Polyprenylated Benzoylphloroglucinols with DNA Polymerase

Jul 13, 2016 - Chemical investigation of the fruits of Garcinia schomburgkiana collected in Vietnam led to the isolation of eight new schomburgkianone...
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Polyprenylated Benzoylphloroglucinols with DNA Polymerase Inhibitory Activity from the Fruits of Garcinia schomburgkiana Duy Hoang Le,† Katsumi Nishimura,† Yukiko Takenaka,† Yoshiyuki Mizushina,‡ and Takao Tanahashi*,† †

Kobe Pharmaceutical University, Kobe 658-8558, Japan Graduate School of Agriculture, Shinshu University, Kamiina-gun, Nagano 399-4598, Japan

‡

S Supporting Information *

ABSTRACT: Chemical investigation of the fruits of Garcinia schomburgkiana collected in Vietnam led to the isolation of eight new schomburgkianones, A−H (1−8), four known (9−12) polyprenylated benzoylphloroglucinols, and four known biflavonoids. The structures of these compounds were elucidated by spectroscopic and chemical means. The absolute configuration at C40 of 1 and 2 was determined by 1H NMR analyses of their MPA esters. The configuration of the bicyclo[3.3.1]nonane core of the polyprenylated benzoylphloroglucinols was assigned by comparison of their experimental ECD spectra with those of related compounds. The polyprenylated benzoylphloroglucinols exhibited inhibitory activities against mammalian DNA polymerases α and λ, with IC50 values ranging from 5.0 to 8.8 μM. Compounds 1, 2, 4, 5, and 9−11 showed cytotoxic effects against HeLa human cervical cancer cells with median lethal dose values lower than 10 μM.

■

RESULTS AND DISCUSSION The n-hexane-, CHCl3-, and AcOEt-soluble portions of a MeOH extract of the fruits of G. schomburgkiana were purified by column chromatography, preparative TLC, and preparative HPLC to afford eight new polyprenylated benzoylphloroglucinols, schomburgkianones A−H (1−8), four known analogues, guttiferone K (9),8a oblongifolin C (10),9 garciyunnanin A (11),10 and garcicowin B (12),11 and four known biflavonoids, GB-1a,12 GB-2a,13 morelloflavone, and volkensiflavone.14 Guttiferone K (9), oblongifolin C (10), morelloflavone, and volkensiflavone were previously isolated from the wood of G. schomburgkiana.7 Garciyunnanin A, garcicowin B, GB-1a, and GB-2a were detected in other Garcinia plants,4 but were isolated from this species for the first time. The structures of the new polyprenylated benzoylphloroglucinols were determined as follows. Schomburgkianone A (1) was obtained as a yellow gum, and its HRESIMS spectrum showed a molecular formula of C43H58O7, corresponding to 15 indices of hydrogen deficiency. The IR spectrum displayed bands for hydroxy (3422 cm−1) and carbonyl groups (1729 and 1668 cm−1). Its NMR spectroscopic features showed close similarities to those of oblongifolin C (10), a major constituent of this plant material. The 1H NMR data exhibited signals of a 1,2,4-trisubstituted benzene ring [δH 7.20 (1H, d, J = 2.5 Hz, H-12), 6.95 (1H, dd, J = 8.5, 2.5 Hz, H16), and 6.69 (1H, d, J = 8.5 Hz, H-15)], a methyl group [δH

Garcinia, belonging to the family Clusiaceae (Guttiferae), is a large genus of polygamous trees and shrubs that are common in moist, lowland tropical forests in Asia, Africa, and Polynesia.1 Phytochemical investigations of the leaves, twigs, stem barks, fruits, or roots of different Garcinia species have resulted in the isolation of xanthones,2 prenylated phloroglucinols,3 biflavonoids,4 and triterpenoids,5 with a wide range of bioactivities such as antioxidant, antibacterial, cytotoxicity against different human cancer cell lines, and HIV-inhibitory activities.1−6 Prenylated phloroglucinols in particular have attracted considerable attention because of their great structural diversity and interesting biological activities. Garcinia schomburgkiana Pierre is a medium-sized tree distributed in Vietnam, Laos, Cambodia, and Thailand. In folk medicine in these countries, its leaves, roots, and fruits are used for the treatment of cough, menstrual disturbances, and diabetes and as a laxative.1 Previous chemical and biological studies on constituents of the wood and bark of G. schomburgkiana showed the presence of xanthones, biphenyls, biflavonoids, and prenylated phloroglucinols, some of which exhibited cytotoxicity.7 However, the fruits of this species have not been examined so far. In the course of a search for bioactive compounds from Vietnamese medicinal plants, the fruits of G. schomburgkiana collected in the Dong Thap Province, Vietnam, were investigated. Eight new and four known prenylated phloroglucinols together with four known biflavonoids were obtained. The structural determination and biological evaluation of the isolated phloroglucinols are reported herein. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 21, 2016

A

DOI: 10.1021/acs.jnatprod.6b00255 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

δ 41.9 supported that the C-6 substituent was in an equatorial orientation, since the C-6 resonance with an axial substituent is located at δ 46−48.8 The absolute configuration at C-40 of 1 was determined by esterification with (R)- and (S)-methoxyphenylacetic acids to yield (R)- and (S)-MPA esters (1a and 1b, respectively).15 The differences of the 1H NMR chemical shifts [ΔδRS values (δR − δS)] of both MPA esters established a 40R configuration in 1 (Figure 3). Thus, the structure of 1 was defined except for the absolute configuration of the bicyclo[3.3.1]nonane core, which is discussed below. Schomburgkianones B (2) and C (3) had the same molecular formula of C43H58O7 as 1, as deduced from the HRESIMS and 13C NMR data. The UV maxima, IR absorption bands, and NMR data of 2 and 3 were similar to those of 1, suggesting that these compounds were closely related polyprenylated benzoylphloroglucinols (Tables 1 and 2). The COSY, HSQC, and HMBC interactions of 2 implied that it possessed the same 2D structure as 1. However, slight differences in their 13C NMR spectra were observed in the resonances of C-40, suggesting that the difference between 1 and 2 was the configuration at C-40. The 1H NMR data of H227, H3-28, H2-29, H2-42, and H3-43 of an (R)-MPA ester of 2 (2a) was nearly consistent with those of the (S)-MPA ester of 1 (1b), thereby verifying that 2 possessed a 40S configuration. The NMR spectroscopic features of schomburgkianone C (3) suggested that 3 differed from 1 and 2 only in the structure of the side chain at C-6. The NMR spectra of 3 showed signals for geminal dimethyl groups [δH 1.23 (6H, s), δC 30.0] and two olefinic resonances of a trans double bond [δH 5.53 (dt, J =

