Design, synthesis, and evaluation of curcumin analogues as potential inhibitors of bacterial sialidase

Article abstract of DOI:10.1080/14756366.2018.1488695

Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was the most potent inhibitor of S. pneumoniae sialidase (IC₅₀ = 0.2 ± 0.1 μM). This compound exhibited a 3.0-fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin derivatives could be potentially used to treat sepsis by bacterial infections.

Full text of DOI:10.1080/14756366.2018.1488695

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis, and evaluation of curcumin
analogues as potential inhibitors of bacterial
sialidase
Bo Ram Kim, Ji-Young Park, Hyung Jae Jeong, Hyung-Jun Kwon, Su-Jin Park,
In-Chul Lee, Young Bae Ryu & Woo Song Lee
To cite this article: Bo Ram Kim, Ji-Young Park, Hyung Jae Jeong, Hyung-Jun Kwon, Su-Jin
Park, In-Chul Lee, Young Bae Ryu & Woo Song Lee (2018) Design, synthesis, and evaluation of
curcumin analogues as potential inhibitors of bacterial sialidase, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 1256-1265, DOI: 10.1080/14756366.2018.1488695
To link to this article: https://doi.org/10.1080/14756366.2018.1488695
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1256–1265
https://doi.org/10.1080/14756366.2018.1488695
RESEARCH PAPER
Design, synthesis, and evaluation of curcumin analogues as potential inhibitors
of bacterial sialidase
Bo Ram Kima†, Ji-Young Parkb†, Hyung Jae Jeonga, Hyung-Jun Kwonb, Su-Jin Parkb, In-Chul Leeb,
Young Bae Ryub and Woo Song Leeb
aBio-processing Technology Development and Support Team, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Republic of
b
Korea; Natural Product Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received 18 September 2017
Revised 5 June 2018
Accepted 12 June 2018
Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking
receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents
for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase
using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies
have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was
the most potent inhibitor of S. pneumoniae sialidase (IC50 ¼ 0.2 0.1 mM). This compound exhibited a 3.0-
fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These
results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin
derivatives could be potentially used to treat sepsis by bacterial infections.
KEYWORDS
Curcumin; sepsis; sialidase;
Nan A
1. Introduction
in the respiratory tract and sepsis in mouse models7,11,12,33
.
Therefore, high-affinity inhibitors that can block Nan A are poten-
tial agents for prevention and treating sepsis. In the last few years,
several studies have reported the discovery of viral or bacterial
sialidase inhibitors from an isolated natural product such as flavo-
noids, coumarins, diplacone, mimulone, pterocarpans, and phloro-
tannins. However, these compounds are known inhibitors as
Clostridium perfringens (Cp-Nan I) or viral sialidase9,14–17. Recently,
some studies have reported to inhibition of S. pneumoniae sialid-
ase such as diazenylaryl sulphonic acids, malabaricone C,
Artocarpin, and anthraquinone glycosides9,10,18–20. Therefore, to
develop novel bacterial sialidase inhibitors, we focused on the nat-
ural product, Turmeric, because it had not yet been evaluated.
Turmeric has been used as a traditional medicine for conditions
such as liver disease21, indigestion22, rheumatoid arthritis23, and
insect bites24 and is consumed daily by millions of people for the
treatment of various diseases. Curcumin is the primary component
Sepsis, a life-threatening organ dysfunction caused by a dysregu-
lated host response to infection, is caused by an overwhelming
immune response to an existing bacterial infection1. It commonly
occurs in the ageing population and results in ꢀ20–30 million
cases annually worldwide. Overall, sepsis remains one of the top
five causes of death worldwide2, also the mortality rate is
ꢀ20–50% for severe sepsis and 40–80% for septic shock.
Especially, bacterial sepsis is a major cause of mortality of hospi-
talised patients, thus the development of drugs for bacterial sepsis
is urgently needed and many efforts have been undertaken in the
medicinal and pharmaceutical industry.
The Gram-positive bacterium, Streptococcus pneumoniae, is one
of the causes of sepsis. It also major human pathogen and causes
a variety of diseases, including bacterial meningitis, otitis media,
pneumonia, conjunctivitis3–6. Several virulence factors contribute
to colonisation and early infection processes, above all sialidases
from bacteria are considered key virulence factors7. Sialidase
removes the terminal sialic acid residues from host cell surface
glycans, unmasking receptors that facilitate bacterial adherence
and colonisation8. This process causes resistance to penicillin and
other antibiotics that are used to treat S. pneumoniae infection9.
According to known literature, all clinical isolates of S. pneumoniae
have sialidases activity known to be involved in sepsis10. S. pneu-
moniae sialidase genes in clinical pneumococcal isolates deter-
mined that Nan A, Nan B, and Nan C are present in 100%, 96%,
and 51% of these strains. Among these sialidases, Nan A has been
of Turmeric and has
a feruloyl methane group containing
methoxy, hydroxyl, and heptadienyl with a 1,3-diketone moiety.
Curcumin has been extensively studied in the past few decades as
an important therapeutic compound. In addition, it still receives a
lot of attention for its biological properties, including anti-inflam-
matory, anti-viral, anti-bacterial, anti-cancer, anti-oxidant, and anti-
carcinogenic activities25, and its use in debilitating diseases such
as Crohn’s disease, ulcerative colitis26, and Alzheimer’s disease27,28
Therefore, many studies evaluating the biological activity of curcu-
min have been performed and potential curcuminoids have been
.
shown to play an essential role in host–pneumococcal interactions developed for several diseases.
CONTACT Woo Song Lee
wslee@kribb.re.kr; Young Bae Ryu
ybryu@kribb.re.kr
Natural Product Material Research Center, Korea Research Institute of
Bioscience and Biotechnology, Jeongeup 580-185, Republic of Korea
Supplemental data for this article can be accessed here.
†These authors contributed equally to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1257
Scheme 1. Designed strategies for the synthesis of curcumin analogues.
In this study, we report that Turmeric and curcumin derivatives 2.3 Enzyme inhibition activity
can targeting the S. pneumoniae Nan A. Designed strategies for
As described, the inhibitory effects of compounds on S. pneumo-
niae Nan A were measured using a fluorescence (FL)-based assay.
In this assay, the 4-methylumbelliferyl-a-D-N-acetylneuraminic acid
(Sigma Chemical Co., St. Louis, MO) was used as a substrate, and
the enzyme activity was determined by measuring the increase in
fluorescence by continuously monitoring the reactions at 450/
synthesis of curcumin analogues are shown Scheme 1.
2. Materials and methods
2.1 General
40 nm with excitation at 365 nm using
a SpectraMax M2e
All the chemical reagents used in this work and curcumin (4a)
were purchased from commercial suppliers (Aldrich, St. Louis, MO;
TCI, Japan; Alfa Aesar, Haverhill, MA or Acros Organics, USA com-
panies) and used without further purification. The 1H and 13 C
NMR spectra were recorded using a JEOL ECA-500 spectrometer,
Japan at 500 MHz and 125 MHz, respectively, with chemical shift
Multimode Reader (Molecular Devices Co.). The IC50 values of the
synthesised compounds were measured in a reaction mixture con-
taining enzyme (final concentration of Nan A, 2.2 nM), the test
compounds (from 0 to 200 mM), and 50 mM of substrate in 20 mM
Sodium phosphate buffer (pH 7.5, containing 300 mM NaCl). To
determine the enzyme activity, the experimental data were fit to a
(d) values reported in ppm unit. Multiplicities are describes as logistic curve with Equation (1), a time-drive protocol was used
and the initial velocity was recorded over a range of concentra-
tions, and the data were analysed using a nonlinear regression
program (Sigma Plot; SPCC Inc., Chicago, IL).
singlet (s), doublet (d), doublet of doublet (dd), triplet (t), multiplet
(m), and broad exchangeable proton (bs). High resolution mass
spectra were obtained on a GC Mate 2, JEOL. A CEM Discover sys-
tem (No. 908005) fitted with a temperature controller was used
for microwave reactions. Irradiation was initiated at 300 W to raise
the temperature to the set point (150 ꢁC). Reactions were moni-
tored by thin-layer chromatography (TLC) with Merck’s DC-
Fertigiplatterm Kiegel 60 GE254 plates. Visualisation was accom-
plished with either UV light or by immersion in a solution of phos-
phomolybdic acid (PMA) followed by heating on a hot plate for
ꢀ10 s. The reaction products were purified by open column chro-
matography using silica gel produced by Merck (Darmstadt,
Germany) (Silica gel 60; 63–200 mesh, ASTM) or Cosmosil 140 C-18
OPN produced by Nacalai Tesque, Inc., USA.
