Synthesis and biological evaluation of asymmetrical diarylpentanoids as antiinflammatory, anti-α-glucosidase, and antioxidant agents

Article abstract of DOI:10.1007/s00044-019-02430-5

A series of seven new (1, 3, 6, 7, 10, 12, and 13) and six (2, 4, 5, 8, 9, and 11) known diarylpentanoid analogs were synthesized and assessed for their nitric oxide (NO) and α-glucosidase inhibitory activities as well as their antioxidant capacity. Nine compounds (2, 3, 4, 5, 6, 8, 11, 12, and 13) were found to exhibit comparable activity to that of curcumin (IC₅₀= 13.0 μM), in which compound 8 has displayed strongest NO inhibitory activity with the IC₅₀ values of 17.5 μM. Meanwhile, four compounds (1, 7, 12, and 13) were found to possess better α-glucosidase inhibitory activity than that of curcumin (30.9 μM), with the IC₅₀ values ranging from 19.4 to 24.9 μM. On the other hand, none of the synthesized compounds has achieved better DPPH scavenging activities than that of curcumin, indicating the relatively poor antioxidant potential of desired diarylpentanoid structure. Structure-activity relationship (SAR) study disclosed that the existence of meta-hydroxyphenyl and bromo groups is crucial for antiinflammatory and anti-α-glucosidase activities, respectively. Molecular docking study showed that bromo moiety could form additional hydrophobic contacts with α-glucosidase, thereby increased the inhibition of diarylpentanoid against α-glucosidase. The overall results suggested that diarylpentanoids with poly-meta-hydroxylated phenyl ring and multiple bromo groups may lead to the discovery of new diarylpentanoids with both antiinflammatory and anti-α-glucosidase activities.

Full text of DOI:10.1007/s00044-019-02430-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02430-5
ORIGINAL RESEARCH
Synthesis and biological evaluation of asymmetrical
diarylpentanoids as antiinflammatory, anti-α-glucosidase, and
antioxidant agents
Sze Wei Leong1,2 Tahani Awin
Faridah Abas
1,3 ●
Siti Munirah Mohd Faudzi
1,4 ●
M. Maulidiani Khozirah Shaari
5 ●
1,4 ●
●
1,6
Received: 29 May 2019 / Accepted: 17 August 2019
Springer Science+Business Media, LLC, part of Springer Nature 2019
©
Abstract
A series of seven new (1, 3, 6, 7, 10, 12, and 13) and six (2, 4, 5, 8, 9, and 11) known diarylpentanoid analogs were
synthesized and assessed for their nitric oxide (NO) and α-glucosidase inhibitory activities as well as their antioxidant
capacity. Nine compounds (2, 3, 4, 5, 6, 8, 11, 12, and 13) were found to exhibit comparable activity to that of curcumin
(
IC = 13.0 µM), in which compound 8 has displayed strongest NO inhibitory activity with the IC values of 17.5 µM.
50 50
Meanwhile, four compounds (1, 7, 12, and 13) were found to possess better α-glucosidase inhibitory activity than that of
curcumin (30.9 µM), with the IC50 values ranging from 19.4 to 24.9 µM. On the other hand, none of the synthesized
compounds has achieved better DPPH scavenging activities than that of curcumin, indicating the relatively poor antioxidant
potential of desired diarylpentanoid structure. Structure-activity relationship (SAR) study disclosed that the existence of
meta-hydroxyphenyl and bromo groups is crucial for antiinflammatory and anti-α-glucosidase activities, respectively.
Molecular docking study showed that bromo moiety could form additional hydrophobic contacts with α-glucosidase, thereby
increased the inhibition of diarylpentanoid against α-glucosidase. The overall results suggested that diarylpentanoids with
poly-meta-hydroxylated phenyl ring and multiple bromo groups may lead to the discovery of new diarylpentanoids with
both antiinflammatory and anti-α-glucosidase activities.
●
●
●
●
Keywords Antiinflammatory α-glucosidase Antioxidant Diarylpentanoids Docking
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02430-5) contains supplementary
material, which is available to authorized users.
Introduction
Curcumin is an important chemical constituent, which
found mostly in the Zingiberaceae family with specific
emphasis on Curcuma species (Akram et al. 2010). Being
an excellent free radical scavenger and superoxide inhibitor,
curcumin has attracted the interest of numerous researchers
for its wide range of biological activities including but not
limited to antiinflammatory, anticancer, and antioxidant
*
Faridah Abas
faridah_abas@upm.edu.my
1
2
Laboratory of Natural Products, Institute of Bioscience, Universiti
Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Department of Microbiology, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, 43400
Serdang, Selangor, Malaysia
(Bisht et al. 2010, Wu et al. 2010, Ben et al. 2011, Naik
3
4
5
6
et al. 2011, Gupta et al. 2013). In addition, curcumin has
been reported to be a respectable antimalarial and anti-
bacterial agent due to its cytotoxic effect on numerous
parasites and bacteria, such as Plasmodium falciparum and
Helicobacter pylori (Cui et al. 2007, De et al. 2009).
