Cyclic diarylheptanoids as inhibitors of NO production from Acer nikoense

Article abstract of DOI:10.1007/s11418-012-0660-0

We prepared a series of acerogenins A and B derivatives as inhibitors of nitric oxide (NO) production in vitro. Our results suggested that an ester group at a hydroxyl at C-2 improved inhibitory effects without cytotoxicity. A benzoyl ester derivative of acerogenin C showed the most potent inhibitory activity of NO production from lipopolysaccharide-activated macrophages.

Full text of DOI:10.1007/s11418-012-0660-0

J Nat Med (2013) 67:234–239
DOI 10.1007/s11418-012-0660-0
NOTE
Cyclic diarylheptanoids as inhibitors of NO production
from Acer nikoense
•
Jun Deguchi Yusuke Motegi Asami Nakata
•
•
•
Takahiro Hosoya Hiroshi Morita
Received: 20 December 2011 / Accepted: 8 March 2012 / Published online: 29 March 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract We prepared a series of acerogenins A and B
derivatives as inhibitors of nitric oxide (NO) production in
vitro. Our results suggested that an ester group at a
hydroxyl at C-2 improved inhibitory effects without cyto-
toxicity. A benzoyl ester derivative of acerogenin C
showed the most potent inhibitory activity of NO produc-
tion from lipopolysaccharide-activated macrophages.
for oxidative stress-induced inflammatory diseases and
septic shock [9].
The barks of Acer nikoense (Aceraceae) are used as a
Japanese folk medicine for hepatic disorders and eye dis-
eases, and contain a series of acerogenins and acerosides
[10] with a range of biological activities such as anti-cancer
[11, 12], anti-inflammatory [13], and anti-bacterial effects
[14]. In a previous report, our efforts at identifying inhib-
itors of SGLT1 and SGLT2 resulted in the isolation of two
cyclic diarylheptanoids, acerogenins A (1) and B (2) from
the barks of Acer nikoense [15]. Recently, Yoshikawa et al.
[16] reported that 1 and 2 showed inhibition of NO pro-
duction from LPS-activated macrophages without cyto-
toxic effects. Therefore, 1 and 2 may be valuable leads as
inhibitors of NO production and have potential applications
in inflammatory diseases. In this paper, we prepared a
series of their derivatives to investigate the effects of two
hydroxyl moieties of 1 and 2 against inhibition of NO
production in vitro (Fig. 1).
Keywords Acerogenin Á Acer nikoense Á Aceraceae Á
Nitric oxide
Introduction
Nitric oxide (NO) is an important intracellular and inter-
cellular signaling molecule as a mediator in cardiovascular,
nervous, and immunological systems [1]. NO is involved in
various biological reactions including vasorelaxation [2],
inhibition of platelet aggregation [3], neurotransmission
[4], inflammation [5], and immunoregulation [6]. In
mammalian cells, NO is synthesized from L-arginine
(L-Arg) by NO synthase (NOS), which is classified into
three homologues; inducible-NOS (iNOS), endothelial-
NOS (eNOS), and neuronal-NOS (nNOS) [7]. iNOS pro-
duces large amounts of NO in macrophages stimulated
with lipopolysaccharide (LPS) and/or proinflammatory
cytokines such as tumor necrosis factor (TNF) and inter-
feron-c (IFN-c) [8]. Therefore, inhibition of excess NO
production by iNOS might have potential therapeutic value
Results and discussion
Acerogenins A (1) [17, 18] and B (2) [19], and acerosides I
(3) [20] and VII (4) [21] were isolated from barks of Acer
nikoense and purified using a procedure described previ-
ously [15]. Oxidation of 1 and 2 by Swern’s procedure
gave the acerogenins C (5) [22, 23] and L (6) [24],
respectively. Dehydroxy derivative 7 was prepared by
hydrogenation of the double bond from a dehydrated
derivative. The esters (8–16) were synthesized using stan-
dard acylation procedures. Acerogenins A (1), C (5), and 7
were treated with acetic anhydride or benzoyl chloride in
pyridine to give 8–11 and 16. Esters 12–15 were formed
from 5 and the corresponding chloride in pyridine.
