Synthesis and evaluation of curcumin derivatives toward an inhibitor of beta-site amyloid precursor protein cleaving enzyme 1

Article abstract of DOI:10.1016/j.bmcl.2013.11.039

To research a new non-peptidyl inhibitor of beta-site amyloid precursor protein cleaving enzyme 1, we focused on the curcumin framework, two phenolic groups combined with an sp₂ carbon spacer for low-molecular and high lipophilicity. The structure-activity relationship study of curcumin derivatives is described. Our results indicate that phenolic hydroxy groups and an alkenyl spacer are important structural factors for the inhibition of beta-site amyloid precursor protein cleaving enzyme 1 and, furthermore, non-competitive inhibition of enzyme activity is anticipated from an inhibitory kinetics experiment and docking simulation.

Full text of DOI:10.1016/j.bmcl.2013.11.039

Bioorganic & Medicinal Chemistry Letters 24 (2014) 685–690
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of curcumin derivatives toward
an inhibitor of beta-site amyloid precursor protein
cleaving enzyme 1
a
a
a
a
b
Hiroyuki Konno a, , Hitoshi Endo , Satomi Ise , Keiki Miyazaki , Hideo Aoki , Akira Sanjoh ,
⇑
Kazuya Kobayashi c, Yasunao Hattori c, Kenichi Akaji c,
a Department of Biochemical Engineering, Graduate School of Science and Technology, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
⇑
b R&D Center, Protein Wave Co., Nara 631-0006, Japan
c Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To research a new non-peptidyl inhibitor of beta-site amyloid precursor protein cleaving enzyme 1, we
focused on the curcumin framework, two phenolic groups combined with an sp2 carbon spacer for low-
molecular and high lipophilicity. The structure–activity relationship study of curcumin derivatives is
described. Our results indicate that phenolic hydroxy groups and an alkenyl spacer are important struc-
tural factors for the inhibition of beta-site amyloid precursor protein cleaving enzyme 1 and, furthermore,
non-competitive inhibition of enzyme activity is anticipated from an inhibitory kinetics experiment and
docking simulation.
Received 16 October 2013
Revised 13 November 2013
Accepted 15 November 2013
Available online 23 November 2013
Keywords:
Curcumin
BACE1
Ó 2013 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
Structure–activity relationship
Alzheimer’s disease (AD)1 is caused by the aggregation of amy-
loid-beta (Ab),2 which is produced from amyloid precursor protein
(APP), a transmembrane protein expressed in many tissues and or-
gans, cleaved by the both b-secretase (b-site amyloid precursor
and/or an unknown mechanism of action for the target enzyme
in exchange for oral bioavailability and brain penetration.
Curcumins11 are components of turmeric, which is consumed as
a curry spice and is especially used in traditional Indian medicine
to treat biliary disorders, anorexia, coughs, etc. around South Asia.
The main ingredient of curcumins is curcumin 1 (1) [1,7-bis(4-hy-
droxy-3-methoxyphenyl)heptane-1,6-diene-3,5-dione], so-called
curcumin, and other compounds exist as curcumin 2 (2) [desmeth-
oxycurcumin] and curcumin 3 (3) [bis-desmethoxycurcumin].
These curcumins form stable enols and are responsible for the yel-
low color. A variety of extensive investigations in past decades
have indicated that curcumin 1 (1) is an antioxidant and anti-
inflammatory and has promising anti-Alzheimer’s disease activ-
ity.12–14
protein cleaving enzyme 1, BACE13) and
c-secretase in the brain.
BACE1 has been recognized as a valuable target for the treatment
of AD. Therefore, BACE1 inhibitors have potential to be developed
as anti-dementia drugs. A variety of inhibitors against BACE1 have
been reported in the literature in past decades, however it has not
been permitted to give BACE1 inhibitors as AD therapeutic agents
to date. Most peptidomimetic inhibitors with potent activity are
promisingly based on the cleaving site of the APP sequence,4–7
but these inhibitors tend to be P-glycoprotein substrates and have
restricted brain penetration. Although non-peptidyl BACE1 inhibi-
tors rationally designed from fragment-based screening techniques
have also been reported by several groups,8,9 the cytotoxicity of the
candidate compounds is frequently a serious problem. In contrast,
natural products are promising bioactive libraries from which
anti-AD agents from microorganisms or food plants have been
isolated,10 although they tend to offer low inhibitory activity
In the course of a research program on BACE1 inhibitors,15 we
focused on the curcumin16,17 framework as non-peptidyl com-
pounds with low-molecular weight and high lipophilicity. Two
crucial structural features of curcumins have been associated with
BACE1 inhibitors: phenolic rings and an alkenyl spacer to join the
two rings.18,19
In the present study, the inhibitory activities of natural curcumins
1, 2 and 3 (1), (2) and (3) were evaluated against recombinant b-site
amyloid precursor protein cleaving enzyme 1 (rBACE1)15 and subse-
quently, the effects of phenolic hydroxy groups, double bonds and
ketone groups were examined. Furthermore, a structure–activity
⇑
Corresponding authors. Tel./fax: +81 (0)238 26 3131 (H.K.).