0.82 (s, H3-22)], a methylene [δ 2.05, 1.46 (H2-7)], and a methine [δH 1.78 (s, H-6)] and signals attributed to a 4methylpent-3-enyl and two 3-methylbut-2-enyl groups (Table 1). The 13C NMR and DEPT spectra also showed the characteristic signals due to a 3,4-dihydroxybenzoyl group and a bicyclo[3.3.1]nonane-2,4,9-trione ring system substituted with a methyl, a 4-methylpent-3-enyl, and two 3-methylbut-2-enyl groups as in 10 (Table 2). The difference in their NMR spectra was that the signals due to a geranyl group in 10 were replaced by the resonances assigned to a 6-hydroxy-3,7-dimethylocta2,7-dienyl group [δH/δC 5.04 (m, H-25)/123.8; 4.86, 4.78 (each br s, H2-42)/111.6; 3.92 (t, J = 7.0 Hz, H-40)/76.1; 2.09, 1.78 (each m, H2-24)/29.9; 2.00 (m, H2-27)/36.8; 1.66 (s, H343)/17.6; 1.58 (m, H2-39)/34.3; 1.58 (s, H3-28)/16.5; δC 148.7 (C-41), 138.0 (C-26)] in 1. The structure of the side chain was confirmed by the 1H−1H COSY correlations of H2-27/H2-39 and H2-39/H-40 as well as the HMBC interactions from the exomethylene protons (H2-42) and methyl (H3-43) to an oxygenated secondary carbon at δ 76.1 (C-40) and from the oxymethine proton (H-40) to C-27, C-39, C-42, and C-43 (Figure 1). The relative configuration of 1 was determined using 1H−1H coupling constants and NOESY correlations. The coupling constant J = 13.0 Hz of H-6/H-7ax and the interactions in the NOESY spectrum between H-7ax/H3-22, H-7ax/H2-29, H217/H3-22, H3-22/H2-24, and H-25/H2-27 suggested an axial orientation of H-6 and H3-22, equatorial orientation of CH2-17, CH2-24, and CH2-29, and an E-configuration of the Δ25,26 double bond (Figure 2). The 13C NMR chemical shift of C-6 at B

DOI: 10.1021/acs.jnatprod.6b00255 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Data (500 MHz, Methanol-d4 + 0.1% TFA-d) for Compounds 1−7 and 10 1

2

3

4

4a

5a

6

7

10b

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

δH, mult. (J in Hz)

6 7eq

1.78, m 2.05, m

1.78, m 2.05, m

2.40, m 1.97, m

1.45, t (13.0) 7.20, d (2.0) 6.69, d (8.0) 6.95, dd (8.0, 2.0) 2.73, br dd (13.5, 8.0) 2.65, m

18

1.46, t (13.0) 7.20, d (2.5) 6.69, d (8.5) 6.95, dd (8.5, 2.5) 2.73, dd (14.0, 8.5) 2.65, br dd (14.0, 5.0) 4.86, br

2.35, m 2.07, dd (13.0, 4.5) 1.52, t (13.0) 7.69, br s 6.75, d (8.5) 7.04, br d (8.5)

2.38, m 2.08, m

7ax 12 15 16

1.78, m 2.04, dd (13.5, 3.5) 1.45, t (13.5) 7.20, d (2.0) 6.69, d (8.5) 6.95, dd (8.5, 2.0) 2.72, dd (14.0, 8.5) 2.64, m

4.86, m

4.85, m

3.06, br dd (15.5, 5.5) 2.42, dd (15.5, 7.5) 4.85, br t (5.5)

1.53, t (13.0) 7.66, d (2.0) 6.76, d (8.0) 7.06, dd (8.0, 2.0) 3.06, br dd (15.5, 5.0) 2.42, br dd (15.5, 8.5) 4.85, br t (6.0)

1.74, m 2.03, br dd (13.5, 3.0) 1.47, t (13.5) 7.18, d (2.0) 6.68, d (8.0) 6.94, dd (8.0, 2.0) 2.74, dd (14.0, 8.0) 2.63, br dd (14.0, 3.0) 4.85, br

1.73, m 2.03, br dd (13.0, 3.0) 1.45, t (13.0) 7.20, d (2.0) 6.69, d (8.0) 6.95, dd (8.0, 2.0) 2.73, dd (13.5, 8.5) 2.63, br dd (13.5, 4.0) 4.84, br

20 21 22 23a

1.69, 1.62, 0.82, 1.68,

1.69, 1.62, 0.82, 1.68,

1.69, 1.62, 0.82, 1.68,

1.74, s 1.68, s 0.79, s 2.08, dd (15.0, 6.0) 1.83, br t (15.0) 2.18, br 1.72, m 5.16, br 1.61, s

1.69, 1.62, 0.81, 2.06,

1.69, 1.62, 0.81, 2.07,

1.69, 1.62, 0.81, 1.70,

1.69, 1.62, 0.81, 1.60,

1.77, br t (14.0) 2.10, m 1.70, m 5.07, br t (5.5) 1.56, s

1.76, br t (14.5) 2.13, m 1.72, m 5.09, br t (8.0) 2.01, m

1.72, s 2.43, dd (14.5, 7.5) 2.37, dd (14.5, 7.0) 5.11, br t (7.0)

1.67, s 2.48, m

1.56, s 2.48, m

5.13, br t (7.0)

5.13, br t (7.0)

1.63, 1.65, 1.98, 1.64, 4.12, 0.79, 0.79,

1.62, 1.69, 1.86, 1.90, 4.12, 1.05, 1.02,

1.62, 1.67, 1.85, 1.92, 4.11, 1.06, 1.03, 2.06,

H

17a 17b

s s s m

s s s m

s s s m

23b 24

1.58, s 2.52, dd (14.0, 8.5) 2.45, br dd (14.0, 5.5) 5.11, br t (7.0)

