DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas

Article abstract of DOI:10.1016/j.molstruc.2015.06.024

Four new nitrosubstituted acylthioureas i.e. 1-acetyl-3-(4-nitrophenyl)thiourea (TU1), 1-acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2), 1-acetyl-3-(2-methoxy-4-nitrophenyl)thiourea (TU3) and 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4) have been sy

Full text of DOI:10.1016/j.molstruc.2015.06.024

Accepted Manuscript
DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas
Shaista Tahir, Amin Badshah, Raja Azadar Hussain, Muhammad Nawaz Tahir, Saira
Tabassum, Jahangir Ali Patujo, Muhammad Khawar Rauf
PII:
S0022-2860(15)30050-8
10.1016/j.molstruc.2015.06.024
MOLSTR 21576
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 April 2015
Revised Date: 3 June 2015
Accepted Date: 5 June 2015
Please cite this article as: S. Tahir, A. Badshah, R.A. Hussain, M.N. Tahir, S. Tabassum, J.A. Patujo,
M.K. Rauf, DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas, Journal
of Molecular Structure (2015), doi: 10.1016/j.molstruc.2015.06.024.
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ACCEPTED MANUSCRIPT
DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas
Shaista Tahira, Amin Badshah*a, Raja Azadar Hussaina, Muhammad Nawaz Tahirc, Saira
Tabassumb, Jahangir Ali Patujo and Muhammad Khawar Raufa
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad (45320) Pakistan
bDepartment of Biotechnology, Quaid-i-Azam University, Islamabad (45320) Pakistan
cDepartment of Physics, University of Sargodha, Punjab Pakistan
*a aminbadshah@yahoo.com
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DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas
Abstract:
Four new nitro substituted acylthioureas i.e. 1-acetyl-3-(4-nitrophenyl)thiourea (TU1), 1-
acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2), 1-acetyl-3-(2-methoxy-4-
nitrophenyl)thiourea (TU3) and 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4) have been
synthesized and characterized (by C13 and H1 nuclear magnetic resonance, Fourier
transform infrared spectroscopy and single crystal X-ray diffraction). As a preliminary
investigation of the anti-cancer potencies of the said compounds, DNA interaction studies
have been carried out using cyclic voltammetry and UV-vis spectroscopy along with
verification from computational studies. The drug-DNA binding constants are found to be
in the order, KTU3 9.04 x 106 M-1 > KTU4 8.57 x 106 M-1 > KTU2 6.05 x 106 M-1 > KTU1 1.16
x 106 M-1. Furthermore, the antioxidant, cytotoxic, antibacterial and antifungal activities
have been carried out against DPPH (1,1-diphenyl-2-dipicrylhydrazyl), Brine shrimp eggs,
gram positive (M. luteus, S. aureus) and gram negative (B. bronchiseptica, S. typhimurium,
E. aerogens) and fungal cultures (F. fumigatus, F. Mucor, F. niger, F. flavus) respectively.
Keywords: Thiourea, DNA binding studies, cyclic voltammetry, UV-vis spectroscopy,
antimicrobial activities, cytotoxicity
1. Introduction:
Thioureas, having a considerably wide range of applications, are the cousin
compounds of ureas in which oxygen has been replaced with sulfur. The properties of
urea and thiourea differ significantly because of the difference in electronegativity
between sulfur and oxygen [1]. Thioureas possess broad spectrum of biological activities
including antiviral [2], antibacterial [3], antifungal [4], antitubercular [5], herbicidal [6],
insecticidal [7], and pharmacological properties [8], act as corrosion inhibitors, antioxidants
and are polymer components [9].
Thiourea derivatives can be easily synthesized in good yield [10], and substituted
thioureas have recently gained much interest in the preparation of wide variety of
biologically active compounds [11]. Acylthiourea derivatives are well known for their
biological activities like bactericidal, fungicidal, herbicidal, insecticidal and regulation of
plant growth [12]. Herein, we have reported, the synthesis of four new nitrosubstituted
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acylthioureas with complete characterization (FTIR, multinuclear NMR, single crystal
XRD) and biological activities (DNA-binding, antioxidant, cytotoxic and antimicrobial
activities).
As we previously reported in our review article on the biological applications of
selenoureas and their homologues [8], thioureas are an important class of organic
compounds with effective anti-cancer applications. However, their UV-vis spectra and
cyclic voltammograms are not very helpful in the investigation of their ability to bind
with DNA. So, we have synthesized nitro substituted thioureas with an aim to quantify
the DNA interaction of the synthesized compounds from the results of their UV-vis
spectroscopic and cyclic voltammetric analyses.
