Synthesis and characterization of halogenated bis(acylthiourea) derivatives and their antibacterial activities

Article abstract of DOI:10.1080/10426507.2017.1315421

A total of 12 bis(acylthiourea) derivatives with different pharmacophores have been synthesized via nucleophilic substitution reaction of benzene-1,4-dicarbonyl isothiocyanate intermediate with aromatic amine bearing halogens at the ortho, meta and para p

Full text of DOI:10.1080/10426507.2017.1315421

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Synthesis and characterisation of halogenated
bis(acylthiourea) derivatives and its antibacterial
activities
Ainaa Nadiah Abd Halim & Zainab Ngaini
To cite this article: Ainaa Nadiah Abd Halim & Zainab Ngaini (2017): Synthesis and
characterisation of halogenated bis(acylthiourea) derivatives and its antibacterial activities,
Phosphorus, Sulfur, and Silicon and the Related Elements, DOI: 10.1080/10426507.2017.1315421
To link to this article: http://dx.doi.org/10.1080/10426507.2017.1315421
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Date: 12 April 2017, At: 05:21

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Synthesis and characterisation of halogenated bis(acylthiourea) derivatives and its
antibacterial activities
Ainaa Nadiah Abd Halim, Zainab Ngaini¥
Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
¥Corresponding email: nzainab@unimas.my
ABSTRACT
A total of 12 bis(acylthiourea) derivatives with different pharmacophores have been synthesized
via nucleophilic substitution reaction of benzene-1,4-dicarbonyl isothiocyanate intermediate with
aromatic amine bearing halogens at the ortho, meta and para positions. The structures of the
1
13
synthesised compounds were confirmed by CHN elemental analysis, FT-IR, H NMR and C
NMR spectroscopies. Antibacterial studies of the compounds via the Kirby Bauer disc diffusion
method against Escherichia coli (E.coli) ATCC 25922 and Staphylococcus aureus (S. aureus)
S48/81 demonstrated that bis(acylthiourea) N1,N4-bis[(2-chlorophenyl)carbamothioyl]terephthalamide
(4) and N1,N4-bis[(2-bromophenyl)carbamothioyl]terephthalamide (7) bearing Cl and Br at the
ortho position exhibited excellent activities against both bacteria strains compared to ampicillin
standard.
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acetonitrile
acetonitrile
1
2
3
4
5
6
7
8
9
= Ortho-F
= Meta- F
= Para- F
= Ortho-Cl
= Meta-Cl
= Para-Cl
= Ortho-Br
= Meta-Br
= Para-Br
X:
10 = Ortho-I
11 = Meta-I
12 = Para-I
KEYWORD
bis(acylthiourea), antibacterial activity, aromatic amine, halogen
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INTRODUCTION
The emergence of new drugs has become a priority in recent decades due to alarming cases
of many infectious diseases caused by antimicrobial resistance.1,2 Concomitant with this
phenomenon, thiourea and its derivatives have been studied by researchers to widen the usages
particularly for biological purposes.2 This is due to the ability of C=S and N-H groups in
thiourea moieties that are easily protonated under acidic conditions and reacts with the carboxyl
and phosphate groups of the bacterial surface for antibacterial activities.3 Thiourea is a diamide
of thiocarbonic acid that resembles urea, with C=S instead of C=O.4 Thiourea consists of reactive
functional groups, namely amino, imino and thiol, and is also an excellent precursor in the
synthesis of organic molecules for various biological applications.5 Several biological activities
reported for thiourea derivatives are antioxidant,6 anti-HIV,7 antituberculosis agents,8 anticancer,9
antibacterial,10-13 antifungal and antiparasitic activities.14
In a previous study, we have reported on a series of symmetrical bis(thiourea) derivatives for
their antibacterial activity.15 The symmetrical position of 1,4-bis(thiourea) showed better activity
compared to 1,2-bis(thiourea)16 and 1,3-bis(thiourea)15,17 due to less steric hindrances, thus
increased the contact between the active site of compound and the bacteria receptor.18
1,4-Bis(thiourea) bearing an aryl side chain showed good antibacterial activity against E.coli
ATCC 8739 due to the lipophilicity of the aromatic group which correlates well with the
bioactivity of the compounds.15,19-20
A halogen is yet another substituent that is commonly introduced onto thiourea moieties,
which may lead to an improvement in the biological activities.1,10 Structure-activity relationship
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studies indicated that halogens (Cl, Br, F) at the ortho, meta and para positions of aromatic
groups attributed to excellent activities against microorganisms.21 Saeed and co-workers reported
that thiourea derivatives bearing fluorine atoms at the ortho position in an aromatic group
demonstrated excellent activities against different fungal strains R. oryzae, F. oxysporum, A.
