Structure-activity relationships of biflavonoids for β-secretase (BACE-1) inhibitory activity

Article abstract of DOI:10.3987/COM-12-12561

Bioactive natural products are useful sources of new pharmaceutics. Their analogues are also important for evaluating structure-activity relationships. In this study the structure-activity relationships of 2,3-dihydro-6-methylginketin (1) for BACE-1 inhibitory activity were studied. Biflavonoids consisting of flavanone and flavone, and also the presence of a methoxy group at the C-4' position, were found to be important for strong activity. 2,3-Dihydro-6- methylbilobetin (2) showed strong activity with an IC₅₀ value of 0.56 μM.

Full text of DOI:10.3987/COM-12-12561

HETEROCYCLES, Vol. 85, No. 11, 2012
2749
HETEROCYCLES, Vol. 85, No. 11, 2012, pp. 2749 - 2756. © 2012 The Japan Institute of Heterocyclic Chemistry
Received, 1st August, 2012, Accepted, 5th September, 2012, Published online, 10th September, 2012
DOI: 10.3987/COM-12-12561
STRUCTURE-ACTIVITY RELATIONSHIPS OF BIFLAVONOIDS FOR
-SECRETASE (BACE-1) INHIBITORY ACTIVITY
Hiroaki Sasaki,a Yuki Kitoh, b Kazuhiko Miki,b Kaoru Kinoshita,b Kiyotaka
Koyama,b Miyuki Kaneda,a and Kunio Takahashi* b
aSchool of Pharmacy, Shujitsu University, Nishigawara 1-6-1, Naka-ku,
b
Okayama-shi, Okayama 703-8516, Japan. Department of Pharmacognosy and
Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi,
Tokyo 204-8588, Japan. E-mail address: diamonds@my-pharm.ac.jp
Abstract – Bioactive natural products are useful sources of new pharmaceutics.
Their analogues are also important for evaluating structure-activity relationships.
In this study the structure-activity relationships of 2,3-dihydro-6-methylginketin
(1) for BACE-1 inhibitory activity were studied. Biflavonoids consisting of
flavanone and flavone, and also the presence of a methoxy group at the C-4’
position,
were
found
to
be
important
for
strong
activity.
2,3-Dihydro-6-methylbilobetin (2) showed strong activity with an IC50 value of
0.56M.
Biflavonoids are flavonoid dimers connected by C-C or C-O-C bonds and exhibit anti-inflammatory1 and
anti-malarial2 activities. We have investigated the biological activities of biflavonoids, and found that
amentoflavone-type biflavonoids showed anti-influenza3,4 and -secretase (BACE-1: -site APP cleaving
enzyme-1) inhibitory activities.5 Amyloid  (A) peptides, involved in the development of Alzheimer’s
disease (AD), are generated from the cleavage of the -amyloid precursor protein (APP) by consecutive
action of BACE-1. Hence, BACE-1 has been recognized as an effective and safe therapeutic for AD.
There are few compounds that function like BACE-1 inhibitors from natural resources.5-10 We found that
flavanone-flavone dimeric biflavonoid, 2,3-dihydro-6-methylginkgetin (1), showed the strongest
inhibitory activity among amentoflavone-type biflavonoids,5 and we later isolated new biflavonoids,
2,3-dihydro-6-methylbilobetin (2) and 2,3-dihydro-6-methylsequoiaflavone (3), from Cephalotaxus
harringtonia var. fastigiata (Cephalotaxaceae).11 In this paper we report the BACE-1 inhibitory activities
and structure-activity relationships of compounds 1-3 and 4 (methylation product of 1,

2750
HETEROCYCLES, Vol. 85, No. 11, 2012
2,3-dihydro-6-methylamentoflavone 7,4’,7’’,4’’’-tetramethyl ether), and some flavonoids, apigenin,
naringenin, and their derivatives (5-10) (Figure 1 and Scheme 1).
OH
HO
O
2S
O
OH
O
3'
OH
8''
HO
1a
OH O
Figure 1. Structures of 1-3 from Cephalotaxus harringtonia var. fastigiata, methylated product of 1 (4)
and 1a from Cumninghamia lanceolata
OMe
OMe
7
7
Me
HO
Me
HO
6
6
O
O
2S
2S
O
O
4'
4' OMe
O
OMe
O
OH
OMe
4'''
4'''
TMS-diazomethane
CH2Cl2, MeOH
7''
7''
HO
MeO
OH O
OH O
1
4
Scheme 1. Methylation procedures of 1, 5, 7 and 9 and products (4, 6, 8 and 10)