However, several reports revealed that curcumin is poor in
stability and absorbability in body’s circulating fluids,
which resulting in low bioavailability, thus reducing its
usefulness in clinical trials (Ravindranath and
Department of Chemistry, Faculty of Science, University of
Benghazi, Benghazi, Libya
Department of Chemistry, Faculty of Science, Universiti Putra
Malaysia, 43400 Serdang, Selangor, Malaysia
School of Fundamental Science, Universiti Malaysia Terengganu,
2
1030 Kuala Nerus, Terengganu, Malaysia
Department of Food Science, Faculty of Food Science and
Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor,
Malaysia

Medicinal Chemistry Research
Chandrasekhara 1981, Shoba et al. 1998, Pan et al. 1999).
Therefore, several laboratories have entered intensive stu-
dies of synthetic modifications of curcumin in order to
overcome the aforementioned challenges. The synthetic
modification of curcumin has led to the discovery of dia-
rylpentanoids, a 5-carbon spacer series of molecules with
curcumin-like medicinal properties and enhanced stability
regularly performed on TLC plate (0.20 mm Merck silica gel
60 F254) in each reaction stage. Purification processes were
performed by using column chromatography on silica gel 60
(mesh 70–230, Merck). Mass spectra were measured using a
gas chromatography-mass spectrometer GCMS- QP2010
Ultra (Shimadzu, Kyoto, Japan) Mass Spectrometer. Nuclear
Magnetic Resonance spectra were recorded using a Varian
500 MHz NMR Spectrometer (Varian Inc., Palo Alto, CA).
(
Lee et al. 2009, Ahmad et al. 2014).
Diarylpentanoids have been reported to display notable
antiinflammatory potential due to their positive inhibitions
on a diverse range of enzymes, pro-inflammatory cytokines,
and mediators such as prostaglandins, nitric oxide (NO),
and tumor necrosis factor alpha (Liang et al. 2008, Zhao
et al. 2010, Fang et al. 2013). On top of these, dia-
rylpentanoids were found to be more chemically and
metabolically stable in both physiological pH and rat liver
microsomes, making them a potent scaffold that worth to be
developed as stable bioactive molecules (Zhang et al. 2014,
Leong et al. 2015).
NO is a short lived free radical molecule, which syn-
thesized endogenously from arginine, molecular oxygen,
and NADPH during a complex reaction catalyzed by
several nitric oxide synthases (NOS). Although NO is
released as one of the primary defensive mechanisms in
human’s body in response to pathogen invasion, the
overproduction of such molecules may promote tissue
injuries through cell lipids peroxidation and deoxyr-
ibonucleic acid mutation, which in turn lead to diverse
diseases including arthritis, cardiovascular disorders,
ulcerative colitis, and cancer (Rachmilewitz et al. 1995,
Sharma et al. 2007, Choudhari et al. 2013). Therefore,
suppressing NO production may be an efficient strategy in
the search of multi-functional drugs. Interestingly, pre-
vious study showed that the inhibition of NOS could
reduce the glucose uptake in diabetes patients, which
suggests that dual inhibition of NO and α-glucosidase, a
key enzyme of glucose metabolism, could be an alter-
native strategy in treating diabetes (Kingwell et al. 2002).
Based upon these, the aim of this study is to synthesize a
series of stable 2,6-dibenzylidenecyclohexanone analogs
and evaluate for their antiinflammatory, anti-α-glucosi-
dase, and antioxidant activities.
General procedure for the synthesis of
diarylpentanoid analogs
Catalytic amount of p-toluenesulphonic acid was added
into the 30 mL toluene that contains a mixture of
20 mmol each of cyclohexanone and pyrrolidine. The
solution was kept in 100 mL SNRB flask at room tem-
perature with subsequent refluxed on a Dean & Stark
apparatus for 2 h in order to produce (I). Upon comple-
tion, 20 mL of toluene containing 20 mmol of 4-
hydroxybenzaldehyde or 3-hydroxybenzaldehyde was
added dropwise into the reaction mixture and stirred for
24 h. Subsequently, 10 mL of deionised water was added
into the solution and refluxed for 30 min. The resulting
solution was extracted thrice with 3 M HCl and once with
water. The toluene layer obtained was then dried over
anhydrous MgSO and concentrated in vacuo to yield the
4
crudes of II and III. After purification with column
chromatography, II and III were dissolved in 20 mL of
acetic acid followed by the addition of appropriate ben-
zaldehydes and catalytic amount of sulfuric acid. The
resulting reaction mixtures were stirred at room tem-
perature overnight. Upon completion, the reaction mix-
tures were added with 100 mL of distilled water and
stirred for 5 min, followed by three times extractions with
100 mL of ethyl acetate. The organic layer obtained was
then washed with saturated sodium bicarbonate solution
(NaHCO ) and dried over anhydrous MgSO to obtain
3
4
the crude of targeted compounds (IV). The crude pro-
ducts were then purified on column chromatography
(Leong et al. 2018a, 2018b).