E-mail addresses: konno@yz.yamagata-u.ac.jp (H. Konno), akaji@mb.
kyoto-phu.ac.jp (K. Akaji).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.039

686
H. Konno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 685–690
relationship study of the aromatic substituents of curcumin deriva-
tives and determination of the inhibition mechanism were at-
tempted. Therefore, 37 curcumin derivatives were prepared from
curcumin 1 (1) or benzaldehyde derivatives and subsequently
screened for their effect on anti-BACE1 activity at a concentration
of 1.3 or 0.67 mM. Then IC50 values of the selected inhibitors were
determined and interactions between the inhibitors and rBACE1
was estimated (Fig. 1).
organic solvents. Although the selected protecting groups were
TBS, MEM, MOM ethers, chemical yields of 4a–4f remained moder-
ate or low because of insolubility in reaction solvents. In addition,
treatment of curcumin 1 (1) with methyl iodide and potassium car-
bonate in acetone afforded
a,a-dimethyl-di-O-methylcurcumin
(4g) in 47% yield. Attempts to deprotect phenolic methyl ether of
4g under several conditions were unsuccessful to give complexed
mixtures. A three step sequence with acetylation of phenolic hy-
To investigate the importance of phenolic hydroxy groups, dou-
ble bonds and keto carbonyl groups, we prepared a variety of cur-
cumin derivatives from curcumin 1 (1). First of all, phenolic
hydroxy groups of curcumin 1 (1) were protected using several
protecting groups because of the insolubility of the curcumins in
droxyl groups/methylation of a,a-carbon on the spacer/deacetyla-
tion afforded 4h in a trace amount (Table 1).
As depicted in Table 2, curcumin derivatives (5a)–(9b) were
prepared by hydrogenolysis of double bonds and reduction of ke-
tones. As the above-mentioned results, using the reductive reac-
tions of curcumin derivatives it was difficult to afford one or
more products in satisfied yield. Although reactions at 0 °C or
À20 °C were attempted, insolubility of the substrates prevented
use of the reactants. In contrast, conjugated alkenyl groups of the
curcumin framework often gave the over-reduced products. In en-
try 1, hydrogenation of curcumin 1 (1) with Pd/C in MeOH/AcOEt at
H2 atmosphere afforded the expected compound (5a) and ketone-
reduced compounds (5b) and (5c) in 69%, 10% and 8% yields,
respectively. On the other hand, treatment of 4c with NaBH4 in
MeOH/AcOEt gave 9a in 30% yield with the recovered material
(4c) in ca. 50% yield (entry 5). Stereochemistry of hydroxyl groups
of these products was not still determined in this case (Table 2).
Figure 1. Structure of curcumins.
Table 1
Protected curcumin derivatives (4a)–(4h)
Entry
1
Conditions
Product
R1
R2
R3
R4
Yield (%)
TBSCl, imidazole, DMF
4a
4b
4c
TBS
TBS
H
H
H
H
H
MEM
H
H
H
H
H
H
H
H
Me2
Me2
45
33
49
32
5
30
47
1
TBS
MEM
H
MEM
MOM
Me
2
MEMCl, iPr2NEt, CH2Cl2
MEM
MEM
MEM
MOM
Me
4d
4e
4f
3
4
5
MOMCl, iPr2NEt, CH2Cl2
MeI, K2CO3, acetone
(a) Ac2O, py.; (b) MeI, K2CO3; (c) 1M NaOH
4ga
4ha
H
H
H
a
4g and 4h were isolated with keto form.
