Synthesis and anticancer activities of 4-[(Halophenyl)diazenyl]phenol and 4-[(Halophenyl)diazenyl]phenyl aspirinate derivatives against nasopharyngeal cancer cell lines

Article abstract of DOI:10.1155/2017/6760413

Aspirin and azo derivatives have been widely studied and have drawn considerable attention due to diverse biological activities. In this study, a series of 4-[(halophenyl)diazenyl]phenyl aspirinate derivatives were synthesized from the reaction of aspirin

Full text of DOI:10.1155/2017/6760413

Hindawi
Journal of Chemistry
Volume 2017, Article ID 6760413, 7 pages
https://doi.org/10.1155/2017/6760413
Research Article
Synthesis and Anticancer Activities of
4-[(Halophenyl)diazenyl]phenol and
4-[(Halophenyl)diazenyl]phenyl Aspirinate Derivatives
against Nasopharyngeal Cancer Cell Lines
Boon Kui Ho,1 Zainab Ngaini,1 Paul Matthew Neilsen,2,3 Siaw San Hwang,2
Reagan Entigu Linton,2 Ee Ling Kong,2 and Boon Kiat Lee2
1Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota
Samarahan, Sarawak, Malaysia
2Cancer Research and Advanced erapeutic Group (CREATE), Faculty of Engineering, Computing and Science,
Swinburne University of Technology Sarawak Campus, Jalan Simpang Tiga, 93350 Kuching, Sarawak, Malaysia
3School of Health, Medical and Applied Sciences, Central Queensland University, North Rockhampton, QLD 4702, Australia
Correspondence should be addressed to Zainab Ngaini; nzainab@unimas.my
Received 15 February 2017; Accepted 18 June 2017; Published 25 July 2017
Academic Editor: Liviu Mitu
Copyright © 2017 Boon Kui Ho et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aspirin and azo derivatives have been widely studied and have drawn considerable attention due to diverse biological activities.
In this study, a series of 4-[(halophenyl)diazenyl]phenyl aspirinate derivatives were synthesized from the reaction of aspirin
with 4-[(halophenyl)diazenyl]phenol via esterification, in the presence of DCC/DMAP in DCM with overall yield of 45–54%. 4-
[(Halophenyl)diazenyl]phenol was prepared prior to esterification from coupling reaction of aniline derivatives and phenol in basic
solution. All compounds were characterized using elemental analysis, FTIR, and 1H and 13C NMR spectroscopies. All compounds
were screened for their anticancer activities against nasopharyngeal cancer (NPC) HK-1 cell lines and the viability of cultured
cells was determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-
based colorimetric assay. 4-[(E)-(Fluorophenyl)diazenyl]phenol showed the highest anticancer activity against NPC HK-1 cell lines
compared to other synthesized compounds. 4-[(Halophenyl)diazenyl]phenyl aspirinate showed low cytotoxicity against NPC HK-1
cell lines compared to 4-[(halophenyl)diazenyl]phenol but better anticancer activity than aspirin alone.
1. Introduction
therapeutic strategy to inhibit the HK-1 cells [6]. e usage of
drugs could stop the growth of the malignant cells in one’s
body by inhibiting its EGFR pathway of the cancer cells itself
[7].
e development of new drug for cancer treatment has
gained much interest in recent years. e modification of
pharmacologically active compounds may lead to a new
finding of novel drugs with diverse pharmacological activities
[8]. Chemical modification of aspirin, for instance, has been
reported to reduce gastrointestinal toxicity and exhibited
diverse biological activities such as anticancer [9], anti-
inflammatory [10], and antibacterial [11, 12] activities. Aspirin
Cancer has become a serious clinical problem worldwide [1].
It is because cancer cells have the ability to grow rapidly and
develop into malignancy tumor [2]. Nasopharyngeal cancer
is the most common malignant tumor and occurs in the
nasopharynx, the upper part of the throat near to the neck [3].
Nasopharyngeal cancer (NPC) is commonly developed from
infection with Epstein-Barr virus, foods, and family history
[4]. NPC, which also known as HK-1, was found to have high
level of epidermal growth factor receptor (EGFR) protein [5].