2-(4-Hydroxybenzylidene)-6-(3-bromobenzylidene)
cyclohexanone (1)
1
Materials and methods
Yellow amorphous powder (19.02%), m. p. 83–85 °C. H-
NMR (500 MHz, C D O, δ ppm): 8.83 (s, 1H), 7.68 (s, 1H),
3
6
Chemistry
7.67 (s, 1H), 7.61 (s, 1H), 7.56 (d, J = 8 Hz, 1H), 7.52 (d,
J = 8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8 Hz,
1H), 6.95 (d, J = 8.5 Hz, 2H), 2.97–2.92 (m, 4H),
All chemicals and reagents were obtained from Sigma-
Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Ger-
many) companies. Solvents were purchased from common
commercial suppliers, were dried and distilled before use. To
observe the reaction progress, thin-layer chromatography was
1
3
1.83–1.79 (m, 2H). C-NMR (125 MHz, C D O, δ ppm):
3
6
191.2, 158.4, 139.8, 139.2, 136.8, 136.0, 132.4, 131.9,
131.3, 130.2, 129.5, 128.2, 127.3, 121.3, 115.4, 28.1, 28.0,
+
22.7. EIMS, m/z: 369.30 (M ).

Medicinal Chemistry Research
2
,6-Bis(4-hydroxybenzylidene)cyclohexanone (2)
(t, J = 8 Hz, 1H), 7.00 (d, J = 5 Hz, 2H), 6.94 (d, J = 9 Hz,
H), 6.87 (dd, J = 8.5, 2 Hz, 2H), 2.96–2.92 (m, 4H),
2
1
3
Yellow amorphous powder (53.72%), m. p. 209–210 °C.
1.82–1.77 (m, 2H). C-NMR (125 MHz, C3D6O, δ ppm):
188.4, 158.2, 157.3, 137.4, 136.6, 136.3, 135.3, 133.7,
132.4, 129.5, 127.4, 121.7, 116.7, 115.6, 115.5, 28.4, 28.3,
1
H-NMR (500 MHz, C D O, δ ppm): 8.71 (s, 2H), 7.64 (s,
3
6
2
2
H), 7.45 (d, J = 8.5 Hz, 4H), 6.93 (d, J = 8.5 Hz, 4H),
.95–2.92 (m, 4H), 1.82–1.77 (m, 2H).
13
+
C-NMR
22.8. EIMS, m/z: 306.12 (M ).
(
1
125 MHz, C D O, δ ppm): 188.5, 158.1, 135.8, 133.7,
32.4, 129.4, 115.5, 28.3, 22.9. EIMS, m/z: 306.11 (M ).
3
6
+
2-(4-Hydroxybenzylidene)-6-(4-bromobenzylidene)
cyclohexanone (7)
2
-(4-Hydroxybenzylidene)-6-(3-methoxybenzylidene)
cyclohexanone (3)
Yellow amorphous powder (20.51%), m. p. 230–231 °C.
1
H-NMR (500 MHz, C D O, δ ppm): 8.80 (s, 1H),
3
6
Yellow amorphous powder (26.31%), m. p. 119–121 °C.
7.66–7.62 (m, 4H), 7.49–7.45 (t, J = 9 Hz, 4H), 6.94 (d,
J = 9 Hz, 2H), 2.97–2.91 (m, 4H), 1.82–1.78 (m, 2H). C-
1
13
H-NMR (500 MHz, C D O, δ ppm): 8.81 (s, 1H), 7.66 (t,
3
6
J = 5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 7.38 (t, J = 7.4 Hz,
NMR (125 MHz, C D O, δ ppm): 191.0, 158.4, 136.0,
3
6
1
H), 7.10 (d, J = 8 Hz, 1H), 7.06 (s, 1H), 6.96 (d, J =
.5 Hz, 1H), 6.94 (d, J = 9 Hz, 2H), 3.85 (s, 3H), 2.96–2.93
133.7, 132.4, 131.2, 130.2, 129.5, 128.2, 121.3, 115.5,
27.6, 20.9. EIMS, m/z: 369.05 (M ).
+
2
(
m, 4H), 1.82–1.77 (m, 2H). 13C-NMR (125 MHz, C3D6O,
δ ppm): 191.7, 159.2, 158.3, 138.8, 137.9, 135.3, 135.1,
2,6-Bis(3-hydroxybenzylidene)cyclohexanone (8)
1
5
32.5, 132.3, 129.4, 128.5, 122.0, 115.4, 114.6, 113.3,
4.6, 28.3, 28.2, 22.8. EIMS, m/z: 320.05 (M ).
+
Yellow amorphous powder (32.24%), m. p. 205–206 °C.
