Synthesis and degranulation-inhibiting activities of the proposed apteniols B, C, and G

Article abstract of DOI:10.1080/09168451.2015.1061421

The synthesis of compounds with the structures proposed for the oxyneolignan apteniols B, C, and G is described. The diphenyl ether skeletons of the proposed apteniols were formed via Ullmann ether synthesis. In particular, the spectral data for the synth

Full text of DOI:10.1080/09168451.2015.1061421

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Synthesis and degranulation-inhibiting activities of
the proposed apteniols B, C, and G
Toshiro Noshita, Akihiro Tai, Hikaru Nishikawa, Kaoru Ikeda, Hidekazu
Ouchi, Yoshitomo Hamada, Akiko Saito & Teiko Yamada
To cite this article: Toshiro Noshita, Akihiro Tai, Hikaru Nishikawa, Kaoru Ikeda, Hidekazu
Ouchi, Yoshitomo Hamada, Akiko Saito & Teiko Yamada (2015) Synthesis and degranulation-
inhibiting activities of the proposed apteniols B, C, and G, Bioscience, Biotechnology, and
Biochemistry, 79:11, 1743-1749, DOI: 10.1080/09168451.2015.1061421
To link to this article: http://dx.doi.org/10.1080/09168451.2015.1061421
Published online: 15 Jul 2015.
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Date: 07 October 2015, At: 00:56

Bioscience, Biotechnology, and Biochemistry, 2015
Vol. 79, No. 11, 1743–1749
Synthesis and degranulation-inhibiting activities of the proposed apteniols B,
C, and G
1
,2,
1,2
2
2
3
Toshiro Noshita *, Akihiro Tai , Hikaru Nishikawa , Kaoru Ikeda , Hidekazu Ouchi ,
4
4
5
Yoshitomo Hamada , Akiko Saito and Teiko Yamada
1
Faculty of Life and Environmental Sciences, Department of Life Sciences, Prefectural University of Hiroshima,
2
Hiroshima, Japan; Program in Biological System Sciences, Graduate School of Comprehensive Scientific Research,
Prefectural University of Hiroshima, Hiroshima Japan; Faculty of Pharmaceutical Education, Department of
Pharmacy, Kinki University, Osaka, Japan; Graduate School of Engineering, Osaka Electro-Communication
University, Osaka Japan; Department of Bioscience and Biotechnology for Future Bioindustry, Graduate School of
3
4
5
Agricultural Science, Tohoku University, Sendai Japan
Received April 17, 2015; accepted May 2, 2015
http://dx.doi.org/10.1080/09168451.2015.1061421
The synthesis of compounds with the structures
proposed for the oxyneolignan apteniols B, C, and
G is described. The diphenyl ether skeletons of the
proposed apteniols were formed via Ullmann ether
synthesis. In particular, the spectral data for the
synthesized apteniols B, C, and G did not agree with
those previously reported for the isolated com-
pounds. Furthermore, the synthesized proposed
apteniol B did not show degranulation-inhibiting
activity, while the prepared proposed apteniols C
and G exhibited activities considerably weaker than
that of the methyl ester of proposed apteniol A.
If the characterization data for a compound synthe-
sized using a method different from that employed by
Jung and Bräse agrees with the data reported for natu-
ral apteniol C, then it could be concluded that the pro-
posed structure of apteniol C (3) is correct. However, if
the analytical data for two compounds prepared using
different synthetic methods do not agree with the data
for isolated 3, then it could be concluded that the pro-
posed structure for the natural product is incorrect.
Here we describe the synthesis of compounds with the
structures proposed for apteniols B (2), C (3), and G
(7), using a method different from that employed by
Jung and Bräse. Furthermore, we describe the degran-
ulation-inhibiting activities of selected compounds syn-
thesized during the present study, because the methyl
ester of the compound proposed to be apteniol A
Key words: Aptenia cordifolia; oxyneolignan; Ull-
mann ether synthesis; apteniol, degran-
ulation-inhibiting activity
4
)
showed strong degranulation-inhibiting activity.
Apteniols A–G (1–7, Fig. 1) were first isolated by
DellaGreca et al. as phytotoxic metabolites from Apte-
nia cordifolia (Aizoaceae), a perennial herb native to
South Africa that has now spread throughout Europe
and become a well-known groundcover or creeping
Experimental
General experimental procedures.
The melting
points were measured using an MP-J3 (Yanaco, Kyoto,
Japan) and are uncorrected. The IR spectra were
obtained using a Nicolet iS10 FT-IR spectrometer
(Thermo Fisher Scientific, Waltham, MA, USA) with a
diamond horizontal attenuated total reflectance (ATR)
accessory and co-addition of 16 interferograms. The
calibration models were generated using OMNIC
9.2.98 software. The H and C nuclear magnetic reso-
nance (NMR) spectra were recorded using ECA-500
(JEOL, Tokyo, Japan) and Agilent 400-MR DD2 (Agi-
lent, Santa Clara CA, USA) spectrometers, respectively,
with tetramethylsilane as the internal standard. The
mass spectra were recorded using a JMS-700 (JEOL)
mass spectrometer. Column chromatography was per-
formed on silica gel 60 N (100–210 mesh, Kanto
Chemical Co., Tokyo, Japan).
