Synthesis and evaluation of chalcone derivatives as inhibitors of neutrophils' chemotaxis, phagocytosis and production of reactive oxygen species

Article abstract of DOI:10.1111/cbdd.12226

Inhibitory effects on neutrophils' chemotaxis, phagocytosis and production of reactive oxygen species (ROS) are among the important targets in developing anti-inflammatory agents and immunosuppressants. Eight series of chalcone derivatives including five newly synthesized series were assessed for their inhibitory effects on chemotaxis, phagocytosis and ROS production in human polymorphonuclear neutrophils (PMNs). Inhibition of PMNs' chemotaxis and phagocytosis abilities were investigated using the Boyden chamber technique and the Phagotest kit, respectively, while ROS production was evaluated using luminol- and lucigenin-based chemiluminescence assay. The new derivatives (4d and 8d), which contain 4-methylaminoethanol functional group were active in all the assays performed. It was also observed that some of the compounds were active in inhibiting chemotaxis while others suppressed phagocytosis and ROS production. The information obtained gave new insight into chalcone derivatives with the potential to be developed as immunomodulators. Chalcone derivatives were synthesised and evaluated for their inhibitory effects on PMNs chemotaxis, phagocytosis and intracellular and extracellular ROS productions. It was observed that phenyl-4-methylaminoethanol and dimethoxy substituents contributed to these effects.

Full text of DOI:10.1111/cbdd.12226

Chem Biol Drug Des 2014; 83: 198–206
Research Article
Synthesis and Evaluation of Chalcone Derivatives as
Inhibitors of Neutrophils’ Chemotaxis, Phagocytosis
and Production of Reactive Oxygen Species
the site of injury (1). Phagocytes, primarily neutrophils, will
then engulf the intruders through phagocytosis. This process
is made possible by the presence of Fc receptors and b2 in-
tegrins, which will bind to immunoglobulins (IgG) or comple-
ment-coated particles, respectively (2). Activation of
neutrophils’ killing mechanisms, which involve the enzymatic
and oxidative processes follows (3). In the oxidative process,
a rise in oxygen consumption by neutrophils led to respiratory
burst where a variety of microbiostatic and microbiocidal
reactive oxygen species (ROS) are generated, namely super-
oxide (O2À) (4), hydrogen peroxide (H2O2), hypochlorous acid
(HOCl) and hydroxyl radical (OH•) (5). Nonetheless, the exact
mechanism on how these ROS evoke their antimicrobial
function is still not fully understood.
Syed N. A. Bukhari, Yasmin Tajuddin, Vannessa
J. Benedict, Kok W. Lam , Ibrahim Jantan,
Juriyati Jalil and Malina Jasamai*
Drugs and Herbal Research Centre, Faculty of Pharmacy,
Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd.
Aziz, 50300, Kuala Lumpur, Malaysia
*Corresponding author: Malina Jasamai,
malina@pharmacy.ukm.my, malina_jasamai@yahoo.co.uk
Inhibitory effects on neutrophils’ chemotaxis, phagocy-
tosis and production of reactive oxygen species (ROS)
are among the important targets in developing anti-
inflammatory agents and immunosuppressants. Eight
series of chalcone derivatives including five newly syn-
thesized series were assessed for their inhibitory
effects on chemotaxis, phagocytosis and ROS produc-
tion in human polymorphonuclear neutrophils (PMNs).
Inhibition of PMNs’ chemotaxis and phagocytosis abili-
ties were investigated using the Boyden chamber tech-
nique and the Phagotest kit, respectively, while ROS
production was evaluated using luminol- and lucige-
nin-based chemiluminescence assay. The new deriva-
tives (4d and 8d), which contain 4-methylaminoethanol
functional group were active in all the assays
performed. It was also observed that some of the com-
pounds were active in inhibiting chemotaxis while oth-
ers suppressed phagocytosis and ROS production.
The information obtained gave new insight into chal-
cone derivatives with the potential to be developed as
immunomodulators.
Although the above processes are desired to preserve
host tissues from infections and injuries, overexpression
on these reactions is undesirable and can lead to many
pathological conditions such as rheumatoid arthritis (6),
tumour (7) and atherosclerosis (8). Anti-inflammatory
agents such as non-steroidal anti-inflammatory drugs
(NSAIDs) and immunosuppressants such as tacrolimus
and cyclosporin are currently used to treat these condi-
tions. Nevertheless, problems such as gastric ulceration
caused by NSAIDs (9) and poor bioavailability of tacrolimus
(10) are still remain unresolved, and hence, drugs with bet-
ter safety profiles and bioavailability are much needed.
Synthesis of chalcone (1,3-diaryl-2-propen-1-one) has been
a considerable interest due to various pharmacological
activities exhibited by natural chalcones, namely anticarcin-
ogenic properties of xanthohumol (11), chemopreventive
effect of isoliquiritigenin (12) and anti-adipogenic of butein
(13). On the other hand, for synthetic chalcone derivatives
among the activities reported are antitumour (14), antimicro-
bial (15) and anti-inflammatory (16). These activities mainly
due to their a,b-unsaturated ketone moiety and various sub-
stituents introduced to the two aryl rings (17). The role of
chalcone derivatives in inhibiting different steps in the inflam-
matory cascades has been explored extensively (18). Meth-
oxy and hydroxyl groups in the diaryl rings are among the
functional groups of interest in promoting the anti-inflamma-
tory effect (19–21). Nevertheless, the study on inhibitory
effects of chalcone derivatives on chemotaxis, phagocytosis
and oxidative burst of neutrophils is still lacking.
