected to reversed-phase ODS column chromatography (20 × 100 mm) eluted with 500 mL of MeOH−H2O (4:1) (fraction 41A, 1.05 g) and 1 L of MeOH (fraction 41B, 0.97 g). Fraction 41A was loaded onto an ODS column (TSKgel ODS-120T) and eluted with CH3CN−H2O (7:3) containing 0.2% trifluoroacetic acid (TFA) (fractions 43A−43H) and CH3CN−H2O (9:1) containing 0.2% TFA (fractions 43I−43J). Compound 8 (4.3 mg) [Develosil C30-UG-5, CH3CN−H2O (1:1, v/ v) containing 0.2% TFA] was purified from fraction 43E. Compounds 2 (3.2 mg) and 12 (2.9 mg) [Capcell Pak C8, CH3CN−H2O (1:1, v/ v) containing 0.2% TFA] were obtained from fraction 43C. Compound 9 (7.0 mg) [Develosil C30-UG-5, CH3CN−H2O (1:1, v/v) containing 0.2% TFA] and coniferyl isovalerate (0.6 mg) [Develosil C30-UG-5, CH3CN−H2O (3:2, v/v) containing 0.2% TFA] were purified from fraction 43F. Compounds 3 (8.3 mg) [Develosil C30-UG-5, CH3CN−H2O (7:3, v/v) containing 0.2% TFA] and 4 (3.8 mg) [Capcell Pak C8, CH3CN−H2O (3:2, v/v) containing 0.2% TFA] were obtained from fraction 43H. Compound 1 (8.4 mg) [Develosil C30-UG-5, CH3CN−H2O (7:3, v/v) containing 0.2% TFA] was purified from fraction 43J. Fraction 19 was loaded on an ODS column (20 × 100 mm) and eluted with 500 mL of MeOH− H2O (4:1) (fraction 44A, 1.2 g) and 1 L of MeOH (fraction 44B, 0.85 g). Fraction 44A was applied on an ODS column (TSKgel ODS120T) and eluted with CH3CN−H2O (4:1) containing 0.2% TFA (fractions 45A−45E). Compounds 5 (2.6 mg) [Develosil C30-UG-5, CH3CN−H2O (1:1, v/v) containing 0.2% TFA], 10 (1.3 mg) [Mightysil RP-18 GP, CH3CN−H2O (7:13, v/v) containing 0.2% TFA], 11 (3.7 mg) [Capcell Pak C8, CH3CN−H2O (1:1, v/v) containing 0.2% TFA], and 13 (4.2 mg) [Develosil C30-UG-5, CH3CN−H2O (2:3, v/v) containing 0.2% TFA] were obtained from fraction 45A. Compound 6 (3.1 mg) [Develosil C30-UG-5, CH3CN− H2O (2:3, v/v) containing 0.2% TFA] and trans-ferulic acid (2.2 mg) [Develosil C30-UG-5, CH3CN−H2O (3:7, v/v)] were purified from fraction 45C. Fraction 17 was loaded on an ODS column (20 × 100 mm) and eluted with 500 mL of MeOH−H2O (4:1) (fraction 46A, 311 mg) and 1 L of MeOH (fraction 46B, 292 mg). Fraction 46A was applied to an ODS column (TSKgel ODS-120T) and eluted with CH3CN−H2O (3:7) containing 0.2% TFA (fractions 47A−47H) and CH3CN−H2O (2:3) containing 0.2% TFA (fractions 47I−47M). 4Hydroxy-3-methoxycinnamaldehyde (7.8 mg) [Develosil C30-UG-5, CH3CN−H2O (7:13, v/v) containing 0.2% TFA] was obtained from fraction 47F, and vanillin (5.4 mg) [Develosil C30-UG-5, CH3CN− H2O (7:13, v/v) containing 0.2% TFA] was purified from fraction 47D. Fraction 20 was subjected to reversed-phase ODS column chromatography (20 × 100 mm) eluted with 500 mL of MeOH− H2O (4:1) (fraction 48A, 1.3 g) and 1 L of MeOH (fraction 48B, 650 mg). Fraction 48A was loaded on an ODS column (TSKgel ODS120T) and eluted with CH3CN−H2O (3:7) containing 0.2% TFA (fractions 49A−49I) and CH3CN−H2O (2:3) containing 0.2% TFA (fractions 49J−49T). Compound 14 (1.6 mg) [Mightysil RP-18 GP, CH3CN−H2O (7:13, v/v) containing 0.2% TFA] was obtained from fraction 49H, and compound 7 (4.3 mg) [Mightysil RP-18 GP, CH3CN−H2O (7:13, v/v) containing 0.2% TFA] was isolated from fraction 49D. Compound 1 (3.9 mg) was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane−EtOH (9:1), flow rate: 1.0 mL/min] to afford 1a (0.7 mg, tR 18.3 min) and 1b (0.8 mg, tR 16.1 min). Compound 2 (2.6 mg) was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane−EtOH (4:1), flow rate: 1.0 mL/ min] to afford 2a (0.9 mg, tR 14.0 min) and 2b (1.4 mg, tR 9.0 min). Compound 3 (1.2 mg) was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane−EtOH (9:1), flow rate: 1.0 mL/min] to afford 3a (0.3 mg, tR 18.7 min) and 3b (0.3 mg, tR 16.9 min). Compound 5 (2.6 mg) was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane−EtOH (4:1), flow rate: 1.0 mL/min] to afford 5a (1.0 mg, tR 9.4 min) and 5b (1.0 mg, tR 5.8 min). Compound 8 (3.2 mg) was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane−EtOH (3:2), flow rate: 1.0 mL/min] to afford 8a (0.9 mg, tR 15.9 min) and 8b (0.9 mg, tR 12.2 min).

