Mononuclear copper(I) complexes with triphenylphosphine and N,N′-disubstituted thioureas: synthesis, characterization, and biological evaluation

Article abstract of DOI:10.1080/00958972.2018.1538504

Twelve new complexes, of the general formula CuCl(TPP)₂Tu^1–12 (Tu = thiourea), were synthesized by the reaction of CuCl(TPP)₃ (TPP = triphenylphosphine) and various N,N′-disubstituted thioureas. The structures of the synthesized complexes were characterized by different techniques such as Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy (¹H, ¹³C, ³¹P, and ¹⁹F), and the representative complexes (1, 2 and 12) were analyzed via single crystal X-ray diffraction. The single crystal X-ray analysis revealed that copper(I) is coordinated with chlorine, two TPP, and the thiourea ligands through the sulfur atom in a mononuclear distorted tetrahedral mode. The compounds were tested for antibacterial, antifungal, cytotoxicity, antileishmanial, and antioxidant activities. The results showed that the synthesized complexes are significantly more active than the free ligands and the commercial reference compounds. The high biological activities of the complexes versus free ligands can be attributed to the copper(I) chloride complexation with thiourea ligands. The synthesized complexes were also evaluated, both experimentally and theoretically, for DNA binding studies. The UV-visible spectroscopic and molecular docking studies demonstrated that the complexes are conjugating with DNA through a groove binding mode.

Full text of DOI:10.1080/00958972.2018.1538504

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Mononuclear copper(I) complexes with
triphenylphosphine and N,N′-disubstituted
thioureas: Synthesis, characterization and
biological evaluation
Syed Ishtiaq Khan, Inayat Ali Khan, Amin Badshah, Fouzia Parveen Malik,
Saira Tabassum, Ikram Ullah, Davit Zargarian & Muhammad Khawar Rauf
To cite this article: Syed Ishtiaq Khan, Inayat Ali Khan, Amin Badshah, Fouzia Parveen
Malik, Saira Tabassum, Ikram Ullah, Davit Zargarian & Muhammad Khawar Rauf (2018):
Mononuclear copper(I) complexes with triphenylphosphine and N,N′-disubstituted thioureas:
Synthesis, characterization and biological evaluation, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2018.1538504
To link to this article: https://doi.org/10.1080/00958972.2018.1538504
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Mononuclear copper(I) complexes with triphenylphosphine and
N,Nʹ-disubstituted thioureas: Synthesis, characterization and
biological evaluation
SYED ISHTIAQ KHANa, INAYAT ALI KHANa,c, AMIN BADSHAH*a, FOUZIA PARVEEN MALIKb,
SAIRA TABASSUMd, IKRAM ULLAHd, DAVIT ZARGARIANe and MUHAMMAD KHAWAR RAUF*a
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
bResearch Center for Modeling and Simulations, National University of Science Technology (NUST), H-12,
Islamabad 44000, Pakistan
cAl-Farabi Research Complex, SA-Center for Interdisciplinary Research in Basic Sciences,
International Islamic University (IIU), H-10, Islamabad 44000, Pakistan
dDepartment of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
eDepartment of Chemistry, University of the Montreal, Montréal (Québec) H3C 3J7, Canada
Twelve new complexes, of the general formula CuCl(TPP)2Tu1-12 (Tu = thiourea), were
synthesized by the reaction of CuCl(TPP)3 (TPP = triphenylphosphine) and various N,Nʹ-
disubstituted thioureas. The structures of the synthesized complexes were characterized by
different techniques such as Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic
resonance (NMR) spectroscopy (1H, 13C, 31P and 19F) and the representative complexes (1, 2 and
12) were analyzed via single crystal X-ray diffraction. The single crystal X-ray analysis revealed
that copper(I) is coordinated with chlorine, two TPP and the thiourea ligands through the sulfur
atom in a mononuclear distorted tetrahedral mode. The compounds were tested for antibacterial,
antifungal, cytotoxicity, antileishmanial and antioxidant activities. The results showed that the
synthesized complexes are significantly more active than the free ligands and the commercial
reference compounds. The high biological activities of the complexes versus free ligands can be
attributed to the copper(I) chloride complexation with thiourea ligands. The synthesized
complexes were also evaluated, both experimentally and theoretically, for DNA binding studies.
The UV-visible spectroscopic and molecular docking studies demonstrated that the complexes
are conjugating with DNA through a groove binding mode.
Keywords: Complexes; Fourier transform infrared; Biological activities; Antibacterial; DNA-
Binding
*Corresponding authors. Email: aminbadshah@yahoo.com (A. Badshah); mkhawarrauf@yahoo.co.uk (M.K. Rauf)

