Total synthesis of apios isoflavones and investigation of their tyrosinase inhibitory activity

Article abstract of DOI:10.1016/j.tet.2019.130589

Apios isoflavone glucosides 1 and 2 were synthesized for the first time via Friedel–Crafts reaction, Bischler–Napieralski-type cyclization, and phase-transfer catalyzed glycosylation as the key steps. In addition, aglycones 4 and 5 and related natural iso

Full text of DOI:10.1016/j.tet.2019.130589

Tetrahedron 75 (2019) 130589
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of apios isoflavones and investigation of their
tyrosinase inhibitory activity
Nana Asebi, Ken-ichi Nihei*
Department of Applied Biological Chemistry, School of Agriculture, Utsunomiya University, Tochigi 321-0943, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Apios isoflavone glucosides 1 and 2 were synthesized for the first time via FriedeleCrafts reaction,
BischlereNapieralski-type cyclization, and phase-transfer catalyzed glycosylation as the key steps. In
addition, aglycones 4 and 5 and related natural isoflavone cajanin (6) were synthesized in short steps.
Evaluation of the inhibitory activity of these compounds toward tyrosinase indicated that all the com-
pounds were active. In particular, the half-maximal inhibitory concentration of compound 1 toward
Received 16 August 2019
Accepted 31 August 2019
Available online 5 September 2019
Keywords:
Isoflavone
Glycoside
tyrosinase was measured to be 729 mM.
© 2019 Elsevier Ltd. All rights reserved.
BischlereNapieralski-type cyclization
Tyrosinase inhibitor
1. Introduction
(8) [21,22].
Apios americana (American groundnut) is classified as Fabaceae,
which is particularly cultivated in the eastern area of Aomori pre-
fecture in Japan. In 2013, novel isoflavone glycosides 1 and 2 were
isolated from the edible tuber of A. americana along with several
derivatives [1e3]. The structure of 1 and 2, as shown in Fig. 1, dis-
plays high similarity to that of bibenzyl glucoside 3 that has been
developed as a potent and hydrophilic tyrosinase inhibitor [4,5].
Tyrosinase (EC 1.14.8.1) and its related copper-containing
oxidoreductase catalyze the oxidation of monophenol, such as
tyrosine, to produce o-quinones via the production of o-diphenols
as reaction intermediates [6,7]. o-Quinones are further reacted to
generate several biopolymers, including melanin and insect cuticle
[8,9]. Thus, the control of this enzymatic oxidation led to the
development of effective cosmetics and insecticides [10e15].
To envision the discovery of novel tyrosinase inhibitors, the total
synthesis of 1 and 2 was designed, including FriedeleCrafts reac-
tion, a BischlereNapieralski-type cyclization, and a phase-transfer
catalyzed (PTC) glycosylation as the key steps [16e19]. To the best
of our knowledge, for the first time, we report herein the chemical
synthesis of 1 and 2 and the concise synthesis of their aglycones 4
and 5 and related natural isoflavone cajanin (6) [20]. In addition,
their tyrosinase inhibitory activities were evaluated and compared
with those of known tyrosinase inhibitors, morin (7) and kojic acid
2. Results and discussion
2.1. Synthesis of 1, 4, and 6
As shown in Scheme 1, FriedeleCrafts reaction was performed
on commercially available carboxylic acid and 1,3,5-
9
trimethoxybenzene (TMB) at 70 ꢀC using boron trifluoride ether-
ate (BF3$OEt2) as the Lewis acid [16]. Consequently, phenyl ketone
10 was solely isolated in 79% yield. Notably, the fully methylated
product could not be detected by TLC. Under the described condi-
tions, the removal of the methyl group at 20-OH preferentially
occurred, which may be due to the coordination of Lewis acid be-
tween 20-OMe and carbonyl groups [23]. The chemical synthesis of
10 starting from 9 and TMB was reported to occur in three steps:
carboxylic halogenation, FriedeleCrafts reaction, and selective
deprotection, although experimental details for those steps were
unclear [17]. In our approach, the three reaction steps were reduced
to one and the synthetic yield of 10 from 9 was significantly
improved from 30% to 79%.
The BischlereNapieralski-type cyclization of 10 with N,N-
dimethylformamide dimethyl acetal (DMFDMA) afforded methyl-
ated isoflavone 11 in 73% yield [24]. During the treatment of
methanesulfonyl chloride, DMF, and BF3$OEt2 for the aforemen-
tioned step [18], the synthetic yield was poor (30%) and several
unidentified side products were observed by TLC. In addition,
triethyl orthoformate and morpholine were not utilized to perform
* Corresponding author.
E-mail address: nihei98@cc.utsunomiya-u.ac.jp (K.-i. Nihei).
https://doi.org/10.1016/j.tet.2019.130589
0040-4020/© 2019 Elsevier Ltd. All rights reserved.

2
N. Asebi, K.-i. Nihei / Tetrahedron 75 (2019) 130589
SuzukieMiyaura coupling was previously used as a key step to
synthesize 4; however, this route required four steps [25].
Remarkably, when boron trichloride (BCl3) was treated under a
cooling condition (0 ꢀC), 6 was alternatively synthesized from 11 in
78% yield [26,27]. The presence of the methyl residue of 6 was
confirmed via NMR spectroscopy experiments. In contrast, 6 was
solely obtained by the BBr3 treatment of 11, although the detailed
experimental conditions were unclear [17]. The selectivity dis-
played in this case might be the result of poor nucleophilicity of 7-
OMe in 11, which brought a clue for the next regioselective
glycosylation.