niger and A. fumigates.22 Similar findings were also reported by Wang et al. on the influence of
halogens to the biological activities.23
Herein, we report on the synthesis of halogenated bis(acylthiourea) derivatives 1-12 from the
reaction of benzene-1,4-dicarbonyl isothiocyanate with various aromatic amine bearing halogens
at ortho, meta and para positions. Compounds 4, 5, 6, 9 and 12 in the series were earlier reported
with no spectroscopic analysis and were evaluated for antifungal activities.24 In this study, the
synthesised compounds 1-12 were screened for their antibacterial activity against gram-negative
bacteria E.Coli (ATCC 25222) and gram-positive bacteria S.aureus (S48/81) in which the effect
of halogens at different positions was evaluated.
RESULTS AND DISCUSSION
Synthesis
The bis(acylthiourea) derivatives 1–12 were prepared by introducing intermediate
benzene-1,4-dicarbonyl isothiocyanate, which was prepared from benzene-1,4-dioylchloride with
potassium thiocyanate in acetonitrile, onto aromatic amine bearing halogens at different
positions. The preparation of 1-12 is depicted in Scheme 1.
The IR spectra illustrated the formation of 1-12 by the presence of the expected absorption
frequency at 3393-3168 cm-1 attributed to vNH. The disappearance of the NCS absorption band at
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2000 cm−1 and appearance of an NH absorption band indicates the formation of target
compounds.25 The strong absorption band at 1680-1663 cm-1 were attributed to vC=O. The
carbonyl absorption observed was lower than the expected value (1700 cm-1) due to
intra-molecular hydrogen bond that causes a slight downshift in the frequency.26,27 The aromatic
ring was represented by the absorption band at 1542-1502 cm-1, while vC=S and vC-N bands were
observed at 1280-1248 cm-1 and 1170-1140 cm-1, respectively.13,22
The formations of 1-12 were supported by 1H and 13C NMR spectroscopy analyses. Findings
from 1H NMR demonstrated the formation of thiourea moieties in the compound by the existence
of broad signals at δH 12.60-12.32 and δH 12.07-11.83 attributed to CSNH and CONH,
respectively.28 The CSNH signal was represented at higher chemical shift due to the downfield
effect caused by possible hydrogen bonds.25,28 Multiple peaks observed at δH 8.17-7.13 were
assigned as aromatic protons. The 13C NMR spectra exhibit thiol C=S and C=O peaks that
resonated at δC 180.5-179.1 and δC 167.9-167.3, respectively. The C=S peak appeared at higher
chemical shift than C=O due to its lower excitation energy n→π.29,30 The resonance of
unresolved aromatic carbons was represented by series of signals at δC 163.1-97.5.
Antibacterial activities
The antibacterial activities of bis(acylthiourea) derivatives 1-12 were initially performed
against gram-negative bacteria (E.coli) and gram-positive bacteria (S.aureus) via Turbidimetric
Kinetic method.31,32 Compounds 1-12, however, experienced solubility limitation and dissolution
of compounds during administration into the culture media that lead to the precipitation of the
vehicle–insoluble particles.33 This phenomenon has hindered a quantitative determination of the
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biological activities. The molecular features of the compounds that highly lipophilic was
envisaged to reduce the solubility of the compounds in the culture media.34
Alternatively, the antibacterial activities of 1-12 were performed employing Kirby Bauer
disc diffusion method following standard methods by the Clinical and Laboratories Standards
Institute (CLSI).35 Ampicillin was used as a standard drug, while dimethyl sulfoxide (DMSO)
was used as a negative control. The inhibition zones after incubation are shown in Table S 1
(Supplemental Materials). The results were assigned to three categories, susceptible (6-13mm),
intermediate (14-16mm), or resistant (>17mm).35 The relative inhibition of E. coli and S. aureus
were calculated via inhibition zone of 1-12 compared to the inhibition zone of ampicillin.
