Synthesis and antibacterial activity of acetoxybenzoyl thioureas with aryl and amino acid side Chains

Article abstract of DOI:10.1080/10426507.2011.562398

A series of acetoxybenzoylthioureas derivatives with aryl and amino acid ester side chains were prepared by reaction of acetoxybenzoyl isothiocyanate, an acyloxy benzyl ester-based derivative of aspirin, with aryl amines or amino-functionalized amino acids with overall yields of 46-73%. The products that display a thiourea segment as a linker showed improved antibacterial properties in comparison with aspirin. The structures of the synthesized compounds were characterized by infra red spectroscopy, ¹³C nuclear magnetic resonance (NMR), and ¹H NMR spectroscopy. The compounds were screened for their antibacterial activity by using gram-negative bacteria (E. coli ATCC 8739). [2-(phenylcarbamothioylcarbamoyl)phenyl] acetate showed the highest antibacterial activity against E. coli compared with other synthesized compounds. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/10426507.2011.562398

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Synthesis and Antibacterial Activity of
Acetoxybenzoyl Thioureas with Aryl and
Amino Acid Side Chains
Zainab Ngaini a , Maya Asyikin Mohd Arif a , Hasnain Hussain b , Er Su
Mei a , Donna Tang a & Dyg Halimatulzahrah Abang Kamaluddin a
a Department of Chemistry
b Department of Molecular Biology, Faculty of Resource Science and
Technology, University Malaysia Sarawak, Kota Samarahan, Sarawak,
Malaysia
Version of record first published: 06 Dec 2011.
To cite this article: Zainab Ngaini , Maya Asyikin Mohd Arif , Hasnain Hussain , Er Su Mei , Donna
Tang & Dyg Halimatulzahrah Abang Kamaluddin (2012): Synthesis and Antibacterial Activity of
Acetoxybenzoyl Thioureas with Aryl and Amino Acid Side Chains, Phosphorus, Sulfur, and Silicon and
the Related Elements, 187:1, 1-7
To link to this article: http://dx.doi.org/10.1080/10426507.2011.562398
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Phosphorus, Sulfur, and Silicon, 187:1–7, 2012
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2011.562398
SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF
ACETOXYBENZOYL THIOUREAS WITH ARYL AND AMINO
ACID SIDE CHAINS
Zainab Ngaini,1 Maya Asyikin Mohd Arif,1 Hasnain Hussain,2
Er Su Mei,1 Donna Tang,1 and Dyg Halimatulzahrah
Abang Kamaluddin1
1Department of Chemistry,
2Department of Molecular Biology, Faculty of Resource Science and Technology,
University Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
GRAPHICAL ABSTRACT
Abstract A series of acetoxybenzoylthioureas derivatives with aryl and amino acid ester side
chains were prepared by reaction of acetoxybenzoyl isothiocyanate, an acyloxy benzyl ester-
based derivative of aspirin, with aryl amines or amino-functionalized amino acids with overall
yields of 46–73%. The products that display a thiourea segment as a linker showed improved an-
tibacterial properties in comparison with aspirin. The structures of the synthesized compounds
were characterized by infra red spectroscopy, 13C nuclear magnetic resonance (NMR), and
1H NMR spectroscopy. The compounds were screened for their antibacterial activity by using
gram-negative bacteria (E. coli ATCC 8739). [2-(phenylcarbamothioylcarbamoyl)phenyl] ac-
etate showed the highest antibacterial activity against E. coli compared with other synthesized
compounds.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.
Keywords Thiourea; aspirin; amino acid; antibacterial activity
INTRODUCTION
Aspirin has been widely used as an analgesic and anti-inflammatory drug. It also plays
an important role in the prevention of cardiovascular diseases and cancer. Modification
Received 23 November 2010; accepted 30 January 2011.
The authors wish to thank the University Malaysia Sarawak and the Ministry of Science, Technol-
ogy, and Innovation (MOSTI) for the financial support through research grants 01(S27)684/2008(19) and
FRGS/01(14)/743/2010(29), respectively. The authors would also like to thank Associate Professor Dr. Ling
Teck Yee for providing technical help for this manuscript.
Address correspondence to Dr. Zainab Ngaini, Associate Professor, Department of Chemistry, Faculty of
Resource Science and Technology, University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
E-mail: nzainab@frst.unimas.my
1