Table 2
Protected curcumin derivatives (5a)–(9b)
Entry
1
Substrate
Conditions
Product
R1
R2
R3
R4
R5
Yield (%)
1
H2/Pd-C
MeOH/AcOEt
5a
5b
5c
6a
H
H
H
TBS
H
H
H
H
H
H
H
H
H
H
H
O
O
OH, H
D3
69
10
8
2
3
4
4b
4c
4g
H2/Pd-C
MeOH/AcOEt
H2/Pd-C
MeOH/AcOEt
H2/Pd-C
MeOH/AcOEt
TBS
O
D3
D3
23
7a
7b
8aa
8b
8c
MEM
MEM
Me
Me
Me
MEM
MEM
Me
Me
Me
H
H
—
H
H
H
H
H
H
Me2
Me2
Me2
H
O
O
O
O
OH, H
OH, H
O
45
25
28
7
3
30
3
5
4c
NaBH4
MeOH/AcOEt
9a
9b
MEM
MEM
MEM
MEM
D1,3
D3
H
a
Chemical structure of 8a was shown diketone derivative.

H. Konno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 685–690
687
For the structure–activity relationship study of aromatic sub-
stituents of curcumin derivatives, we attempted to synthesize cur-
cumin derivatives (22)–(36) by an aldol reaction. As depicted in
Table 3, diketone building blocks (16)–(21) were constructed using
Pabon’s protocols,20,21 although the conventional conditions of the
aldol reaction afforded 3-substituted-2,4-diketone derivatives
mainly. Treatment of benzaldehyde (10) and 2,4-pentanedione
with (EtO)3B/B2O3/piperidine in dioxane gave phenyl diketone
(16) and curcumin 1 (1) in 22% and 8% yields, respectively. The
spectral data (1H NMR, MS) of synthetic curcumin 1 (1) were iden-
tified with those of commercially available curcumin 1 (1). Unfor-
tunately, it is difficult for us to increase the chemical yields using
these several conditions as efficiently as the preparation of 16
and/or 1. Similarly, the aldol reaction using the hydroxy or meth-
oxy substituted benzaldehyde (11)–(15) with 2,4-pentanedione
gave the corresponding diketone detivatives (17)–(21) and sym-
metrical curcumin derivatives (22)–(25) and curcumin 3 (3) in
low yields. As a consequence, the combination of several phenolic
aldehydes with 2,4-pentanedione could be directly converted to
the symmetric curcumin frameworks by this methodology
(Table 3).
Subsequently, we attempted to prepare the asymmetrical cur-
cumin analogues using diketone (16) and (21) and substituted
benzaldehydes under the above-mentioned condition. Diketone
(21) was subjected to a second aldol reaction with benzaldehyde
(11) to give curcumin derivative (26) in 7% yield with abundant
starting material after purification. In spite of the poor yield, the
Pabon’s protocol was employed to prepare asymmetrical curcumin
derivatives because other conditions to perform the aldol reaction
did not isolate the desired products. As a result, asymmetrical cur-
cumin derivatives (26)–(35)22 were prepared for the evaluation of
BACE1 inhibitors (Table 4).
The inhibition of rBACE1 activity was determined by the previ-
ous procedure using a synthetic dodecapeptide with the BACE1
cleavage sequence of the Swedish type as a substrate.23 Commer-
cially available natural curcumins 1, 2 and 3 (1), (2) and (3) and
37 synthetic curcumin analogues from several structural subclasses
were investigated, including hydroxy group-protected curcumin
(4a)–(4h), curcumin-reduced derivatives (5a)–(9b), and symmetri-
cal and asymmetrical curcumin analogues obtained by the
Pabon–aldol reaction (22)–(36). The inhibition potency of curcumin
derivatives was screened for rBACE1 inhibition at 1.3 or 0.67 mM
Table 3
Synthesis of symmetric curcumin derivatives by Pabon’s reaction of benzaldehyde (10)–(15) with 2,4-pentanedione
Entry
Benz-aldehyde
X
Y
Phenyl diketone
Yield (%)
Curcumin derivative
Yield (%)
1
2
3
4
5
6
10
11
12
13
14
15
OMe
H
OMe
H
OH
OH
OH
H
OMe
OH
H
16
17
18
19
20
21
22
17
24
20
17
35
1
8
50
4
10
12
38
22
23
3
24
25
OH
Table 4
Synthesis of curcumin derivatives (26)–(35) by Pabon’s reaction
Entry
1
Diketone
Aldehyde
Product
Yield (%)
Entry
6
Diketone
Aldehyde
Product
Yield (%)
7
21
11
26
7
21
21
31
32
2
21
13
27
10
7
6
3
4
16
21
28
29
21
2
8
9
21
21
33
34
3
4
5
21
30
2
10
21
35
15

688
H. Konno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 685–690
Table 7
IC50 values against rBACE1
concentration and subsequently, the IC50 values were determined
only for the selected compounds. As depicted in Table 5, many com-
pounds showed relatively less potent inhibitory effects of rBACE1
activity by the comparison with naturally curcumin 2 (2). It is sug-
gested that phenolic hydroxyl groups and the planer structure with
an sp2 carbon spacer are essential and, additionally, the water sol-
ubility of compounds is important for this assay. It is noted that
symmetrical curcumins 1 and 3 (1) and (3) showing no inhibitory
activities were particularly interesting (Table 5).