HETEROCYCLES, Vol. 85, No. 11, 2012
2751
Table 1. 1H NMR and 13C NMR spectral data of 4 in DMSO-d6
position
C
H
2
79.17, 78.45
5.60, 5.62 (each 1H, dd, J = 13.2, 2.9 Hz)
2.79, 2.83 (each 1H, dd, J = 17.2, 2.9 Hz)
5.60, 5.62 (each 1H, dd, J = 17.2, 13.2 Hz)
3eq
41.97, 42.32
3ax
4
196.94
5
159.42
6
7
104.10*1
165.15
8
91.13
161.20*2
6.20, 6.21 (each 1H, s)
9
10
102.23
1’
130.09, 130.24
130.83
2’
7.50, 7.52 (each 1H, d, J = 2.3 Hz)
3’
120.32
4’
157.48, 157.54
111.20, 111.25
128.24, 128.33
163.35
5’
7.22 (2H, d, J = 8.6 Hz)
6’
7.60, 7.62 (each 1H, dd, J = 8.6, 2.3 Hz)
2’’
3’’
103.03
6.947, 6.950 (each 1H, s)
6.65 (2H, s)
4’’
182.18, 182.36
161.20*2
95.50
5’’
6’’
7’’
162.35
8’’
105.42
9’’
153.34
10’’
1’’’
104.10*1
122.70
2’’’, 5’’’
3’’’, 6’’’
4’’’
128.03, 128.09
114.53
7.64, 7.65 (each 2H, d, J = 8.6 Hz)
6.99, 7.00 (each 2H, d, J = 8.6 Hz)
162.61
6-CH3
7-OCH3
4’-OCH3
7’’-OCH3
4’’’-OCH3
5-OH
5’’-OH
6.90
1.885, 1.892 (each 1H, s)
3.775, 3.780 (each 3H, s) *4
3.71 (6H, s)
3.797, 3.806 (each 3H, s) *4
3.816, 3.823 (each 3H, s) *4
12.06, 12.07 (each 1H, s)
13.085, 13.088 (each 1H, s)
56.42*3
55.61
56.02, 56.04*3
55.56*3
*1-4may be exchanged within the same column.

2752
HETEROCYCLES, Vol. 85, No. 11, 2012
Table 2. Inhibitory assay results for compounds 1-10
BACE-1 Inhibition IC50
compounds
(M)
2,3-dihydro-6-methylginkgetin (1)
2,3-dihydro-6-methylbilobetin (2)
2,3-dihydro-6-methylsequoiaflavone (3)
2,3-dihydro-6-methylamentoflavone 7,4’,7’’,4’’’-tetramethyl ether (4)
apigenin (5)
0.44
0.56
3.05
>10
>10
>10
>10
>10
>10
>10
0.07
apigenin 7,4’-dimethyl ether (6)
naringenin (7)
naringenin 7,4’-dimethyl ether (8)
hesperetin (9)
7,3'-dimethoxy hesperetin (10)
-Secretase Inhibitor (positive control)
We have already reported strong BACE-1 inhibitory compounds, 2,3-dihydro-6-methylginkgetin (1) and
2,3-dihydroamentoflavone (1a), among amentoflavone-type biflavonoids by using the BACE-1 FRET
assay kit.5 These results showed that flavanone-flavone dimeric biflavonoids (IC50, 0.35 - 0.75 M)
showed stronger BACE-1 inhibitory activities than flavone-flavone dimers (IC50, 1.54 - >10 M). Thus,
the former is more important than the latter for significant activity. Differences in the structures of 1 and
1a were the presence of 6-methyl and 7,4’-dimethoxy groups in 1 instead of 6-H and 7,4’-dihydroxy
groups in 1a. The substitution of the 6-methyl group with 7 and 4’ methoxy groups on the flavanone
moiety does not affect the inhibitory activity. We have also isolated new biflavonoids,
3-dihydro-6-methylbilobetin (2), 2,3-dihydro-6-sequoiaflavone (3), with 2,3-dihydro-6-methylginkgetin
(1) from Cephalotaxus harringtonia var. fastigiata (Cephalotaxaceae).11 In this paper, the correlation
between methoxy positions and activities were discussed among some 6-methylflavanone-flavone
biflavonoids. Compound 1 was methylated by trimethylsilyldiazomethane (TMS-diazomethane) in
CH2Cl2 and MeOH to afford 4 as a mixture of diastereoisomers due to the presence of a chiral center on
C-2 and atropisomerism induced along the C3’-C8’’ bond. Therefore, the resultant doubling of proton and
carbon resonances was observed in the spectra (Table 1).11 Since the diastereoisomers of 4 could not be
separated from each other, they were used in the assay without further purification. In addition, flavonoid
monomers 5, 7 and 9 and their methylated compounds 6, 8 and 10 were used and compared to
biflavonoids 1-4 in the assay. As shown in Table 2, 1 and 2 showed similar strong inhibitory activities,