The average diameter of the inhibition zones varied from 8 to 18 mm for both bacteria.
ortho-Substituted halogens (4 and 7) demonstrated higher antibacterial activities than meta- (5
and 8) and para- (6, 9 and 12) substituted halogens. Compounds 4 and 7 bearing Cl and Br at
ortho positions exhibited better activities with the relative inhibition zone >100% in comparison
to ampicillin (standard drug). The higher antibacterial activity of ortho-substituted halogen was
due to the strategic position that is favourable for binding process to specific sites of bacterial
cells by interaction with outer cellular components.36,37 The halogen atoms stabilised the
non-hydrophobic interactions in bacteria through halogen bond interactions with surrounding
amino acids, leading to an improvement of bioavailability.35 The presence of two thiourea
moieties offered more reactive moieties of C=S, N-H and C=O that easily protonated under
acidic condition and reacted with the bacterial surfaces, therefore further enhance the biological
activity.3
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CONCLUSION
A series of bis(acylthiourea) derivatives 1-12 were successfully synthesised and fully
characterised by typical selected spectroscopic methods. The antibacterial studies of the
synthesised bis(acylthiourea) derivatives elucidated that ortho-substituted halogens 4 and 7
possess excellent antibacterial activity than reference drug against both bacteria E. coli and S.
aureus. This concludes that substituent at ortho position gave better activity compared to meta
and para positions in the molecular framework of the synthesised molecules.
EXPERIMENTAL
All chemicals were purchased from Merck kGaa or Acros Company and used as received
without any further purification. The solvent acetonitrile was dried and distilled over calcium
hydride.
Physical Measurement: Melting points were determined by using Stuart SMP3 melting
point apparatus and uncorrected. The elemental CHNS analyses were provided by Universiti
Teknologi MARA (UiTM), Shah Alam, Malaysia using Thermo Scientific™ FLASH 2000
CHNS/O Analyzers. Infra-red (IR) spectra (ν/cm−1) were recorded as KBr pellets on Perkin
Elmer Thermoscientific Smart Omni Transmission Nicolet IS10 Fourier Transform Infrared
1
Spectrometer (FTIR). H NMR and 13C NMR spectra were recorded using JEOL ECA 500
spectrometer at 500 MHz (1H) and 125 MHz (13C) with the chemical shifts δH/δC (ppm) reported
relative to DMSO-d6 as standards.
General procedure for the preparation of bis(acylthiourea) derivatives 1-12
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A solution of benzene-1,4-dioylchloride (2 mmol) in acetonitrile (15 mL) was added to a
suspension of potassium thiocyanate (4 mmol) in acetonitrile (15 mL). The mixture was stirred at
room temperature to form KCl as a white precipitate. The mixture was filtered and KCl was
removed. A series of aromatic amine bearing halogens (4 mmol) in acetonitrile (15 mL) was
slowly added to the filtrate. The resulting mixture was stirred at room temperature until a
precipitate formed. The mixture was filtered and the solid crude was purified by recrystallisation
from hot ethanol to afford yellow or white solids of the title compounds.
N1,N4-bis[(2-fluorophenyl)carbamothioyl]terephthalamide (1)
Compound 1 was obtained from o-fluoroaniline (0.5 mL, 4 mmol). Yield 0.73 g, 77 % as light
yellow solids; m.p: 249-250 ˚C; Anal.Calcd for C22H16F2N4O2S2 (470 g) : C, 56.16; H, 3.43; N,
11.91; S, 13.63%. Found: C, 56.66; H, 3.50; N, 12.09; S, 13.36. ); IR (KBr/ cm-1): 3246 (NH),
3138 (CH), 1679 (C=O amide), 1530 (Ar-C), 1253 (C=S), 1165 (C-N). 1H NMR (500 MHz,
DMSO-d6) 7.27 (m, 2H, Ar-H), 7.36 (d, 4H, J = 8.4 Hz, Ar-H), 8.03 (t, 2H, J = 7.6 Hz, Ar-H),
8.10 (s, 4H, Ar-H), 12.01 (s, 2H, 2xNH), 12.46 (s, 2H, 2xNH). 13C NMR (125 MHz, DMSO-d6)
115.7, 124.3, 126.0, 127.2, 128.4, 135.9, 154.5, 156.4 (Ar-C), 167.8 (C=O), 180.1 (C=S).