1
H-NMR (500 MHz, C D O, δ ppm): 8.50 (s, 2H), 7.62 (s,
3
6
2
-(4-Hydroxybenzylidene)-6-(3,4-dimethoxybenzylidene)
2H), 7.31 (t, J = 8 Hz, 2H), 7.01 (d, J = 7 Hz, 4H), 6.88
(dd, J = 9.5, 2.5 Hz, 2H), 2.96–2.93 (m, 4H), 1.82–1.77 (m,
cyclohexanone (4)
1
3
2
H). C-NMR (125 MHz, C D O, δ ppm): 192.1, 157.4,
3
6
Yellow amorphous powder (17.80%), m. p. 180–182 °C.
135.6, 134.5, 129.4, 128.6, 121.0, 115.6, 114.9, 28.2, 22.7.
EIMS, m/z: 306.00 (M ).
1
+
H-NMR (500 MHz, C D O, δ ppm): 8.76 (s, 1H), 7.65 (d,
3
6
J = 2 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.14 (dd, J = 8.5,
3
2
1
1
1
2
.5 Hz, 2H), 7.04 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 8.5 Hz,
H), 3.87 (s, 3H), 3.86 (s, 3H), 2.99–2.93 (m, 4H),
.82–1.77 (m, 2H). 13C-NMR (125 MHz, C3D6O, δ ppm):
86.4, 157.3, 149.7, 149.5, 137.3, 136.2, 135.5, 135.2,
32.5, 129.5, 128.5, 121.6, 115.4, 111.5, 110.7, 55.9, 28.4,
2-(3-Hydroxybenzylidene)-6-(3-methoxybenzylidene)
cyclohexanone (9)
Yellow amorphous powder (20.63%), m. p. 147–148 °C.
1
H-NMR (500 MHz, C D O, δ ppm): 8.51 (s, 1H), 7.66 (s,
3
6
+
8.2, 22.9. EIMS, m/z: 350.05 (M ).
1H), 7.63 (s, 1H), 7.38 (t, J = 8 Hz, 1H), 7.30 (t, J = 8 Hz,
H), 7.11 (d, J = 8 Hz, 1H), 7.07 (s, 1H), 7.02 (d, J =
1
2
-(4-Hydroxybenzylidene)-6-(3,4,5-trimethoxybenzylidene)
8.5 Hz, 2H), 6.97(dd, J = 8.5, 2.5 Hz, 1H), 6.88 (dd, J = 9,
1.5 Hz, 1H), 3.85 (s, 3H), 2.98–2.94 (m, 4H), 1.82–1.80 (m,
cyclohexanone (5)
1
3
2
H). C-NMR (125 MHz, C D O, δ ppm): 192.1, 159.7,
3
6
1
Yellow amorphous powder (11.29%), m. p. 78–79 °C. H-
NMR (500 MHz, C D O, δ ppm): 8.79 (s, 1H), 7.65 (s, 1H),
7
2
4
156.8, 135.6, 135.4, 134.7, 134.4, 129.5, 129.4, 128.7,
128.6, 121.9, 120.9, 116.6, 116.0, 115.6, 115.3, 54.6, 28.4,
3
6
+
.62 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 9 Hz,
H), 6.84 (s, 2H), 3.89 (s, 6H), 3.77 (s, 3H), 3.01–2.93 (m,
H), 1.82–1.78 (m, 2H). 13C-NMR (126 MHz, C D O, δ
24.3, 22.7. EIMS, m/z: 320.20 (M ).
2-(3-Hydroxybenzylidene)-6-(4-methoxybenzylidene)
cyclohexanone (10)
3
6
ppm): 191.0, 158.4, 153.1, 139.7, 139.3, 136.8, 136.0,
1
2
35.9, 132.5, 130.2, 128.3, 115.5, 111.3, 58.9, 55.5, 28.3,
8.0, 22.8. EIMS, m/z: 380.15 (M ).
+
Yellow amorphous powder (23.75%), m. p. 145–146 °C.
1
H-NMR (500 MHz, C D O, δ ppm): 8.51 (s, 1H), 7.67 (s,
3
6
2
-(4-Hydroxybenzylidene)-6-(3-hydroxybenzylidene)
1H), 7.62 (s, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 8 Hz,
1H), 7.03 (m, 4H), 6.87 (m, 1H), 3.86 (s, 3H), 2.97–2.92 (m,
cyclohexanone (6)
1
3
4
H), 1.82–1.77 (m, 2H). C-NMR (125 MHz, C D O, δ
3
6
Yellow amorphous powder (19.00%), m. p. 200–202 °C.
ppm): 191.7, 160.1, 156.9, 138.2, 136.5, 135.2, 134.7,
1
H-NMR (500 MHz, C D O, δ ppm): 8.80 (s, 1H), 8.49 (s,
132.2, 131.7, 130.9, 128.6, 120.9, 116.1, 113.9, 113.0, 54.8,
3
6
+
1
H), 7.65 (s, 1H), 7.61 (s, 1H), 7.46 (d, J = 5 Hz, 2H), 7.32
28.2, 28.3, 22.8. EIMS, m/z: 320.10 (M ).