PTC glycosylation of 4 was conducted in a two-phase system in
the presence of Aliquat 336 and K2CO3 [28]. 2,3,5,6-Tetra-O-piv-
aloyl-a-D-glucopyranosyl bromide [(Pv)4GlcBr] was selected as the
glucose donor because the pivaloyl (Pv) group showed high resis-
tance under basic conditions [19,29]. As a consequence, mono-
glycosylated isoflavone 12 was obtained in 55% yield. Thus, no
regioisomers of 12 or poly-glycosylated isoflavones were isolated
by silica gel chromatography. Schmidt glycosylation of 4 using
2,3,4,6-tetra-O-acetyl-a-D-glycosyl trichloroacetimidate as the
glucose donor was sluggish and did not proceed completely [4,11].
The glycosylation position in 12 was determined by extensive NMR
experiments, especially the significant HMBC correlations depicted
in Scheme 1. This regioselective glycosylation was explained on the
basis of the high acidity of 7-OH in 4 as implied by the demethy-
lation of 11.
Fig. 1. Structure of compounds 1e8.
The removal of the Pv groups from 12 achieved through trans-
esterification with NaOMe afforded the natural glycoside 1 quan-
titatively. The obtained spectral data were consistent with those
reported previously [1]. Thus, total synthesis of 1 was achieved for
the first time in 28% yield over five steps starting from 9. In addi-
tion, 4 and 6 were synthesized from 9 over three steps in 51% and
45%, respectively.
2.2. Synthesis of 2 and 5
Scheme 2 depicts the synthetic route leading to the formation of
natural glycoside 2 from glycosylated isoflavone 12. Because of the
hydrogen bond between 5-OH and carbonyl oxygen at C-4 in 12, 5-
OH was less reactive. Thus, the selective acylation at 20-OH and 40-
OH was achieved using PvCl in the presence of pyridine as a weak
base [30]. As a result, the hexa-acylated isoflavone 13 was obtained
in 84% yield. After a few unsuccessful attempts to introduce a
methyl group at 5-OH [31], the methylation of 13 was achieved
using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an organic base
to afford the methyl ether 14 in moderate yield (58%). Finally, the
natural product isoflavone 2 was afforded in 58% yield via trans-
esterification with NaOMe. Thus, 2 was synthesized for the first
time over seven steps from 9 with a recorded total yield of 8%.
Scheme 1. Synthesis of compounds 1, 4, and 6. Curved arrows indicate key HMBC
correlations.
this BischlereNapieralski-type cyclization because of the previ-
ously reported low yield (48%) [17].
Use of boron tribromide (BBr3) as an ether-cleaving agent
transformed 11 into isoflavone
4
in high yield (89%).
Scheme 2. Synthesis of compounds 2 and 5.

N. Asebi, K.-i. Nihei / Tetrahedron 75 (2019) 130589
3
Table 1
activity that was similar in magnitude to that of 7, a known
flavonoid-type tyrosinase inhibitor.
Tyrosinase inhibitory activities of compounds 1e8.
Compounds tested
Inhibition (%)a,b
IC50 (m
M)b
4. Experimental
1
2
3
4
5
6
7
8
61.9 0.06
24.3 0.35
-
729.3 127.7
c
-
0.77 0.04d
c
4.1. General
c
37.3 0.28
25.8 0.64
31.2 0.06
60.9 0.06
-
-
-
c
c
RP-HPLC was performed on a Hitachi LaChrome Elite instrument
equipped with Inertsil ODS-3 column (7.6 mm ꢁ 250 mm). A flow
rate and a detection wavelength were used as 2.0 mL/min and
254 nm, respectively. Optical rotations were recorded on a Jasco P-
1020 polarimeter. IR spectra were measured with a Perkin-Elmer
Frontier FT-IR spectrometer. NMR spectra were recorded in CDCl3,
CD3OD, or DMSO‑d6 on a JEOL EX-400 spectrometer (1H at 400 MHz
and 13C at 100 MHz). Chemical shifts were described as ppm rela-
tive to the solvent signal for CDCl3 (7.24 ppm for 1H NMR, 77.0 ppm
for 13C NMR), CD3OD (3.30 ppm for 1H NMR, 49.0 ppm for 13C NMR),
or DMSO‑d6 (2.49 ppm for 1H NMR, 39.7 ppm for 13C NMR). HRMS
spectra were measured on an AB SCIEX TripleTOF 5600 mass
spectrometer fitted with an electrospray ion source in positive
ionization mode.
706.7 89.3
11.3 1.03
c
-
a
The data are expressed as % of control using a solution containing 1 mM of the
compound.
b
c
The IC50 value is represented as mean value SE of three different experiments.
This value was not obtained.
This IC50 value is drawn from a previous report [4].
d
The synthesis of methylated isoflavone 5 as the aglycone part of
2 is shown in Scheme 2. Isoflavone 4 was transformed into tri-
acylated aglycone 15 in 87% yield by the action of PvCl and pyri-
dine. The subsequent methylation of 15 with iodomethane in the
presence of DBU afforded methyl ether 16 in 43% yield. Finally, 5
was obtained in 83% yield from 16 by the treatment of NaOMe. The
aforementioned synthetic route comprised six steps from 9 in 16%
overall yield.