5.11, br t (7.0)

2.10, m 1.78, m 5.07, m 2.70, m 2.66, d (5.5) 1.56, s 2.51, dd (14.0, 8.5) 2.44, br dd (14.0, 5.0) 5.10, br t (7.0)

32 33 34a 34b 35 37 38 39

1.67, s 1.71, s 1.98, m

1.68, s 1.71, s 1.98, m

1.66, s 1.71, s 1.98, m

5.05, 1.60, 1.67, 1.58,

m s s m

5.06, 1.60, 1.67, 1.58,

m s s m

40 42

3.92, 4.86, 4.78, 1.66,

t (7.0) br s br s s

3.93, 4.90, 4.78, 1.68,

t (6.5) br s br s s

5.06, m 1.60, s 1.67, s 5.53, dt (15.5, 5.5) 5.57, d (15.5) 1.23, s

25 27 28 29

30

43 a

2.09, 1.78, 5.04, 2.00,

m m m m

2.09, 1.78, 5.05, 2.01,

m m m m

1.58, s 2.51, br dd (14.0, 9.0) 2.43, m

1.60, m 7.35, d (1.5) 6.75, d (8.5) 7.12, dd (8.5, 1.5) 3.10, br dd (15.0, 4.0) 2.60, dd (15.0, 7.5) 4.88, br

s s m m d (10.5) s s

1.23, s

s s s m

s s m m d (10.0) s s

s s s m

s s m m d (10.0) s s m

s s s m

s s s m

1.58, m 1.48, m 3.90, t (6.5) 4.91, br s 4.81, br s 1.70, s 2.51, dd (14.0, 9.0) 2.44, br dd (14.0, 3.0) 5.10, br t (7.0)

1.58, m 1.48, m 3.91, t (5.5) 4.90, br s 4.82, br s 1.69, s 2.50, dd (14.0, 8.0) 2.44, br dd (14.0, 5.0) 5.10, br t (7.0)

1.66, 1.70, 2.08, 1.73, 4.99, 1.58, 1.67,

1.66, 1.70, 2.08, 1.73, 5.00, 1.57, 1.67,

s s m m br t (7.0) s s

s s m m br t (7.0) s s

1.77, m 2.07, m 1.45, t (12.6) 7.21, d (2.1) 6.69, d (8.4) 6.96, dd (8.4, 2.1) 2.74, dd (13.5, 8.1) 2.65, dd (13.5, 5.0) 4.87, br t (5.1) 1.70, s 1.63, s 0.82, s 1.68, m

2.10, 1.70, 5.00, 1.98,

m m m m

1.57, s 2.54, dd (14.1, 8.4) 2.45, dd (14.1, 5.4) 5.12, br t (5.1) 1.67, s 1.72, s 2.06, m 5.05, 1.57, 1.65, 1.98,

m s s m

5.06, br t (8.0) 1.67, s

5.05, m 1.67, s

1.58, s

1.60, s

b

Measured in CDCl3. Measured at 300 MHz.

15.5, 5.5 Hz) and 5.57 (d, J = 15.5 Hz), δC 125.8, 140.8] and an oxygenated tertiary carbon [δC 71.2] instead of the signals due to a 2-hydroxy-3-methylbutenyl group connected to C-27 in 1 and 2. These signals were attributable to a 3-hydroxy-3methylbut-1-enyl group from the HMBC correlations of dimethyl groups at δH 1.23 to an oxygenated tertiary carbon and olefinic carbon at δC 140.8. The connection of the group to C-27 was confirmed by further COSY, HSQC, HMBC, and NOESY correlations, demonstrating that the C-6 side chain was a (2E,5E)-7-hydroxy-3,7-dimethylocta-2,5-dienyl group. The relative configuration of the bicyclo[3.3.1]nonane core in 2 and 3 was determined to be the same as that in 1 using the

coupling constant of H-6/H-7ax, chemical shift of C-6, and ROESY correlations. Another set of closely related phloroglucinol derivatives composed schomburgkianones D (4) and E (5). The HRESIMS and 13C NMR data of 4 and 5 exhibited molecular formulas of C38H50O7 and C43H58O7, one oxygen atom more than those of 9 and 10, respectively. The NMR spectroscopic features suggested that the structures of these compounds were closely related. However, the NMR spectra of 4 and 5 in CDCl3 afforded simple spectra without broad signals caused by tautomerism of the 5-hydroxycyclohex-4-ene-1,3-dione moiety. The spectra of 9 and 10 had to be measured in methanol-d4 + 0.1% TFA-d to simplify the NMR spectra (Tables 1 and 2). C

DOI: 10.1021/acs.jnatprod.6b00255 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data (125 MHz, Methanol-d4 + 0.1% TFA-d) for Compounds 1−7a and 10 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 a

1

2

3

4

4b

5b

6

7

10c

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

194.0, 119.7, 192.4, 69.3, 51.4, 41.9, 43.0, 63.8, 209.0, 196.5, 130.2, 117.3, 146.3, 152.5, 115.1, 124.9, 26.5, 121.3, 135.0, 18.3, 26.3, 16.2, 37.4, 29.9, 123.8, 138.0, 36.8, 16.5, 31.6, 120.9, 135.5, 18.2, 26.3, 25.2, 125.5, 132.5, 17.9, 25.9, 34.3, 76.1, 148.7, 111.6, 17.6,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH2 CH3

194.4, 119.6, 192.5, 69.1, 51.4, 41.7, 43.0, 63.9, 208.9, 196.5, 130.1, 117.3, 146.3, 152.5, 115.1, 124.9, 26.5, 121.3, 135.0, 18.3, 26.3, 16.4, 37.4, 30.0, 123.9, 137.9, 36.8, 16.5, 31.6, 120.9, 135.5, 18.2, 26.3, 25.2, 125.4, 132.5, 17.9, 25.9, 34.2, 75.9, 148.8, 111.5, 17.7,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH2 CH3