Medicinal Chemistry Research
2
-(3-Hydroxybenzylidene)-6-(3,4-dimethoxybenzylidene)
NO inhibition assay
cyclohexanone (11)
The RAW 264.7 cells at 90–95% confluence were detached
Yellow amorphous powder (15.67%), m. p. 163–165 °C.
and seeded into a 96-well culture plate at a density of 5 ×
10 cells/well and allowed to attach for 24 h. Then, the cells
1
4
H-NMR (500 MHz, C D O, δ ppm): 8.52 (s, 1H), 7.66 (s,
3
6
1
6
6
3
H), 7.62 (s, 1H), 7.29 (t, J = 8.5 Hz, 1H), 7.16 (d, J =
Hz, 2H), 7.05 (d, J = 9 Hz, 1H), 7.00 (d, J = 7 Hz, 2H),
.87 (dd, J = 7.5, 2 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H),
were stimulated with 1 µg/mL of LPS (Escherichia coli,
serotype 0111:B4) and 1 ng/mL of interferon-gamma (IFN-
γ) in the presence or absence of the test compounds for 17 h.
The nitrite production was then determined by Griess assay.
Concisely, 50 μL of Griess reagent (1% sulfanilamide and
0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in
2.5% phosphoric acid) was added to 50 μL of cell culture
supernatant for 15 min. The optical density was recorded at
13
.00–2.93 (m, 4H), 1.83–1.78 (m, 2H).
C-NMR
(
125 MHz, C D O, δ ppm): 191.7, 159.2, 149.8, 138.7,
3
6
1
1
37.9, 136.1, 135.3, 132.5, 131.3, 129.9, 128.6, 127.4,
15.4, 114.6, 114.2, 113.3, 54.6, 54.5, 28.4, 28.2, 22.8.
+
EIMS, m/z: 350.10 (M ).
550 nm using a microplate reader Spectramax Plus (Mole-
2
-(3-Hydroxybenzylidene)-6-(3-bromobenzylidene)
cular Devices LLC, Sunnyvale, CA, USA) (Mohd Faudzi
et al. 2015).
cyclohexanone (12)
Yellow amorphous powder (13.42%), m. p. 157–159 °C.
Cytotoxicity determination (MTT assay)
1
H-NMR (500 MHz, C D O, δ ppm): 8.53 (s, 1H), 7.69 (s,
3
6
1
7
H), 7.63 (d, J = 5.5 Hz, 2H), 7.57 (d, J = 5.5 Hz, 1H),
.54 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 7.30 (t,
In each well, cell culture medium was replaced with 100 µL
of 0.5 mg/ml MTT-containing DMEM followed by 4 h
J = 8 Hz, 1H), 7.02 (dd, J = 7, 1 Hz, 2H), 6.88 (m, 1H),
incubation at 37 °C in a 95% air and 5% CO atmosphere.
2
13
2
.97–2.93 (m, 4H), 1.82–1.79 (m, 2H).
C-NMR
Upon completion, the supernatants in all wells were then
removed, and the formazan salts formed were dissolved in
100% dimethyl sulfoxide (DMSO). After 15 min of incu-
bation at room temperature, the absorbance was recorded at
570 nm using a microplate reader (Mohd Faudzi et al.
(
125 MHz, C D O, δ ppm): 191.5, 156.9, 136.0, 135.3,
3
6
1
1
34.0, 132.6, 132.5, 131.6, 131.1, 130.3, 129.5, 128.7,
28.2, 121.6, 120.9, 116.1, 115.0, 28.4, 28.3, 22.6. EIMS,
+
m/z: 369.05 (M ).
2015).
2
-(3-Hydroxybenzylidene)-6-(4-bromobenzylidene)
cyclohexanone (13)
α-Glucosidase inhibitory activity assay
Yellow amorphous powder (12.63%), m. p. 192–193 °C.
The α-glucosidase inhibitory assay was measured based on
the previously reported method [27]. The samples were
prepared by dissolving compounds 1–13 in DMSO-
containing buffer. The substrate, p-nitrophenyl-α-D-gluco-
pyranoside (PNPG) was prepared by dissolving it in 50 mM
phosphate buffer (pH 6.5). For the assay, 96-well micro-
plate that contains 10 µL of samples, 10 µL of enzyme, and
130 µL of buffer was incubated for 5 min at room tem-
perature. Subsequently, 50 µL PNPG was added to each
well and the plate was further incubated for 15 min at room
temperature. Upon the completion, the reactions were
stopped by adding 50 µL of 2 M glycine (pH 10) and left for
5 min in room temperature before the absorbance was
recorded at 405 nm using SPECTRAmax PLUS (Leong
et al. 2018a, 2018b).