Compound
IC50
M)
mp (°C)
CLogP
(
l
1600
2200
1800
1400
250
NDa 3.676
225–227 2.047
186–189 0.019
156–158 3.230
111–115 2.644
Next, the structure–activity relationship study of phenolic ana-
logues (22)–(35) with an sp2 carbon spacer against rBACE1 was at-
tempted at 0.67 mM concentration of the inhibitors shown in
Table 6. Curcumin derivatives (24), (25), (26), (27), (31) and (32)
with the attachment of multiple hydroxy groups exhibited potent
activity. Nitrogen-containing compounds (33), (34) and (35) also
showed inhibitory activity against rBACE1. In contrast, symmetri-
cal biphenyl-(22) and tetramethoxy-(23) and p-bromo phenyl
derivative (28) were inactive; therefore, non-substituted phenyl
groups and methoxy and p-halogenated groups had a tendency
to decrease inhibitory activities markedly. The results for these
analogues suggest that regio-specific phenolic hydroxy groups
interact site-specifically with the pharmacophore or the periphery
of BACE1 using the hydrogen bond network (Table 6).
Additionally, the inhibitory activities of six selected curcumin
analogues were evaluated based on IC50 values. The IC50 value,
melting point and ClogP of selected inhibitors are summarized in
Table 7. The naturally occurring curcumin 2 (2) control exhibited
an IC50 value of 1.6 mM. Symmetrical tetrahydroxy curcumin ana-
logue (25) and dihydroxy-nitro curcumin analogue (29) showed
IC50 values of 2.2 and 1.8 mM, respectively. Surprisingly ortho-
substituted trihydroxy curcumin derivative (32) showed IC50 calue
450
165–168 3.301
a
ND: no data.
To estimate the inhibitory mechanism, an inhibitory kinetics
experiment with 32 was performed by constructing a Linewe-
aver–Burk plot (Fig. 2). The cleavage rate of different amounts of
substrate [S] by rBACE1 in the absence or presence of 32 (0, 200
of 250
lM. Indole-type compounds (35) inhibited increase rBACE1
with an IC50 value of 450
lM. These results are partly proportional
to the melting points and accordingly, water solubility also seems
to be an important factor to access the target enzyme. However,
there is no correlation between the IC50 value and C log P in this
case (Table 7).
and 250 lM) was monitored during the initial 10–15 min reaction
period using HPLC. The enzymatic reaction rate was obtained by
monitoring the decrease in the area corresponding to the substrate,
and the resulting 1/v was plotted against 1/S. The plots resulted in
three straight lines with the close x-axis intercept reflecting non-
competitive inhibition toward the protease (Fig. 2).
The binding mode of 32 to BACE1 was predicted by docking
simulations using MOE from Ryoka Systems Inc. A cylinder-shaped
space near the P3 pocket fitted with the bis-phenyl group of 32.
Table 5
rBACE1 inhibitory activity of curcumin and its analogues (1)–(9b)
Compound
Inhibitiona
Compound
Inhibitiona
1
2
3
NI
5a
5b
5c
6a
7a
7b
8a
8b
8c
9a
9b
7% (1.3 mM)
7% (1.3 mM)
NI
35% (0.67 mM)
9% (0.67 mM)
22% (1.3 mM)
NI
19% (1.3 mM)
17% (1.3 mM)
36% (1.3 mM)
7% (0.67 mM)
4% (0.67 mM)
6% (0.67 mM)
4a
4b
4c
4d
4e
4f
4g
4h
NI
17% (1.3 mM)
15% (1.3 mM)
5% (0.67 mM)
1% (0.67 mM)
9% (0.67 mM)
NI
17% (1.3 mM)
a
NI: no inhibition.
