Gold(I), silver(I) and copper(I) complexes of 2,4,6-trimethylphenyl-3-benzoylthiourea; synthesis and biological applications

Article abstract of DOI:10.1016/j.poly.2020.114485

2,4,6-Trimethylphenyl-3-benzoylthiourea (L) and its gold(I), silver(I) and copper(I) complexes were synthesized. Treatment of L with HAuCl₄, AgNO₃ and CuI in CH₃CN afforded complexes of general representation, M(L)X, where

Full text of DOI:10.1016/j.poly.2020.114485

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Gold(I), Silver(I) and Copper(I) Complexes of 2,4,6-trimethylphenyl-3-ben‐
zoylthiourea; Synthesis and biological applications
Umar Ali Khan, Amin Badshah, Muhammad Nawaz Tahir, Ezzat Khan
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Please cite this article as: U. Ali Khan, A. Badshah, M. Nawaz Tahir, E. Khan, Gold(I), Silver(I) and Copper(I)
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Gold(I), Silver(I) and Copper(I) Complexes of 2,4,6-trimethylphenyl-3-benzoylthiourea;
Synthesis and biological applications
Umar Ali Khan1, Amin Badshah2, Muhammad Nawaz Tahir3, Ezzat Khan1*
1Depatrment of Chemistry, University of Malakand, 18800 Chakdara, Khyber Pakhtunkhwa,
Pakistan
2Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
3Department of Physics, University of Sargodha, 40100 Sargodha, Punjab, Pakistan.
*Corresponding Author: ekhan@uom.edu.pk

Abstract
2,4,6-trimethylphenyl-3-benzoylthiourea (L) and its gold(I), silver(I) and copper(I)
complexes were synthesized. Treatment of L with HAuCl4, AgNO3 and CuI in CH3CN afforded
complexes of general representation, M(L)X, where M/X = Au/Cl (1), Ag/NO3 (2), Cu/I (3).
Reaction with Au3+ salt afforded two products, wherein complex containing Au+ (reduced under
normal and aerobic reaction conditions ca. 66% on NMR time scale) was confirmed by x-ray
diffraction for a single crystal of complex 1. The minor fraction (ca. 33%, from 1H-NMR data) in
the reaction mixture was identified as complex 4 with a general formula [AuL2]Cl3. Reaction with
AgNO3 and CuI exclusively afforded pure products 2 and 3. Ligand L and complexes 1-4 were
characterized by UV/visible, FT-IR and 1H-, 13C-NMR spectroscopy. Complex 1 is crystalline in
nature and good quality crystals were obtained which allowed for collection of diffraction data and
the structure was thus confirmed. Solution state and solid state structures have been compared.
The resultant complexes were tested for their cytotoxicity, antibacterial, antifungal, antioxidant
and DNA binding activities. Complex 1 (Au based) showed promising activities in comparison
with the available selected drugs used as standards.
Keywords: 2,4,6-trimethylphenyl-3-benzoylthiourea; M(I) complexes; Characterization; X-ray
structure; DNA binding.

1. Introduction
Transition metals have tremendous role in the development of new metal based drugs to
combat several human ailments. Transition metal complexes exist both in anionic and neutral
forms. Metal ion is normally surrounded by ligands which determine the reactivity in a desired
direction and many of metal complexes are in field to fight against several human related diseases
like carcinomas, lymphomas, diabetes, neurological disorder and anti-inflammatory disease.[1, 2]
Transition metal complexes exist in a variety of oxidation states and this characteristic enable them
to accommodate a variety of organic molecules or ligands around them. The incorporation of
different ligands can tune the properties of metal ions in a desired manner thus making metal based
drugs promising pharmacological candidates. A number of metal complexes are in field which
encourage studies for exploration of new metallodrugs with attractive properties like biological
potency, easy accessibility, least toxicity and improved physical profile. Among transition metals
gold, silver, copper, nickel and zinc have been in use for their non-biological and biological
applications[3, 4].
The pharmacological applications of metal complexes depend upon the type of metal, its
oxidation state and nature of ligand(s). Different metal ions with same ligands and same metal
with different ligands exhibit far different biological properties. Currently a number of metal based
drugs have been investigated showing promising in vitro results, but during in vivo studies they
suffer from some limitations and very less are recommended for clinical trial. The challenge for a
metallodurg is its in vivo stability and compatibility with biological system which entirely depends
on the selection of an adequate ligand and its counter metal ion[5, 6]. Among ligands thiourea
derivatives enriched with O and N donors in addition to its S functionality, have attracted much
interest because of their diverse and strong coordination ability, variety of coordination modes,
biocompatibility, accessibility and ease of modification. Thiourea derivatives exist both in neutral