195.4, 119.7, 191.0, 69.5, 51.4, 41.9, 43.0, 64.0, 208.9, 196.5, 130.1, 117.3, 146.3, 152.5, 115.1, 124.9, 26.5, 121.3, 135.0, 18.3, 26.3, 16.2, 37.4, 29.9, 124.4, 137.3, 43.4, 16.6, 31.6, 120.9, 135.5, 18.2, 26.3, 25.2, 125.5, 132.5, 17.9, 25.9, 125.8, 140.8, 71.2, 30.0, 30.0,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH CH C CH3 CH3

195.7, 130.0, 175.0, 65.9, 46.0, 37.1, 41.2, 65.0, 207.2, 195.1, 130.8, 116.1, 146.9, 153.1, 115.6, 125.2, 25.4, 120.5, 135.0, 18.5, 26.3, 17.1, 35.7, 29.0, 123.6, 134.3, 18.0, 26.0, 30.6, 121.2, 135.1, 18.1, 26.2, 24.2, 90.3, 72.9, 27.4, 27.4,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

194.3, 131.7, 173.4, 64.7, 45.1, 35.8, 40.4, 64.1, 206.2, 194.2, 130.0, 114.6, 144.3, 150.8, 114.2, 125.0, 24.6, 118.7, 134.4, 18.3, 26.0, 16.9, 34.7, 28.0, 121.9, 133.7, 18.0, 25.8, 29.6, 119.7, 134.6, 18.1, 26.0, 23.1, 90.1, 73.6, 25.2, 26.2,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

194.2, 131.8, 173.3, 64.7, 45.1, 35.7, 40.4, 64.1, 206.3, 194.1, 130.0, 114.7, 144.2, 150.7, 114.3, 125.0, 24.6, 118.7, 134.6, 18.3, 26.0, 16.9, 34.8, 27.9, 121.9, 137.3, 39.8, 16.2, 29.6, 119.7, 134.4, 18.0, 26.0, 23.2, 90.1, 73.5, 25.3, 26.3, 26.5, 124.0, 131.6, 25.7, 17.7,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

194.2, 119.6, 191.1, 69.5, 51.2, 41.8, 42.9, 64.0, 208.9, 196.5, 130.1, 117.3, 146.3, 152.5, 115.1, 124.9, 26.5, 121.3, 135.0, 18.2, 26.3, 16.6, 33.8, 31.3, 77.6, 148.8, 111.4, 17.9, 31.6, 120.9, 135.5, 18.3, 26.3, 29.9, 123.5, 134.5, 18.1, 26.0,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

195.0, 119.7, 190.9, 69.5, 51.0, 42.3, 42.9, 64.3, 209.0, 196.4, 130.1, 117.3, 146.3, 152.5, 115.1, 124.9, 26.6, 121.3, 135.0, 18.2, 26.3, 16.0, 33.4, 31.3, 77.5, 148.3, 112.1, 17.4, 31.5, 120.9, 135.5, 18.3, 26.3, 29.9, 123.5, 134.5, 18.2, 26.0,

C C C C C CH CH2 C C C C CH C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

194.8, 119.6, 191.4, 69.3, 51.5, 41.9, 43.2, 64.0, 209.1, 196.7, 130.2, 117.4, 146.4, 152.6, 115.2, 125.3, 26.7, 121.4, 135.1, 18.5, 26.5, 16.5, 37.5, 30.1, 123.9, 138.2, 40.9, 16.6, 31.7, 121.1, 135.6, 18.4, 26.5, 27.6, 125.1, 132.4, 18.0, 26.2, 25.3, 125.6, 132.6, 26.1, 18.1,

C C C C C CH CH2 C C C C C C C CH CH CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3

Assignments were based on DEPT, HSQC, and HMBC. bMeasured in CDCl3. cMeasured at 75 MHz.

Figure 1. Key correlations observed in COSY and HMBC spectra of 1. Figure 2. Key correlations observed in the NOESY spectrum of 1.

These findings implied that the 5-hydroxycyclohex-4-ene-1,3dione group in 4 and 5 was modified to hinder keto−enol D

DOI: 10.1021/acs.jnatprod.6b00255 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Values of ΔδRS (δR − δS) (in ppm) obtained from 1a/1b.

interconversion.6a Analysis of 1H and 13C NMR data together with COSY, HSQC, and HMBC spectra revealed that 4 and 5 were closely related to 9 and 10, respectively, but possessed a 2,3-dioxygenated 3-methylbutyl group instead of a prenyl group at C-23 in 9 and 10 (Figure 4). From 2D NMR experiments,

Figure 5. Key correlations observed in ROESY spectra of 4 and 5.

the relative configuration at C-35 of 4 and 5 differed from that of guttiferone S. The UV maxima, IR absorption bands, and NMR data of schomburgkianones F (6) and G (7) closely resembled those of guttiferone K (9) (Tables 1 and 2). The same molecular formula of C38H50O7 deduced by HRESIMS and 13C NMR data indicated that 6 and 7 were isomeric and possessed one oxygen atom more than guttiferone K (9).8 The structural difference of 6/7 and 9 involved the C-6 substituent. COSY correlations observed between H2-24/H-25 and H2-27/H3-28 and the HMBC interactions from H2-27 and H3-28 to the oxymethine carbon (C-25) and from H-25 to C-27 confirmed the assignment of the 2-hydroxy-3-methylbut-3-enyl group. Thus, compounds 6 and 7 differed in their C-25 configurations. The relative configuration of the bicyclo[3.3.1]nonane core in 6 and 7 was deduced to be the same as in 1−3 by analysis of the 1 H−1H coupling constants and ROESY correlations. The absolute configuration at C-25 of 6 and 7 still remains to be determined because of the limited amount of the samples. The isolated phloroglucinols except for 4 and 5 showed broad NMR signals in CDCl3 as a result of tautomerism of the 5-hydroxycyclohex-4-ene-1,3-dione system.6a This phenomenon was confirmed by methylation of (+)-guttiferone K (9) with TMS-CHN2 to yield two trimethyl ethers, 9a and 9b (Figure 6). HMBC experiments confirmed that 9a and 9b possessed methoxy groups at C-1 and C-3, respectively. Recently, experimental and calculated ECD were utilized to determine the absolute configurations of polyprenylated phloroglucinols with a bicyclo[3.3.1]nonane skeleton.17−19 Therefore, the experimental ECD spectra of compounds 1−7, 9, 10, 9a, and 9b were obtained to compare with reported data of experimental and calculated ECD curves for related compounds. Compound 9a showed an opposite ECD curve to 9b (Figure 7), although both compounds possessed the same absolute configurations at C-4 and C-8. This was explained by the fact that the structure 9a′ could be recognized as related to a mirror image of 9b, supposing that the substituents at C-5 could be ignored. The ECD curve of 9a was similar to that reported for oblongifolins R (14) and S (15)17 with an ether ring at C-1, whereas 9b showed similar Cotton effects to those of oxyguttiferone K (16),18 which possessed an ether linkage at C-3. These results suggested that the keto−enol tautomerism of the bicyclo[3.3.1]nonane core could affect the ECD spectra (Figures 6 and 7 and Table 3). The similarity of experimental ECD curves of 1−3, 9, and 10 to that of 9b (Figures S1 and S2, Supporting Information,