2. Materials and methods:
2.1 General
Melting points were determined in a capillary tube using electrothermal melting
point apparatus model MP-D Mitamura Riken Kogyo (Tokyo, Japan). Infrared spectra were
taken on a Thermo Scientific NICOLET 6700 Fourier transform infrared spectroscope
(FT-IR). H1 and C13 nuclear magnetic resonance (NMR) were recorded on a Jeol JNM-
LA 500 FT-NMR. Si(CH3)4 was used as internal reference. Suitable single crystal of each
nitrosubstituted acyl thiourea was mounted on a glass fiber and the intensity data were
collected on a Brucker kappa APEXII CCD diffractometer using graphite monochromator
having Mₒ Kα radiation (λ = 0.71073 Å) at 296 K. The structures were solved by direct
methods and refined by full-matrix least squares against F2 of data using SHELXL97
(Sheldrick, 1997) software [13]. Basic crystal data and description of diffraction
experiment are given in Table 1.
Table 1. Crystal diffraction data of TU1, TU2, TU3 and TU4
TU1
TU2
TU3
TU4
Empirical
C9H9N3O3S
C10H11N3O3S
C20H22N6O8S2
C9H8ClN3O3S
formula
Formula weight
Temperature (K) 296(2)
239.25
253.28
296(2)
538.55
296(2)
273.69
296(2)
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Wavelength (Å)
a [Å]
0.71073
5.1519(4)
0.710173
8.7665(11)
0.710173
3.9876(12)
0.710173
4.484(2)
b [Å]
9.0702(7)
7.7560(7)
8.973(3)
23.617(11)
c [Å]
α [degree]
β [degree]
11.9097(9)
71.175(4)
82.499(4)
86.865(5)
522.20(7)
triclinic
17.384(2)
90
100.680(3)
90
1161.5(2)
monoclinic
P 21/c
16.899(6)
81.083(12)
88.322(13)
79.505(13)
587.3(3)
triclinic
10.749(5)
90
97.151(14)
90
1129.5(9)
monoclinic
P 21/c
γ [degree]
Volume (°A3)
Crystal system
Space group
Index ranges
P -1
P -1
–6 ≤ h ≤ 6, –11 –11≤ h ≤ 11, –9 ≤ –5≤ h ≤ 4, –11 ≤ –5≤ h ≤ 5, –29 ≤
≤ k ≤11,
k ≤10,
k ≤11,
k ≤21,
–15 ≤ l ≤15
–22 ≤ l ≤21
–22 ≤ l ≤21
–13 ≤ l ≤13
Absorption
coefficient (µ)
F(000)
0.305
0.279
0.287
0.522
248
528
280
560
Goodness-of-fit
on F2 (S)
1.044
1.035
0.922
0.968
R factor (%); R1, 0.0652, 0.0425
0.0602, 0.0422
0.2252, 0.0650
0.1452, 0.0556
R2.
The computational study (DFT) was carried out using GaussView 5.0 software.
2.2 Synthesis:
All chemicals, organic solvents and reagents were purchased from Sigma-Aldrich,
Fluka and E. Merck. Organic solvents acetone (99.9 % pure), and n-hexane (95 % pure)
were distilled, purified & dried according to reported methods. Potassium thiocyanate (98
% pure), acetyl chloride (99 % pure), 4-nitroaniline (99 % pure), 4-chloro-3-nitroaniline
(99 % pure), 2-methyl-4-nitroaniline (99 % pure) and 2-methoxy-4-nitroaniline (99 %
pure) were used without further purification.
Acetyl chloride was added to the solution containing potassium thiocyanate in dry
acetone and stirred for about 3 hours then respective nitroaniline was added into the
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reaction mixture and further refluxed for about 5 hours to obtain the desired thioureas
[14].
2.3 DNA-binding study by cyclic voltammetry:
Commercial Salmon DNA was solubilized in doubly distilled water to prepare a
stock solution of 6 × 10−4 M from which working concentrations of DNA were prepared.
Concentration of the stock solution was measured by UV absorbance (with Shimadzu
1800 spectrphotometer) at 260 nm using an epsilon value of 6600 M−1cm−1. This DNA
was protein free because A260/A280 > 1.8. Solution preparation for DNA binding studies
was carried out according to our previously reported method [15, 16]. Cyclic voltammetry
was performed on Biologic SP-300 cyclic voltammeter running with EC-Lab Express V
5.40 software (made in France). Before every reading, the working electrode was
polished with alumina powder and rinsed with distilled water. Analytical grade KCl was
used as supporting electrolyte and nitrogen gas (99.9 %) was purged through the mixture
to avoid interference from oxygen. A setup having three electrodes system, i.e. working
(platinum disc electrode with a geometric area of 0.071 cm2s−1), reference Ag/AgCl and
auxiliary electrode (platinum electrode with geometric area much greater than working
electrode) was used for cyclic voltammetric studies. The decrease in the peak current
provided information about DNA binding constant whereas the shifts in the peak
potentials were useful for the determination of the mode of interaction of the drug with
DNA. Drug-DNA binding constant was determined with the help of following equation:
log (1/ [DNA]) = log K + log (I/ Iₒ − I)
(1)
where K is the binding constant and Iₒ and I are the peak currents of free drug and
DNA-bound drug respectively [14].
