Four new ginkgolic acids from Ginkgo biloba

Article abstract of DOI:10.1016/j.tetlet.2014.05.076

Four new compounds, 2-hydroxy-6-(12′-hydroxyheptadec-13′(E)-en- 1-yl)benzoic acid (1), 2-hydroxy-6-(13′-hydroxyheptadec-11′(E)-en-1- yl)benzoic acid (2), 2-hydroxy-6-(10′-hydroxypentadec-11′(E)-en-1- yl)benzoic acid (3), and 2-hydroxy-6-(11′-hydroxypentad

Full text of DOI:10.1016/j.tetlet.2014.05.076

Tetrahedron Letters 55 (2014) 3788–3791
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Four new ginkgolic acids from Ginkgo biloba
Jun Deguchi, Yuki Hasegawa, Ayana Takagi, Shihoko Kutsukake, Mizue Kono, Yusuke Hirasawa,
⇑
Chin Piow Wong, Toshio Kaneda, Hiroshi Morita
Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Four new compounds, 2-hydroxy-6-(120-hydroxyheptadec-130(E)-en-1-yl)benzoic acid (1), 2-hydroxy-
6-(130-hydroxyheptadec-110(E)-en-1-yl)benzoic acid (2), 2-hydroxy-6-(100-hydroxypentadec-110(E)-en-
1-yl)benzoic acid (3), and 2-hydroxy-6-(110-hydroxypentadec-90(E)-en-1-yl)benzoic acid (4) were
isolated from the leaves of Ginkgo biloba and the structures of new ginkgolic acids were deduced on
the basis of spectroscopic methods and chemical means. Compounds 1 and 2, and 3 and 4 examined
as an inseparable mixture of hydroxyl and double bond positional isomers, were ultimately defined by
total synthesis. Compounds 1–4 showed moderate lipid droplets accumulation inhibitory activity on
mouse pre-adipocyte cell line, MC3T3-G2/PA6.
Article history:
Received 19 March 2014
Revised 24 April 2014
Accepted 16 May 2014
Available online 23 May 2014
Keywords:
Ginkgo biloba
Ginkgoaceae
Ginkgolic acid
Structure elucidation
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
OR2
OR2
n
Ginkgo biloba L., although grown mainly in China and Japan, is
currently well-known in various European countries. Various
chemical components have been isolated from the plant, including
flavonoids,1 terpenoids (ginkgolides, bilobalides),2 and organic
acids (ginkgolic acid, cardanol).3 While ginkgolic acids possess
strong allergenic properties,4 they show interesting biological
activities, such as inhibition of SUMOylation,5 insecticidal activity,6
antibacterial properties against Gram-positive bacteria,7 and inhib-
itory activity of glycerol-3-phosphate dehydrogenase.8 Our efforts
on identifying new natural products from the leaves of Ginkgo
biloba resulted in the isolation of four new ginkgolic acids (1–4).
Herein we would like to report the structure elucidation of 1–4
on the basis of spectroscopic data, chemical means, and total syn-
thesis, and its anti-lipid droplets accumulation (LDA) activity.