Hence, by targeting the EGFR protein, it might become a new

2
Journal of Chemistry
bearing active functional groups such as diethyl phosphate
and nitro groups has been reported to have anticancer activity
against pancreatic and colon cancers [13, 14].
11.98% C H N OCl Requires C, 61.94; H, 3.87; N, 12.04%);
12
9
2
ꢀꢀ 0.57 (1 : 4 EA/hexane) IR: Vmax (thin film/cm−1) 3186 (OH),
1587 (C=C aromatic), 1474 (N=N), 1221 (C-N), 1080 (C-O),
1
836 (C-Cl). H NMR (500 MHz, CDCl ) ppm 5.46 (1H, s,
Azo is another active compound reported for its pharma-
ceutical importance as antidiabetic [15], antineoplastic [16],
antibacterial [17], and anticancer [18] agent. Azo molecules
are known to be involved in the inhibition of DNA, RNA,
carcinogenesis, and protein synthesis [19]. e presence of
-N=N- in the molecular structure of azo is responsible for
the interaction with the active site of protein [20]. e
presence of halogen substituents in azo molecule also played
an important role in the inhibition of receptor enzyme [21].
In this study, a series of azo derivatives (1a–d) and aspirin
azo derivatives (2a–d) have been synthesized and demon-
strated for potential anticancer agent against nasopharyngeal
cancer cell lines. e viability of the cultured cells was
determined by MTS-based colorimetric assay and discussed.
3
OH) 6.96 (2H, d, ꢁ = 8 Hz, Ar-H
耠
) 7.46 (2H, d, ꢁ = 8 Hz, Ar-
3
H
耠
) 7.82 (2H, d, ꢁ = 8 Hz, Ar-H ) 7.86 (2H, d, ꢁ = 8 Hz, Ar-
2
2
H ) 13C NMR (125 MHz, CDCl ) 116.0 (Ar-C
耠
), 123.98 (Ar-
3
3
3
C
耠
), 125.25 (Ar-C ), 129.42 (Ar-C ), 136.46 (Ar-C ), 147.17
2
2
3
4
(Ar-C
耠
), 151.42 (Ar-C ), 158.76 (Ar-C
耠
).
1
1
4
4-[(E)-(Bromophenyl)diazenyl]phenol (1c) (0.89 g, 64%)
as red solid; m.p. 165-166∘C. (Found: C, 51.20; H, 3.16; N,
10.05% C H N OBr Requires C, 51.99; H, 3.25; N, 10.11%); ꢀꢀ
12
9
2
0.56 (1 : 4 EA/hexane) IR: Vmax (thin film/cm−1) 3076 (OH),
1588 (C=C aromatic), 1473 (N=N), 1225 (C-N), 1101 (C-O),
1
833 (C-Br). H NMR (500 MHz, CDCl ) ppm 5.54 (1H, s,
3
OH) 6.94 (2H, d, ꢁ = 8.6 Hz, Ar-H
耠
) 7.62 (2H, d, ꢁ = 8.6 Hz
3
Ar-H
耠
) 7.74 (2H, d, ꢁ = 8.6 Hz, Ar-H ) 7.86 (2H, d, ꢁ =
2
2
8.6 Hz, Ar-H ). 13C NMR (125 MHz, CDCl ) ppm 116.02 (Ar-
3
3
2. Materials and Methods
C
耠
), 124.23 (Ar-C
耠
), 124.79 (Ar-C ), 125.28 (Ar-C ), 132.39
3
2
4
2
(Ar-C ), 147.10 (Ar-C
耠
), 151.48 (Ar-C ), 158.76 (Ar-C
耠
).
2.1. Materials. Chemical agents used in the present study in-
cluded dimethyl sulfoxide (DMSO, Sigma-Aldrich), penicil-
lin-streptomycin (GIBCO), fetal bovine serum (FBS, Sigma-
Aldrich), and RPM1 1640 (GIBCO).