J. Deguchi Á Y. Motegi Á A. Nakata Á T. Hosoya Á
H. Morita (&)
Faculty of Pharmaceutical Sciences, Hoshi University,
Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan
e-mail: moritah@hoshi.ac.jp
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J Nat Med (2013) 67:234–239
235
3
1
Table 1 Effects of acerogenins, acerosides, and their derivatives on
NO production inhibitory activity
7
11
9
4
5
10
19
14
8
3
2
6
12
Cell viability (%)a
18
1
Compounds
Inhibition (IC50, lM)
2
13
17
16
15
1
162.3
[200
[100
[100
61.4
106
106
100
100
87
β
1
1
1
1
1
1
1
1
2
3
2
3
2
2
2
2
3
β
3
2
4
2
3
2
5
2
2
2
3
3
2
2
6
[100
23.9
122
6
β
2
3
2
7
β
2
3
2
8
[100
68.2
86
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
2
2
9
106
88
10
11
12
13
14
15
16
17
18
19
20
21
22
23
a
47.4
3
2
5
3
2
13.0
86
3
2
3
2
3
2
92.7
47
3
3
2
69.0
27
2
2
3
2
[100
[100
66.5
27
2
2
3
2
110
73
β
2
2
β
2
3
3
2
74.6
90
2
2
2
3
2
[100
39.0
107
106
95
2
2
3
2
3
2
82.6
35.9
90
Fig. 1 Structures of acerogenins A (1) and B (2) and acerosides I (3)
and VII (4) and their derivatives 5–23
65.7
96
68.7
100
Incorporation of ethers at C-2 was accomplished by treat-
ment of 1 and 5–7 with the corresponding alkyl halides in
the presence of K2CO3 in acetone to provide compounds
17–22. Reaction of 5 with hydroxylamine hydrochloride
provided the oxime 23 [26].
All compounds were evaluated at 50 lM
increased the inhibitory activity. Introduction of the
methoxy and chloro substituents as representative for
electro-withdrawing and electro-donor moiety at the para
position resulted in a completely loss of the inhibitory
activity. Derivatives 16 and 17 showed no improvement to
the inhibitory activity though they weakened the cytotoxic
effects.
The NO inhibitory activities of acerogenins A (1) and B
(2), acerosides I (3) and VII (4) and their derivatives 5–23
were evaluated as shown in Table 1. Although acerogenins
A (1) and B (2) inhibited NO production at IC50 74 and
88 lM, respectively, in the previous report [16], only ac-
erogenin A (1) showed a weak inhibitory activity of NO
production from LPS-activated macrophages in our assay
system (1: IC50 162.3 lM; 2: IC50 [200 lM). No inhibi-
tory activity of acerosides I (3) and VII (4) indicated that a
hydrophilic group such as glucose diminished the activity.
Similar patterns were observed in the ether derivatives
18–22. Methyl ethers 18, 20, and 22 showed little effects
on the inhibitory activity. A benzyl group of 19 and 21 led
to an increase in the inhibitory activity similar to the
benzoyl group. In comparison with 5 and 23, the oxime
group had no influence on the activity.
Acerogenin
C (5) showed moderate activity (IC50
61.4 lM) along with 1 and 2. On the other hand, acerog-
enin L (6), possessing a carbonyl group at C-9, showed no
inhibitory activity ([100 lM) even though the structure
was very similar to that of 5. Removing a hydroxyl group
at C-11 of 1 built the cytotoxic derivative 7 (Table 1).
