Structure-activity relationship of clovamide and its related compounds for the inhibition of amyloid β aggregation

Article abstract of DOI:10.1016/j.bmc.2018.04.044

Alzheimer's disease (AD), a neurodegenerative disorder, is characterized by aggregation of amyloid β-protein (Aβ). Aβ aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids and phenylethanoid glycosides, which possess two or more catechol moieties, strongly inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ aggregation, we synthesized 1 and related compounds 2–11 through reaction between L-DOPA, D-DOPA, L-tyrosine, or L-phenylalanine and caffeic acid, p-coumaric acid, or cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–13, those containing one or two catechol moieties exhibited potent anti-aggregation activity, whereas the non-catechol-type related compounds showed little or no activity. This suggests that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this activity increases depending on the number of catechol moieties. Consequently, clovamide (1) and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated pathology in AD.

Full text of DOI:10.1016/j.bmc.2018.04.044

Accepted Manuscript
Structure–Activity Relationship of Clovamide and Its Related Compounds for
the Inhibition of Amyloid β Aggregation
Tatsuhiko Tsunoda, Mio Takase, Hideyuki Shigemori
PII:
S0968-0896(18)30663-1
https://doi.org/10.1016/j.bmc.2018.04.044
BMC 14327
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2018
18 April 2018
20 April 2018
Please cite this article as: Tsunoda, T., Takase, M., Shigemori, H., Structure–Activity Relationship of Clovamide
and Its Related Compounds for the Inhibition of Amyloid β Aggregation, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.04.044
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Tatsuhiko Tsunoda, Mio Takase, Hideyuki Shigemori*

Bioorganic & Medicinal Chemistry
Structure–Activity Relationship of Clovamide and Its Related Compounds for
the Inhibition of Amyloid β Aggregation
Tatsuhiko Tsunoda,a Mio Takase,a and Hideyuki Shigemorib,*
aGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
bFaculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of
amyloid β-protein (Aβ). A aggregates through β-sheet formation and induces cytotoxicity
against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a
promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids
and phenylethanoid glycosides, which possess two or more catechol moieties, strongly
inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao
beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To
investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ
aggregation, we synthesized 1 and related compounds 2–11 through reaction between L-
DOPA, D-DOPA, L-tyrosine, or L-phenylalanine and caffeic acid, p-coumaric acid, or
cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–
13, those containing one or two catechol moieties exhibited potent anti-aggregation activity,
whereas the non-catechol-type related compounds showed little or no activity. This suggests
that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this
activity increases depending on the number of catechol moieties. Consequently, clovamide (1)
and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated
pathology in AD.
Available online
Keywords:
Alzheimer’s disease
Amyloid aggregation
Clovamide
Structure-activity relationship
Catechol
2018 Elsevier Ltd. All rights reserved.
1. Introduction
respect to Aβ42 aggregation. The aim of this study was to
evaluate the potential role of 1 in the treatment of AD. For this
Many age-related degenerative diseases are characterized by
the accumulation of amyloid deposits derived from a variety of
proteins. Alzheimer’s disease (AD) is the most common
progressive neurodegenerative disorder.1–4 The deposits mainly
consist of 40- and 42-mer amyloid β-proteins (Aβ40 and Aβ42),
which are produced from amyloid β-protein precursor (APP) by
two proteases, β- and γ-secretases.5,6 Aβ42 plays a more
important role in the pathogenesis of AD than Aβ40 because of
its stronger aggregative ability and neurotoxicity.4 Accumulated
evidence shows that Aβoligomers (intermediates of Aβ
aggregates), but not the monomers nor fibrils, induce cognitive
dysfunction and synaptic impairment during AD progression.7,8
Recently, several small molecules were reported to inhibit Aβ-
related pathologies in vitro and in vivo, especially polyphenols,
some of which (resveratrol and epigallocatechin gallate) are
currently in preclinical or clinical trials.9
We previously reported that caffeoylquinic acid derivatives,
contained in coffee beans and sweet potato, and phenylethanoid
glycosides inhibit aggregation of Aβ42. These compounds, which
inhibited Aβ42 aggregation, possessed a catechol moiety as a
common structure.10–13 Clovamide (1), containing two catechol
moieties, was isolated from red clover (Trifolium pratense)14 as a
constituent of cacao liquor (Theobroma cacao).15 Various
effective properties of 1 were reported: antioxidant activity,16
anti-inflammatory properties,17 and neuroprotective effects.18
However, nothing is known about the inhibitory activity with
purpose, we synthesized 1 and related compounds 2–13 and
investigated the structure–activity relationship of 1–13 with
respect to Aβ42 aggregation using thioflavin T (Th-T) assay and
transmission electron microscopy (TEM).
2. Results
2.1. Syntheses of L-clovamide (1) and related compounds 2–
13
L-clovamide (1) has five key structural groups: two phenolic
hydroxyl groups, a carboxyl group, a stereoisomer, and an
unsaturated C-C bond.
L-clovamide (1) and related compounds 2–11 containing
different numbers and structures of catechol moieties were
synthesized in three steps via a modification of a previously
reported method.16 Firstly, L-DOPA, L-tyrosine, and L-
phenylalanine were methylated with SOCl2 in anhydrous MeOH.
Each aromatic amino acid hydrochloride was reacted with
available caffeic acid, p-coumaric acid, and cinnamic acid in
CH2Cl2/DMF to obtain the corresponding amide. Hydrolysis of
methoxy groups with LiOH in THF/H2O afforded clovamide (1)
and related compounds 2–10. Compound 11, the stereoisomer of
1, was synthesized using D-DOPA as a starting material instead
of L-DOPA. However, all hydroxyl groups of 1 were methylated
with MeI and K2CO3 in DMF to afford 12 in 85% yield, and the
unsaturated C-C bond of 1 was reduced with H2 and Pd/C,

Table 1. Effects of compounds 1–13 on Aβ42 aggregation
Compounds
IC50 (µM)a
L-Clovamide (1)
5.7
p-Coumaroyl-DOPA (2)
>100
Cinnamoyl-DOPA (3)
Caffeoyl-Tyrosine (4)
22.7
87.8
>100
>100
72.9
>100
>100
4.9
p-Coumaroyl-Tyrosine (5)
Cinnamoyl-Tyrosine (6)
Caffeoyl-Phenylalanine (7)
p-Coumaroyl-Phenylalanine (8)
Cinnamoyl-Phenylalanine (9)
Clovamide-Me (10)
D-Clovamide (11)
1.6
Clovamide-5Me (12)
>100
Dihydroclovamide (13)
8.7
aIC50 values were calculated from the inhibitory rate (%) of each
concentration of derivatives for Aβ42 aggregation estimated using Th-T
assay at 24 hours.