1. Introduction
Medicinal inorganic chemistry offers a wide range of opportunities for the design of therapeutic
agents which are not accessible to organic compounds [1-3]. Some properties of the cationic
metal ion and the ligand such as wide range of coordination numbers, geometries, various
oxidation states, thermodynamic and kinetic properties, and intrinsic properties of the metal and
ligand attract the medicinal chemist to exploit their reactivity for the development of new drugs.
The success of cisplatin in the clinical treatment of various types of neoplasias has placed
coordination chemistry in the frontline in the fight against cancer due to metal-based drugs [1, 2].
Treatment of cancer and other infectious diseases faces serious difficulties due to the
development of resistance to present anticancer/antibiotic drugs. There are many anticancer and
antibiotic agents having thiourea functionality of natural and synthetic origin. Polyfunctional
thioureas are physiologically active substances possessing a wide range of activities such as
herbicides [3], insecticides [4], plant-growth regulators [5, 6], antihypertensive agents [7],
antitubercular, antimalarial, anticancer [8], antithyroid, antihelmintic and TRPV1 receptor
antagonists [11-13]. The copper complexes, due to their unusual structural features, have attained
considerable importance due to their application in luminescence, solar cells, supramolecular
devices, biological activities and catalytic properties [14-17].
DNA is the pharmacological target for many drugs that are in clinical use or in advanced
clinical trials. Small ligand molecules interact with the DNA and alter its function. These small
molecules act as drugs when DNA alteration is required to cure or control the disease [18]. Since
1969 it has been found that copper has a high affinity for DNA binding [19] analogous to the
phenomenon illustrated for cisplatin [20]. The crystal structure of CuCl2 and DNA was published
for the first time in 1991 in which it was found that copper is bound to a guanine N7 residue.
This binding is dependent on the electron affinity, complex size and geometry of the adduct
formed. It induces irreversible modifications in the DNA conformational structure. Due to these
observations, large numbers of copper complexes for targeting DNA were tested and still
progress is going on. Furthermore, it was found that the copper complexes interacted non-
covalently with DNA rather than coordinating through covalent adduct formation with DNA.
The copper complexes interact non-covalently with DNA through electrostatic, intercalative and
groove binding, including the major or minor groove [21]. Moreover, it was found that
physiochemical features such as planarity, hydrophobicity, size of the ligand, nature of co-ligand

and the geometry of metal complex all play vital roles in determining the binding/intercalating
power with DNA.
A series of copper(I) complexes with N,Nʹ-disubstituted thiourea ligands have been
screened for in vitro cytotoxic activity in several human cancer cell lines (A498, EVSAT, H226,
IGROV, M19, MCF-7 and WiDr), showing a moderate cytotoxicity which is comparable to that
of cisplatin and etoposide [22]. The N-(alkyl/aryl)-Nꞌ-acylthiourea and the N-di(alkyl/aryl)-Nꞌ-
acylthiourea act as the versatile ligands and they can coordinate through the S, N and oxygen
atom [23, 24]. The thiourea can exhibit monodentate behavior with various transition metals ions
such as Ag(I) [25], Au(I) [26], Pt(II) [27], Hg(II) [28] and Cu(I) [29]. Triphenylphosphine is
widely used in the synthesis of complexes due to its reducing properties and nucleophilic
character. In the present work we have used triphenylphosphine as supporting ligand along with
thiourea as the main ligand.
In this present work we have synthesized a series of complexes with CuCl using
triphenylphosphine as co-ligand and various disubstituted thioureas of the general formula
ArNHCSNHCOPh, shown in figure 1. The synthesized complexes were characterized by
spectroscopic techniques and elemental analyses. Complexes 1, 2 and 12 were also analyzed by
X-ray crystallography. After successful characterization, the complexes were tested for DNA-
binding and selected complexes for different biological activities such as cytotoxicity,
antioxidant and antimicrobial behavior. All the selected complexes have demonstrated
significant activities.
2. Experimental
2.1. Chemicals
All chemicals, reagents and organic solvents were purchased from Sigma-Aldrich, Fluka and
E. Merck. CuCl, organic acids, and triphenylphosphine (TPP) were used as received without
purification. The organic solvents acetone, dichloromethane, chloroform, alcohols, petroleum
ether and n-hexane were distilled, purified and dried according to reported methods [30],
saturated with nitrogen, stored over molecular sieves 4 Å and degassed before use.