(S donor only) and anionic (N donor, N, S donor, N, S, O donor) state[7]. To date, a number of
metal complexes of thiourea derivatives especially those containing Cu, Ag and Au as central
metal ions have been investigated with excellent biological applications like DNA binding,
antimicrobial, cytotoxic, anti-arthritic, and antioxidant agents[8-10].
In continuation to our previous work [11] on thiourea ligands and their complexes with a
variety of metal ions, here we report synthesis and complexation of 2,4,6-trimethylphenyl-3-
benzoylthiourea ligand with Au(I), Ag(I), Cu(I) and Au(III) metal ions. Structure of complex 1,
was determined with the help of X-ray diffraction and experimental data were calculated and
compared. The ligands and its complexes were tested for their preliminary biological evaluation
of DNA binding, cytotoxicity, free radical scavenging and antibacterial potency. Activities of all
complexes are promising as compared to corresponding standard drug.
2. Experimental
2.1
General
All chemical reagents and metal salts were purchased from Sigma Aldrich and Merck and
were used without further treatment to purify them. All organic solvents were distilled prior to use.
The NMR spectra were recorded on BRUKER AVANCE 300 MHz spectrometer in deuterated
solvents as mentioned against each compound. The FT-IR spectra were recorded with Shimadzu
8300 spectrometer as KBr discs in the frequency range of 4000-200 cm-1. The UV spectra were
recorded using Shimadzu UVPC-1800 UV-visible scanning spectrometer (sample path length 1.0
cm) in the range of 800-200 nm.
2.2
Synthesis of 2,4,6-trimethylphenyl-3-benzoylthiourea, L
Ligand L was prepared by following the literature procedure[12]. The white precipitates
obtained were recrystallized in ethanol/chloroform mixture (1:1 v/v). Yield, 78%; m.p = 154-

156°C; 1H-NMR (Acetone-D6); δ (ppm) = 11.80 (br, 1H, NH ), 10.20 (br, 1H, NH), 7.99-7.96 (m,
2H, Ar-H), 7.57-7.38 (m, 3H, Ar-H), 6.80 (s, 2H, Ar-H), 2.72 (s, 3H, CH3), 1.92 (s, 6H, 2CH3);
13C-NMR (Acetone-D6); δ (ppm) = 181.1, 168.2, 137.5, 136.0, 135.4, 134.1, 133.6, 132.7, 130.0,
127.8, 20.5, 17.6; FT-IR (cm-1) = 3245w, 3156w, 2917w, 1689m, 1500s, 1475s, 1209m, 1139s,
699s, 653s, 514m.
2.3
Synthesis of 2,4,6-trimethylphenyl-3-benzoylthiouragold(I) chloride, 1
A solution containing L (100 mg, 0.33 mmol, in 15 mL acetonitrile) was added drop wise
to a solution of HAuCl4 (113 mg, 0.33 mmol), in the same solvent (15 mL). The reaction mixture
was allowed to stir overnight at room temperature, during this time the pale yellow color of the
solution mixture turned colorless. The solution was filtered, solvent and other readily volatile
materials were evaporated under reduced pressure and NMR was measured in acetone-D6, which
indicated two products in a ratio 66% (1) and 33% (4). A portion of solid was dissolved in
acetonitrile and the resulting solution was allowed to evaporate slowly. After few days colorless
needle like crystals appeared, they were separated from the mother liquor and a suitable crystal
was selected for single crystal analysis, confirming complex 1, the major fraction in the mixture.
1
Complex 1: H-NMR (Acetone-D6) δ (ppm) = 10.09 (br, NH), 9.85 (br, NH), 8.18-8.10,
7.73-7.64, 7.61-7.56 (m, m, m, CH, Ar), 2.34 (s, Me), 2.33 (s, Me); FT-IR (cm-1) = 3210w(br),
3130w(br), 2916w(br), 1658m(br), 1498s(br), 1259m(br), 1146s(br), 686s(br).
2.4
Synthesis of Complex 2,4,6-trimethylphenyl-3-benzoylthiourea silver(I) nitrate, 2
Ligand (100 mg, 0.33 mmol) was treated with AgNO3 (56.5 mg, 0.33 mmol) in acetonitrile
in the same way as stated for complex 1 above. During the course of overnight stirring, a color
change from pale yellow to colorless was observed, which indicated reaction completion. Solid
unreacted salt material were separated through filtration and the resultant solution was kept for

slow evaporation. After few days solid powder of the proposed compound were obtained and were
accordingly analyzed. Yield = 70%; m. p = 290-300ᵒC; 1H-NMR (Acetone-D6) δ (ppm) = 10.10
(br, 1H, NH ), 9.88 (br, 1H, NH), 8.18-8.15, 7.73-7.67, 7.61-7.52 (m, m, m, 5H, Ph), 6.94 (s, 2H,
CH), 3.03 (s, 3H, Me), 2.27 (s, 6H, 2Me); FT-IR (cm-1) = 3244w, 3128w, 2917w, 1688s, 1523s,
1500s, 1474s, 1288m, 1209s, 710s, 515m.
2.5
Synthesis of 2,4,6-trimethylphenyl-3-benzoylthioureacopper(I) iodide, 3
CuI (63 mg, 0.33mmol) and ligand (100 mg, 0.33 mmol) were mixed together in 15 mL
acetonitrile and the reaction was proceeded in the same way as stated for compounds 1 and 2. After
2h, white precipitate were obtained, were filtered off, washed with ether and dried in open air.
Crystallization in various solvents did not lead to the formation of crystallographically suitable
crystals. Yield = 68%; 1H-NMR (CDCl3) δ (ppm) = 12.11 (br, 1H, NH), 10.37 (br, 1H, NH), 7.99-
7.92, 7.62-7.47 (overlapped multiplet, 5H, Ph), 6.96 (s, 2H, CH), 2.32 (br, 3H, Me), 1.36 (s, 6H,
Me); FT-IR, (cm-1) = 3270br, 2916w, 2851w, 1673m, 1639m, 1504s, 1486s, 1258m, 709m, 655s,
435m.
2.6. Synthesis of bis(2,4,6-trimethylphenyl-3-benzoylthiourea)gold(III) chloride, 4
Complex 4 was obtained as block crystals together with needles of complex 1 and the
crystals were separated under microscope. UV/Visible, CHCl3 solution, max(nm)/(M-1cm-1):
303/15228. 1H-NMR (Acetone-D6) δ (ppm) = 12.40 (br, 1H, NH ), 10.98 (br, 1H, NH), 8.10 (m,
CH, Ar), 7.79 (m, CH, Ar), 7.65 (m, CH, Ar), 7.03 (s, 2H, Ar), 2.87 (s, 3H, CH3), 2.29 (s, 6H,
2CH3); FT-IR, (cm-1) = 3303m, 3131w, 3015w, 2915w, 1679s, 1556s, 1508s, 1485s, 1236s,
1171m, 1066m, 832m, 585m.