Scheme 1. Syntheses of compounds 1–13
(a) SOCl2, MeOH, 0℃ → RT, 43–45 h; (b) 2, 4, 5, 6, 7, 8, 9: EDC, HOBt,
DIEA, CH2Cl2/DMF (3:1), RT, 8–17 h / 1, 3, 10: EDC, HOBt, Et3N,
CH2Cl2/DMF (3:1), RT, 9–19 h; (c) LiOH, THF/H2O (2:1), RT, 6 h; (d) MeI,
K2CO3, DMF, RT, 24 h; (e) H2, Pd/C, MeOH, RT, 9 h
providing 13 in 96% yield (Scheme 1)
2.2. Structure–activity relationship of clovamide (1) and
related compounds 2–13 on Aβ42 aggregation
To evaluate the activity of clovamide (1) and related
compounds 2–13, thioflavin T (Th-T) fluorescence assays were
performed for each compound. Compounds 1 (IC50 = 5.7 µM), 3
(IC50 = 22.7 µM), 4 (IC50 = 87.8 µM), 7 (IC50 = 72.9 µM), 10
(IC50 = 4.9 µM), 11 (IC50 = 1.6 µM), and 13 (IC50 = 8.7 µM)
prevented the aggregation of Aβ42 in a dose-dependent manner
(Figs 1 and S1), while compounds 2, 5, 6, 8, 9, and 12 exhibited
little or no activity (Fig. S1). The IC50 values of these compounds
are shown in Table 1, in which the ability to prevent Aβ42
aggregation follows the order: 1 ≈ 10 ≈ 11 ≈ 13 > 3 > 4 ≈ 7 > 2, 5,
6, 8, 9, and 12. In addition, compounds possessing two catechol
moieties such as 1, 10, 11, and 13 exhibited more potent
inhibition of aggregation than those containing only one catechol
moiety. These results demonstrate the significance of the
catechol moiety in the inhibition of Aβ42 aggregation.
To ensure that the compounds inhibited Aβ42 fibril formation,
we observed Aβ42 fibrils directly using TEM (Figs 2 and S2).
Typical Aβ42 fibril formation was observed in the presence of
Aβ42 alone. Compound
2 and non-catechol-type related
compounds 5, 6, 8, 9, and 12 exhibited little or no activity. On
the other hand, Aβ42 fibrils were reduced in the presence of 1, 3,
4, 7, 10, 11, and 13 in a dose-dependent manner. Remarkably, 1,
10, 11, and 13 containing two catechol moieties significantly
reduced Aβ42 fibrils.
Figure 1. Effect of compounds 1, 9, 10, 11, 12, and 13 on aggregation of
Aβ42. Fibril formation of Aβ42 (25 µM ） was monitored by Th-T
fluorescence and presence of 1, 10, and 100 µM of (A) Clovamide (1), (B)
Cinnamoyl-Phenylalanine (9), (C) Clovamide-Me (10), (D) D-Clovamide (11),
(E) Clovamide-5Me, and (F) Dihydroclovamide (13). Fluorescence intensity
was measured at an excitation wavelength 420 nm and emission wavelength
of 485 nm. Values represent the mean ± SD (n = 6).
3. Discussion
In this study, we investigated the effects of clovamide (1) and
related compounds 2–13 on fibril formation (Figs 1 and 2). The
Th-T assay and TEM results showed that at least one catechol
moiety must be present for inhibition of Aβ42 aggregation.
Moreover, the other chemical moieties of 1, a carboxyl group, a
stereoisomer, and an unsaturated C-C bond, did not contribute to
the activity. The activity tends to increase according to the
number of catechol moieties (Table 1). This tendency is
consistent with the results of previous studies showing that
polyphenols possessing multiple catechol moieties exhibit higher
inhibitory activity against Aβ42 aggregation.10, 19 The catechol
moiety easily undergoes autoxidation to form o-quinone.20 Such
covalent modification may destabilize the β-sheet structure in
amyloidogenic polypeptides.21 The reason for 1 and compounds

bonded to the 7-position carbon atom can rotate in a three-
dimensional manner. Since the catechol moiety directly binds to
Aβ42, it can inhibit Aβ42 aggregation. Taken together, it is
possible that a more flexible catechol moiety was involved in
inhibiting Aβ42 aggregation. It is known from the structure–
activity relationships of curcumin and curcumin related
compounds that the presence of aromatic rings and the number of
phenolic hydroxyl groups on aromatic rings affect inhibition of
24
Aβ42 aggregation.23,
Moreover, rosmarinic acid, the amide
isostere of clovamide (1), and its derivatives also indicated the
importance of phenolic hydroxyl groups in inhibiting Aβ42
aggregation.25 Taxifolin, a flavonoid compound containing a
catechol moiety, was reported to inhibit Aβ aggregation, while
dihydrokaempferol, a compound related to taxifolin having a
phenolic hydroxyl group only at the 4'-position, exhibited
relatively low inhibitory activity against Aβ42 aggregation.21
From these reports, the weak activity of 2, despite its catechol
moiety, is presumed to originate from the phenolic hydroxyl
group at the 4'-position. Further structural analysis of clovamide
and related compounds to elucidate the mechanism of inhibitory
activity against Aβ42 aggregation could help to develop more
potent inhibitors for AD therapy.
4. Materials and methods
4.1. General Procedure
NMR spectra were recorded on an AVANCE 500
1
spectrometer (Bruker, USA), operating at 500 MHz for H and
125 MHz for 13C NMR in CD3OD and DMSO-d6. The
resonances at δH 3.35 and δH 2.49 of the residual CD2HOD and
CD3SOCD2H and those at δC 49.0 and δC 39.5 of CD3OD and
1
DMSO-d6 were used as internal references for the H and 13C
NMR spectra, respectively. ESI-MS was performed on a Synapt
G2 mass spectrometer (Waters, USA). Optical rotations were
recorded on a Jasco DIP-1000 digital polarimeter, while UV
Figure 2. Effects of compounds 1, 9, 10, 11, 12, and 13 on Aβ fibrillogenesis
by TEM. The fibril formation was observed after 24 h incubation in 50 mM
PBS buffer. Scale bars: 500 nm. (A) Aβ42 (25 µM) + 1 (100 µM), (B) Aβ42
(25 µM) + 9 (100 µM), (C) Aβ42 (25 µM) + 10 (100 µM), (D) Aβ42 (25 µM)
+ 11 (100 µM), (E) Aβ42 (25 µM) + 12 (100 µM), (F) Aβ42 (25 µM) + 13
(100 µM), and (G) Aβ42 (25 µM)
spectra were recorded on
a
Hitachi double beam
spectrophotometer U-2000A. Syntheses were conducted under a
nitrogen atmosphere.
