Article pubs.acs.org/jnp
Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa Delfly B. Abdjul,†,‡ Hiroyuki Yamazaki,*,† Ohgi Takahashi,† Ryota Kirikoshi,† Kazuyo Ukai,† and Michio Namikoshi† †
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan Faculty of Fisheries and Marine Science, Sam Ratulangi University, Kampus Bahu, Manado 95115, Indonesia
‡
S Supporting Information *
ABSTRACT: Three new sesquiterpene hydroquinones, avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), were isolated from a Dysidea sp. marine sponge collected in Okinawa together with five known congeners: avarol (4), neoavarol (5), 20-O-acetylavarol (6), 20-O-acetylneoavarol (7), and 3′-aminoavarone (8). The structures of 1−3 were assigned on the basis of their spectroscopic data. Compounds 1−3 inhibited the activity of protein tyrosine phosphatase 1B with IC50 values of 11, 9.5, and 6.5 μM, respectively, while known compounds 4−8 gave IC50 values of 12, >32, 10, 8.6, and 18 μM, respectively. In a preliminary investigation on structure−activity relationships, six ester and methoxy derivatives (9−14) were prepared from 4 and 5. μg/mL). Bioassay-guided separation led to the isolation of three new sesquiterpene hydroquinones, named avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), together with five known congeners: avarol (4),6a neoavarol (5),6b 20-Oacetylavarol (6),6c 20-O-acetylneoavarol (7),6d and 3′-aminoavarone (8).6e Furthermore, six chemical derivatives (9−14) were prepared from 4 and 5 in order to investigate preliminary structure−activity relationships. We herein describe the isolation, structure elucidation including absolute configurations, and biological activities of sesquiterpene hydroquinones 1−14.
T
he protein tyrosine phosphatase (PTP) family catalyzes key processes related to various cell functions, and dysfunctions in PTP activity can cause major health issues such as diabetes, cancer, and autoimmune and neurological diseases.1 This enzyme family comprises more than 100 members, which have been classified into four groups (classes I−IV).1 Of these members, protein tyrosine phosphatase 1B (PTP1B) is known to be expressed in the brain, liver, muscles, and adipose tissue and plays a significant role in negatively regulating signals from insulin and leptin receptors.2 Accordingly, PTP1B is regarded as an attractive drug target for the treatment of type 2 diabetes and obesity. Moreover, recent studies have indicated that PTP1B has potential as a target for the treatment of breast cancer.3 Although research and trials have been performed to develop clinically useful PTP1B inhibitors, a drug candidate has not been discovered due to insufficient activity and selectivity.4 Therefore, continuing efforts to identify novel types of PTP1B inhibitors are still needed. In the course of our screening program targeting PTP1B inhibitors from marine organisms (sponges, ascidians, and microorganisms), we reported the activities of polybromodiphenyl ethers,5a dehydroeuryspongin,5b hyattellactones,5c trichoketides,5d verruculides,5e and 26-O-ethylstrongylophorine14.5f Further investigations on extracts from marine organisms revealed that the EtOH extract of a Dysidea sp. marine sponge, collected at Iriomote Island, exhibited marked inhibitory activity against PTP1B (approximately 80% inhibition at 50 © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The EtOH extract of the marine sponge was separated by an ODS column and repeated HPLC (ODS column) to yield compounds 1 (0.8 mg), 2 (2.8 mg), 3 (1.5 mg), 4 (255.2 mg), 5 (1.2 mg), 6 (1.9 mg), 7 (1.5 mg), and 8 (1.8 mg). Compounds 4−8 were identified by comparing their spectroscopic data with the reported values for avarol, neoavarol, 20-O-acetylavarol, 20-O-acetylneoavarol, and 3′aminoavarone, respectively.6 The molecular formula of compound 1 was deduced as C21H28O2 from HREIMS and NMR data (Table 1). The 1H and 13C NMR spectra (CDCl3) of 1 showed 27 proton and 21 carbon signals, which were classified into four methyl, five Received: April 25, 2016
A
DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1
Table 1. 1H and 13C NMR Data for Avapyran (1) in CDCl3 Avapyran (1) C#
δC, type
1
18.4,
CH2
2
26.8,
CH2
3 4 5 6
120.4, 143.8, 37.6, 31.0,
CH C C CH2
7
31.5,
CH2
8 9 10 11 12 13 14 15
79.1, 36.8, 41.6, 17.9, 20.3, 22.8, 21.3, 35.1,
C C CH CH3 CH3 CH3 CH3 CH2
16 17 18 19 20 21
121.2, 147.1, 116.8, 114.2, 148.2, 114.9,
C C CH CH C CH
δH, mult. (J in Hz)
COSY
1.54, 1.79, 1.71, 1.95, 5.05,
m m m m brs
2a
1.46, 1.75, 1.67, 1.92, 1.50,
m m m m m
7b
1.52, 1.55, 1.04, 1.15, 0.99, 2.45, 2.76,
m s s s s d (17.4) d (17.4)
6.59, d (8.8) 6.54, dd (8.8, 2.9) 6.44, d (2.9)
HMBC
1a, 3 3
Figure 1. (a) COSY and key HMBC correlations of 1 and (b) key NOESY correlations of 1 based on the energy-minimized conformer of 1.