2.2. Instrumentation
Melting points were determined using a melting point apparatus, model Bio Cote SMP10- UK;
all the values are uncorrected. NMR spectra were recorded on Bruker AV-300 MHz and Bruker
1
AV-400 MHz spectrometers using deuterated solvents. H NMR (300 MHz): internal standard
CDCl3 {7.28 ppm from TMS (Si(CH3)4)}; 13C NMR (75.47 MHz): internal standard
solvent
solvent CDCl3 (77.0 ppm from TMS). The 31P chemical shift was measured relative to 83%
19
orthophosphoric acid (H3PO4) in water at 121.49 MHz (operating frequency). The F chemical
shift was measured at 282.23 MHz (operating frequency). The splitting of proton resonance in
the 1H NMR spectra are defined as s = singlet, d = doublet, t = triplet and m = multiplet. Infrared
(FT-IR) absorption measurements were carried out on an Alpha Bruker Model 10018394 in the
range of 4000-400 cm-1 using attenuated total reflection (ATR). Elemental analyses were
performed on a Fisons EA1108 CHNS analyzer while the metal (copper) contents were
determined using an atomic absorption spectrophotometer (AAS), Perkin Elmer A800. Single
crystal analysis of the synthesized thiourea phosphine copper(I) halide complexes were
conducted on Bruker Smart APEX diffractometers. A suitable crystal was selected and kept at
100 K during data collection. The structures were solved and refined using Olex2 software.
2.3. Synthesis
2.3.1. Synthesis and characterization of chlorotris[triphenylphosphinecopper(I)]
[Cu(TPP)3]Cl precursor. All the reactions were carried out in inert atmosphere. The synthesis
of the precursor [Cu(TPP)3]Cl according to the procedure is given below. The
triphenylphosphine (TPP) was dissolved in acetonitrile and was added to the suspension of
copper(I) chloride in 3:1 giving a white solid of the precursor formed after a few minutes
(scheme 1 equation 1). The resulting suspension was stirred for at least 1 h. The solution was
filtered and the solid was washed with ethanol several times. The white solid was dissolved in
dichloromethane (DCM) and filtered to ensure the removal of insoluble salts. The resulting
solution was kept for recrystallization and transparent crystalline solids formed after 24 h with
very high yield. The melting point was determined and compared with the literature value. The
melting point of the resulting precursor was found to be 176 °C, which falls in the melting point
range given in the literature (175 °C).

885.87;
The precursor was insoluble in water and the yield was 77%; molecular weight =
FT-IR (ATR, υ in cm-1): 3048 aromatic (C-H), 1584 (aromatic C=C), 1431, 1090, 740 (TPP);
1
(300 MHz,
): 7.16-7.36 (m, 45H, Ar-H), 13
C NMR (75 MHz, CDCl
H NMR
CDCl δ in ppm
,
,
3
3
3
1
δ in ppm): 128.52 (d, J=8.66 Hz, P-C coupling, Ar-C), 129.49, 133.45 (d, J=20.36 Hz, P-C
31P NMR (121.49 MHz, 83%
2
coupling, Ar-C), 133.87 (d, J=15.95 Hz, P-C coupling, Ar-C);
aqueous H3PO4
): -3.53(s), elemental analyses for C H ClCuP (885.87): C, 73.22; H,
55 45 3
δ in ppm
,
5.12; Cu, 7.17. Found: C, 73.02; H, 5.06; Cu, 7.01%.
2.3.2. Synthesis and characterization of N,Nʹ-disubstituted thioureas. The general synthetic
route for the synthesis of ligands 1-12 is shown in scheme 1, while the proposed chemical
structures are shown in figure 1. The ligands 1-benzoyl-3-(3-trifluoromethyl)phenyl)thiourea
(Tu1),
1-benzoyl-3-(2-methoxyphenyl)thiourea
(Tu2),
1-benzoyl-3-(2,3,5,6-
(Tu4),
tetrachlorophenyl)thiourea (Tu3), 1-benzoyl-3-(2-trifluoromethyl)phenyl)thiourea
1-benzoyl-3-(3,5-dichlorophenyl)thiourea (Tu5), 1-benzoyl-3-(2,4,6-trichlorophenyl)thiourea
(Tu6), 1-benzoyl-3-cyclohexylthiourea (Tu7), 1-(benzo[d]thiazol-2-yl)-3-benzoylthiourea (Tu8),
1-(2-chlorobenzoyl)-3-phenylthiourea (Tu9), 1-(2-chlorobenzoyl)-3-(2-chlorophenyl)thiourea
(Tu10), 1-(2-chlorobenzoyl)-3-(2,3-dichlorophenyl)thiourea (Tu11) and 1-(2-chlorobenzoyl)-3-(3-
(trifluoromethyl)phenyl)thiourea (Tu12) are shown in figure 1. These ligands were synthesized
according to our previous reported work, scheme 1 and equation 2 [31]. The general synthetic
scheme used for the complexes is given in scheme 1 equation 3. All the ligands and complexes
were insoluble in aqueous media.
2.3.3. Synthesis and characterization of [CuCl(TPP)2Tu1-12] complexes. To a 100 mL double
necked round bottomed flask containing the appropriate thiourea ligand (1 mmol) dissolved in
20 mL of methanol, was added the salt [Cu(TPP)3]Cl (1 mmol) dissolved in chloroform. The
color of the solution changed to yellow immediately and a solid formed after a few minutes. The
suspension was stirred for 1 h. The yellow solid was separated by filtration and washed twice
with methanol. The product was dissolved in chloroform and filtered, from which 5 ml solution
was separated to stand at 25 °C in which prism-like light yellow crystals of complexes 1, 2 and
12 were grown.