Â
Ã
ð Þ
%
ð Þ
S
Inhibition activity
¼ 100 –
–ðS0Þ=ðC ꢂC0Þ ꢃ 100; (1)
where C is the fluorescence of the control (enzyme, buffer, and
substrate) after 60 min of incubation, C0 is the fluorescence of the
control at 0 min, S is the fluorescence of the tested samples
(enzyme, sample solution, and substrate) after incubation, and S0
is the fluorescence of the tested samples at 0 min.
2.4 Other sialidases assay
Clostridium perfringens sialidase (Catalogue No. 2876; Sigma) and
Vibrio cholerae sialidase (Catalogue No. 72197; Sigma) activities
were evaluated according to a method described by Lee et al.9
using 4-MUNANA as the substrate, where 10 mL of enzyme solu-
tion was mixed with 20 mL of 0.4 mM MUNANA in 20 mM sodium
acetate buffer at pH 5.5 with 4 mM CaCl2 150 mM NaCl. For the
inhibition studies, C. perfringens sialidase and 0–200 mM of
the individual compounds were mixed with MUNANA at 37 ꢁC.
The production of 4-methylumbelliferone was measured by moni-
toring the fluorometric determination at excitation wavelength
365 nm/emission wavelength 450 nm.
2.2 Expression, purification, and preparation of S. pneumoniae
Nan A
We have synthesised and expressed the full-length genes for the
S. pneumoniae sialidase (Nan A) in Escherichia coli. The gene
encoding Nan A (Figure S28, GenBank accession no. COT45929.1,
PDB: 2VVZ) of S. pneumoniae TIGR4 was synthesised (Thermo
Fisher Scientific GENEART GmbH, Regensburg, Germany). The syn-
thesised gene was inserted into the cloning sites of a pET151/d-
TOPO vector (Invitrogen, Carlsbad, CA) containing a 6x His-tag at
the C-terminus. S. pneumoniae sialidase was expressed and puri-
fied from E. coli BL21 (DE3) (HIT; Real biotech Co., Taipei, Taiwan).
The purified Nan A was detected at ꢀ56.6 kDa with greater than
90% purity using sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) (Figure S29). The purified sialidase had
specific activities (Km values) of 43.9 mM using 4-methylumbelli-
feryl-a-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA;
Catalogue No. M8639; Sigma) as substrate (Figure S30).
2.5. Enzyme kinetic study
The inhibition mechanism was determined, and the apparent
inhibition constants (Ki) for the respective sialidase (Nan A) were
performed on the test compounds, for which the IC50 values
were below 25 mM. The test compounds were studied at three
different concentrations that were chosen based on the IC50
values obtained with each sialidases (ꢀ1/2 ꢄ IC50, IC50, 2 ꢄ IC50).

1258
B. R. KIM ET AL.
The concentrations of marker substrates were chosen (ꢀ1/4 Km,
1/2 Km, Km) with regard to their Michaelis–Menten kinetics. The Ki
values were calculated by nonlinear regression analysis by fitting
different models of enzyme inhibition to the kinetic data using
SigmaPlot Enzyme Kinetics Module 1.3 (SPSS Inc., Chicago, IL). The
inhibition mechanism of the compounds was determined by com-
paring the statistical results, including the Akaike’s information cri-
terion values, of different inhibition models and selecting the one
with the best fit.
7-(3,4-Dimethoxyphenyl)-5-hydroxy-1-(3-hydroxy-4-methox-
yphenyl)hepta-1,4,6-trien-3-one (5b): Yield: 274 mg (56%); dark
orange solid; mp: 155–158 ꢁC (Lit34. 157–158 ꢁC); 1H NMR
(500 MHz, Acetone-d6) d 3.83 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 5.98
(s, 1H), 6.64 (d, 1H, J ¼ 15.5 Hz), 6.73 (d, 1H, J ¼ 15.0 Hz), 6.98 (dd,
2H, J1 ¼ 8.2 Hz, J2 ¼ 1.2 Hz), 7.11 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz),
7.17 (d, 1H, J ¼ 1.5 Hz), 7.21 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz), 7.31
(d, 1H, J ¼ 2.0 Hz), 7.54 (d, 1H, J ¼ 16.0 Hz), 7.58 (d, 1H, J ¼ 16.0 Hz),
7.86 (bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.20, 55.46,
101.1, 110.3, 111.5, 111.6, 113.4, 121.7, 122.0, 122.8, 128.1, 128.5,
140.2, 140.3, 146.9, 149.7, 149.8, 151.6, 183.3. HRMS (EI) m/z: [M]þ
calcd. for C22H22O6 382.1416; Found: 382.1412.
2.6 General procedures for the synthesis of curcumins and the
characterisation of synthesised compounds
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(3-hydroxy-4-
methoxyphenyl)hepta-1,4,6-trien-3-one (5c): Yield: 212 mg
(45%); red orange solid; mp: 137–140 ꢁC; 1H NMR (500 MHz,
Acetone-d6) d 3.87 (s, 3H), 3.89 (s, 3H), 5.96 (s, 1H), 6.63 (d, 1H,
J ¼ 16.0 Hz), 6.69 (d, 1H, J ¼ 15.0 Hz), 6.85 (d, 1H, J ¼ 8.0 Hz), 6.97
(d, 1H, J ¼ 8.5 Hz), 7.10 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 2.5 Hz), 7.15 (dd,
1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz), 7.17 (d, 1H, J ¼ 2.5 Hz), 7.32 (d, 1H,
J ¼ 1.5 Hz), 7.53 (d, 1H, J ¼ 16.0 Hz), 7.57 (d, 1H, J ¼ 16.0 Hz), 7.83
(bs, 1H), 8.18 (bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.44,
101.0, 110.9, 111.4, 113.5, 115.3, 121.4, 121.6, 122.0, 123.1, 140.1,
140.7, 146.8, 146.9, 147.9, 149.1, 149.2, 149.7, 183.3, 183.9. HRMS
(EI) m/z: [M]þ calcd. for C21H20O6 368.1260; Found: 368.1262.
1-(3,4-Dihydroxyphenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphe-
nyl)hepta-1,4,6-trien-3-one (5d): Yield: 100 mg (20%); orange solid;
mp: 160 ꢁC (decomposed); 1H NMR (500 MHz, Acetone-d6) d 3.89
(s, 3H), 5.94 (s, 1H), 6.58 (d, 1H, J ¼ 13.0 Hz), 6.68 (d, 1H,
J ¼ 15.5 Hz), 6.86 (s, 2H), 7.03 (s, 1H), 7.15 (d, 1H, J ¼ 4.0 Hz), 7.31
(s, 1H), 7.51 (d, 1H, J ¼ 12.0 Hz), 7.56 (d, 1H, J ¼ 16.0 Hz); 13 C NMR
(125 MHz, Acetone-d6) d 55.48, 100.9, 110.6, 114.3, 115.3, 115.6,
121.2, 121.4, 121.8, 123.0, 127.3, 127.5, 140.4, 140.5, 145.5, 147.8,
147.9, 149.1, 183.0, 183.2. HRMS (EI) m/z: [M]þ calcd. for C20H18O16
354.1103; Found: 354.1105.
2.6.1 Synthesis of curcumin derivatives using Pabon’s reaction
(compounds 3, 4b, 4e, 5a–5p, 5r, 5s)
Boron trioxide (43.44 mmol) was added to a solution of 2,4-peta-
dione (65.16 mmol for 3, 4) or monophenyl intermediate (3,
21.7 mmol) in ethyl acetate (100 ml) at ambient temperature. After
stirring for 1 h at 90 ꢁC, the corresponding benzaldehydes
(21.7 mmol) and triethyl borate (21.7 mmol) in ethyl acetate were
added to the reaction mixture. The mixture was stirred for 2 h at
90 ꢁC, then n-BuNH2 (21.7 mmol, 1 equiv., 7% solution in ethyl
acetate) was slowly added, and the mixture was stirred at 90 ꢁC
until the aldehydes disappeared on TLC monitoring. The reaction
mixture was then cooled to 50 ꢁC and 1 M HCl (aq.) was added.
After stirring the mixture for an additional 1 h and cooling to
room temperature, water (30 ml) and ethyl acetate (20 ml) were
added. The reaction mixture was washed with water and brine
until it was neutralised. The organic phase was dried over anhyd-
rous MgSO4 and evaporated under reduced pressure. The residue
was purified using open-bed column chromatography on silica gel
or Cosmosil 140 C–18 OPN to yielded curcumin derivatives.