3
1
1
4
4-[(E)-(Iodophenyl)diazenyl]phenol (1d) (0.68 g, 42%) as
purple solid; m.p. 168–170∘C. (Found: C, 44.06; H, 2.76; N,
8.44% C H N OI Requires C, 44.44; H, 2.78; N, 8.64%); ꢀꢀ
12
9
2
0.54 (1 : 4 EA/hexane) IR: Vmax (thin film/cm−1) 3172 (OH),
2.2. Characterizations. Melting point was measured by Stuart
SMP3 melting point apparatus and the elemental analysis
was determined by flash EA1112 analyzer. IR spectra (v/cm−1)
were recorded on a Perkin Elmer GX spectrophotometer able
to show infrared spectra of functional groups, potassium
1591 (C=C aromatic), 1471 (N=N), 1242 (C-N), 1074 (C-O),
1
837 (C-I) H NMR (500 MHz, DMSO-d ) ppm 6.93 (2H,
6
d, ꢁ = 8.6 Hz, CH, Ar-H
耠
) 7.58 (2H, d, ꢁ = 8 Hz, CH,
3
Ar-H ) 7.79 (2H, d, ꢁ = 8.6 Hz, CH, Ar-H
耠
) 7.92 (2H, d,
2
1
2
ꢁ = 8 Hz, CH, Ar-H ) 10.41 (1H, s, OH) 13C NMR (125 MHz,
1
3
bromide pellet (KBr) solid material. H NMR spectra were
DMSO-d ) ppm 97.45 (Ar-C ), 116.07 (Ar-C
耠
), 124.06 (Ar-
6
4
3
recorded on a JEOL.ECA 500 spectrometer at 500 MHz and
13C NMR at 125 MHz. Tetramethylsilance (TMS) was used as
a reference for both spectra while CDCl and DMSO-d were
C
耠
), 125.11 (Ar-C ), 138.29 (Ar-C ), 145.15 (Ar-C
耠
), 151.46
2
2
3
1
(Ar-C ), 161.32 (Ar-C
耠
).
4
3
6
used as solvents.
2.4. General Method for the Synthesis of Aspirin-Halogenated
Azo Derivatives 2a–d. Azo (1a) (0.65 g, 3 mmol) was dis-
solved in dry DCM (20 mL) and added to a round bottom
flask containing acetylsalicylic acid (0.54 g, 3 mmol) in dry
DCM (20 mL) at 0–10∘C. DCC (0.62 g, 3 mmol) and DMAP
(0.37 g, 3 mmol) were added to the mixture and stirred for 4 h
at 0–10∘C. e mixture was filtered and evaporated. e crude
was recrystallized from hot ethanol to give (2a) (0.58 g, 51%)
as pale yellow solid; m.p. 147-148∘C. (Found: C, 66.65; H, 3.96;
N, 7.22% C H N O F Requires C, 66.67; H, 3.97; N, 7.41%);
2.3. General Method for the Synthesis of Halogenated Azo
Compounds 1a–d. A mixture of 4-fluoroaniline (0.56 g,
5 mmol) and HCl (2 M, 6 mL) was added slowly to NaNO
2
in water (1 M, 2 mL) at 0–5∘C. Phenol (0.47 g, 5 mmol) was
dissolved in NaOH (2 M, 10 mL) and cooled to 0–5∘C. e
diazo salt was added slowly to phenol solution at 0–5∘C and
stirred for 40 min. HCl (2 M, 10 mL) was added to form pre-
cipitate and filtered and washed with cold water (2 × 20 mL).
