Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa

Article abstract of DOI:10.1021/acs.jnatprod.6b00367

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.

Full text of DOI:10.1021/acs.jnatprod.6b00367

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, ava-
pyran (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-O-
acetylavarol (6),6c 20-O-acetylneoavarol (7),6d and 3′-amino-
avarone (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 configura-
tions, and biological activities of sesquiterpene hydroquinones
1−14.
he protein tyrosine phosphatase (PTP) family catalyzes
Tkey 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 Accord-
ingly, 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 polybromodi-
phenyl ethers,5a dehydroeuryspongin,5b hyattellactones,5c tri-
choketides,5d verruculides,5e and 26-O-ethylstrongylophorine-
14.5f 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
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
1
C21H28O2 from HREIMS and NMR data (Table 1). The H
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
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00367
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Chart 1
1
Table 1. H and 13C NMR Data for Avapyran (1) in CDCl3
Avapyran (1)
C#
δC, type
18.4,
δH, mult. (J in Hz)
COSY
2a
HMBC
1
CH2
CH2
1.54, m
1.79, m
1.71, m
1.95, m
5.05, brs
2
26.8,
1a, 3
3
3
4
5
6
120.4,
143.8,
37.6,
CH
C
C
31.0,
CH2
1.46, m
1.75, m
1.67, m
1.92, m
1.50, m
7b
6a
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.
7
31.5,
CH2
8
79.1,
36.8,
41.6,
17.9,
20.3,
22.8,
21.3,
35.1,
C
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 conforma-
tional 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 (δC 152.3 and 144.3,
respectively).6c
9
C
10
11
12
13
14
15
CH
CH3
CH3
CH3
CH3
CH2
1.52, m
1
1.55, s
3, 4, 5
1.04, s
4, 5, 6, 10
7, 8, 9
1.15, s
0.99, s
8, 10, 15
9, 10, 14, 16
8, 16, 17, 21
2.45, d (17.4)
2.76, d (17.4)
16
17
18
19
20
21
121.2,
147.1,
116.8,
114.2,
148.2,
114.9,
C
C
CH
CH
C
6.59, d (8.8)
19
18
16, 20
17
6.54, dd (8.8, 2.9)
CH
6.44, d (2.9)
17, 19
methylene, one sp3 methine, two sp3 quaternary, one
oxygenated sp3 quaternary, four sp2 methine, two sp2
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 (C-
1−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 C-
8 and C-17 positions were expected to be connected through
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
B
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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.
1
Table 3. H 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#
δC, type
23.1,
δH, mult. (J in Hz)
COSY
HMBC
1
CH2
CH2
CH2
1.54, m
1.90, m
1.36, m
1.88, m
2.08, m
2.28, m
Table 2. 1H and 13C NMR Data for 17-O-Acetylavarol (2) in
CDCl3
5
2
3
28.2,
32.9,
3b
2a
17-O-Acetylavarol (2)
1, 2
C#
δC, type
δH, mult. (J in Hz) COSY
HMBC
4
5
6
159.7,
40.2,
36.4,
C
1
19.8,
CH2
1.62, m
1.89, m
1.74, m
2.07, m
5.13, brs
2b
C
CH2
1.19, m
1.44, m
1.40, m
1.38, m
7
2
26.5,
CH2
1a, 3
2b
7
27.6,
36.1,
CH2
CH
C
6a
3
4
5
6
120.4,
144.3,
38.3,
CH
C
8
10
9
42.0,
C
10
11
48.0,
CH
CH2
0.98, m
35.7,
CH2
0.96, m
1.37, m
1.31, m
1.43, m
1.58, m
7a
6a
13
103.0,
4.37, brs
3, 4, 5
4.42, brs
3, 4, 5
7
27.6,
CH2
12
13
14
15
20.6,
17.8,
17.6,
37.6,
CH3
CH3
CH3
CH2
1.04, s
4, 5, 6, 10
7, 8
0.95, d (5.9)
0.82, s
8
35.8,
41.7,
45.8,
18.1,
20.0,
17.6,
17.7,
37.8,
CH
C
9, 10, 15
9, 10, 16, 21
8, 16, 17, 21
9
2.40, d (14.4)
2.46, d (14.4)
10
11
12
13
14
15
CH
CH3
CH3
CH3
CH3
CH2
1.16, m
1, 2, 5, 9
1.49, s
3, 4, 5
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
0.99, s
4, 5, 6, 10
7, 8, 9
C
0.94, d (6.3)
0.81, s
8
CH
CH
C
6.81, d (8.8)
19
18
16, 20
17, 21
8, 9, 10, 15
9, 10, 16, 17, 21
8, 9, 16, 17, 21
6.64, dd (8.8, 2.9)
2.45, d (14.4)
2.50, d (14.4)
CH
C
6.57, d (2.9)
2.26, s
17, 19
22
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
CH3
CH
CH
C
6.81, d (9.3)
19
18
16, 17, 19, 20
17, 21
6.64, dd (9.3, 2.9)
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
CH
C
6.62, brs
2.28, s
17
22
CH3
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-O-
acetylavarol.
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-O-
acetylneoavarol (7).6d Therefore, the structure of 3 was
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.
C
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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 PTP1B-
inhibitory 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.
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 J-