2
Z. NGAINI ET AL.
of aspirin has been reported to inhibit the growth of various human cancer cell lines
originating from colon, lung, liver, pancreas, and breast.1 Due to its significant biological
properties, extensive effort has been exerted to improve the pharmacological properties of
aspirin.2,3
It is well known that thioureas play important roles in many biological processes,4
which stimulated our interest in the synthesis of a series of compounds containing as-
pirin associated with thiourea moiety. Presently, the search for new thiourea derivatives
with reduced undesirable side effects and improved oral bioavailability has intensified.
For example, 3-benzoyl-1-butyl-1-methyl-thiourea was reported to have antifungal activ-
ities and successfully inhibited the growth of Penicillium digitatum and Saccharomyces
cerevisiae.5
In this paper, we report the synthesis of acetoxybenzoyl thioureas with aryl and
amino acid side chains (1–8) by reaction of acetoxybenzoyl isothiocyanates with aniline,6
aminophenol,6 glycine, beta-alanine, alpha-alanine, phenylalanine, methionine, and aspar-
tic acid, respectively. The preparation of thiourea derivatives 1 and 2 via 2-acetoxybenzoyl
isothiocyanate has been independently described in a recent Chinese patent.6 The antibac-
terial activities of synthesized thiourea derivatives toward E. coli ATCC 8739 have also
been characterized.
RESULTS AND DISCUSSION
The acetoxybenzoyl thiourea derivatives 1–8 exhibiting aryl and an amino acid ester
side chains were prepared by first reacting aspirin with oxalyl chloride under nitrogen
atmosphere with dimethylformamide as catalyst to yield 2-(chlorocarbonyl)phenyl acetate.
The thiourea derivatives of aspirin were prepared by reacting acyl chloride with potassium
thiocyanate under basic conditions, followed by the reaction of the acetoxybenzoyl isoth-
iocyanate with an aryl amine or an amino-functionalized amino acid to afford 1–8 with
overall yields of 46–73%. The low yields of thiourea might be due to the possible forma-
tion of by-products such as urethanes.7 The preparation of compounds 1–8 is depicted in
Scheme 1.
O
O
O
O
O
O
S
R- NH2
Oxalyl chloride, DMF
KSCN, Acetone
OH
O
N
H
N
H
R
NCS
O
Acetone reflux
O
1
8
R
MIC (ppm)
Thiourea
1
2
3
4
5
6
7
8
-C6H5
-C6H4OH
-CH2COOH
-CH2CH2COOH
-CH(CH3)COOH
-CH(CH2C6H5)COOH
-CH(CH2CH2SCH3)COOH
-CH(CH2COOH)COOH
87
>200
200
210
145
>200
195
>200
Scheme 1 Synthesis of thiourea derivatives 1–8.