6a
1 3, 4, 7, 8, 9, 8,
19 18
an ether linkage. Thus, compound 1 was assigned to have a tetracyclic structure and was named avapyran (Figure 1a). The relative configuration of 1 was defined by the NOESY spectrum. The correlations between H-7b (δH 1.92)/H3-12 (1.04), H-7b/H3-14 (0.99), H3-13 (1.15)/H-15a (2.45), and H3-14/H-15a revealed the stereostructure shown in Figure 1b. On the basis of the NOE data for 1, the most stable conformer was calculated using Spartan’14 by a Monte Carlo conformational analysis with an MMFF94 force field (Figure 1b). The absolute configuration of 1 was investigated by comparing its experimental electronic circular dichroism (ECD) spectrum with the calculated ECD spectra of (5S,8R,9S,10R)-1 and (5S,8S,9S,10R)-1, a C-8 epimer. The experimental ECD spectrum of 1 (green line) was closer to the calculated ECD spectrum of the (5S,8R,9S,10R)-isomer (black solid line) than that of the (5S,8S,9S,10R)-isomer (black dashed line) (Figure 2). Consequently, the absolute configuration of 1 was assigned as shown in the scheme. The molecular formula of 2 was deduced as C23H32O3 from HREIMS and NMR data (Table 2), which was the same as that of 20-O-acetylavarol (6). 1H and 13C NMR data for 2 were also very similar to those for 6,6c and an analysis of the COSY and HMBC spectra of 2 indicated that compound 2 was a monoacetylated derivative of avarol (Figure 3a). The location of the O-acetyl group was assigned to C-17 by comparing chemical shifts at C-17 and C-20 of 2 (δC 143.7 and 152.5, respectively) with those of 6 (δ C 152.3 and 144.3, respectively).6c The relative configuration of 2 at the bicyclic terpene moiety was determined from NOESY correlations between H-8 (δH 1.58)/H-10 (1.16), H3-12 (0.99)/H3-14 (0.81), H3-13 (0.94)/ H-15a (2.45), and H3-14/H-15a (Figure 3b), which were the same as those of avarol. The most stable conformer of 2 was
4, 5 5, 6, 10 8, 9 10, 15 10, 14, 16 16, 17, 21
16, 20 17 17, 19
methylene, one sp3 methine, two sp3 quaternary, one oxygenated sp 3 quaternary, four sp 2 methine, two sp 2 quaternary, and two oxygenated sp2 quaternary carbons by an analysis of DEPT and HMQC spectra. Three aromatic proton signals at δH 6.59 (1H, d, J = 8.8 Hz), 6.54 (1H, dd, J = 8.8, 2.9 Hz), and 6.44 (1H, d, J = 2.9 Hz) suggested the presence of a 1,2,4-trisubstituted benzene ring, which was confirmed by the COSY and HMBC spectra of 1 (Figure 1a). Analyses of COSY and HMBC correlations for 1 established a rearranged drimane skeleton similar to avarol (4). The aromatic ring (C-16−C-21) and sesquiterpene moiety (C1−C-15) were subsequently connected by HMBC correlations from H-15a (δ 2.45) to C-16 (121.2) and from H-15b (2.76) to C-16, C-17 (147.1), and C-21 (114.9) (Figure 1a). Based on the molecular formula and the 13C chemical shift at C-8, the C8 and C-17 positions were expected to be connected through B
DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 3. (a) COSY and key HMBC correlations of 2 and (b) key NOESY correlations of 2 based on the energy-minimized conformer of 2.