Figure 4. Key correlations observed in COSY and HMBC spectra of 4 and 5.

the oxymethine carbon [4 and 5: δC 90.1] and oxygenated tertiary carbon [4: δC 73.6, 5: δC 73.5] were assigned to C-35 and C-36. The chemical shifts of C-35, C-36, C-1, and C-3 and the molecular formula implied the ether ring closure between C-35 and C-3 and the substitution of the hydroxy group at C36 to construct the 2-(1-hydroxy-1-methylethyl)-2,3,4,5tetrahydrooxepine ring as seen in guttiferone S (13), which was isolated from Garcinia cochinchinensis.16 The relative configuration of 4 was determined by analysis of 1 H−1H coupling constants and ROESY correlations. The coupling constant of 13.0 Hz between H-7ax (δH 1.52)/H-6 showed their diaxial orientation. The ROESY correlations between H-7ax/H3-22, H-7ax/H2-29, H-17a (δH 3.06)/H3-22, and H3-22/H2-24 confirmed the axial orientation of H3-22 and the equatorial orientation of the prenyl groups at C-4, C-6, and C-8 on the six-membered ring. The coupling constants of 14.0 Hz for H-23b (δH 1.77)/H-34b (δH 1.90) and 10.0 Hz for H35/H-34b and the ROESY interactions between H-17b (δH 2.42)/H-23b, H-17b/H-35, H3-22/H-23b, and H-23b/H-35 revealed the quasi-axial orientation of H-23b, H2-17, and H-35 on the same face of the seven-membered-ring system (Figure 5). These findings suggested the relative configuration of 4 to be (4R*, 5S*, 6R*, 8R*, 35R*). The similarities in 1H−1H coupling constants and ROESY correlations of 4 and 5 suggested that 5 shared the same configuration as 4. The oxymethine proton H-35 of 4 and 5 resonated as a doublet (J = 10.0 Hz in CDCl3), although the corresponding signal of guttiferone S was observed as a doublet of doublets (J = 11.3, 7.8 Hz in acetone-d6).16 These findings suggested that E

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Figure 6. Methylation of (+)-guttiferone K (9).

and 9b (Figure S3, Supporting Information, and Table 3) and assigned as (4R, 5S, 6R, 8R). The high-amplitude positive Cotton effect in the 300−350 nm region compared with its low amplitude in 1−3 could be explained by ether formation to interfere with tautomerism of the 5-hydroxycyclohex-4-ene-1,3dione system in 4 and 5. The experimental ECD spectra of 6 and 7 exhibited similar Cotton effects to those of 1−3, 9, and 10 at wavelengths around 210 and 320 nm, in spite of the different features at 275 nm (Figure S3, Supporting Information, and Table 3). From these findings, it was tentatively concluded that 6 and 7 possessed the same (4R, 5S, 6R, 8S) absolute configurations as 1−3. Schomburgkianone H (8) was isolated as a yellow gum. Its positive-ion HRESIMS revealed a protonated molecule consistent with the molecular formula C33H40O6. Its IR spectrum exhibited bands at 3430 and 1740 cm−1, indicating the presence of hydroxy and carbonyl groups, respectively. Its 1 H and 13C NMR spectra demonstrated signals assignable to a geranyl, a prenyl, and a 3,4-dihydroxybenzoyl group. Furthermore, the signals of geminal dimethyl groups [δH 1.467, 1.473 (each s), δC 28.9, 29.0], a cis double bond [δH 5,45, 6.41 (each d, J = 10.0 Hz), δC 115.7, 124.9], and seven quaternary carbons [δC 55.8, 82.1, 109.0, 109.5, 170.7, 184.5, 190.5] were observed. The presence of a 2,2-dimethyl-2Hpyran ring was indicated by the HMBC correlations from H-7 to C-2, C-4, and C-9; from H-8 to C-3; from H3-10 and H3-11 to C-8 and C-9; and from H2-12 to C-2. Further HMBC interactions confirmed that both the prenyl and geranyl groups were located at C-1 (Figure 8). The remaining three quaternary carbons constituted a 5-hydroxycyclohex-4-ene-1,3-dione group. These spectroscopic data were closely similar to those of machuone (17),20 excluding the appearance of two additional hydroxy groups at C-20 and C-21 and a prenyl group at C-15. Accordingly, the structure of 8, schomburgkianone H, was established as shown except for the absolute configuration at C-1. Biological Activities. Compounds 1−6 and 8−11 were evaluated for their inhibition of mammalian DNA polymerases (pols) α and λ. The investigations on pols were initiated in a search for specific inhibitors because these enzymes are essential for DNA replication, repair, and cell division.21 Selective inhibitors of mammalian pols have been considered as a group of potentially useful chemotherapy agents for anticancer, antivirus, and anti-inflammation treatments.22 The

Figure 7. ECD spectra of 9a and 9b.