Compound 9 (2.7 mg) was subjected to a chiral-phase column [Daicel Chiralpak AS-H, n-hexane−EtOH (3:2), flow rate: 1.0 mL/ min] to afford 9a (0.7 mg, tR 10.6 min) and 9b (1.0 mg, tR 6.9 min). Brachangobinan A (1): yellowish, amorphous solid; [α]22D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.31), 278 (3.88) nm; 1 H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 1; HRFABMS (positive) m/z 549.2458 [M + Na]+ (calcd for C30H38O8Na, 549.2464). (7′R,8′S)-Brachangobinan A (1a): colorless, amorphous solid; [α]25D −17 (c 0.07, MeOH); ECD (c 0.000 40, MeOH) ([θ]) 202 (+15 300), 216 (0), 233 (+2000), 289 (−5500) nm. (7′S,8′R)-Brachangobinan A (1b): colorless, amorphous solid; [α]25D +15 (c 0.07, MeOH); ECD (c 0.000 60, MeOH) ([θ]) 202 (−11 000), 232 (−2700), 272 (+3000), 288 (+4600) nm. Brachangobinan B (2): yellowish, amorphous solid; [α]22D −4 (c 0.3 MeOH); UV (MeOH) λmax (log ε) 282 (3.68) nm; 1H NMR (methanol-d4, 400 MHz), see Table 2; 13C NMR (methanol-d4, 100 MHz), see Table 2; HRFABMS (positive) m/z 465.1901 [M + Na]+ (calcd for C25H30O7Na, 465.1889). (8R)-Brachangobinan B (2a): yellowish, amorphous solid; [α]28D −54 (c 0.07, MeOH); ECD (c 0.000 50, MeOH) ([θ]) 213 (−19 500), 242 (+3500), 257 (−1000), 270 (0), 294 (−5900) nm. (8S)-Brachangobinan B (2b): yellowish, amorphous solid; [α]28D +32 (c 0.1, MeOH); ECD (c 0.000 90, MeOH) ([θ]) 215 (+11 800), 241 (−1600), 257 (+2600), 271 (+1700), 294 (+6100) nm. Brachangobinan C (3): yellowish, amorphous solid; [α]23D 0 (c 0.8 MeOH); UV (MeOH) λmax (log ε) 203 (4.06), 268 (3.67) nm; 1H NMR (methanol-d4, 400 MHz), see Table 2; 13C NMR (methanol-d4, 100 MHz), see Table 2; HREIMS (positive) m/z 544.2660 [M]+ (calcd for C30H40O9, 544.2673). (7′R,8′R)-Brachangobinan C (3a): colorless, amorphous solid; [α]24D +7 (c 0.03, MeOH). (7′S,8′S)-Brachangobinan C (3b): colorless, amorphous solid; [α]24D −7 (c 0.03, MeOH). Brachangobinan D (4): yellowish, amorphous solid; [α]22D +1 (c 0.4 MeOH); UV (MeOH) λmax (log ε) 206 (4.15), 270 (3.84) nm; 1 H NMR (CDCl3, 400 MHz), see Table 2; 13C NMR (CDCl3, 100 MHz), see Table 2; HREIMS m/z 542.2521 [M+] (calcd for C30H38O9, 542.2516). Brachangobinan E (5): yellowish, amorphous solid; [α]22D +5 (c 0.3 MeOH); UV (MeOH) λmax (log ε) 203 (4.28), 231 (3.79), 281 (3.61) nm; 1H NMR (methanol-d4, 400 MHz), see Table 3; 13C NMR (methanol-d4, 100 MHz), see Table 3; HREIMS m/z 312.1577 [M+] (calcd for C16H24O6, 312.1573). (7R,8R)-Brachangobinan E (5a): colorless, amorphous solid; [α]25D −14 (c 0.1, MeOH); ECD (c 0.000 36, MeOH) ([θ]) 204 (−3200), 216 (+300), 234 (−700), 277 (+500) nm. (7S,8S)-Brachangobinan E (5b): colorless, amorphous solid; [α]25D +14 (c 0.1, MeOH); ECD (c 0.000 36, MeOH) ([θ]) 203 (+8900), 216 (+1500), 230 (+3000) nm. Brachangobinan F (6): yellowish, amorphous solid; [α]22D +1 (c 0.3 MeOH); UV (MeOH) λmax (log ε) 284 (3.48) nm; 1H NMR (methanol-d4, 400 MHz), see Table 3; 13C NMR (methanol-d4, 100 MHz), see Table 3; HREIMS m/z 296.1271 [M+] (calcd for C15H20O6, 296.1260). Brachangobinan G (7): yellowish, amorphous solid; [α]22D −3 (c 0.4 MeOH); UV (MeOH) λmax (log ε) 203 (4.18), 230 (3.65), 280 (3.41) nm; 1H NMR (methanol-d4, 400 MHz), see Table 3; 13C NMR (methanol-d4, 100 MHz), see Table 3; HREIMS (positive) m/z 298.1421 [M+] (calcd for C15H22O6, 298.1417). Brachangobinan H (8): yellowish, amorphous solid; [α]22D 0 (c 0.4 MeOH); UV (MeOH) λmax (log ε) 203 (3.95), 230 (3.58), 289 (3.30), 338 (3.49) nm; 1H NMR (methanol-d4, 400 MHz), see Table 1; 13C NMR (methanol-d4, 100 MHz), see Table 1; HREIMS m/z 440.1823 [M+] (calcd for C25H28O7, 440.1835). (7′R,8′S)-Brachangobinan H (8a): yellowish, amorphous solid; [α]25D −16 (c 0.09, MeOH); ECD (c 0.000 80, MeOH) ([θ]) 212 (−1500), 236 (+2500), 255 (−900), 277 (+500), 327 (−1000) nm. (7′S,8′R)-Brachangobinan H (8b): yellowish, amorphous solid; [α]25D +11 (c 0.09, MeOH); ECD (c 0.000 80, MeOH) ([θ]) 204 I


