Nerve Growth Factor-Potentiating Benzofuran Derivatives from the

Dec 10, 2012 - Four new benzofuran derivatives, ribisin A (1), ribisin B (2), ribisin C (3), and ribisin D (4), were isolated from the MeOH extract of...
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Nerve Growth Factor-Potentiating Benzofuran Derivatives from the Medicinal Fungus Phellinus ribis Yuhong Liu,†,‡ Miwa Kubo,† and Yoshiyasu Fukuyama*,† †

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250355, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Four new benzofuran derivatives, ribisin A (1), ribisin B (2), ribisin C (3), and ribisin D (4), were isolated from the MeOH extract of the fruiting bodies of Phellinus ribis. Their structures including their absolute configurations were determined by NMR and CD exciton chirality methods. Compounds 1−4 were found to promote neurite outgrowth in NGF-mediated PC12 cells at concentrations ranging from 1 to 30 μM. The structure−activity relationships of these compounds are also discussed.

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chirality method. In this paper, we report the structures of 1−4 and their NGF-potentiating activity in PC12 cells.

he most pathological symptom of neurodegenerative diseases is the progressive loss of neuronal cells in the brain. It might be possible to prevent such cell loss via the localized delivery of neurotrophic factors. Nerve growth factor (NGF), a protein that promotes growth in certain neuronal populations, exerts neurotrophic effects and protects neurons from death.1,2 Thus, inducing NGF expression via pharmacological intervention during the critical period after injury might ameliorate the extent of neuronal damage after acute events, such as stroke and trauma, and could even have therapeutic effects in Alzheimer’s disease.3 Recently, we reported that a number of natural molecules such as ptychonal hemiacetal and ptychonal from Ptychopetalum olacoides,4 neovibsanin L and (8Z)-neovibsanin M from Viburnum sieboldii,5 and (2R)hydroxynorneomajucin from Illicium jiadifengpi6 exhibited neurotrophic activity in NGF-mediated PC12 cells or primary cultured rat cortical neurons. Mushrooms are important sources of physiologically beneficial molecules and, hence, are considered to be functional foods. In addition, they produce various classes of secondary metabolites with interesting biological activities and, thus, have the potential to be valuable chemical resources for drug discovery.7−9 Phellinus ribis (Schumach.) Quél. (Hymenochaetaceae), which is distributed in East Asia including China, Japan, and Korea, is a white-rot fungus that prefers to live on stumps of Rosa polyantha and Weigela subsessilis. The fruiting body of P. ribis is used as a traditional medicine for enhancing immunity and treating gastrointestinal cancer.10,11 As part of our efforts to discover natural products with neurotrophic properties, we investigated the MeOH extract of the fruiting bodies of P. ribis, which was previously found to promote the neurite outgrowth of NGF-mediated PC12 cells, resulting in the isolation of four new benzofuran derivatives, designated ribisin A (1), ribisin B (2), ribisin C (3), and ribisin D (4). The absolute configurations of 1−4 were also determined by subjecting their p-bromobenzoate derivatives to the CD exciton © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The air-dried and powdered fruiting bodies of P. ribis were extracted with MeOH at room temperature. The MeOH extract was fractionated by silica gel chromatography eluted with CH2Cl2− EtOAc−MeOH in order of increasing polarity. The CH2Cl2− EtOAc (5:5) fraction afforded compounds 1−4. Compound 1 was obtained as a white, amorphous powder. Its molecular formula was determined to be C13H12O5 by HREIMS. Its IR spectrum displayed absorption bands at 3401, 1684, and 1587 cm−1 due to hydroxy, carbonyl, and aromatic groups, respectively. Analysis of its 1H NMR and COSY spectra (Table 1) revealed the presence of a 1,2-disubstituted benzene ring at δH 7.79 (1H, ddd, J = 7.5, 1.4, 0.7 Hz), 7.52 (1H, ddd, J = 7.5, 1.0, 0.7 Hz), 7.40 (1H, td, J = 7.5, 1.4 Hz), and 7.36 (1H, td, J = 7.5, 1.0 Hz); three contiguous oxymethines at δH 4.97 (1H, d, J = 7.5 Hz), Received: August 20, 2012