5-Hydroxy-1,7-bis(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-
trien-3-one (4b): Yield: 556 mg (23%); orange solid; mp:
182–183 ꢁC (Lit34. 181–183 ꢁC); 1H NMR (500 MHz, Acetone-d6) d
3.83 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 5.98 (s, 1H), 6.64 (d, 1H,
J ¼ 15.5 Hz), 6.73 (d, 1H, J ¼ 15.0 Hz), 6.97 (d, 1H, J ¼ 1.5 Hz), 6.99
(d, 1H, J ¼ 1.5 Hz), 7.11 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz), 7.17 (d,
1H, J ¼ 1.5 Hz), 7.21 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz), 7.31 (d, 1H,
J ¼ 2.0 Hz); 13 C NMR (125 MHz, Acetone-d6) d 55.28, 55.46, 101.1,
110.3, 111.5, 111.6, 113.4, 121.7, 122.0, 122.8, 128.1, 128.5, 140.2,
140.3, 146.9, 149.7, 149.8, 151.6, 100.3, 114.1, 115.5, 183.3. HRMS
(EI) m/z: [M]þ calcd. for C21H20O6 368.1260; Found: 368.1261.
5-Hydroxy-1,7-bis(4-hydroxyphenyl)hepta-1,4,6-trien-3-one
(4e): Yield: 378 mg (15%); orange solid; mp: 234–235 ꢁC (Lit35.
1-(3,4-Dihydroxyphenyl)-5-hydroxy-7-(3-hydroxy-4-methoxyphe-
nyl)hepta-1,4,6-trien-3-one (5e): Yield: 127 mg (28%); brown solid;
1
mp: 203–204 ꢁC; H NMR (500 MHz, Acetone-d6) d 3.87 (s, 3H), 5.97
(s, 1H), 6.58 (d, 1H, J ¼ 15.5 Hz), 6.64 (d, 1H, J ¼ 16.0 Hz), 6.85 (s,
1H), 6.97 (d, 1H, J ¼ 7.5 Hz), 7.04 (s, 1H), 7.11 (d, 1H, J ¼ 7.0 Hz),
7.18 (s, 2H), 7.51 (d, 1H, J ¼ 15.0 Hz), 7.52 (d, 1H, J ¼ 16.0 Hz); 13 C
NMR (125 MHz, Acetone-d6) d 55.46, 101.0, 111.5, 113.4, 115.5,
121.2, 121.6, 121.8, 122.0, 127.4, 128.5, 140.0, 140.6, 145.5, 146.9,
147.8, 149.7, 182.7, 183.6. HRMS (EI) m/z: [M]þ calcd. for C20H18O6
354.1103; Found: 354.1104.
1-(3-Ethoxy-4-hydroxyphenyl)-5-hydroxy-7-(4-hydroxy-3-
methoxyphenyl)hepta-1,4,6-trien-3-one (5f): Yield: 284 mg
(58%); yellow solid; mp: 182–183 ꢁC; 1H NMR (500 MHz, Acetone-
d6) d 1.37 (t, 3H, J ¼ 7.0 Hz) , 3.89 (s, 3H), 4.15 (m, 2H), 5.94 (s, 1H),
6.66 (d, 1H, J ¼ 2.5 Hz), 6.69 (d, 1H, J ¼ 3.0 Hz), 6.85 (d, 2H,
J ¼ 8.5 Hz), 7.15 (dd, 2H, J1 ¼ 10.0 Hz, J2 ¼ 2.5 Hz), 7.30 (dd, 2H, J1
1
232–233 ꢁC); H NMR (500 MHz, Acetone-d6) d 5.95 (s, 1H), 6.63 (s,
1H), 6.66 (s, 1H), 6.86–6.89 (m, 4H), 7.53 (s, 2H), 7.55 (s, 2H), 7.56
(s, 1H), 7.59 (s, 1H), 8.99 (bs, 2H); 13 C NMR (125 MHz, Acetone-d6)
d 100.9, 115.9, 121.2, 126.9, 130.1, 140.2, 159.6, 183.7. HRMS (EI)
m/z: [M]þ calcd. for C19H16O4 308.1049; Found: 308.1047.
¼
7.0 Hz, J2 ¼ 1.5 Hz), 7.54 (d, 1H, J ¼ 3.0 Hz), 7.58 (d, 1H,
J ¼ 3.0 Hz), 8.21 (d, 1H, J ¼ 10.5 Hz); 13 C NMR (125 MHz, Acetone-
d6) d 14.64, 55.93, 64.77, 101.3, 111.1, 112.1, 115.8, 121.9, 121.8,
123.3(4), 123.3(6), 123.4(3), 123.4(6), 127.7(4), 127.7(9), 141.0(0),
141.0(8), 147.5, 148.3, 149.5, 149.7, 184.0. HRMS (EI) m/z: [M]þ
calcd. for C22H22O6 382.1416; Found: 382.1417.
7-(3,4-Dimethoxyphenyl)-5-hydroxy-1-(4-hydroxy-3-methox-
yphenyl)hepta-1,4,6-trien-3-one (5a): Yield: 245 mg (50%); dark
orange solid; mp: 136–137 ꢁC; 1H NMR (500 MHz, Acetone-d6) d
3.83 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 5.95 (s, 1H), 6.70 (t, 2H,
J ¼ 6.7 Hz), 6.86 (d, 1H, J ¼ 8.0 Hz), 6.97 (d, 1H, J ¼ 8.0 Hz), 7.15 (dd,
1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz), 7.20 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz),
7.30 (dd, 2H, J1 ¼ 4.7 Hz, J2 ¼ 1.2 Hz), 7.57 (d, 2H, J ¼ 16.0 Hz), 8.25
(bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.27, 55.45, 100.9,
110.3, 110.6, 111.6, 122.0, 122.7, 123.0, 128.1, 140.2, 140.7, 147.9,
149.2, 149.7, 151.6, 183.3, 183.9. HRMS (EI) m/z: [M]þ calcd. for
C22H22O6 382.1416; Found: 382.1416.
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(pyridin-3-yl)
hepta-1,4,6-trien-3-one (5g): Yield: 33 mg (8%); orange solid;
mp: 185 ꢁC (decomposed); 1H NMR (500 MHz, DMSO-d6) d 3.80 (s,
3H), 6.12 (s, 1H), 6.81–6.84 (m, 2H), 7.15 (d, 1H, J ¼ 8.0 Hz), 7.22 (d,
1H, J ¼ 16.0 Hz), 7.32 (s, 1H), 7.59 (d, 1H, J ¼ 16.0 Hz), 7.66 (d, 1H,
J ¼ 16.0 Hz), 7.89 (d, 1H, J ¼ 4.5 Hz), 8.62–8.69 (m, 1H), 8.77 (d,
1H, J ¼ 3.5 Hz), 9.13 (s, 1H), 9.80 (bs, 1H); 13 C NMR (125 MHz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1259
DMSO-d6) d 56.23, 102.9, 111.9, 116.2, 121.6, 124.3, 126.5, 126.7, (m, 2H), 7.17 (d, 1H, J ¼ 7.5 Hz), 7.33 (s, 1H), 7.56–7.63 (m, 6H), 8.28
129.2, 133.8, 141.0, 143.1, 144.4, 144.8, 148.5, 150.4, 179.2, 187.2. (bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.48, 110.7, 115.3,
HRMS (EI) m/z: [M]þ calcd. for C19H17NO4 323.1158; Found: 323.1156. 110.7, 115.3, 121.5, 123.3, 123.5, 125.1, 127.2, 129.8, 132.1, 134.6,
138.0, 141.4, 147.9, 149.3, 180.7, 185.1. HRMS (EI) m/z: [M]þ calcd.
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhepta-
1,4,6-trien-3-one (5h): Yield: 45 mg (11%); red brown solid; mp: for C20H17BrO4 400.0310; Found: 430.0309.