Journal of Natural Products (−11 000), 213 (+1100), 238 (−1500), 257 (+2400), 287 (+200), 399 (1800) nm. Brachangobinan I (9): yellowish, amorphous solid; [α]22D −4 (c 0.6 MeOH); UV (MeOH) λmax (log ε) 233 (4.11), 288 (3.88) nm; 1 H NMR (methanol-d4, 400 MHz) see Table 1; 13C NMR (methanold4, 100 MHz), see Table 1; HREIMS m/z 414.1669 [M+] (calcd for C23H26O7, 414.1679). (7′R,8′S)-Brachangobinan I (9a): colorless, amorphous solid; [α]28D −51 (c 0.14, MeOH); ECD (c 0.000 70, MeOH) ([θ]) 215 (−800), 232 (+1900), 245 (−3100), 258 (+300), 297 (−2700) nm. (7′S,8′R)-Brachangobinan I (9b): colorless, amorphous solid; [α]28D +64 (c 0.09, MeOH); ECD (c 0.000 70, MeOH) ([θ]) 214 (+1400), 231 (−1300), 245 (+5400), 258 (+1200), 296 (+4600) nm. Brachangobinan J (10): yellowish, amorphous solid; [α]22D −5 (c 0.14 MeOH); UV (MeOH) λmax (log ε) 205 (4.35), 263 (3.77) nm; 1 H NMR (methanol-d4, 400 MHz), see Table 1; 13C NMR (methanol-d4, 100 MHz), see Table 1; HREIMS m/z 430.1623 [M+] (calcd for C23H26O8, 430.1628). Brachangobinan K (11): yellowish, amorphous solid; [α]22D 0 (c 0.36 MeOH); UV (MeOH) λmax (log ε) 203 (4.09), 232 (3.92), 284 (3.76), 308 (3.72) nm; 1H NMR (CDCl3, 400 MHz), see Table 2; 13 C NMR (CDCl3, 100 MHz), see Table 2; HREIMS m/z 430.1623 [M+] (calcd for C23H26O8, 430.1629). HPLC Analysis of Compounds 7, 10, and 12−14. Compound 7 was subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, nhexane−EtOH (4:1), flow rate: 1.0 mL/min], and two peaks were observed at tR 5.8 and 9.4 min. Similarly, 10 and 12−14 were subjected to chiral-phase HPLC [Daicel Chiralpak AS-H, n-hexane− EtOH (3:2), flow rate: 1.0 mL/min], and two peaks were observed (10: tR 6.2 and 7.4 min; 12: tR 9.0 and 10.2 min; 13: tR 19.7 and 21.6 min; 14: tR 16.2 and 21.5 min). Evaluation of Trypanocidal Activity. The IL3000 strain of the bloodstream form (BSF) of T. congolense was used to evaluate the trypanocidal activities of the isolated compounds. BSF was cultured daily on Hirumi’s modified Iscove’s medium (HMI)-9 prepared according to a reported method.31 Each compound was serially diluted 5× with HMI-9 medium from 25 μg/mL to 1.6 ng/mL. BSF trypanosomes were cultured with several concentrations of various compounds in a 96-well optical bottom cell culture plate (Thermo Fisher Scientific, Waltham, MA, USA). After 72 h, 25 μL of CellTiterGlo reagent (Promega Corp. Madison, WI, USA) was added to each well, and the luminescence was measured using a GloMax-Multi+ Detection System plate reader (Promega Corp. Madison, WI, USA). The IC50 value for each compound was calculated using GraphPad PRISM v.5 software (GraphPad Software, Inc., La Jolla, CA, USA). Pentamidine (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as the positive control.