4.2. 1-(20-Hydroxy-40,60-dimethoxyphenyl)-2-(200,400-
dimethoxyphenyl)ethanone (10)
Under Ar atmosphere, 9 (1.20 g, 6.12 mmol) and TMB (6.00 g,
35.7 mmol) were dissolved with BF3$OEt2 (16.0 mL, 127 mmol) at
room temperature (rt). After being stirred overnight at 70 ꢀC, the
resultant solution was cooled to rt. Saturated aqueous NaHCO3
solution (50 mL) was slowly added into the stirred solution. The
resultant mixture was stirred for 1 h and extracted with EtOAc
(50 mL ꢁ 3). The organic layer was washed with saturated aqueous
NaHCO3 solution (50 mL ꢁ 3) and brine (50 mL ꢁ 3). The resultant
aqueous layers were extracted with EtOAc (30 mL ꢁ 3). The com-
bined organic layers were dried over Na2SO4. Filtration and con-
centration followed by silica gel chromatography (25% EtOAc in
hexane) gave the title compound 10 (1.60 g, 4.81 mmol, 79% yield
2.3. Tyrosinase inhibitory activity
The tyrosinase inhibitory activity of 1e8 was evaluated using
DOPA as a substrate, as summarized in Table 1. A 1 mM solution of 1
showed 62% inhibition of tyrosinase-catalyzed oxidation. Further
dose-response experiments were conducted that identified 729 mM
as the half-maximal inhibitory concentration (IC50), which is nearly
equivalent to that of 7, a known flavonoid-type tyrosinase inhibitor
[21]. The IC50 of methylated glycoside 2 could not be estimated
because its 1 mM solution showed only 24% tyrosinase inhibition.
These findings indicated that a hydrogen bond between 5-OH and
C-4 in the apios isoflavone glucoside might be required for tyrosi-
nase inhibitory activity to be displayed.
Likewise, 5 that is the aglycone part of 2 without the hydrogen
bond, possessed very weak inhibitory activity (26% inhibition).
Notably, the methylated isoflavone 6 (31% inhibition) proved to be a
less effective tyrosinase inhibitor than aglycone 4 (37% inhibition),
although both contained the intramolecular hydrogen bond. This
suggests that the introduction of a hydrophilic substituent such as
glucose at 7-OH may positively influence the tyrosinase inhibitory
activity. The IC50 of 1 is about a thousand times less than that of
bibenzyl glucoside 3. This gap can be attributed to the structural
rigidity of the isoflavone skeleton. The C-ring (see Fig. 1) induced a
quasi-planar structure, which may hinder the binding of the
resorcinolic B-ring toward the active site of tyrosinase [19]. Further
chemical investigations to develop potent tyrosinase inhibitors
based on the isoflavone skeleton are underway.
from 9) as a colorless solid. IR (film) nmax 2932,1732, 875, 815 cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
6.98 (d, J ¼ 8.2 Hz 1H, H-600), 6.48 (d,
J ¼ 2.3 Hz 1H, H-300), 6.45 (dd, J ¼ 8.2, 2.3 Hz 1H, H-500), 6.06 (d,
J ¼ 2.4 Hz, 1H, H-50), 5.93 (d, J ¼ 2.4 Hz, 1H, H-30), 4.23 (s, 2H, H-2),
3.85 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.74 (s, 3H,
OMe); 13C NMR (100 MHz, CDCl3)
d
203.2 (C-1), 167.4 (C-40), 165.8
(C-60), 162.8 (C-400), 159.9 (C-20), 158.5 (C-200), 131.2 (C-600), 116.7 (C-
10), 105.8 (C-100), 104.0 (C-500), 98.6 (C-300), 93.5 (C-50), 90.7 (C-30),
55.51 (OMe), 55.49 (OMe), 55.4 (OMe), 55.3 (OMe), 44.9 (C-2);
ESIHRMS m/z 355.1161 [MþNa]þ (calcd for C18H20O6Na, 355.1158).
4.3. 5,7,20,40-Tetramethoxyisoflavone (11)
Under Ar atmosphere, 10 (1.60 g, 4.81 mmol) and DMFDMA
(1.20 mL, 9.06 mmol) were dissolved with anhydrous PhMe
(40 mL). The resultant solution was refluxed overnight and cooled
to rt. Water (50 mL) was slowly poured into the solution and the
resultant mixture was extracted with EtOAc (100 mL). The organic
layer was washed with H2O (50 mL ꢁ 3) and brine (50 mL ꢁ 3). The
resultant aqueous layers were extracted with EtOAc (30 mL ꢁ 3).
The combined organic layers were dried over Na2SO4. Filtration and
concentration followed by silica gel chromatography (20e80%
EtOAc in hexane) gave the title compound 11 (1.20 g, 3.51 mmol,
73% yield) as a colorless solid. IR (film) nmax 1644, 1028, 864,
3. Conclusion
To the best of our knowledge, we synthesized 1 and 2 for the
first time. The synthetic routes for 1 and 2 comprised five and seven
steps from 9, respectively, and they were characterized by higher
overall yields than those of the known processes. In addition,
compounds 4 and 5, the aglycone derivatives of 1 and 2, and the
related isoflavone cajanin 6 were synthesized in three, six, and
three steps from 9, respectively. The isoflavones thus obtained
proved weak inhibitors of the tyrosinase-catalyzed DOPA oxidation.