Key words: chalcones, chemotaxis, inhibitors, phagocytosis,
reactive oxygen species, structure-activity relationship
Received 10 June 2013 and accepted for publication 22
August 2013
Immune system is a complex network of cells involves in an
organized reactions to preserve host tissues from external
aggressions. Among the cascade of events that happen in
respond to these external stimuli are chemotaxis, phagocyto-
sis and oxidative burst. The endogenous and exogenous
mediators for instance interleukin-8 (IL-8) and formyl-methio-
nyl-leucyl-phenylalanine (fMLP) released during tissue injury
act as chemo-attractants in the recruitment of phagocytes to
198
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12226

Immunomodulatory Effects of Chalcones
This study aimed to synthesize five series of new chalcone
derivatives. The inhibitory activities of these new derivatives
and another three series, which were synthesized previ-
ously on polymorphonuclear neutrophils (PMNs) chemo-
taxis, suppression of PMNs phagocytosis, and inhibition of
intracellular and extracellular ROS production in PMNs and
human whole blood were also examined. Results from
these assays help in designing better derivatives, which
can be potentially used to modulate immune response
during inflammation.
(E)-3-(4-methyaminoethanolphenyl)-1-(5-methyl-2-
furyl)-2-propen-1-one (4d)
This compound was obtained by reacting 2-acetyl-5-meth-
ylfuran (1.24 g, 10 mmol) with N-methyl-N-(2-hydroxyace-
tyl)-4-aminobezaldehyde (2.15 g, 12 mmol) to give clear
crystals (0.91 g, 32%). RF 0.22 (EtOAc-PE 2:3 v/v); mp:
131–132 °C; 1H NMR (500 MHz, CDCl3): d = 7.79 (d,
J = 16 Hz, 1H), 7.53 (d, J = 8 Hz, 2H), 7.21 (d, J = 1.8 Hz,
1H), 7.19 (d, J = 10.2 Hz, 1H), 6.75 (d, J = 9 Hz, 2H), 6.19
(d, J = 3 Hz, 1H), 3.86 (t, J = 6 Hz, 2H), 3.58 (t, J = 6 Hz,
2H), 3.07 (s, 3H), 2.44 (s, 3H), 1.81 (s, 1H), 13C NMR
(500 MHz, CDCl3): d = 177.71, 157.43, 152.93, 151.37,
143.97, 130.47, 123.16, 118.54, 116.41, 112.05, 109.07,
60.23, 54.60, 38.97, 14.18; IRVmax/cm-1 (ATR): 3374.1,
1624.80, 1581.48, 1261.58; HRMS (ESI) m/z: 286.14
[M + H]+, 308.12 [M + Na]+.
Methods and Materials
General methods
1H and 13C NMR spectra were recorded with a JEOL ECP
spectrometer operating at 500 MHz, with Me4Si as internal
standard and CDCl3 or DMSO-d6 as solvent. High resolu-
tion mass spectra (HRMS) were determined by the
electrospray ionization mass spectrometry (ESI-MS) on
MicroTOF-Q mass spectrometer (Bruker, Coventry, UK).
Microanalyses data were obtained from the Fison EA 1108
elemental analyser. Infrared spectra were recorded using
KBr disc on a Perkin Elmer 400 (FTIR) spectrometer. Flash
column chromatography was performed with silica gel 60
(230–400 mesh) (Merck, Kuala Lumpur, Malaysia), and thin
layer chromatography (TLC) was carried out on precoated
silica plates (kiesel gel 60 F254, BDH). Melting points were
determined on an electrothermal instrument and are uncor-
rected. Compounds were visualized by illumination under
ultraviolet (UV) light (254 nm) or by the use of vanillin stain
followed by charring on a hotplate.
(E)-3-(4-methyaminoethanolphenyl)-1-(2-furyl)-2-
propen-1-one (5d)
This compound was obtained by reacting 2-furyl methyl
ketone (1 mL, 10 mmol) with N-methyl-N-(2-hydroxyace-
tyl)-4-aminobezaldehyde (1.79 g, 12 mmol) to give dark
brown crystals (2.2 g, 81%). RF 0.61 (EtOAc-PE 1:3 v/v);
mp: 150–151 °C; 1H NMR (500 MHz, CDCl3) d = 7.84 (d,
J = 13.0 Hz, 1H), 7.63 (dd, J = 0.5 Hz, 1H), 7.55 (d,
J = 7.5 Hz, 2H), 7.28 (d, J = 0.5 Hz, 1H), 7.27 (d, J =
4.0 Hz, 1H), 6.75 (d, J = 7.5 Hz, 2H), 6.57 (d, J = 1.5 Hz,
1H), 3.87 (t, J = 4.5 Hz, 2H), 3.59 (t, J = 5.0 Hz, 2H),
3.09 (s, 3H), 1.59 (s, 1H), 13C NMR (500 MHz, CDCl3)
d = 178.33, 154.23, 151.52, 145.89, 144.65, 130.60,
123.10, 116.46, 116.13, 112.29, 112.08, 109.07, 60.26,
54.58, 38.96; HRMS (ESI) m/z: 294.09 [M + Na]+.
Syntheses of compounds 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b,
3c, 4a, 4b, 4c, 4e, 5b, 5c and 6e were reported previ-
ously (22,23), whereas compounds 4d, 5d, 6d, 6f, 6g,
7d, 7e, 7h, 7i, 8d and 8e were prepared in this study
using the sodium hydroxide-catalysed Claisen–Schmidt
condensation reaction.