4.2. Syntheses of clovamide (1) and related compounds 2–13
10, 11, and 13, which contain two catechol moieties, exhibiting
the highest activity could be attributed to the number of
autoxidized catechol moieties. Gazit proposed that blocking of β-
sheet formation by polyphenols could originate from π-π stacking
interactions between Aβ42 and polyphenols.22 The π-orbital of
the aromatic ring of the catechol moiety in 1 would also
contribute to the π-π stacking effects, inhibiting Aβ42
aggregation.
According to the result for 3, the catechol moiety derived
from L-DOPA exhibits a greater contribution than those in 4 and
7, which are derived from caffeic acid, with respect to inhibitory
activity against Aβ42 aggregation (Table 1). This result might be
related to the flexibility of the catechol unit (hybrid orbits of the
catechol moiety bonding carbon atom). Based on the structure–
activity relationship in the inhibitory activity against Aβ42
agglutination of curcumin and curcumin related compounds, it is
known that the length of the linker region and the flexibility of
the substructure are related to the intensity of the inhibitory
activity against Aβ42 aggregation.23 It is relevant to consider the
flexibility of the catechol moiety, which is important in the
expression of activity. The orbital of the 7’-position carbon atom
in the catechol moiety derived from caffeic acid is sp2-hybridized,
while that of the 7-position carbon atom in the catechol moiety
derived from L-DOPA is sp3-hybridized. The catechol moiety
L-DOPA methyl ester hydrochloride (a)
SOCl2 (947 µL, 12.7 mmol) was added to anhydrous MeOH
(18 mL) in an ice bath. After 30 min, L-3,4-
dihydroxyphenylalanine (1.00 g, 5.07 mmol) was added, and the
mixture was stirred at room temperature for 56 h. After removal
of the solvent, the residue was purified by silica gel
chromatography (CHCl3:MeOH = 14:1) to afford L-DOPA
methyl ester hydrochloride (a, 1.03 g, 93%) as a white powder.
L -Tyrosine methyl ester hydrochloride (b)
SOCl2 (906 µL, 12.5 mmol) was added to anhydrous MeOH
(18 mL) in an ice-water bath. After 30 min, L-tyrosine (0.906 g,
5.00 mmol) was added, and the mixture was stirred at room
temperature for 36 h. After removal of the solvent, the residue
was purified by silica gel chromatography (CHCl3:MeOH = 14:1
→
12:1 → 5:1 → 0:1) to afford L-tyrosine methyl ester
hydrochloride (b, 0.876 g, 90%) as a white powder.
L -Phenylalanine methyl ester hydrochloride (c)
SOCl2 (723 µL, 10.0 mmol) was added to anhydrous MeOH
(12 mL) in an ice-water bath. After 30 min, L-phenylalanine

(0.661 g, 4.0 mmol) was added, and the mixture was stirred at
room temperature for 45 h. The residue was evaporated to afford
L-phenylalanine methyl ester hydrochloride (c, 0.867 g, quant.)
as a white powder.
poured into H2O (50 mL), extracted with EtOAc (50 mL × 3),
washed with 5% NaHCO3 (150 mL) and brine (150 mL), dried
over MgSO4, filtered, and the solvent removed in vacuo. The
residue was purified by silica gel column chromatography to
afford p-coumaroyl-DOPA methyl ester (17.1 mg, 7%) as a
yellow powder.
Synthesis of L -clovamide (1)
This crude ester (11.2 mg, 0.03 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (4.0 mg,
0.09 mmol) and stirring at 45°C for 6 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified using Sep-Pak C18 (12 cc, MeOH:H2O
= 40:60 → 100:0) to afford p-coumaroyl-DOPA (2, 5.0 mg,
47%) as a dark brown powder: 1H NMR (CD3OD) δ: 7.40 (1H, d,
J = 15.7 Hz, H-7’), 7.39 (2H, d, J = 8.5 Hz, H-2’, 6’), 6.77 (2H,
d, J = 8.5 Hz, H-3’, 5’), 6.67 (1H, d, J = 2.0 Hz, H-2), 6.65 (1H,
d, J = 8.1 Hz, H-5), 6.55 (1H, dd, J = 8.1, 2.0 Hz, H-6), 6.45 (1H,
d, J = 15.7 Hz, H-8’), 4.65 (1H, m, H-8), 3.08 (1H, dd, J = 13.9,
5.2 Hz, H-7a), 2.93 (1H, dd, J = 13.9, 7.8 Hz, H-7b). 13C NMR
(CD3OD) δ 177.5 (CH, C-3’, 5’), 176.9 (C, C-9), 169.5 (C, C-9’),
161.3 (C, C-4’), 146.9 (C, C-3), 145.9 (C, C-4), 142.9 (CH, C-7’),
131.4 (CH, C-2’, 6’), 131.0 (C, C-1), 128.6 (C, C-1’), 122.6 (CH,
C-6), 119.2 (CH, C-8’), 118.3 (CH, C-2), 117.0 (CH, C-5), 57.0
(CH, C-8), 39.1 (C, C-7). UV λmax (MeOH) nm (ε): 204 (19800),
Caffeic acid (140.7 mg, 0.78 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL), and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide (EDC) (247 µL, 147 mmol) and 1-hydroxy-
benzotriazole (HOBt) (75.4 mg, 0.56 mmol) were added to the
solution. After the mixture was cooled in an ice-water bath for 15
min, Et3N (156 µL, 1.12 mmol) and compound a (138.3 mg, 0.56
mmol) were added to the mixture. After stirring at room
temperature for 19 h, the mixture was poured into H2O (50 mL),
extracted with EtOAc (50 mL × 3), washed with 5% NaHCO3
(150 mL) and brine (150 mL), dried over MgSO4, filtered, and
the solvent removed in vacuo. The residue was purified by silica
gel column chromatography (CHCl3:MeOH = 9:1) to afford
clovamide methyl ester (clovamide-Me, 10, 84.7 mg, 40%) as a
1
yellow powder: H NMR (CD3OD) δ 7.36 (1H, d, J = 15.7 Hz,
H-7’), 6.99 (1H, d, J = 1.9 Hz, H-2), 6.89 (1H, dd, J = 8.2, 1.9
Hz, H-6), 6.75 (1H, d, J = 8.2 Hz, H-5), 6.64 (1H, d, J = 1.9 Hz,
H-2’), 6.67 (1H, d, J = 8.1 Hz, H-5’), 6.53 (1H, dd, J = 8.1, 1.9
Hz, H-6’), 6.41 (1H, d, J = 15.7 Hz, H-8’), 4.69 (1H, m, H-8),
3.68 (3H, s, MeO-9’), 3.02 (1H, dd, J = 13.9, 5.9 Hz, H-7a), 2.93
(1H, dd, J = 13.9, 6.6 Hz, H-7b); 13C NMR (CD3OD) δ 174.6 (C,
C-9), 169.8 (C, C-9’), 149.5 (C, C-4’), 147.3 (C, C-3’), 146.8 (C,
C-3), 145.9 (C, C-4), 143.8 (CH, C-7’), 130.2 (C, C-1), 128.9 (C,
C-1’), 123.1 (CH, C-6’), 122.4 (CH, C-6), 118.4 (CH, C-8’),
118.0 (CH, C-2), 117.2 (CH, C-5), 117.1 (CH, C-5’), 115.9 (CH,
C-2’), 56.5 (CH, C-8), 53.4 (CH3, CO-9) 39.1 (C, C-7); UV λmax
(MeOH) nm (ε): 208 (12400), 220 (12100), 291 (9700), 324
(10700); HR-ESI-MS (negative ion) m/z: 372.1100 [M-H]- (calcd
291 (10800), 309 (10300), 399 (500). HR-ESI-MS (negative ion)
29
m/z: 342.0998 [M-H]- (calcd for C18H16NO6, 342.0978). [α]D
-
10˚ (c = 1.0, MeOH).