Some of the nine esters 8–16 showed improvement of
the inhibitory activity. Derivatives 8, 12, and 13 with bis-
esters and an alkyl ester such as acetyl, isobutyl, and
heptanoyl group diminished the inhibitory activity. Inter-
estingly, a benzoyl derivative 11 of acerogenin C (5)
In conclusion, we synthesized and evaluated a series of
acerogenin derivatives. Of them, the benzoyl ester 11
derived from acerogenin C (5) exhibited potent inhibitory
activity of NO production from LPS-activated macro-
phages without cytotoxic effect. In addition, we found that
the presence of the substituents on the side chain at C-9 and
C-11 and the hydroxyl at C-2, and the position of each
substituent, have influence on the potency of inhibition of
NO production as well as cytotoxicity.
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J Nat Med (2013) 67:234–239
Further studies on 11, such as the mechanisms of inhi-
bition of NO production, are necessary to develop a unique
anti-inflammatory drug.
(1 atm). The reaction mixture was stirred under H2 for 1 h,
then filtered over a plug of silica gel topped with Celite
(MeOHeluent)togive7(17 mg, 60 %).UV(MeOH)kmax (e)
279.5 (1500) and 203 (27000); IR (KBr) 3442, 1510, and
1
1214 cm-1; H NMR (400 MHz, CDCl3) d 0.64 (2H, m),
Experimental section
0.78 (2H, m), 1.03 (2H, m), 1.23 (2H, m), 1.50 (2H, m), 2.36
(2H, m), 2.60(2H, m), 5.67(1H, d, J = 2.3 Hz), 6.52(1H, dd,
J = 2.3, 8.0 Hz), 6.76 (1H, d, J = 8.0 Hz), 6.95 (2H, d,
J = 8.6 Hz), 7.17 (2H, d, J = 8.6 Hz); ESIMS m/z 305
(M ? Na)?. HRESIMS m/z 305.15228 [calcd. for C19H22
NaO2 (M ? Na)?, 305.15175].
General experimental procedures
Optical rotations were measured on a JASCO P-1030
polarimeter. UV spectra were obtained on an Ultrospec
2100 pro spectrophotometer, and IR spectra on a JASCO
1
FTIR-230 spectrometer. H NMR spectrum was recorded
Synthesis of acerogenin A diacetate (8)
on a Bruker AV 400 spectrometer at 300 K, and chemical
shifts were reported using residual CHCl3 (dH 7.26) as
internal standard. Mass spectra were obtained with a
Micromass LCT spectrometer.
Ac2O (500 lL) was added to a pyridine solution of 1
(4.4 mg, 14.8 lmol) and the solution was stirred at room
temperature for 30 min. The reaction mixture was worked
up in the usual way and the product was purified by column
chromatography (hexane/ethyl acetate = 9:1) to give 8
(5 mg, 88 %) as a white powder. Spectroscopic data
corresponded to those in Ref. [24].
Synthesis of acerogenin C (5)
DMSO (141 lL, 2.0 mmol) was added to a stirred solution
of oxalyl chloride (113 lL, 1.3 mmol) in CH2Cl2 (7 mL) at
-78 °C under argon atmosphere. After the mixture had
been stirred for 5 min, a solution of 1 (200 mg, 0.67 mmol)
in CH2Cl2 was added, and stirring was continued for an
additional 15 min. Et3N (472 lL, 3.4 mmol) was added
and the mixture was stirred for 1 h at 0 °C. H2O was then
added and extracted with ethyl acetate. The combined
organic phase was dried with Na2SO4. Purification with
column chromatography (hexane/ethyl acetate = 9:1) gave
5 (129.2 mg, 65 %) as a white powder. Spectroscopic data
corresponded to those in Ref. [23].
Synthesis of acerogenin A dibenzoate (9)
Benzoyl chloride (6.0 lL, 52 lmol) was added to a stirred
CHCl3 solution of 1 (4.7 mg,15.7 lmol) andDMAP(catalytic
amount) at 0 °C. The reaction mixture was allowed to warm to
room temperature and stirred for 30 min. Ice-cold water
(5 mL) and ethyl acetate were added to the reaction mixture.