(E)-3-(4-methyaminoethanolphenyl)-1-(naphthalen-
1-yl)-2-propen-1-one (6d)
This compound was obtained by reacting 1-acetonaph-
thone (1.52 mL, 10 mmol) with N-methyl-N-(2-hydroxyeth-
yl)-4-aminobenzaldehyde (2.15 g, 12 mmol) to give
reddish-orange oil (1.76 g, 53%). RF 0.44 (EtOAc-PE 1:1
v/v); 1H NMR (500 MHz, CDCl3): d = 8.26 (dd, J = 6.6 &
4.2 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.91 (dd, J = 4.8 &
5.4 Hz, 1H), 7.71 (dd, J = 7.2 & 6.6 Hz, 1H), 7.52 (m, 4H),
7.46 (d, J = 11.4 Hz, 2H), 7.10 (d, J = 15.6 Hz,1H), 6.74
(dd, J = 12.0 & 11.4 Hz, 2H), 3.85 (t, J = 5.4 Hz, 2H), 3.58
(t, J = 6.0 Hz, 2H), 3.07 (s, 3H), 2.06 (s, 1H); 13C NMR
(500 MHz, CDCl3): d = 196.41, 151.56, 147.15, 138.06,
133.81, 130.80, 130.58, 128.34, 127.11, 126.48, 126.31,
125.83, 124.61, 122.67, 112.05, 60.25, 54.52, 39.00;
HRMS (ESI) m/z: 354.14 [M + Na]+, 685.30 [2M + Na]+.
General procedure for the synthesis of chalcone
derivatives
Synthesis of the chalcone derivatives was achieved by the
steps outlined in Scheme 1. An amount of 10 mmol of the
respective ketones was added to a solution of the respec-
tive aldehydes (10 mmol) in ethanol (15 mL). A solution of
50% NaOH was added dropwise, and the reaction mixture
was stirred at room temperature (27 °C) for 2–24 h accord-
ingly. The appearance of precipitate and colour changes of
the reaction mixture are an indicative of product formation,
and reaction completion was monitored by TLC. Upon
completion, the reaction mixture was poured into ice
(50 mL), which has been acidified with concentrated HCl
(1 mL), extracted with ethyl acetate (50 mL), washed with
water (2 9 150 mL), dried and concentrated in vacuo to
give oils and solids. The crude products were further puri-
fied either by column chromatography or recrystallization.
(Z)-3-(5-methyl-2-furyl)-1-(naphthalen-1-yl)-2-
propen-1-one (6f)
This compound was obtained by reacting 1-acetonaphthone
(1.52 mL, 10 mmol) with 5-methyl furfural (1 mL, 10 mmol)
to give brown crystals (2.2 g, 84%). RF 0.38 (EtOAc-PE 1:3 v/
Chem Biol Drug Des 2014; 83: 198–206
199

Bukhari et al.
R
O
O
1a 2-hydroxyphenyl
1b 3-hydroxyphenyl
1c 4-hdroxyphenyl
R
N
N
O
O
O
O
O
O
2a 2-hydroxyphenyl
2b 3-hydroxyphenyl
2c 4-hdroxyphenyl
S
S
Cl
Cl
R
R
R
3a 2-hydroxyphenyl
3b 3-hydroxyphenyl
3c 4-hdroxyphenyl
O
O
4a 2-hydroxyphenyl
4b 3-hydroxyphenyl
4c 4-hdroxyphenyl
4d Phenyl-4-methylaminoethanol
4e 2,3-dimethoxy-benzene
O
O
O
O
O
O
O
O
5b 3-hydroxyphenyl
5c 4-hdroxyphenyl
5d Phenyl-4-methylaminoethanol
O
i
R
R
+
H
R
6d Phenyl-4-methylaminoethanol
6e 2,3-dimethoxy-benzene
6f
2-methyl-furan
6g Tert-butyl-benzene
O
O
7d Phenyl-4-methylaminoethanol
7e 2,3-dimethoxy-benzene
7h 2-methyl-benzene
R
7i Phenyl-4-diethylamine
O
O
O
O
O
O
8d Phenyl-4-methylaminoethanol
8e 2,3-dimethoxy-benzene
R
O
O
Scheme 1: Reagents and
conditions: (i) NaOH, EtOH, r.t.
v); mp: 138–139 °C; 1H NMR (500 MHz, CDCl3): d = 8.39
(d, J = 7.5 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.91 (d,
J = 17.5 Hz, 1H), 7.77 (d, J = 18.5 Hz, 1H), 7.55 (m, 3H),
7.31(d, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 6.65 (s,
1H), 6.14 (s, 1H), 2.34 (s, 3H); 13C NMR (500 MHz, CDCl3):
d = 195.20, 156.29, 149.93, 137.45, 133.85, 131.85,
131.36, 130.53, 128.40, 127.32, 126.89, 126.39, 125.81,
124.58, 122.73, 118.40, 109.47, 14.01; HRMS (ESI) m/z:
285.06 [M + Na]+; Anal. calcd for C18H14O2: C 82.42, H
5.38, found C 82.76, H 5.45.
(500 MHz, CDCl3): d=196.03, 154.47, 146.06, 137.32,
133.86, 131.87, 131.44, 130.55, 127.38, 127.01, 126.46,
126.00, 125.72, 124.56, 34.98, 31.16; HRMS (ESI) m/z:
337.12 [M + Na]+; Anal. calcd for C23H22O: C 87.86, H
7.05, found C 88.22, H 7.27.