Table 3. 1H and 13C NMR Data for 17-O-Acetylneoavarol (3) in CDCl3 17-O-Acetylneoavarol (3)
Figure 2. Experimental ECD spectrum of 1 (a) and calculated ECD spectra of (5S,8R,9S,10R)- (b) and (5S,8S,9S,10R)-isomers (c).
C#
Table 2. 1H and 13C NMR Data for 17-O-Acetylavarol (2) in CDCl3 17-O-Acetylavarol (2) C#
δC, type
1
19.8,
CH2
2
26.5,
CH2
3 4 5 6
120.4, 144.3, 38.3, 35.7,
CH C C CH2
7
27.6,
CH2
8 9 10 11 12 13 14 15
35.8, 41.7, 45.8, 18.1, 20.0, 17.6, 17.7, 37.8,
CH C CH CH3 CH3 CH3 CH3 CH2
16 17 18 19 20 21 22 23
132.2, 143.7, 123.1, 114.0, 152.5, 119.3, 170.1, 21.2,
C C CH CH C CH C CH3
δH, mult. (J in Hz)
COSY
1.62, 1.89, 1.74, 2.07, 5.13,
2b
m m m m brs
0.96, 1.37, 1.31, 1.43, 1.58,
m m m m m
1.16, 1.49, 0.99, 0.94, 0.81, 2.45, 2.50,
m s s d (6.3) s d (14.4) d (14.4)
6.81, d (9.3) 6.64, dd (9.3, 2.9)
δC, type
1
23.1,
CH2
2
28.2,
CH2
3
32.9,
CH2
HMBC
1a, 3 2b
7a
4 5 6
159.7, 40.2, 36.4,
C C CH2
7 8 9 10 11
27.6, 36.1, 42.0, 48.0, 103.0,
CH2 CH C CH CH2
12 13 14 15
20.6, 17.8, 17.6, 37.6,
CH3 CH3 CH3 CH2
16 17 18 19 20 21 22 23
132.1, 143.8, 123.1, 114.1, 152.4, 119.0, 170.0, 21.1,
C C CH CH C CH C CH3
6a 13
8
19 18
1, 3, 4, 7, 8, 9, 8,
2, 5, 9 4, 5 5, 6, 10 8, 9 9, 10, 15 10, 16, 17, 21 9, 16, 17, 21
16, 17, 19, 20 17, 21
6.62, brs
17
2.28, s
22
δH, mult. (J in Hz) 1.54, 1.90, 1.36, 1.88, 2.08, 2.28,
m m m m m m
1.19, 1.44, 1.40, 1.38,
m m m m
0.98, 4.37, 4.42, 1.04, 0.95, 0.82, 2.40, 2.46,
m brs brs s d (5.9) s d (14.4) d (14.4)
6.81, d (8.8) 6.64, dd (8.8, 2.9)
COSY
HMBC 5
3b 1, 2 2a
7 6a 10
3, 3, 4, 7, 9, 9, 8,
19 18
4, 5 4, 5 5, 6, 10 8 10, 15 10, 16, 21 16, 17, 21
16, 20 17, 21
6.57, d (2.9)
17, 19
2.26, s
22
presumed to be a 17-O-acetyl isomer of 7, and this was confirmed by COSY and HMBC data for 3 (Figure 4a) with comparisons of the chemical shifts at C-17 and C-20 of 3 (δC 143.8 and 152.4, respectively) with those of 7 (δC 152.3 and 143.6, respectively).6d The NOESY correlations between H-8 (δH 1.38)/H-10 (0.98), H3-12 (1.04)/H3-14 (0.82), H3-13 (0.95)/H-15a (2.40), and H3-14/H-15a established the relative configurations at the C-5, C-8, C-9, and C-10 positions of 3 (Figure 4b), which were the same as those of neoavarol (5). According to the NOESY spectrum of 3, a Monte Carlo conformational analysis was performed with an MMFF94 force field utilizing Spartan’14 (Figure 4b). Consequently, the structure of 3 was assigned as 17-O-acetylneoavarol.