2.4 DNA-binding study by UV-vis spectroscopy:
For the DNA binding constants with the help of UV-vis spectroscopy at first the
spectra of the analytes before the addition of DNA were recorded by taking solvent in
the reference cell and solution of the analyte in the sample cell. Then the spectra were
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recorded by adding different concentration of DNA, in solutions having constant
concentration of the compound. The whole experiment was carried out by keeping the
volume and concentration of the compound constant while varying the amount of DNA
[16]. The equilibrium constants (binding constant) is calculated by fitting data in the
Benesi-Hildebrand equation (2).
Aₒ/ A-Aₒ = (εG/ εH-G - εG) + (εG/ εH-G - εG) (1/ K[DNA])
(2)
Where Aₒ and A are the absorbance of the free compound and of the compound–DNA
complex, εG and εH-G are the molar extinction coefficients of free compound and of
the compound–DNA complex respectively.
2.5 Antioxidant assay:
Oxidant reducing abilities of all the four nitrosubstituted thiureas were determined
with the help of 1,1-diphenyl-2-picrylhydrazyl radical in DMSO to produce 1,1-diphenyl-
2-picrylhydrazine. The decrease in the absorption of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
was monitored to calculate the percentage scavenging according to the following formula
[17]:
Scavenging activity (%) = Aₒ − A/Aₒ × 100
(3)
Where Aₒ is the absorbance of free DPPH and A is the absorption of DPPH–drug
mixture with increasing concentration of drug. To a solution of DPPH (3.9 mg of DPPH
in 100 mL DMSO) were added the increasing concentrations (1 mM) of nitrosubstituted
thioureas. The decrease in absorption was monitored after 5 min at a wavelength of 517
nm of DPPH with the help of spectrophotometer. All the readings were taken in triplicate
and average of all the readings was used.
2.6 Cytotoxic assay:
Cytotoxic effect of synthesized thioureas was determined using brine shrimp assay
following the method as described by McLaughlin and Rogers [18]. 50 mg of compound
was dissolved in 1 mL of DMSO to the prepared stock solution of each sample. Further
1000 µg/mL, 100 µg/mL and 10 µg/mL dilutions were prepared from the stock solution.
Terbinafine was used as the standard drug. 1 mg of standard was dissolved in 1 mL of
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DMSO to prepare stock solution. Three concentrations i.e. 10 µg/mL, 1 µg/mL and 0.1
µg/mL were used. Sea water produced by dissolving 34 g of sea salt in 1000 mL of
water. Solution was stirred constantly, sea water was aerated in open mouth beaker for
sufficient time. Hatching of shrimp eggs was carried out in compartmentalized rectangular
dish (22 × 23 cm), which was filled with sea water. One large and other small
compartment with separating wall containing holes were present in the dish. Eggs were
poured in large compartment. Lamp light was showered on smaller compartment, after
covering larger compartment with aluminum foil. Newly hatched nauplii moved towards
small compartment due to light. These were picked with pasture pipette and dispensed in
small beaker having sea water. Initially all vials were labeled, followed by addition of 2
mL sea water in all vials. After that 0.1 mL sample solution from their respective stock
solutions were added in the respective vials. Ten nauplii were counted using pasture
pipette and 3X magnify glass and transferred to vials. Simultaneously, volume was raised
up to 5 mL with sea water, so that it does not affect the shrimps and desired
concentration of sample is attained. These vials were incubated for 24 hours at room
temperature. After that shrimps were taken out from vials using pasture pipette and
counted under magnifying glass. Finally lethal dose (LD50) of the thioureas was
determined using finny (1971) software.
2.7 Antibacterial assay:
Antibacterial activity was determined by using disc diffusion method as described
by Islam et al. (2013) [19], against two gram positive (M. luteus ATCC 10240, S. aureus
ATCC 6538) and three gram negative strains (B. bronchiseptica ATCC 4617, S.
typhimurium ATCC 14028, E. aerogens ATCC 13048). Nutrient broth was used for the
maintenance of bacterial cultures for 24 hours at 4 ˚C. Next day cold cultures of these
strains in nutrient broth (Merck, Germany) were used for assay. 4 mg of each
nitrosubstituted thioureas was dissolved in 1 mL of DMSO, for the production of stock
solution of each complex. Final concentration of 30 µg/disc of each complex was used
in the assay. Cefixime-USP and roxythromycin were used as standard drugs. Stock of each
standard drug was prepared by dissolving 4 mg 1 mL of DMSO. DMSO was used as
negative control.