1
,2)
plant; their structures were also determined.
more, Devi et al. reported the isolation of apteniol A
1) using the marine bacterium Bacillus licheniformis
Further-
(
3)
SAB1 as an antimicrobial metabolite. In 2014, we
synthesized the compound reported as apteniol A (1),
and Jung and Bräse synthesized the compound reported
4
)
1
13
5
)
as apteniol C (3) in 2009. These two studies revealed
that the spectral data for the isolated apteniols A (1)
and C (3) slightly differed from those of the synthe-
sized compounds. Moreover, DellaGreca et al.
described the conversion of apteniol B (2) to apteniol
C (3) via esterification. On the other hand, the synthe-
sis of apteniols B and G is not reported previously.
Therefore, it is necessary to evaluate the structure of
apteniol B.
*Corresponding author. Email: noshita@pu-hiroshima.ac.jp
©
2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

1744
T. Noshita et al.
Fig. 1. Proposed structures of apteniols A–G.
Chemicals.
Both p-nitrophenyl-2-acetamido-2-
buffer (pH 4.5), and the mixture was incubated in a
96-well plate at 37 °C for 90 min. The reaction was
terminated by adding a 2 M glycine buffer (pH 10.4,
40 μL), and the absorbance at 405 nm was measured
using a microplate reader.
deoxy-β-D-glucopyranoside and a penicillin–strepto-
mycin mixed solution were obtained from Nacalai Tes-
que, Kyoto, Japan. The antibody monoclonal anti-
dinitrophenyl and DNP-labeled human serum albumin
were purchased from Sigma-Aldrich Co., St. Louis,
MO, USA. Dulbecco’s modified Eagle’s medium was
purchased from Corning, NY, USA. Fetal bovine serum
was acquired from HyClone, Logan, UT, USA. Wort-
mannin was purchased from Wako Pure Chemical
Industries, Osaka, Japan. All other chemicals were of
at least reagent grade and used as received without fur-
ther purification.
Data analysis of the degranulation-inhibiting
assays.
assays are expressed as means and standard deviations
SDs) of three independent cultures. Multiple data com-
The results of the degranulation-inhibiting
(
parisons were performed by analyzing the variance
using Dunnett’s test. P-values of less than 5% were
regarded as significant. One asterisk (*) indicates a p-
value <0.05, and two asterisks (**) indicate a p-value
<0.01.
Evaluation of degranulation-inhibiting activity.
The inhibitory activity against the release of β-hex-
osaminidase from RBL-2H3 cells was evaluated using
a modified version of the method reported by Watanabe
Synthesis
6
)
et al. RBL-2H3 cells were purchased from the JCRB
Cell Bank (Osaka, Japan). Dulbecco’s modified Eagle’s
medium containing 10% heat-inactivated fetal bovine
serum, 100 U/mL penicillin, and 100 μg/mL strepto-
mycin was used as the growth medium. The cells were
Coupling of bromobenzene with phenols (general
coupling procedure).
A mixture of bromobenzene
(6.60 mmol), phenol (7.40 mmol), Cs CO (3.92 g,
2
3
12.0 mmol), copper iodide (140 mg, 0.72 mmol), N,N-
dimethylglycine HCl salt (250 mg, 1.81 mmol), and
DMF (10 mL) was heated to 100 °C in a sealed tube
under a nitrogen atmosphere for 4 days with stirring.
The cooled mixture was then poured into water and
extracted three times with CH Cl . The combined
4
cultured in a 96-well plate (5.0 × 10 cells/well) for 24
h at 37 °C under a humidified 5% CO atmosphere and
incubated in a growth medium containing 50 ng/mL
mouse monoclonal anti-dinitrophenyl (DNP) IgE for 2
h. The cells were then washed with modified Tyrode’s
2
2
2
organic layers were washed with brine and dried over
(
(
MT) buffer before a test compound or wortmannin
2.5 μM) was added. The test compounds and wortman-
Na SO Evaporation of the solvent and purification of
2
4.
the residue by column chromatography (n-hexane/ethyl
acetate = 4:1) on silica gel yielded the desired dialde-
hyde.
nin were first dissolved in dimethyl sulfoxide (DMSO)
and diluted with MT buffer to obtain a final DMSO
concentration of 0.25%. After 20 min of incubation,
DNP-labeled human serum albumin (50 ng/mL final
concentration) was added to the cells, and the culture
was incubated for 1 h. The supernatant was subse-
quently collected, and the cells were lysed with MT
buffer containing 0.1% Triton X-100. The β-hex-
osaminidase activities of the supernatant and cell lysate
were determined using the method reported by Demo
4-(4-Formylphenoxy)-3-methoxybenzaldehyde
(7;
proposed apteniol G). According to the general cou-
pling procedure, the coupling of vanillin (1.130 g, 7.40
mmol) and 4-bromobenzaldehyde (1.370 g, 6.60 mmol)
produces compound 7 (proposed apteniol G) as a pale
yellow powder with a 31.4% yield (530 mg, 2.07
mmol). Mp 127.0–127.3 °C; IR νmax (diamond ATR)
7
)
et al. The supernatant or cell lysate (20 μL) was
mixed with 3.3 mM p-nitrophenyl-2-acetamide-2-
deoxy-β-D-glucopyranoside (40 μL) in a 100 mM citrate
−
1
cm : 1683, 1577, 1500, 1274, 1231, and 1149. The
1
13
H and C NMR spectral data are reported in Table 1.