predicted using Spartan’14 by a Monte Carlo conformational analysis with an MMFF94 force field based on the relative configuration (Figure 3b). Thus, the structure of compound 2 was elucidated as shown in the scheme and named 17-Oacetylavarol. The molecular formula of 3 (C23H32O3) determined from HREIMS and NMR data for 3 (Table 3) was identical to that of 2. The 1H and 13C NMR spectra of 3 resembled those of 20-Oacetylneoavarol (7).6d Therefore, the structure of 3 was C
DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Diacetyl avarol (9) and 20-O-methylavarol (10) were inactive, whereas the diacetyl and 20-O-methyl derivatives of neoavarol (12 and 13) showed slightly stronger activities than that of neoavarol (5) (Table 4). Moreover, the inhibitory activities of dipropionyl derivatives (11 and 14) were more potent than those of the diacetyl derivatives (9 and 12). A synthetic study on ester derivatives with longer acyl chains is now in progress. Dysidine and 21-dehydroxybolinaquinone (Figure 5), isolated from a Dysidea sp. marine sponge, possess a different Figure 4. (a) COSY and key HMBC correlations of 3 and (b) key NOESY correlations of 3 based on the energy-minimized conformer of 3.
The absolute configurations of avarol and neoavarol have been determined by total syntheses7 and X-ray crystallographic analyses.8 The specific rotations of the known compounds (4− 8) isolated in this study showed similar signs and magnitudes to previously reported values.6 The absolute configurations of the rearranged drimane sesquiterpene moieties in new compounds 2 and 3 were considered to be the same as those of avarol (4) and neoavarol (5), respectively, because these sesquiterpene hydroquinones (2−5) were obtained from the same marine sponge. Marine sesquiterpene hydroquinones belonging to the avarol family have been reported to exhibit various biological properties including cytotoxic, anti-HIV, anti-inflammatory, and antimicrobial activities.9 In the present study, the PTP1Binhibitory activities of 1−8 were tested, and their IC50 values are listed in Table 4.
Figure 5. Structures of dysidine and 21-dehydroxybolinaquinone.
rearranged drimane skeleton and have previously been reported to inhibit PTP1B activity with IC50 values of 6.7 and 39.5 μM, respectively.10a Cell-based experiments demonstrated that dysidine activated the insulin signaling pathway and promoted glucose uptake by inhibiting PTP1B activity.10b The three new compounds (1−3) obtained in this study also displayed similar PTP1B-inhibitory properties. Therefore, the development of a new type of antidiabetes agent may be possible with continued evaluation of these drimane-type sesquiterpene hydroquinones.
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Table 4. PTP1B Inhibitory Activities of Compounds 1−14 compound
IC50 (μM)
avapyran (1) 17-O-acetylavarol (2) 17-O-acetylneoavarol (3) avarol (4) neoavarol (5) 20-O-acetylavarol (6) 20-O-acetylneoavarol (7) 3′-aminoavarone (8) 17,20-O-diacetylavarol (9) 17-O-methylavarol (10) 17,20-O-dipropionylavarol (11) 17,20-O-diacetylneoavarol (12) 17-O-methylneoavarol (13) 17,20-O-dipropionylneoavarol (14) oleanolic acid (positive control)11
11 9.5 6.5 12 35% inhibition at 32 μM 10 8.6 18 5% inhibition at 25 μM 0% inhibition at 31 μM 8.8 14 50% inhibition at 31 μM 9.4 1.1
EXPERIMENTAL SECTION