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Bacterial inoculum was prepared in nutrient broth, by dissolving 2 g of broth in 100
mL of water and pH was maintained at 7. Nutrient agar medium was used for the
growth of bacterial strains that was produced by dissolving 2 g in 100 mL of water.
Filter paper discs of 6 mm in size ware prepared from Whatman no. 1 filter paper.
Media, filter paper discs along with other apparatus required in this assay were
autoclaved for sterilization. After autoclaving, whole experiment was carried out in
microbiological safety cabinet. Solidified plates of nutrient agar were labeled and respective
bacterial strain was streaked. Then 7.5 µL of each complex solution was absorbed on disc.
These discs were placed on respective places in petri plate. These petri plates were incubated
for 24 hours at 28 ˚C. Zones of inhibitions were measured after 24 hours. The following
formula was used to calculate the percent bacterial inhibition.
% inhibition = A/B x 100
(4)
Where A is the linear growth (mm) in test sample and B is the linear growth (mm) in
control.
2.8 Antifungal assay:
Antifungal assay was performed by following disc diffusion method as described
by Islam e t al. (2013) [19]. Fungal cultures (F. fumigates FCBP 66, Mucorspecies FCBP
0300, F. niger FCBP 0198, F. flavus FCBP 0064) were maintained on SDA at 4 ˚C. 4 mg
of each synthesized thiourea was dissolved in 1 mL of DMSO to prepare stock solution
of each sample. Terbinafine was used as a standard drug (positive control) and DMSO
was used as negative control. Stock solution of standard was prepared by dissolving 4
mg of standard drug in 1 mL of DMSO and 10 µg/disc concentration of standard drug
was used. Fungal cultures were grown on SDA medium at 6.5 pH. Sabouraud dextrose agar
(SDA) media was prepared by dissolving 6.5 g in 100 mL of distilled water and the pH
was adjusted to 6.5. Whatman no.1 filter paper was used for the preparation of circular
discs of 6 mm size. All apparatus required in the assay were autoclaved for sterilization.
Experiment was carried out in microbiological safety cabinet. SDA media was poured in petri
plates and left for solidification. Filter paper discs were used for the absorption of sample
solution and 7.5 µL of compound solution absorbed on disc. After absorption of complex
solution on disc, were placed in their respective places on solidified SDA plates.
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Contamination was prevented by wrapping petri plates with parafilm. After that plates were
˚
incubated for 24 hours at 28 C. On next day inhibition zones were measured by using
vernier caliper. Percent fungal inhibition was calculated by using equation 4.
3. Experimental:
3.1 1-acetyl-3-(4-nitrophenyl)thiourea (TU1)
TU1 was synthesized by the reaction of 0.2 g of potassium thiocyanate (0.00206
mol) with 0.15 mL (0.00206 mol) of acetylchloride, in dry acetone at continuous stirring
for 3 hours. After the formation of white precipitates of potassium chloride (KCl), 0.427g
(0.00206 mol) of 4-nitroaniline was added into the reaction mixture. The reaction was
kept on stirring under reflux for further 5 hours and monitored with the help of thin
layer chromatography (Scheme.1).
Scheme 1.
The reaction mixture was then poured in cold water and again stirred for at least
two hours to get rid of the byproduct and impurities. The obtained solid product was
filtered, washed with n-hexane and recrystallized in acetone.
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Yield 92 %; mp 194 ˚C; H1 NMR (acetone) δ (ppm) 13.092 (s, 1H), 10.587 (s, 1H),
8.328-8.122 (4H), 2.306 (s, 3H); C13 NMR (acetone) δ (ppm) 179.12, 172.83, 144.83,
144.02, 124.20, 123.26, 123.11, 23.33; FTIR υ (cm−1) 3188.3 (N-H, stretching), 2999.5
(Aromatic C-H, stretching), 1689.2 (C=O, stretching), 1578 (N-O, bending), 1507.5 (C-C,
stretching), 1411.6 (C-H, bending), 1332.99 (N-O, bending), 1250.8 (C=S, stretching).
3.2 1-acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2)
TU2 was synthesized following the same procedure as used for the synthesis of
TU1, except that 0.4712 g (0.00206 mol) of 2-methyl-4-nitroaniline were added after
stirring, for 2 hours instead of 4-nitroaniline.