Synthesis of Cinnamoyl-DOPA (3)
Cinnamic acid (88.1 mg, 0.59 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL). EDC (105 µL, 0.89 mmol) and
HOBt (80.3 mg, 0.59 mmol) were added to the solution. After the
mixture was cooled in an ice-water bath for 15 min, Et3N (166
µL, 1.18 mmol) and compound a (147.2 mg, 0.59 mmol) were
added to the mixture. After stirring at room temperature for 9 h,
the mixture was poured into H2O (50 mL), extracted with EtOAc
(50 mL × 3), washed with 5% NaHCO3 (150 mL) and brine
(150 mL), dried over MgSO4, filtered, and the solvent removed in
vacuo. The residue was purified by silica gel column
chromatography to afford cinnamoyl-DOPA methyl ester (83.7
mg, 41%) as a yellow powder.
This crude ester (64.3 mg, 0.19 mmol) was dissolved in
THF/H2O (2:1, 2.0 mL) before adding LiOH・H2O (23.7 mg,
0.56 mmol) and stirring at 45°C for 6 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified by silica gel column chromatography
using hexane:EtOAc = 7:3 → 6:4 → 5:5 to remove some non-
polar compounds, then CHCl3:MeOH = 8:2 was used to afford
cinnamoyl-DOPA (3, 20.0 mg, 33%) as a dark brown powder: 1H
NMR (CD3OD) δ: 7.52 (2H, d, J = 8.5 Hz, H-2’, 6’), 7.49 (1H, d,
J = 15.7 Hz, H-7’), 7.35 (3H, m, H-3’, 4’, 5’), 6.69 (1H, d, J =
2.0 Hz, H-2), 6.67 (1H, d, J = 8.0 Hz, H-5), 6.65 (1H, d, J = 15.7
Hz, H-8’), 6.56 (1H, dd, J = 8.0, 2.0 Hz, H-6), 4.72 (1H, m, H-8),
3.10 (1H, dd, J = 14.1, 5.2 Hz, H-7a), 2.93 (1H, dd, J = 14.1, 8.4
Hz, H-7b). 13C NMR (CD3OD) δ: 175.7 (C, C-9), 169.2 (C, C-9’),
147.0 (C, C-3), 146.0 (C, C-4), 143.0 (CH, C-7’), 137.0 (C, C-1’),
131.7 (CH, C-4’), 130.7 (C, C-3’, 5’), 130.6 (C, C-1), 129.7 (CH,
C-2’, 6’), 122.5 (CH, C-6), 122.2 (CH, C-8’), 118.1 (CH, C-5),
117.1 (CH, C-2), 56.4 (CH, C-8), 38.8 (C, C-7). UV λmax
(MeOH) nm (ε): 207 (11500), 220 (11000), 272 (9600). HR-ESI-
MS (negative ion) m/z: 326.1026 [M-H]- (calcd for C18H16NO5,
326.1028). [α]D29 -28˚ (c= 1.0, MeOH).
20
for C19H18NO7, 372.1083); [α]D +20˚ (c= 1.0, MeOH).
This crude ester (60.0 mg, 0.16 mmol) was dissolved in
THF/H2O (2:1, 5.0 mL) before adding LiOH・H2O (16.9 mg,
0.40 mmol) and stirring at 45°C for 9 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified by silica gel column chromatography
using hexane:EtOAc = 1:4 to remove some non-polar compounds,
then CHCl3:MeOH = 2:1 → 1:1 was used to obtain clovamide (1,
1
26.9 mg, 47%) as a pale yellow powder: H NMR (CD3OD) δ:
7.31 (1H, d, J = 15.7 Hz, H-7’), 6.98 (1H, d, J = 1.9 Hz, H-2),
6.88 (1H, dd, J = 8.2, 1.9 Hz, H-6), 6.74 (1H, d, J = 8.2 Hz, H-5),
6.66 (1H, d, J = 2.0 Hz, H-2’), 6.62 (1H, d, J = 8.0 Hz, H-5’),
6.53 (1H, dd, J = 8.0, 2.0 Hz, H-6’), 6.38 (1H, d, J = 15.7 Hz, H-
8’), 4.58 (1H, m, H-8), 3.08 (1H, dd, J = 13.8, 4.9 Hz, H-7a),
2.93 (1H, dd, J = 13.8, 6.6 Hz, H-7b). 13C NMR (CD3OD) δ:
177.5 (C, C-9), 169.5 (C, C-9’), 149.5 (C, C-4’), 147.4 (C, C-3’),
146.8 (C, C-3), 145.8 (C, C-4), 143.2 (CH, C-7’), 131.2 (C, C-1),
129.1 (C, C-1’), 123.0 (CH, C-6’), 122.6 (CH, C-6), 119.1 (CH,
C-8’), 118.3 (CH, C-2), 117.2 (CH, C-5’), 117.0 (CH, C-5),
115.8 (CH, C-2’), 57.4 (CH, C-8), 39.1 (C, C-7). UV λmax
(MeOH) nm (ε): 208 (22100), 290 (14700), 322 (15000). HR-
ESI-MS (negative ion) m/z: 358.0907 [M-H]- (calcd for
30
C18H16NO7, 358.0927). [α]D +23˚ (c = 1.0, MeOH).