Scheme 1. General synthetic route for the synthesis of [Cu(TPP)3]Cl, N,Nʹ-disubstituted
thioureas ligands (Tu1-12) and their Cu(I) complexes.

2.3.3.1. Synthesis of the complex chloro(1-benzoyl-3-(3-trifluoromethyl)phenyl)thiourea)
bis(triphenylphosphine)copper(I) (1). Quantities used were 0.075 g of the ligand (Tu1)
(0.22 mmol) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state:
light yellow solid; Yield: 0.149 g (78%); M.P: 145 °C; FT-IR (ATR, υ in cm-1): 3229 (N-H),
3110 (C-H), 1666.7 (C=O), 1528 and 1478 (aromatic C=C), 1454 (C-F), 1433 (C-P), 1266
(C=S), 1091 (C-N), 742 (TPP); 1H NMR (300 MHz, CDCl3, δ in ppm): 7.14-7.78 (m, 37 H, Ar-
1
H), 8.19-8.21 (d, J=7.41 Hz, 2H, Ar-H) 10.98 (s, 1H, NH protons), 11.57 (s, 1H, NH proton);
3
13C NMR (75 MHz, CDCl3, δ in ppm): 121.52, 123.35, 127.88, 128.31-128.42 (d, J=8.38 Hz,
4
due to splitting of P-C coupling, Ar-C), 128.56, 128.72, 129.30, 131.42-131.94 (t, J=39.02 Hz,
2
due to splitting of F-C coupling, Ar-C), 132.02-132.15 (d, J=9.75 Hz, due to splitting of P-C
1
coupling, Ar-C), 133.24, 133.2, 133.72-133.92 (d, J=15 Hz, due to splitting of P-C coupling,
31
Ar-C), 134.03, 134.29, 168.9, 184.8; P NMR (121.49 MHz, 83% aqueous H3PO4, δ in ppm):
(s) -3.07; 19F NMR (282.23 MHz, CFCl3, δ in ppm): (s) -63.99, Anal. Calcd for
C51H41ClCuF3N2OP2S (947.89): C, 64.62; H, 4.36; N, 2.96; S, 3.38; Cu, 6.70. Found: C, 64.11;
H, 4.06; N, 2.16; S, 3.11; Cu, 6.12%.
2.3.3.2.
Synthesis
of
the
complex
chloro(1-benzoyl-3-(2-methoxyphenyl)thiourea)
bis(triphenylphosphine)copper(I) (2). Quantities used were 0.0646 (0.22 mmol) of the ligand
(Tu2) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state: light
yellow solid; Yield 0.168 g (82%); M.P.: 180 °C; FT-IR (ATR, υ in cm-1):3226 (N-H), 3112 (C-
1
(300
H), 1667(C=O), 1528 (C=C), 1434 (C-P), 1260 (C=S), 1093 (C-N), 743 (TPP); H NMR
MHz,
):
CDCl δ in ppm 3.89 (s, due to CH protons), 6.88-7.62 (m, 36H, Ar-H), 8.10-8.20 (d,
,
3
3
1J=8.13 Hz, 3H, Ar-H), 11.30 (s, NH proton), 11.87 (s, NH proton), 13C NMR (75 MHz, CDCl
,
3
δ in ppm): 55.90 (s, OCH3 carbon), 110.79, 120.00, 124.61, 126.57-127.21 (d, 1J=48.41 Hz, due
3
to splitting of P-C coupling, Ar-C), 128.32-128.44 (d, J=9.04 Hz, due to splitting of P-C
2
coupling, Ar-C), 129.08, 129.43, 131.63, 133.30, 133.45, 133.79, 133.85-134.04 (d, J=14.80
31P NMR (121.49 MHz,
Hz, due to splitting of P-C coupling, Ar-C), 151.47, 168.78, 177.59;
83% aqueous H3PO4
):
δ in ppm (s) -3.60, Anal. Calcd for C51H44ClCuN2O2P2S (909.92): C,
,
67.32, H, 4.87; N, 3.08; S, 3.52; Cu, 6.98. Found: C, 66.98; H, 4.01; N, 2.91; S, 2.98; Cu, 6.04%.