Synthesis of the proposed apteniols B, C, and G
1745
+
HREIMS m/z (M ): Calcd. for C H O : 256.0736,
of diester 9 (234 mg, 0.58 mmol) with a small amount
1
5 12 4
Found: 256.0739.
of NaOH (100 mg, 2.5 mmol) in THF/H O (1:2; 10
2
mL) was stirred at room temperature for 8 h. The solu-
tion was then poured into 4% aqueous HCl and
extracted three times with ethyl acetate. The combined
organic layers were washed with water and brine and
then dried over Na SO Evaporation of the extract and
Ethyl (E)-3-(4-(4-((E)-3-ethoxy-3-oxoprop-1-en-1-yl)-
-methoxyphenoxy)phenyl) acrylate (8). Sodium
hydride (60% dispersion in mineral oil, 253 mg, 6.33
mmol) was washed with dry n-hexane and suspended
in dry benzene (10 mL). This suspension was added
dropwise to a solution of triethyl phosphonoacetate
2
2
4.
purification of the residue by preparative TLC (CHCl3/
methanol = 10:1) yielded 2 as a white powder (172 mg,
0
.50 mmol, 86.2%). Mp. 84.5–85.0 °C. IR ν
(dia-
max
−
1
(
1.16 g, 5.17 mmol) at ice-cooled temperature. The
mond ATR) cm : 3001, 2933, 1700, 1505, 1445,
1
13
solution was then stirred at room temperature until gas
evolution ceased. The resultant yellow solution was
added dropwise to a solution of 7 (521 mg, 2.00 mmol)
in dry benzene at ice-cooled temperature. The solution
was then stirred at room temperature for 4 h and subse-
quently poured into water and extracted three times
with ethyl acetate. The combined organic layers were
washed with brine and dried over Na SO Evaporation
1414, 1274, and 1217. H and C NMR spectral data
+
are reported in Table 2. HREIMS m/z (M ): Calcd. for
C H O : 344.1260, Found: 344.1262.
1
9 20 6
Methyl
3-(4-(2-methoxy-4-(3-methoxy-3-oxopropyl)
phenoxy)phenyl)propanoate (3; proposed apteniol C).
A solution of the proposed apteniol B (2) (50 mg, 0.15
mmol) was refluxed for 24 h in the presence of a cat-
alytic amount of H SO in dry methanol (5 mL). The
2
4.
of the extract and purification of the residue by column
chromatography (CHCl /benzene = 1:1) on silica gel
3
2
4
yielded diester 8 (790 mg, 2.00 mmol, 99.6%). IR ν
cooled solution was then evaporated in vacuo. The dry
residue was diluted with ethyl acetate, and this solution
max
−
1
(
diamond ATR) cm : 1704, 1634, 1591, 1499, 1259,
1
1
155. H NMR δ (400 MHz in CD OD): 1.30 (3H, t, J
was washed with saturated aqueous NaHCO and brine
3
3
=
7.1 Hz), 1.32 (3H, t, J = 7.1 Hz), 3.79 (3H, s), 4.21
and then dried over Na SO . Evaporation of the solvent
2
4
(
2H, q, J = 7.1 Hz), 4.24 (2H, q, J = 7.1 Hz), 6.38 (1H,
and purification by preparative TLC (n-hexane/ethyl
acetate = 2:1) yielded diester 3 (proposed apteniol C)
d, J = 16.1 Hz), 6.51 (1H, d, J = 15.9 Hz), 6.86 (2H, d,
J = 8.5 Hz), 7.04 (1H, dd, J = 8.3, 2.0 Hz), 7.20 (1H, d,
J = 8.3 Hz), 7.35 (1H, d, J = 2.0 Hz), 7.52 (2H, d,
J = 8.5 Hz), 7.62 (1H, d, J = 16.1 Hz), 7.66 (1H, d,
as a pale yellow oil (42 mg, 0.11 mmol, 73%). IR ν
max
−
1
(diamond ATR) cm : 1734, 1505, 1271, 1226, 1167,
1
13
and 1155. H and C NMR spectral data are reported
1
3
+
J = 15.9 Hz). C NMR δ (100 MHz in CD OD): 14.7
in Table 3. HREIMS m/z (M ): Calcd. for C H O :
3
21 24 6
(
1
1
overlapped), 56.5, 61.6, 61.7, 113.4, 117.4, 117.7,
19.0, 123.0, 123.2, 130.1, 130.9, 133.7, 145.41,
45.43, 147.0, 153.3, 161.2, 168.6, 168.8. HREIMS m/
372.1573, Found: 372.1575.