3. X-ray crystallography
The X-ray diffraction data of complex 1, were collected with the help of Bruker kappa APEXII
CCDC diffractometer (Chemical formula C17H18AuN2ClOS, Mr 530.81, crystal system
monoclinic, space group P21/c, temperature 296, a 8.059(7) Å, b 14.254(13) Å, c 15.795(15) Å, 
102.22°, volume 1773.3(3) Å3, Z 4, radiation type Mo-Kα,  8.57 mm-1, crystal size
0.38×0.34×0.30 mm, absorption correction multi-scan, Tmin 0.160, Tmax 0.215, number of
measured reflections 11252, independent reflections 3842, observed reflections with [I > 2(I)]
3201, Rint 0.042, R[F2 > 2(F2)] 0.027, wR(F2) 0.067, S 1.02 and number of parameter 211).
Structure solution and refinements were accomplished by taking advantage of software PLATON,
WinGx, SHELXL and SAINT[13-16].
3.1 Theoretical Studies
The complex 1 was investigated by DFT using B3LYP method and LANLD2Z basis set [17-
19], while the ligand L by the same method but 6-31G basis set was used instead, employing
Gaussian view 09 package with the support of Gaussian view visualization program [20].
Optimized geometrical parameters such as bond angles and lengths, for ligand L and complex 1
have been calculated[21]. The highest occupied molecular orbitals (HOMO) and Lowest
Unoccupied molecular orbitals (LUMO) and electrostatic potentials of the complex were also
analyzed[18]. Global reactivity parameters such as electronegativity (A), ionization potential (I),
hardness (η), softness (σ) electronegativity (X), chemical potential (μ) and electrophilicity (ω) by
using the following equations[22].

4. Cytotoxicity Studies
4.1
Brine shrimp lethality bioassay
The selected compounds were screened for bioactivity using shrimp lethality test to check
the cytotoxic activity of ligand and its complexes 1-3. Lethality test was performed in a 96 well
plate against brine shrimp (Artemia salina) larvae as previously described at 30-32°C in simulated
seawater (38 g/L supplemented with 6 mg/L dried yeast) in a specially designed two-compartment
plastic tray.[23] The assay system was performed by taking 10 bijoux bottles for different
concentrations of test compounds. A 100 mg of each test compound (ligand and respective
complexes, each) were weighed and dissolved in mixture of solvents containing 1 mL DMSO and
2 mL of seawater. Two-fold dilution was carried out to prepare a series of concentrations ranging
from 100 to 0.195 mg/mL. Afterwards, 10 mature phototropic nauplii were harvested with the help
of Pasteur pipette and transferred to each bijoux bottle using magnifying glass for correct counting
of nauplii. Positive and negative control wells included serial concentrations of doxorubicin and
1% DMSO, respectively. The setup was allowed to stay for 24 h at normal conditions of
temperature. After 6 and 24 h the bottles were observed using magnifying glass and the number of
survived larvae were counted and recorded. Percent of mortality of nauplii was calculated for each
concentration of the respective sample. The median lethal concentration (LC50) was determined
by plotting graph of mean percent mortality versus log of concentration. The LC50 value greater
than 1.0 mg/mL, suggests that the compound is nontoxic. Percent mortality can be calculate by
using the following formula [24, 25].
% Mortality = 푁0 ― 푁푖 × 100
푁표
(Where, N0 = number of nauplii taken, Ni = number of napulii dead)

5. Antibacterial activity
Susceptibility of extracts against bacterial species was tested according to formerly described
procedure[26]. Three Gram negative strains (Bordetella bronchiseptica, Salmonella typhimurium
and Klebsiella aerogenes also known as Enterobacter aerogenes, ATCC # 4617, 14028 and 13048,
respectively) were selected during this study. The strains were cultured in nutrient broth media and
incubated for 24 h at 37°C. Sterilized deionized water was used to adjust the turbidity to 104
CFU/mL by comparing with McFarland 0.5 BaSO4 turbidity standards. The refreshed inoculum
(100 µL) was then swabbed onto Petri plates containing 20 mL nutrient agar. Test samples (5 µL
of 20 mg/mL DMSO; 100µg/disc) were infused on sterile filter paper discs (6 mm) and placed on
seeded nutrient agar plate. Cefixime-USP at a concentration of 20 µg/disc and DMSO impregnated
discs were included as positive and negative controls, respectively in order to assess the activity
in a better way. After 24 h incubation, clear zones of growth inhibition were measured manually.
The following formula was used to calculate the percent bacterial inhibition.
% Inhibition = 퐵퐴 × 100
(A = linear growth (mm) in test sample and B = linear growth (mm) in control)
6. Antifungal activity
Antifungal activity of compounds 1-3 and L was evaluated by following the described
standard protocol[26]. The fungal strains (Aspergillus fumigatus, Fusarium solani and Mucor
specie, commercially available under FCBP # 66, 0291 and 0300, respectively) were purchased
from fungal culture bank of Pakistan, they were accordingly cultured on SDA. Prior to the
sensitivity determination, the spores were harvested in 0.02% Tween 20 solution and turbidity was
adjusted according to McFarland 0.5 turbidity standard. A 100 µL sample of each harvested spores
was swabbed on plates containing 25 mL sterilized SDA. Filter paper discs loaded with 5 µL of