Table 6
rBACE1 inhibitory activity of synthesized curcumin analogues (22)–(35)
Compound
Inhibitiona (%)
Compound
Inhibitiona (%)
22
23
24
25
26
27
28
1
0
29
30
31
32
33
34
35
18
21
47
100
21
30
71
33
25
26
18
5
Figure 2. (a) Incubated without inhibitor; (b) incubated with 200
incubation of 250 M of 32.
lM of 32; (c)
a
Inhibitory activity was measured by 0.67 mM.
l

H. Konno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 685–690
689
A
B
C
Figure 3. Docking simulation of inhibitor (32) bound to BACE1 (PDB code 2ZHT) using MOE from Ryoka Systems Inc. Molecular graphics image showed using PyMol from
Schrödinger; oxygen (red) and carbon (blue) of 32; Asp32 and Asp228 (purple) of BACE1. (A) Surface mode. (B) Cartoon mode to eliminate the side chains of BACE1. (C) Model
of interaction.
Two polar phenolic hydroxy groups and a ketone of 32 were in-
volved in hydrogen bonding interactions (Gly230 and Glu339).
Inhibitor 32 did not directly interact the active site of two aspartic
acids (Asp32, Asp228) of BACE1, but binding of the substrate of the
P3 pocket partially interfered with the mode of action. Therefore,
non-competitive inhibition of enzyme activity is expected. The cyl-
inder-shaped space near the P3 pocket might be unique and a new
potential target for anti-BACE1 agents (Fig. 3).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11.
039.
References and notes
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The present study indicated that free hydroxy groups in the
place of bis-phenols were preferable for the activity but were not
potently effective compared with those of curcumin 2 (2). In addi-
tion, both ketones and double bonds in the spacer were essential
motifs and the corresponding reductive compounds showed no
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HCl was added to the reaction mixture and extracted with AcOEt. The organic
phase was washed with brine, dried over MgSO4, and evaporated in vacuo. The
residue was purified by chromatography on silica gel (hexane/AcOEt) to give
25 (73.6 mg, 0.216 mmol, 19%) as
a
brown powder. 1H NMR (DMSO-d6,
400 MHz): d = 5.91 (s, 1H), 6.49 (d, 2H, J = 16.0 Hz), 6.76 (d, 2H, J = 8.4 Hz), 6.95
(dd, 2H, J = 8.0, 1.6 Hz), 7.05 (d, 2H, J = 16.0 Hz), 7.46 (d, 2H, J = 16.0 Hz); 13C
NMR (CDCl3, 100 MHz): d = 113.9, 115.2, 120.6, 121.6, 127.2, 140.9, 145.5,
148.1, 183.4; IR (film)
1354, 1286, 1265, 1205, 1115, 976, 822, 739; ESI–HRMS [M+Na]+ calcd for
19H16O6Na 363.0845, found: 363.0869.
m
max cmÀ1: 3485, 3400, 3390, 3100, 1601, 1514, 1442,
C
19. Congreve, M.; Aharony, D.; Albert, J.; Callaghan, O.; Campbell, J.; Carr, R.;
Chessari, G.; Cowan, S.; Edwards, P.; Frederickson, M.; McMenamin, R.; Murray,
C.; Patel, S. J. Med. Chem. 2007, 50, 1124.
20. Pabon, H. J. J. Recl. Trav. Chim. Pays-Bas 1964, 83, 379.
21. Wisut, W.; Nimmannit, U.; Wacharasindhu, S.; Rojsitthisak, P. Molecules 2011,
16, 1888.
22. General procedure for the preparation of the curcumin framework: To a solution of
21 (250 mg, 1.14 mmol) in 1,4-dioxane was added triethyl borate (250 mg,
3.6 mmol) at room temperature. After stirring for 30 min at 85 °C,
23. Measurement of inhibitory activity: Enzyme assays were carried out using
recombinant BACE1 at an enzyme concentration of 120 nM. The reaction
mixture (40 mM AcONa buffer, pH 4.0, containing 10% grecerol, 10 mM DTT
and 4 M NaCl) was analyzed on a Cosmosil 5C18-AR-II column (4.6 Â 150 mm),
employing a linear gradient of MeCN (10–40%, 30 min) in aq 0.1% TFA. Each
IC50 value was obtained from a sigmoidal dose–response curve obtained from
the decrease of the substrate in the reaction mixture. Each experiment was
repeated three times.