Notably, the natural glucoside 1 displayed tyrosinase inhibitory
821 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.71 (s, 1H, H-2), 7.21 (d,
J ¼ 8.9 Hz, 1H, H-60), 6.50 (m, 2H, H-30, H-50), 6.42 (d, J ¼ 2.4 Hz, 1H,
H-8), 6.33 (d, J ¼ 2.4 Hz, 1H, H-6), 3.89 (s, 3H, OMe), 3.86 (s, 3H,

4
N. Asebi, K.-i. Nihei / Tetrahedron 75 (2019) 130589
OMe), 3.81 (s, 3H, OMe), 3.73 (s, 3H, OMe); 13C NMR (100 MHz,
CDCl3)
175.3 (C-4), 163.7 (C-7), 161.4 (C-5), 160.9 (C-40), 160.0 (C-
8a), 158.5 (C-20), 151.7 (C-2), 132.5 (C-60), 123.2 (C-3), 113.6 (C-10),
110.0 (C-4a), 104.1 (C-50), 98.8 (C-30), 96.0 (C-6), 92.5 (C-8), 56.3
(OMe), 55.69 (OMe), 55.67 (OMe), 55.4 (OMe); ESIHRMS m/z
343.1185 [MþH]þ (calcd for C19H19O6, 343.1182).
combined organic layers were dried over Na2SO4. Filtration and
concentration followed by silica gel chromatography (30% EtOAc in
hexane) gave the title compound 12 (0.38 g, 0.48 mmol, 55% yield)
d
as a colorless solid. [
a]
;
23-25.7 (c 0.42, CHCl3); IR (film) nmax 2973,
D
1742, 1135, 753 cmꢂ1
1H NMR (400 MHz, CDCl3)
d 12.13 (s, 1H, 5-
OH), 8.16 (bs, 1H, 20-OH), 7.94 (s, 1H, H-2), 7.01 (d, J ¼ 8.4 Hz, 1H,
H-60), 6.60 (d, J ¼ 2.5 Hz,1H, H-30), 6.51 (dd, J ¼ 8.4, 2.5 Hz,1H, H-50),
60.48 (d, J ¼ 2.2 Hz, 1H, H-8), 6.40 (d, J ¼ 2.2 Hz, 1H, H-6), 6.17 (bs, 1H,
4 -OH), 5.38 (t, J ¼ 9.6 Hz, 1H, H-300), 5.28 (dd, J ¼ 9.6, 7.8 Hz, 1H, H-
200), 5.15 (t, J ¼ 9.6 Hz, 1H, H-400), 4.98 (d, J ¼ 7.8 Hz, 1H, H-100), 4.27
(dd, J ¼ 12.4, 1.6 Hz, 1H, H-600), 4.04 (dd, J ¼ 12.4, 6.6 Hz, 1H, H-600),
3.91 (ddd, J ¼ 9.6, 6.6, 1.6 Hz, 1H, H-500), 1.22 (s, 9H, Pv), 1.17 (s, 9H,
Pv), 1.14 (s, 9H, Pv), 1.13 (s, 9H, Pv); 13C NMR (100 MHz, CDCl3)
4.4. 20-Hydroxygenistein (4)
To the solution of 11 (0.28 g, 0.82 mmol) in anhydrous CH2Cl2
(5 mL), 1 M BBr3 in CH2Cl2 (15.0 mL, 15.0 mmol) was slowly added
at ꢂ15 ꢀC under Ar atmosphere. After being stirred overnight at rt,
the resultant solution was poured into ice water (100 mL) and
extracted with EtOAc (50 mL). The organic layer was washed with
water (30 mL ꢁ 3) and brine (30 mL ꢁ 3). The resultant aqueous
layers were extracted with EtOAc (30 mL ꢁ 3). The combined
organic layers were dried over Na2SO4. Filtration and concentration
followed by silica gel chromatography (3% MeOH in CHCl3) gave the
title compound 4 (0.21 g, 0.73 mmol, 89% yield) as a colorless solid.
d
182.0 (C-4),178.3 (Pv),177.6 (Pv),176.7 (Pv),176.6 (Pv),162.9 (C-7),
162.4 (C-5), 158.7 (C-40), 157.3 (C-8a), 157.1 (C-20), 155.3 (C-2), 130.8
(C-60), 123.4 (C-3), 111.7 (C-10), 109.0 (C-50), 106.7 (C-4a), 106.6 (C-
30), 100.6 (C-6), 98.4 (C-100), 94.9 (C-8), 73.0 (C-500), 71.7 (C-300), 70.3
(C-200), 67.6 (C-400), 62.1 (C-600), 38.9 (Pv), 38.8 (Pv), 27.12 (Pv), 27.10
(Pv), 27.02 (Pv), 27.00 (Pv); ESIHRMS m/z 785.3384 [MþH]þ (calcd
for C41H53O15, 785.3385).
IR (film) nmax 3225, 1650, 1171, 870, 808 cmꢂ1 1H NMR (400 MHz,
;
CD3OD)
d
8.00 (s, 1H, H-2), 7.03 (d, J ¼ 8.2 Hz, 1H, H-60), 6.39 (d,
J ¼ 2.4 Hz, 1H, H-30), 6.36 (dd, J ¼ 8.2, 2.4 Hz, 1H, H-50), 6.35 (d,
4.7. 7-(b
-Glucopyranosyl)-20-hydroxygenistein (1)
J ¼ 2.1 Hz, 1H, H-8), 6.22 (d, J ¼ 2.1 Hz, 1H, H-6); 13C NMR (100 MHz,
CD3OD) d
182.7 (C-4), 166.1 (C-7), 163.7 (C-5), 160.3 (C-40), 159.8 (C-
To a solution of 12 (0.10 g, 0.13 mmol) in MeOH (2 mL), 5 M
NaOMe in MeOH (0.10 mL, 0.50 mmol) was added. The resultant
solution was refluxed for 2 h, cooled to rt, and neutralized with
Amberlite IR-120H. Filtration and concentration followed by crys-
8a), 157.8 (C-20), 156.7 (C-2), 133.2 (C-60), 122.6 (C-3), 110.8 (C-10),
108.1 (C-50), 106.2 (C-4a), 104.2 (C-30), 100.2 (C-6), 94.8 (C-8);
ESIHRMS m/z 287.0551 [MþH]þ (calcd for C15H11O6, 287.0556). The
spectral data (NMR and MS) that mentioned above were consistent
with those that were previously reported [25].