Table 3. Comparison of the ECD Spectra of 1−7, 9, 9a, 9b, and 10 Δεnm maxima and minima in the CD spectra

compound 1 2 3 4 5 6

Δε210 +13.44 Δε209.5 +20.31 Δε210 +13.44 Δε217 +13.27 Δε216.5 +18.83 Δε215.5 +20.57

Δε252.5 −6.81 Δε253.5 −14.62 Δε252.5 −6.81 Δε269 −6.08 Δε266 −8.32 Δε249 −4.69

7

Δε214.5 +6.57

Δε243 −0.07

9 9a 9b 10

Δε205 Δε215 Δε216 Δε211

Δε253 Δε252 Δε262 Δε253

+18.20 −13.78 +19.28 +27.79

−12.13 +10.27 −13.72 −17.67

Δε274 Δε297 Δε275 Δε297

+5.99 −10.24 +5.99 −6.25

Δε308 −0.85 Δε308 +0.95

Δε333.5 +1.76 Δε328.5 +2.70 Δε333.5 +1.76 Δε325.5 +10.51 Δε325.5 +11.54 Δε320 +3.07 Δε322 +1.10 Δε328.5 +2.22 Δε335 −4.38 Δε336 +4.47 Δε333 +2.89

Table 3) suggested that the absolute configuration of 1, 2, 3, (+)-guttiferone K (9), and (+)-oblongifolin C (10) could be assigned as (4S, 5S, 6R, 8S), and these compounds exist mainly as a C-1 keto and C-3 enol form in ECD measurements. (+)-Guttiferone K (9) isolated in this study could be an enantiomer of guttiferone K, with a small but negative optical rotation that was first isolated from the fruits of Rheedia calcicola.8 The absolute configurations of the bicyclo[3.3.1]nonane core in 4 and 5 were implied to be the same as those in 1−3 by their similar experimental ECD spectra compared to those of 1−3 F

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inhibitory activity against two mammalian pols, calf pol α and human pol λ, of each compound was investigated. All test compounds inhibited pol α and pol λ with half-maximum inhibitory concentrations (IC50’s) ranging from 5.0 to 8.8 μM and showed stronger inhibition than aphidicolin and dideoxyTTP, which are known inhibitors of mammalian pol α and pol λ, respectively23 (Table 4).

and 5 showed stronger inhibitory effects toward mammalian pol α and also stronger cytotoxic effects on HeLa cell growth than 9 and 10, respectively. These results suggested that the cyclization of the side chain to make the bicyclo[3.3.1]nonane core more stable in the phloroglucinol skeleton may play a crucial role in the pol α inhibition and the cytotoxic effects.

Table 4. IC50 Values of Compounds 1−11, Aphilidocolin, and DideoxyTTP on Mammalian Pol α and λ Activitiesa and on the Proliferation of HeLa Cellsb

General Experimental Procedures. Optical rotations were measured using a Jasco DIP-370 digital polarimeter. UV absorption spectra were recorded using a Shimadzu UV-240 spectrophotometer. ECD spectra were recorded using a JASCO J-820 spectropolarimeter. IR spectra were measured using a Shimadzu FTIR-8200 infrared spectrophotometer. The NMR experiments were performed with Varian System-500, INOVA-500, and Varian Mercury-300 spectrometers, with TMS as an internal standard (CDCl3) or the solvent peaks (methanol-d4 + 0.1% TFA-d: δH 3.30 and δC 49.0). HRESIMS and HRSIMS were obtained with an Exactive mass spectrometer (Thermo Fisher Scientific). HPLC was performed using a Waters system (600E multisolvent delivery system, 2487 dual λ absorbance detector). Silica gel 60 (Merck) was used for column chromatography (CC). TLC was performed on precoated Kieselgel 60F254 plates (Merck), and spots were visualized under UV light. Plant Material. The fruits of G. schomburgkiana, Bua Dong in Vietnamese, were collected in Lap Vo District, Dong Thap Province, Vietnam, in 2013 and identified by Dr. Hoang Viet, Department of Botany, Faculty of Biology, University of Science, Ho Chi Minh City, Vietnam. A voucher specimen (No. KPU-VMP01F) was deposited at Kobe Pharmaceutical University, Japan. Extraction and Isolation. The air-dried fruits (199 g) were extracted with MeOH (4 × 600 mL) under reflux for 30 min each to yield a MeOH extract (80 g). The MeOH extract was suspended in H2O and successively partitioned with n-hexane, CHCl3, EtOAc, and n-BuOH (each 3 × 300 mL) to obtain n-hexane (8.52 g), CHCl3 (2.18 g), EtOAc (8.17 g), and n-BuOH (31.29 g) extracts, respectively. TLC examination of the n-hexane and CHCl3 extracts showed identical compounds, which were pooled. The combined extract was separated by silica gel CC (CHCl3/MeOH) to afford five fractions: C1 (1.40 g), C2 (6.26 g), C3 (0.79 g, 1−10% MeOH), C4 (0.47 g, 10% MeOH), and C5 (0.83 g, 20−50% MeOH). Fraction C2 was separated and purified by silica gel CC (CHCl3/MeOH; n-hexane/EtOAc; n-hexane/ acetone), preparative TLC (n-hexane/EtOAc, 4/1, 2/1; n-hexane/ acetone, 4/1, 3/1; toluene/MeOH, 9/1), and preparative HPLC (5C18-AR-II, 20 × 250 mm, MeOH/(H2O + 0.1% HOAc), 9/1) to give 1 (11.4 mg), 2 (9.5 mg), 3 (15.3 mg), 4 (18.9 mg), 5 (6.3 mg), 6 (4.9 mg), 7 (4.2 mg), 8 (5.4 mg), 9 (950 mg), a mixture of 9 and 10 (967 mg), 10 (916 mg), 11 (19.7 mg), and 12 (4.8 mg). Fraction C3 was separated by silica gel CC (n-hexane/acetone, 4/1) and preparative TLC (toluene/MeOH, 9/1) to give 1 (10.7 mg), 2 (8.0 mg), 6 (3.1 mg), and 7 (2.4 mg). Separation and purification of the EtOAc extract by CC (CHCl3/MeOH) and preparative TLC (nhexane/EtOAc, 1/1, 1/2; n-hexane/acetone, 1/1; CHCl3/acetone, 3/ 1; CHCl3/MeOH, 19/1, 9/1, 17/3) led to GB-1a (221 mg), GB-2a (40 mg), morelloflavone (18 mg), and volkensiflavone (102 mg). Schomburgkianone A (1): yellow gum, [α]27 D +15 (c 0.7, CHCl3); UV (MeOH), λmax (log ε) 280 (4.19), 229.5 (4.09) nm; ECD (5.83 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3422, 2926, 1729, 1668, 1520, 1443, 1378, 1293, 1197 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 687.4247 [M + H]+ (calcd for C43H59O7, 687.4263), 709.4063 [M + Na]+ (calcd for C43H58O7Na, 709.4083). Schomburgkianone B (2): yellow gum, [α]27 D +40 (c 1.0, CHCl3); UV (MeOH), λmax (log ε) 280.5 (4.22), 229.5 (4.12) nm; ECD (5.53 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3420, 2926, 1730, 1653, 1521, 1443, 1385, 1293, 1197 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 687.4252 [M + H]+ (calcd for C43H59O7, 687.4263), m/z 709.4067 [M + Na]+ (calcd for C43H58O7Na, 709.4083). Schomburgkianone C (3): yellow gum, [α]27 D +25 (c 0.8, CHCl3); UV (MeOH), λmax (log ε) 278 (4.24), 230.5 (4.25) nm; ECD (5.69 ×