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ACKNOWLEDGMENTS

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REFERENCES

The authors would like to thank Dr. N. Inoue, Obihiro University of Agriculture and Veterinary Medicine, for assistance with the experiments and discussion and Mr. S. Sato and Mr. T. Matsuki, Tohoku Medical and Pharmaceutical University, for assistance with the MS measurements. This work was supported by a Grant-in-aid from the Mongolian Foundation of Science and Technology (2018/58), the JICA M-JEED project, a Cooperative Research Grant (28-joint-12, 29-joint-6, 30-joint-11) from the National Research Center for Protozoan Diseases Obihiro University of Agriculture and Veterinary Medicine, the Kanno Foundation of Japan, and a grant from the JSPS KAKENHI (grant no. 26860068). The trypanocidal inhibitory experiments were supported by AMED/JICA SATPERS. Rotary Yoneyama Scholarship (Tsukidate Rotary Club and Mongolian Rotary Yoneyama Graduation Association) and Monbukagakushio Honors Scholarship (JASSO) given to O.B. are gratefully acknowledged.

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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.8b00670. NMR spectroscopy data for 1−11 including 1H NMR, C NMR, 1H−1H COSY, HMQC, and HMBC (PDF)

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Corresponding Author

*Tel: +81 22 727 0086. Fax: +81 22 727 0220. E-mail: [email protected]. ORCID

Toshihiro Murata: 0000-0001-7778-3822 Notes

The authors declare no competing financial interest. J



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