tallization from 50% MeOH in H2O gave the title compound 1
23
(60 mg, 0.13 mmol, 100% yield) as a colorless solid. [
a
]
D
-60.1 (c
0.17, MeOH); IR (film) nmax 2973, 1742, 1135, 753 cmꢂ1
(400 MHz, DMSO‑d6)
;
1H NMR
d
13.00 (s, 1H, 5-OH), 9.39 (s, 1H, 40-OH), 9.31
4.5. Cajanin (6)
(s, 1H, 20-OH), 8.24 (s, 1H, H-2), 6.98 (d, J ¼ 8.3 Hz, 1H, H-60), 6.70 (d,
J ¼ 2.2 Hz, 1H, H-8), 6.46 (d, J ¼ 2.2 Hz, 1H, H-6), 6.36 (d, J ¼ 2.4 Hz,
1H, H-30), 6.26 (dd, J ¼ 8.3, 2.4 Hz, 1H, H-50), 5.40 (d, J ¼ 4.8 Hz, 1H,
200-OH), 5.13 (d, J ¼ 4.6 Hz, 1H, 400-OH), 5.06 (d, J ¼ 5.2 Hz, 1H, 300-
OH), 5.05 (d, J ¼ 7.4 Hz, 1H, H-100), 4.60 (m, 1H, 600-OH), 3.70 (m, 1H,
H-600), 3.47 (m, 1H, H-600), 3.43 (m, 1H, H-500), 3.29 (m, 1H, H-300),
3.26 (m, 1H, H-200), 3.15 (m, 1H, H-400); 13C NMR (100 MHz,
To the solution of 11 (0.11 g, 0.32 mmol) in anhydrous CH2Cl2
(3 mL), 1 M BCl3 in CH2Cl2 (7.0 mL, 7.0 mmol) was slowly added
at ꢂ15 ꢀC under Ar atmosphere. After being stirred overnight at rt,
the resultant solution was poured into ice water (50 mL) and
extracted with EtOAc (30 mL). The organic layer was washed with
water (20 mL ꢁ 3) and brine (20 mL ꢁ 3). The resultant aqueous
layers were extracted with EtOAc (20 mL ꢁ 3). The combined
organic layers were dried over Na2SO4. Filtration and concentration
followed by silica gel chromatography (3% MeOH in CHCl3) gave the
title compound 6 (75 mg, 0.25 mmol, 78% yield) as a colorless solid.
DMSO‑d6)
d
180.9 (C-4), 163.1 (C-7), 161.7 (C-5), 158.9 (C-40), 157.4
(C-8a),156.6 (C-20), 156.1 (C-2),132.4 (C-60), 121.0 (C-3),108.6 (C-10),
106.5 (C-50), 106.3 (C-4a), 102.8 (C-30), 100.1 (C-100), 99.7 (C-6), 94.7
(C-8), 77.4 (C-500), 76.6 (C-300), 73.3 (C-200), 69.8 (C-400), 60.8 (C-600);
ESIHRMS m/z 449.1084 [MþH]þ (calcd for C21H21O11, 449.1084). The
spectral data (specific rotation, NMR, and MS) that mentioned
above were consistent with those that were previously reported
[1].
IR (film) nmax 3452, 1652, 1503, 1145 cmꢂ1 1H NMR (400 MHz,
;
CD3OD)
d
8.06 (s, 1H, H-2), 7.04 (d, J ¼ 8.2 Hz, 1H, H-60), 6.55 (d,
J ¼ 2.3 Hz, 1H, H-8), 6.39 (d, J ¼ 2.3 Hz, 1H, H-30), 6.363 (dd, J ¼ 8.2,
2.3 Hz,1H, H-50), 6.362 (d, J ¼ 2.3 Hz,1H, H-6), 3.88 (s, 3H, OMe); 13C
NMR (100 MHz, CD3OD) d 182.8 (C-4), 167.3 (C-7), 163.5 (C-5), 160.3
(C-8a),159.7 (C-40), 157.8 (C-20), 157.0 (C-2), 133.2 (C-60), 122.8 (C-3),
110.6 (C-10), 108.1 (C-50), 107.1 (C-4a), 104.2 (C-30), 99.3 (C-6), 93.2
(C-8), 56.5 (OMe); ESIHRMS m/z 301.0717 [MþH]þ (calcd for
4.8. 7-(200,300,400,600-Tetra-O-pivaloyl- -glucopyranosyl)-20,4'-(di-O-
b
pivaloyl)-20- hydroxygenistein (13)
C
16H13O6, 301.0712). The spectral data (NMR and MS) that
To a solution of 12 (0.25 g, 0.32 mmol) in CH2Cl2 (3 mL), pyridine
(1 mL) and PvCl (0.10 mL, 0.81 mmol) were added. After being
stirred for 2 h at rt, water (20 mL) was poured into the solution and
the resultant mixture was extracted with EtOAc (20 mL ꢁ 3). The
organic layer was washed with saturated aqueous CuSO4 solution
(20 mL ꢁ 4), water (20 mL) and brine (20 mL) and was dried over
Na2SO4. Filtration and concentration followed by silica gel chro-
matography (10e15% EtOAc in hexane) gave the title compound 13
mentioned above were consistent with those that were previously
reported [20].