N1,N4-bis[(3-fluorophenyl)carbamothioyl]terephthalamide (2)
Compound 2 was obtained from m-fluoroaniline (0.5 mL,4 mmol). Yield 0.59 g, 62 % as light
yellow solids; m.p: 247-248 ˚C; Anal.Calcd for C22H16F2N4O2S2 (470 g): C, 56.16; H, 3.43; N,
11.91; S, 13.63%.Found: C, 56.45; H, 3.30; N, 11.61; S, 13.56.; IR (KBr/ cm-1) 3168 (NH), 3032
1
(CH), 1676 (C=O amide), 1532 (Ar-C), 1280 (C=S), 1154 (C-N). H NMR (500 MHz,
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DMSO-d6) 7.13 (t, 2H, J = 7.6 Hz Ar-H), 7.46 (d, 2H, J = 3.8 Hz , Ar-H), 7.48 (s, 2H, Ar-H),
7.81 (d, 2H, J = 8.4 Hz , Ar-H), 8.08 (s, 4H, Ar-H), 11.88 (s, 2H,2xNH), 12.55 (s, 2H, 2xNH).
13C NMR (125 MHz, DMSO-d6) 113.5, 120.8, 129.2, 130.9, 136.4, 140.1, 161.2, 163.1 (Ar-C),
167.9 (C=O), 179.6 (C=S).
N1,N4-bis[(4-fluorophenyl)carbamothioyl]terephthalamide (3)
Compound 3 was obtained from p-fluoroaniline (0.5 mL 4 mmol). Yield (0.74 g, 78 %) as
white solids; m.p: 247-248 ˚C; Anal.Calcd for C22H16F2N4O2S2 (470 g) : C, 56.16; H, 3.43; N,
11.91; S, 13.63%.Found: C, 56.35; H, 3.50; N, 12.00; S, 13.76.; IR (KBr/ cm-1) 3238 (NH), 3150
(CH), 1678 (C=O Amide), 1502 (Ar-C), 1161 (C-N), 1248 (C=S).. 1H NMR (500 MHz,
DMSO-d6) 7.27 (t, 4H, J = 8.8 Hz, Ar-H), 7.67 (m, 4H, Ar-H), 8.08 (s, 4H, Ar-H), 11.83 (s, 2H,
2xNH), 12.38 (s, 2H,2xNH). 13C NMR (125 MHz, DMSO-d6) 115.8, 127.5, 129.2, 134.8, 136.5,
159.3 (Ar-C), 167.9 (C=O), 179.9 (C=S).
N1,N4-bis[(2-chlorophenyl)carbamothioyl]terephthalamide (4) 24
Compound 4 was obtained from o-chloroaniline (0.5 mL, 4 mmol). Yield (0.87 g, 87 %) as white
solids; m.p: 230-231 ˚C (lit24 215 ºC); Anal.Calcd for C22H16Cl2N4O2S2 (502 g) : C, 52.49; H,
3.20; N, 11.13; S, 12.74%.Found: C, 52.53; H, 3.34; N, 10.97; S, 12.83.; IR (KBr/ cm-1) 3247
(NH), 3140 (CH), 1678 (C=O amide), 1525 (Ar-C), 1250 (C=S), 1170 (C-N). 1H NMR (500
MHz, DMSO-d6) 7.35 (t, 2H, J = 9.2 Hz Ar-H), 7.43 (t, 2H, J = 7.3 Hz Ar-H), 7.61 (d, 2H, J =
7.6 Hz , Ar-H), 8.04 (d, 2H, J = 8.4 Hz, Ar-H), 8.11 (s, 4H, Ar-H), 12.05 (s, 2H, 2xNH), 12.60 (s,
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13
2H, 2xNH). C NMR (125 MHz, DMSO-d6) 127.3, 128.0, 128.2, 128.3, 128.7, 129.53, 135.3,
135.8 (Ar-C), 167.6 (C=O), 180.0 (C=S).