+
4-(4-Formylphenoxy)-2-methoxybenzaldehyde (10).
z (M ): Calcd. for C H O : 396.1573, Found:
3
2
3 24 6
According to the general coupling procedure, the cou-
pling of 4-bromo-2-methoxybenzaldehyde (1.420 g,
96.1577.
6.60 mmol) and 4-hydroxybenzaldehyde (900 mg, 7.40
Ethyl 3-(4-(2-methoxy-4-(3-methoxy-3-oxopropyl)phe-
noxy)phenyl)propanoate (9). A suspension of diester
(790 mg, 2.00 mmol) and 5% Pd/C (50.0 mg) in dry
mmol) produced compound 10 as a colorless crystals
with a 14.5% yield (246 mg, 0.96 mmol). Mp. 107.5–
−
1
8
109.3 °C. IR νmax (diamond ATR) cm : 1691, 1576,
1
methanol (8 mL) was stirred at room temperature under
hydrogen for 2 h. The reaction mixture was then fil-
tered and concentrated in vacuo. The dry residue was
purified by preparative thin-layer chromatography (p-
TLC) to produce diester 9 as a pale yellow oil (380
1222, 1159, and 838. H NMR δ (400 MHz in
CD OD): 3.95 (3H, s), 6.68 (1H, dd, J = 9.5, 2.0 Hz),
3
6.86 (1H, d, J = 2.0 Hz), 7.25 (2H, d, J = 8.6 Hz), 7.82
(1H, d, J = 9.5 Hz), 7.98 (2H, d, J = 8.6 Hz), 9.95 (1H,
1
3
s), 10.31 (1H, s). C NMR δ (100 MHz in CD OD):
3
−
1
mg, 0.95 mmol, 47.5%). IR νmax (diamond ATR) cm :
55.2, 102.7, 110.6, 119.2, 119.7, 130.2, 131.7, 132.8,
1
1
733, 1505, 1272, 1227, 1155. H NMR δ (400 MHz
161.0, 162.8, 164.0, 188.4, and 191.2. HRFABMS m/z
+
in CD OD): 1.19 (3H, t, J = 7.1 Hz), 1.21 (3H, t,
(MH ): Calcd. for C H O : 257.0814, Found:
3
15 13 4
J = 7.1 Hz), 2.57 (2H, t, J = 7.6 Hz), 2.64 (2H, t,
J = 7.6 Hz), 2.85 (2H, t, J = 7.6 Hz), 2.91 (2H, t,
J = 7.6 Hz), 3.74 (3H, s), 4.08 (2H, q, J = 7.1 Hz), 4.10
257.0818.
3-(4-Formylphenoxy)-6-methoxybenzaldehyde (11).
(
2H, q, J = 7.1 Hz), 6.72 (2H, d, J = 8.6 Hz), 6.77 (1H,
According to the general coupling procedure, the cou-
pling of 5-hydroxy-2-methoxybenzaldehyde (1.130 g,
dd, J = 8.1, 2.0 Hz), 6.85 (1H, d, J = 8.1 Hz), 6.95 (1H,
1
3
d, J = 2.0 Hz), 7.08 (2H, d, J = 8.6 Hz). C NMR δ
100 MHz in CD OD): 14.5, 14.6, 31.2, 31.8, 36.9,
7.40 mmol) and 4-bromobenzaldehyde (1.370 g, 6.60
(
3
mmol) produced compound 11 as a white powder with
a 7.8% yield (131 mg, 0.51 mmol). Mp. 89.0–90.5 °C.
IR νmax (diamond ATR) cm : 1677, 1485, 1261, 1229,
3
1
1
4
7.1, 56.3, 61.5, 61.6, 114.5, 117.4, 121.9, 122.6,
30.4, 135.6, 139.4, 144.3, 152.9, 158.3, 174.66,
−
1
+
74.69. HREIMS m/z (M ): Calcd. for C H O :
2
3 28 6
1
1158, 1023, and 826. H NMR δ (400 MHz in
00.1886, Found: 400.1889.
CD OD): 3.99 (3H, s), 7.07 (2H, d, J = 8.9 Hz), 7.28
3
(1H, d, J = 9.1 Hz), 7.41 (1H, dd, J = 9.1, 3.1 Hz), 7.46
3
-(4-(4-(2-Carboxyethyl)-2-methoxyphenoxy)phenyl)
(1H, d, J = 3.1 Hz), 7.90 (2H, d, J = 8.9 Hz), 9.89 (1H,
1
3
propanoic acid (2; proposed apteniol B). A solution
s), 10.40 (1H, s). C NMR δ (100 MHz in CD OD):
3

1746
T. Noshita et al.
1
3
5
1
6.9, 102.7, 115.4, 118.0, 118.3, 120.3, 129.7, 133.2,
and 192.7. C NMR δ (100 MHz in CDCl ): 56.3,
3
54.6, 164.8, 168.9, 190.2, and 192.7. HRFABMS m/z
112.5, 116.6, 122.3, 129.5, 130.4, 131.5, 131.9, 143.8,
156.8, 162.7, 190.0, and 190.7. {lit.
+
8) 13
(
MH ): Calcd. for C H O : 257.0814, Found:
,
C NMR δ
1
5 13 4
2
57.0815.