e crude product was recrystallized from hot ethanol to give
(1a) (0.56 g, 52%) as purple solid; m.p. 158-159∘C. (Found:
C, 66.58; H, 4.12; N, 12.80% C H N OF Requires C 66.67;
21 15
2
4
ꢀꢀ 0.62 (1 : 4 EA/hexane) IR: Vmax (thin film/cm−1) 1755, 1745
(C=O), 1590 (C=C aromatic), 1482 (N=N), 1222 (C-N), 1184
1
12
9
2
(C-O), 841 (C-F) H NMR (500 MHz, DMSO-d ) ppm 2.27
6
H, 4.17; N, 12.96%); ꢀꢀ 0.54 (1 : 4 EA/hexane) IR: Vmax (thin
(3H, s, -CH ) 7.35 (1H, d, ꢁ = 7.80 Hz, Ar-H 耠耠 ) 7.44 (2H, d,
3
5
film/cm−1) 3135 (OH), 1591 (C=C aromatic), 1471 (N=N), 1235
ꢁ = 8.60 Hz, Ar-H ) 7.47 (2H, d, ꢁ = 8.60 Hz, Ar-H
耠
) 7.51
3
3
1
(C-N), 1102 (C-O), 839 (C-F) H NMR (500 MHz, DMSO-
(1H, t, ꢁ = 7.55 Hz, Ar-H 耠耠 ) 7.79 (1H, t, ꢁ = 7.8 Hz, Ar-H 耠耠
)
)
4
3
d ) ppm 6.94 (2H, d, ꢁ = 6.85 Hz, Ar-H
耠
) 7.37 (2H, d, ꢁ =
7. 98–8. 01 (4H, m, Ar-H 耠 ) 8.20 (1H, d, ꢁ = 7.80 Hz, Ar-H 耠耠
6
3
2,2
2
13C NMR (125 MHz, DMSO-d ) 20.73 (-CH ), 116.43 (Ar-
8.78 Hz, Ar-H ) 7.78–7.88 (4H, m, Ar-H 耠 ) 10.32 (1H, s, OH)
3
2,2
6
3
13C NMR (125 MHz, CDCl ) 116.00 (Ar-C ), 116.21 (Ar-C
耠
),
C ), 116.61 (Ar-C 耠耠 ), 121.98 (Ar-C
耠
), 122.98 (Ar-C
耠
), 123.98
3
3
3
3
1
3
2
124.58 (Ar-C
耠
), 124.65 (Ar-C ), 125.13 (Ar-C
耠
), 147.02 (Ar-
(Ar-C 耠耠 ), 124.23 (Ar-C ), 125.03 (Ar-C 耠耠 ), 126.57 (Ar-C 耠耠 ),
2
2
1
5
2
3
2
C ), 149.32 (Ar-C
耠
), 158.63 (Ar-C ). e others, 1b–d, were
131.87 (Ar-C 耠耠 ), 135.33 (Ar-C
耠
), 148.57 (Ar-C ), 149.70 (Ar-
1
4
4
4
1
1
synthesized by repeating the same procedure as 1a.
C
耠耠 ), 150.49 (Ar-C
耠
), 152.43 (Ar-C ), 162.41 (Ca=O), 169.24
6
4
4
4-[(E)-(Chlorophenyl)diazenyl]phenol (1b) (0.62 g, 53%)
(Cb=O). e others, 2b–d, were synthesized following the
same procedure as 2a with yields given below.
as yellow solid; m.p. 154-155∘C. (Found: C, 61.68; H, 3.86; N,

Journal of Chemistry
3
4-[(E)-(Chlorophenyl)diazenyl]phenyl aspirinate (2b)
(0.54 g, 46%) as orange solid; m.p. 161-162∘C. (Found: C,
63.58; H, 3.73 N, 7.44% C H N O Cl Requires C, 63.88;
of 0.5 ꢂL (0.1% DMSO) was added with cells as a control.
e final drug concentrations were ranging from 0.39 to
50 ꢂM at 200 ꢂL. e cells were incubated for 72 h at 37∘C.
21 15
2
4
H, 3.80; N, 7.10%); ꢀꢀ 0.64 (1 : 4 EA/hexane) IR: Vmax
Afer incubation, the media were discarded from each well
and added with 10 ꢂL MTS solution and 50 ꢂL fresh media.
e plates were further incubated for 30 minutes at 37∘C.
e absorbance was measured by a multiwell plate reader at
490 nm. is experiment was performed in triplicate [22].
(thin film/cm−1) 1766, 1740 (C=O), 1588 (C=C aromatic),
1
1479 (N=N), 1246 (C-N), 1182 (C-O), 833 (C-Cl) H NMR
(500 MHz, DMSO-d ) ppm 2.27 (3H, s, -CH ) 7.35 (1H, d,
6
3
ꢁ = 8.6 Hz, Ar-H 耠耠 ) 7.49 (2H, d, ꢁ = 9.2 Hz, Ar-H
耠
) 7.53 (1H,
5
3
t, ꢁ = 7.5 Hz, Ar-H 耠耠 ) 7.68 (2H, d, ꢁ = 8.6 Hz, Ar-H ) 7.81
4
3
2.6. Statistical Analyses. e quantitative data are expressed
(1H, t, ꢁ = 7.7 Hz, Ar-H 耠耠 ) 7.93 (2H, d, ꢁ = 9.2 Hz, Ar-H ) 8.01
3
2
13
as mean
standard deviation. e cell viability against
(2H, d, ꢁ = 8.6 Hz, Ar-H
耠
) 8.20 (1H, d, ꢁ = 8 Hz, Ar-H 耠耠
)
C
2
2
log IC values of all compounds was determined using
NMR (125 MHz, DMSO-d ) 20.74 (-CH ), 121.96 (Ar-C 耠耠 ),
50
6
3
1
Graphpad prism 6 program.