The leaves of G. biloba (10 kg) were extracted with MeOH, and
the extract was partitioned between CHCl3 and H2O. CHCl3-soluble
materials were subjected to a silica gel column, an ODS column,
and an ODS HPLC to give mixtures of compounds 1 and 2
(0.0048%), and 3 and 4 (0.0093%) with common ginkgolic acids
(17:1, 9), (15:1, 10), and (13:0, 11).3
Me
R1O
Me
R1O
m
COOR1
COOR1
m = 10 R1 = R2 = H
1 : n = 11 R1 = R2 = H
2
:
4 : m = 8 R1 = R2 = H
n = 9
R1 = R2 = H
3 :
:
m = 10 R1 = Me, R2 = (S)-MTPA
m = 10 R1 = Me, R2 = (R)-MTPA
m = 8 R1 = Me, R2 = (S)-MTPA
m = 8 R1 = Me, R2 = (R)-MTPA
5a n = 11 R1 = Me, R2 = (S)-MTPA
6a :
n = 11 R1 = Me, R2 = (R)-MTPA
5b :
6b
:
R1 = Me, R2 = (S)-MTPA
8a :
8b :
n = 9
n = 9
7a :
7b
R
1 = Me, R2 = (R)-MTPA
:
IR absorptions implied the presence of hydroxyl (3742 cmꢀ1) and
carboxylic acid (1683 cmꢀ1) functionalities. 1H and 13C NMR spectra
are presented in Table 1. Although multiple HPLC attempts were
made, this mixture could not be separated. The molecular formula
of 1 indicated the addition of 16 mass units to the formula for
ginkgolic acid (17:1) (9), thus indicating an additional hydroxyl
group. The gross structures of 1 and 2 were deduced from extensive
analysis of HMBC spectrum in CD3OD (Fig. 1). In the structure elu-
cidation of 1, the analysis of the 1Hꢀ H COSY spectrum revealed
1
three partial structures, a (C-3 to C-5), b (C-10 and C-20), and c
(C-110 to C-170), and the existence of allyl alcohol (C-120 to C-140)
in the alkyl chain. Connection between partial structures a and b,
which form 5-substituted salicylic acid, could be assigned by HMBC
correlations of H-3 (dH 6.72) to C-1 (dC 117.5) and H-4 (dH 7.20) to
C-2 (dC 162.2) and C-6 (dC 146.9), and H-5 (dH 6.69) to C-1 and C-
10 (dC 36.6). The same partial structure, 5-substituted salicylic acid
was revealed for 2 by the same manner. The HMBC correlations of
H-120 (dH 3.95) to C-100 (dC 26.6) and C-140 (dC 132.2) and H-130
(dH 5.41) to C-110 (dC 38.5) and C-150 (dC 35.4) and H-140 (dH 5.60)
Compounds 1 and 29 were isolated as pale yellow oils that con-
tained isometric component. Both showed the molecular formula,
C
24H38O4, which was determined by HRESIMS [m/z 389.2692,
(MꢀH)ꢀ, +0.4 mmu].
⇑
Corresponding author. Tel./fax: +81 354985778.
E-mail address: moritah@hoshi.ac.jp (H. Morita).
http://dx.doi.org/10.1016/j.tetlet.2014.05.076
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

J. Deguchi et al. / Tetrahedron Letters 55 (2014) 3788–3791
3789
Table 1
1H NMR Data [dH (J, Hz)] and 13C NMR Data [dC] of compounds 1–4 in CD3OD at 300 K
Position
1
2
3
4
1H
13C
1H
13C
1H
13C
1H
13C
1
2
3
4
117.5
162.2
115.5
133.5
122.7
146.9
36.6
33.3
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
117.5
162.2
115.5
133.5
122.7
146.9
36.6
33.3
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
118.4
162.2
115.3
132.2
122.5
147.1
36.5
33.3
118.4
162.2
115.3
132.2
122.5
147.1
36.5
6.72 (1H, d, 7.8)
7.20 (1H, dd, 7.8, 7.8)
6.69 (1H, d, 7.8)
6.72 (1H, d, 7.8)
7.20 (1H, dd, 7.8, 7.8)
6.69 (1H, d, 7.8)
6.67 (1H, d, 7.8)
7.14 (1H, dd, 7.8, 7.8)
6.64 (1H, d, 7.8)
6.67 (1H, d, 7.8)
7.14 (1H, dd, 7.8, 7.8)
6.64 (1H, d, 7.8)
5
6
10
2.93 (2H, br)
1.57 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
2.93 (2H, br)
1.57 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.30 (2H, m)
3.00 (2H, t, 7.7)
1.57 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.41 (1H, m)
1.51 (1H, m)
3.95 (1H, dt, 6.8, 6.4)
5.41 (1H, m)
3.00 (2H, t, 7.7)