(100 MHz in CDCl ): 56.3, 112.5, 116.6, 122.3, 129.5,
3
1
1
2
30.4, 131.5, 132.0, 143.8, 156.8, 162.6, 190.0, and
+
90.7} HRFABMS m/z (MH ): Calcd. for C H O :
1
5 13 4
2
-(4-Formylphenoxy)-5-methoxybenzaldehyde (12).
57.0814, Found: 257.0811.
According to the general coupling procedure, the cou-
pling of 2-hydroxy-5-methoxybenzaldehyde (1.130 g,
7
.40 mmol) and 4-bromobenzaldehyde (1.370 g, 6.60
2-(4-Formylphenoxy)-4-methoxybenzaldehyde (14).
mmol) produced compound 12 as a white powder with
a 64.7% yield (1.090 g, 4.27 mmol). Mp. 71.5–72.5 °C.
According to the general coupling procedure, the cou-
pling of 2-hydroxy-4-methoxybenzaldehyde (1.130 g,
7.40 mmol) and 4-bromobenzaldehyde (1.370 g, 6.60
mmol) produced compound 14 as colorless crystals
with a 27.7% yield (470 mg, 1.83 mmol). Mp. 90.0–
−
1
IR νmax (diamond ATR) cm : 1676, 1482, 1223, 1156,
1
1
026, and 827. H NMR δ (400 MHz in CD OD): 3.88
3
(
3H, s), 7.12 (2H, d, J = 8.8 Hz), 7.14 (1H, d, J = 9.0
−
1
Hz), 7.30 (1H, dd, J = 9.0, 3.3 Hz), 7.44 (1H, d,
J = 3.3 Hz), 7.93 (2H, d, J = 8.8 Hz), 9.90 (1H, s),
91.5 °C. IR νmax (diamond ATR) cm :1678, 1601,
1
1214, 1155, 1023, and 826. H NMR δ (400 MHz in
1
3
1
5
1
0.19 (1H, s). C NMR δ (100 MHz in CD OD):
CD OD): 3.83 (3H, s), 6.61 (1H, d, J = 2.2 Hz), 6.95
3
3
4.9, 111.0, 111.5, 115.0, 116.9, 122.8, 123.0, 131.8,
57.3, 163.7, 164.4, 188.2, and 191.3. HRFABMS m/z
MH ): Calcd. for C H O : 257.0814, Found:
(1H, dd, J = 8.8, 2.2 Hz), 7.21 (2H, d, J = 8.6 Hz), 7.92
(1H, d, J = 8.8 Hz), 7.96 (2H, d, J = 8.6 Hz), 9.93 (1H,
+
13
(
s), 10.12 (1H, s). C NMR δ (100 MHz in CD OD):
1
5
13
4
3
2
57.0817.
55.1, 105.4, 111.4, 117.9, 121.3, 130.6, 131.8, 132.4,
1
59.7, 162.5, 166.5, 187.3, and 191.2. HRFABMS m/z
+
(MH ): Calcd. for C H O : 257.0814, Found:
15 13 4
3
-(4-Formylphenoxy)-4-methoxybenzaldehyde (13).
257.0813.
According to the general coupling procedure, the cou-
pling of 3-hydroxy-4-methoxybenzaldehyde (1.130 g,
7
.40 mmol) and 4-bromobenzaldehyde (1.370 g, 6.60
Results and discussion
mmol) produced compound 13 as a white powder with
a 68.4% yield (1.160 g, 4.51 mmol). Mp. 63.8–64.2 °C.
IR νmax (diamond ATR) cm : 1677, 1482, 1224, 1157,
The synthetic plan for the preparation of the pro-
posed apteniols B, C, and G is shown in Fig. 2. Forma-
tion of the diphenyl ether, which was the key step in
this synthesis, was performed via Ullmann ether syn-
thesis, which was previously used for the preparation
−
1
1
8
58, and 827. H NMR δ (400 MHz in CD OD): 3.87
3
(
3H, s), 6.99 (2H, d, J = 8.6 Hz), 7.35 (1H, d, J = 8.3
Hz), 7.65 (1H, d, J = 1.7 Hz), 7.87 (2H, d, J = 8.6 Hz),
.88 (1H, dd, J = 8.3, 1.7 Hz), 9.86 (1H, s), 9.87 (1H,
4
)
7
of apteniol A (1). Ullmann etherification of vanillin
and 4-bromobenzadehyde produced the compound with
the proposed structure for apteniol G (7) with a 31.4%
yield. Both of the formyl groups in 7 were then con-
verted to α,β-unsaturated diethyl ester substituents in 8
via the Horner–Wadsworth–Emmons reaction. Catalytic
hydrogenation and subsequent hydrolysis of these ester
groups afforded the desired dicarboxylic acid corre-
sponding to the proposed structure for apteniol B (2),
and methylation of 2 gave the proposed structure for
apteniol C (3). The chemical structures of all of the
1
s). H NMR δ (400 MHz in CDCl ): 3.90 (3H, s), 7.01
3
(
2H, d, J = 8.8 Hz), 7.16 (1H, d, J = 8.6 Hz), 7.63 (1H,
d, J = 1.8 Hz), 7.79 (1H, dd, J = 8.6, 1.8 Hz), 7.85 (2H,
d, J = 8.8 Hz), 9.89 (1H, s), 9.93 (1H, s). {lit. , H
NMR δ (400 MHz in CDCl ): 3.90 (3H, s), 7.01 (2H,
d, J = 8.7 Hz), 7.16 (1H, d, J = 8.4 Hz), 7.63 (1H, d,
J = 2.0 Hz), 7.79 (1H, dd, J = 8.4, 2.0 Hz), 7.85 (2H, d,
8
) 1
3
1
3
J = 8.7 Hz), 9.88 (1H, s), 9.92 (1H, s)} C NMR δ
100 MHz in CD OD): 56.8, 114.3, 117.3, 123.6,
(
3
1
31.2, 132.0, 132.8, 133.1, 144.8, 158.6, 164.6, 192.2,
Fig. 2. Synthesis of proposed apteniols B (2), C (3), and G (7).