123.04 (Ar-C
耠
), 124.14 (Ar-C
耠
), 124.23 (Ar-C ), 124.31 (Ar-
3
2
2
C
耠耠 ), 126.57 (Ar-C 耠耠 ), 128.78 (Ar-C ), 129.67 (Ar-C 耠耠 ), 131.88
5
3
3
2
(Ar-C 耠耠 ), 134.99 (Ar-C ), 136.09 (Ar-C
耠
), 149.71 (Ar-C ),
4
4
1
1
3. Results and Discussion
150.50 (Ar-C 耠耠 ), 152.50 (Ar-C
耠
), 162.38 (Ca=O), 169.35
6
4
e synthesis of 4-[(halophenyl)diazenyl]phenyl aspirinate
(2a–d) was carried out via esterification of aspirin and
4-[(halophenyl)diazenyl]phenol (1a–d) in the presence of
DCC/DMAP in DCM. 4-[(Halophenyl)diazenyl]phenol
(1a–d) was earlier synthesized from the reaction of ani-
line derivatives and phenol via direct coupling reaction. e
synthesis of 2a–d is depicted in Scheme 1. Characteristic
spectroscopic data of the synthesised compounds are pro-
vided in the supplementary data in Supplementary Mate-
rial available online at https://doi.org/10.1155/2017/6760413.
(Cb=O).
4-[(E)-(Bromophenyl)diazenyl]phenyl aspirinate (2c)
(0.59 g, 45%) as yellow solid; m.p. 149–151∘C. (Found: C,
57.31; H, 3.39; N, 6.02% C H N O Br Requires C, 57.40;
21 15
2
4
H, 3.42; N, 6.38%); ꢀꢀ 0.62 (1 : 4 EA/hexane) IR: Vmax
(thin film/cm−1) 1763, 1734 (C=O), 1581 (C=C aromatic),
1
1477 (N=N), 1250 (C-N), 1179 (C-O), 829 (C-Br) H NMR
(500 MHz, DMSO-d ) ppm 2.27 (3H, s, -CH ) 7.34 (1H, d,
6
3
ꢁ = 8.6 Hz, Ar-H 耠耠 ) 7.48 (2H, d, ꢁ = 9.15 Hz, Ar-H
耠
) 7.51 (1H,
5
3
t, ꢁ = 8.3 Hz, Ar-H 耠耠 ) 7.79–7.87 (5H, m, Ar-H 耠耠 ) 8.01 (2H,
4
2,3,3
All compounds were characterized using CHN, FTIR,
13
d, ꢁ = 6.9 Hz, Ar-H
耠
) 8.20 (1H, d, ꢁ = 9.15 Hz, Ar-H 耠耠
)
C
2
2
1
and H and 13C NMR spectroscopy. FTIR spectra of 1a–d
NMR (125 MHz, DMSO-d ) 20.78 (-CH ), 121.91 (Ar-C 耠耠 ),
6
3
1
and 2a–d showed the absorption peaks at 1474–1471 cm−1
123.09 (Ar-C
耠
), 124.20 (Ar-C
耠
), 124.26 (Ar-C 耠耠 ), 124.55 (Ar-
3
2
5
and 1591–1587 cm−1 attributed to V(-N=N-) and V(C=C)
,
C ), 125.14 (Ar-C ), 126.62 (Ar-C 耠耠 ), 131.92 (Ar-C 耠耠 ), 132.耠6耠 6
2
4
3
2
respectively. e formation of 2a–d was confirmed by the
disappearance of V(OH) peak at 3186–3076 cm−1 and the
appearance of new absorption peaks at 1765–1745 cm−1
corresponded to V(C=O) ester. e absorption frequencies
at 841–826 cm−1 in both FTIR spectra of 1a–d and 2a–d
corresponded to para-halogenated substitution [23].