General Experimental Procedures. Specific rotations were determined with a JASCO P-2300 digital polarimeter. UV spectra were measured on a U-3310 UV−visible spectrophotometer (Hitachi), and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer. ECD spectra were measured with a JASCO J720 spectropolarimeter. NMR spectra were recorded on a JEOL JNMAL-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) in CDCl3 (δH 7.24, δC 77.0) or CD2Cl2 (δH 5.32, δC 53.8). EIMS and HREIMS were performed using a JMS-MS 700 mass spectrometer (JEOL). Preparative HPLC was carried out with a Hitachi L-6200 system. PTP1B was purchased from Enzo Life Sciences. p-Nitrophenyl phosphate (pNPP) was purchased from Sigma-Aldrich. Oleanolic acid was purchased from Tokyo Chemical Industry. Plastic plates (96-well) were purchased from Corning Inc. All other chemicals including organic solvents were purchased from Wako Pure Chemical Industries Ltd. Marine Sponge and Isolation of Compounds 1−8. The marine sponge was collected by scuba diving at Iriomote Island (Okinawa, Japan) in 2013 and identified by Dr. K. Ogawa (Z. Nakai Laboratory) as Dysidea sp. A voucher specimen is deposited at the Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, as 13-9-7=2-2. The diagnoses of the marine sponge were as follows: irregularly lobate, surface coarsely conulose, forming irregular reticulation with deep angular depressions; light brown in alcohol; fibers irregular, entirely filled with detritus, and only light collagen deposition. The frozen Dysidea sp. sponge (98.9 g, wet weight) was cut into small pieces and extracted three times with EtOH (1.0 L). The EtOH extract was evaporated, and the residue (3.6 g) was separated into nine fractions (Frs. 1−9) by an ODS column (100 g) with the stepwise elution of CH3OH−H2O. Frs. 5 and 6 exhibited PTP1B-inhibitory activities at 10 μg/mL. Active Fr. 6 (314.5 mg, eluted with 85% CH3OH) was purified by preparative HPLC [column, PEGASIL ODS (Senshu Sci. Co., Ltd.), i.d. 10 mm × 250 mm; solvent, 78% CH3OH in H2O; flow rate, 2.0 mL/min; detection, UV 210 nm] to afford compounds 5 (1.2 mg, tR = 27.0 min), 4 (255.2 mg, tR = 32.0 min), 1 (0.8 mg, tR = 39.0 min), 3 (1.5 mg, tR = 45.0 min), 2 (2.8 mg, tR = 50.0
Avarol (4) inhibited PTP1B activity with an IC50 value of 12 μM, while neoavarol (5) showed only 35% inhibition at 32 μM. Therefore, the avarol-type bicyclic sesquiterpene moiety appears to be more favorable for inhibitory activity than the neoavarol-type skeleton. However, the monoacetyl derivatives of neoavarol (3 and 7) were more potent than similar derivatives of avarol (2 and 6). Compound 3 was the strongest PTP1B inhibitor (IC50 = 6.5 μM) among these sesquiterpene hydroquinones. In order to investigate the preliminary structure−activity relationships of the sesquiterpene hydroquinones, the ester and 20-O-methyl derivatives (9−14) of avarol (4) and neoavarol (5) were prepared as described in the Experimental Section. D
DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