A

dx.doi.org/10.1021/np300566y | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data for Compounds 1 and 2 1a,c

a

position

δC

1 1a 2 3 4 4-OH 4a 5a 6 7 8 9 9a 2-OMe 3-OMe

193.1 116.1 87.1 78.8 70.3 169.2 157.4 112.7 127.1 125.9 122.8 124.3 60.8

2b,d

δH mult. (J in Hz)

4.01 d (9.8) 3.97 dd (9.8, 7.5) 4.97 d (7.5)

HMBC

δC

C-1, C-3, C-4, 2-OMe C-1, C-2, C-4 C-3, C-4a, C-1a

190.2 115.3 81.8 81.6 63.7

7.52 ddd (7.5, 1.0, 0.7) 7.40 td (7.5, 1.4) 7.36 td (7.5, 1.0) 7.97 ddd (7.5, 1.4, 0.7)

C-8, C-5a, C-9a C-9, C-5a C-6, C-7, C-9a C-7, C-5a, C-1a

3.71 s

C-2

165.2 155.8 111.8 126.1 124.9 122.3 123.0 59.7 59.4

δH mult. (J in Hz)

HMBC

4.16 d (6.6) 4.00 dd (6.6, 4.1) 5.30 dd (7.0, 4.1) 3.05 d (7.0)

C-1, C-3, C-4, 2-OMe C-1, C-2, C-4, 3-OMe C-4a C-4, C-4a

7.56 dd (7.4, 1.8) 7.39 td (7.4, 1.7) 7.37 td (7.4, 1.8) 8.07 dd (7.4, 1.7)

C-8 C-6 C-6, C-9a C-7, C-5a

3.61 s 3.62 s

C-2 C-3

Recorded at 600 MHz. bRecorded at 500 MHz. cRecorded in methanol-d4. dRecorded in chloroform-d1.

Figure 1. CD and UV spectra of the p-bromobenzoate derivatives of (A) 1, (B) 2, and (C) 3.

from 1H−1H coupling constants and NOESY experiments. First, the NOE correlation from H-2 to H-4 suggested that a 1,3-diaxial relationship existed between H-2 and H-4. In addition, the large coupling constants between H-2 and H-3 (J = 9.8 Hz) and between H-3 and H-4 (J = 7.5 Hz) indicated that H-3 was also in an axial position. Thus, 2-OCH3, 3-OH, and 4-OH were considered to occupy equatorial positions in a half-chair conformation. In order to determine its absolute configuration, 1 was converted into the corresponding p-bromobenzoyl ester, which was then subjected to the CD exciton chirality method.12,13 The resultant CD spectrum (Figure 1, A) showed a positive Cotton effect at 261 nm and a negative Cotton effect at 245 nm (UV: λmax 250 nm), which was indicative of positive chirality between the two chromophores (the two p-bromobenzoyl groups). This result suggested that 1 contains groups with 3R and 4R configurations, and thus, the absolute configuration of C-2 was concluded to be S. On the basis of the above evidence, the structure of 1 was elucidated to be (2S,3R,4R)-3,4-dihydroxy-2-methoxy-3,4-dihydro-1(2H)dibenzofuranone and was named ribisin A. Compound 2 was obtained as a white, amorphous powder. Its molecular formula was determined to be C14H14O5 by HREIMS. The IR spectrum of 2 indicated the presence of hydroxy (3431 cm−1),

4.01 (1H, d, J = 9.8 Hz), and 3.97 (1H, dd, J = 9.8, 7.5 Hz); and a methoxy group at δH 3.71 (3H, s). On the basis of DEPT and HMQC spectra, the 13C NMR spectrum of 1 showed 13 carbon signals, which were assigned to a ketone carbonyl carbon at δC 193.1 (C-1); a benzofuran ring at δC 112.7 (C-6), 127.1 (C-7), 125.9 (C-8), 122.8 (C-9), 157.4 (C-5a), 124.3 (C-9a), 116.1 (C-1a), and 169.2 (C-4a); three oxygen-bearing methines at δC 87.1 (C-2), 78.8 (C-3), and 70.3 (C-4); and a methoxy carbon at δC 60.8. The HMBC correlations from H-2 (δH 4.01) to C-1, C-3, and C-4 and from H-3 (δH 3.97) to C-1, C-2, and C-4 were indicative of a connection between C-1 and C-2. The HMBC correlations from H-4 (δH 4.97) to C-4a and C-1a indicated that the structural unit (C1−C2−C3−C4) was linked to the C-4a of the benzofuran ring. Taking into account the eight degrees of unsaturation of 1, we considered that the ketone carbonyl carbon C-1 was connected to the quaternary carbon C-1a to form a hydrogenated dibenzofuranone skeleton. Finally, the cross-peak demonstrated by the HMBC between the methoxy protons and C-2 indicated that the methoxy group was located at C-2. In addition, the HMBC correlations from H-6 to C-8, C-5a, and C-9a and from H-9 to C-7, C-5a, and C-1a (Table 1) suggested a planar structure for 1. The relative configuration of 1 was established B