Yield 92 %; mp 215 ˚C; H1 NMR (acetone) δ (ppm) 12.70 (s, 1H), 10.607 (s, 1H),
8.469-8.109 (3H), 2.878 (s, 3H), 2.452 (s, 3H); C13 NMR (acetone) δ (ppm) 179.70,
172.75, 142.99-121.26, 23.25, 17.30; FTIR υ (cm−1) 3233.6 (N-H, stretching), 3001.0
(Aromatic C-H, stretching), 1693.1 (C=O, stretching), 1574 (N-O, bending), 1511.9 (C-C,
stretching), 1332 (N-O, bending), 1257.8 (C=S, stretching).
3.3 1-acetyl-3-(2-methoxy-4-nitrophenyl)thiourea (TU3)
TU3 was also synthesized following the same procedure as used for the synthesis
of TU1, except that 0.5208 g (0.00206 mol) of 2-methoxy-4-nitroaniline were added after
stirring, for 2 hours instead of 4-nitroaniline.
Yield 90 %; mp 227 ˚C; H1 NMR (acetone) δ (ppm) 13.188 (s, 1H), 10.491 (s, 1H),
8.172-8.132 (3H), 4.128 (s, 3H), 2.303 (s, 3H); C13 NMR (acetone) δ (ppm) 178.18,
172.25, 140.36-121.77, 56.63, 23.22; FTIR υ (cm−1) 3201.9 (N-H, stretching), 3000.6
(Aromatic C-H, stretching),
1702.1 (C=O, stretching), 1573 (N-O, bending), 1516.4 (C-C,
stretching), 1421 (C-H, bending), 1349.9 (N-O, bending), 1243 (C=S, stretching), 1156 (C-O,
stretching).
3.4 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4)
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Again, TU4 was synthesized following the same procedure as used for the synthesis of
TU1, except that 0.535 g (0.00206mol) of 4-chloro-3-nitroaniline were added after stirring, for 2
hours instead of 4-nitroaniline.
Yield 90 %; mp 149 ˚C; H1 NMR (acetone) δ (ppm) 12.870 (s, 1H), 10.598 (s, 1H), 8.725-
7.747 (3H), 2.299 (s, 3H); C13 NMR (acetone) δ (ppm) 131.67, 128.87, 120.53, 23.28; FTIR
υ (cm−1) 3185.3 (N-H, stretching), 3019.8 (Aromatic C-H, stretching),
1686.0 (C=O,
1241
stretching), 1524.7 (N-O, bending), 1419.5 (C-H, bending), 1370.8 (N-O, bending),
(C=S, stretching).
4. Results and Discussion:
The title compounds TU1-TU4 were prepared using previously reported method,
[14] as depicted in Scheme 1.
The synthesized nitrosubstituted acyl thioureas have been characterized on the
basis of their spectroscopic data. H1 and C13 NMR spectra were recorded in deuterated acetone.
Three different types of protons gave their distinct signals in H1 NMR, in case of TU1
and TU4 i.e. singlets for each of the two –NH protons, aromatic protons and methyl
protons. The methyl and methoxy protons, of TU2 and TU3 respectively, gave an
additional singlet, in their H1 NMR spectra. In all of the synthesized thioureas, the -NH
proton situated in between phenyl ring and C=S, is maximum desheilded owing to the
intramolecular hydrogen bonding and thus gave a singlet at around 13 ppm and the -NH
proton located in between the C=O and C=S is deshielded to a lesser extent thus
providing a singlet in the region of 10-11 ppm [8, 15-17]. The aromatic ring protons gave
their signals between 8 and 9 ppm. In C13 NMR, carbon bonded with sulfur provided a
weak signal at about 180 ppm and carbonyl carbon appeared in the range of 172-175
ppm. Signals for aromatic carbons are visible in the region between 120-140 ppm [8, 15-17].
In FTIR, -NH of thioureas gave a broad band above 3100 cm−1 owing to the
intramolecular hydrogen bonding between the oxygen of the carbonyl and -NH. Just
around 3000 cm−1 Ar-H stretch was evident and carbonyl group appeared as an intense
band in the region of 1700-1600 cm-1. C=S was available between 1050 cm-1 and
1250 cm−1 [8, 15-17], for all the compounds (Figure 1.).
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Figure 1. FTIR spectra of the synthesized thioureas.
4.1 Crystallography:
The crystal structures of TU1, TU2, TU3 and TU4, along with their nonbonding
interactions, have been depicted in Figure 2-5 respectively and the selected bond lengths
and bond angles for the crystal structures of TU1, TU2, TU3 and TU4 have been
provided in the Tables 2-5.
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Figure 2. (a) Molecular diagram of TU1 with ball and stick arrangement, non-hydrogen
atoms represented by 30 % probability boundary spheres and hydrogen atoms are spheres
of arbitrary size, (b) expanded (red) and short contacts (magenta) of TU1 at vdW radii
of 0.1 Å, with labeled contact atoms and (c) π-stacked layered supramolecular structure
of TU1.