ACETOXYBENZOYL THIOUREAS AS ANTIBACTERIAL AGENTS
3
The Fourier transform-infra red (FT-IR) spectra showed bands at 3269–3289 cm−1
and 1159–1231 cm−1, which were attributed to v(NH) and v(C S). The peaks at
1602–1608 cm−1and 1659–1732 cm−1 were attributed to v(C N) and v(C O), respec-
tively. Disappearance of the NCS peak at 2000 cm−1 and appearance of a NH peak
at 3269–3289 cm−1 proved that the reaction was complete. The expected normal car-
bonyl absorption at 1659–1672 cm−1 (C O) suggested a possible hydrogen bond forma-
tion between the hydrogen atom of the NH group and the oxygen atom of the carbonyl
group.8
The chemical structures of all synthesized compounds were further confirmed by
1H and 13C nuclear magnetic resonance (NMR) spectroscopy, which exhibited the sig-
nals corresponding to the structures. The presence of aromatic protons was observed in
1
the range of 6.70–8.38 ppm in the H NMR spectra, whereas CONH and CSNH peaks
were observed at 11.38–11.65 ppm and 8.91–11.40 ppm, respectively. The CONH signal
was shifted to higher frequency due to the downfield effect.8 The 13C NMR spectrum
showed the presence of CONH at 168.46–172.88 ppm, whereas CSNH was observed at
178.87–206.55 ppm. The resonance at 115.79–157.48 ppm was attributed to aromatic
carbons.
The inhibition activity of compounds 1–8 against E. coli ATCC 8739 is shown in
Figure 1. Compound 1 showed complete inhibition against E. coli at all concentrations
compared to that of aspirin, which has no antibacterial activity (Figure 1, compound 1).
Compounds 2–8, on the other hand, showed varying degrees of antibacterial activity against
the tested bacteria.
Different effects of the synthesized acetoxybenzoyl thiourea derivatives at various
concentrations are further shown by their minimum inhibitory concentrations (MIC). The
MIC value of these compounds were determined by extrapolating the concentration at the
zero growth rate of E. coli (µ = 0)9 based on the specific growth rate of E. coli ATCC 8739.
The series for MIC observed was 87, >200, 200, 210, 145, >220, 195, and >200 ppm (as
shown in Scheme 1). Compound 1 exhibited the best bacteriostatic activities followed by
compounds 5 and 7. Other compounds showed MIC values of 200 and above due to very
weak antibacterial activities.
These results indicated that C S, NH, and C O groups in all synthesized
compounds play an important role with respect to bacteriostatic activities, as these
important groups can be protonated under acidic condition, reacted with the carboxyl
and phosphate groups of the bacterial surface, and thus show antibacterial activity.10
Lipophilicity, which correlates well with the bioactivity of chemicals, is a very im-
portant molecular descriptor, and different lipophilic behavior of compounds plays
an important role in their biological activity mechanisms. Compounds with phenyl
groups showed more lipophilic character compared with the compounds without phenyl
groups.11
Compounds 5 and 7 with one phenyl group exhibit lower antibacterial activity whereas
compound 1 with two phenyl groups showed higher antibacterial activity. Due to their lower
lipophilicity, compounds 4 and 7 did not penetrate into the microorganisms as easily as
compound 1 with two phenyl groups.11 Increasing the length or bulkiness of the side chain
afforded varied effects and comparable results.12 Although compound 6 possesses two
phenyl groups, it does not show as potent antibacterial properties as compound 1. This
might be due to the presence of bulky groups in the structure, which led to a dramatic
decrease in activity. The occurrence of steric hindrance prevents the compounds from
reaching the active site.12

4
Z. NGAINI ET AL.
Figure 1 Inhibition activity of aspirin and 1–8 against E. coli shown as ln Nt for E. coli growth versus time.