N1,N4-bis[(3-chlorophenyl)carbamothioyl]terephthalamide (5)24
Compound 5 was obtained from m-chloroaniline (0.5 mL, 4 mmol). Yield (0.88 g, 87 %) as
white solids; m.p: 256-257 ˚C (lit24 240 ºC); Anal.Calcd for C22H16Cl2N4O2S2 (502 g): C, 52.49;
H, 3.20; N, 11.13; S, 12.74%.Found: C, 52.79; H, 3.34; N, 11.67; S, 12.63.; IR (KBr/ cm-1) 3225
(NH), 3035 (CH), 1680 (C=O amide), 1529 (Ar-C), 1258 (C=S), 1161 (C-N).. 1H NMR (500
MHz, DMSO-d6) 7.35 (d, 2H, J = 7.6 Hz, Ar-H), 7.45 (t, 2H, J = 8.0 Hz Ar-H), 7.58 (d, 2H, J =
8.4Hz, Ar-H), 7.93 (s, 2H, Ar-H), 8.07 (s, 4H, Ar-H), 11.87 (s, 2H, 2xNH), 12.47 (s, 2H, 2xNH).
13C NMR (125 MHz, DMSO-d6) 123.2, 124.0, 126.2, 128.7, 130.4, 132.7, 136.0, 139.5 (Ar-C),
167.3 (C=O), 179.3 (C=S).
N1,N4-bis[(4-chlorophenyl)carbamothioyl]terephthalamide (6)24
Compound 6 was obtained from p-chloroaniline (0.6 mL,4 mmol). Yield (0.77 g, 77 %) as light
yellow solids; m.p: 257-258 ˚C (lit24 230 ºC); Anal.Calcd for C22H16Cl2N4O2S2 (502 g): C, 52.49;
H, 3.20; N, 11.13; S, 12.74%. Found: C, 51.79; H, 3.43; N, 11.23; S, 12.98.; IR (KBr/ cm-1) 3378
1
(NH), 3054 (CH), 1673 (C=O amide), 1546 (Ar-C), 1261 (C=S), 1148 (C-N). H NMR (500
MHz, DMSO-d6) 7.49 (d, 4H, J = 8.4 Hz, Ar-H), 7.73 (d, 4H, J = 8.4 Hz, Ar-H), 8.08 (s, 4H,
Ar-H), 11.83 (s, 2H, 2xNH), 12.44 (s, 2H, 2xNH). 13C NMR (125 MHz, DMSO-d6) 126.3, 128.6,
128.7, 130.3, 135.9, 137.0 (Ar-C), 167.5 (C=O), 179.1 (C=S).
N1,N4-bis[(2-bromophenyl)carbamothioyl]terephthalamide (7)
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Compound 7 was obtained from o-bromoaniline (0.70g, 4 mmol). Yield (0.94 g, 78 %) as white
solids; m.p: 254-255 ˚C; Anal.Calcd for C22H16Br2N4O2S2 (591 g) : C, 44.61; H, 2.72; N, 9.46; S,
10.83%. Found: C, 44.53; H, 2.82; N, 9.48; S, 10.35.; IR (KBr/ cm-1) 3242 (NH), 3158 (CH),
1677 (C=O amide), 1576 (Ar-C), 1250 (C=S), 1170 (C-N). 1H NMR (500 MHz, DMSO-d6) 7.29
(t, 2H,J = 8.0 Hz Ar-H), 7.48 (t, 2H,J = 7.7 Hz Ar-H), 7.76 (d, 2H, J = 7.7 Hz , Ar-H), 7.89
(d, 2H,J =6.9 Hz , Ar-H), 8.10 (s, 4H,Ar-H), 12.04 (s, 2H,2xNH), 12.50 (s, 2H,2xNH). 13C NMR
(125 MHz, DMSO-d6) 119.5, 128.0, 128.8, 132.8, 135.9, 136.9 (Ar-C), 167.8 (C=O), 180.3
(C=S).