Table 2. Selected bond lengths and bond angles for TU1
Bond lengths (Å)
Bond angles (˚)
O3 C8 1.219(2) O2 N1 O1 123.7(2)
N3 C8 1.376(3) O1 N1 C4 118.5(2)
N3 H3A 0.860(2) C1 N2 H2A 113.8(2)
N3 C7 1.389(3) H2A N2 C7 113.8(2)
N2 C7 1.337(2) C7 N3 S1 118.9(1)
N2 H2A 0.860(2) C7 N3 H3A 115.3(2)
N2 C1 1.402(3) H3A N3 C8 115.3(2)
N1 C4 1.461(3) N3 C8 O3 122.1(2)
N1 O1 1.217(2) C9 C8 O3 123.2(2)
S1 C7 1.656(2)
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Figure 3. (a) Molecular diagram of TU2 with ball and stick arrangement, non-hydrogen
atoms represented by 30 % probability boundary spheres and hydrogen atoms are spheres
of arbitrary size, (b) expanded (red) and short contacts (magenta) of TU2 at vdW radii
of 0.1 Å, with labeled contact atoms and (c)
of TU2.
π-stacked layered supramolecular structure
Table 3. Selected bond lengths and bond angles for TU2
Bond lengths (Å)
Bond angles (˚)
O3 C9 1.217(2) O2 N1 O1 122.7(2)
C9 N3 1.375(2) C3 C4 CN1 118.5(2)
N3 H3 0.860(1) N1 C4 C5 113.8(2)
N3 C8 1.391(2) C5 C6 C7 113.8(2)
N3 H3 0.860(1) C6 C1 N2 118.9(1)
N2 H2A 0.860(2) C1 N2 H2A 115.3(2)
N2 C1 1.407(2) H2A N2 C8 115.3(2)
N1 C4 1.461(3) N3 C8 S1 122.1(2)
N1 O1 1.214(2) S1 C8 N2 123.2(2)
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S1 C8 1.655(2) C8 N3 H3 115.4(2)
N2 C8 1.341(2) H2 C3 N3 115.3(2)
C6 C7 1.505(3) O3 C9 N3 121.8(2)
Figure 4. (a) Molecular diagram of TU3 with ball and stick arrangement, non-hydrogen
atoms represented by 30 % probability boundary spheres and hydrogen atoms are spheres
of arbitrary size, (b) expanded (red) and short contacts (magenta) of TU3 at vdW radii
of 0.1 Å, with labeled contact atoms and (c)
of TU3.
π-stacked layered supramolecular structure
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Table 4. Selected bond lengths and bond angles for TU3
Bond lengths (Å)
Bond angles (˚)
N1 O1
N1 C4
C4 C5
C4 C3
C5 C6
C6 O3
C7 O3
C6 C1
C1 N2
1.208(6) O1
1.460(6) N1
N1 C4
118.5(4)
118.6(4)
118.2(4)
124.2(4)
118.6(4)
115.0(4)
C4 C3
C4 C5
C6 O3
O3 C7
C1 C6
1.367(6) N1
1.368(7) C5
1.375(6) C6
1.355(5) O3
1.423(6) C1
N2 H2A 114.2(4)
1.394(7) H2A N2 C8
1.400(6) S1
114.2(4)
117.3(3)
127.9(4)
C8 N3
C8 S1
N2 H2A 0.860(4) N2
N2 C8
1.332(6) C8
N3 H3A 115.4(4)
N3 C8
1.380(5) C8
N3 C9 115.4(4)
Figure 5. (a) Molecular diagram of TU4 with ball and stick arrangement, non-hydrogen
atoms represented by 30 % probability boundary spheres and hydrogen atoms are spheres
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of arbitrary size, (b) expanded (red) and short contacts (magenta) of TU4 at vdW radii
of 0.1 Å, with labeled contact atoms and (c)
of TU4.
π-stacked layered supramolecular structure
Table 5. Selected bond lengths and bond angles for TU4
Bond lengths (Å)
Bond angles (˚)
N1 O1 1.207(6) C9 C8 O3 122.7(4)
N1 C3 1.463(6) C9 C8 N3 115.2(4)
C3 C2 1.370(6) C8 N3 H3 115.0(4)
C3 C4 1.379(6) H3 N3 C7 115.0(4)
C4 Cl1 1.721(5) N3 C7 S1 120.4(3)
C4 C5 1.371(7) S1 C7 N2 124.4(3)
C1 C6 1.382(6) C7 N2 H2A 180.0(4)
C1 N2 1.422(6) C1 N2 H2A 118.0(4)
N2 H2A 0.860(3) N2 C1 C2 120.9(4)
N2 C7 1.338(5) N2 C1 C6 119.2(4)
C7 N3 1.374(6) C2 C3 N1 115.9(4)
H3 N3 0.861(3) N1 C3 C4 123.0(4)
It can be seen that both inter and intramolecular hydrogen bonding are present in
all of these four nitrosubstituted acyl thioureas rendering them remarkable supramolecular
chemistry. TU1 forms a
acetyl moiety of one layer situated at so called trans position to the methyl of the acetyl
functionality of the adjacent layer. -stacking of phenyl rings is also evident in the zig-
π-stacked layered supramolecular structure with methyl of the
π
zag patterned layers of TU2. TU3 forms a bamboo fence like supramolecular structure
with methoxy substituent of each layer pointed outwardly towards the next layer. TU4
forms a beautiful motif like supramolecular structure.