Synthesis of p-Coumaroyl-DOPA (2)
EDC (152 µL, 0.86 mmol), p-coumaric acid (141.0 mg, 0.86
mmol), and HOBt (116.1 mg, 0.86 mmol) were dissolved in
CH2Cl2/DMF (1:3, 3.0 mL). The solution was cooled in an ice-
water bath. After 15 min, Et3N (149 µL, 0.86 mmol) and
compound a (129.6 mg, 0.61 mmol) were added to the solution.
After stirring at room temperature for 8 h, the mixture was
Synthesis of Caffeoyl-Tyrosine (4)

Caffeic acid (144.0 mg, 0.80 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL). EDC (142 µL, 0.80 mmol), HOBt
(108.0 mg, 0.80 mmol), DIEA (230 µL, 1.33 mmol), and
compound b (130.0 mg, 0.67 mmol) were added to the solution.
After stirring at room temperature for 10 h, the mixture was
poured into H2O (50 mL), extracted with EtOAc (50 mL × 3),
washed with 5% NaHCO3 (150 mL) and brine (150 mL), dried
over MgSO4, filtered, and the solvent removed in vacuo. The
residue was purified by silica gel column chromatography and
with Sep-Pak C18 to afford caffeoyl-tyrosine methyl ester (26.4
mg, 10%) as a yellow powder.
Cinnamic acid (99.8 mg, 0.67 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL). EDC (119 µL, 0.67 mmol) and
HOBt (91.0 mg, 0.67 mmol) were added to the solution. After the
solution was cooled in an ice-water bath for 15 min, DIEA (194
µL, 1.12 mmol) and compound b (130.0 mg, 0.56 mmol) were
added. After stirring at room temperature for 3 h, the mixture was
poured into H2O (50 mL), extracted with EtOAc (50 mL × 3),
washed with 5% NaHCO3 (150 mL) and brine (150 mL), dried
over MgSO4, filtered, and the solvent removed in vacuo. The
residue was purified with Sep-Pak C18 to afford caffeoyl-
tyrosine methyl ester (35.5 mg, 20%) as a white powder.
This crude ester (24.1 mg, 0.07 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (15.5 mg,
0.37 mmol) and stirring at 45°C for 4.5 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated to
afford cinnamoyl-tyrosine (6, 22.2 mg, 96%) as a white powder:
1H NMR (CD3OD) δ: 7.54 (2H, d, J = 8.0 Hz, H-2’, 6’), 7.49 (1H,
d, J = 15.8 Hz, H-7’), 7.36 (3H, m, H-3’, 4’, 5’), 7.06 (2H, d, J =
8.6 Hz, H-2, 6), 6.77 (2H, d, J = 8.6 Hz, H-3, 5), 6.65 (1H, d, J =
15.8 Hz, H-8’), 4.69 (1H, m, H-8), 3.15 (1H, dd, J = 14.1, 5.2 Hz,
H-7a), 2.93 (1H, dd, J = 14.1, 8.7 Hz, H-7b). 13C NMR (CD3OD)
δ: 175.6 (C, C-9), 169.2 (C, C-9’), 158.1 (C, C-4), 143.1 (CH, C-
7’), 137.0 (C, C-1’), 132.1 (CH, C-2, 6), 131.7 (C, C-1), 130.7
(CH, C-3’, 5’), 129.8 (CH, C-4’), 129.7 (CH, C-2’, 6’), 122.2
(CH, C-8’), 117.0 (CH, C-3, 5), 56.4 (CH, C-8), 38.6 (C, C-7).
UV λmax (MeOH) nm (ε): 203 (16000), 216 (11200), 276 (9300),
392 (300). HR-ESI-MS (negative ion) m/z: 310.1044 [M-H]-
(calcd for C18H16NO4, 310.1079). [α]D29 -24˚ (c = 1.0, MeOH).
This crude ester (9.9 mg, 0.03 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (5.8 mg,
0.14 mmol) and stirring at 45°C for 9 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified using Sep-Pak C18 (0.7 cc, MeOH:H2O
= 3:7 → 5:5 → 1:0) to afford caffeoyl-tyrosine (4, 4.5 mg, 47%)
1
as a brown powder: H NMR (CD3OD) δ: 7.34 (1H, d, J = 15.7
Hz, H-7’), 7.05 (2H, d, J = 8.7 Hz, H-2, 6), 6.99 (1H, d, J = 2.0
Hz, H-2’), 6.89 (1H, dd, J = 8.2, 2.0 Hz, H-6’), 6.77 (2H, d, J =
8.7 Hz, H-3, 5), 6.75 (1H, d, J = 8.2 Hz, H-5’), 6.40 (1H, d, J =
15.7 Hz, H-8’), 4.69 (1H, m, H-8), 3.13(1H, dd, J = 14.1, 5.3 Hz,
H-7a), 2.93 (1H, dd, J = 14.1, 8.4 Hz, H-7b). 13C NMR (CD3OD)
δ: 175.1 (C, C-9), 169.8 (C, C-9’), 158.1 (C, C-4), 149.6 (C, C-
4’), 147.5 (CH, C-3’), 143.6 (CH, C-7’), 132.1 (C, C-2, 6), 130.0
(C, C-1), 129.1 (C, C-1’), 123.0 (CH, C-6’), 118.8 (CH, C-8’),
117.2 (CH, C-5’), 117.0 (CH, C-3, 5), 115.9 (CH, C-2’), 56.5
(CH, C-8), 39.7 (C, C-7). UV λmax (MeOH) nm (ε): 203 (18600),
287 (6200), 294 (6200), 321 (6700); HR-ESI-MS (negative ion)
29
m/z: 342.0980 [M-H]- (calcd for C18H16NO6, 342.0978). [α]D
-
17˚ (c = 1.0, MeOH).
Synthesis of Caffeoyl-Phenylalanine (7)
Synthesis of p-Coumaroyl-Tyrosine (5)
Caffeic acid (156.8 mg, 0.87 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL). EDC (154 µL, 0.87 mmol) and
HOBt (117.6 mg, 0.87 mmol) were added to the solution. After
the solution was cooled in an ice-water bath for 15 min, DIEA
(251 µL, 1.45 mmol) and compound c (130.0 mg, 0.73 mmol)
were added. After stirring at room temperature for 17 h, the
mixture was poured into H2O (50 mL), extracted with EtOAc (50
mL × 3), washed with 5% NaHCO3 (150 mL) and brine (150
mL), dried over MgSO4, filtered, and the solvent removed in
vacuo. The residue was purified by silica gel column
chromatography and with Sep-Pak C18 to afford caffeoyl-
tyrosine methyl ester (12.8 mg, 5%) as a yellow powder.