N1,N4-bis[(3-bromophenyl)carbamothioyl]terephthalamide (8)
Compound 8 was obtained from m-bromoaniline (0.5 mL, 4 mmol). Yield (0.83g, 70 %) as white
solids; m.p: 244-245 ˚C; Anal.Calcd for C22H16Br2N4O2S2 (591 g) : C, 44.61; H, 2.72; N, 9.46; S,
10.83%. Found: C, 44.53; H, 2.91; N, 9.44; S, 10.62.; IR (KBr/ cm-1) 3228 (NH), 3036 (CH),
1677 (C=O amide), 1529 (Ar-C), 1258 (C=S), 1159 (C-N).1H NMR (500 MHz, DMSO-d6) 7.40
(t, 2H, J = 8.0 HzAr-H), 7.48 (d, 2H, J = 8.4 Hz , Ar-H), 7.62 (d, 2H, J = 7.7 Hz , Ar-H), 8.06 (s,
2H, Ar-H), 8.07 (s, 4H, Ar-H), 11.87 (s, 2H, 2xNH), 12.45 (s, 2H, 2xNH). 13C NMR (125 MHz,
DMSO-d6) 121.0, 123.6, 126.8, 128.7, 129.0, 130.6, 136.0, 139.6 (Ar-C), 167.3 (C=O), 179.3
(C=S).
N1,N4-bis[(4-bromophenyl)carbamothioyl]terephthalamide (9)24
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Compound 9 was obtained from p-bromoaniline (0.71 g, 4 mmol). Yield (0.82 g, 69 %) as dusty
yellow solids; m.p: 257-258 ˚C (lit24 200 ºC); Anal.Calcd for C22H16Br2N4O2S2 (591 g): C, 44.61;
H, 2.72; N, 9.46; S, 10.83%. Found: C, 44.43; H, 2.69; N, 9.45; S, 10.61. IR (KBr/ cm-1) 3369
1
(NH), 3050 (CH), 1663 (C=O amide), 1537 (Ar-C), 1263 (C=S), 1145 (C-N). H NMR (500
MHz, DMSO-d6) 7.63 (d, 4H, J = 8.4 Hz, Ar-H), 7.68 (d, 4H, J = 9.2 Hz, Ar-H), 8.07 (s, 4H,
Ar-H), 11.85 (s, 2H, 2xNH), 12.44 (s, 2H, 2xNH). 13C NMR (125 MHz, DMSO-d6) 118.6, 126.5,
128.6, 131.5, 135.9, 137.3 (Ar-C), 167.3 (C=O), 179.1 (C=S).
N1,N4-bis[(2-iodophenyl)carbamothioyl]terephthalamide (10)
Compound 10 was obtained from o-iodoaniline (0.9 g, 4 mmol). Yield (0.99 g, 72%) as white
solids; m.p: 247-249 ˚C; Anal.Calcd for C22H16I2N4O2S2, (685 g): C, 38.50; H, 2.35; N, 8.16; S,
9.34%. Found: C, 38.95; H, 2.18; N, 8.05; S, 9.57.; IR (KBr/ cm-1) 3163 (NH), 3058 (CH), 1675
(C=O amide), 1522 (Ar-C), 1250 (C=S) 1166 (C-N). 1H NMR (500 MHz, DMSO-d6) 7.09 (t, 2H,
J = 7.7 Hz Ar-H), 7.47 (t, 2H,J = 6.9 Hz Ar-H), 7.66 (d, 2H, J = 7.7 Hz, Ar-H), 7.95 (d, 2H, J =
7.7 Hz, Ar-H), 8.11 (s, 4H, Ar-H), 12.07 (s, 2H,2xNH), 12.32 (s, 2H, 2xNH). 13C NMR (125
MHz, DMSO-d6) 97.5, 128.6, 128.7, 128.8, 129.0, 135.9, 138.9, 140.3 (Ar-C), 167.6 (C=O),
180.5 (C=S).
N1,N4-bis[(3-iodophenyl)carbamothioyl]terephthalamide (11)
Compound 11 was obtained from m-iodoaniline (0.5 mL,4 mmol). Yield (1.09 g, 79 %) as dusty
brown solids; m.p: 246-247 ˚C; Anal.Calcd for C22H16I2N4O2S2, (685 g): C, 38.50; H, 2.35; N,
8.16; S, 9.34%. Found: C, 38.38; H, 2.27; N, 8.44; S, 9.26.; IR (KBr/ cm-1) 3392 (NH), 3041
1
(CH), 1670 (C=O amide), 1534 (Ar-C), 1254 (C=S), 1145 (C-N). H NMR (500 MHz,
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DMSO-d6) 7.24 (t, 2H, J = 8.0 Hz Ar-H), 7.65 (d, 4H, J = 7.7 Hz, Ar-H), 8.07 s, 4H, Ar-H), 8.17
(s, 2H, Ar-H), 11.85 (s, 2H, 2xNH), 12.44 (s, 2H, 2xNH). 13C NMR (125 MHz, DMSO-d6) 94.4,
124.5, 129.2, 131.1, 133.2, 135.5, 136.4, 139.8 (Ar-C), 167.5 (C=O), 179.7 (C=S).