dx.doi.org/10.1021/np300566y | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. NMR Spectroscopic Data for Compounds 3 and 4 3a,c

a

position

δC

1 1a 2 3 4 4-OH 4a 5a 6 7 8 9 9a 2-OMe 3-OMe

189.9 114.6 83.8 82.9 66.1 165.9 155.7 111.8 126.1 124.8 122.3 123.0 59.8 58.8

4b,d

δH mult. (J in Hz)

3.97 dd (6.1, 0.7) 3.99 dd (6.1, 4.3) 4.99ddd (8.3, 4.3, 0.7) 3.41 d (8.3)

HMBC

C-1, C-1a, C-3, C-4, 2-OMe C-1, C-2, C-4, C-4a, 3-OMe C-2, C-3, C-4a, C-1a C-3, C-4, C-4a

7.55 ddd (7.4, 1.3, 0.6) 7.39 td (7.4, 1.6) 7.37 td (7.4, 1.3) 8.07 ddd (7.4, 1.6, 0.6)

C-8, C-5a, C-9a C-6, C-9, C-5a C-6, C-7, C-9a C-7, C-5a, C-1a

3.61 s 3.59 s

C-2 C-3

δC

δH mult. (J in Hz)

HMBC

192.8 116.4 87.0 88.8 69.9

4.11 d (9.8) 3.69 dd (9.8, 7.6) 5.06 d (7.6)

C-1, C-3, 2-OMe C-4, 3-OMe C-3, C-4a, C-1a

6.83 dd (7.7, 1.0) 7.16 t (7.7) 7.41 dd (7.7, 1.0)

C-9, C-5a C-6, C-9a C-7, C-5a

3.71 s 3.71 s

C-2 C-3

168.7 146.4 144.5 113.7 126.9 113.0 125.4 61.0 61.5

Recorded at 600 MHz. bRecorded at 500 MHz. cRecorded in methanol-d4. dRecorded in chloroform-d1.

Scheme 1. Plausible Biosynthesis of Benzofurans 1−4

carbonyl (1682 cm−1), and aromatic (1593 cm−1) groups. A comparison of the 1H and 13C spectra of 2 with those of 1 showed the presence of another methoxy group, which was located at C-3 according to the HMBC correlation from the methoxy protons (δH 3.62) to C-3 (δC 81.6). Both H-2 and H-4 are axial according to the NOE correlation from H-2 to H-4, while the coupling constants between H-2 and H-3 (J = 6.6 Hz) and between H-3 and H-4 (J = 4.1 Hz) indicated that H-3 is equatorial. The CD spectrum of the p-bromobenzoate derivative of 2 (Figure 1, B) exhibited a positive Cotton effect at 248 nm (UV: λmax 248 nm), which was suggestive of positive helicity between the p-bromobenzoate and conjugated enone chromophores corresponding to a (2S, 3S, 4R) configuration. Thus, the structure of compound 2 was elucidated to be (2S,3S,4R)-2,3-dimethoxy-4-hydroxy-3,4-dihydro-1(2H)dibenzofuranone and was named ribisin B. Compound 3 was obtained as a white, amorphous powder. Its molecular formula was determined to be C14H14O5 by HREIMS. Its IR spectrum displayed absorption bands at 3310 (OH), 1684 (CO), and 1587 (aromatic group) cm−1. The molecular formula of 3 was the same as that of 2. On the basis of COSY, HMQC, and HMBC data, it was elucidated that C-2 and C-3 both possessed methoxy groups, whereas C-4 had a hydroxy group. These groups were arranged in the same manner as 2. However, the chemical shifts of H-2 and H-4 in 3 were different from those seen in 2 (Tables 1 and 2), which was not the case for the two axial protons in 2 when compared to those of 3. Additionally, the W-type long-range coupling (J = 0.7 Hz) observed between H-2 and H-4 was also supportive of a 1,3diequatorial relationship. The chemical shift of H-3 in 3 was