2.3.3.3. Synthesis of the complex chloro(1-benzoyl-3-(2,3,5,6-tetrachlorophenyl)thiourea)
bis(triphenylphosphine)copper(I) (3). Quantities used were 0.0895 g (0.22 mmol) of the ligand
(Tu3) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state: light
yellow solid; Yield 0.181 g (79%); M.P.: 200 °C; FT-IR (ATR, υ in cm-1): 3229 (N-H), 3114
(C-H), 1664.8 (C=O), 1526 (C=C), 1433 (C-P), 1265 (C=S), 1092 (C-N), 741.9 (TPP), 692
1
1
(300 MHz,
):
CDCl δ in ppm 7.11-7.66 (m, 34H, Ar-H), 8.16-8.32 (d, J=8.29
(C-Cl); H NMR
,
3
Hz, 2H, Ar-H), 12.05 (s, NH proton), 12.62 (s, NH proton; 13C NMR (75 MHz, CDCl δ in
,
3
3
ppm): 128.31-128.43 (d, J=9.00 Hz, due to splitting of P-C coupling, Ar-C), 128.55, 129.47,
130.47, 130.97, 132.02, 132.10, 133.28-133.62 (d, 1J=24.00 Hz, due to splitting of P-C coupling,
2
Ar-C), 133.82-134.02 (d, J=15 Hz, due to splitting of P-C coupling, Ar-C), 135.61, 169.76,
31P NMR (121.49 MHz, 83% aqueous H3PO4
): (s)
-3.51, Anal. Calcd for
181.12;
δ in ppm
,
C50H38Cl5CuN2OP2S (1017.67): C, 59.01; H, 3.76; N, 2.75; S, 3.15; Cu, 6.24. Found: C, 58.76;
H, 3.08; N, 2.12; S, 2.79; Cu, 6.02%.
2.3.3.4. Synthesis of the complex chloro(1-benzoyl-3-(2-trifluoromethyl)phenyl)thiourea)
bis(triphenylphosphine)copper(I) (4). Quantities used were 0.091 (0.28 mmol) of the ligand
(Tu4) and 0.25 g (0.28 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state: light
-1
;
yellow solid; Yield 0.164 g (77%); M.P.: 165 °C FT-IR (ATR, υ in cm ): 3223 (N-H), 3116 (C-
H), 1669.8 (C=O), 1523.6 (C=C), 1433 (C-P), 1320 (C-F, 1262 (C=S), 1092(C-N), 741(TPP);
1
1
(300 MHz,
):
CDCl δ in ppm 6.99-8.25 (m, 37H, Ar-H) 8.16-8.19 (d, J=7.26 Hz, 2H,
H NMR
,
3
Ar-H), 11.88 (s, due to NH proton); 13C NMR (75 MHz, CDCl δ in ppm): 126.27, 127.42,
,
3
2
128.36-128.42 (d, J=8.63 Hz, due to splitting of P-C coupling, Ar-C), 129.43, 130.20, 131.17-
131.94 (d, 1J=58.39 Hz, due to splitting of P-C coupling, Ar-C), 132.04, 132.17, 133.60, 133.82-
31P
3
134.02 (d, J=15.02 Hz, due to splitting of P-C coupling, Ar-C), 135.20, 169.57, 181.02;
19
NMR (121.49 MHz, 83% aqueous H PO ,
): (s)
282.23 MHz,
CFCl δ
,
3
δ in ppm
-3.75; F NMR (
3
4
in ppm): (s) -62.61, Anal. Calcd for C51H41ClCuF3N2OP2S (947.89): C, 64.62; H, 4.36; N, 2.96;
S, 3.38; Cu, 6.70. Found: C, 63.99; H, 4.09; N, 2.49; S, 3.19; Cu, 6.22%.
2.3.3.5. Synthesis of the complex chloro(1-benzoyl-3-(3,5-dichlorophenyl)thiourea)
bis(triphenylphosphine)copper(I) (5). Quantities used were 0.0734 (0.22 mmol) of the ligand
(Tu5) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state: light