N1,N4-bis[(4-iodophenyl)carbamothioyl]terephthalamide (12)24
Compound 12 was obtained from p-iodoaniline (0.91 g, 4 mmol). Yield (1.14 g, 83 %) as
yellowish solids; m.p: 257-258 ˚C (lit24 225 ºC); Anal.Calcd for C22H16I2N4O2S2, (685 g): C,
38.50; H, 2.35; N, 8.16; S, 9.34%. Found: C, 38.63; H, 2.29; N, 8.41; S, 9.39.; IR (KBr/ cm-1)
3350 (NH), 3047 (CH), 1664 (C=O amide), 1542 (Ar-C), 1262 (C=S), 1147 (C-N). 1H NMR
(500 MHz, DMSO-d6) 7.50 (d, 4H, J = 8.4 Hz, Ar-H), 7.73 (d, 4H, J = 9.2 Hz, Ar-H), 8.08 (s,
4H,Ar-H), 11.87 (s, 2H,2xNH), 12.45 (s, 2H,2xNH). 13C NMR (125 MHz, DMSO-d6) 90.9,
126.5, 128.7, , 137.4, 137.6, 137.9 (Ar-C), 170.7 (C=O), 179.0 (C=S).
Antibacterial Screening
Turbidimetric Kinetic Method
The synthesised bis(acylthiourea) compounds were initially tested for their antibacterial
activities against two types of bacteria; E.coli ATCC 25922 and S.aureus S48/81 using the
turbidimetric kinetic method.31,32 The bacteria were cultured on Luria-Bertani (LB) agar for 1 d
at 37 ºC. The inoculums were prepared by allowing bacteria to grow on media containing
nutrient broth at 37 ºC with permanent stirring at 250 rpm for 18 h. The 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 ºC. Inoculums with DMSO were
used as a control. Aliquots of each replicate were taken at every 1 h interval for 6 h. The
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transmittances (T) were recorded using a UV-visible spectrophotometer (Optima SP-300). The
antibacterial activities were determined by plotting a graph of In Nt versus time. The ln Nt, is
defined as transmittance value, which represents the number colony forming units (CFU) mL−1
for bacteria versus time following expressions: E. coli In Nt= 27.1-8.56T and S. aureus ln Nt =
27.4 – 10.3T.31
Kirby-Bauer Disc Diffusion Method
Kirby-Bauer disc diffusion method was employed due to the dissolution of compounds
during administration into the culture media. The bacteria E.coli ATCC 25922 and S.aureus
S48/81 were grown on media with Mueller-Hinton (MH) broth at 37 °C. The inoculums were
stirred at 120 rpm for 18 h. The MH agar was poured into a plate and left five minutes to be
solid. The surface of the Mueller Hinton plate was inoculated by streaking the swab over the
entire agar surface. The plate was rotated approximately 60° each time and swabbed a few times
to ensure even inoculums distribution. Filter paper discs were placed on the surface of agar plate
by using forceps. Each disc was pressed down softly to make sure complete contact with the
surface. The synthesised compounds were impregnated on the discs. The plates were incubated
aerobically for 24 h and the diameter of zone inhibition was measured.35 In this study, ampicillin
was used as a control due to its ability to interact with both positive and negative bacteria in
comparison to other antibiotic such as penicillin.39
ACKNOWLEDGEMENT
The authors would like to thank Universiti Malaysia Sarawak and Ministry of Education for
research funds F07(DPP42)/1262/2015(17) and FRGS/ST01(01)/1298/2015(15).
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acetonitrile
acetonitrile
X:
1
2
3
4
5
6
7
8
9
= Ortho-F
= Meta- F
= Para- F
= Ortho-Cl
= Meta-Cl
= Para-Cl
= Ortho-Br
= Meta-Br
= Para-Br
10 = Ortho-I
11 = Meta-I
12 = Para-I
Scheme 1: The synthesis of bis(acylthiourea) 1-12
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