1
H-NMR (500 MHz, C D O, δ ppm): 8.53 (s, 1H), 7.65 (s,
3
6
2
7
2
H), 7.63 (d, J = 3.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H),
.30 (t, J = 8 Hz, 1H), 7.01 (m, 2H), 6.88 (dd, J = 9.5,
Hz, 1H), 2.97–2.92 (m, 4H), 1.83–1.78 (m, 2H). C-
1
3
NMR (125 MHz, C D O, δ ppm): 191.0, 158.5, 139.7,
3
6
1
1
3
39.3, 136.8, 135.6, 133.7, 133.3, 131.9, 131.7, 128.9,
27.3, 121.1, 115.5, 114.7, 28.3, 28.0, 22.8. EIMS, m/z:
+
69.05 (M ).
Cell culture
RAW 264.7 murine macrophage cells acquired from the
American Type Culture Collection (Rockville, MD) were
2
cultured as monolayer cultures in T75 cell culture flasks
in Dulbecco’s Modified Eagle’s Medium (DMEM) with
phenol red containing HEPES, L-glutamine supple-
mented with 10% fetal bovine serum, and 1% antibiotic
solution (Gibco/BRL Life Technologies Inc, Eggenstein,
DPPH radical scavenging activity assay
In 96-well microplate, 100 µL of DPPH solution was
added to 100 µL of different samples at different con-
centrations, of which both DPPH and samples were
Germany) under 5% CO at 37 °C (Mohd Faudzi et al.
2
2
015).

Medicinal Chemistry Research
O
O
O
Scheme 1 General synthetic
steps for compounds 1–13.
Chemical reagents and
conditions: a p-toluene-sulfonic
acid, toluene, reflux (2 h); b
benzaldehyde, rt (24 h); c H2O,
reflux (0.5 h); d benzaldehyde,
acetic acid, H2SO4, rt
Ar
Ar
H
N
O
d
HO
HO
HO
HO
O
N
I
II
Ar
H
IV (1-7)
O
OR
a
b,c
(
overnight)
III
IV (8-13)
dissolved in 100% DMSO. The mixtures were then incu-
bated in the dark for 30 min at a room temperature. Upon
completion, the absorbance of each reaction mixture was
measured at 517 nm using SPECTRAmax PLUS (Leong
et al. 2016).
Results and discussion
Chemistry
Compounds 1–13 (IV) were synthesized successfully via
acidic aldol condensation as shown in Scheme 1. In detail,
Intermediate I (N-(1-cyclohexenyl)pyrrolidine) was first
achieved by reacting cyclohexanone and pyrrolidine on a
Dean–Stark setup. In this step, Dean–Stark apparatus was
used to remove the water side product formed during the
enamine formation as water molecules could hydrolyse the
desired intermediate I back to its precursors. Without any
purification, the crude of intermediate I was directly reacted
with 3- and 4-hydroxybenzaldehyde to form intermediates II
and III, respectively. Upon purifications, II and III were
further reacted with various benzaldehydes to produce the
targeted compounds IV via acid-catalyzed aldol condensa-
tion reaction. The crudes of all compounds were purified
using column chromatography and their chemical structures
Molecular docking
Molecular docking studies were carried out using Dis-
®
covery Studio 3.1 (Accelrys, San Diego, USA) on an Intel
(
TM)2 Quad CPU Q8200 @2.33 GHz running under a
Windows XP Professional environment. Since the crystal
structure of Saccharomyces cerevisiae α-glucosidase is
still not available, a homology model of α-glucosidase was
built according to our previosly reported method. The
crystal structure of isomaltase (PDB: 3A4A) from Sac-
charomyces cerevisiae and α-glucosidase sequence
(
P53341) of Saccharomyces cerevisiae were obtained from
the Protein Data Bank and UniProt (www.uniprot.org),
respectively. Sequence alignment of P53341 on 3A4A was
performed and homology models of α-glucosidase were
built and validated using Modeler program. The model
with the lowest modeler objective function was finally
selected for ramachandran plot validation prior to mole-
cular docking. Then, compound 7 and co-crystallized
ligand (alpha-D-glucose) were drawn with ChemDraw
Ultra 12.0 followed by ligands preparation protocol with
the default setting recommended by Accelrys. The pre-
pared ligands were then subjected to ligands minimization
with CHARMm force field before being used for docking
analyses. Minimized co-crystallized ligands were re-
docked into their respective enzymes with several sets of
amino acids as flexible residues. The top ranked con-
formations resulted from the docking experiment were
compared with their original crystallographic confirmation
in terms of RMSD. The parameters with lowest RMSD
values were selected for the flexible docking of compound
1
13
were confirmed through the H-NMR, C-NMR (supple-
mentary data) and mass spectrometry-based characterization.
NO inhibitory activity of diarylpentanoids
The synthesized 2,6-dibenzylidenecyclohexanone analogs
(1–13) were assessed for their ability in inhibiting NO
activity in IFN-γ/LPS-stimulated RAW 264.7 murine
macrophage. The preliminary studies at 50 µM testing
concentration revealed that nine compounds (2, 3, 4, 5, 6, 8,
11, 12, and 13) have significantly suppressed the production
of NO with the percentages of inhibition >50%. These nine
bioactive compounds were further tested to determine their
IC50 values, and the values obtained were compared with
that of curcumin as a positive control. MTT assay was
subsequently performed to confirm that NO inhibition was
not caused by the cytotoxicity of the synthesized analogs.