1
135–137 ꢁC (Lit36. 137–140); H NMR (500 MHz, Acetone-d6) d 3.90
N-(4–(5-Hydroxy-7–(4-hydroxy-3-methoxyphenyl)-3-oxo-
(s, 3H), 6.02 (s, 1H), 6.83 (d, 1H, J ¼ 21.5 Hz), 6.86 (d, 1H, hepta-1,4,6-trien-1-yl)phenyl)acetamide (5n): Yield: 281 mg
J ¼ 13.0 Hz), 7.17 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 1.7 Hz), 7.33 (d, 1H, (58%); yellow solid; mp: 183–185 ꢁC; 1H NMR (500 MHz,
J ¼ 2.5 Hz), 7.38–7.43 (m, 3H), 7.52 (d, 1H, J ¼ 16.0 Hz), 7.63 (d, 1H, Acetone-d6) d 2.07 (s, 3H), 3.89 (s, 3H), 5.98 (s, 1H), 6.69 (d, 1H,
J ¼ 8.5 Hz), 7.65 (d, 1H, J ¼ 1.5 Hz), 7.67 (s, 1H), 8.18 (bs, 1H); 13 C J ¼ 10.5 Hz), 6.73 (d, 1H, J ¼ 10.5 Hz), 6.86 (d, 1H, J ¼ 8.5 Hz),
NMR (125 MHz, Acetone-d6) d 55.48, 101.3, 110.7, 115.4, 121.4, 7.16 (dd, 1H, J1 ¼ 7.7 Hz, J2 ¼ 1.7 Hz), 7.32 (d, 1H, J ¼ 2.5 Hz),
123.2, 124.3, 127.2, 128.1, 129.0, 130.0, 135.3, 139.6, 141.2, 147.9, 7.57–7.61 (m, 4H), 7.68 (s, 1H), 7.70 (s, 1H), 8.15 (bs, 1H); 13 C
149.3, 182.1. HRMS (EI) m/z: [M]þ calcd. for C20H18O4 322.1205; NMR (125 MHz, Acetone-d6) d 23.58, 23.62, 55.56, 101.2, 110.7,
Found: 322.1205.
115.4, 119.1, 119.2, 121.5, 122.7, 123.2, 127.3, 129.0, 130.1,
Methyl 4-(4-(5-hydroxy-7–(4-hydroxy-3-methoxyphenyl)-3- 139.6, 141.0, 141.4, 141.5, 148.0, 149.3, 168.3, 168.4, 182.8,
oxohepta-1,4,6-trien-1-yl)-2-methoxyphenoxy)-butanoate (5i): 184.3. HRMS (EI) m/z: [M]þ calcd. for C22H21NO5 379.1420;
Yield: 48 mg (8%); yellow solid; mp: 100–101 ꢁC; 1H NMR Found: 379.1419.
(500 MHz, Acetone-d6) d 2.04–2.09 (m, 2H), 2.51 (t, 2H, J ¼ 7.2 Hz),
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxy-
3.61 (s, 3H), 3.85 (s, 3H), 3.88 (s, 3H), 4.07 (t, 2H, J ¼ 6.0 Hz), 5.96 phenyl)hepta-1,4,6-trien-3-one (5o): Yield: 320 mg (66%); yel-
(s, 1H), 6.70 (t, 2H, J ¼ 15.7 Hz), 6.85 (d, 1H, J ¼ 8.0 Hz), 6.97 (d, low solid; mp: 143–145 ꢁC; 1H NMR (500 MHz, Acetone-d6) d 3.01
1H, J ¼ 7.5 Hz), 7.15 (d, 1H, J ¼ 9.0 Hz), 7.19 (d, 1H, J ¼ 8.0 Hz), (s, 6H), 3.89 (s, 3H), 5.90 (s, 1H), 6.55 (d, 1H, J ¼ 15.0 Hz), 6.67 (d,
7.30 (s, 2H), 7.57 (d, 1H, J ¼ 16.0 Hz), 8.29 (bs, 1H); 13 C NMR 1H, J ¼ 16.0 Hz), 6.73 (d, 2H, J ¼ 9.0 Hz), 6.85 (d, 1H, J ¼ 7.5 Hz), 7.14
(125 MHz, Acetone-d6) d 24.58, 50.87, 55.37, 55.46, 67.58, 100.9, (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz), 7.31 (d, 1H, J ¼ 2.0 Hz), 7.50 (d,
110.6(6), 110.6(9), 112.9, 115.4, 121.4, 122.0, 122.7, 123.0, 127.2, 1H, J ¼ 9.0 Hz), 7.55 (d, 1H, J ¼ 1.5 Hz), 7.58 (s, 1H), 8.19 (bs, 1H);
128.3, 140.2, 140.7, 147.9, 149.3, 149.9, 150.7, 172.9, 183.2, 184.0. 13 C NMR (125 MHz, Acetone-d6) d 54.96, 55.46, 101.0, 110.6, 114.4,
HRMS (EI) m/z: [M]þ calcd. for C26H28O8 468.1784; 115.4, 121.4, 121.9, 123.1, 127.3, 127.8, 129.9, 139.8, 140.7, 147.9,
Found: 468.1784.
149.2, 161.5, 183.3, 184.0. HRMS (EI) m/z: [M]þ calcd. for C21H20O5
352.1311; Found: 352.1314.
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-nitrophe-
nyl)hepta-1,4,6-trien-3-one (5j): Yield: 249 mg (53%); orange
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxy-
1
solid; mp: 204–205 ꢁC; H NMR (500 MHz, Acetone-d6) d 3.89 (s, 3H), phenyl)hepta-1,4,6-trien-3-one (5p): Yield: 281 mg (65%);
6.10 (s, 1H), 6.76 (d, 1H, J¼ 16.0 Hz), 6.87 (d, 1H, J¼ 8.5 Hz), 7.05 (d, orange solid; mp: 169–170 ꢁC (Lit37. 170–172 ꢁC); 1H NMR
1H, J¼ 16.0 Hz), 7.19 (dd, 2H, J1 ¼ 8.2 Hz, J2 ¼ 2.2 Hz), 7.35 (d, 1H, (500 MHz, Acetone-d6) d 3.89 (s, 3H), 5.94 (s, 1H), 6.64 (d, 1H,
J¼ 1.5 Hz), 7.65 (d, 1H, J¼ 16.0 Hz), 7.69 (d, 1H, J¼ 16.0 Hz), 7.93–7.96 J ¼ 16.0 Hz), 6.69 (d, 1H, J ¼ 15.5 Hz), 6.84–6.88 (m, 3H), 7.15
(m, 2H), 8.25–8.28 (m, 2H), 8.31 (bs, 1H); 13 C NMR (125 MHz, Acetone- (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 2.5 Hz), 7.31 (d, 1H, J ¼ 1.5 Hz),
d6) d 55.48, 102.2, 110.7, 115.3, 121.6, 123.5, 124.0, 127.0, 128.2, 7.53–7.56 (m, 3H), 7.59 (d, 1H, J ¼ 3.5 Hz), 8.21 (bs, 1H); 13 C
128.9, 136.3, 141.7, 142.1, 149.9, 148.2, 149.5, 178.8, 186.6. HRMS (EI) NMR (125 MHz, Acetone-d6) d 55.45, 101.9, 110.5, 115.4, 115.9,
m/z: [M]þ calcd. for C20H17NO6 367.1056; Found: 367.1056.
121.2, 121.4, 123.0, 126.8, 127.3, 130.1, 140.2, 140.5, 147.9,
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-(trifluoro- 149.2, 159.7, 183.6. HRMS (EI) m/z: [M]þ calcd. for C20H18O5
methyl)phenyl)hepta-1,4,6-trien-3-one (5k): Yield: 114 mg 338.1154; Found: 338.1159.
(23%); yellow solid; mp: 158–160 ꢁC; 1H NMR (500 MHz, Acetone-
1-(4-(Dimethylamino)phenyl)-5-hydroxy-7-(4-hydroxy-3-
d6) d 3.89 (s, 3H), 6.07 (s, 1H), 6.75 (d, 1H, J ¼ 16.5 Hz), 6.86 (d, 1H, methoxyphenyl)hepta-1,4,6-trien-3-one (5r): Yield: 220 mg
J ¼ 7.5 Hz), 6.98 (d, 1H, J ¼ 16.0 Hz), 7.18 (dd, 1H, J1 ¼ 8.0 Hz, (48%); red solid; mp: 174–175 ꢁC; 1H NMR (500 MHz, Acetone-
J2 ¼ 1.5 Hz), 7.34 (d, 1H, J ¼ 1.5 Hz), 7.63 (d, 1H, J ¼ 10.5 Hz), 7.66 d6) d 3.01 (s, 6H), 3.89 (s, 3H), 5.90 (s, 1H), 6.55 (d, 1H,
(d, 1H, J ¼ 10.5 Hz), 7.75 (d, 1H, J ¼ 7.5 Hz), 7.89 (d, 1H, J ¼ 7.5 Hz), J ¼ 15.0 Hz), 6.67 (d, 1H, J ¼ 16.0 Hz), 6.73 (d, 2H, J ¼ 9.0 Hz),
8.35 (bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.51, 101.8, 6.82 (d, 1H, J ¼ 7.5 Hz), 7.14 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz),
110.8, 115.3, 121.5, 123.4, 125.8, 126.9, 127.1, 128.5, 130.4, 137.3, 7.31 (d, 1H, J ¼ 2.0 Hz), 7.50 (d, 2H, J ¼ 9.0 Hz), 7.55 (d, 1H,
139.2, 141.8, 147.9, 149.4, 179.7, 185.9. HRMS (EI) m/z: [M]þ calcd. J ¼ 1.5 Hz), 7.58 (s, 1H), 8.19 (bs, 1H); 13 C NMR (125 MHz,
for C21H17F3O4 390.1079; Found: 390.1080.