test sample (20 mg/mL DMSO; 100 µg/disc) as well as standard available antifungal terbinafine
(50 µg/disc) and DMSO were placed on seeded SDA plates. The plates were incubated at 28°C for
24-48 h. Thereafter, clear zones of inhibition around discs were observed and measured using
Vernier caliper.
7. Antioxidant assay
Free radical scavenging activity of complexes 1-3 was determined by previously reported
methods against a robust radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH)[27]. The scavenging or
reducing activities of metal complexes due to hydrogen atom or electron donation were measured
by color change of DPPH from purple to pale yellow. Absorbance of free DPPH solution (50 mM
DMSO) was measured at 517 nm represented by A(blank) in the following equation. The DPPH (1.0
mL) was added to each sample solution of complexes and the resultant mixtures were incubated
for 30 min in dark, at ambient temperature (23±1°C). The absorbance of each solution was
measured (represented by Asample in the equation given below) and percent scavenging activity was
calculated as below.
Percent Inhibition = (Abalnk - Asample/Ablank) ×100
The IC50 of each sample was determined by plotting %I vs concentration. All measurements were
carried out in triplicate and their mean was considered for calculations [28-31].
8. DNA Binding studies
8.1Spectrophotometric method
The binding activities of complexes 1-4 with CT-DNA were measured in 5 mM Tris-HCl
buffer at pH 7.2. Stock solution of DNA in double distilled water was prepared and stored at
4±1℃. The absorbance of solution containing DNA was measured at 260 and 280 nm, the ratio

1.9 indicates that the DNA is sufficiently free from protein. The concentration of the stock solution
was determined by measuring absorption at 260 nm using its molar absorption coefficient (ɛ260
=
6600 M-1cm-1). The absorption titration was performed for solutions of 100 μM of each complex
in the presence of various concentrations of DNA (0.001 μM - 0.032 μM). The spectra were
recorded after the addition of equal amounts of different concentrations of DNA to different
solutions of complex 1-4 of same concentration followed by equilibration for 10 min. The intrinsic
binding constant, Kb was calculated as per equation below [32, 33].
[DNA]/[ɛa-ɛf] = [DNA]/[ɛb- ɛf] + 1/Kb [ ɛb- ɛf]
Where, [DNA] = concentration of DNA, ɛa = the extinction coefficient for the absorption band at
the given DNA concentration and ɛf, ɛb = the extrinsic coefficients of free metal complex and of
the complex fully bounded to DNA. A plot of [DNA]/[ ɛa- ɛf] vs [DNA] gives a slop 1/[ ɛa- ɛf] and
intercept Y equal to 1/Kb [ ɛb- ɛf]. The binding constant is regarded as the ratio of the slop to
intercept.
8.2
Viscosity measurements
Viscometric titration was carried out using ubbelodhe viscometer maintained at 25℃ immersed in
water bath. The concentration of DNA was kept constant and concentration of the tested complexes
was increased gradually. Different concentration of the respective complex was introduced into
DNA solution present in viscometer and equilibrated for some time. Flow time was measured with
stop watch. Each experiment was performed in triplicate and average flow time was calculated.
Relative viscosity of DNA in the presence and absence of complex was determined using the
following relation.
ηʹsp/ηsp = {(tcomplex-t0)/t0}/{(tcontrol-t0)t0}

where, ηʹsp and ηsp are the specific viscosity of DNA-Compound complex and DNA, respectively;
tcomplex, tcontrol and t0 are the flow time of DNA-compound complex, pure DNA and buffer,
respectively. Data were presented as (η/ ηo)1/3 versus [complex]/[DNA] where, η is the viscosity
of DNA with complex and ηo is the viscosity of free DNA.[33, 34]
9. Result and Discussion
9.1
General considerations
Thiourea derivative L and its Complexes 1-4 were characterized by UV-visible, FT-IR,
NMR spectroscopy and structure of complex 1 was confirmed by x-ray diffraction for a single
crystal. The UV-visible spectra of the free ligand and the respective complexes provided enough
information regarding product formation. An absorption peak at 243 nm with a broad shoulder at
282 nm was observed for the free ligand (Figure S1). The shoulder at 282 nm may be
predominantly assigned to C=S, π-π* transition. In complexes the shoulder disappeared, thus
revealing the stabilization of -electron as a result of electron flow towards metal ion. The
observation also supports the coordination through S atom[6]. The UV/Visible spectrum of
complex 4 is different from other three complexes. The shoulder peak at higher wavelength was
nor resolved however, the deformed structure of the parent peak indicate additional electronic
excitations such as M-X in complexes 1-3. The absence of shoulder peak in the UV spectrum of
complex 4, indicates that the molecule is lacking of M-X moiety as has been reported for Au(III)
complexes [35]. Molar absorptivity, calculated from the same data was also very close to the data
for structurally analogous compounds.
The FT-IR spectra of ligand L give a typically broad peak at 3245, 3156 cm-1, corresponds
to stretching frequency of N-H bonds, 2917 cm-1 for C-H, a strong peak at 1689 cm-1 for C=O and
at 1209 cm-1 for C=S bond. In complexes 1, the existence of peak around 1658 cm-1 typically broad