EDC (170 µL, 0.96 mmol) and HOBt (129.6 mg, 0.96 mmol)
were added to a solution of p-coumaric acid (157.4 mg, 0.96
mmol) in CH2Cl2/DMF (1:3, 3.0 mL). After cooling the solution
in an ice-water bath for 15 min, DIEA (166 µL, 0.96 mmol) and
compound b (156.0 mg, 0.80 mmol) were added. After stirring at
room temperature for 10 h, the mixture was poured into H2O (50
mL), extracted with EtOAc (50 mL × 3), washed with 5%
NaHCO3 (150 mL) and brine (150 mL), dried over MgSO4,
filtered, and the solvent removed in vacuo. The residue was
purified by silica gel column chromatography and with Sep-Pak
C18 to afford caffeoyl-tyrosine methyl ester (54.0 mg, 20%) as a
yellow powder.
This crude ester (12.8 mg, 0.04 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (5.8 mg,
0.19 mmol) and stirring at 45°C for 3 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified using Sep-Pak C18 (6 cc, MeOH:H2O =
5:5 → 1:0) to afford caffeoyl-phenylalanine (7, 4.6 mg, 37%) as
a brown powder: 1H NMR (CD3OD) δ: 7.34 (1H, d, J = 15.7 Hz,
H-7’), 7.23 (5H, m, H-2, 3, 4, 5, 6), 6.98 (1H, d, J = 2.1 Hz, H-
2’), 6.88 (1H, dd, J = 8.7, 2.1 Hz, H-6’), 6.74 (1H, d, J = 8.7 Hz,
H-5’), 6.39 (1H, d, J = 15.7 Hz, H-8’), 4.77 (1H, m, H-8), 3.24
(1H, dd, J = 14.0, 5.2 Hz, H-7a), 3.02 (1H, dd, J = 14.0, 8.8 Hz,
H-7b). 13C NMR (CD3OD) δ: 175.7 (C, C-9), 169.8 (C, C-9’),
143.6 (CH, C-7’), 132.3 (C, C-1), 131.1 (CH, C-3, 5), 130.2 (CH,
C-2, 6), 129.0 (C, C-1’), 128.6 (C, C-4, 4’), 123.0 (CH, C-6’),
118.7 (CH, C-8’), 117.2 (CH, C-5’), 115.9 (CH, C-2’), 56.2 (CH,
C-8), 39.4 (C, C-7). UV λmax (MeOH) nm (ε): 204 (18800), 296
(9800), 323 (10900), 428 (700). HR-ESI-MS (negative ion) m/z:
This crude ester (21.1 mg, 0.06 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (7.8 mg,
0.19 mmol) and stirring at 45°C for 6 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated to
afford p-coumaroyl-tyrosine (5, 20.2 mg, quant.) as a white
1
powder: H NMR (CD3OD) δ: 7.41 (1H, d, J = 15.7 Hz, H-7’),
7.37 (2H, d, J = 8.6 Hz, H-2’, 6’), 7.03 (2H, d, J = 8.6 Hz, H-2,
6), 6.77 (1H, d, J = 8.6 Hz, H-3, 5), 6.70 (1H, d, J = 8.6 Hz, H-3’,
5’), 6.45 (1H, d, J = 15.7 Hz, H-8’), 4.70 (1H, m, H-8), 3.13 (1H,
dd, J = 14.1, 5.3 Hz, H-7a), 2.93 (1H, dd, J = 14.1, 8.5 Hz, H-7b).
13C NMR (CD3OD) δ 175.7 (C, C-9), 169.8 (C, C-9’), 161.4 (C,
C-4), 158.1 (C, C-4’), 143.2 (CH, C-7’), 132.1 (CH, C-2, 6),
131.5 (CH, C-2’, 6’), 129.9 (C, C-1), 128.5 (C, C-1’), 118.7 (CH,
C-8’), 117.5 (CH, C-3’, 5’), 117.0 (CH, C-3, 5), 56.3 (CH, C-8),
38.6 (C, C-7). UV λmax (MeOH) nm (ε): 223 (10200), 293 (7900),
309 (7900), 444 (400). HR-ESI-MS (negative ion) m/z: 326.1026
29
326.1047 [M-H]- (calcd for C18H16NO5, 326.1028). [α]D -20˚ (c
[M-H]- (calcd for C18H16NO5, 326.1028). [α]D -40˚ (c = 1.0,
= 1.0, MeOH).
30
MeOH).
Synthesis of p-Coumaroyl-Phenylalanine (8)
Synthesis of Cinnamoyl-Tyrosine (6)

EDC (128 µL, 0.72 mmol) and HOBt (97.7 mg, 0.72 mmol)
were added to a solution of p-coumaric acid (118.7 mg, 0.72
mmol) in CH2Cl2/DMF (1:3, 3.0 mL). The solution was cooled in
an ice-water bath. After 15 min, DIEA (209 µL, 1.21 mmol) and
compound c (130.0 mg, 0.60 mmol) were added to the solution.
After stirring at room temperature for 8 h, the mixture was
poured into H2O (50 mL), extracted with EtOAc (50 mL × 3),
washed with 5% NaHCO3 (150 mL × 1) and brine (150 mL ×
1), dried over MgSO4, filtered, and the solvent removed in vacuo.
The residue was purified with Sep-Pak C18 to afford caffeoyl-
tyrosine methyl ester (31.4 mg, 16%) as a white powder.
This crude ester (27.1 mg, 0.08 mmol) was dissolved in
THF/H2O (2:1, 0.80 mL) before adding LiOH・H2O (17.5 mg,
0.42 mmol) and stirring at 45°C for 3 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated.
The residue was purified using Sep-Pak C18 (35 cc, MeOH:H2O
= 40:60 → 100:0) to afford p-coumaroyl-phenylalanine (8, 27.2
mg, quant.) as a white powder: 1H NMR (CD3OD) δ: 7.41 (1H, d,
J = 15.7 Hz, H-7’), 7.38 (2H, d, J = 8.6 Hz, H-2’, 6’), 7.23 (6H,
m, H-1, 2, 3, 4, 5, 6), 6.77 (2H, d, J = 8.6 Hz, H-3’, 5’), 6.44 (1H,
d, J = 15.7 Hz, H-8’), 4.77 (1H, m, H-8), 3.24 (1H, dd, J = 14.0,
5.1 Hz, H-7a), 2.93 (1H, dd, J = 14.0, 8.8 Hz, H-7b). 13C NMR
(CD3OD) δ: 175.6 (C, C-9), 169.8 (C, C-9’), 161.4 (C, C-4),
143.3 (CH, C-7’), 139.2 (C, C-1), 131.5 (CH, C-2, 6), 131.1 (CH,
C-3, 5), 130.2 (C, C-2’, 6’), 128.6 (C, C-1’), 128.4 (C, C-4),
118.7 (CH, C-8’), 117.5 (CH, C-3’ 5’), 56.1 (CH, C-8), 39.4 (C,
C-7). UV λmax (MeOH) nm (ε): 203 (15800), 299 (8700), 310
(8600), 404 (500). HR-ESI-MS (negative ion) m/z: 310.1044 [M-
H]- (calcd for C18H16NO4, 342.1079). [α]D30 -37˚ (c = 1.0, MeOH).
mixture was stirred at room temperature for 42 h. After removal
of the solvent, the residue was purified by silica gel column
chromatography (CHCl3:MeOH = 14:1 → 0:1) to afford D-
DOPA methyl ester hydrochloride (316.9 mg, quant.) as a white
powder.