■

IC50 value (μM) compound 1 2 3 4 5 6 8 9 10 11 aphidicolin ddTTP

Pol α 6.3 6.4 8.8 5.6 5.4 8.6 7.9 6.2 5.7 6.5 19.6 >200

± ± ± ± ± ± ± ± ± ± ±

Pol λ 0.3 0.3 0.5 0.3 0.3 0.4 0.4 0.3 0.3 0.3 1.0

5.0 5.8 5.6 7.4 6.9 8.8 6.2 5.4 5.2 5.7 >200 28.7

± ± ± ± ± ± ± ± ± ±

HeLa cells 0.3 0.3 0.3 0.4 0.4 0.4 0.3 0.3 0.3 0.3

± 1.5

9.8 9.9 73.4 6.5 5.9 66.5 50.8 9.7 7.3 10.2 22.0 >200

± ± ± ± ± ± ± ± ± ± ±

1.0 1.1 7.2 0.7 0.6 6.7 5.1 1.0 0.8 1.1 2.4

Data are shown as mean ± SD of three independent experiments. b Data are shown as mean ± SD of five independent experiments. a

Compounds 1−6 and 8−11 were also assayed for their cytotoxic effects against HeLa human cervical cancer cultured cells. Compounds 1, 2, 4, 5, and 9−11 showed cytotoxic effects against the HeLa cells with a median lethal dose (LD50) lower than 10 μM. The suppression of HeLa cell growth by these compounds showed almost the same tendency as the inhibitory activity of mammalian pols, especially pol α, suggesting that the suppression of human cancer cell proliferation by these compounds might be related to inhibition of the activities of DNA replicative pol α.

Figure 8. Key correlations observed in COSY and HMBC spectra of 8.

In conclusion, from the fruits of G. schomburgkiana, 16 compounds including eight new and four known polyprenylated benzoylphloroglucinols together with four known biflavonoids were isolated. Many phloroglucinols are well known for exhibiting cytotoxic or antitumor effects on human cancer cell lines. The benzoyl and prenyl groups play a crucial role in their cytotoxic effects upon cancer cell lines.24 The biological actions of guttiferone K (9)25 and oblongifolin C (10)26−28 have been studied in detail. In the present study, the isolated polyprenylated benzoylphloroglucinols were evaluated for their inhibitory activities against mammalian DNA pol α and pol λ and cytotoxic effects against the HeLa human cervical cancer cells. The new polyprenylated benzoylphloroglucinols 4 G