(Z)-3-(4-methyaminoethanolphenyl)-1-diphenyl-
2-propen-1-one (7d)
This compound was obtained by reacting 1,1-diphenylace-
tone (2.10 g, 10 mmol) with N-methyl-N-(2-hydroxyethyl)-4-
aminobenzaldehyde (1.8 g, 10 mmol) to give light brown
solids (1.25 g, 34%). RF 0.48 (EtOAc-PE 1:3 v/v); mp: 128–
(Z)-3-(4-tert-butyl-phenyl)-1-(naphthalen-1-yl)-2-
propen-1-one (6g)
1
129 °C; H NMR (500 MHz, CDCl3): d=7.83 (d, J = 7.5 Hz,
This compound was obtained by reacting 1-acetonaph-
thone (1.52 mL, 10 mmol) with 4-tert-butylbenzaldehyde
(1.7 mL, 10 mmol) to give light yellow crystals (2.4 g,
76%). RF 0.47 (EtOAc-PE 2:3 v/v); mp: 112–113 °C; 1H
NMR (500 MHz, CDCl3): d=8.33 (d, J = 7.0 Hz, 1H), 8.02
(d, J = 8.50 Hz, 1H), 7.94 (d, J = 7.0 Hz, 1H), 7.78 (d,
J = 6.5 Hz, 1H), 7.57 (m, 6H), 7.45 (d, J = 8.5 Hz, 2H),
7.30 (d, J = 16.5 Hz, 1H), 1.36 (s, 9H); 13C NMR
1H), 7.61 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 7.50 Hz 2H),
7.35 (m, 10H), 6.70 (d, J = 9.5 Hz, 2H), 5.40 (s, 1H), 3.58
(m, 4H), 2.30 (s, 3H). 13C NMR (500 MHz, CDCl3):
d = 197.93, 154.08, 152.45, 145.21, 130.48, 130.08,
129.28, 128.60, 128.49, 128.30, 111.97, 62.97, 60.17,
54.51, 38.93; HRMS (ESI) m/z: 372.19 [M + H]+; Anal.
calcd for C25H25NO2: C 80.83, H 6.78, N 3.77, found C
80.85, H 6.60, N 3.73.
200
Chem Biol Drug Des 2014; 83: 198–206

Immunomodulatory Effects of Chalcones
(E)-3-(2,4-dimethoxyphenyl)-1-diphenyl-2-propen-
1-one (7e)
d = 194.85, 162.58, 159.02, 153.25, 148.63, 139.17,
130.43, 129.51, 124.39, 113.96, 111.96, 90.86, 61.49,
56.06, 55.65; IRVmax/cm-1 (ATR) 3381.86, 1641.06,
1565.82, 1088.80, 1176.93; HRMS (ESI) m/z: 394.11
[M + Na]+, 765.23 [2M + Na]+.
This compound was obtained by reacting 1,1-diphenylace-
tone (2.10 g, 10 mmol) with 2,3-dimethoxybenzaldehyde
(1.7 g, 10 mmol) to give clear crystals (2.8 g, 78%). RF
0.45 (EtOAc-PE 2:3 v/v); mp: 121–122 °C; 1H NMR
(500 MHz, CDCl3): d = 8.02 (d, J = 16.0 Hz, 1H), 7.35 (m,
10H), 7.12 (d, J = 8.0, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.94
(d, J = 8 Hz, 1H), 6.88 (d, J = 16.0 Hz, 1H), 5.48 (s, 1H),
3.88 (s, 3H), 3.75 (s, 3H). 13C NMR (500 MHz, CDCl3):
d = 197.83, 153.16, 148.86, 138.75, 138.27, 129.29,
128.70, 127.15, 126.81, 124.17, 119.41, 114.23, 62.89,
61.26, 55.90; HRMS (ESI) m/z: 381.09 [M + Na]+; Anal.
calcd for C24H22O3: C 80.42, H 6.19, found C 80.22,
H 6.08.
(E)-2′,4′,6′-trimethoxy-4-(2,3-dimethoxy)-chalcone
(8e)
This compound was obtained by reacting 2,4,6-trimeth-
oxyacetophenone (2.10 g, 10 mmol) with 2,3-dimethoxy-
benzaldehyde (1.99 g, 12 mmol) to give yellow crystals
(1.52 g, 42%). RF 0.37 (EtOAc-PE 1:2 v/v); mp:
119–124 °C; 1H NMR (500 MHz, CDCl3): d=7.68 (d,
J = 16.0 Hz, 1H), 7.19 (d, J = 19.0 Hz, 1H), 6.96 (m, 3H),
6.16 (s, 2H), 3.86 (s, 6H), 3.83 (s, 6H), 3.05 (s, 3H); 13C
NMR (500 MHz, CDCl3): d = 195.07, 162.20, 158.77,
151.41, 146.01, 130.53, 124.78, 123.19, 112.16, 90.88,
56.10, 55.63, 54.73; IRVmax/cm-1 (ATR) 1666.52, 1583.83,
1120.92; HRMS (ESI) m/z: 381.08 [M + Na]+, 739.18
[2M + Na]+.