Caffeic acid (143.3 mg, 0.80 mmol) was dissolved in
CH2Cl2/DMF (1:3, 3.0 mL), to which was added EDC (252 µL,
1.42 mmol) and HOBt (76.8 mg, 0.57 mmol). After the mixture
was cooled in an ice-water bath for 15 min, Et3N (158 µL, 1.14
mmol) and D-DOPA methyl ester hydrochloride (120.0 mg, 0.57
mmol) were added. After stirring at room temperature for 12 h,
the mixture was poured into H2O (50 mL), extracted with EtOAc
(50 mL × 3), washed with 5% NaHCO3 (150 mL) and brine
(150 mL), dried over MgSO4, filtered, and the solvent removed in
vacuo. The residue was purified by silica gel column
chromatography (hexane:EtOAc = 1:2 → MeOH) to afford D-
clovamide methyl ester (32.9 mg, 16%) as a yellow powder.
This crude ester (17.2 mg, 0.05 mmol) was dissolved in
THF/H2O (2:1, 2.0 mL) before adding LiOH・H2O (5.8 mg, 0.14
mmol) and stirring at 45°C for 6 h. The mixture was acidified
with Dowex® 50W×8 resin, filtered, and evaporated. The residue
was purified by silica gel column chromatography using
hexane:EtOAc = 3:7 to remove some non-polar compounds,
before using CHCl3:MeOH = 3:7 → 0:1 and Sep-Pak C18 (12 cc,
MeOH:H2O = 1:9 → 1:0) to obtain D-clovamide (11, 3.6 mg,
20%) as a brown powder: 1H NMR (CD3OD) δ: 7.33 (1H, d, J =
15.7 Hz, H-7’), 6.98 (1H, d, J = 1.9 Hz, H-2), 6.88 (1H, dd, J =
8.2, 1.9 Hz, H-6), 6.74 (1H, d, J = 8.2 Hz, H-5), 6.67 (1H, d, J =
1.9 Hz, H-2’), 6.64 (1H, d, J = 8.1 Hz, H-5’), 6.54 (1H, dd, J =
8.1, 1.9 Hz, H-6’), 6.39 (1H, d, J = 15.7 Hz, H-8’), 4.62 (1H, m,
H-8), 3.08 (1H, dd, J = 13.9, 4.8 Hz, H-7a), 2.93 (1H, dd, J =
13.9, 7.5 Hz, H-7b). 13C NMR (CD3OD) δ: 177.5 (C, C-9), 169.4
(C, C-9’), 149.5 (C, C-4’), 147.4 (C, C-3’), 146.8 (C, C-3), 145.8
(C, C-4), 143.1 (CH, C-7’), 131.3 (C, C-1), 129.2 (C, C-1’),
123.0 (CH, C-6’), 122.7 (CH, C-6), 119.3 (CH, C-8’), 118.3 (CH,
C-2), 117.2 (CH, C-5’), 117.0 (CH, C-5), 115.8 (CH, C-2’), 57.6
(CH, C-8), 39.2 (C, C-7). UV λmax (MeOH) nm (ε): 207 (22000),
290 (12300). HR-ESI-MS (negative ion) m/z: 358.0907 [M-H]-
(calcd for C18H16NO7, 358.0927). [α]D30 -23˚ (c = 1.0, MeOH).
Synthesis of Cinnamoyl-Phenylalanine (9)
Cinnamic acid (111.3 mg, 0.75 mmol) was dissolved in 3 mL
CH2Cl2/DMF (1:3, 3.0 mL), to which was added EDC (217 µL,
0.75 mmol) and HOBt (101.5 mg, 0.75 mmol). The solution was
cooled in an ice-water bath. After 15 min, DIEA (217 µL, 1.25
mmol) and compound c (135.0 mg, 0.63 mmol) were added.
After stirring at room temperature for 17 h, the mixture was
poured into H2O (50 mL), extracted with EtOAc (50 mL × 3),
washed with 5% NaHCO3 (150 mL) and brine (150 mL), dried
over MgSO4, filtered, and the solvent removed in vacuo. The
residue was purified by silica gel column chromatography to
afford caffeoyl-tyrosine methyl ester (86.2 mg, 45%) as a white
powder.