HETEROCYCLES, Vol. 85, No. 11, 2012
2753
with IC50 values of 0.44, and 0.56 M, respectively, while 3 with an IC50 value of 3.05 M showed lower
activity than both 1 and 2. From comparing 1 and 2, the substitution of a methoxy instead of a hydroxy
group at C-7 does not affect the activity. Comparing 1, 2 and 3, a methoxy instead of a hydroxy group at
C-4’ increases the activity. Comparing 1 to 4, methoxy instead of hydroxy groups at both C-7’’ and 4’’’
caused the activity to disappear (IC50 values of 0.44, 0.56, 3.05 and >10 M). Compounds 4-10 showed
no activities. On the basis of these results, it was revealed that the structure of a flavanone-flavone dimer
with a methoxy group at the 4’ position as in 1 and 2 is important for strong BACE-1 inhibitory activities.
It was concluded that structures consisting of flavanone-flavone and a 4’-methoxy on a flavanone moiety
are important basic structures for significant BACE-1 inhibitory activity.
EXPERIMENTAL
Melting points were measured with a Yanaco MP-J3 micro melting point apparatus. IR spectra were
measured as KBr disks with a JASCO IR Report-100 infrared spectrometer. UV spectra were measured
with a HITACHI U-1800 spectrometer. MS spectra were measured with a JEOL JMS-700 spectrometer.
NMR spectra were measured with a JEOL JNM-500 FT NMR spectrometer. Circular dichroism (CD)
measurements were performed under dry N2 with a JASCO-820 spectropolarimeter at 25 °C.
Materials. Compounds 1-3 were isolated from Cephalotaxus harringtonia var. fastigiata.11 Apigenin
(Tokyo Chemical Industry, Japan) was used as Compound 5. Naringenin (LKT Laboratories, USA) was
used as Compound 7. Hesperetin (Tokyo Chemical Industry, Japan) was used as Compound 9.
2,3-Dihydro-6-methylamentoflavone 7,4’,7’’,4’’’-tetramethyl ether (4): 2,3-Dihydro-6-methyl-
ginkgetin (1) (11.0 mg, 18.9 mmol) was dissolved in CH2Cl2/MeOH (6 mL, 5:1 v/v). TMS-diazomethane
as a 0.6 M solution in hexanes (2 mL) was added. After stirring at rt for 12 h, acetic acid was added. The
solution was evaporated and the residue was purified by column chromatography on silica gel
(CHCl3-MeOH) to give 4 (4.4 mg, 34%) as a white solid; mp 85-87 °C; IR 3500 (sh), 2990, 2920, 1770,
1750, 1690, 1670, 1570, 1540, 1490, 1410, 1160 cm-1; UV max () 215 (4.40), 278 (4.13), 324 (3.94)
1
13
nm; CD max () 330 (+1.9), 290 (-5.7), 250 (+0.2), 241 (-0.3), 212 (+6.1) nm; H NMR and C NMR
see Table 1; EIMS m/z 610 (M+, 100), 430 (16); HREIMS 610.1842 (M+, Calcd for C35H30O10,
610.1839).
Apigenin 7,4'-dimethyl ether (6): Apigenin (5) (50.1 mg, 185 mmol) was dissolved in CH2Cl2/MeOH (6
mL; 5:1 v/v). TMS-diazomethane as a 0.6 M solution in hexanes (2 mL) was added. After stirring at rt for
12 h, acetic acid was added. The solution was evaporated and the residue was purified by column
chromatography on silica gel (CHCl3-MeOH) to give 6 (44.5 mg, 81%) as a yellow solid; mp
175-176 °C; IR 3425 (sh), 1670, 1600, 1505, 1440, 1380, 1340, 1270, 1160, 1020, 835 cm-1; UV max