(Z)-1,1,5-triphenyl-3-hexen-2-one (7h)
This compound was obtained by reacting 1,1-diphenylace-
tone (2.10 g, 10 mmol) with 2-phenylpropionaldehyde
(1.34 mL, 10 mmol) to give white powder (2.8 g, 86%). RF
0.72 (EtOAc-PE 2:3 v/v); mp: 156–157 °C; 1H NMR
(500 MHz, CDCl3): d = 7.66 (d, J = 8.0 Hz, 1H), 7.30 (m,
10H), 7.11 (m, 5H), 6.88 (d, J = 8.0 Hz, 1H), 4.95 (s, 1H),
3.65 (m, 1H), 1.61 (d, J = 6.0 Hz, 3H); 13C NMR
(500 MHz, CDCl3): d=207.71, 153.41, 143.06, 140.14,
128.65, 128.60, 127.26, 126.94, 126.21, 125.92, 122.98,
63.00, 41.82, 16.97; Anal. calcd for C24H22O: C 88.31, H
6.79, found C 88.15, H 6.97.
Isolation of human polymorphonuclear neutrophils
Polymorphonuclear neutrophils (PMNs) used in this study
were isolated as described in our previous work (24).
Briefly, blood obtained from healthy volunteers (aged
> 18 years old) was centrifuged to get white cells, which
were then diluted with phosphate buffer saline (PBS). Dex-
tran was added, and the mixture was left to sediment. The
supernatant was centrifuged again and washed with dis-
tilled water, PMNs pellets were then collected. The use of
human blood was approved by the Ethics Committee of
the Universiti Kebangsaan Malaysia (approval no: FF-220-
2008).
(Z)-3-(4-diethylaminophenyl)-1-diphenyl-2-propen-
1-one (7i)
This compound was obtained by reacting 1,1-diphenylace-
tone (2.10 g, 10 mmol) with 4-diethylamino benzaldehyde
(1.76 g, 10 mmol) to give light red solids (1.25 g, 34%). RF
0.55 (EtOAc-PE 1:3 v/v); mp: 106–107 °C; 1H NMR
(500 MHz, CDCl3): d = 7.84 (d, J = 7.5 Hz, 2H), 7.74 (d,
J = 8.50, 1H), 7.61 (d, J = 7.0 Hz, 1H), 7.35 (m, 10H),
6.70 (d J = 7.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 5.41 (s,
1H), 3.45 (q, J = 7.0 Hz 4H), 1.23 (t, J = 10.0 Hz, 6H).
13C NMR (500 MHz, CDCl3): d=190.10, 149.68, 144.40,
141.32, 130.74, 129.30, 128.57, 128.49, 128.09, 126.93,
110.60, 62.91, 44.50, 12.57; HRMS (ESI) m/z: 370.21
[M + H]+; Anal. calcd for C26H27NO: C 84.51, H 7.37, N
3.79, found C 84.53, H 7.67, N 3.81.
Cell viability
Cell viability was determined by the standard trypan blue
exclusion method. The PMNs and macrophages cells
(1 9 106/mL) were incubated with 6.25 or 100 lg/mL of
test compounds, each in triplicate at room temperature
(27 °C) for 1–2 h. The blue dye uptake was an indication of
cell death. The percentage viability was calculated from the
total cell counts. The concentration of compounds at which
viability was > 90% and was used for the assays (24).
(Z)-2’,4’,6’-trimethoxy-4-methylaminoethanol-
chalcone (8d)
Chemotaxis assay
This assay was performed as described in our previous
work (24). Briefly, formyl-methionyl-leucyl-phenylalanine
(fMLP), a chemotaxin was added to the modified 48-well
Boyden lower chamber whereas various concentrations of
test compounds (10.00, 5.00, 2.50, 1.25 and 0.63 lg/mL)
in DMSO were added to the upper chamber. The inhibitory
activity of test compounds on the movement of PMN cells
towards chemotaxin was calculated from the distance of
the cell migration.
This compound was obtained by reacting 2,4,6-trimeth-
oxyacetophenone (2.10 g, 10 mmol) with N-methyl-N-
(2-hydroxyacetyl)-4-aminobezaldehyde (2.15 g, 12 mmol)
to give orange solids (1.15 g, 31%). RF 0.21 (EtOAc-PE
1:1 v/v); mp: 140–141 °C; 1H NMR (500 MHz, CDCl3):
d=7.34 (d, J = 9.0 Hz, 1H), 7.25 (d, J = 7.7 Hz, 1H), 7.00
(m, 4H), 6.16 (s, 2H), 3.86 (m, 7H), 3.77 (s, 9H), 0.71 (t,
J = 14.3 Hz, 1H), 13C NMR (500 MHz, CDCl3):
Chem Biol Drug Des 2014; 83: 198–206
201

Bukhari et al.
Phagocytosis assay
tives (Scheme 1). The respective aldehydes and ketones
used in the reaction were obtained commercially. Com-
pounds 4d, 5d, 6d, 7d and 8d were synthesized without
prior protection of the hydroxyl group on the N-methyl-
N-(2-hydroxyethyl)-4-aminobenzaldehyde as about similar
yield was obtained when the hydroxyl was protected with
p-toulenesulphonic acid prior to the synthesis of com-
pound 4d. The compounds synthesized were character-
ized by 1H and 13C NMR, IR and MS. Purity of these
compounds were verified by microanalysis, and melting
point data obtained. E and Z configurations assigned at
position 2 and 3 of the 2-propen-1-one were based on
the J values of 7.19–16.5 Hz obtained from the 1H NMR
(26).