Synthesis of clovamide-5Me (12)
L-clovamide (1, 55.6 mg, 0.15 mmol) was dissolved in DMF
(3.0 mL). K2CO3 (213.9 mg, 1.55 mmol) and MeI (96.4 µL, 1.55
mmol) were added to the mixture. After stirring at room
temperature for 24 h, the mixture was poured into H2O (50 mL),
extracted with EtOAc (50 mL × 3), washed with 5% NaHCO3
(150 mL) and brine (150 mL), dried over MgSO4, filtered, and
the solvent removed in vacuo. The residue was purified by silica
gel column chromatography (CHCl3:MeOH = 9:1) to afford
clovamide-5Me (12, 32.9 mg, 16%) as a white powder: 1H NMR
(DMSO-d6) δ: 8.38 (1H, d, J = 7.8 Hz, NH), 7.35 (1H, d, J =
15.8 Hz, H-7’), 7.15 (1H, d, J = 1.9 Hz, H-2), 7.11 (1H, dd, J =
8.4, 1.9 Hz, H-6), 6.98 (1H, d, J = 8.4 Hz, H-5), 6.85 (1H, d, J =
2.2 Hz, H-2’), 6.84 (1H, d, J = 8.1 Hz, H-5’), 6.75 (1H, dd, J =
8.1, 2.2 Hz, H-6’), 6.60 (1H, d, J = 15.8 Hz, H-8’), 4.60 (1H, m,
H-8), 3.79 (3H, s, MeO-3), 3.78 (3H, s, MeO-4), 3.72 (3H, s,
MeO-3’), 3.70 (3H, s, MeO-4’), 3.64 (3H, s, MeO-9’), 2.93 (1H,
dd, J = 13.9, 5.5 Hz, H-7b), 2.88 (1H, dd, J = 13.9, 4.7 Hz, H-
7b). 13C NMR (DMSO-d6) δ: 172.2 (C, C-9), 165.2 (C, C-9’),
150.2 (C, C-4), 148.9 (C, C-3), 148.4 (C, C-3’), 147.5 (C, C-4’),
139.5 (CH, C-7’), 129.5 (C, C-1), 127.5 (C, C-1’), 121.5 (CH, C-
6’), 121.5 (CH, C-6), 119.1 (CH, C-8’), 109.9 (CH, C-2), 111.7
(CH, C-5’), 111.6 (CH, C-5), 112.9 (CH, C-2’), 53.9 (CH, C-8),
36.4 (C, C-7). UV λmax (MeOH) nm (ε): 233 (6200), 287 (5300),
329 (5500), 439 (500). HR-ESI-MS (negative ion) m/z: 428.1693
This crude ester (50.5 mg, 0.16 mmol) was dissolved in
THF/H2O (2:1, 0.8 mL) before adding LiOH・H2O (20.6 mg,
0.49 mmol) and stirring at 45°C for 6 h. The mixture was
acidified with Dowex® 50W×8 resin, filtered, and evaporated to
afford cinnamoyl-phenylalanine (9, 25.3 mg, 53%) as a white
powder: 1H NMR (CD3OD) δ: 7.53 (2H, d, J = 8.6 Hz, H-2’, 6’),
7.48 (1H, d, J = 15.8 Hz, H-7’), 7.36 (3H, m, H-3’, 4’, 5’), 7.24
(5H, m, H-2, 3, 4, 5, 6), 6.63 (1H, d, J = 15.8 Hz, H-8’), 4.79 (1H,
m, H-8), 3.24 (1H, dd, J = 14.0, 5.2 Hz, H-7a), 3.02 (1H, dd, J =
14.0, 8.8 Hz, H-7b). 13C NMR (CD3OD) δ: 175.4 (C, C-9), 169.2
(C, C-9’), 143.1 (CH, C-7’), 139.2 (C, C-1), 137.0 (C, C-1’),
131.1 (CH, C-3, 5), 130.7 (CH, C-3’, 5’), 130.2 (CH, C-2, 6),
128.6 (C, C-4), 122.1 (CH, C-8’), 56.1 (CH, C-8), 39.3 (C, C-7).
UV λmax (MeOH) nm (ε): 216 (23400), 263 (21000). HR-ESI-MS
(negative ion) m/z: 294.1154 [M-H]- (calcd for C18H16NO3,
294.1130). [α]D30 -23˚ (c = 1.0, MeOH).
Synthesis of D-clovamide (11)
SOCl2 (230 µL, 3.17 mmol) was added to anhydrous MeOH
(4.5 mL) in an ice bath. After 30 min, D-3,4-dihydroxy-
phenylalanine (250.0 mg, 1.27 mmol) was added, and the

26
[M-H]- (calcd for C23H26NO7, 428.1709). [α]D +73˚ (c = 1.0,
MeOH).
1.
2.
3.
4.
5.
Hardy, J.; Allsop, D. Trends Pharmacol. Sci. 1991, 12, 383.
Hardy, J., Selkoe, D. J. Science 2002, 297, 353.
Synthesis of Dihydroclovamide (13)
Hamley, I. W. Chem. Rev. 2012 112, 5147.
Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101.
Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald,
B. L.; Beyreuther, K. Proc. Natl. Acad. Sci. USA 1985, 82, 4245.
Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res. Commun. 1984,
120, 885.
L-clovamide (1, 55.6 mg, 0.15 mmol) was dissolved in
anhydrous MeOH (1.5 mL). Pd/C (4.1 mg) was added to the
mixture. After stirring at room temperature for 9 h under a H2
atmosphere, Pd/C was removed with Celite and the filtrate was
evaporated to afford dihydroclovamide (13, 40.0 mg, 96%) as a
6.
7.
8.
9.
Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.;
Wolfe, M. S.; Rowan, M. J.; Selkoe, D. J. Nature 2002, 416, 535.
Roychaudhuri, R.; Yang, M.; Hoshi, M. M.; Teplow, D. B. J. Biol.
Chem. 2009, 284, 4749.
1
brown powder: H NMR (DMSO-d6) δ: 7.74 (1H, d, J = 7.5 Hz,
NH), 6.74 (1H, d, J = 7.9 Hz, H-5), 6.60 (1H, m, H-2) 6.59 (1H,
J = 7.2 Hz, H-5’), 6.57 (1H, m, H-2’), 6.41 (1H, m, H-6’), 6.39
(1H, m, H-6), 4.24 (1H, m, H-8), 2.93 (1H, dd, J = 13.7, 8.3 Hz,
H-7b), 2.87 (1H, dd, J = 13.7, 4.7 Hz, H-7a), 2.53 (1H, t, J = 8.5
Hz, H-7’), 2.26 (1H, t, J = 8.5 Hz, H-8’). 13C NMR (DMSO-d6)
δ: 174.2 (C, C-9), 171.2 (C, C-9’), 145.1 (C, C-4), 144.8 (C, C-
3), 143.6 (C, C-3’), 143.4 (C, C-4’), 132.2 (C, C-1’), 129.2 (C, C-
1), 120.0 (CH, C-6’), 118.7 (CH, C-6), 115.3 (CH, C-2), 115.8
(CH, C-5’), 115.6 (CH, C-5), 116.8 (CH, C-2’), 54.7 (CH, C-8),
36.8 (CH, C-8’), 37.6 (C, C-7), 30.7 (CH, C-7’). UV λmax
(MeOH) nm (ε): 209 (14100), 283 (4100). HR-ESI-MS (negative
ion) m/z: 360.1088 [M-H]- (calcd for C18H18NO7, 360.1083).
[α]D20 +20˚ (c = 1.0, MeOH).
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Acknowledgements
28. Murakami, K.; Murata, N.; Noda, Y.; Tahara, S.; Kaneko, T.;
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We thank Professor Kazuhiro Irie, Associate Professor
Kazuma Murakami, and Dr. Mizuho Hanaki, Graduate School of
Agriculture, Kyoto University for preparing Aβ42. We thank
Professor Masaki Kita, Graduate School of Bioagricultural
Sciences, Nagoya University for kind support of optical rotations.
This work was partially supported by JSPS KAKENHI Grant
Number JP24580156.
Supplementary material
Supplementary material associated with this article can be
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References and notes