EXPERIMENTAL SECTION

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10−5 M, MeOH), Table 3; IR (KBr) νmax 3428, 2926, 1728, 1673, 1560, 1443, 1385, 1292, 1202 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 709.4072 [M + Na]+ (calcd for C43H58O7Na, 709.4083). Schomburgkianone D (4): pale yellow gum, [α]27 D +125 (c 0.9, CHCl3); UV (MeOH), λmax (log ε) 318 sh (3.90), 279 (4.17), 234.5 (4.15), 200.5 (4.47) nm; ECD (8.09 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3430, 2928, 1731, 1662, 1593, 1521, 1442, 1374, 1293, 1175 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 619.3615 [M + H]+ (calcd for C38H51O7, 619.3637), m/z 641.3432 [M + Na]+ (calcd for C38H50O7Na, 641.3456). Schomburgkianone E (5): pale yellow gum, [α]28 D +104 (c 0.4, CHCl3); UV (MeOH), λmax (log ε) 317 sh (3.89), 278 (4.15), 234 (4.15), 200 (4.57) nm; ECD (5.90 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3430, 2926, 1731, 1662, 1594, 1521, 1442, 1374, 1294, 1175 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 687.4238 [M + H]+ (calcd for C43H59O7, 687.4263), m/z 709.4056 [M + Na]+ (calcd for C43H58O7Na, 709.4083). Schomburgkianone F (6): yellow gum, [α]26 D −13 (c 0.4, CHCl3); UV (MeOH), λmax (log ε) 281 (4.27), 231 (4.12) nm; ECD (4.85 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3421, 2925, 1728, 1559, 1520, 1444, 1378, 1292, 1197 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 619.3633 [M + H]+ (calcd for C38H51O7, 619.3637), m/z 641.3452 [M + Na]+ (calcd for C38H50O7Na, 641.3456). Schomburgkianone G (7): yellow gum, [α]26 D +6 (c 0.3, CHCl3); UV (MeOH), λmax (log ε) 281 (4.28), 230.5 (4.16) nm; ECD (6.47 × 10−5 M, MeOH), Table 3; IR (KBr) νmax 3405, 2925, 1720, 1558, 1444, 1379, 1292, 1196 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 619.3627 [M + H]+ (calcd for C38H51O7, 619.3637), m/z 641.3446 [M + Na]+ (calcd for C38H50O7Na, 641.3456). Schomburgkianone H (8): yellow gum, [α]27 D −4 (c 0.5, CHCl3); UV (MeOH), λmax (log ε) 276.5 (4.06), 231 (4.12) nm; IR (KBr) νmax 3430, 2926, 1740, 1603, 1508, 1442, 1378, 1290, 1199 cm−1; 1H NMR (500 MHz, methanol-d4 + 0.1% TFA-d) δH 7.09 (1H, d, J = 2.0 Hz, H19), 6.98 (1H, dd, J = 8.5, 2.0 Hz, H-23), 6.71 (1H, d, J = 8.5 Hz, H22), 6.41 (1H, d, J = 10.0 Hz, H-7), 5.45 (1H, d, J = 10.0 Hz, H-8), 4.99 (1H, br t, J = 7.0, H-25), 4.92 (1H, br t, J = 7.0, H-13), 4.90 (1H, m, H-13′), 2.70 (2H, dd, J = 14.0, 7.5 Hz, H-12 and H-12′), 2.64, 2.62 (each 1H, m, H-12 and H-12′), 1.98 (2H, m, H2-24), 1.92 (2H, m, H216), 1.63 (3H, s, H3-15′), 1.61 (6H, s, H3-15 6′), 1.59 (3H, s, H3-28), 1.51 (3H, s, H3-27), 1.473 (3H, s, H3-11), 1.467 (3H, s, H3-10); 13C NMR (125 MHz, methanol-d4 + 0.1% TFA-d) δC 197.0 (C-17), 190.5 (C-6), 184.5 (C-4), 170.7 (C-2), 151.3 (C-21), 145.8 (C-20), 140.0 (C-14), 136.4 (C-14′), 132.4 (C-26), 131.3 (C-18), 125.1 (C-25), 124.9 (C-8), 123.9 (C-23), 119.1 (C-13′), 118.8 (C-13), 117.2 (C-19), 115.7 (C-7), 115.1 (C-22), 109.5 (C-3), 109.0 (C-5), 82.1 (C-9), 55.8 (C-1), 40.7 (C-16), 37.8 (C-12), 37.7 (C-12′), 29.0 (C-11), 28.9 (C10), 27.5 (C-24), 26.1 (C-15′), 25.9 (C-28), 18.3 (C-16′), 17.8 (C27), 17.0 (C-15); HRESIMS m/z 533.2894 [M + H]+ (calcd for C33H41O6, 533.2905), m/z 555.2712 [M + Na]+ (calcd for C33H40O6Na, 555.2724). 26 (+)-Guttiferone K (9): yellow gum, [α]24 D +5 (c 0.6, CHCl3), [α]D 8 +1.8 (c 0.51, MeOH) [lit: [α]23 −2 (c 0.35, CHCl ) ]; ECD (8.31 × D 3 10−5 M, MeOH), Table 3; 1H and 13C NMR (500 MHz, methanol-d4 + 0.1% TFA-d) data were identical to published data;8 HRESIMS m/z 603.3687 [M + H]+ (calcd for C38H51O6, 603.3688), m/z 625.3503 [M + Na]+ (calcd for C38H50O6Na, 625.3507). (+)-Oblongifolin C (10): yellow gum, [α]23 D +13 (c 0.5, CHCl3), 9 20 [α]25 D +4.2 (c 0.53, MeOH) [lit: [α]D +14.5 (c 0.21, CHCl3) ]; ECD (5.97 × 10−5 M, MeOH), Table 3; 1H and 13C NMR (500 MHz, methanol-d4 + 0.1% TFA-d) data were identical to published data;9 HRESIMS m/z 671.4315 [M + H]+ (calcd for C43H59O6, 671.4314), m/z 693.4133 [M + Na]+ (calcd for C43H58O9Na, 693.4134). (−)-Garciyunnanin (11): yellow gum, [α]27 D −19 (c 1.4, CHCl3) [lit. 10a [α]D −34.3 (c 1.75, MeOH)10b]; 1H [α]26.2 D −3.0 (c 0.11, CHCl3); and 13C NMR (500 MHz, methanol-d4 + 0.1% TFA-d) data were identical to published data;10a HRESIMS m/z 587.3740 [M + H]+

(calcd for C38H51O5, 587.3739), m/z 609.3559 [M + Na]+ (calcd for C38H50O5Na, 609.3558). (+)-Garcicowin B (12): yellow gum, [α]28 D +43 (c 0.1, CHCl3) [lit. 11 1 13 C NMR (500 MHz, [α]15 D −16.0 (c 0.21, CHCl3) ]; H and methanol-d4 + 0.1% TFA-d) data were identical to published data;11 HRESIMS m/z 655.4367 [M + H]+ (calcd for C43H59O5, 655.4365), m/z 677.4185 [M + Na]+ (calcd for C43H58O5Na, 677.4185). Preparation of (R)- and (S)-MPA Esters of 1 and 2. To a solution of 1 (5.0 mg) in dry CH2Cl2 (0.3 mL) was added (R)-MPA (12 mg), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (14 mg), and a catalytic amount of 4-pyrrolidinopyridine, and the mixture was stirred at room temperature for 48 h. The reaction mixture was poured into aqueous 10% HCl and extracted with CHCl3. The CHCl3 layer was washed with brine, dried, and concentrated in vacuo. The residue was purified by preparative TLC (toluene/MeOH, 9/1) to give monoacylated compound 1a (0.8 mg) and 1a′ (2.2 mg) together with a negligible amount of other acylated products (for the structure and spectroscopic data of 1a′, see Supporting Information). Compound 1 (5.0 mg) was treated with (S)-MPA (12 mg) as described above to yield 1b (2.1 mg). Compound 2 (5.0 mg) was treated with (R)-MPA (12 mg) as described above to obtain 2a (2.2 mg). 1a: 1H NMR (500 MHz, methanol-d4 + 0.1% TFA-d) δH 7.500− 7.328 (5H, m, MPA-Ph), 7.228 (1H, d, J = 2.0 Hz, H-12), 6.981 (1H, dd, J = 8.5, 2.0 Hz, H-16), 6.715 (1H, d, J = 2.0 Hz, H-15), 5.080 (1H, m, H-30), 5.050 (1H, m, H-40), 4.765 (1H, m, H-25), 4.733 (1H, s, MPA-CH