The NO inhibition of synthesized 2,6-dibenzylidenecyclo-
hexanones is shown in Table 1.
7
in the homology model. The flexible docking results
were analyzed using Discovery Studio Visualizer
v4.1.0.14169 (Accelrys, San Diego, USA) (Leong et al.
Among the tested analogs, compound 8 was found to
exhibit the strongest NO inhibitory activity with the IC50
value of 17.5 µM. Meanwhile, the rest of eight compounds
2
018a, 2018b).

Medicinal Chemistry Research
Table 1 DPPH radical scavenging, NO, and α-glucosidase inhibitory activity of compounds 1–13
(Ring A)
(Ring B)
Compounds Ar (ring A)
R 1 (ring B)
NO inhibition
%) ± SD
NO inhibition
IC50 (µM) ± SD
Cytotoxicity
IC50 (µM) ± SD
α-Glucosidase
IC50 (µM)
DPPH
inhibition inhibition
%) 50 µM IC50 (µM)
DPPH
(
(
Curcumin
–
–
97.5 ± 0.4
12.6 ± 3.8
91.5 ± 0.6
85.1 ± 2.0
74.3 ± 1.2
80.5 ± 4.2
93.4 ± 2.3
17.2 ± 1.3
97.6 ± 1.9
77.9 ± 0.9
65.3 ± 6.8
69.5 ± 0.9
31.4 ± 1.3
25.7 ± 2.6
13.0 ± 0.7
ND
97.3 ± 4.5
ND
30.9 ± 5.2
21.9 ± 3.5
34.9 ± 4.6
53.7 ± 6.6
94.8 ± 7.1
50.7 ± 3.4
25.0 ± 0.5
19.4 ± 1.4
35.2 ± 1.7
50.3 ± 2.2
59.1 ± 2.5
95.2 ± 6.7
24.9 ± 4.3
23.8 ± 2.1
82.6 ± 4.5 25.8 ± 3.6
9.8 ± 0.4 ND
1
2
3
4
5
6
7
8
9
3-Bromophenyl
4-Hydroxyphenyl
3-Methoxyphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
3-Hydroxyphenyl
4-Bromophenyl
3-Hydroxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
3-Bromophenyl
4-Bromophenyl
4′-OH
4′-OH
4′-OH
4′-OH
4′-OH
4′-OH
4′-OH
3′-OH
3′-OH
3′-OH
3′-OH
3′-OH
3′-OH
21.3 ± 0.8
28.5 ± 0.6
26.1 ± 0.5
22.8 ± 1.1
20.5 ± 0.4
ND
>100
87.8 ± 0.8
>100
>100
>100
ND
31.1 ± 1.2 ND
10.8 ± 0.6 ND
28.2 ± 1.5 ND
26.6 ± 2.3 ND
31.2 ± 0.8 ND
15.4 ± 0.8 ND
19.7 ± 0.4 ND
10.6 ± 0.7 ND
12.5 ± 1.4 ND
11.5 ± 0.5 ND
22.0 ± 0.4 ND
14.7 ± 1.9 ND
17.5 ± 0.9
27.1 ± 1.0
31.5 ± 3.2
26.8 ± 1.4
ND
>100
>100
>100
>100
ND
1
1
1
1
0
1
2
3
ND
ND
ND Not Determine
were found to display slightly weaker NO inhibitory
activity as compared with that of compound 8, with the IC50
values ranging from 19.7 to 31.5 µM. Structure-activity
relationship (SAR) study revealed that hydroxyl group is
crucial for antiinflammatory activity as all of the hydro-
xylated compounds (6 and 8) were found to possess better
NO inhibitory activity than their respective methoxylated (3
and 9) and halogenated (1 and 12) analogs. On the other
hand, comparison of meta- and para-substituted analogs
revealed that meta-substitution is preferable as all of the
meta-substituted diarylpentanoids (8 and 9) exhibit better
NO inhibitory effects than their respective para-substituted
analogs (6 and 10), regardless to the substituted functional
groups. These results are in agreement with our previous
studies, in which the meta-hydroxylated phenyl rings pos-
sess the strongest antiinflammatory activity. In addition to
substitution pattern and substituted functional groups,
electron density may also affect the NO inhibitory activity
of synthesized compounds. This can be seen by comparing
the NO inhibitory activity of all methoxylated compounds
α-Glucosidase inhibitory activity of diarylpentanoids
Apart from antiinflammatory activity, all synthesized com-
pounds were also tested for their α-glucosidase inhibitory
activity. Based on the results obtained, four compounds (1,
7, 12, and 13) were found to exhibit strong α-glucosidase
inhibitory activities in which compound 7 has displayed the
strongest activity with the IC50 value of 19.4 µM. Mean-
while, three other compounds (2, 6, and 8) have displayed
moderate α-glucosidase inhibition, with the IC50 values
ranging from 25 to 35 µM (Table 1). SAR study revealed
that bromo group is important for α-glucosidase inhibitory
activities as all of the brominated analogs (1, 7, 12, and 13)
are strong α-glucosidase inhibitors. Unlike NO inhibitory
activity, the substitution position on the phenyl ring does
not affect the anti-α-glucosidase potential of targeted com-
pounds. This can be observed by comparing the meta-
substituted compounds (1, 2, and 12) to their respective
para-substituted analogs (7, 8, and 13) of which both meta-
and para-substituted analogs possess similar activity.