similar to that seen in 2, suggesting that H-3 was also in an equatorial position. Finally, 3 was determined to have a (2R, 3S, 4S) configuration from the negative Cotton effect observed at 248 nm (UV: λmax 249 nm) in the CD spectrum of its p-bromobenzoate derivative (Figure 1, C). Therefore, the structure of compound 3 was assigned as (2R,3S,4S)-2,3-dimethoxy-4-hydroxy-3,4-dihydro1(2H)-dibenzofuranone and was named ribisin C. Compound 4 was obtained as a white, amorphous powder and displayed absorption bands observed at 3373 (OH), 1680 (CO), and 1595 (aromatic group) cm−1 in its IR spectrum. The molecular formula of 4 was determined to be C14H14O6 by HREIMS, which was indicative of an additional hydroxy group being present in 4 compared with 2. The aromatic protons at δH 7.41 (1H, dd, J = 7.7, 1.0 Hz), 7.16 (1H, t, J = 7.7 Hz), and 6.83 (1H, dd, J = 7.7, 1.0 Hz) in its 1H NMR spectrum revealed a 1,2,3-trisubstituted aromatic system, suggesting the presence of a hydroxy group in the phenyl ring. The upfield shift of C-5a (δC 146.4) indicated that the additional hydroxy group was located at the C-6 position, which was further confirmed by the HMBC correlation from the proton at δH 7.16 (t, J = 7.7 Hz, H-8) to C-9a (δC 125.4). The NOE correlation detected between H-2 and H-4 revealed that they were in axial positions, and the large coupling constants between H-2 and H-3 (J = 9.8 Hz) and between H-3 and H-4 (J = 7.6 Hz) indicated that H-3 was also in an axial position. Finally, by comparing the [α]D value (−22.0) and CD data (198 nm, Δε +2.1; 204, Δε −0.6) of 4 with those of compound 1 ([α]D −21.9; CD 195 nm, Δε +1.0, and 205 nm, Δε −0.9), the absolute configuration of 4 was determined to be 2S, 3R, and 4R. Thus, the structure of 4 was elucidated to be C

dx.doi.org/10.1021/np300566y | J. Nat. Prod. XXXX, XXX, XXX−XXX

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The above-mentioned results demonstrated that compounds 1−4, which have a hydrogenated dibenzofuranone skeleton, possess strong NGF-potentiating activity in PC12 cells. However, when a hydroxy group is added to the phenyl ring as in compound 4, it decreased the NGF-potentiating activity of the compound. In a comparison of the mean neurite length between the cells treated with 2 and 3, 3 seemed to be a more potent NGF potentiator. It should be noted that in 3 only the absolute configurations of C-2, C-3, and C-4 are different from those of 2. This suggests that in addition to the hydrogenated dibenzofuranone skeleton, which is regarded as a structural requirement for NGF-potentiating activity, the stereochemistry of the groups at the C-2, C-3, and C-4 positions plays an important role in the NGF-potentiating effects on PC12 cells. In conclusion, as compounds 1−4 have the ability to enhance the activity of NGF, which is capable of stimulating neurite outgrowth in PC12 cells, they might be useful lead candidates to produce drugs for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

(2S,3R,4R)-4,6-dihydroxy-2,3-dimethoxy-3,4-dihydro-1(2H)-dibenzofuranone and was named ribisin D. Although a number of naturally occurring dibenzofurans are known,14 highly oxygenated benzofurans such as compounds 1− 4 are not. A plausible biosynthetic route from 2-hydroxycinnamic acid (5)15 to 1−4 is proposed in Scheme 1.16 The 2-hydroxyphenylacetyl-CoA 6, which is derived from 5, is condensed successively with two units of malonyl-CoA, producing aromatic polyketide 7. The subsequent Claisen and acetal cyclization of 7 following dehydration would lead to 9, which could be converted to 1−4 by oxidations on the carbons α to carbonyl groups and reduction of the ketone, whereas dibenzofuran derivatives would be formed if 9 is solely aromatized. The effects of compounds 1−4 on the neurite outgrowth of PC12 cells were evaluated according to previously reported methods.17−20 None of the compounds had morphological effects on PC12 cells in the absence of NGF, whereas in the presence of NGF (1 ng/mL), ribisins A (1), B (2), C (3), and D (4) showed marked neurite outgrowth-promoting activity. The strength of their effects on the neurite outgrowth of PC12 cells was assessed by morphological observations (Figure 2) and a quantitative