1.57 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.29 (2H, m)
1.30 (2H, m)
1.57 (2H, m)
5.60 (1H, dt, 15.4, 6.7)
20
33.3
30
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
26.6
30.4–31.0
30.4–31.0
30.4–31.0
30.4–31.0
30.2
40
50
60
70
80
33.3
132.6
90a
90b
100
110a
110b
120a
120b
130
140a
140b
150
160
170
COOH
38.5a
1.30 (2H, m)
1.41 (1H, m)
1.50 (1H, m)
3.95 (1H, dt, 5.9, 5.4)
26.6
1.57 (2H, m)
5.60 (1H, dt, 15.3, 6.8)
33.3
132.2
73.8
5.41 (1H, m)
3.95 (1H, dt, 6.8, 6.4)
134.5a
73.8
38.5a
134.7a
73.8
5.41 (1H, m)
134.5a
5.60 (1H, dt, 15.4, 6.7)
132.5
1.41 (1H, m)
1.51 (1H, m)
1.30 (2H, m)
1.33 (2H, m)
38.2a
5.41 (1H, m)
5.60 (1H, dt, 15.3, 6.8)
134.7a
132.2
3.95 (1H, dt, 5.9, 5.4)
1.41 (1H, m)
1.50 (1H, m)
1.31 (2H, m)
1.33 (2H, m)
73.8
2.02 (2H, m)
1.33 (2H, m)
35.4
28.9
38.2a
23.5a
23.7a
2.02 (2H, m)
1.33 (2H, m)
0.91 (3H, t, 7.4)
35.4
23.5a
14.0a
175.4
28.9
23.7a
14.4a
175.4
0.91 (3H, t, 7.4)
14.0a
175.9
0.90 (3H, t, 7.4)
14.4a
175.9
0.90 (3H, t, 7.4)
a
The 13C NMR data on 1–4 were defined on the synthetic samples.
were confirmed by the modified Mosher method.10 Treatment of
mixture of 1 and 2 with iodomethane in acetone followed by reac-
tion with (R)- and (S)-a-methoxy-a-(trifluoromethyl)phenylacetyl
chloride (MTPACl) afforded (S)- and (R)-MTPA esters. Both the (S)-
MTPA and (R)-MTPA esters (5a, 5b, 6a, and 6b) showed an identical
NMR spectrum, indicating that the compounds 1 and 2 were
racemate.
a
4
5
3
2
OH 14'
2'
16'
10'
15'
15'
6
17'
17'
HO
HO
12'
Me
13'
1
1'
11'
b
COOH
1
c
4
5
3
2
12'
Compounds 3 and 411 were isolated as pale yellow oils that
contained isometric component. The molecular formula for both
constituents was established by HRESIMS as C22H34O4 ([MꢀH]ꢀ
m/z 361.2379). This molecular formula of 3 and 4 indicated the
addition of 16 mass units to the formula for ginkgolic acid (15:1)
(10) and lack of two methylenes from 1 and 2. The 1D and 2D
NMR spectroscopic data of 3 and 4 were indistinguishable from
those of 1 and 2. According to the above data, the structures of 3
10'
2'
16'
9'
6
14'
Me
13'
1
1'
11'
COOH
OH
2
1H-1H COSY
HMBC
Figure 1. 1H–1H COSY and key HMBC correlations used to establish the structures
of 1 and 2.
and
4
were assigned as 2-hydroxy-6-(100-hydroxypentadec-
110(E)-en-1-yl)benzoic acid and 2-hydroxy-6-(110-hydroxypenta-
to C-160 (dC 23.5), and H3-170 (dH 0.91) to C-150 and C-160 for 1 and
HMBC correlations of H-110 (dH 5.60) to C-90 (dC 30.4–31.0) and
C-100 (dC 33.3) and H-120 (dH 5.41) to C-100 and H-130 (dH 3.95) to
C-110 (dC 132.2), C-140 (dC 38.2), and C-150 (dC 28.9), and H-170 (dH
0.90) to C-150 and C-160 (dC 23.7) for 2 indicated the position of each
hydroxyl group and double bond, which indicated that the two-
component mixture was realized to be composed of hydroxyl group
and double bond positional isomers. The characteristic signals,
dec-90(E)-en-1-yl)benzoic acid.
The absolute configurations at C-10 of 3 and C-11 of 4 were con-
firmed by the modified Mosher method, which generated esters
(7a, 7b, 8a, and 8b) showed an identical NMR spectrum, indicating
that the compounds 3 and 4 were racemate.
While some new ginkgolic acids or related natural products,
anacardic acids, which possessing new length alkyl chains, have
been isolated from plant resources,12 ginkgolic acids possessing
hydroxyl group and E double bond on its alkyl chain were still on
few report. Each new compound might be biosynthetically pro-
duced by an oxidative transformation of known ginkgolic acids
(17:1) and (15:1).