The organic layer was concentrated in vacuo and then purified
by column chromatography (hexane/ethyl acetate = 9:1) to
give 9 (2.0 mg, 25 %) as a white powder. [a]D ?33; UV
(MeOH) kmax (e) 270 (3100), 225 (25000), and 200.5 (41000);
IR (KBr) 1738, 1649, 1598, 1509, and 1421 cm-1; 1H NMR
(400 MHz, CDCl3) d 1.00(2H, m), 1.42(2H, m), 1.70(3H, m),
2.08 (1H, m), 2.56 (2H, m), 2.81 (1H, m), 2.98 (1H, m), 4.64
(1H, m), 5.90 (1H, d, J = 2.0 Hz), 6.73 (1H, dd, J = 2.0,
8.1 Hz), 7.05 (1H, d, J = 8.1 Hz), 7.06 (1H, dd, J = 2.4,
8.3 Hz), 7.21 (1H, dd, J = 2.4, 8.3 Hz), 7.28 (1H, dd, J = 2.4,
8.3 Hz), 7.33 (1H, dd, J = 2.4, 8.3 Hz), 7.45 (2H, dd, J = 7.0,
7.0 Hz), 7.53 (2H, dd, J = 7.0, 7.0 Hz), 7.57 (1H, over-
lapped), 7.64 (1H, dddd, J = 1.3, 1.3, 7.0, 7.0 Hz), 8.05 (2H,
brd, J = 7.0 Hz), 8.30 (2H, brd, J = 7.0 Hz); ESIMS m/z 529
(M ? Na)?. HRESIMS m/z 529.19909 [calcd. for
C33H30NaO5 (M ? Na)?, 529.20154].
Synthesis of acerogenin L (6)
The compound was prepared in a manner similar to that of
5 from 2 (26.8 mg) with 65 % yield (13 mg) as a white
powder. Spectroscopic data corresponded to those in
Ref. [24].
Synthesis of 11-dehydroxy acerogenin A (7)
p-TsOH (5.7 mg, 0.03 mmol) was added to a stirred toluene
solution of 1 (100 mg, 0.34 mmol) and the reaction mixture
was stirred at 100 °C for 1 h. Saturated aqueous NaHCO3
was added to the mixture and extracted with ethyl acetate.
The combined organic phases were washed with brine, dried
over Na2SO4, and concentrated in vacuo. Purification with
column chromatography (hexane/ethyl acetate = 9:1) gave
dehydrated compound (84 mg, 88 %) as a white powder. The
dehydrated compound obtained (32 mg, 0.1 mmol) and
10 % Pd/C (3 mg) were combined in MeOH (8 mL) and the
reaction vessel was evacuated and back-filled with H2
Synthesis of acerogenin C acetate (10)
The compound was prepared in a manner similar to that of
8 from 5 (2.0 mg) with 96 % yield (2.2 mg) as a white
powder. UV (MeOH) kmax (e) 365.5 (10000) and 200.5
1
(10000); IR (KBr) 1766, 1707, 1510, and 1201 cm-1; H
NMR (400 MHz, CDCl3) d 1.06 (2H, m), 1.38 (2H, m),
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J Nat Med (2013) 67:234–239
237
Synthesis of acerogenin C p-chlorobenzoate (14)
1.89 (2H, m), 2.37 (3H, s), 2.50 (2H, m), 2.60 (2H, m), 2.98
(2H, m), 5.75 (1H, d, J = 2.0 Hz), 6.70 (1H, dd, J = 2.0,
8.5 Hz), 6.92 (1H, d, J = 8.5 Hz), 7.02 (2H, d,
J = 8.5 Hz), 7.16 (2H, d, J = 8.5 Hz); ESIMS m/z 361
(M ? Na)?. HRESIMS m/z 361.14060 [calcd. for
C21H22NaO4 (M ? Na)?, 361.14158].