2754
HETEROCYCLES, Vol. 85, No. 11, 2012
() 236 (4.48), 271 (4.37), 322 (3.24) nm; 1H NMR (500 MHz, DMSO-d6):  3.86 (3H, s), 3.87 (3H, s),
6.37 (1H, d, J = 2.3 Hz), 6.78 (1H, d, J = 2.3 Hz), 6.93 (1H, s), 7.11 (2H, d, J = 9.2 Hz), 8.05 (2H, d, J =
9.2 Hz), 12.91 (1H, s); 13C NMR (DMSO-d6):  55.6, 56.0, 92.7, 98.0, 103.7, 104.7, 114.6, 122.7, 128.4,
157.2, 161.2, 162.4, 163.6, 165.2, 182.0; EIMS m/z 298 (M+, 100), 297 (10), 269 (12), 255 (10);
HREIMS 298.0840 (M+, Calcd for C17H14O5, 298.0841).
Naringenin 7,4'-dimethyl ether (8): Naringenin (7) (50.4 mg, 185 mmol) was dissolved in
CH2Cl2/MeOH (6 mL; 5:1 v/v). TMS-diazomethane as a 0.6 M solution in hexanes (2 mL) was added.
After stirring at rt for 12 h, acetic acid was added. The solution was evaporated and the residue was
purified by column chromatography on silica gel (CHCl3-MeOH) to give 8 (42.6 mg, 77%) as a white
solid; mp 117-118 °C; IR 3425 (sh), 1655, 1630, 1555, 1505, 1305, 1210, 1160, 1090, 1030, 830 cm-1;
1
UV max () 237 (4.24), 290 (4.39) nm; H NMR (500 MHz, DMSO-d6):  2.76 (1H, dd, J = 17.2, 2.9
Hz), 3.34 (1H, dd, J = 17.2, 12.6 Hz), 3.77 (3H, s), 3.79 (3H, s), 5.56 (1H, dd, J = 12.6, 2.9 Hz), 6.09 (1H,
d, J = 2.3 Hz), 6.12 (1H, d, J = 2.3 Hz), 6.98 (1H, d, J = 9.2 Hz), 7.45 (1H, d, J = 9.2 Hz), 12.11 (3H, s);
13C NMR (DMSO-d6):  42.0, 55.2, 55.9, 78.4, 93.8, 94.7, 102.6, 113.9, 128.3, 130.4, 159.5, 162.8, 163.2,
167.4, 196.8; EIMS m/z 300 (M+, 100), 299 (47), 134 (74), 121 (40); HREIMS 300.0994 (M+, Calcd for
C17H16O5, 300.0998).
7,3'-Dimethoxy hesperetin (10): Hesperetin (9) (56.0 mg, 185 mmol) was dissolved in CH2Cl2/MeOH (6
mL; 5:1 v/v). TMS-diazomethane as a 0.6 M solution in hexanes (2 mL) was added. After stirring at rt for
12 h, acetic acid was added. The solution was evaporated and the residue was purified by column
chromatography on silica gel (CHCl3-MeOH) to give 10 (30.5 mg, 50%) as a yellow solid; mp
136-137 °C; IR 1680, 1620, 1570, 1355, 1320, 1260, 1210, 1140, 1080, 860 cm-1; UV max () 238
(4.22), 288 (4.50) nm; 1H NMR (500 MHz, DMSO-d6):  2.76 (1H, dd, J = 17.2, 2.9 Hz), 3.39 (1H, dd, J
= 17.2, 12.6 Hz), 3.77 (3H, s), 3.78 (3H, s), 3.79 (3H, s), 5.53 (1H, dd, J = 12.6, 2.9 Hz), 6.09 (1H, d, J =
1.7 Hz), 6.14 (1H, d, J = 1.7 Hz), 6.98 (1H, d, J = 8.0 Hz), 7.04 (1H, dd, J = 8.0, 1.7 Hz), 7.15 (1H, d, J =
13
1.7 Hz), 12.12 (1H, s); C NMR (DMSO-d6):  42.1, 55.5, 55.6, 55.9, 78.7, 93.8, 94.7, 102.6, 110.6,
111.5, 119.3, 130.8, 148.7, 149.1, 162.8, 163.2, 167.4, 196.8; EIMS m/z 330 (M+, 100), 329 (26), 164
(63), 151 (76); HREIMS 330.1096 (M+, Calcd for C18H18O6, 330.1103).
BACE-1 Assay Method. BACE-1 assays were performed on black 384-well plates using a BACE-1
FRET Assay kit (Invitrogen Co., USA). The assay was carried out according to the supplied manual with
modifications.5 Samples were dissolved in the assay buffer (50 mM sodium acetate, pH 4.5) with DMSO
(final concentrations were 10%). 10 L of test samples, 10 L of BACE-1 substrate (750 nM
Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate), and 10 L of BACE-1 enzyme (1.0
U/mL) were mixed in the wells, and incubated 60 min in the dark at 25 °C. The fluorescence intensities of