Notes: Reagents and conditions: (i) Cs CO , CuI, N,N-dimethylglycine HCl salt, DMF, 100 °C (31.4%); (ii) NaH, triethyl phosphonoacetate,
benzene, rt (99.6%); (iii) Pd/C, H , MeOH, rt (47.5%); (iv) NaOH, THF/H O, rt (86.2%); and (v) H SO , MeOH, reflux (73.3%).
2
3
2
2
2
4

Synthesis of the proposed apteniols B, C, and G
1
1747
synthesized compounds were determined by H and
synthesized 7 and the isolated compound, despite the
fact that the same analysis conditions (solvent and
measurement frequency) were used.
1
3
C NMR spectroscopy and high-resolution mass spec-
1
13
trometry (HRMS) analyses; the H and C NMR spec-
tral data for the synthesized compounds and isolated
apteniols B (2), C (3), and G (7) are shown in Tables
For synthesized compound 2 and isolated apteniol B,
there are only slight differences of less than 0.2 ppm in
1
1
–3, respectively.
It can be seen from Table 1 that the H and
the shifts of the H NMR signals. However, the C-1,
1
13
C
C-3, and C-5 carbon signals for the two compounds
each differ by more than 6 ppm, as shown in Table 2.
NMR spectral data for the synthesized compound 7
with the proposed structure and isolated apteniol G do
not agree but are similar. For example, the H-6 proton
signal is observed at 7.61 ppm in the spectrum for syn-
thesized 7 and at 6.91 ppm in the spectrum of the iso-
lated compound proposed to have the structure in 7.
Furthermore, the C-1, C-6, and C-1’ carbon signals
differ by 3.5, 5.3, and 3.2 ppm, respectively, for
1
3
The C NMR spectral data for the three groups in
apteniol C (3) are also listed in Table 3. In particular,
1
3
the C NMR data for synthesized compound 3 are in
complete agreement with those previously reported for
the apteniol C (3) that was synthesized by Jung and
5
)
Bräse using a different synthetic approach. However,
the spectral data for both synthetic compounds (3) dif-
fer from those reported for natural apteniol C (3). Fur-
thermore, DellaGreca et al. reported that apteniol C
was obtained via methylation of apteniol B.
1
13
a
Table 1.
H and C NMR data for synthesized and reported
OD, 500/125 MHz).
apteniol G (CD
3
The results described above suggest that the struc-
tures of isolated apteniols B, C, and G differ from the
proposed structures. Therefore, additional compounds
that could potentially have the actual structure of apte-
niol G were synthesized. As shown in Fig. 3, bisalde-
hyde ethers 10 to 14 were prepared from 1,2,4-
trisubstituted benzaldehydes and 4-bromo(or 4-hy-
droxy)benzaldehyde. In the case of preparation of com-
pounds 10, 11 and 14, the yields decreased due to the
formation of uncharacterized polar by-products.
The chemical shifts of the typical functional groups
of these compounds are listed in Table 4. Unfortu-
nately, the spectral data for all of compounds 10–14
did not completely agree with those reported for iso-
lated apteniol G. For example, DellaGreca et al.
reported that the chemical shifts of both aldehyde car-
bons in the natural compound are 193.3 ppm; however,
the chemical shifts for the two aldehyde carbons in
each of compounds 10–14 differed by more than 0.5
ppm. Furthermore, for compound 13, while the alde-
hyde carbon chemical shifts were relatively close to
one another, the chemical shifts for many hydrogen
atoms were very different than those in the spectrum of
natural apteniol G. For example, the H-2, H-5, and H-6
proton signals observed at 7.66, 7.35, and 7.87 ppm in
b
Synthesized apteniol G
Reported apteniol G
Position
δ (H)
δ (C)
δ (H)
δ (C)
1
2
3
4
–
125.8
113.4
149.8
153.7
117.0
123.5
**192.7
136.3
133.1
117.9
164.1
117.9
133.1
**192.9
56.5
–
129.3
111.7
149.7
156.0
117.1
128.8
193.3
133.1
134.0
117.6
164.6
117.6
134.0
193.3
56.9
7.62 (d, 1.7)
7.43 (s)
–
–
–
–
5
6
7
1
2
3
4
5
6
7
3
7.61 (dd, 8.1, 1.7)
7.29 (d, 8.1)
6.91 (d, 7.0)
7.41 (d, 7.0)
9.72 (s)
–
7.77 (d, 8.8)
6.88 (d, 8.8)
–
6.88 (d, 8.8)
7.77 (d, 8.8)
9.75 (s)
3.92 (s)
c
*9.87 (s)
′
′
′
′
′
′
′
–
7.89 (d, 8.8)
7.04 (d, 8.8)
–
7.04 (d, 8.8)
7.89 (d, 8.8)
*9.96 (s)
3.85 (s)
-OMe
a
The coupling constants (J in Hertz) are in parentheses.