The compound was prepared in a manner similar to that of
9 from 5 (3.8 mg) with 81 % yield (4.2 mg) as a white
powder. UV (MeOH) kmax (e) 273.5 (2800), 236 (25000),
and 200.5 (62000); IR (KBr) 1742, 1594, 1504, and
1
1262 cm-1; H NMR (400 MHz, CDCl3) d 1.09 (2H, m),
1.42 (2H, m), 1.90 (2H, m), 2.53 (2H, m), 2.60 (2H, m),
2.97 (2H, m), 5.80 (1H, d, J = 2.0 Hz), 6.75 (1H, dd,
J = 2.0, 8.0 Hz), 7.04 (2H ? 1H, overlapped), 7.15 (2H,
d, J = 8.6 Hz), 7.49 (2H, d, J = 8.6 Hz), 8.21 (2H, d,
J = 8.6 Hz); ESIMS m/z 457 (M ? Na)?. HRESIMS m/z
435.13780 [calcd. for C26H24ClO4 (M ? H)?, 435.13631].
Synthesis of acerogenin C benzoate (11)
The compound was prepared in a manner similar to that of
9 from 5 (2.0 mg) with 96 % yield (2.7 mg) as a white
powder. UV (MeOH) kmax (e) 273.5 (2900), 228.5 (25000),
and 201 (40000); IR (KBr) 1741, 1707, 1510, and
1
1201 cm-1; H NMR (400 MHz, CDCl3) d 1.08 (2H, m),
Synthesis of acerogenin C p-methoxybenzoate (15)
1.41 (2H, m), 1.92 (2H, m), 2.53 (2H, m), 2.60 (2H, m),
2.96 (2H, m), 5.82 (1H, d, J = 2.1 Hz), 6.75 (1H, dd,
J = 2.1, 8.1 Hz), 7.05 (1H, d, J = 8.1 Hz), 7.06 (2H,
d, J = 8.5 Hz), 7.15 (2H, d, J = 8.5 Hz), 7.52 (2H, dd,
J = 7.1, 7.1 Hz), 7.63 (1H, dd, J = 7.1, 7.1 Hz), 8.28 (2H,
d, J = 7.1 Hz); ESIMS m/z 423 (M ? Na)?. HRESIMS
m/z 423.15791 [calcd. for C26H24NaO4 (M ? Na)?,
423.15723].
The compound was prepared in a manner similar to that of
9 from 5 (3.0 mg) with 79 % yield (3.2 mg) as a white
powder. UV (MeOH) kmax (e) 260 (17000) and 203.5
1
(43000); IR (KBr) 1734, 1601, 1507, and 1260 cm-1; H
NMR (400 MHz, CDCl3) d 1.08 (2H, m), 1.42 (2H, m),
1.89 (2H, m), 2.46 (2H, m), 2.59 (2H, m), 3.00 (2H, m),
3.90 (3H, s), 5.80 (1H, d, J = 2.0 Hz), 6.74 (1H, dd,
J = 2.0, 8.3 Hz), 6.99 (2H, d, J = 9.0 Hz), 7.04 (1H,
d, J = 8.3 Hz), 7.05 (2H, d, J = 8.5 Hz), 7.15 (2H, d,
J = 8.5 Hz), 8.25 (2H, d, J = 9.0 Hz); ESIMS m/z 453
(M ? Na)?. HRESIMS m/z 431.18780 [calcd. for
C27H27O5 (M ? H)?, 431.18585].
Synthesis of acerogenin C heptanoate (12)
The compound was prepared in a manner similar to that of
9 from 5 (4.7 mg) with 6 % yield (0.4 mg) as a white
powder. UV (MeOH) kmax (e) 274 (400) and 200.5
1
(17000); IR (KBr) 1762, 1707, 1508, and 1208 cm-1; H
Synthesis of 11-dehydroxy acerogenin A benzoate (16)
NMR (400 MHz, CDCl3) d 0.87 (3H, t, J = 7.0 Hz), 1.06
(2H, m), 1.31 (4H, m), 1.41 (4H, m), 1.80 (2H, m), 1.88
(2H, m), 2.49 (2H, m), 2.63 (4H, m), 2.98 (2H, m), 5.75
(1H, d, J = 2.0 Hz), 6.69 (1H, dd, J = 2.0, 8.5 Hz), 6.92
(1H, d, J = 8.5 Hz), 7.03 (2H, d, J = 8.1 Hz), 7.15 (2H, d,
J = 8.1 Hz); ESIMS m/z 431 (M ? Na)?. HRESIMS m/z
431.22202 [calcd. for C26H32NaO4 (M ? Na)?,
431.21983].