Phagotest kit was used to determine the suppressive effect
of test compounds on PMNs phagocytic activity. This assay
was carried out on compounds 4d, 4e, 5d, 6d, 6e, 6f, 6g,
7d, 7e, 7h, 7i, 8d and 8e only, according to the protocol
given by the manufacturer (25). Briefly, heparinized whole
blood was mixed using a vortex mixture, and 100-lL aliqu-
ots were incubated in an ice bath (0 °C) for 10 min prior to
the addition of E. coli. The mixture was mixed well incubated
further at 37 °C for 10 min after the addition of E. coli
(20 lL) and test compounds (20 lL). Phagocytosis was
quenched by adding 100 lL of ice-cold quenching solution
to the mixture at the end of the incubation period. The mix-
ture was washed twice with washing solution (3 mL), and
the supernatant was discarded. The whole blood was lysed
by adding the lysing solution (2 mL), spun (250 g at 4 °C)
for 5 min, the supernatant was discarded and the sample
was washed. Finally, DNA staining solution (200 lL) was
added, the mixture was mixed and incubated for 10 min on ice.
The cell suspension was analysed using the flow cytometry
blue–green excitation light (488 nm argon-ion laser, FACS
Canto II/V96101153) within 60 min of the last procedure under-
taken. A control was used to set marker for fluorescence, and
percentage of phagocytosis above the marker was determined.
Pharmacology
The chalcone derivatives synthesized were evaluated for
their immunomodulatory properties by chemotaxis, lumi-
nol- and lucigenin-amplified chemiluminescence and
phagotest assays. These assays were carried out on
PMNs and human whole blood using ibuprofen (27) and
aspirin (28) that were used as reference compounds
based on reports on their roles in inflammatory process.
The effect of test compounds on PMNs and human whole
blood cells’ viability were assessed at concentrations of
6.25 and 100.0 lg/mL. The compounds were rendered
non-toxic if more than 90% of the cells were viable after
2 hours incubation. Structure-activity relationships (SAR) of
the derivatives were deduced based on their activities in
the assays performed.
Chemiluminescence assay
This assay was performed as described in our previous
work (24). Briefly, PMNs suspended in Hanks’ balance salt
solution (HBSS++) or blood diluted with PBS was incu-
bated with the test compounds, luminol and opsonized
zymosan in dimethyl sulphoxide (DMSO) and distilled
water. The inhibitory effect of test compounds on intracel-
lular ROS production by PMNs and blood cells was calcu-
lated from the luminometer readings. The inhibitory effect
of test compounds on extracelluar ROS production was
studied using similar procedure as described above, but
luminol was replaced with lusigenin, and the assay was
performed on human whole blood.
Inhibition of PMN chemotaxis
The inhibitory effects of the test compounds on PMNs
towards exogenous chemo-attractant, N-formyl-methionyl-
leucyl-phenylalanine (fMLP) were investigated at 10 lg/mL,
and test compounds which gave more than 50% inhibition
were investigated further at serial concentrations of 5.00,
2.50, 1.25 and 0.63 lg/mL to determine their IC50 values
(Table 1).
Statistical analysis
All data were analysed using the Statistically Package for
Social Sciences (SPSS, IBM, Armonk, NY, USA). Each sam-
ple was measured in triplicate, and the data are presented as
means Æ standard error of mean (SEM). GraphPad Prism 5
Software (GraphPad Software Inc., San Diego, CA, USA)
was used to determine the IC50 values for the active test
compounds. The values were obtained from at least three
determinations. Data were analysed using a one-way analy-
sis of variance (ANOVA) for multiple comparisons, and p < 0.05
was considered to be statistically significant.
Compounds 2a, 2b, 2c, 3b, 3c, 4a, 4d, 4e, 5c, 5d, 6d,
7d, 7e, 7i, 8d and 8e displayed strong inhibition of PMNs
migration towards fMLP with percentage inhibition ranging
from 64.2 to 80.8%. Compound 4d was the most active
with 80.0% inhibition but with a moderate IC50 value of
9.5 lM. Compounds 3b and 7i on the other hand dis-
played strong inhibition, 79.1 and 80.0%, respectively, and
IC50 values (6.7 and 7.5 lM) comparable to that of ibupro-
fen. Moreover, compounds 2c, 4a, 4d, 4e, 5c, 8d and 8e
exhibited strong inhibition than control (>68%) but have
moderate IC50 values (8.7-26.4 lM).