4.2 DNA binding studies:
4.2(a) By UV-vis spectroscopy and cyclic voltammetry:
The DNA-binding constants calculated by cyclic voltammetry using Equation 2, are
found to be in close agreement with the ones calculated by UV-vis spectroscopy. The
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voltammograms of all the synthesized thioureas give oxidation, reduction peak in a region
between -1V-1V. Compounds show quasi reversible behavior which is evident by:
•
•
•
Change of potential of the oxidation and reduction peaks at different scan rates.
Ratio of oxidation and reduction peak currents not exactly equal to one.
Difference of potential between the oxidation and reduction peaks not close to 60
mV.
The representative cyclic voltammograms for TU3 have been depicted in Figure
7(a).
Figure 7. (a) Representative cyclic voltammograms for sample TU3 solution free of DNA
and TU3 solution with different concentrations of DNA (b) Graphical representation of
the plot of log 1/DNA vs. log I/I
ₒ
-I for the calculation of DNA binding constant KTU3
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for TU3 and (c) cyclic voltammograms of TU1 (1-Acetyl-3-(4-nitrophenyl)thiourea) in
pink, TU2 (1-Acetyl-3-(2-methyl-4-nitrophenyl)thiourea) in red, TU3 (1-Acetyl-3-(2-
methoxy-4-nitrophenyl)thiourea) in bright green and TU4 (1-Acetyl-3-(4-chloro-3-
nitrophenyl)thiourea) in plum.
The peak at –0.79713 V for nitro was scanned with different additions of the
DNA. By the addition of the DNA (7-49 µM) there is a shift in the peak potential to -
0.8072 V, -0.9719 V, -0.9881 V, -0.9981 V, -0.9982 V, -0.9983 V and -1.007 V with a
decrease in the peak current. This decrease in the peak current confirms the formation of
TU3-DNA adduct which diffuses comparatively slowly towards the electrodes and
decrease the current. Negative shift in the potential is in favor of electrostatic mode of
interaction. All the compounds show similar type of peaks (Figure 7-b) and same mode
of interaction with the DNA. Representative UV-vis spectroscopic data of DNA binding
for TU4 confirms the cyclic voltammetric results by showing hypochromism and blue
shift of the TU4-DNA adducts relative to free TU4 [8, 15-17], (Figure 6).
Figure 6. (a) Representative plot of absorbance vs. wavelength for TU4 sample without
DNA and with addition of 10 µM, 20 µM, 30 µM, 40 µM and 50 µM DNA solution
(b) Graphical representation of Aₒ/A-Aₒ vs. 1/DNA to calculate binding constant KTU4.
DNA binding parameters for all the compounds have been presented in Table 6.
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Table 6. Calculations of DNA binding constant by UV-vis spectroscopy and cyclic
voltammetry for TU1, TU2, TU3 and TU4
Samples
UV-vis data
In K
Cyclic voltammetry data
K(M-1)
∆G(kJM-1)
K(M-1)
In K
15.464
∆G(kJM-1)
TU1
TU2
TU3
TU4
1.16 x 106
13.964
-34.6
5.20 x 106
1.24 x 106
1.51 x 106
2.40 x 106
-38.7
-34.8
-35.2
-36.4
6.05 x 106
9.04 x 106
8.57 x 106
15.616
16.017
15.964
-38.7
-39.7
-39.5
14.031
14.228
14.691
4.3 Computational study:
The oxidation and reduction potential of TU3 is observed to be least among the
four thioureas and is in accordance with the computed HOMO and LUMO values (Table
7), which can be attributed to the presence of methoxy group at the ortho position of
phenyl ring in TU3.
Table 7. Observed oxidation and reduction potentials in cyclic voltammetry and
computed HOMO, LUMO values for TU1, TU2, TU3 and TU4.