indicates traces of compound 4, due to which the peaks corresponding to C=O do not resolve.
Further a shift to lower wave number supports coordination of ligand with the metal ion. The signal
corresponds to the same functional group appear at 1688 and 1673 cm-1 in complex 2 and 3,
respectively. Slight change in wave number of C=O group (1688 in comparison to 1689 cm-1 of
the free ligand) in compound 2 rules out the involvement of this group in coordination, and it can
be clarified that the ligand is monodentate S-donor. Further shift of the peak 1673 cm-1 indicates
that the ligand predominantly affords a chelate through S and O atoms, affording three coordinated
complex, 3. In complex 4, the C=O bond stretching appear at 1679 cm-1 which is in close
agreement with bidentate (S,O-donor) behavior of the ligand. Other peaks in IR spectra of
compounds agree well with the proposed structure as given in Scheme 1. The C=S bonds in
complexes 1-4, show negative shift indicating coordination of ligands with metal ion via C=S site.
The NMR spectroscopy is a reliable technique which provides enough information regarding the
proposed structure of the ligand, L and its complexes. Characteristic broad signals at 11.80 and
10.20 ppm can be assigned qualitatively to two protons bonded to N atoms. A sharp singlet in the
aromatic region and two sharp signals in aliphatic region can be assigned unambiguously to CH
and Me protons of the 2,4,6-trimethylphenyl group of the unsymmetrical thiourea. Similarly 13C-
NMR spectra contains signals at 181.1 and 168.2 ppm, correspond to CS and CO carbons,
respectively. Two signals in aliphatic region with relative intensities of 1:2, clearly indicate the
presence of chemically different Me groups. The 1H–NMR signals of complexes in characteristic
regions provide enough information regarding formation of the proposed complexes. These data
of gold complex reveal formation of two products where chemical shifts of NH groups of the
ligand and CH and Me groups of 2,4,6-trimethylpheny moiety are quite helpful in proposing
structure of these complexes. Structure of minor (ca. 33%) fraction can be proposed as compound
4, shown in Scheme 1, which is crystalline in nature and can easily be separated under microscope

from the major (66%) unexpected product, complex 1. The NMR spectrum of compound 2 with
signals at 10.10 and 9.88 ppm, indicate formation of a single product and the same can be supported
by the appearance of aromatic CH protons (at 6.94 ppm) of 2,4,6-trimethylphenyl group. The same
signals for compound 3 were observed at 12.10 and 10.37 ppm for NH groups and at 6.96 ppm for
CH of 2,4,6-trimethylphenyl group. From a consistent IR and NMR data sets it can be concluded
that Cu is predominately three coordinated with trigonal planar geometry as shown in Scheme 1.
The chemical shift values of compound 1, 2 and 3, 4 are in support of monodentate and bidentate
nature of the ligand, respectively. The presence of two NH signals in NMR spectra of ligand and
complexes confirm that no coordination through N-atom has occurred. [36, 37]
NH2
O
H
H
O
N
N
KSCN
Cl
NCS
S
O
Acetone
Acetone, -KCl
L
H
H
N
H
N
H
N
N
S
O
S
O
NH
NH
S
O
M
Cu
Au
Cl3
HN
HN
X
I
O
S
3
1
2
M/X = Au/Cl( ), Ag/NO3( )
4
Scheme 1
9.2
Synthesis of ligand, L and proposed structures of its complexes, 1-4.
Single Crystal Analysis and comparison of experimental and theoretical
parameters of complex 1
Complex 1 was isolated from the mixture as colorless needles and structure for a single
crystal with suitable dimensions was determined as shown in Figure 1. The compound crystalizes

as monoclinic system with space group P21/c, a=8.0593(7) (Å), b=14.2537(13) (Å),
c=15.7948(15) (Å), β=102.224(4). The reaction was carried out in CH3CN in open air therefore
reduction of Au ion from +3 to +1 was observed wherein the major component of the mixture was
found to be a linear complex, 1 [37]. The metal ion in complex 1 accommodates the ligand through
it S site and O remains uncoordinated. The Au-S and Au-Cl bond lengths are 2.265 and 2.252 Å,
respectively. The Au-S bond with considerably shorter distance as compared with other
homoleptic thiourea complexes [38, 39] is predominantly because of the presence of chloro
function in trans position, across metal ion. This observation also clarifies the nature of M-Cl bond
being expected to be labile in further relevant studies. The Cl-Au-S fragment is considerably bent
with a bond angle of 174.87°. A close view of intermolecular interactions of non-covalent nature
did not give any clue to explain the deviation from linearity as we have reported recently for a Ag
complex, where the geometry around metal ion was pseudo-five coordinated.[40] The inter-
/intramolecular non-covalent interactions can be found in molecules of compound 1, wherein no
aurophilic interactions were detected. The most important interaction is Cl…C=O with a separation
distance between Cl…C 3.410 Å and the same function also interacts with C4H with a separation
distance of 3.626 Å. The N2…O1 within the same molecule interacts with each other through
intramolecular H-bonding (2.626 Å). This interaction inhibits the ligand up to some extent to act
as bidentate and also the donor sites S and O are stabilized in a trans manner with respect to each
other at 168.67°. Other interactions were also found due to which the supramolecular structure
extends in a 2D fashion, a representative dimmer unit is shown in Figure 2 with selected
interactions. Similarly, the compound is also lacking agostic interactions as has been reported for
linear Au-thiourea complexes.[41]
Geometry optimization of ligand L and the respective complex 1 was carried out with the
intent to correlate solid state (experimental) and gas phase structure (optimized). The optimized