In order to confirm the position of each substituent and spectral
data, and evaluate their each biological activity, we synthesized
compounds 1–4. Our retrosynthetic analysis of 1–4 is outlined in
Scheme 1. Generation of E geometry double bond and allyl alcohol
could be constructed by Horner–Wadsworth–Emmons (HWE)13
reaction and the reduction under Luche conditions.14 Preparing a
0
0
which appeared as a doublet of triplet at dH 5.60 (dt, J13 ,14 = 15.3
Hz, J14 ,15 = 6.8 Hz) for
0
0
0
0
1 and dH 5.60 (dt, J11 ,12 = 15.3 Hz,
0
0
J10 ,11 = 6.8 Hz) for 2 were assigned E configuration for their double
bonds, respectively. Therefore, the structures of compounds 1 and 2
were assigned as 2-hydroxy-6-(120-hydroxyheptadec-130(E)-en-1-
yl)benzoic acid and 2-hydroxy-6-(130-hydroxyheptadec-110(E)-en-
1-yl)benzoic acid, respectively.
Based on the no optical rotation of mixture of 1 and 2, the abso-
lute configurations at C-120 for 1 and C-140 for 2 could be deduced
as racemic forms. The absolute configurations at C-120 and C-140

3790
J. Deguchi et al. / Tetrahedron Letters 55 (2014) 3788–3791
H
O
Me
OH
O
n
O
P
OMe
OMe
MeO
Me
HO
HO
Me
Me
n
COOH
COOH
COOEt
O
OEt
1 or 3
O
P
O
P
MeO
MeO
MeO
OEt
COOEt
12
O
OH
MeO
H
m
m
COOEt
2 or 4
Scheme 1. Retrosynthetic analysis for compounds 1–4.
1) t-BuOK, HMPA,
O
P
THF, -78 °C, 30 min
1) K2CO3, MeOH, rt, 3h
2) H2, PtO2, EOAc, rt, 3h
OEt
OR
n
MeO
MeO
OEt
2)
13a :
COOEt
10 OBz
OHC
COOEt
13b :
13c :
13d :
9
8
7
OAc
OAc
OHC
OHC
12
14a : n = 10, R = Bz (48%)
14b : n = 9, R = Ac (45%)
14c : n = 8, R = Ac (40%)
14d : n = 7, R = Ac (43%)
OAc
OHC
, THF, rt, overnight
O
(COCl)2, DMSO, Et3N
-78 °C, 1h
OH
n
MeO
H
MeO
n
COOEt
COOEt
16a : n = 11 (60%)
16b : n = 10 (66%)
16c : n = 9 (64%)
16d : n = 8 (62%)
15a : n = 12 (85%, 2 steps)
15b : n = 11 (82%, 2 steps)
15c : n = 10 (85%, 2 steps)
15d : n = 9 (84%, 2 steps)
Scheme 2. Synthesis of aldehydes 16a, 16b, 16c, and 16d.
O
H
OMe
N2
O
P
O
n
O
P
O
Me
LiBr, Et3N,
THF, 0 °C,
16 h
OMe
OMe
16a
16c
MeO
HO
Me
MeO
n
OMe
COOEt
18a
SnCl2, CH2Cl2,
rt, 3 h
COOEt
17a
: n = 11 (50%)
18c : n = 9 (46%)
: n = 11 (70%)
17c : n = 9 (62%)
1) 20% NaOH aq.,
MeOH, reflux,
12 h
OH
OH
n
NaBH4, CeCl3
MeO
Me
Me
n
MeOH, 0 °C,
1 h
2) NaH, ethanethiol,
DMF, 0 °C, reflux,
1 h
COOEt
COOH
19a
19c
: n = 11 (quant.)
: n = 9 (quant.)
1 : n = 11 (40%, 2 steps)
: n = 9 (45%, 2 steps)
3
Scheme 3. Synthesis of 1 and 3.
O
O
MeO
MeO
P
O
Me
NaBH4, CeCl3
Me
16b
16d
MeO
n
MeOH
0 °C, 1 h
NaH, THF, 0 °C
COOEt
20b
: n = 10 (56%)
20d : n = 8 (54%)
1) 20% NaOH aq.,
OH
OH
MeOH, reflux,
12 h
Me
Me
MeO
HO
n
n
2) NaH, ethanethiol,
DMF, 0 °C, reflux,
1 h
COOEt
COOH
21b
21d
: n = 10 (quant.)