-1
;
yellow solid; Yield 0.171 g (80%), M.P.: 165 °C FT-IR (ATR, υ in cm ):3221(N-H), 3121(C-
H), 1663.6 (C=O), 1522 (C=S), 1433 (C-P), 1263 (C=S), 1092 (C-N), 741.5 (TPP), 691.6 (C-Cl);
1
1
(300 MHz,
):
H NMR
CDCl δ in ppm 7.00-8.29 (m, 36H, Ar-H), 8.10-8.34 (d, J=7.20 Hz, 2H,
,
3
13
Ar-H), 11.74 (s, 1H, NH proton), 12.54 (s, 1H, NH proton); C NMR (75 MHz, CDCl δ in
,
3
2
ppm): 123.44, 126.98, 128.36-128.48 (d, J=9.00 Hz, due to splitting of P-C coupling, Ar-C),
128.63, 129.27, 129.50, 131.06, 133.33, 133.68, 133.79-133.98 (d, 1J=14.25 Hz, due to splitting
31P NMR (121.49 MHz, 83% aqueous
of P-C coupling, Ar-C), 134.87, 138.92, 169.57, 179.40;
H3PO4
): (s)
δ in ppm
-3.23, Anal. Calcd for C50H40Cl3CuN2OP2S (948.78): C, 63.30; H, 4.25; N,
,
2.95; S, 3.38; Cu, 6.70. Found: C, 63.29; H, 4.02; N, 2.39; S, 3.11; Cu, 6.24%.
2.3.3.6.
trichlorophenyl)thiourea)bis(triphenylphosphine)copper(I) (6). Quantities used were 0.0811
(0.22 mmol) of the ligand and 0.20 (0.22 mmol) of
(Tu6)
Synthesis
of
the
complex
chloro(1-benzoyl-3-(2,4,6-
g
chlorotris(triphenylphosphine)copper(I). Physical state: light yellow solid; Yield 0.179 g (81%),
M.P.: 180 °C; FT-IR (ATR, υ in cm-1): 3228 (N-H), 3119 (C-H), 1673.7 (C=O), 1519 (C=C),
1
(300 MHz,
1434 (C-P), 1254 (C=S), 1070 (C-N), 742.3 (TPP), 694.9 (C-Cl); H NMR
CDCl δ
,
3
13
):
in ppm 7.19-7.75 (m, 39H, Ar-H), 11.67 ((s, 1H, NH proton), 12,52 (s, 1H, NH proton); C
NMR (75 MHz, CDCl δ in ppm): 127.73, 128.60-128.73 (d, 2J=9.75 Hz due to splitting of P-C
,
3
coupling, Ar-C), 129.67, 129.83, 130.02, 131.95, 132.34, 132.76, 133.80-133.87 (d, 1J=12.75 Hz
31P NMR
due to splitting of P-C coupling, Ar-C), 134.00, 134.21, 134.81, 168.91, 181.41;
(121.49 MHz, 83% aqueous H3PO4
): (s)
-1.36, Anal. Calcd for C50H39Cl4CuN2OP2S:
δ in ppm
,
C, 61.08; H, 4.00; N, 2.85; S, 3.26; Cu, 6.46. Found: C, 52.75; H, 3.09; N, 2.26; S, 3.98; Cu,
8.41%.
2.3.3.7.
Synthesis
of
the
complex
chloro(1-benzoyl-3-
cyclohexylthiourea)bis(triphenylphosphine)copper(I) (7). Quantities used were 0.059 (0.22
mmol) of the ligand (Tu7) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I).
Physical state: light yellow solid; Yield 0.148 g (74%), M.P.: 140 °C; FT-IR (ATR, υ in cm-1):
3312 (N-H), 3126 (C-H), 1664 (C=O), 1525.8 (C=C), 1433.6 (C-P), 1262 (C=S), 1093 (C-N),
1
(300 MHz,
):
CDCl δ in ppm 1.14-1.83 (m, 10H, cyclohexylproton), 5.24
741 (TPP); H NMR
,
3