ACETOXYBENZOYL THIOUREAS AS ANTIBACTERIAL AGENTS
5
CONCLUSION
A series of acetoxybenzoyl thioureas with aryl or amino acid ester side chains (1–8)
has been synthesized and screened for their antibacterial activities. The acetoxybenzoyl
thioureas showed antibacterial activity against gram-negative bacteria in contrast to the
standard aspirin. Compounds containing C S, C O, and NH groups have been demon-
strated to possess antibacterial properties. It is well established that the C S, C O, and
NH groups induce antibacterial activity by reacting with the carboxyl and phosphate groups
of the bacterial surface.
EXPERIMENTAL
Aspirin, oxalyl dichloride, aniline, aminophenol, glycine, beta-alanine, alpha-alanine,
phenylalanine, methionine, and aspartic acid were obtained from Merck and used without
further purification. All other reagents and solvents were used as received.
Measurements: Melting points were determined by the open tube capillary method
and are uncorrected. Infra red (IR) spectra (ν/cm−1) were recorded as KBr pellets on a
Perkin Elmer 1605 FTIR spectrophotometer. 1H and 13C NMR spectra were recorded on a
JEOL ECA 500 spectrometer at 500MHz (1H) and 126 MHz (13C) with the chemical shifts
δ (ppm) reported relative to acetone-d6 and DMSO-d6 as standards.
General Procedure for the Preparation of Aspirin–Thiourea Derivatives
Oxalyl chloride (2.5 mmol) was added to aspirin (2.5 mmol) in 20 mL of
dichloromethane. Few drops of dimethylformamide (DMF) were added to initiate the
reaction. The solution was stirred for 1 h at room temperature under N2. The mixture was
added into potassium thiocyanate (KSCN) (2.5 mmol) in dry acetone (20 mL) at room
temperature to form a precipitate. The mixture was filtered and the solid was removed. A
solution of amine (2.5 mmol) in dry acetone (20 mL) was added to the filtrate. The reaction
mixture was refluxed for 6 h, cooled to room temperature, and filtered. The filtrate was
poured into a beaker with ice to form a solid, which was recrystallized in EtOH/CH3CN
(1:1) to give 1–8.
N-(2-acetoxybenzoyl)-Nꢁ-phenylthiourea (1):6 Compound 1 was obtained as a light
brown solid. Yield: 0.57 g (73%); mp 172 ◦C–175 ◦C; ν 3269 (NH), 3072 (Ar), 1732 (C O
ester), 1659 (C O carbonyl), 1604 (C N), 1201 (C S). δH (DMSO-d6) 2.08 (3H, s, CH3),
7.05–8.00 (9H, m, Ar H), 11.68 (1H, s, NH), 12.65 (1H, s, NH). δC (acetone-d6) 30.11,
117.65, 118.11, 121.67, 124.52, 127.04, 129.49, 132.37, 136.28, 139.21, 157.42, 166.35,
171.55, and 179.49.
N-(2-acetoxybenzoyl)-Nꢁ-(4-hydroxyphenyl)thiourea (2):6 Compound 2 was ob-
◦
◦
tained as a dark brown solid. Yield: 0.50 g (60%); mp 169 C–178 C; ν 3276 (NH),
1608 (C N), 1208 (C S), 3453 (OH), 1659 (C O amide). δH (acetone-d6) 1.32 (3H, s,
CH3), 6.70–8.08 (8H, m, Ar H), 8.91 (1H, m, NH), 11.38 (1H, s, NH). δC (acetone-d6)
23.93, 115.79, 115.91, 118.07, 121.53, 121.66, 126.23, 126.31, 132.29, 136.15, 154.17,
and 156.60.
[Nꢁ-(2-acetoxybenzoyl)thioureido]-ethanoi◦c acid (3): Compound 3 was obtained as
◦
a yellow solid. Yield: 0.43 g (57.5%); mp 148 C–154 C; ν 3261 (NH), 1602 (C N),
1194 (C S), 3347 (OH), 1716 (C O ester), 1677 (C O amide). δH (DMSO-d6) 2.28 (3H,
s, CH3), 4.21 (2H, d, J 4.0, CH2), 7.26–7.67 (4H, m, Ar H), 10.83 (1H, s, NH), 11.40