structures with complete atom labelling and numbering scheme, of L and complex 1 are shown in
Figure S2 and S3, respectively. The calculated structural parameters (bond lengths and angles) are
summarized together with experimental data in Table 1. Considering bond lengths, the identical
value was found to be 1.480 Å for (C1-C7). The least deviated value was found to be 0.0087 Å for
(N2-C9) and the most deviated values was found for Au1-S1 with 0.2005 Å. In case of bond angles
the identical value was found for N1-C8-S1 to be 119.9°. The least deviation was observed for
theoretical and experimental value of C2-C3-C4 with a difference of 0.18o and the most deviated
angle was Cl1-Au1-S1 (2.59o). Critical overview of the calculated and experimental data are in
close agreement with each other.
Figure 1 Solid state structure of Complex 1, with 50% thermal ellipsoids, partial numbering
scheme, all hydrogen atoms are omitted for clarity.

Figure 2 Extended structure of complex 1, (a) short contacts wherein Cl function and carbonyl
carbon (C=O) are involved, Hydrogen atoms of methyl belong to 2,4,6-
trimethylphenyl and CH of phenyl moiety are also linked. Hanging contacts which
extend the structure further are deleted for clarity reasons. (b) Same view of molecules
without short contacts.
Table 1 Summarized bond lengths (Å) and angles (°) obtained experimentally and theoretical
Calculations using B3LYP/LANLD2Z
Bond lengths
Experimental
Bond angles
Experimental Calculated
Calculated
2.3900
2.4654
1.7808
1.2650
1.3887
1.4059
1.3396
1.4527
1.4801
1.4158
1.4178
1.4018
1.4101
1.4085
Au1-Cl1
Au1-S1
S1-C8
O1-C7
N1-C8
N1-C7
N2-C8
N2-C9
C1-C7
C1-C2
C1-C6
C2-C3
C3-C4
C4-C5
2.2517(10)
2.2649(10)
1.701(4)
1.213(4)
1.355(4)
1.378(5)
1.313(4)
1.444(4)
1.480(5)
1.375(5)
1.380(5)
1.379(6)
1.355(7)
1.370(7)
Cl1-Au1-S1 174.87(4)
172.28
101.39
127.66
125.36
119.54
116.77
122.98
120.20
123.69
119.98
121.76
119.9
C8-S1-Au1
C8-N1-C7
C8-N2-C9
C2-C1-C6
C2-C1-C7
O1-C7-C1
O1-C7-N1
C7-C1-C6
C2-C3-C4
N2-C8-S1
N1-C8-S1
104.39(12)
128.7(3)
126.5 (3)
119.0 (4)
117.8 (4)
123.3 (3)
120.8
123.2 (3)
119.8
122.4(3)
119.9(3)
9.3.
Frontier Molecular Analysis and Global parameters

The energy of LUMO and HOMO molecular orbitals and other related parameters are of prime
interest for chemists, physicists and material scientists. Energies of frontiers orbitals decide
whether the molecule is soft or hard. The frontier orbitals also predict the most reactive position
in π-electron system and energy gap between HOMO and LUMO are also used for interpreting the
chemical stability of a molecule. The HOMO has the ability to donate electrons, while LUMO
accepts electrons and these two levels are very important for coordination chemists. These orbitals
predict the coordination behavior of an organic molecule to act as ligand and/or to accept electrons
from the metal ion during back bonding. The position of HOMO and LUMO in a molecule also
determine the softness or hardness of the ligand. The ionization potential is the energy of HOMO,
while the energy of LUMO is concerned with electron affinity. The energy difference between
HOMO and LUMO is the band gap or energy gap and it corresponds to the kinetic stability and
chemical reactivity of molecules. The energy gap calculated for ligand L is -0.144 eV and for
complex 1 is -0.1.37 eV. Other global reactivity parameters are calculated using frontier orbital
energies, given in Table 2. The frontier orbitals HOMO and LUMO of both ligand and complex 1
are given in Figure S4 and S5. The electronic cloud in HOMO is localized on C=S moiety in the
ligand and in complex 1 these electrons are localized between metal ion and the C=S fragment.
The LUMO of the ligand are localized on aromatic rings and S atom, while the same orbitals in
metal complex are localized on aromatic rings.[42] These observations reveal that the ligand is
considerably soft and has the ability to act as -acceptor and to afford stable complexes with soft
metals.
9.4.
Electrostatic potential analysis
Molecular electrostatic potential (MEP) is generally used to explore molecular interactions. It
is the potential experienced by a unit positive charge which surrounds the molecule due to the