(Ar-C ), 135.34 (Ar-C 耠耠 ), 149.67 (Ar-C
耠
), 150.12 (Ar-C ),
3
4
1
6
150.63 (Ar-C
耠
), 152.31 (Ar-C ), 162.38 (Ca=O), 169.31 (Cb=O).
4
1
3-[(E)-(Iodophenyl)diazenyl]phenyl aspirinate (2d)
(0.79 g, 54%) as orange solid; m.p. 180-181∘C. (Found: C,
51.63; H, 3.06; N, 5.43% C H N O I Requires C, 51.85;
21 15
2
4
H, 3.09; N, 5.76%); ꢀꢀ 0.63 (1 : 4 EA/hexane) IR: Vmax
1H NMR spectra of 1a–d and 2a–d showed the presence
(thin film/cm−1) 1764, 1732 (C=O), 1578 (C=C aromatic),
1
of aromatic protons at 8.20–6.93 ppm, while CH in 2a–d was
1475 (N=N), 1250 (C-N), 1176 (C-O), 826 (C-I) H NMR
3
observed at 2.27 ppm. 13C NMR spectra of 1a–d and 2a–d
(500 MHz, DMSO-d ) 2.27 (3H, s, -CH ) ppm 7.35 (1H, d,
6
3
showed resonance of aromatic carbons at 152.7–99.4 ppm.
ꢁ = 8.0 Hz, Ar-H 耠耠 ) 7.48 (2H, d, ꢁ = 8.60 Hz, Ar-H
耠
) 7.51 (1H,
5
3
e presence of C=O and CH in 2a–d was observed at
t, ꢁ = 7.45 Hz, Ar-H 耠耠 ) 7.69 (2H, d, ꢁ = 8.0 Hz, Ar-H ) 7.79
3
4
2
169.4–162.4 ppm and 20.8–20.7 ppm, respectively.
(1H, t, ꢁ = 8.0 Hz, Ar-H 耠耠 ). 7.99–8.03 (4H, m, Ar-H 耠 ) 8.20
3
3,2
(1H, d, ꢁ = 8.0 Hz, Ar-H 耠耠
)
13C NMR (125 MHz, DMSO-d )
2
6
3.1. Anticancer Activity. Compounds 1a–d and 2a–d were
screened for anticancer activity against NPC HK-1 cell lines.
e anticancer activities of 1a–d and 2a–d on NPC HK-
1 cell lines were studied by MTS based on colorimetric
assay. e cells were seeded onto 96-well culture plates and
incubated with different concentrations of compounds (up
to 50 ꢂM) for 72 h. e relative influence of 1a–d and 2a–d
was determined via viability of the NPC cell line against
20.78 (-CH ), 99.40 (Ar-C ), 121.96 (Ar-C 耠耠 ), 123.08 (Ar-
3
4
1
C
耠
), 124.18 (Ar-C
耠
), 124.48 (Ar-C 耠耠 ), 126.62 (Ar-C ), 131.92
3
2
5
2
(Ar-C 耠耠 ), 135.33 (Ar-C 耠耠 ), 137.56 (Ar-C 耠耠 ), 138.54 (Ar-C ),
3
2
4
3
149.60 (Ar-C
耠
), 150.43 (Ar-C 耠耠 ), 151.11 (Ar-C
耠
), 152.72
1
6
4
(Ar-C ), 162.21 (Ca=O), 169.17 (Cb=O).