HETEROCYCLES, Vol. 85, No. 11, 2012
2755
the mixtures were measured by Fluoroskan Ascent (Thermo Scientific) for excitation at 544 nm and
emission at 590 nm. The inhibition ratio was calculated by the following equation: inhibition (%) =
[1-{(S-S0)-(B-B0)/(C-C0)-(B-B0)}]×100, where C was the fluorescence of a control [enzyme, substrate,
and assay buffer concentration with DMSO (final concentrations were 10%)] after 60 min of incubation,
C0 was the initial fluorescence of a control [enzyme, substrate, and assay buffer concentration with
DMSO (final concentrations were 10%)], B was the fluorescence of a control [substrate and assay buffer
concentration with DMSO (final concentrations were 10%)] after 60 min of incubation, B0 was the initial
fluorescence of a control [substrate and assay buffer concentration with DMSO (final concentrations were
10%)], S was the fluorescence of the tested samples (enzyme, sample solution, and substrate) after 60 min
of incubation, and S0 was the initial fluorescence of the tested samples (enzyme, sample solution, and
substrate). To check the quenching effect of the tested samples, the sample solution was added to reaction
mixture C, and any reduction in fluorescence by the sample was investigated. -Secretase Inhibitor
(Wako, Japan) was used as a positive control.
ACKNOWLEDGEMENT
This work was supported in part by a grant from the High-Tech Research Center Project, the Ministry of
Education, Culture, Sports, Science and Technology (MEXT), Japan (No. S0801043).
REFERENCES
1. W.-J. Kwak, C. K. Han, K. H. Son, H. W. Chang, S. S. Kang, B. K. Park, and H. P. Kim, Planta
Med., 2002, 68, 316.
2. C. Ichino, H. Kiyohara, N. Soonthornchareonnon, W. Chuakul, A. Ishiyama, H. Sekiguchi, M.
Namatame, K. Otoguro, S. Omura, and H. Yamada, Planta Med., 2006, 72, 611.
3. K. Miki, T. Nagai, K. Suzuki, R. Tsujimura, K. Koyama, K. Kinoshita, K. Furuhata, H. Yamada, and
K. Takahashi, Bioorg. Med. Chem. Lett., 2007, 17, 772.
4. K. Miki, T. Nagai, T. Nakamura, M. Tuji, K. Koyama, K. Kinoshita, K. Furuhata, H. Yamada, and K.
Takahashi, Heterocycles, 2008, 75, 879.
5. H. Sasaki, K. Miki, K. Kinoshita, K. Koyama, L. D. Juliawaty, S. A. Achmad, E. H. Hakim, M.
Kaneda, and K. Takahashi, Bioorg. Med. Chem. Lett., 2010, 20, 4558.
6. S. Y. Jeon, K. H. Bae, Y. H. Seong, and K. S. Song, Bioorg. Med. Chem. Lett., 2003, 13, 3905.
7. E. M. Hwang, Y. B. Ryu, H. Y. Kim, D.-G. Kim, S.-G. Hong, J. H. Lee, M. J. Curtis-Long, S. H.
Jeong, J.-Y. Park, and K. H. Park, Bioorg. Med. Chem., 2008, 16, 6669.
8. S. Marumoto and M. Miyazawa, Phytother. Res., 2010, 24, 510.
9. J. K. Cho, Y. B. Ryu, M. J. Curtis-Long, J. Y. Kim, D. Kim, S. Lee, W. S. Lee, and K. H. Park,

2756
HETEROCYCLES, Vol. 85, No. 11, 2012
Bioorg. Med. Chem. Lett., 2011, 21, 2945.
10. S. Marumoto and M. Miyazawa, Bioorg. Med. Chem., 2012, 20, 784.
11. H. Sasaki, K. Miki, K. Koyama, K. Kinoshita, and K. Takahashi, Heterocycles, 2008, 75, 939.