The compound was prepared in a manner similar to that of
9 from 7 (2.0 mg) with 99 % yield (2.7 mg) as a white
powder. UV (MeOH) kmax (e) 273.5 (3600) and 228.5
(23000); IR (KBr) 2928, 1742, 1593, 1505, and
1
1260 cm-1; H NMR (400 MHz, CDCl3) d 0.72 (2H, m),
0.87 (2H, m), 1.12 (2H, m), 1.36 (2H, m), 1.57 (2H, m),
2.52 (2H, m), 2.66 (2H, m), 5.93 (1H, d, J = 2.1 Hz), 6.73
(1H, dd, J = 2.1, 8.3 Hz), 7.04 (1H, d, J = 8.3 Hz), 7.07
(2H, dd, J = 2.0, 8.6 Hz), 7.23 (2H, dd, J = 2.0, 8.6 Hz),
7.53 (2H, dd, J = 7.3, 7.3 Hz), 7.64 (1H, dd, J = 2.0,
7.3 Hz), 8.29 (2H, d, J = 7.3 Hz); ESIMS m/z 409
(M ? Na)?. HRESIMS m/z 409.17819 [calcd. for
C26H26NaO3 (M ? Na)?, 409.17796].
Synthesis of acerogenin C isobutylate (13)
The compound was prepared in a manner similar to that of
9 from 5 (5.0 mg) with 58 % yield (3.6 mg) as a white
powder. UV (MeOH) kmax (e) 276.5 (1400) and 202.5
(35000); IR (KBr) 1759, 1708, 1593, 1504, and
1
1253 cm-1; H NMR (400 MHz, CDCl3) d 1.05 (2H, m),
Synthesis of 11-dehydroxy acerogenin A benzyl ether
1.35–1.39 (6H ? 2H, overlapped), 1.88 (2H, m), 2.50 (2H,
m), 2.59 (2H, m), 2.90 (1H, m), 2.97 (2H, m), 5.76 (1H,
d, J = 1.8 Hz), 6.69 (1H, d, J = 8.0 Hz), 6.91 (1H, dd,
J = 1.8, 8.0 Hz), 7.02 (2H, d, J = 8.3 Hz), 7.15 (2H, d,
J = 8.3 Hz); ESIMS m/z 389 (M ? Na)?. HRESIMS m/z
389.17312 [calcd. for C23H26NaO4 (M ? Na)?,
389.17288].
(17)
K2CO3 (3 mg) and BnBr (3 lL, 27 lmol) were added to a
stirred acetone solution of 7 (2.0 mg, 7.1 lmol) and the
reaction mixture was refluxed for 24 h, concentrated under
reduced pressure and purified by column chromatography
(hexane/ethyl acetate = 9:1) to give 17 (1.2 mg, 45 %) as
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J Nat Med (2013) 67:234–239
a white powder. UV (MeOH) kmax (e) 276.5 (1500) and 203
(29000); IR (KBr) 2925, 2857, 1584, 1509, and
powder. Spectroscopic data corresponded to those in
Ref. [24].