4.6. 7-(200,300,400,600-Tetra-O-pivaloyl- -glucopyranosyl)-20-
b
hydroxygenistein (12)
K2CO3 (0.24 g, 1.74 mmol) and 4 (0.25 g, 0.87 mmol) were dis-
solved with water (10 mL) and DMF (10 mL). To the solution,
(Pv)4GlcBr (1.70 g, 2.93 mmol) and Aliquat 336 (0.31 g, 0.77 mmol)
in CHCl3 (10 mL) were added. After being stirred for 24 h at 45 ꢀC,
water (50 mL) was poured into the solution and the resultant
mixture was extracted with EtOAc (20 mL ꢁ 3). The organic layer
was washed with water (10 mL ꢁ 3) and brine (10 mL ꢁ 3). The
(0.26 g, 0.27 mmol, 84% yield) as a colorless solid. [
CHCl3); IR (film) nmax 2973, 1744, 1111, 760 cmꢂ1
(400 MHz, CDCl3)
a]
23 -22.6 (c 0.38,
D
;
1H NMR
12.58 (s, 1H, 5-OH), 7.77 (s, 1H, H-2), 7.30 (d,
d
J ¼ 8.4 Hz, 1H, H-60), 7.03 (dd, J ¼ 8.4, 2.3 Hz, 1H, H-50), 6.96 (d,
J ¼ 2.3 Hz, 1H, H-30), 6.48 (d, J ¼ 2.2 Hz, 1H, H-8), 6.40 (d, J ¼ 2.2 Hz,
1H, H-6), 5.41 (t, J ¼ 9.4 Hz,1H, H-300), 5.30 (dd, J ¼ 9.4, 7.9 Hz,1H, H-

N. Asebi, K.-i. Nihei / Tetrahedron 75 (2019) 130589
5
200), 5.18 (d, J ¼ 7.9 Hz, 1H, H-100), 5.16 (t, J ¼ 9.4 Hz, 1H, H-400), 4.26
(dd, J ¼ 12.3, 1.7 Hz, 1H, H-600), 4.06 (dd, J ¼ 12.3, 6.8 Hz, 1H, H-600),
3.96 (ddd, J ¼ 9.4, 6.8, 1.7 Hz, 1H, H-500), 1.32 (s, 9H, Pv), 1.21 (s, 9H,
159.0 (C-8a),158.5 (C-40),156.6 (C-20),152.1 (C-2),132.3 (C-60),123.5
(C-3), 110.5 (C-10), 109.7 (C-4a), 106.4 (C-50), 103.0 (C-30), 100.1 (C-
100), 97.3 (C-6), 95.8 (C-8), 77.5 (C-500), 76.8 (C-300), 73.3 (C-200), 70.þ0
(C-400), 60.9 (C-600), 56.3 (5-OMe); ESIHRMS m/z 463.1243 [MþH]
(calcd for C22H23O11, 463.1240). The spectral data (specific rotation,
NMR, and MS) that mentioned above were consistent with those
that were previously reported [1].
Pv),1.16 (s, 9H, Pv),1.15 (s, 9H, Pv),1.12 (s, 9H, Pv),1.11 (s, 9H, Pv); 13
C
NMR (100 MHz, CDCl3)
d 179.9 (C-4), 178.1 (Pv), 177.1 (Pv), 176.49
(Pv), 176.46 (Pv), 176.2 (Pv), 162.7 (C-7), 162.4 (C-5), 157.5 (C-8a),
154.5 (C-2), 152.0 (C-40), 149.9 (C-20), 131.6 (C-60), 120.8 (C-3), 120.7
(C-10), 119.1 (C-50), 116.6 (C-30), 107.2 (C-4a), 100.0 (C-6), 98.4 (C-100),
94.9 (C-8), 73.0 (C-500), 71.7 (C-300), 70.5 (C-200), 67.7 (C-400), 62.1 (C-
600), 39.14 (Pv), 39.06 (Pv), 38.81 (Pv), 38.78 (Pv), 38.7 (Pv), 27.09
(Pv), 27.05 (Pv), 27.0 (Pv), 26.9 (Pv); ESIHRMS m/z 953.4523
[MþH]þ (calcd for C51H69O17, 953.4535).
4.11. 7,20,4'-(Tri-O-pivaloyl)-20-hydroxygenistein (15)
To a solution of 4 (86 mg, 0.30 mmol) in CH2Cl2 (2 mL), pyridine
(1 mL) and PvCl (0.23 mL, 1.87 mmol) were added. After being
stirred for 2 h at rt, water (20 mL) was poured into the solution and
the resultant mixture was extracted with EtOAc (20 mL ꢁ 3). The
organic layer was washed with saturated aqueous CuSO4 solution
(20 mL ꢁ 4), water (20 mL) and brine (20 mL) and was dried over
Na2SO4. Filtration and concentration followed by silica gel chro-
matography (20% EtOAc in hexane) gave the title compound 15
(0.14 g, 0.26 mmol, 87% yield) as a colorless solid. IR (film) nmax
4.9. 5-(O-Methyl)-7-(200,300,400,600-tetra-O-pivaloyl-
b-
glucopyranosyl)-20,4'-(di-O- pivaloyl)-20-hydroxygenistein (14)
MeI (0.20 mL, 3.21 mmol) and DBU (0.60 mL, 4.02 mmol) were
added to a solution of 13 (0.15 g, 0.16 mmol) in DMF (3 mL) under Ar
atmosphere. After being stirred overnight at rt, water (20 mL) was
poured into the solution and the resultant mixture was extracted
with EtOAc (30 mL). The organic layer was washed with water
(10 mL ꢁ 3) and brine (10 mL ꢁ 3) and was dried over Na2SO4.