: n = 8 (quant.)
2 : n = 10 (35%, 2 steps)
4 : n = 8 (41%, 2 steps)
Scheme 4. Synthesis of 2 and 4.

J. Deguchi et al. / Tetrahedron Letters 55 (2014) 3788–3791
3791
common starting intermediate 12, we applied the improved meth-
Supplementary data
odology described by Yamagiwa et al.15
The Horner–Emmons olefination was carried out in order to
extend the side chain at the C-6 position; phosphonate 12 was
treated with n-BuLi at ꢀ78 °C and reacted with different aldehydes
(13a,16 13b,17 13c,18 and 13d19) to afford the products 14a–d.
Furthermore, hydrolysis of these esters and hydrogenation of the
double bond using 5% platinum on carbon as catalyst gave satu-
rated alcohols 15a, 15b, 15c, and 15d. Oxidation of the alcohol
by Swern’s procedure20 afforded the aldehydes 16a, 16b, 16c,
and 16d (Scheme 2).
We set the stage for a daring two-step sequence consisting
of (1) Roskamp reaction21 for the introduction of the desired
b-ketophosphonate, and (2) coupling to the other fragment by a
HWE reaction to afford E geometry double bond of 1 and 3. Reac-
tion of 16a and 16c with dimethyl(diazomethyl) phosphonate22
and SnCl2 gave 17a and 17c, and then reaction with butanal in
the presence of LiBr and triethylamine in THF produced 18a and
18c in 50% and 46% yields, respectively. Enones 18a and 18c were
regioselectively reduced to 19a and 19c under Luche condition in
quantitative yield. The two protecting groups were removed by
successive treatment with sodium hydroxide in MeOH and with
sodium ethanethiolate in DMF,23 to give the desired compounds
1 and 3 in an overall yield of 3.4% from 12 for 1 (9 steps) and
3.0% from 12 for 3 (9 steps) (Scheme 3). The spectral data of
synthetic compounds 1 and 3 were identical to those of authentic
samples and we have achieved the supplement of samples for
biological evaluation.
Supplementary data (scanned copies of NMR spectra including
1H NMR, 13C NMR, 1H–1H COSY, HSQC, HMBC, and ROESY spectra)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.tetlet.2014.05.076.
References and notes
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9. Compounds 1 and 2: colorless oil; UV (MeOH) kmax 246 (e 2600), and 201 (e
4700) nm; 1H and 13C NMR, see Table 1 ; HRESIMS m/z 389.2689 [(MꢀH)ꢀ,
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and 201 (e
4600) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 361.2375
For the synthesis of other compounds, 2 and 4, enones 20b and
20d were synthesized from aldehydes 16b and 16d and a known
b-keto phosphonate24 by HWE reaction, which was then regiose-
lectively reduced to 21b and 21d under Luche condition in quanti-
tative yield. The protecting groups were removed by the same
manner as 1 and 3 to give the desired compounds 2 and 4 in an
overall yield of 5.4% from 12 for 2 (8 steps) and 5.8% from 12 for
4 (8 steps) (Scheme 4). The spectroscopic data of the synthetically
obtained compounds 2 and 4 were identical to those of authentic
samples.
[(MꢀH)ꢀ, calcd for C22H33O4, 361.2379].
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During our investigation for LDA inhibitors from plant natural
products, we found a promising small molecule, ceramicine B from
C. ceramicus,25–27 showing anti-LDA activity on mouse pre-adipo-
cyte cell line, MC3T3-G2/PA6.28 Each synthetic compound (1–4)
was tested for its anti-LDA activity on mouse pre-adipocyte cell
line, MC3T3-G2/PA6 and was found to show moderate LDA inhib-
itory activity (IC50 1: 57.6
lM, 2: 60.7
lM, 3: 74.0
l
M, 4: 76.7
lM)
as compared to positive control berberine (15.1
lM). LDA inhibi-
tory assay procedure was performed as previously described.25,27
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Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research from Japan Society for the Promotion of Science (JSPS).