Acetone-d6) d 39.36, 55.52, 100.8, 110.6, 111.9, 115.3, 118.7,
1-(4-Fluorophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphe-
121.5, 122.8, 127.4, 130.0, 139.8, 141.3, 147.8, 148.8, 152.0,
nyl)hepta-1,4,6-trien-3-one (5l): Yield: 231 mg (53%); orange 182.0, 184.4. HRMS (EI) m/z: [M]þ calcd. for C22H23NO4
solid; mp: 146–147 ꢁC; 1H NMR (500 MHz, Acetone-d6) d 3.89 (s, 365.1627; Found: 365.1624.
3H), 6.00 (s, 1H), 6.72 (d, 1H, J ¼ 16.0 Hz), 6.79 (d, 1H, J ¼ 16.0 Hz),
6.86 (d, 1H, J ¼ 8.5 Hz), 7.16–7.20 (m, 3H), 7.33 (d, 1H, J ¼ 2.5 Hz),
7.59 (d, 1H, J ¼ 3.0 Hz), 7.62 (d, 1H, J ¼ 3.5 Hz), 7.73 (d, 1H,
5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-(piperidin-
1-yl)phenyl)hepta-1,4,6-trien-3-one (5s): Yield: 56 mg (12%);
red solid; mp: 204–205 ꢁC; 1H NMR (500 MHz, Acetone-d6) d
J ¼ 5.0 Hz), 7.74 (d, 1H, J ¼ 5.0 Hz), 8.27 (bs, 1H); 13 C NMR 1.61–1.63 (m, 6H), 3.22–3.31 (m, 4H), 3.89 (s, 3H), 5.92 (s, 1H),
6.58 (d, 1H, J ¼ 15.5 Hz), 6.67 (d, 1H, J ¼ 15.5 Hz), 6.85 (d, 1H,
(125 MHz, Acetone-d6) d 55.48, 110.7, 115.3, 115.8, 116.0, 121.4,
J ¼ 8.0 Hz), 6.93 (d, 2H, J ¼ 9.5 Hz), 7.14 (dd, 1H, J1 ¼ 8.2 Hz,
123.2, 124.2, 127.2, 130.1, 130.2, 130.3, 131.8, 138.2, 141.2, 147.9,
149.2, 162.7, 164.6, 181.4, 184.5. HRMS (EI) m/z: [M]þ calcd. for J2 ¼ 1.7 Hz), 7.31 (d, 1H, J ¼ 2.0 Hz), 7.50 (d, 2H, J ¼ 9.5 Hz), 7.54
(d, 1H, J ¼ 5.0 Hz), 7.57 (d, 1H, J ¼ 4.5 Hz), 8.19 (bs, 1H); 13 C NMR
C20H17FO4 340.1111; Found: 340.1115.
1-(4-Bromophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphe- (125 MHz, Acetone-d6) d 24.22, 25.36, 48.70, 55.46, 100.81, 110.58,
nyl)hepta-1,4,6-trien-3-one (5m): Yield: 164 mg (32%); yellow 114.7, 115.3, 119.7, 121.5, 122.9, 124.3, 127.4, 129.8, 140.1, 140.7,
solid; mp: 148–149 ꢁC; 1H NMR (500 MHz, Acetone-d6) d 3.89 (s, 147.9, 149.1, 153.0, 182.9, 184.3. HRMS (EI) m/z: [M]þ calcd. for
3H), 6.02 (s, 1H), 6.73 (dd, 1H, J1 ¼ 16.2 Hz, J2 ¼ 2.2 Hz), 6.85–6.88
C25H27NO4 405.1940; Found: 405.1943.

1260
B. R. KIM ET AL.
2.6.2 Synthesis of 1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5- The reaction mixture was evaporated under reduced pressure, then
purified further by silica gel column chromatography (n-hexane:
ethyl acetate: MeOH ¼60:38:2 (v/v)) to give 2-(3-hydroxy-4-methoxy-
benzylidene)-6-(3-(3-hydroxy-4-methoxyphenyl)acryloyl)cyclohexan-
1-one (4f, 390 mg, 45%) as an orange red solid.
dione (4c)
Curcumin (1.0 g, 2.71 mmol) was dissolved in dry acetone (30 ml)
then anhydrous K2CO3 (1.12 g, 8.14 mmol) and CH3I (1.69 ml,
27.14 mmol) were added. The reaction mixture was refluxed for
24h and monitored by TLC. The reaction mixture was then cooled
to ambient temperature and filtrated. The resulting filtrate was
evaporated under reduced pressure and ethyl acetate (30ml) and
water (20ml) were added. The organic layer was washed with water
and brine, and then dried over anhydrous MgSO4. After evaporating
under reduced pressure, the residue was purified using a Cosmosil
140 C-18 OPN column (CH3CN:H2O ¼ 3:2 (v/v)) to yield 1,7-bis(3, 4-
dimethoxyphenyl)hepta-1,6-diene-3,5-dione (4c, 150 mg, 14%) and
1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-
diene-3,5-dione (5a, 210mg, 20%) as orange solids.
2–(1-Hydroxy-3-(4-hydroxy-3-methoxyphenyl)allylidene)-6-
(4-hydroxy-3-methoxybenzylidene)cyclohexan-1-one (4f): Yield:
390 mg (45%); orange red solid; mp: 182–183 ꢁC (Lit40.
175–176 ꢁC); 1H NMR (500 MHz, MeOH-d4) d 1.75–1.80 (m, 2H),
2.67 (t, 2H, J ¼ 6.2 Hz), 2.77 (t, 2H, J ¼ 5.5 Hz), 3.86 (s, 3H), 3.90 (s,
3H), 6.80 (d, 1H, J ¼ 3.5 Hz), 6.82 (d, 1H, J ¼ 4.0 Hz), 6.95 (dd, 1H, J1
¼ 8.5 Hz, J2 ¼ 1.5 Hz), 7.03 (d, 1H, J ¼ 1.5 Hz), 7.07 (d, 1H, J ¼ 15 Hz),
7.13 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 2.2 Hz), 7.22 (d, 1H, J ¼ 2.5 Hz), 7.58
(s, 1H), 7.63 (d, 1H, J ¼ 15.5 Hz); 13 C NMR (125 MHz, MeOH-d4) d
22.90, 24.03, 27.08, 55.06, 108.3, 110.6, 113.5, 114.8, 115.2, 117.3,
122.9, 123.8, 127.4, 128.2, 130.6, 133.2, 142.5, 147.1, 147.4, 148.0,
149.2, 177.6, 185.7. HRMS (EI) m/z: [M]þ calcd. for C24H24O6
408.1573; Found: 408.1572.
1,7-Bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-
one (4c): Yield: 150 mg (14%); orange solid; mp: 134–136 ꢁC (Lit38.
1
132–133 ꢁC); H NMR (500 MHz, Acetone-d6) d 3.84 (s, 6H), 3.85 (s,
6H), 5.97 (s, 1H), 6.70 (s, 1H), 6.73 (s, 1H), 6.97 (s, 1H), 6.98 (s, 1H),
7.11 (s, 1H), 7.21 (s, 1H), 7.29 (s, 2H), 7.57 (s, 1H), 7.60 (s, 1H); 13 C
NMR (125 MHz, Acetone-d6) d 55.30, 101.2, 110.4, 111.6, 122.0,
122.8, 123.0, 123.7, 128.1, 140.4, 142.8, 149.8, 151.6, 183.6. HRMS
(EI) m/z: [M]þ calcd. for C23H24O6 396.1573; Found: 396.1572.
2.6.5 Synthesis of 1-(4-aminophenyl)-7-(4-hydroxy-3-methoxyphe-
nyl)hepta-1, 6-diene-3,5-dione (5q)
Compound 5n (100 mg, 0.26 mmol) was dissolved in tetrahydro-
furan (5 ml), then ethanol (20 ml) and 1 M HCl (aq., 20 ml) were
added. The reaction mixture was stirred at reflux for 22 h and
monitored by TLC, then cooled to ambient temperature and
evaporated under reduced pressure. Ethyl acetate (20 ml) was
added to the residue and the mixture was neutralised using
NaHCO3 (aq, sat.). The organic phase was washed with water
(20 ml ꢄ3 times) and brine, dried over MgSO4, and the solvent
was evaporated under reduced pressure. The residue was applied
to the top of an open-bed silica gel column (n-hexane: ethyl acet-
ate: methanol ¼60:35:5 (v/v)) to yield 1-(4-aminophenyl)-7-(4-
hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5q, 75 mg,
85%) as a red solid.