1
2.5. Cell Culture. e cell viability of nasopharyngeal cancer
cell lines HK-1 was determined by MTS-based colorimetric
assay. e cells were kept at 37∘C in a 5% CO in RPMI 1640
log [concentration, ꢂM] as shown in Figure 1. e IC
50
2
(GIBCO) supplemented with 10% fetal bovine serum (FBS;
Sigma-Aldrich). e NPC cells were assayed with a volume of
of 1a–d and 2a–d is summarized in Table 1. Among four
halogen substituents of 1a–d, 1a substituted with fluorine
100 ꢂL to each well plate at a concentration of 5000 cells/well.
e drug dissolved in DMSO and a compound dilution 96-
well plate was prepared at different concentrations of which
100 ꢂL/well of the diluted mixtures was then added. A volume
showed good anticancer activity against HK-1 cell lines with
IC of 19.1 0.9 ꢂM. In comparison to IC of positive
50
50
control (standard drug), 5-fluorouracil, and cisplatin with
12.5 1.4 ꢂM and 8.9 1.9 ꢂM, respectively, IC of 1a
50

4
Journal of Chemistry
3
4
R
2
OH
．（2
1
3
N
N
HCl, ．；．／2
NaOH, 0–5∘C
2
2
+
1
2
3
4
1a–d
R
HO
3
O
O
OH
DCC, DMAP
DCM, 0–10∘C
O
3
2
2
4
R
1
3
N
2
1
3
N
O
2
4
1
3
4
2
a
O
3
6
2a, ２ = ＆
2b, ２ = ＃Ｆ
2c, ２ = ＂Ｌ
2d, ２ = ）
O
5
b
O
2a–d
Scheme 1: Synthesis of 1a–d and 2a–d.
Table 1: IC of 1a–d and 2a–d, aspirin, 5-fluorouracil, and cisplatin
was comparable to 5-fluorouracil. Compound 1a–d bearing
halogen substituents has good lipophilic property and thus
improved the binding properties towards the active site of
cells enzyme [21, 24]. Among all halogens, fluorine is the most
selectively bound halogen to specific enzyme and remains
covalently and irreversibly bound to the active site of enzyme,
hence providing good anticancer activity [25]. In addition,
the presence of -N=N- group in the molecular structure has
also contributed to anticancer activity by interaction with the
active site of protein via hydrogen bonding resulting in the
inhibition growth of cells [20]. e presence of phenyl ring
is also important to provide more lipophilic character [26].
Compound with high lipophilicity has potential to inhibit
proteins for cell growth [27].
Anticancer activity of 2a–d against NPC HK-1 cell lines
only showed 2a (38.7 0.8 ꢂM) and 2d (44.9 5.4 ꢂM)
with average anticancer activity compared to the aspirin
alone. e presence of azo moiety in the molecular structure
has contributed to the anticancer activity [20]. e weak
inhibition on NPC HK-1 cell lines on 2c-2d was due to
higher molecular weight with larger size of bromine and
50
against NPC HK-1 cells lines.
Compound
Aspirin
cisplatin
5-Fluorouracil
1a
1b
1c
1d
2a
2b
2c
2d
IC (ꢂM)
50
>50
8.9 3.2
12.5 2.5
19.1 1.5
36.7 8.2
21.9 7.3
20.8 6.6
38.7 1.5
44.9 9.4
>50
>50
iodine atoms resulting in bulky molecules [28] and gave
low cytotoxicity towards cancer cells. High molecular weight

Journal of Chemistry
5
Cisplatin
Aspirin
100
50
100
50
0
−12
0
−7
−6
−5
−4
−10
−8
−6
−4
log [concentration] (M)
log [concentration] (M)
5-Fluorouracil
1a
100
50
100
50
0
0
−12
−10
−8
−6
−4
−12
−10
−8
−6
−4
log [concentration] (M)
log [concentration] (M)
1c
1b
100
50
100
50
0
0
−7
−6
−5
−4
−7
−6
−5
−4
log [concentration] (M)
log [concentration] (M)
1d
2a
150
100
50
100
50
0
0
−7
−6
−5
−4
−12
−10
−8
−6
−4
log [concentration] (M)
log [concentration] (M)
Figure 1: Continued.

6
Journal of Chemistry
2c
2b
100
50
0
100
50
0
−7
−6
−5
−4
−12
−10
−8
−6
−4
log [concentration] (M)
log [concentration] (M)
2d
100
50
0
−12
−10
−8
−6
−4
log [concentration] (M)
Figure 1: Cell viability of NPC cells, HK1 treated with 1a–d and 2a–d, aspirin, and standards (cisplatin and 5-fluorouracil).
and bulky molecules contributed to steric hindrance, thus
preventing 2a–d from reaching the active site of enzyme [29].
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Compound 1a–d demonstrated good anticancer activities
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