(
3, 4, 5, 10, and 11), in which the multi-metoxylated dia-
In order to gain functional and structural insights into the
binding mode of the most active analog (7) in α-glucosi-
dase, molecular docking was performed with Discovery
Studio 3.1. Since the crystal structure of Saccharomyces
cerevisiae α-glucosidase is unavailable, a homology model
of α-glucosidase was first built and validated using Dis-
covery Studio 3.1 and Ramachandran plot, respectively,
rylpentanoids (4, 5, and 11) showed improved NO inhibi-
tory activity as compared with that of mono-methoxylated
analogs (3 and 10). Since methoxy moiety is an electron
donating functional group, this result is therefore implying
that the higher the electron density of phenyl ring, the better
the antiinflammatory activity of the compounds.

Medicinal Chemistry Research
Fig. 1 Binding interactions of
compound 7 with the active site
residues of α-glucosidase
receptor
Table 2 Data resulted from the molecular docking of compounds 7 in α-glucosidase
Compound structure
Interacting
Bond type
Bonding distance (Å)
amino acid residue
ASP68
Hydrogen bonding
Pi–Pi shaped
Pi–Alkyl
2.25
5.21
5.27
4.42
5.25
3.17
TRY71
PHE300
ARG312
Pi–Alkyl
Pi–Alkyl
Pi–donar
followed by re-docking of co-crystallized ligands into the
validated homology model. According to the result obtained
glucosidase inhibitory effects of brominated compounds
(1, 7, 12, and 13) as compared with the rest of the syn-
thesized analgos. The molecular docking results of com-
pounds 7 are summarized in Table 2.
(
supplementary data), both homology model and flexible
docking parameters were found to be acceptable as 100% of
residues in homology built are located found in either
favored (97.9%) or acceptable (2.1 %) regions while a
RMSD value of 0.7147 Å was observed in the re-docking
procedure. Figure 1 illustrated the binding interactions of
compounds 7 in α-glucosidase.
As shown in Fig. 1, the hydroxyl and phenyl moieties
of 4-hydroxyphenyl fragment of compound 7 interact with
ASP68 and TYR71 residues through hydrogen bonding
DPPH scavenging activities of diarylpentanoids
Lastly, all synthesized compounds were evaluated for their
antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) scavenging assay. Noteworthy, none of the tested
compounds showed significant DPPH scavenging activity at
the testing concentration of 100 µM. This indicates that
mono- and di-hydroxylated diarylpentanoids are poor anti-
oxidant. The result obtained are summarized in Table 1.
(
green dashed line) and Pi–Pi shaped (purple dashed line)
interactions, respectively. Meanwhile, the cyclohexanone
fragment of the respective compound was found to
interact with PHE 330 residue through Pi–Alkyl (light
purple dashed line) hydrophobic interaction. Interestingly,
both bromo and phenyl moieties of bromophenol frag-
ment have displayed strong hydrophobic contacts
including Pi–Alkyl (light purple dashed line) and
Pi–donar (light green dashed line) interactions with ARG
Conclusions
In summary, a series of seven new (1, 3, 6, 7, 10, 12, and
13) and six (2, 4, 5, 8, 9, and 11) diarylpentanoids were
synthesized and evaluated for their antiinflammatory, anti-
α-glucosidase, and antioxidant activities. Compounds 7 and
8 were found to exhibit strongest anti-α-glucosidase and
3
12 residue. The multiple hydrophobic interactions of
bromophenyl fragment thus explained the much better α-

Medicinal Chemistry Research
antiinflammatory activity, respectively. SAR studies showed
that meta-hydroxyphenyl is crucial for NO inhibitory
activity, while bromo moiety is essential for enhanced anti-
α-glucosidase activity. Subsequent molecular docking ana-
lysis revealed that additional hydrophobic interactions by
bromo group is responsible for the enhanced anti-α-gluco-
sidase activity of compound 7. Since hydroxyl moiety is
preferable to be substituted at the meta-position for NO
inhibitory activity, while bromo moiety is independent to the
substitution position for better α-glucosidase inhibition, we
therefore concluded that 4-bromo-3-hydroxyphenyl-con-
taining diarylpentanoid may be a new lead compounds with
both antiinflammatory and anti-α-glucosidase properties.
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Acknowledgements The authors thank the Ministry of Higher Edu-
cation (MOHE) of Malaysia for financial support under the project
FRGS/1/2014/ST01/UPM/02/1 (5538011).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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