2.6.3 Synthesis of 1,7-bis(3,4-dihydroxyphenyl)hepta-1,6-diene-3,5-
dione (4d)34
Curcumin (0.3 g, 0.81 mmol) was suspended in dry CH2Cl2 (30 ml),
then stirred for 10 min at –20 ꢁC under N2 (g) atmosphere.
Tribromoborane (0.25 ml ꢄ5 times) was slowly added. The reac-
tion mixture was allowed to warm up to ambient temperature
and stirred overnight. Therefore, the reaction mixture was slowly
poured into the saturated NaHCO3 solution (30 ml) with ice pow-
der, and then stirred for 2 h. The water layer was separated and
acidified with 1 M HCl (aqueous), then extracted with ethyl acetate
(30 ml ꢄ3 times). The organic layer was washed with water and
brine, and then dried over anhydrous MgSO4. After evaporating
the solvent under reduced pressure, the residue was applied to
the top of an open-bed silica gel column (n-hexane: ethyl acetate:
MeOH ¼60:38:2 (v/v)) to yield 1,7-bis(3,4-dihydroxyphenyl)hepta-
1,6-diene-3,5-dione (4d, 27 mg, 10%) as an orange amorph-
ous solid.
1-(4-Aminophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphe-
nyl)hepta-1,4,6-trien-3-one (5q): Yield: 75 mg (85%); red solid;
mp: 99–100 ꢁC; 1H NMR (500 MHz, Acetone-d6) d 3.89 (s, 3H), 5.29
(bs, 2H), 5.89 (s, 1H), 6.51 (d, 1H, J ¼ 15.5 Hz), 6.65–6.68 (m, 3H),
6.85 (d, 1H, J ¼ 8.5 Hz), 7.13 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz), 7.30
(d, 1H, J ¼ 2.0 Hz), 7.39 (d, 2H, J ¼ 9.5 Hz), 7.52 (d, 1H, J ¼ 2.5 Hz),
7.55 (d, 1H, J ¼ 2.5 Hz), 8.22 (bs, 1H); 13 C NMR (125 MHz, Acetone-
d6) d 55.46, 100.6, 110.5, 114.2, 115.3, 118.6, 120.0, 121.4, 122.9,
123.4, 127.4, 129.2, 130.2, 139.9, 141.4, 147.9, 149.0, 151.3, 182.4,
184.8. HRMS (EI) m/z: [M]þ calcd. for C20H19NO4 337.1314;
Found: 337.1316.
1,7-bis(3,4-dihydroxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-
one (4d): Yield: 27 mg (10%); orange solid; mp: 300 ꢁC (decom-
posed, Lit39. 304–305); 1H NMR (500 MHz, Acetone-d6) d 5.95 (d,
1H, J ¼ 6.0 Hz), 6.58 (dd, 2H, J1 ¼ 15.5 Hz, J2 ¼ 6.0 Hz), 6.85 (s, 2H),
7.04 (s, 2H), 7.16 (d, 2H, J ¼ 3.0 Hz), 7.51 (dd, 2H, J1 ¼ 15.2 Hz,
J2 ¼ 6.2 Hz), 8.39 (bs, 4H); 13 C NMR (125 MHz, Acetone-d6) d 100.9,
114.4, 115.6, 121.2, 121.8, 127.5, 140.5, 145.5, 147.9, 183.5. HRMS
(EI) m/z: [M]þ calcd. for C19H16O6 340.0947; Found: 340.0949.
2.6.6 Synthesis of 1,7-bis(3-hydroxy-4-methoxyphenyl)heptane-3,5-
dione (6)
Pd-C (10%, 50 mg) was added to a solution of curcumin (0.5 g,
0.27 mmol) in methanol (10 ml). After degassing, the mixture was
stirred at 0 ꢁC for 10 min, and then hydrogenated in a hydrogen
atmosphere (balloon) for 12 h. The mixture was filtered through a
Celite 545 and the solvent was evaporated under reduced pres-
sure. The residue was applied to the top of an open-bed silica gel
column (CH2Cl2:MeOH gradient elution) to yield 1,7-bis(3-hydroxy-
2.6.4 Synthesis of 2–(3-hydroxy-4-methoxybenzylidene)-6–(3-(3-
hydroxy-4-methoxyphenyl)acryloyl)cyclohexa-n-1-one (4f)
2-Acetyl-cyclohexan-1-one (0.3g, 2.14 mmol), boron trioxide (0.14g,
2.14 mmol), morpholine (0.037 ml, 0.42 mmol), acetic acid (0.024 ml,
0.42 mmol), and vanillin (0.651 g, 4.28 mmol) were placed in a
capped vial without solvent at ambient temperature. The resulting
mixture was irradiated in a microwave oven (300 W output,
1378KPa) at 150 ꢁC for 40min until the 2-acetyl-cyclohexan-1-one
4-methoxyphenyl)heptane-3,5-dione (6, 140 mg, 28%) as
a
white solid.
1,7-Bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (6):
or vanillin was consumed. The reaction was monitored by TLC. Yield: 140 mg (28%); white solid; mp: 94–96 ꢁC (Lit41. 95–97 ꢁC); H
1
After the reaction vial cooled, the product was dissolved in MeOH. NMR (500 MHz, Acetone-d6) d 2.55 (t, 2H, J1 ¼ 7.5 Hz, J2 ¼ 8.5 Hz),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1261
2.76–2.80 (m, 6H), 3.78 (s, 6H), 5.62 (s, 1H), 6.59–6.64 (m, 2H),
6.68–6.70 (m, 2H), 6.78 (d, 1H, J ¼ 1.5 Hz), 6.81 (d, 1H, J ¼ 1.5 Hz);
13 C NMR (125 MHz, Acetone-d6) d 30.99, 40.03, 45.07, 55.34, 99.49,
111.8, 114.8, 120.6, 132.2, 144.9, 147.5, 197.6. HRMS (EI) m/z: [M]þ
calcd. for C21H24O6 372.1573; Found: 372.1574.
4,4'-(1H-Pyrazole-3,5-diyl)bis(ethene-1,2-diyl))bis(2-methox-
yphenol) (8): Yield: 130 mg (68%); ivory solid; mp: 220–222 ꢁC (Lit.
218 ꢁC); 1H NMR (500 MHz, Acetone-d6) d 3.86 (s, 6H), 6.65 (s, 1H),
6.79 (s, 1H), 6.81 (s, 1H), 6.96 (d, 1H, J¼ 3.0 Hz), 6.98 (d, 1H,
J¼ 1.5 Hz), 7.00 (s, 1H), 7.09 (s, 1H), 7.12 (s, 1H), 7.17 (d, 2H,
J¼ 2.5Hz), 8.21 (bs, 1H); 13 C NMR (125 MHz, Acetone-d6) d 55.38,
99.21, 109.0, 115.2, 115.8, 120.3, 129.2, 129.9, 146.9, 147.1, 147.8.
HRMS (EI) m/z: [M]þ calcd. for C21H20N2O4 364.1423; Found: 364.1423.
2.6.7
Synthesis
of
(3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-
methoxy-1,4-phenylene) diacetate (7)
N,N-dimethylaminopyridine (0.13 g, 1.08 mmol) was added to a
solution of curcumin (2 g, 5.42 mmol) in anhydrous pyridine
(10 ml) at ambient temperature. Acetic anhydride (1.02 ml,
10.8 mmol) was dropped into the reaction mixture in a 0 ꢁC ice
bath, and then stirred overnight at ambient temperature. The
reaction mixture was then poured into crashed ice and extracted
with EtOAc (3 ꢄ 30 ml). The organic layer was washed with water,
dried over anhydrous Na2SO4, and the solvent was evaporated
under reduced pressure. The crude product was purified by flash
column chromatography (CH2Cl2) to yield di-(3,5-dioxohepta-1,6-
diene-1,7-diyl)bis(2-methoxy-1,4-phenylene) diacetate7 (1.9 g, 77%)
as a yellow solid.
3. Results and discussion
3.1 Chemistry
The synthetic route of the curcumin derivatives is shown in
Schemes 2–4. The 6-(3, 4-substituted-phenyl)-hex-5-ene-2,4-diones
(3) were prepared using acetylacetone with 1 equivalent of corre-
sponding aldehydes by Pabon’s method. From this reaction, the
symmetrical curcumins, 4a–4e, were obtained as by-products
(Scheme 2)39. The symmetrical curcumin derivatives, 4, were syn-
thesised by combining 1 with 2 equivalents of corresponding
aldehydes 2, except of compounds 4c and 4d owing to trace
yields. The synthesis of 4c and 4d is shown in Scheme 4.