1
1264 cm-1; H NMR (400 MHz, CDCl3) d 0.67 (2H, m),
0.82 (2H, m), 1.06 (2H, m), 1.25 (2H, m), 1.52 (2H, m),
2.36 (2H, m), 2.62 (2H, m), 5.19 (2H, s), 5.73 (1H, d,
J = 2.1 Hz), 6.50 (1H, dd, J = 2.1, 8.8 Hz), 6.76 (1H,
d, J = 8.8 Hz), 6.99 (2H, d, J = 8.4 Hz), 7.19 (2H, d,
J = 8.4 Hz), 7.27 (1H, dd, J = 7.1, 7.1 Hz), 7.33 (2H, dd,
J = 7.1, 7.1 Hz), 7.47 (2H, d, J = 7.1 Hz); ESIMS m/
z 395 (M ? Na)?. HRESIMS m/z 395.20065 [calcd. for
C26H28NaO2 (M ? Na)?, 395.19870].
Synthesis of acerogenin C benzyl ether (21)
The compound was prepared in a manner similar to that of
17 from 5 (2.0 mg) with 35 % yield (0.9 mg) as a white
powder. UV (MeOH) kmax (e) 199.5 (8300); IR (KBr)
1
1706, 1514, and 1261 cm-1; H NMR (400 MHz, CDCl3)
d 1.06 (2H, m), 1.36 (4H, m), 2.43 (2H, m), 2.60 (2H, m),
2.98 (2H, m), 5.22 (2H, s), 5.67 (1H, d, J = 2.0 Hz), 6.58
(1H, dd, J = 2.0, 8.2 Hz), 6.84 (1H, d, J = 8.2 Hz), 7.01
(2H, d, J = 8.4 Hz), 7.17 (2H, d, J = 8.4 Hz), 7.32 (1H,
dd, J = 7.4, 7.4 Hz), 7.39 (2H, dd, J = 7.4, 7.4 Hz), 7.50
(2H, d, J = 7.4 Hz); ESIMS m/z 409 (M ? Na)?. HRE-
SIMS m/z 409.18009 [calcd. for C26H26NaO3 (M ? Na)?,
409.17796].
Synthesis of acerogenin A methyl ether (18)
K2CO3 (10 mg) and MeI (4.0 lL, 67 lmol) were added to a
stirred acetone solution of 1 (4.0 mg, 13.4 lmol) and the
reaction mixture was refluxed for 24 h, concentrated under
reduced pressure and purified by column chromatography
(hexane/ethyl acetate = 9:1) to give 18 (3.7 mg, 88 %) as a
white powder. [a]D -25; UV (MeOH) kmax (e) 271.5 (1100)
and 200.5 (16000); IR (KBr) 2880, 1509, 1419, and
Synthesis of acerogenin L methyl ether (22)
The compound was prepared in a manner similar to that of
18 from 6 (2.0 mg) with 72 % yield (1.5 mg) as a white
powder. Spectroscopic data corresponded to those in
Ref. [25].
1
1215 cm-1; H NMR (400 MHz, CDCl3) d 0.95 (2H, m),
1.08 (2H, m), 1.25 (1H, m), 1.48 (1H, m), 1.66 (1H, m), 1.88
(1H, m), 2.45 (2H, m), 2.69 (1H, m), 2.99 (1H, m), 3.30
(1H, m), 3.95 (3H, s), 5.62 (1H, d, J = 2.1 Hz), 6.64 (1H, dd,
J = 2.1, 8.2 Hz), 6.81 (1H, d, J = 8.2 Hz), 6.97 (1H, dd,
J = 2.2, 8.3 Hz), 7.17 (1H, dd, J = 2.2, 8.3 Hz), 7.20 (1H,
dd, J = 2.2, 8.3 Hz), 7.28 (1H, dd, J = 2.2, 8.3 Hz); ESIMS
m/z 335 (M ? Na)?. HRESIMS m/z 335.16027 [calcd. for
C20H24NaO3 (M ? Na)?, 335.16231].