Filtration and concentration followed by silica gel chromatography
2975, 1753, 1088, 752 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 12.58 (s,
1H, 5-OH), 7.84 (s, 1H, H-2), 7.33 (d, J ¼ 8.4 Hz, 1H, H-60), 7.04 (dd,
J ¼ 8.4, 2.2 Hz, 1H, H-50), 6.98 (d, J ¼ 2.2 Hz, 1H, H-3ꞌ), 6.72 (d,
J ¼ 2.1 Hz, 1H, H-8), 6.55 (d, J ¼ 2.1 Hz, 1H, H-6), 1.35 (s, 9H, Pv), 1.34
(s, 9H, Pv), 1.16 (s, 9H, Pv); 13C NMR (100 MHz, CDCl3)
d 180.3 (C-4),
(30% EtOAc in hexane) gave the title compound 14 (89 mg, 92
m
mol,
23
176.5 (Pv), 176.2 (Pv), 176.0 (Pv), 162.2 (C-5), 156.8 (C-7), 156.7 (C-
8a), 154.9 (C-2), 152.1 (C-40), 149.9 (C-20), 131.7 (C-60), 120.8 (C-3),
120.7 (C-10), 119.1 (C-50), 116.6 (C-30), 108.9 (C-4a), 105.7 (C-6), 100.9
(C-8), 39.3 (Pv), 39.2 (Pv), 39.1 (Pv), 27.1 (Pv), 27.0 (Pv), 26.9 (Pv);
ESIHRMS m/z 539.2286 [MþH]þ (calcd for C30H35O9, 539.2281).
58% yield) as a colorless solid. [
a]
-65.5 (c 0.33, CHCl3); IR (film)
D
nmax 2973,1745, 1113, 754 cmꢂ1; 1H NMR (400 MHz, CDCl3)
d
7.62 (s,
1H, H-2), 7.32 (d, J ¼ 8.4 Hz,1H, H-60), 6.99 (dd, J ¼ 8.4, 2.3 Hz,1H, H-
50), 6.91 (d, J ¼ 2.3 Hz, 1H, H-30), 6.55 (d, J ¼ 2.2 Hz, 1H, H-8), 6.34 (d,
J ¼ 2.2 Hz, 1H, H-6), 5.42 (t, J ¼ 9.4 Hz, 1H, H-300), 5.30 (dd, J ¼ 9.4,
7.9 Hz, 1H, H-200), 5.18 (d, J ¼ 7.9 Hz, 1H, H-100), 5.17 (t, J ¼ 9.4 Hz, 1H,
H-400), 4.28 (dd, J ¼ 12.2, 1.5 Hz, 1H, H-600), 4.06 (dd, J ¼ 12.2, 6.6 Hz,
1H, H-600), 3.97 (ddd, J ¼ 9.4, 6.6, 1.5 Hz, 1H, H-500), 3.85 (s, 3H, 5-
OMe), 1.32 (s, 9H, Pv), 1.20 (s, 9H, Pv), 1.16 (s, 9H, Pv), 1.14 (s, 9H,
Pv), 1.120 (s, 9H, Pv), 1.116 (s, 9H, Pv); 13C NMR (100 MHz, CDCl3)
4.12. 5-(O-Methyl)-7,20,4'-(tri-O-pivaloyl)-20-hydroxygenistein (16)
MeI (0.10 mL, 1.61 mmol) and DBU (0.30 mL, 2.01 mmol) were
added to a solution of 15 (0.20 g, 0.37 mmol) in DMF (2 mL) under
Ar atmosphere. After being stirred overnight at rt, water (20 mL)
was poured into the solution and the resultant mixture was
extracted with EtOAc (30 mL). The organic layer was washed with
water (10 mL ꢁ 3) and brine (10 mL ꢁ 3) and was dried over Na2SO4.
Filtration and concentration followed by silica gel chromatography
(30% EtOAc in hexane) gave the title compound 16 (86 mg, 0.16 mol,
43% yield) as a colorless solid. IR (film) nmax 2975, 1752, 1094,
d
178.0 (Pv), 177.1 (Pv), 176.48 (Pv), 176.46 (Pv), 176.4 (Pv), 174.1 (C-
4), 161.5 (C-5), 160.9 (C-7), 159.4 (C-8a), 151.6 (C-2), 151.5 (C-40),
149.6 (C-20), 132.2 (C-60), 122.6 (C-3), 122.1 (C-10), 118.8 (C-50), 116.1
(C-30), 111.2 (C-4a), 98.7 (C-100), 97.6 (C-6), 95.3 (C-8), 73.1 (C-500),
71.6 (C-300), 70.6 (C-200), 67.6 (C-400), 62.1 (C-600), 56.4 (5-OMe), 39.1
(Pv), 39.0 (Pv), 38.80 (Pv), 38.79 (Pv), 38.7 (Pv), 27.08 (Pv), 27.07
(Pv), 27.05 (Pv), 27.0 (Pv), 26.9 (Pv); ESIHRMS m/z 967.4690 [MþH]þ
(calcd for C52H71O17, 967.4691).