Compound 4f, which has an inserted cyclohexyl group in 1,3-dike-
tone, was prepared by combining 2-acetylcyclohexanone with
vanillin under microwave irradiation40.
3-Hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2-methoxy-
4,1-phenylene) diacetate (7): Yield: 1.9 g (77%); yellow solid; mp:
1
158–159 ꢁC (Lit38. 156–158); H NMR (500 MHz, Acetone-d6) d 2.23
(s, 6H), 3.86 (s, 6H), 6.04 (s, 1H), 6.82 (s, 1H), 6.86 (s, 1H), 7.08 (s,
1H), 7.10 (s, 1H), 7.24 (d, 1H, J ¼ 2.5 Hz), 7.26 (d, 1H, J ¼ 1.5 Hz),
7.41 (d, 2H, J ¼ 2.0 Hz); 13 C NMR (125 MHz, Acetone-d6) d 19.65,
55.54, 101.6, 111.7, 121.1, 123.3, 124.4, 134.0, 139.8, 141.7, 151.8,
168.0, 183.6. HRMS (EI) m/z: [M]þ calcd. for C25H24O8 352.1472;
Found: 352.1472.
Twenty asymmetrical curcumin derivatives, 5, were prepared
using Pabon’s reaction with 3 and the corresponding aldehydes
(Scheme 3)39. Compound 5q was synthesised by deacetylation of
5n using 1 M HCl aqueous solution.
Symmetrical curcumin derivatives 4c, 4d, and 6–8 were pre-
pared by treating the corresponding reagent with curcumin
(Scheme 4). Compound 4c was prepared with MeI/K2CO3 in acet-
one under reflux conditions and compound 4d was produced by
a demethylation reaction after treating tribromoborane with dry
dichloromethane41. Tetrahydrocurcumin (6) was prepared by
hydrogenation using 10% Pd-C as a catalyst42 and acetylated
curcumin 7 was prepared using acetic anhydride with DMAP as
a catalyst with anhydrous pyridine34. Compound 8 was prepared
by condensation the 1, 3-diketone in curcumin with hydrazine
hydrate under reflux condition28. The structures of all com-
pounds were characterised using 1H NMR, 13 C NMR, and
EI-HRMS.
2.6.8 Synthesis of 4,4'-((1H-pyrazole-3,5-diyl)bis(ethene-1,2-diyl))-
bis(2-methoxyphenol) (8)
Hydrazine hydrate (0.07 ml, 0.81 mmol) was added to a solution of
curcumin (0.2 g, 0.54 mmol) in glacial acetic acid at room tempera-
ture, then the mixture was stirred for 2 h under reflux and the
solvent was evaporated under reduced pressure. EtOAc (30 ml)
was added to the residue and the organic layer was washed with
water and brine, and then dried over anhydrous MgSO4. After the
solvent was evaporated under reduced pressure, the residue was
applied to the top of an open-bed silica gel column (CH2Cl2:
MeOH ¼95:5 (v/v)) to yield 4,4'-((1H-pyrazole-3, 5-diyl)bis(ethene-
1,2-diyl))bis(2-methoxyphenol) (8, 130 mg, 68%) as an ivory solid.
R1
H
H
H
H
H
R2
H
H
H
H
H
R3
R4
R1
H
H
H
H
H
R2
H
H
H
H
H
R3
R4
3
4
OH
OMe
OH
OMe
OMe
OH
OH
OH
OMe
OH
OMe
OH
H
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
OMe OH
OMe OMe
OH
OH
OH
H
cyclohexyl
OMe
Scheme 2. Reagent and conditions for synthesis of 3: (i) 1, B2O3, ethyl acetate, 90 ꢁC; (ii) 2, n-(BuO)3B, ethyl acetate, 90 ꢁC; (iii) n-BuNH2, ethyl acetate, 90 ꢁC; (iv)
hydrochloric acid (1M, aq.), 50ꢁC; synthesis of 4: (i) 3, B2O3, ethyl acetate, 90 ꢁC; (ii) 2, n-(BuO)3B, ethyl acetate, 90 ꢁC; (iii) n-BuNH2, ethyl acetate, 90ꢁC; (iv) hydro-
chloric acid (1M, aq.), 50 ꢁC; synthesis of 4f: 1, 2, B2O3, morpholine, AcOH, 40 min, microwave irradiation (300 W).

1262
B. R. KIM ET AL.
X
C
C
C
C
C
C
N
C
C
C
R1
R2
R3
R4
X
C
C
C
C
C
C
C
C
C
R1
R2
R3
R4
H
5
5
OMe OMe OH
OMe OMe OMe
OH OMe OMe
OH OMe OH
OMe OH OH
OH OMe OH
OMe
OH
OH
OH
OH
OEt
H
OH OMe
OH OMe
OH OMe
OH OMe
OH OMe
OH OMe
OH OMe
OH OMe
OH OMe
CF3
5a
5b
5c
5d
5e
5f
5g
5h
5i
5k
5l
F
H
Br
H
H
5m
5n
5o
5p
5q
5r
5s
NHAc
OMe
OH
H
H
H
H
H
OH OMe
OH OMe
H
H
NH2
N(CH3)2
c-C5H10N
H
OH OMe O(CH2)2COOMe
OMe
OH OMe NO2
H
5j
Scheme 3. Reagent and conditions for synthesis of asymmetrical curcumin derivatives 5: (i) 3, B2O3, ethyl acetate, 90 ꢁC; (ii) 2, n-(BuO)3B, ethyl acetate, 90 ꢁC; (iii) n-
BuNH2, ethyl acetate, 90 ꢁC; (iv) hydrochloric acid (1M, aq.), 50 ꢁC.
Scheme 4. Reagent and conditions for synthesis of curcumin derivatives 4c, 4d, and 6–8: (A) 4a, CH3I, K2CO3, acetone, reflux; (B): 4a, BBr3, –78 ꢁC to ambient tempera-
ture, N2 (g), H2O; (C) 4a, H2/Pd-C (10wt. % of palladium), MeOH, 0 ꢁC to ambient temperature; (D) 4a, Ac2O, DMAP, pyridine, ambient temperature; (E) 4a,
.
NH2NH2 H2O, AcOH, reflux.
3.2 Biological evaluation
IC50 ¼ 0.6 0.1 mM), demethoxycurcumin (5p, IC50 ¼ 0.6 0.2 mM),
and bisdemthoxycurcumin (4e, IC50 ¼ 4.0 1.2 mM).
Hydrolytic activity of S. pneumoniae Nan A was confirmed using
DANA (Neu5Ac2en). The IC50 value of DANA with respect to
S. pneumoniae sialidase inhibition was 4.8 1.1 mM12. To identify a
sialidase inhibitor of S. pneumoniae, the inhibitory activity of
Turmeric ethanol extract and its three major components was
compared. For the methanol extract, the Nan A activity was 88%
at 30 mg/mL. The sialidase inhibitory activity of the major compo-
Among these compounds, 4e, in which two of the methoxy
groups were removed from curcumin showed diminished inhibi-
tory activity. Based on this result, we predicted that curcumin
derivatives would have inhibitory activity against sialidase from S.
pneumoniae Nan A and that the methoxy group played an import-
ant role in the inhibitory activity. Therefore, we modified curcumin
and evaluated the inhibitory effects on S. pneumoniae Nan A
(Table 1).
nents
of
Turmeric
was
as
follows;
curcumin
(4a,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1263
To evaluate the functionalisation of the curcumin skeleton to group (5 g, IC50 ¼ 4.4 0.1 mM) was more effective than the phenyl
find a suitable backbone. Hydrogenation of the heptadienyl group
group (5 h, IC50 ¼7.1 0.1 mM), but both compounds showed
(6, IC50 ¼ 82.1 7.6 mM) resulted in significantly diminished inhibi-
lower inhibitory activity than curcumin. To investigate the effect
tory activity. Next, to confirm the phenyl group at the 7-position,
inhibitory activity after the addition of a pyridinyl (5g) or phenyl
ring (5h) was compared. The results indicated that the pyridinyl
of 1,3-diketone, 4-hydroxy or 3-methoxy groups, synthesised and
compared with six kinds of curcumins. First, to investigate the
effect of the 1, 3-diketone moiety, pyrazole (8, IC50 ¼ 6.2 1.5 mM)
or carbocyclic 1, 3-diketone (4f, IC50 ¼ 4.7 0.5 mM) was substi-
tuted. Second, to investigate the effect of the 4-hydroxyl groups
in curcumin, acetyl (7, IC50 ¼ 2.6 0.6 mM) or methyl groups (4c,
IC50 ¼ 3.4 1.0 mM) were substituted and conversion of the
hydroxy at the para position to ester (5i, IC50 ¼ 1.0 0.5 mM). Third,
to investigate the 3-methoxy groups, elimination of the 3-methoxy
groups (4d, IC50 ¼ 1.5 0.7 mM) and conversion of the methoxy at
the meta position to ethoxy (5f, IC50 ¼ 1.5 0.8 mM) were com-
pared. Inhibitory activity of these compounds was diminished
than curcumin (4a). This suggested that 1,3-diketone and a hepta-
dienyl group were essential functional groups for sialidase inhib-
ition and either a methoxy or a hydroxyl group was required.