6
Z. NGAINI ET AL.
(1H, s, NH). δC (acetone-d6) 29.49, 47.28, 124.52, 126.64, 127.10, 131.34, 134.52, 149.52,
165.74, 169.10, and 181.16.
3-[Nꢁ-(2-acetoxybenzoyl)thioureido]propanoic acid (4): Compound 4 was obtained
as a yellow solid. Yield: 0.42 g (57%); mp 185 ◦C–188 ◦C; ν 3289 (NH), 3199 (CH), 1703
(C O ester), 1665 (C O carbonyl), 1606 (C N), 1222 (C S). δH (DMSO-d6) 2.08 (3H,
s, CH3), 2.64 (2H, t, J 6.6, CH2), 3.44 (1H, s, OH), 3.82 (2H, t, J 6.3, CH2), 7.01–7.92
(4H, m, Ar H), 10.92 (1H, t, J 5.7, NH), 11.48 (1H, s, NH). δC (DMSO-d6) 30.65, 32.31,
40.70, 116.49, 117.26, 120.14, 131.11, 135.20, 156.63, 164.53, 172.86, and 179.32.
2-[Nꢁ-(2-acetoxybenzoyl)thioureido]propanoic acid (5): Compound 5 was obtained
as a pale yellow solid. Yield: 0.41 g (53%); mp 173 ◦C–178 ◦C; ν 3278 (NH), 1605 (C N),
1181 (C S), 3406 (OH), 1701 (C O ester) 1662 (C O amide). δH (DMSO-d6) 1.36 (3H,
d, J 7.1, CH3), 1.82 (3H, s, CH3), 4.82 (1H, m, CH), 6.99–8.14 (4H, m, Ar H), 11.18
(1H, d, J 7.5, NH), 11.65 (1H, s, NH). δC (DMSO-d6) 17.27, 39.66, 53.32, 116.39, 117.42,
120.02, 131.13, 135.33, 157.10, 165.02, 172.88, and 178.91.
2-[Nꢁ-(2-acetoxybenzoyl)thioureido]-3-phenylpropanoic acid (6): Compound 6 was
obtained as a light yellow solid. Yield: 0.50 g (51%); mp 158 ◦C–160 ◦C; ν 3272 (NH), 3160
(C-H), 3028 (Ar), 1704 (C O ester), 1666 (C O carbonyl), 1604 (C N), 1226 (C S).
δH (DMSO-d6) 2.08 (3H, s, CH3), 2.24 (2H, d, J 4.5, CH2), 3.46 (1H, s, OH), 5.04 (1H,
t, J 5.0, CH), 7.00–7.91 (9H, m, Ar H), 11.07 (1H, d, J 5.0, NH), 11.60 (1H, s, NH). δC
(DMSO-d6) 22.33, 36.05, 58.77, 116.30, 117.35, 120.19, 126.94, 128.44, 129.27, 131.19,
135.43, 136.15, 156.82, 164.90, 171.32, and 179.28.
2-[Nꢁ-(2-acetoxybenzoyl)thioureido]-4-(methylthio)butanoic acid (7): Compound 7
was obtained as a yellow solid. Yield: 0.57 g (59%); mp 155 ◦C–158 ◦C; ν 3277 (NH), 3079
(CH), 1720 (C O ester), 1672 (C O carbonyl), 1604 (C N), 1231 (C S), 715 (C-S). δH
(DMSO-d6) 2.06 (3H, s, CH3), 2.14 (2H, m, CH2), 2.23 (2H, m, CH2), 2.54 (3H, s, CH3),
3.57 (1H, s, OH), 4.97 (1H, t, J 5.0, CH), 7.02–7.96 (4H, m, Ar H), 11.17 (1H, d, J 10.0,
NH), 11.60 (1H, s, NH). δC (DMSO-d6) 14.70, 29.21, 29.71, 30.55, 56.75, 116.39, 117.34,
120.22, 131.20, 135.41, 156.80, 164.99, 171.81, and 179.54.
2-[Nꢁ-(2-acetoxybenzoyl)thiourido]butanedioic acid (8): Compound 8 was obtained
as yellow crystals. Yield: 0.40 g (46%); mp 133 ◦C–140 ◦C; ν 3269 (NH), 1604 (C N), 1159
(C S), 3448 (OH), 1715 (C O ester), 1649 (C O amide), 1206 (C-O-C). δH (DMSO-d6)
1.94 (3H, s, CH3), 2.39 (2H, d, J 10.0, CH2), 2.99 (1H, s, CH), 6.95–8.38 (4H, m, Ar H),
11.40 (1H, d, J 7.3, NH), 11.56 (1H, s, NH). δC (DMSO-d6) 25.78, 30.64, 39.67, 117.22,
117.46, 118.42, 119.81, 128.17, 130.32, 130.87, 134.13, 134.62, 157.05, 161.19, 161.35,
172.00, 172.21, and 206.55.
Antibacterial Studies
The antibacterial activities of the synthesized compounds against E. coli ATCC 8739
were investigated by the turbidimetric kinetic method. The inoculums was prepared by
◦
allowing E. coli to grow on media containing nutrient broth at 37 C with permanent
stirring at 250 rpm for 18 h. Inoculums (0.2 mL) were inoculated with 10 mL of culture
medium (with increasing concentration of the compounds dissolved in DMSO). The mixture
◦
was shaken at 180 rpm at 37 C. Inoculums with DMSO solvent were used as controls.
Aliquots of each replicate were taken at every 1-h interval for 7 h. The transmittance (T) was
registered in a UV-visible spectrophotometer (Optima SP-300). The antibacterial activities
were determined by a graph as ln Nt, define as transmittance value, which was related to
the number colony forming units (CFU) mL−1 for E. coli versus time.9

ACETOXYBENZOYL THIOUREAS AS ANTIBACTERIAL AGENTS
7
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