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was measured on a JASCO P-2200 digital polarimeter. UV and CD spectra were measured on a Shimadzu UV-1650PC spectrophotometer and a JASCO J-725 instrument, respectively. IR spectra were recorded on a JASCO FT-IR 410 infrared spectrophotometer. The NMR experiments were performed on a Varian Unity 600 or 500 instrument. Deuterated solvent peaks were used as references for the 1H and 13C NMR spectra. HRFABMS, HREIMS, and HRCIMS were performed on an MStation JMS-700 or a JMX-AX 500 spectrometer. TLC was carried out with silica gel 60 F254 and PR-18 F254 plates. HPLC was performed on a JASCO PU-1580 pump equipped with a JASCO UV-1575 detector, and all peaks were detected at 210 nm. All solvents used for extraction and isolation were of analytical grade. Fungus Material. The fruiting bodies of P. ribis were collected in Jinan, Shandong Province, People's Republic of China, in October 2010. A voucher specimen (1807FB) was identified by Prof. Lingchuan Xu at Shandong University of Traditional Chinese Medicine and was deposited at the Institute of Pharmacognosy, Tokushima Bunri University. Extraction and Isolation. The dried fruiting bodies of P. ribis (1.5 kg) were powdered and extracted with MeOH (5 L) at room temperature for one month to give 17 g of crude extract following solvent removal. The MeOH extract (17 g) was chromatographed on a Si gel column (Kanto silica gel 60N, 63−210 μm, 350 g) eluted with a step gradient of CH2Cl2 (A: 100%, 3 L), CH2Cl2−EtOAc (B: 9:1, 3 L; C: 5:5, 4 L), EtOAc (D: 100%, 4 L), and EtOAc−MeOH (E: 9:1, 4 L; F: 7:3, 4 L; G: 5:5, 4 L) to yield seven fractions (A−G). Fraction C (2.3 g) was first subjected to Sephadex LH-20 chromatography (GE Healthcare Bio-Sciences, Uppsala, Sweden, 260 mL) eluted with MeOH to give fractions 1−8. Fraction 4 (563 mg) was further separated by reversed-phase HPLC (Cosmosil C18-AR-II, 20 × 250 mm) eluted with MeOH−H2O (42:58) at a flow rate of 8 mL/min to give fractions 9−15. Fraction 12 (tR = 25.0 min, 22.7 mg) was purified by reversed-phase HPLC (Cosmosil C18-AR-II, 10 × 250 mm) eluted with MeOH−H2O (35:65) at a flow rate of 2 mL/min to give compounds 1 (tR = 38.5 min, 9.8 mg) and 4 (tR = 43.0 min, 1.4 mg). Fraction 15 (tR = 37.2 min, 27.6 mg) was purified by normal-phase HPLC (Cosmosil 5SL-II, 10 × 250 mm) eluted with n-hexane−EtOAc (6:4) at a flow rate of 2 mL/min to afford compounds 2 (tR = 25.5 min, 1.6 mg) and 3 (tR = 22.0 min, 2.5 mg). Ribisin A (1): amorphous, white powder; [α]25D −21.9 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 203 (4.49), 228 (4.31) nm; IR (film) νmax 3401 (OH), 1684 (CO), 1587, 1484, 1450, 1092, 1020, 860, 749 cm−1; CD (MeOH) λmax (Δε) 195 (+1.0), 205 (−0.9) nm; 1H NMR (methonal-d4, 600 MHz) and 13C NMR (methonal-d4, 600 MHz) data (Table 1); EIMS m/z (rel int) 248 (33), 218 (53), 187 (37), 174 (100),

Figure 2. Morphological changes observed in PC12 cells treated with (A) NGF 1 ng/mL, (B) 1 (30 μM) + NGF 1 ng/mL, (C) 2 (30 μM) + NGF 1 ng/mL, (D) 3 (30 μM) + NGF 1 ng/mL, and (E) 4 (30 μM) + NGF 1 ng/mL.

analysis of neurite length (Figure 3). All of the compounds caused the mean neurite length of the NGF-mediated PC12 cells to increase in a dose-dependent manner at concentrations ranging from 1 to 30 μM, and all of these increases were significant (Figure 3).