720 spectropolarimeter. NMR spectra were recorded on a JEOL JNM-
Table 4. PTP1B Inhibitory Activities of Compounds 1−14
compound
avapyran (1)
IC50 (μM)
11
17-O-acetylavarol (2)
9.5
6.5
12
AL-400 NMR spectrometer (400 MHz for H and 100 MHz for 13C)
1
17-O-acetylneoavarol (3)
avarol (4)
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 Pharma-
ceutical 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
neoavarol (5)
35% inhibition at 32 μM
20-O-acetylavarol (6)
10
20-O-acetylneoavarol (7)
3′-aminoavarone (8)
8.6
18
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
5% inhibition at 25 μM
0% inhibition at 31 μM
8.8
14
50% inhibition at 31 μM
9.4
1.1
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
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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].
43%) was obtained by a similar method to that for 5 (3.0 mg, 0.0096
mmol).
1
20-O-Methylavarol (10): colorless oil; H 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).
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.
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).
Avapyran (1): colorless oil; [α]24 +8.0 (c 0.08, CHCl3); UV
D
(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
1
cm−1; H and 13C NMR (CDCl3), Table 1; EIMS m/z 312 [M]+;
Preparation of Dipropionyl Derivatives (11 and 14). Triethyl-
amine (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).
HREIMS m/z 312.2099 (calcd for C21H28O2, 312.2089).
17-O-Acetylavarol (2): reddish oil; [α]24 +4.5 (c 0.27, CHCl3);
D
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,
1
2374, 1760, 1637, 1497, 1453, 1384, 1233, 1186, 1029 cm−1; H and
13C NMR (CDCl3), Table 3; EIMS m/z 356 [M]+; HREIMS m/z
356.2349 (calcd for C23H32O3, 356.2351).
Avarol (4): yellowish oil; [α]21 +13.0 (c 0.40, CHCl3); lit. [α]D
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]21 +13.0 (c 0.14, CHCl3);
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).
D
lit. [α]25 +11.0 (c 2.3, CHCl3).6c
D
20-O-Acetylneoavarol (7): reddish oil; [α]23 −40.2 (c 0.02,
D
CHCl3); lit. [α]25 −31.9 (c 0.10, CHCl3).6d
D
3′-Aminoavarone (8): reddish oil; [α]21 +30.7 (c 0.13, CH2Cl2);
D
lit. [α]22 +45.0 (c 0.10, CH2Cl2).6e
D
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.
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 half-
width 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 deter-
mined by measuring the rate of hydrolysis of the substrate, p-
nitrophenyl 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 96-
well 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
1
17,20-O-Diacetylavarol (9): colorless oil; H 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).
1
17,20-O-Diacetylneoavarol (12): colorless oil; H 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,
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
* Supporting Information
■
S
(8) (a) Puliti, R.; De Rosa, S. Acta Crystallogr., Sect. C: Cryst. Struct.
Commun. 1994, C50, 830−33. (b) Ilyin, S. G.; Shubina, L. K.; Stonik,
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00367.
(9) (a) Gordaliza, M. Mar. Drugs 2010, 8, 2849−2870. (b) Namba,
T.; Kodama, R. Mar. Drugs 2015, 13, 2376−2389. (c) Loya, S.; Hizi, A.
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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Bustos, G.; Paya,
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AUTHOR INFORMATION
Corresponding Author
*Tel/fax (H. Yamazaki): +81 22 727 0218. E-mail: yamazaki@
tohoku-mpu.ac.jp.
S.; Terencio, M. C. Life Sci. 2008, 82, 256−264. (f) Pejin, B.; Iodice,
C.; Tommonaro, G.; Stanimirovic, B.; Ciric, A.; Glamoclija, J.; Nikolic,
M.; De Rosa, S.; Sokovic, M. Curr. Pharm. Biotechnol. 2014, 15, 583−
588. (g) Menna, M.; Imperatore, C.; D’Aniello, F.; Aiello, A. Mar.
Drugs 2013, 11, 1602−1643.
■
Notes
(10) (a) Li, Y.; Zhang, Y.; Shen, X.; Guo, Y. W. Bioorg. Med. Chem.
Lett. 2009, 19, 390−392. (b) Zhang, Y.; Li, Y.; Guo, Y. W.; Jiang, H.
L.; Shen, X. Acta Pharmacol. Sin. 2009, 30, 333−345.
(11) Zhang, Y. N.; Zhang, W.; Hong, D.; Shi, L.; Shen, Q.; Li, J. Y.;
Li, J.; Hu, L. H. Bioorg. Med. Chem. 2008, 16, 8697−8705.
(12) Cui, L.; Na, M. K.; Oh, H.; Bae, E. Y.; Jeong, D. G.; Ryu, S. E.;
Kim, S.; Kim, B. Y.; Oh, W. K.; Ahn, J. S. Bioorg. Med. Chem. Lett.
2006, 16, 1426−1429.
The authors declare no competing financial interest.
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 super-
computing 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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J. Nat. Prod. XXXX, XXX, XXX−XXX