distribution of electrons in the respective molecule. Electrostatic potential predicts the chemical
reactivity of the molecule. The site of negative potential corresponds to be site of protonation and
available for nucleophilic attack (electron rich), while the positive potential site is considered to
be the site accessible for electrophilic attack [20, 21]. The 3D representation of complex 1 and the
ligand L is shown in Figure S6 and S7, respectively, where the color scheme ‘red’ is a negative
extreme and ‘blue’ is positive extreme. The order of increasing potential is
red<orange<yellow<green<blue.
Table 2
Global reactivity parameters (eV) for ligand L and complex 1
Σ
Compound I
A
X
Η
μ
Ω
Ligand
Complex 1
5.57
6.53
1.68
2.83
3.22
4.68
1.945
1.85
0.25
0.27
-3.625
-4.68
3.36
5.91
9.5.
Cytotoxicity
Brine shrimp lethality bioassay is one of the rapid preliminary test for the biochemical
activity of compounds. This test is based on the lethality potentials of any metal complex to brine
shrimp nauplii. It is suggested that a test sample containing LC50 less than 1 mg/mL are considered
most damaging to cells[25, 43, 44]. In present study the LC50 value of complexes 1-3 has been
evaluated. Among all the three complexes the complex 1 exhibits maximum lethality after 24 h,
which is quite promising and comparable to standard Doxorubicin (0.1 mg/mL) and may have
anticancer effect on cancerous cells. The summarized data related to complex 1 and the standard
(Doxorubicin) are represented in Table 3. Graphically the relationship of concentration vs
mortality is represented in Error! Reference source not found. (for complex 1).

Figure 3
Brine shrimp lethality of a) complex 1 after 6 h, b) 24 h and c) Doxorubicin as positive
control

Table 3 Summarized values of brine shrimp toxicity as LC50 value determined after 6 and 24 h.
Sample/control
Complex 1
LC50 (mg/mL) after 6h
LC50 (mg/mL) after 24 h
2.06
2.13
2.41
0.1
1.35
1.70
1.87
0.1
Complex 2
Complex 3
Doxorubicin
9.6.
Antibacterial Activities
The in vitro antibacterial activity of complexes 1-3 were tested against three gram negative
bacterial strains namely Bordetella bronchiseptica, Salmonella typhimurium and Enterobacter
aerogenes, the obtained results have been tabulated in

Table 4 and the same has been represented graphically in Figure 4. The activity of
complexes 1-3 was compared with the available standard antibiotic, cefixime. Among complexes
silver based complex was more potent with inhibition zones 17, 17 and 16 mm against E.
aerogenes, B. bronchiseptica and S. typhimurium, respectively. Complex 1 exhibited relatively
higher activity against B. bronchiseptica with inhibited zone of 18 mm (81.8%). The increased
antibacterial activity of metal complex compared to free ligand can be explained as, ligand
delocalizes its π electrons with metal ion and metal ion partially shares its positive charge with the
ligand, which results decrease in polarity of the complex, consequently increase the lipophilicity
of metal complex. This increased lipophilicity enhances penetration of metal complex to lipid
membrane forming hydrogen bond with imine group by the active site which interfere enzyme
activity, DNA damage and cell wall synthesis and finally may lead to cell apoptosis[45-47].

Table 4
Bacterial Inhibition data for complexes 1-3 against three selected strains.
Zone of Inhibition (mm)
Complex/Control
E. aerogenes ±SE
12±1.51
B. bronchiseptica ±SE
18±1.81
S. typhimurium ±SE
15±1.41
1
2
17±1.27
17±1.42
16.2±1.26
3
9.4±1.5
15±1.27
10.7±1.5
Cefixime
22±1.26
25±0.56
22±1.808
Figure 4 Graphical representation of zones of inhibition of complex 1-3 and the standard
available drug, against three strains selected in this study.
9.7.
Antifungal Activity
The in vitro antifungal activity of complexes 1-3 was determined against selected fungal
strains Aspergillus fumigatus, Fusarium solani and Mucor specie. All the complexes are active
against Fusarium solani, with inhibited zones of 17.5 (72.9%), 12.5 and 12 mm, respectively. The
activity of complex 1, was very poor against Mucor specie as compared to complexes 2 and 3. The
activity of complexes against A. fumigatus and Mucor specie was poor to moderate. On the basis
of these results it can be concluded that these complexes cannot be used as better antifungal agents.
The data obtained against the selected strains of fungi are summarized in Table 5 and graphically

represented in Figure 5. The enhanced activity of coordinated ligand can be explained on the basis
of chelation theory as discussed above for antibacterial activity [48-51].
Table 5
Fungal Inhibition data for complexes 1-3:
Zone of Inhibition (mm)
Sample/Control
Aspergillus fumigatus±SE Fusarium solani±SE Mucor specie ±SE
Complex 1
Complex 2
Complex 3
Terbinafine
7.5±0.707
7.0±0.5
17.5±1.11
12.5±0.8
0
7.7±0.85
6.7±0.5
24±1.21
12±0.5
5.5±0.75
22±1.61
21±1.11
Figure 5
Graphical representation of fungal zones of inhibitions by complex 1-3
9.8.
DPPH Scavenging assay
DPPH is a stable free radical and its nature is affected by molecules capable of donating
hydrogen atom or electron. Reactive Oxygen Species, ROS [52] are continuously produced in the
form of free radicals in normal life processes of aerobic organisms. Over production of various
free radicals in the body affect adversely the DNA bases, thus causing serious disorders. In human