Result and Discussion
The presence of aromatic ring A did not govern inhibitory
activity of the test compounds as lack of activity was
observed in series 1 despite having pyridine ring in their
structures. The position of hydroxyl group on ring B also
Chemistry
Claisen–Schmidt base-catalysed condensation reaction
was employed to synthesize five series of chalcone deriva-
202
Chem Biol Drug Des 2014; 83: 198–206

Immunomodulatory Effects of Chalcones
Table 1: Percentage inhibition at 10 lg/mL and IC50 values (lM)
of chalcone derivatives on PMNs chemotaxis
Table 2: Percentage of PMNs phagocytic activity at 100 and
6.25 lg/mL of chalcone derivatives
Compounds
Mean Æ SEM
IC50 values (lM)
Mean Æ SEM
1a
35.2 Æ 1.4
36.8 Æ 2.2
44.3 Æ 0.8
76.6 Æ 1.4
64.2 Æ 1.7
69.2 Æ 0.8
39.2 Æ 0.8
79.1 Æ 2.5
64.9 Æ 1.7
80.0 Æ 1.4
53.4 Æ 1.4
30.9 Æ 2.9
80.8 Æ 0.8
70.8 Æ 0.8
54.2 Æ 0.8
77.4 Æ 0.8
67.5 Æ 2.5
65.0 Æ 1.4
54.2 Æ 0.8
53.3 Æ 1.7
48.3 Æ 3.6
65.8 Æ 0.8
65.0 Æ 1.4
42.5 Æ 2.9
80.0 Æ 1.4
74.2 Æ 0.8
79.2 Æ 0.8
65.8 Æ 2.20
–
–
–
Compounds
100 lg/mL
6.25 lg/mL
1b
1c
4d
4e
21.43 Æ 1.58
31.73 Æ 0.39
36.45 Æ 0.39
51.91 Æ 1.73
47.63 Æ 0.39
63.90 Æ 1.25
42.59 Æ 0.48
46.18 Æ 0.77
44.15 Æ 2.19
50.96 Æ 0.57
24.62 Æ 0.48
19.12 Æ 3.85
39.34 Æ 3.91
97.43 Æ 1.43
86.53 Æ 0.93
89.03 Æ 0.93
88.93 Æ 0.93
86.93 Æ 0.93
90.73 Æ 0.93
87.13 Æ 0.93
90.43 Æ 0.93
89.83 Æ 0.93
91.23 Æ 0.93
89.63 Æ 0.93
85.23 Æ 0.93
93.43 Æ 0.93
85.13 Æ 0.93
2a
10.7 Æ 0.9
14.9 Æ 1.0
26.4 Æ 0.6
–
6.7 Æ 0.3
17.8 Æ 2.4
12.2 Æ 0.6
35.1 Æ 1.2
–
9.5 Æ 0.3
12.7 Æ 0.8
32.0 Æ 1.7
9.6 Æ 1.3
11.0 Æ 0.5
15.3 Æ 1.2
21.5 Æ 1.2
30.0 Æ 3.2
–
9.9 Æ 1.1
12.3 Æ 0.4
–
7.5 Æ 0.4
10.5 Æ 0.3
8.7 Æ 0.3
6.6 Æ 0.8
5d
6d
2b
2c
6e
3a
6f
6 g
3b
3c
7d
7e
4a
4b
4c
7 h
7i
8d
4d
4e
8e
Negative control
5b
5c
5d
6d
6e
Values are presented as mean Æ SD (n = 3).
6f
6 g
7d
7e
observed on test compounds containing this functional
group.
7 h
7i
Inhibition of intracellular and extracellular ROS
productions
8d
8e
Ibuprofen
ROS productions in PMNs and human whole blood were
activated by serum opsonized zymosan (SOZ), and the
ability of the test compounds to inhibit this process was
investigated using a luminol and a lucigenin probes. Lumi-
nol and lucigenin were used to detect the presence of
intracellular and extracellular ROS, respectively. Test com-
pounds which showed percentage inhibition of more than
50% were investigated further at serial concentrations of
12.5, 6.25, 3.13, 1.56 and 0.78 lg/mL to determine their
IC50 values (Table 3).
Values are presented as mean Æ SD (n = 3). (–): IC50 values were
not determined.
was not important as inhibitory activity was observed at
ortho, meta and para substitutions (compounds 2a, 2b,
2c, 3b, 3c, 4a and 5c). On the other hand, 4-methylami-
noethanol and 2,3-dimethoxy substituents on ring B are
viewed important as most compounds with these func-
tional groups displayed strong inhibitory activity towards
PMNs chemotaxis.
In general, these chalcone derivatives were more active
inhibiting both intracellular and extracellular ROS produc-
tions in human whole blood rather than in PMNs. Com-
pounds 4a, 4b, 4d, 7d, 7e, 7i, 8d and 8e showed
lower or comparable IC50 values with controls on intra-
cellular ROS production in human whole blood inhibition
while only compound 4a, 7i and 8d were active in
PMNs. Only five series of test compounds were assayed
for inhibitory effect on extracellular ROS production, and
most of the compounds tested displayed lower IC50
values than that of controls both in human whole blood
and in PMNs. Among all the compounds tested, com-
pound 7i was the most active in inhibiting intracellular
and extracellular ROS productions both in human whole
blood and PMNs.
Phagocytic activity of PMNs
The ability of the test compounds to inhibit the ingestion of
FITC-labelled E. coli suspension by PMNs at the concen-
trations of 100.0 and 6.25 lg/mL was investigated. The
suppressive effect of the test compounds was dose
dependent as the effect was only observed at higher
concentration (100 lg/mL) of the compounds. Strong sup-
pressive effect on FITC-labelled E. coli suspension inges-
tion by PMNs was displayed by compounds 4d and 8d
with phagocytic activity of 21.43% and 19.12%, respec-
tively (Table 2).
Compounds which bore dimethoxy group on ring
B
showed strong suppressive effect on PMNs phagocytic
activity. On the hand, naphthalene group on ring A was
not favourable as only moderate inhibitory activity was
It is gathered from this study that 4-methylaminoethanol
and 2,3-dimethoxy substituents on ring B were favourable
in inhibiting human whole blood extracellular ROS produc-
Chem Biol Drug Des 2014; 83: 198–206
203

Bukhari et al.
Table 3: Inhibitory activity of the synthetic chalcone derivatives
on intracellular and extracellular ROS productions
Intracellular IC50 values
Extracellular IC50 values
(lM)
(lM)
Whole
Whole
Compounds blood
PMNs
blood
PMNs
1a
1b
74.7 Æ 3.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Figure 1: Summary of chalcone derivatives SAR on the assays
carried out.