Figure 3. Quantitative analysis of neurite outgrowth promoted by 1, 2, 3, and 4. PC12 cells were supplemented with NGF (1 ng/mL) and 1, 2, 3, or 4. After 4 days, the neurite lengths of the PC12 cells were quantified. Data are expressed as the mean ± SE (n = 100). **p < 0.01 vs control; Dunnett’s t test. D

dx.doi.org/10.1021/np300566y | J. Nat. Prod. XXXX, XXX, XXX−XXX

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146 (90), 118 (32), 89 (22); HREIMS m/z 248.0705 [M]+ (calcd for C13H12O5, 248.0675). Ribisin B (2): amorphous, white powder; [α]25D −91.1 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 202 (4.45), 228 (4.23) nm; IR (film) νmax 3431 (OH), 1682 (CO), 1593, 1483, 1448, 1095, 1028, 810, 752 cm−1; 1H NMR (chloroform-d1, 500 MHz) and 13C NMR (chloroformd1, 500 MHz) data (Table 1); EIMS m/z (rel int) 262 (47), 247 (53), 229 (37), 201 (23), 187 (16),174 (100), 146 (81), 118 (34), 89 (35); HREIMS m/z 262.0834 [M]+ (calcd for C14H14O5, 262.0841). Ribisin C (3): amorphous, white powder; [α]24D −11.4 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 202 (4.47), 229 (4.29) nm; IR (film) νmax 3310 (OH), 1684 (CO), 1587, 1477, 1447, 1346, 1146, 1022, 756 cm−1; 1H NMR (chloroform-d1, 600 MHz) and 13C NMR (chloroform-d1, 600 MHz) data (Table 2); EIMS m/z (rel int) 262 (39), 247 (16), 232 (52), 201 (23), 187 (18), 174 (100), 146 (83), 118 (32), 89 (34); HREIMS m/z 262.0845 [M]+ (calcd for C14H14O5, 262.0841). Ribisin D (4): amorphous, white powder; [α]24D −22.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.50), 235 (4.15) nm; IR (film) νmax 3373 (OH), 1680 (CO), 1595, 1490, 1259, 1142, 1020, 790 cm−1; CD (MeOH) λmax (Δε) 198 (+2.1), 204 (−0.6) nm; 1H NMR (methonal-d4, 500 MHz) and 13C NMR (methonal-d4, 500 MHz) data (Table 2); EIMS m/z (rel int) 278 (78), 247 (16), 246 (42), 231 (17), 190 (100), 162 (59), 134 (47), 44 (21); HREIMS m/z 278.0794 [M]+ (calcd for C14H14O6, 278.0790). Preparation of the p-Bromobenzoate Derivative of 1. A solution of ribisin A (1) (4.5 mg) in dry pyridine (0.5 mL) was treated with p-bromobenzoyl chloride (40 mg) and 4-dimethylaminopyridine (1 mg) at room temperature for 3 h. The reaction mixture was concentrated in vacuo, and the crude product was purified by TLC (CHCl3−MeOH, 20:1) to give the p-bromobenzoate derivative of 1 (2.7 mg): UV (MeOH) λmax (log ε) 250 (4.79) nm; CD (MeOH) λmax (Δε) 245 (−18.8), 261 (+15.6) nm; 1H NMR (chloroform-d1, 500 MHz) δ 8.14 (1H, m), 7.93 (2H, d, J = 8.5 Hz), 7.87 (2H, d, J = 8.5 Hz), 7.60 (2H, d, J = 8.5 Hz), 7.58 (2H, d, J = 8.5 Hz), 7.54 (1H, m), 7.43 (2H, m), 6.77 (1H, d, J = 7.0 Hz), 6.10 (1H, dd, J = 8.8, 7.0 Hz), 4.28 (1H, d, J = 8.8 Hz), 3.69 (3H, s); HRFABMS m/z 612.9481 [M + 1]+ (calcd for C27H1979Br2O7, 612.9498). Preparation of the p-Bromobenzoate Derivative of 2. A solution of ribisin B (2) (1.0 mg) in dry CH2Cl2 (0.15 mL) was treated with p-bromobenzoyl chloride (4 mg), triethylamine (15 μL), and 4-dimethylaminopyridine (0.3 mg) at room temperature for 2 h. The reaction mixture was concentrated in vacuo, and the crude product was purified by TLC (n-hexane−EtOAc, 8:2) to give the