750 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.69 (s, 1H, H-2), 7.35 (d,
J ¼ 8.4 Hz, 1H, H-60), 7.00 (dd, J ¼ 8.4, 2.3 Hz, 1H, H-50), 6.92 (d,
J ¼ 2.3 Hz, 1H, H-3ꞌ), 6.79 (d, J ¼ 2.1 Hz, 1H, H-8), 6.53 (d, J ¼ 2.1 Hz,
1H, H-6), 3.92 (s, 3H, 5-OMe), 1.37 (s, 9H, Pv), 1.33 (s, 9H, Pv), 1.16 (s,
4.10. 5-(O-Methyl)-7-(b
-Glucopyranosyl)-20-hydroxygenistein (2)
9H, Pv); 13C NMR (100 MHz, CDCl3)
d 176.52 (Pv), 176.50 (Pv), 176.1
To a solution of 14 (0.18 g, 0.19 mmol) in MeOH (4 mL), 5 M
NaOMe in MeOH (0.26 mL, 1.30 mmol) was added. The resultant
solution was refluxed for 1.5 h, cooled to rt, and neutralized with
Amberlite IR-120H. The resin was filtered off and the filtrate was
washed with cyclohexane (1 mL ꢁ 3). The MeOH layer was con-
centered and the residue was purified by solid phase extraction
using InertSep Slim C18-B cartridge (20e50% MeOH in H2O) fol-
lowed by preparative RP-HPLC (35% MeOH in H2O, tR ¼ 12.2 min).
(Pv), 174.4 (C-4), 161.2 (C-5), 158.6 (C-7), 155.2 (C-8a), 152.1 (C-2),
151.5 (C-40), 149.6 (C-20), 132.3 (C-60), 122.6 (C-3), 122.2 (C-10), 118.8
(C-50), 116.2 (C-30), 112.8 (C-4a), 103.0 (C-6), 101.3 (C-8), 56.6 (5-
OMe), 39.4 (Pv), 39.1 (Pv), 39.0 (Pv), 27.1 (Pv), 27.0 (Pv), 26.9 (Pv);
ESIHRMS m/z 553.2441 [MþH]þ (calcd for C31H37O9, 553.2438).
4.13. 5-(O-Methyl)-20-hydroxygenistein (5)
The title compound 2 (51 mg, 0.11 mmol, 58% yield) was obtained as
23
a colorless solid. [
a
]
D
-80.3 (c 0.10, 50% MeOH in H2O); IR (film)
To a solution of 16 (0.16 g, 0.29 mmol) in MeOH (3 mL), 5 M
NaOMe in MeOH (0.17 mL, 0.85 mmol) was added. The resultant
solution was refluxed for 2 h, cooled to rt, and neutralized with
Amberlite IR-120H. Filtration and concentration followed by silica
gel chromatography (10% MeOH in CHCl3) gave the title compound
5 (72 mg, 0.24 mmol, 83% yield) as a colorless solid. IR (film) nmax
nmax 2972, 1745, 1135, 753 cmꢂ1 1H NMR (400 MHz, DMSO‑d6)
;
d
9.24 (m, 2H, 20-OH, 40-OH), 8.00 (s,1H, H-2), 6.90 (d, J ¼ 8.3 Hz,1H,
H-60), 6.72 (d, J ¼ 2.2 Hz, 1H, H-8), 6.59 (d, J ¼ 2.2 Hz, 1H, H-6), 6.32
(d, J ¼ 2.3 Hz, 1H, H-30), 6.23 (dd, J ¼ 8.3, 2.3 Hz, 1H, H-50), 5.41 (d,
J ¼ 3.9 Hz, 1H, 200-OH), 5.17 (d, J ¼ 3.4 Hz, 1H, 400-OH), 5.09 (d,
J ¼ 5.3 Hz, 1H, 300-OH), 5.06 (d, J ¼ 7.4 Hz, 1H, H-100), 4.63 (m, 1H, 600-
OH), 3.82 (s, 3H, 5-OMe), 3.72 (m, 1H, H-600), 3.44 (m, 2H, H-500, H-
600), 3.36 (m, 1H, H-300), 3.28 (m, 1H, H-200), 3.15 (m, 1H, H-400); 13C
3180, 1573, 1085, 844 cmꢂ1 1H NMR (400 MHz, CD3OD)
; d 7.93 (s,
1H, H-2), 7.00 (d, J ¼ 8.1 Hz, 1H, H-60), 6.42 (d, J ¼ 2.4 Hz, 1H, H-3ꞌ),
6.41 (d, J ¼ 2.2 Hz, 1H, H-8), 6.37 (d, J ¼ 2.2 Hz, 1H, H-6), 6.35 (dd,
J ¼ 8.1, 2.4 Hz, 1H, H-5ꞌ), 3.87 (s, 3H, Me); 13C NMR (100 MHz,
NMR (100 MHz, DMSO‑d6)
d 174.6 (C-4), 161.5 (C-7), 160.8 (C-5),

6
N. Asebi, K.-i. Nihei / Tetrahedron 75 (2019) 130589
CD3OD)
d
179.0 (C-4), 166.1 (C-7), 163.0 (C-5), 161.5 (C-8a), 160.2 (C-
References
40), 158.1 (C-20), 154.4 (C-2), 133.0 (C-60), 125.2 (C-3), 112.6 (C-10),
108.6 (C-4a),108.3 (C-50),104.8 (C-30), 98.0 (C-6), 96.3 (C-8), 56.4 (5-
OMe); ESIHRMS m/z 301.0715 [MþH]þ (calcd for C16H13O6,
301.0712).
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We thank Drs. Yoichi Yamada and Michinori Karikomi (Utsu-
nomiya University) for technical assistances of NMR and optical
rotation analyses. This study was supported in part by MAYEKAWA
HOUONKAI FOUNDATION, JSPS KAKENHI Grant Number 15K01797,
and a Grant of Utsunomiya University Exploratory Research for
Young Scientists. N.A acknowledges the scholarship of Japan Stu-
dent Services Organization.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.130589.