DellaGreca et al., Chem. & Biodivers., 4, 118–128 (2007).
May be interchangeable within the same column.
b
c*,**
1
13
a
Table 2.
apteniol B (CD
H and C NMR data for synthesized and reported
OD, 500/125 MHz).
3
b
Synthesized apteniol B
Reported apteniol B
Position
δ (H)
δ (C)
δ (H)
δ (C)
the spectrum for compound 13 were instead observed
1
at 7.43, 6.91, and 7.41 ppm, respectively, in the
H
1
2
3
4
5
6
7
8
9
–
6.97 (d, 1.9)
–
139.6
114.6
152.9
144.4
122.6
121.9
31.9
–
132.1
111.0
146.5
144.0
114.4
NMR spectrum of the isolated compound proposed to
have structure 7. Based on the foregoing results, it can
be concluded that the actual structure of the “apteniol
G” that was isolated by DellaGreca et al. is neither that
of compound 7 nor those of compounds 10–14. Fur-
thermore, the proposed structures for apteniols B and
C, which are related to apteniol G by a biosynthetic
pathway, are also incorrect.
6.72 (d, 1.5)
–
–
–
6.86 (d, 8.1)
6.79 (dd, 8.1, 1.9)
2.92 (t, 7.6)
2.62 (t, 7.6)
–
6.82 (d, 8.2)
6.70 (dd, 8.2, 1.5) 120.8
2.89 (t, 8.0)
2.66 (t, 8.0)
–
30.3
35.9
37.0
c
*176.8
135.8
130.3
117.5
158.3
117.5
130.3
31.3
178.9
132.1
129.4
115.4
154.1
115.4
129.4
30.3
1
2
3
4
5
6
7
8
9
3
′
′
′
′
′
′
′
′
–
–
Next, since the synthesized compounds matching the
proposed structures for apteniols B, C, and G had
already been produced, their degranulation-inhibiting
activities were evaluated, because the methyl ester of the
compound with the structure proposed for apteniol A
had been reported to show potent degranulation-inhibit-
7.11 (d, 8.7)
6.73 (d, 8.7)
–
6.73 (d, 8.7)
7.11 (d, 8.7)
2.85 (d, 7.7)
2.56 (d, 7.7)
–
7.05 (d, 8.5)
6.74 (d, 8.5)
–
6.74 (d, 8.5)
7.05 (d, 8.5)
2.89 (d, 8.0)
2.66 (d, 8.0)
–
36.8
*176.7
56.4
35.8
178.9
55.8
4
)
ing activity. As shown in Fig. 4, synthesized proposed
apteniol B (2) did not show such activity in the concen-
tration range from 50 to 150 μM. On the other hand, the
proposed apteniols C (3) and G (7) showed activities that
were considerably weaker than that of the methyl ester
′
-OMe
3.76 (s)
3.86 (s)
a
The coupling constants (J in Hertz) are in parentheses.
DellaGreca et al., Tetrahedron, 61, 11924–11928 (2005).
May be interchangeable within the same column.
b
c*

1748
T. Noshita et al.
1
13
a
Table 3.
H and C NMR data for synthesized and reported apteniol C (CDCl3, 500/125 MHz).
b
c
Synthesized Apteniol C
Synthesized Apteniol C
Reported Apteniol C
Position
δ (H)
δ (C)
δ (H)
δ (C)
δ (H)
δ (C)
1
2
3
4
5
6
7
8
9
–
137.3
113.0
151.3
143.6
120.9
120.6
30.8
–
137.2
112.9
151.2
143.5
120.8
120.6
30.8
–
137.2
112.9
151.2
143.5
120.8
120.6
30.8
6.84 (d, 2.0)
6.82–6.87 (m)
–
6.70 (d, 1.5)
–
–
–
–
–
6.86 (d, 8.1)
6.73 (dd, 8.1, 2.0)
2.94 (t, 7.6)
2.65 (t, 7.6)
–
6.82–6.87 (m)
6.73 (d, 7.4)
2.92–2.96 (m)
2.63–2.69 (m)
–
6.83 (d, 8.0)
6.68 (dd, 8.0, 1.5)
2.88 (t, 7.5)
2.59 (t, 7.5)
–
35.9
35.9
35.9
*
d
173.4
134.5
129.3
117.2
156.5
117.2
129.3
30.2
173.4
134.4
129.3
117.2
156.4
117.2
129.3
30.2
35.8
173.3
56.0
51.6
51.7
173.4
134.4
129.3
117.2
156.4
117.2
129.3
30.2
35.8
173.3
56.0
51.6
51.7
1
2
3
4
5
6
7
8
9
3
9
9
′
′
′
′
′
′
′
′
–
–
–
7.10 (d, 8.7)
6.85 (d, 8.7)
–
6.85 (d, 8.7)
7.10 (d, 8.7)
2.90 (t, 7.6)
2.60 (t, 7.6)
–
7.10 (d, 8.4)
6.82–6.87 (m)
–
6.82–6.87 (m)
7.10 (d, 8.4)
2.88–2.92 (m)
2.60–2.66 (m)
–
7.05 (d, 9.0)
6.75 (d, 9.0)
–
6.75 (d, 9.0)
7.05 (d, 9.0)
2.88 (t, 7.5)
2.61 (t, 7.5)
–
35.8
*
′
173.3
56.0
-OMe
-OMe
’-OMe
3.82 (s)
3.69 (s)
3.66 (s)
3.82 (s)
3.69 (s)
3.67 (s)
3.87 (s)
3.67 (s)
3.67 (s)
*
*
***
51.6
51.7
**
***
a
The coupling constants (J in Hertz) are in parentheses.