(m, 1H, cyclohexylproton), 7.15-7.96 (m, 35H, Ar-H), 9.08 (s, 1H, NH proton), 11.35 (s, 1H, NH
13
proton); C NMR (75 MHz, CDCl δ in ppm): 25.4 (s,C H ), 25.7 (s, C H ), 32.5 (s, C H ),
,
6
11
6
11
6
11
3
58.70 (s, C6H11), 127.52-127.54 (d, 3J=9.00 Hz, due to splitting of P-C coupling, Ar-C), 128.41-
1
128.66 (d, J=18.00 Hz, due to splitting of P-C coupling, Ar-C), 130.70, 131.10-131.21 (d,
31P NMR
2J=8.25 Hz due to splitting of P-C coupling, Ar-C), 132.1, 133.2, 134.0, 168.5, 183.1;
(121.49 MHz, 83% aqueous H3PO4
): (s)
-3.47, Anal. Calcd for C50H48ClCuN2OP2S
δ in ppm
,
(885.94): C, 67.79; H, 5.46; N, 3.16; S, 3.62; Cu, 7.17. Found: C, 67.13; H, 5.17; N, 2.19; S,
3.10; Cu, 7.01%.
2.3.3.8.
Synthesis
of
the
complex
chloro(1-(benzo[d]thiazol-2-yl)-3-
benzoylthiourea)bis(triphenylphosphine)copper(I) (8). Quantities used were 0.0707 g (0.22
mmol) of the ligand (Tu8) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I).
Physical state: light yellow solid; Yield 0.154 g (73%), M.P.: 165 °C; FT-IR (ATR, υ in cm-1):
3227 (N-H), 3137 (C-H), 1666 (C=O), 1525.5 (C=C), 1434 (C-P), 1260 (C=S), 1091 (C-N), 742
1
1
(300 MHz,
):
CDCl δ in ppm 7.17-8.24 (m, 37H, Ar-H), 8.19-8.26 (d, J=7.53
(TPP); H NMR
,
3
Hz, 2H, Ar-H), 11.63 (s, 1H, NH proton), 12.08 (s, 1H, NH proton); 13C NMR (75 MHz, CDCl
,
3
δ in ppm): 121.04, 121.72, 124.55, 126.45, 128.42-128.53 (d, 3J=8.87 Hz, due to splitting of P-C
1
coupling, Ar-C), 128.65, 129.29, 129.60, 130.88, 132.04, 132.25, 133.10-133.45 (d, J=26.10
Hz, due to splitting of P-C coupling, Ar-C), 133.81-134.00 (d, 2J=14.65 Hz, due to splitting of P-
31P NMR (121.49 MHz, 83% aqueous
C coupling, Ar-C), 148.42, 158.16, 168.82, 176.39;
H3PO4
): (s)
δ in ppm
-3.05, Anal. Calcd for C51H41ClCuN3OP2S2 (936.97): C, 65.38; H, 4.41; N,
,
4.48; S, 6.84; Cu, 6.78. Found: C, 64.43; H, 3.98; N, 4.19; S, 6.30; Cu, 6.01%.
2.3.3.9.
Synthesis
of
the
complex
chloro(1-(2-chlorobenzoyl)-3-phenylthiourea)
bis(triphenylphosphine)copper(I) (9). Quantities used were 0.0065 g (0.22 mmol) of the ligand
(Tu9) and 0.20 g (0.22 mmol) of chlorotris(triphenylphosphine)copper(I). Physical state: light
yellow solid; Yield 0.152 g (74%), M.P.: 180 °C; FT-IR (ATR, υ in cm-1): 3240 (N-H), 3138
(C-H), 1669 (C=S), 1592 (C=C), 1433 (C-P), 1253 (C=S), 1070 (C-N), 740.6 (TPP), 690 (C-Cl);
1
1
(300 MHz,
):
H NMR
CDCl δ in ppm 7.17-7.60 (m, 37H, Ar-H), 7.65-7.73 (d, J=6.24 Hz, 2H,
,
3
13
Ar-H), 12.08 (s, 1H, NH), 12.52 (s, 1H, NH); C NMR (75 MHz, CDCl δ in ppm): 125.04,
,
3
3
126.82, 127.17, 128.33-128.45 (d, J=8.94 Hz, due to splitting of P-C coupling, Ar-C), 128.84,