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min), 7 (1.5 mg, tR = 63.0 min), and 6 (1.9 mg, tR = 67.0 min). Compound 8 (1.8 mg, tR = 25.0 min) was isolated from active Fr. 5 (133.1 mg, eluted with 70% CH3OH) by repeated HPLC [column, PEGASIL ODS, i.d. 10 mm × 250 mm; solvent, 80% CH3OH in H2O containing 0.05% TFA; flow rate, 2.0 mL/min; detection, UV 210 nm]. Compound 5 (12.3 mg) used for the preparation of derivatives for the structure−activity relationship study was purified from the EtOH extract of a Chelonaplysilla sp. marine sponge, collected at Iriomote Island in 2012, by similar separation procedures to those above. Avapyran (1): colorless oil; [α]24D +8.0 (c 0.08, CHCl3); UV (CH3OH) λmax (log ε) 297 (3.2) nm; ECD (3.2 × 10−4 M, CH3CN) λmax (Δε) 202 (+2.5), 233 (−1.7), 282 (+0.3) nm; IR νmax (KBr) 3423, 2930, 2344, 1638, 1497, 1454, 1384, 1210, 1152, 1055, 1032 cm−1; 1H and 13C NMR (CDCl3), Table 1; EIMS m/z 312 [M]+; HREIMS m/z 312.2099 (calcd for C21H28O2, 312.2089). 17-O-Acetylavarol (2): reddish oil; [α]24D +4.5 (c 0.27, CHCl3); UV (CH3OH) λmax (log ε) 283 (3.2) nm; IR νmax (KBr) 3421, 2939, 2365, 1760, 1729, 1628, 1497, 1441, 1384, 1217, 1185, 1030 cm−1; 1H and 13C NMR (CDCl3), Table 2; EIMS m/z 356 [M]+; HREIMS m/z 356.2343 (calcd for C23H32O3, 356.2351). 17-O-Acetylneovarol (3): reddish oil; [α]24D −7.3 (c 0.16, CHCl3); UV (CH3OH) λmax (log ε) 281 (3.1) nm; IR νmax (KBr) 3403, 2932, 2374, 1760, 1637, 1497, 1453, 1384, 1233, 1186, 1029 cm−1; 1H and 13 C NMR (CDCl3), Table 3; EIMS m/z 356 [M]+; HREIMS m/z 356.2349 (calcd for C23H32O3, 356.2351). Avarol (4): yellowish oil; [α]21D +13.0 (c 0.40, CHCl3); lit. [α]D +6.1,5a lit. [α]D +4.7 (c 0.17, CHCl3).6b Neoavarol (5): yellowish oil; [α]20D −28.5 (c 0.15, CHCl3); lit. [α]D −38.6 (c 0.14, CHCl3).6b 20-O-Acetylavarol (6): reddish oil; [α]21D +13.0 (c 0.14, CHCl3); lit. [α]25D +11.0 (c 2.3, CHCl3).6c 20-O-Acetylneoavarol (7): reddish oil; [α]23D −40.2 (c 0.02, CHCl3); lit. [α]25D −31.9 (c 0.10, CHCl3).6d 3′-Aminoavarone (8): reddish oil; [α]21D +30.7 (c 0.13, CH2Cl2); lit. [α]22D +45.0 (c 0.10, CH2Cl2).6e Preparation of Diacetyl Derivatives (9 and 12). Acetic anhydride (100 μL, 1.1 mmol) and 4-(dimethylamino)pyridine (DMAP, 1.0 mg, 0.0080 mmol) were added to a solution of 4 (4.0 mg, 0.0127 mmol) in pyridine (100 μL), and the resulting solution was stirred at room temperature (rt) for 12 h. The reaction mixture was concentrated in vacuo to dryness, and the product was purified by preparative HPLC [column, PEGASIL ODS, i.d. 10 mm × 250 mm; solvent, 83% CH3OH in H2O; flow rate, 2.0 mL/min; detection, UV 210 nm] to give 17,20-O-diacetylavarol (9, 3.3 mg, 0.0083 mmol, 83%). 17,20-O-Diacetylneoavarol (12, 1.9 mg, 0.0048 mmol, 63%) was prepared from 5 (3.0 mg, 0.0096 mmol) using similar procedures. 17,20-O-Diacetylavarol (9): colorless oil; 1H NMR (CDCl3) δH 6.98 (1H, d, J = 8.8 Hz), 6.95 (1H, dd, J = 8.8, 2.4 Hz), 6.89 (1H, d, J = 2.4 Hz), 5.12 (1H, brs), 2.56 (1H, d, J = 14.2 Hz), 2.51 (1H, d, J = 14.2 Hz), 2.29 (3H, s), 2.26 (3H, s), 1.49 (3H, s), 0.99 (3H, s), 0.94 (3H, d, J = 5.9 Hz), 0.82 (3H, s); EIMS m/z 398 [M]+; HREIMS m/z 398. 2458 (calcd for C25H34O4, 398.2457). 17,20-O-Diacetylneoavarol (12): colorless oil; 1H NMR (CDCl3) δ 6.98 (1H, d, J = 8.2 Hz), 6.93 (1H, brd, J = 8.8), 6.92 (1H, brs), 4.70 (1H, brs), 4.67 (1H, brs), 2.60 (1H, d, J = 14.0 Hz), 2.48 (1H, d, J = 14.0 Hz), 2.30 (3H, s), 2.27 (3H, s), 1.53 (3H, s), 1.02 (3H, s), 0.93 (3H, d, J = 6.3 Hz), 0.88 (3H, s); EIMS m/z 398 [M]+; HREIMS m/z 398. 