Jung and Bräse, Eur. J. Org. Chem., 2009, 4494–4502.
DellaGreca et al., Tetrahedron, 61, 11924–11928 (2005).
b
c
d*,**,***
May be interchangeable within the same column.
Fig. 3. Synthesis of compounds 10–14.
Notes: Reagents and conditions: (i) Cs
2
CO
3
, CuI, N,N-dimethylglycine HCl salt, DMF, 100 °C.
a
Table 4. Comparison of chemical shifts of 7- and 7ʹ-aldehyde and methoxy group.
Compounds
b
Position
Reported apteniol G
10
11
12
13
14
*
*
c
*
*
*
*
7
7
7
7
-Aldehyde δ (H)
’-Aldehyde δ (H)
-Aldehyde δ (C)
’-Aldehyde δ (C)
9.72
9.75
193.3
193.3
3.92
9.95
9.89
9.90
9.86
9.93
*
*
*
*
10.31
188.4
191.2
10.40
190.2
192.7
10.19
188.2
191.3
9.87
10.12
187.3
191.2
**
**
**
**
**
**
**
**
192.2
**
**
192.7
Methoxy δ (H)
Methoxy δ (C)
3.95
55.2
3.99
56.9
3.88
54.9
3.87
56.8
3.83
55.1
56.9
a
CD
CD
3
OD, 400/100 MHz.
3
OD, 500/125 MHz, DellaGreca et al., Chemistry & Biodiversty, 4, 118–128 (2007).
b
c*,**
May be interchangeable within the same column.

Synthesis of the proposed apteniols B, C, and G
1749
of data: AT, HO, and AS. Drafting of manuscript: TN.
Critical revision: AT. And all authors read and
approved the final manuscript.
Acknowledgments
The authors would like to thank Enago (www.enago.
jp) for the English language review.
Disclosure statement
No potential conflict of interest was reported by the authors.
Fig. 4. Inhibitory effects of compounds on antigen-induced degran-
ulation in RBL-2H3 cells.
Notes: DNP-IgE-sensitized RBL-2H3 cells were incubated with
indicated sample concentrations for 20 min and stimulated with DNP-
HAS for 1 h, and then the quantity of released β-hexosaminidase was
determined. Data points each represent means of three independent
cultures, and bars indicate SDs. p < 0.05, p < 0.01 (Dunnett’s test),
as compared to control.
Funding
This work was supported by a Sasakawa Scientific Research
Grant from the Japan Science Society and BRAIN; Technol-
ogy Research Promotion Program for Agriculture, Forestry,
Fisheries, and Food Industry; Program for the Promotion of
Basic and Applied Research for Innovations in Bio-oriented
Industry.
*
**
of proposed apteniol A at the same concentration (the
release ratio of β-hexosaminidase at 150 μM for the
methyl ester of proposed apteniol A is ca. 5%, according
References
4
)
to ref. . It should be noted that the hydrophobic com-
pounds showed activity, while the hydrophilic com-
pound did not, suggesting a characteristic behavior.
However, because of the limited number of compounds
evaluated, this characteristic can only be postulated.
In conclusion, the proposed apteniols B (2), C (3), and
G (7) were synthesized, and their H and C NMR data
were found to be different from the corresponding
spectral data for the natural products. Therefore, the
results of this study indicate that the actual structures of
the isolated compounds referred to as apteniols B, C,
and G differ from the proposed structures.
The synthesized proposed apteniol B did not show
degranulation-inhibiting activity, while the prepared,
proposed apteniols C and G exhibited activities con-
siderably weaker than that of the methyl ester of pro-
posed apteniol A. These results suggest that the
hydrophobicity of the compounds in this series may
influence their degranulation-inhibiting activities.
[
[
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[4] Nishikawa H, Noshita T, Tai A, et al. Syntheses and biological
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Author contribution
2
009;11:47–71.
Study conception and design: TN. Acquisition of
data: HN, KI, YH, and TY. Analysis and interpretation