p-bromobenzoate derivative of 2 (0.8 mg): UV (MeOH) λmax (log ε) 248 (4.22) nm; CD (MeOH) λmax (Δε) 248 (+22.2) nm; 1H NMR (chloroform-d1, 500 MHz) δ 8.12 (1H, d, J = 7.3 Hz), 7.96 (2H, d, J = 7.5 Hz), 7.62 (2H, d, J = 7.5 Hz), 7.54 (1H, d, J = 7.8 Hz), 7.40 (2H, m), 6.86 (1H, d, J = 3.7 Hz), 4.26 (1H, d, J = 7.3 Hz), 4.16 (1H, dd, J = 7.3, 3.7 Hz), 3.71 (3H, s), 3.58 (3H, s); HRCIMS m/z 445.0271 [M + 1]+ (calcd for C21H1879BrO6, 445.0287). Preparation of the p-Bromobenzoate Derivative of 3. A solution of ribisin C (3) (1.5 mg) in dry CH2Cl2 (0.2 mL) was treated with p-bromobenzoyl chloride (6 mg), triethylamine (20 μL), and 4-dimethylaminopyridine (0.5 mg) at room temperature for 3 h. The reaction mixture was concentrated in vacuo, and the crude product was purified by TLC (n-hexane−EtOAc, 8:2) to give the p-bromobenzoate derivative of 3 (1.3 mg): UV (MeOH) λmax (log ε) 249 (4.31) nm; CD (MeOH) λmax (Δε) 248 (−31.7) nm; 1H NMR (chloroform-d1, 500 MHz) δ 8.10 (1H, m), 8.00 (2H, d, J = 7.6 Hz), 7.64 (2H, d, J = 7.6 Hz), 7.50 (1H, m), 7.39 (2H, m), 6.55 (1H, d, J = 5.4 Hz), 4.09 (2H, m), 3.75 (3H, s), 3.64 (3H, s); HRCIMS m/z 444.0212 [M]+ (calcd for C21H1779BrO6, 444.0208). Neurite Outgrowth-Promoting Activity. PC12 (pheochromocytoma) cells were cultured in a 24-well plate at a density of 8 × 103 cells/cm2 in DMEM + 10% HS, 5% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C under a humidified atmosphere of 95% air and 5% CO2 for 24 h. The culture medium was then replaced with DMEM + 2% HS, 1% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. At the same time, different concentrations of the test samples with or without 1 ng/mL NGF were added. Each concentration was repeated in three wells.

After being incubated with the test samples for 4 days, the cultures were fixed with 4% paraformaldehyde-PBS and stained with methylene blue. Cell morphology was observed under a phase-contrast microscope, and neurite length was quantified. Ten microscopic fields were randomly selected for each well. Then, 3−5 significantly differentiated cells were selected in each field, and the length of their longest neurite was recorded. At least 100 cells were examined for each concentration. Statistical analyses were performed using Dunnett’s t test. Compounds 1, 2, 3, and 4 had neurite outgrowthpromoting effects on NGF-treated PC12 cells at concentrations of 1, 10, and 30 μM, respectively (Figures 2 and 3).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR, 1H−1H COSY, HMQC, HMBC, and NOESY spectra of compounds 1−4; 1H NMR spetra of the p-bromobenzoate derivatives of 1−4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81 88 602 8435. Fax: +81 88 655 3051. E-mail: fukuyama@ ph.bunri-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. M.Tanaka and Mrs. Y. Okamoto (Tokushima Bunri University) for taking the 600 MHz NMR and MS measurements. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22590029, 23790035) and a grant from MEXT-Senryaku. Y.L. would like to acknowledge Tokushima Bunri University for providing a postdoctoral fellowship.

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