2464 (calcd for C25H34O4, 398.2457). Preparation of Mono-O-methyl Derivatives (10 and 13). TMS-diazomethane (250 μL, 0.150 mmol) was added to a CH3OH solution of 4 (4.0 mg, 0.0127 mmol in 250 μL) and stirred at rt for 14 h. The reaction mixture was concentrated in vacuo and separated by preparative HPLC [solvent, 83% CH3OH in H2O containing 0.05% TFA; flow rate, 2.0 mL/min; detection, UV 210 nm] with an ODS column (PEGASIL ODS) to give mono-O-methylavarol (10, 1.6 mg, 0.0049 mmol, 40%). The structure of 10 was confirmed by a NOESY correlation between H-19 (δH 6.62) and an O-methyl signal (3.72) as 20-O-methylavarol. 20-O-Methylneoavarol (13, 1.3 mg, 0.0040 mmol,
43%) was obtained by a similar method to that for 5 (3.0 mg, 0.0096 mmol). 20-O-Methylavarol (10): colorless oil; 1H NMR (CDCl3) δ 6.64 (1H, d, J = 8.8 Hz), 6.62 (1H, dd, J = 8.8, 2.4 Hz), 6.59 (1H, d, J = 2.4 Hz), 5.12 (1H, brs), 2.68 (1H, d, J = 14.2 Hz), 2.57 (1H, d, J = 14.2 Hz), 3.72 (3H, s) 1.49 (3H, s), 1.00 (3H, s), 0.99 (3H, d, J = 6.3 Hz), 0.84 (3H, s); EIMS m/z 328 [M]+; HREIMS m/z 328. 2401 (calcd for C22H32O2, 328.2402). 20-O-Methylneoavarol (13): colorless oil; 1H NMR (CDCl3) δ 6.66 (1H, d, J = 8.2 Hz, 6.64 (1H, brs), 6.61 (1H, d, J = 2.4 Hz), 4.70 (1H, brs), 4.68 (1H, brs), 3.73 (3H, s), 2.70 (1H, d, J = 14.5 Hz), 2.53 (1H, d, J = 14.5 Hz), 1.54 (3H, s), 1.02 (3H, s), 0.96 (3H, d, J = 6.3 Hz), 0.90 (3H, s); EIMS m/z 328 [M]+; HREIMS m/z 328.2401 (calcd for C22H32O2, 328.2402). Preparation of Dipropionyl Derivatives (11 and 14). Triethylamine (14 μL, 0.1000 mmol), propionyl chloride (100 μL, 0.1000 mmol), and DMAP (1.0 mg, 0.0080 mmol) were added to a solution of 5 (4.0 mg, 0.0127 mmol) in pyridine (100 μL), and the resulting solution was stirred at rt for 12 h. The reaction mixture was concentrated in vacuo to dryness and purified by preparative HPLC [column, PEGASIL ODS, i.d. 10 mm × 250 mm; solvent, 83% CH3OH in H2O; flow rate, 2.0 mL/min; detection, UV 210 nm] to give 17,20-O-dipropionylavarol (11, 3.0 mg, 0.0070 mmol, 75%). 17,20-O-Dipropionylneoavarol (14, 2.1 mg, 0.0049 mmol, 70%) was prepared by a similar reaction to that for 5 (3.0 mg, 0.0096 mmol). 17,20-O-Dipropionylavarol (11): colorless oil; 1H NMR (CDCl3) δ 6.96 (1H, d, J = 8.2 Hz), 6.95 (1H, brs), 6.88 (1H, brs), 5.11 (1H, brs), 2.52−2.62 (6H, m), 1.49 (3H, s), 1.24 (3H, t, J = 7.6 Hz), 1.23 (3H, t, J = 7.6 Hz), 0.99 (3H, s), 0.92 (3H, d, J = 6.3 Hz), 0.82 (3H, s); EIMS m/z 426 [M]+; HREIMS m/z 426.2763 (calcd for C27H38O4, 426.2770). 17,20-O-Dipropionylneoavarol (14): colorless oil; 1H NMR (CDCl3) δ 6.97 (1H, d, J = 8.2 Hz), 6.93 (1H, brd, J = 8.2), 6.91 (1H, d, J = 2.9), 4.70 (1H, brs), 4.66 (1H, brs), 2.53−2.64 (6H, m), 1.53 (3H, s), 1.25 (3H, t, J = 7.5 Hz), 1.24 (3H, t, J = 7.5 Hz), 1.02 (3H, s), 0.93 (3H, d, J = 6.3 Hz), 0.87 (3H, s); EIMS m/z 426 [M]+; HREIMS m/z 426.2763 (calcd for C27H38O4, 426.2770). Conformational Analyses and Calculations of ECD Spectra. Preliminary conformational analysis of each compound was performed by using the MMFF94 force field, followed by full geometry optimization by the density functional theory (DFT) method with the B3LYP functional and the 6-31G(d) basis set. These calculations were performed using Spartan’14 (Wavefunction, Inc.). The ECD spectra of (5S,8R,9S,10R)-1 and (5S,8S,9S,10R)-1 in CH3CN were calculated using Gaussian 09 (Gaussian, Inc., 2009) by the time-dependent DFT method with the CAM-B3LYP functional and the 