Samples Oxidation Potential (V) HOMO (eV) Reduction potential (V) LUMO (eV)
-0.917332
-0.847337
-0.797130
-1.00521
-0.23455
-0.22384
-0.19976
-0.23548
-1.1259
-0.854572
-0.794254
-1.00825
-0.09796
-0.09627
-0.07549
-0.12099
TU1
TU2
TU3
TU4
It can also be seen in Figure 8. that the orbitals are polarized to a much greater
extent in TU3, compared to those in the rest of the thioureas, owing to the presence of a
more polar methoxy group on the phenyl ring of TU3.
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Figure 8. Orbital diagrams of (a) TU1, (b) TU2, (c) TU4 and (d) TU3.
4.4 DPPH scavaenging activity:
The percent scavenging of synthesized compounds against DPPH is shown in
Figure 9.
120
100
80
60
40
20
0
Ascorbic
acid
TU1
TU2
TU3
TU4
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Figure 9. Percent scavenging of TU1, TU2, TU3 and TU4 against reference ascorbic
acid.
The better antioxidant activity of TU1 and TU2 can be attributed to their planar
structures which favor the free radical intermediate structure formation by delocalization
of elcectron over the wider part of the molecule [20]. IC50 value for TU1 which showed
more than 50 % scavenging is found to be 250 µM.
4.5 Cytotoixicity:
Owing to the same functionality, all thioureas show promising but almost same
percent cytotoxicity Figure 10.
14
12
10
1000 ppm
500 ppm
250 ppm
8
6
4
2
0
TU1
TU2
TU3
TU4
Figure 10. Percentage cytotoxicity graph for TU1, TU2, TU3 and TU4.
The LD50 value is found to be 55 ppm for TU1, TU2 and TU3 and 60 ppm for
TU4.
4.6 Antibacterial activity and antifungal activity:
Except TU3, all of the thioureas show moderate antibacterial
activity 36-40 % (Table 8), against gram positive bacterial strains and lower or no
antibacterial activity against gram negative bacterial strains, this could be because of the
well known structural difference in the cell membranes of the gram positive and gram
negative bacteria [21], but the actual mechanism of the drugs against respective bacterial
membranes is not the subject of this research article.
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Table 8. Bacterial inhibition chart of thioureas against gram positive and gram negative
bacterial strains.
Samples
(µg/disc)
TU1
E.Aerogens S.Typhimurium M.Lutues B.Bronchiseptaca S.Auaurea
±SE
6.5±0.075
7.5±0.075
0
±SE
0
0
±SE
±SE
0
6.5±0.075
0
±SE
6.5±0.075
6.5±0.075
0
6.5±0.075
6.5±0.075
7±0.075
TU2
TU3
TU4
6.5±0.075
6.5±0.075
7±0.078
6.5±0.075
6.6±0.075
6.5±0.075
Among all of the synthesized thioureas, TU4 shows better activity against both positive
and negative bacterial strains. This can be attributed to chloro substituent available at the
phenyl ring in TU4, as better antimicrobial activities associated with different compounds
having chloro substitution has been reported in literature as well [22].
All of the synthesized thioureas have shown about 39 % fungal inhibition (Table 9),
against all fungal cultures. This almost similar percent fungal inhibition can be correlated
to the similar functional moiety i.e. thio and nitro substituted phenyl rings available in
all the synthesized compounds which may inhibit fungal growth following similar
mechanism [23].
Table 9. Fungal inhibition data for the four thioureas
Samples(µg/disc) F.Fumagatus F. Mucor
F.Niger
F.Flaws
±SE
±SE
±SE
±SE
7.5±0.722
7.5±0.717
7.5±0.719
7.5±0.715
7.5±0.711 7.5±0.712 7.5±0.712
7.5±0.717 7.5±0.717 7.5±0.717
7.5±0.707 7.5±0.719 7.5±0.719
7.5±0.715 7.5±0.715 7.5±0.715
TU1
TU2
TU3
TU4
5. Conclusions:
In case of all the synthesized thioureas, the observed decrease in peak potential
with increase in DNA concentration in cyclic voltammetry and blue shift with
hypochromism in UV-vis spectroscopy suggest the electrostatic binding mode of
interaction of drug with DNA. The DNA binding constants calculated by both techniques
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are in the order of 106 M-1 for all of the four nitrosubstituted acyl thioureas, showing
their good binding potential with DNA. The planar thiourea i.e. TU1 shows good antioxidant
activity. Moderate biological activities are shown by TU1, TU2 and TU3. TU4 which is
chloro substituted is found to have better biological activities, among all of the
synthesized thioureas.
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ACKNOWLEDGEMENT:
The Higher Education Commission of Pakistan
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Highlights:
•
•
•
Synthesis and characterization of new nitrosubstituted acyl thioureas
DNA-binding studies by UV-vis spectroscopy and cyclic voltammetery
Biological assays of the synthesized thioureas