1c
2a
42.3 Æ 2.0 32.3 Æ 0.4
2b
2c
–
–
–
–
Table 4: Physicochemical properties of compound 8d
3a
3b
25.7 Æ 1.3 13.3 Æ 0.8
32.8 Æ 1.7 25.6 Æ 1.3
33.8 Æ 1.5 24.9 Æ 1.1
Optimal range
(29,30)
Values for
compound 8da
Properties
3c
4a
10.9 Æ 1.0
6.2 Æ 0.6
MW
ClogP
<500
<5
<5
<10
<140
371.43
3.31
1
4b
4c
18.2 Æ 2.0 11.7 Æ 1.2
27.5 Æ 2.1 20.2 Æ 1.5
H-bond donors
H-bond acceptors
Polar surface area (PSA) [A ]
4d
4e
14.6 Æ 0.8 13.6 Æ 0.0 16.4 Æ 2.9 17.5 Æ 3.0
26.7 Æ 1.4 19.4 Æ 0.9 18.5 Æ 0.8 16.3 Æ 0.7
6
ꢀ2
68.24
5b
5c
63.9 Æ 4.0 49.1 Æ 3.0
41.4 Æ 0.7 31.5 Æ 0.5
–
–
–
–
aCalculated with Molinspiration property engine v.2011.04 (http://
www.molinspiration.com).
5d
6d
30.5 Æ 1.6 19.7 Æ 0.9 70.1 Æ 6.4 61.8 Æ 5.6
30.0 Æ 1.3 28.1 Æ 2.1 19.9 Æ 2.0 16.4 Æ 1.6
6e
30.3 Æ 1.8 25.6 Æ 1.1
–
–
34.2 Æ 9.2
both intracellular and extracellular ROS productions were
inhibited exclusively only by compound containing a 4-dieth-
ylamine substituent. Furthermore, an absence of methyl
group on furan in ring A diminished activities of the com-
pound altogether as observed in 5d (absence of methyl
group) in contrast to 4d (with methyl group).
6f
6g
–
–
–
23.7 Æ 5.9 16.5 Æ 3.2 22.2 Æ 2.9 25.7 Æ 3.3
7d
7e
14.5 Æ 0.3 13.5 Æ 1.0 8.6 Æ 0.3 7.0 Æ 0.2
12.2 Æ 1.3 14.8 Æ 0.8 14.9 Æ 6.7 12.1 Æ 5.5
7h
–
–
–
–
7i
8d
6.9 Æ 0.7
4.8 Æ 0.5 7.3 Æ 1.1
17.6 Æ 2.5
17.3 Æ 0.9
9.6 Æ 0.1 10.9 Æ 1.1 12.5 Æ 1.3
8e
14.3 Æ 4.1 13.4 Æ 4.2 12.6 Æ 1.4 10.2 Æ 1.1
Compound 8d was found to have high potency in most of the
assays carried out. It can be depicted from its physicochemi-
cal properties (Table 4) that this compound has potential to
be a new lead compound in developing inhibitors of neu-
trophils’ chemotaxis, phagocytosis and ROS production.
Aspirin
Negative
control
18.7 Æ 0.2
9.6 Æ 0.1 50.2 Æ 0.8
–
0.0
Values are presented as mean Æ SD (n = 3). (–): IC50 values were
not determined.
Conclusion
tion as the activity was retained except for compound 5d,
regardless of functional groups on ring A. Moreover, inhibi-
tory activities of the test compounds diminished with
naphthalene or furan functional groups on ring A.
Five series of chalcone derivatives were successfully
synthesized using the Claisen–Schmidt base-catalysed
condensation reaction. It was observed that phenyl-4-
methylaminoethanol and dimethoxy substituents contribute
to the inhibition of PMNs chemotaxis towards fMLP,
suppressive effect of phagocytic activity of phagocytes
and preventing intracellular and extracellular ROS produc-
tions. Compounds 4d and 8d that posses the 4-methyla-
minoethanol functional group were active in all the four
assays performed. This active compound adds to the res-
ervoir of potential immunomodulatory and anti-inflamma-
tory agents worth investigating.
Summary of SAR
Overall, size of the diaryl rings has little influence on the
activities of the test compounds as weak or strong inhibi-
tions were observed in both bulky substituents (naphthalene
and diphenyl) and small groups (thiophene and furan). Nev-
ertheless, inhibitory activities at different stages of the
immune response were observed with variation of substitu-
ents on the diaryl rings (Figure 1). Migration of PMNs
towards chemo-attractant was mainly inhibited by the pres-
ence of hydroxyl, 4-methylaminoethanol, dimethoxy and 4-
diethylamine groups in ring B. On the other hand, phagocy-
tic activity of PMNs were suppressed primarily only by com-
pounds, which bore trimethoxy and methyl-furan groups in
ring A and 4-methylaminoethanol group in ring B. Moreover,
Acknowledgments
The authors would like to thank the University Kebangsaan
Malaysia and the Ministry of Science, Technology and
204
Chem Biol Drug Des 2014; 83: 198–206

Immunomodulatory Effects of Chalcones
Innovation, Malaysia for the financial support (Grants no.:
UKM-GUP-2011-015 and 02-01-02-SF0016).
cones as potential anti-inflammatory and cancer
chemopreventive agents. Eur J Med Chem;40:103–
112.
15. Nowakowska Z., Kedzia B., Schroeder G. (2008) Syn-
z
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