6-31+G(d,p) basis set. For both compounds, the calculations were performed for the two lowest energy conformers, which differ just in the orientation of the OH group and lie within 0.06 and 0.04 kcal mol−1 of one another for (5S,8R,9S,10R)-1 and (5S,8S,9S,10R)-1, respectively. For (5S,8R,9S,10R)-1, the energies of the other conformers are higher than the most stable one by more than 8.6 kcal mol−1. For (5S,8S,9S,10R)-1, no other conformers were found. The solvent effect was introduced by the polarizable continuum model. Forty low-lying excited states were calculated for both compounds, corresponding to the wavelength region down to about 160 nm. The simulated spectrum for each conformer was generated by using GaussView 5.0.9 (Semichem, Inc., 2009) with the peak halfwidth at half-height being 0.2 eV, and the Boltzmann-averaged spectra at 298.15 K were calculated by using Excel 2013 (Microsoft Co., Redmond, WA, USA). The calculated spectra were red-shifted by 25 nm for matching with the experimental spectrum of (5S,8R,9S,10R)-1. PTP1B-Inhibitory Assay. PTP1B-inhibitory activity was determined by measuring the rate of hydrolysis of the substrate, pnitrophenyl phosphate, according to the reported method with a slight modification.5a,12 Briefly, PTP1B (100 μL of a 0.5 μg/mL stock solution) in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCI, 1 mM dithiothreitol, and 1 mM EDTA was added to each well of a 96well plastic plate. Each sample (2.0 μL in CH3OH) was added to each well to make the final concentration and was then incubated at 37 °C E
DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
for 10 min. The reaction was initiated by the addition of pNPP (100 μL of a 4.0 mM stock solution) to the citrate buffer, incubated at 37 °C for 30 min, and terminated with the addition of 10 μL of a stop solution (10 M NaOH). The optical density of each well was measured at 405 nm using an MTP-500 microplate reader (Corona Electric Co., Ltd.). PTP1B-inhibitory activity (%) was defined as [1 − (ABSsample − ABSblank)/(ABScontrol − ABSblank)] × 100, in which ABSblank is the absorbance of wells containing only the buffer and pNPP, ABScontrol is the absorbance of p-nitrophenol liberated by the enzyme in the assay system without a test sample, and ABSsample is that with a test sample. Oleanolic acid, a known phosphatase inhibitor,11 was used as a positive control. Data are expressed as the mean ± SE (n = 4).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00367. Experimental data for known compounds 4−8, 1D and 2D NMR spectra of 1−3, and 1H NMR spectra of 9−14 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel/fax (H. Yamazaki): +81 22 727 0218. E-mail: yamazaki@ tohoku-mpu.ac.jp. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by Sasakawa Grants for Science Fellows from the Japan Science Society to H.Y. and the Foundation for Japanese Chemical Research to H.Y. The calculations by Gaussian 09 were performed using supercomputing resources at the Cyberscience Center, Tohoku University. We express our thanks to Dr. K. Ogawa of the Z. Nakai Laboratory for identifying the marine sponge, and Mr. T. Matsuki and S. Sato of Tohoku Medical and Pharmaceutical University for measuring mass and NMR spectra.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00367 J. Nat. Prod. XXXX, XXX, XXX−XXX