Synthesis of acerogenin C oxime (23)
Sodium acetate (7.0 mg, 85 lmol) and hydroxylamine
hydrochloride (60 mg, 85 lmol) were added sequentially
to a stirred EtOH solution of 5 (10.0 mg, 34 lmol) under
argon and stirred for 2 h at room temperature. The reaction
mixture was poured into water and the precipitated product
was purified by recrystallization from EtOH to give 23
(1.4 mg, 13 %) as a white powder. UV (MeOH) kmax (e)
276.5 (1000) and 203 (56000); IR (KBr) 3479, 3372, 1599,
1509, and 1222 cm-1; 1H NMR (400 MHz, CDCl3) d 1.20
(2H, m), 1.38 (2H, m), 1.81 (2H, m), 2.51 (2H, m), 2.71
(2H, m), 3.16 (2H, m), 5.71 (1H, d, J = 2.0 Hz), 6.61 (1H,
dd, J = 2.0, 8.3 Hz), 6.86 (1H, d, J = 8.3 Hz), 7.02 (2H,
d, J = 8.6 Hz), 7.21 (2H, d, J = 8.6 Hz); ESIMS m/z
312 (M ? H)?. HRESIMS m/z 312.15748 [calcd. for
C19H22NO3 (M ? H)?, 312.15997].
Synthesis of acerogenin A benzyl ether (19)
The compound was prepared in a manner similar to that of 17
from 1 (4.0 mg) with 88 % yield (4.6 mg) as a white powder.
[a]D ?22; UV (MeOH) kmax (e) 278 (2200) and 203.5
1
(48000); IR (KBr) 1587, 1509, and 1211 cm-1; H NMR
(400 MHz, CDCl3) d 0.90 (2H, m), 1.08 (2H, m), 1.25 (1H,
m), 1.55 (2H, m), 1.88 (1H, m), 2.44 (2H, m), 2.68 (1H, m),
2.98 (1H, m), 3.29 (1H, m), 5.25 (2H, s), 5.66 (1H, d,
J = 2.1 Hz), 6.58 (1H, dd, J = 2.1, 8.2 Hz), 6.82 (1H,
d, J = 8.2 Hz), 6.95 (1H, dd, J = 2.5, 8.3 Hz), 7.17 (1H, dd,
J = 2.5, 8.3 Hz), 7.20 (1H, dd, J = 2.5, 8.3 Hz), 7.28 (1H,
dd, J = 2.5, 8.3 Hz), 7.31 (1H, dd, J = 8.0, 8.0 Hz), 7.38
(2H, dd, J = 8.0, 8.0 Hz), 7.51 (2H, d, J = 8.0 Hz); ESIMS
m/z 411 (M ? Na)?. HRESIMS m/z 411.19661 [calcd. for
C26H28NaO3 (M ? Na)?, 411.19361].
NO inhibition assay
J774.1 cells were cultured in RPMI-1640 medium supple-
mented with 10 % fetal bovine serum and penicillin/
streptomycin. J774.1 cells were seeded onto 96-well
microtiter plates at 1 9 105 cells in 100 lL per well, and
were preincubated for 12 h at 37 °C in a humidified
atmosphere containing 5 % CO2. The cells were cultured
in the medium containing LPS (5 lg/mL) with or without
the test sample at different concentrations for 24 h. The NO
Synthesis of acerogenin C methyl ether (20)
The compound was prepared in a manner similar to that of
18 from 5 (4.7 mg) with 72 % yield (1.5 mg) as a white
123

J Nat Med (2013) 67:234–239
239
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production was then determined by Griess assay: 100 lL of
the supernatant of the cultured medium were transferred to
96-well microtiter plates, and then 100 lL of Griess
reagent (1 % sulfanilamide, 0.1 % N-L-naphthylethylen-
ediamine dihydrochloride in 2.5 % H3PO4) were added.
After incubation at room temperature for 15 min, the
absorbance at 540 and 620 nm was measured with a
microplate reader (Benchmark Plus microplate spectrom-
eter, Bio-Rad). L-NMMA [27] (98 %), an NOS inhibitor,
was used as a positive control (IC50 = 73 lM).
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(2003) Structures of new cyclic diarylheptanoids and inhibitors of
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Acknowledgments This work was partly supported by a Grant-in-
Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, and a grant from the Open
Research Center Project.
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