Based on these results, we substituted a methoxy or hydroxyl
group at the para- or meta- position of the 1,7-diphenylhepta-1,6-
diene-3,5-dione backbone. To confirm the positional tendency of
the methoxy and hydroxyl groups, we reacted it with feruloyl (3a)
or isoferuloyl (3b) acetone or hispolon (3c) with the correspond-
ing aldehyde 2. Because a methoxy or hydroxyl group was
Table 1. Inhibitory effects of synthesised compounds
against S. pneumoniae Nan A.
Compounds
IC50 (lM)a
Compounds
IC50 (lM)
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
0.6 0.1
1.0 0.4
3.4 1.0
1.5 0.7
0.4 1.2
4.7 0.5
1.5 0.7
0.6 0.1
1.0 0.4
0.7 0.2
0.2 0.1
1.5 0.8
4.4 0.1
7.1 0.1
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
6
1.0 0.5
0.9 0.3
1.0 0.1
1.8 0.2
1.0 0.2
1.8 0.4
1.2 0.1
0.6 0.2
1.6 0.1
1.5 0.2
1.5 0.1
82.1 7.6
2.6 0.6
6.2 1.5
5g
5h
7
8
aIC50 values of compounds represent the concentration that
caused 50% enzyme activity loss; all compounds were
examined in a set of triplicates experiment.
(A) 4a
(B) 4e
0.016
0.025
[I] = 0 µM
[I] = 0 µM
[I] = 0.39 µM
[I] = 0.78 µM
[I] = 1.56 µM
0.014
0.012
0.010
0.008
0.006
0.004
0.002
[I] = 0.39 µM
[I] = 0.78 µM
[I] = 1.56 µM
0.020
0.015
0.010
0.005
0.00
0.05
0.10
0.15
0.00
0.05
0.10
0.15
0.20
1/[S], (µM)
1/[S], (µM)
(C) 5q
(D) 5e
0.025
0.04
0.03
0.02
0.01
[I] = 0 µM
[I] = 0 µM
[I] = 0.1 µM
[I] = 0.2 µM
[I] = 0.4 µM
[I] = 0.78 µM
[I] = 1.56 µM
[I] = 3.12 µM
0.020
0.015
0.010
0.005
0.00
0.05
0.10
0.15
0.20
0.00
0.05
0.10
0.15
1/[S], (µM)
1/[S], (µM)
Figure 1. Graphical determination of the inhibition type for compounds 4a, 4e, 5q, and 5e. Lineweaver–Burk (A–D) plots for the inhibitory activity of compounds 4a,
4e, 5q, and 5e, respectively, against S. pneumoniae Nan A hydrolysis activity in the presence of different substrate concentrations.

1264
B. R. KIM ET AL.
Table 2. Inhibitory effects of curcumin derivatives in V. cholera and C. perfrin-
gens sialidase.
2.7-fold improvement in inhibitory activity over that of curcumin.
The IC50 values for sialidase inhibitory activity were 2.7–43.6 mM in
V. Choleara and 0.5–269.4 mM in C. perfringens.
IC50 (lM)a
IC50 (lM)
Compound V. cholerae C. perfringens Compound V. cholerae C. perfringens
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
5.3 0.7
7.6 0.5
21.5 7.0
11.9 2.0
15.1 0.1
24.9 1.8
11.7 0.2
9.2 0.2
7.7 0.7
2.7 0.8
4.8 1.5
6.7 0.6
1.6 0.4
3.2 0.4
13.1 2.2
6.1 0.8
11.0 0.8
13.4 0.1
6.7 0.5
6.7 0.8
3.5 0.2
1.0 0.1
0.5 0.07
2.9 0.5
5g
5i
43.6 0.1
9.5 1.5
6.9 1.8
6.8 0.8
4.2 0.8
4.3 0.7
2.9 0.9
1.9 0.2
NAb
32.9 5.1
3.3 0.4
1.6 0.5
5.9 0.8
2.8 0.3
2.1 0.4
1.7 0.1
1.3 0.2
269.4 31.2
13.8 0.6
9.5 0.1
4. Conclusions
5j
5n
5o
5p
5q
5r
6
In conclusion, we developed, for the first time, inhibitors of sialidase
from S. pneumoniae Nan A, V. cholerae and C. perfringens using curcu-
min derivatives. Of the 28 compounds synthesised, 7-(3,4-dihydroxy-
phenyl)-5-hydroxy-1-(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-
3-one (5e), which is novel compound contained isoferuloyl and cat-
echol moieties, was the most potent inhibitors of S. pneumoniae
(IC50 ¼ 0.2 0.1 mM) and C. perfringens (IC50 ¼ 0.5 0.07 mM). All
major Turmeric components showed noncompetitive inhibition, but
compound 5e exhibited competitive inhibition against S. pneumo-
niae Nan A. In the case of V. cholerae sialidase, 7-(4-(dimethylamino)-
phenyl)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-
3-one (5r), containing N, N-dimethylamino as an electron-donating
group, was the most potent inhibitor (IC50 ¼ 1.9 0.2 mM). The SAR
analysis suggested that the a, b-unsaturated ketone, heptadienyl
and either the methoxy or hydroxy group in curcumin was required
for sialidase inhibition. These results indicated that curcumin ana-
logues may potentially be used for sepsis caused by bacterial sialid-
ase. Further in vivo evaluation of compound 5e and 5r will be
performed in our laboratory.
7
8
37.5 3.5
41.6 18.7
aIC50 values of compounds represent the concentration that caused 50% enzyme
activity loss; all compounds were examined in a set of triplicates experiment.
bNo activity.
essential for sialidase inhibition, compounds 4c and 4d were
excluded from consideration. As shown in Table 1, IC50 values
ranged from 0.2–1.5 mM, indicating there was no positional ten-
dency for inhibition (4a, 4 b, and 5a–5e in Table 1). Among the
examined compounds, 5e, containing the catechol with the isofer-
uloyl moiety, was the most potent inhibitor (IC50 ¼ 0.2 0.1 mM),
with a 3.0-fold improvement in inhibitory activity over that of cur-
cumin. Thereafter, to investigate the electronic effect, we substi-
tuted an electron-donating group (EDG) or electron-withdrawing
group (EWG) into the para position of the phenyl rings, including
nitro (5j), trifluoromethyl (5k), fluoro (5l), bromo (5m), acetamido
(5n), methoxy (5o), hydroxyl (5p), amino (5q), N, N-dimethylamino
(5r), and piperidinyl (5s) groups. The IC50 values ranged from
0.6–1.8 mM and the electronic effect did not influence in Nan A
inhibition. Based on these observations, we investigated the kin-
etic mechanisms of inhibitors with IC50 values of 25 mM or less.
We selected the major components of Turmeric (4a, 4e, 5q) and
compound 5e as the most potent inhibitors for the kinetic study.
We found that the major components of Turmeric (4a, 4e, 5q)
showed noncompetitive inhibition characteristics with a Ki of
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by the KRIBB Research Initiative
Program and part of the project titled “Development of the con-
trol of aquaculture infectious disease using natural resources,”
funded by the Ministry of Oceans and Fisheries, Republic of Korea.
1.3 mM (4a), 1.2 mM (4e), and 0.8 mM (5q), respectively. Conversely, J-. Y. Park and H-. J. Kwon were supported by the Basic Science
compound 5e exhibited a potent competitive inhibition against
Nan A with a Ki of 0.14 mM (Figure 1).
Synthesised compounds were then evaluated for their inhibi-
tory effect on sialidase from Vibrio cholerae and Clostridium perfrin-
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT & Future
Planning [NRF-2016R1C1B2015753]. Also, this study was supported
by Korea Coast Guard.
gens, which also release sialidase and play
a role in the
pathogenesis. The inhibitory assay results are summerised in
Table 2.
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