beings the cancer, aging, chronic and oxidative stress can be associated with the production of free
radicals [10, 53]. It is need of the day to have an efficient supplement as antioxidant in order to
control the production of antioxidants or to neutralize them. Complexes 1-3 were investigated for
their possible antioxidant activity against DPPH free radical. The complexes exhibited IC50 values
11.4, 35, 28 ppm with 69.87, 54.2 and 60.4%, respectively. The ascorbic acid was used as standard
antioxidant during this study. Among complexes the activity of complex 1, is far better than
complex 2 and 3 and is very close to the activity recorded for the standard. The efficiency of
complex 1, is quite impressive in comparison to other metal based antioxidant [53, 54] but still it
needs to be improved so that up to mark activity is achieved in order to make these reagents
commercially viable.

Figure 6
Free radical scavenging efficiency of complex 1, which increases with increase in
concentration of the complex in a dose dependent manner (upper), and plot indicating
relationship between percent inhibition vs concentration of complex 1 used for
calculating IC50 value.

9.9.
9.9.1 UV- Visible studies
The interaction of complex 1-4 with DNA was studied by electronic absorption to
DNA binding activities
determine the binding affinities of the target complexes. The binding mode of the complexes to
DNA can be determined by the spectral changes (either increase or decrease in shift or intensity in
wavelength) of complex upon the addition of DNA. The complex 2 did not show any change upon
addition of DNA and hence was declared as inactive for DNA binding. Upon addition of different
concentration of DNA to the solution of complex 1, 3 and 4 with constant concentration, a
significant decrease in absorption peak was observed at 248, 247, 310 nm (hypochromic) with no
detectable shift in wavelength, respectively. The gradual decrease in absorption shows interaction
of complex with double strand of CT-DNA through intercalation, which was further confirmed
through viscosity measurement (given in the following section). The binding constant Kb of
complexes 1, 3 and 4 were determined by plotting [DNA]/[ɛa-ɛf] vs [DNA] and was found to be
1.270×104, 1.646×104 and 2.178×104, respectively. The spectral change determined during
experiments for complex 1, 3 and 4 are given in Figure 7, Figure 8 and S8, respectively. The poor
DNA bonding of efficiency of complex 4, supports the proposed structure of the complex where
free sites are not accessible for interaction with DNA.
Figure 7
UV-visible spectra of complex 1, indicating DNA binding

Figure 8
DNA binding studies through UV-visible spectroscopy of complex 3
The absorbance for pure complex is maximum (1, 3 and 4) and by addition of the DNA,
the intensity abruptly/gradually changes. The decrease in absorbance is a function of interaction
potentials of the respective complex with DNA. A comparison of complex 1, 3 and 4 reveals that
Au based linear complex 1 is more efficient than complex 3 and 4.
9.9.2 Viscometry
Viscosity measurement provide critical information about the mode of interaction of a
complex/compound with DNA and is considered to be the least ambiguous test. In classical
intercalation mode of binding the complex lengthen the DNA helix by separating the base pair to
accommodate the ligand. The increase in viscosity of DNA was observed in complex 1, 3 and 4
suggesting classical intercalation mode of binding as shown collectively for both the complexes in
Figure 9. The mode and intensity of interaction can thus be confirmed. The efficiency of complex
1 is expectedly prominent as compared with complex 3 and 4, where in complex 3 very slight
change in activity with increase in concentration was observed.

Complex 1
complex 3
complex 4
7
6
5
4
3
2
1
0
0.04
0.08
0.12
[COMPLEX/DNA]
0.16
0.2
Figure 9
Plot of η/ηo)1/3 vs [complex/DNA] of complex 1, 3 and 4.
10. Conclusion
Unsymmetrical thiourea (2,4,6-trimethylphenyl-3-benzoyl thiourea) as ligand is accessible in
the laboratory and readily afford complexes of general formula MLX, where M = Au(I), Ag(I) and
-
Cu(I), X = Cl-, NO3 and I- and [ML2]Cl3. Ligand was expected to give chelating complexes with
S, O donor sites but the spectroscopic data reveal that Au affords mixture of two products. The
products can be identified on the basis of their characteristic H-NMR chemical shifts and could be
separated under microscope on the basis of their different crystalline shapes. Complexes 2 and 3
were pure compounds and formation of side product was not observed, however geometry around
metal ion was different, linear around Au(I), Ag(I), trigonal around Cu(I) and square planar around
Au(III). Complex 1 was isolated as crystalline material from the solid mixture and its structure
was confirmed in solid state with the help of x-ray diffraction. All complexes exhibit promising
antibacterial activities against E. aerogenes, B. bronchiseptica and S. typhimurium. Complex 1
exhibited higher activity (81.8%) against B. bronchiseptica, complex 2 was active against all three
strains with zones of inhibition 17, 17 and 16 mm, respectively. Antifungal activity was in the
same order and complex 1 (Au(I) based) was more active than Ag and Cu complexes. Antioxidant
activities and DNA interaction were also studied and compounds were found to be of moderate
efficiency. Experimental structural parameters of complex 1, were compared with calculated data
and a close agreement was found.
Conflict of Interest