129.48, 130.57-136.94 (d, 1J=240.24 Hz, due to splitting of P-C coupling, Ar-C), 132.10, 132.19,
2
132.77, 133.38, 133.73, 133.85-134.04,(d, J=14.65 Hz, due to splitting of P-C coupling, Ar-C),
31P NMR (121.49 MHz, 83% aqueous H3PO4
): (s)
-3.48, Anal.
136.94, 168.50, 178.74;
δ in ppm
,
Calcd for C50H41Cl2CuN2OP2S (914.34): C, 65.68; H, 4.52; N, 3.06; S, 3.51; Cu, 6.95. Found: C,
65.13; H, 4.01; N, 2.89; S, 3.10; Cu, 6.21%.
2.3.3.10.
chlorophenyl)thiourea)bis(triphenylphosphine)copper(I) (10). Quantities used were 0.0734
(0.22 mmol) of the ligand and 0.20 (0.22 mmol) of
(Tu10)
Synthesis
of
the
complex
chloro(1-(2-chlorobenzoyl)-3-(2-
g
chlorotris(triphenylphosphine)copper(I). Physical state: light yellow solid; Yield 0.162 g (76%),
M.P.: 190 °C; FT-IR (ATR, υ in cm-1): 3237 (N-H), 3129 (C-H), 1669.9 (C=O), 1528 (C=S),
1
(300 MHz,
1433 (C-P), 1250 (C=S), 1091 (C-N), 741.6 (TPP), 691 (C-Cl); H NMR
CDCl δ in
,
3
1
):
ppm 7.18-7.93 (m, 36H, Ar-H), 7.87-7.90 (d, J=9.60 Hz, 2H, Ar-H), 11.67 (s, NH proton),
13
12.64 (s, NH proton); C NMR (75 MHz, CDCl δ in ppm): 126.79, 126.92, 127.14, 128.05,
,
3
3
128.40-128.52 (d, J=8.97 Hz, due to splitting of P-C coupling, Ar-C), 128.71, 129.56, 129.73,
1
130.61, 130.73, 131.95, 132.02, 132.93, 133.16-133.51 (d, J=26.60 Hz, due to splitting of P-C
coupling, Ar-C), 133.81-134.00 (d, 2J=14.84 Hz, due to splitting of P-C coupling, Ar-C), 134.49,
31P NMR (121.49 MHz, 83% aqueous H3PO4
): (s)
-3.46, Anal. Calcd
167.89, 178.94;
δ in ppm
,
for C50H40Cl3CuN2OP2S (948.78): C, 63.30; H, 4.25; N, 2.95; S, 3.38; Cu, 6.70. Found: C, 65.13;
H, 4.01; N, 2.89; S, 3.10; Cu, 6.21%.
2.3.3.11.
dichlorophenyl)thiourea)bis(triphenylphosphine)copper(I) (11). Quantities used were 0.0811
(0.22 mmol) of the ligand and 0.20 (0.22 mmol) of
(Tu11)
Synthesis
of
the
complex
chloro(1-(2-chlorobenzoyl)-3-(2,3-
g
chlorotris(triphenylphosphine)copper(I). Physical state: light yellow solid; Yield 0.168 g (71%),
M.P: 197 °C; FT-IR (ATR, υ in cm-1): 3218 (N-H), 3048.6 (C-H), 1669.9 (C=O), 1523 (C=C),
1
(300 MHz,
1432.6 (C-P), 1265.6 (C=S), 1091.4 (C-N), 748.8 (C-N), 691 (C-Cl); H NMR
13
):
CDCl δ in ppm 7.13-7.74 (m, 37H, Ar-H), 12.03 (s, NH proton), 12.14 (s, NH proton); C
,
3
NMR (75 MHz, CDCl δ in ppm): 125.60, 126.79-126.87 (d, 3J=6.29 Hz, due to splitting of P-C
,
3
2
coupling, Ar-C), 127.86, 128.37-128.49 (d, J=8.39 Hz, due to splitting of P-C coupling, Ar-C),
128.71, 130.61, 130.76, 131.89, 132.07, 132.02, 132.96, 133.25, 133.46, 133.60, 133.82-134.01

31P NMR
(d, 1J=14.65 Hz, due to splitting of P-C coupling, Ar-C), 136.17, 168.20, 179.30;
(121.49 MHz, 83% aqueous H3PO4
): (s)
-3.42, Anal. Calcd for C50H39Cl4CuN2OP2S
δ in ppm
,
(983.23): C, 61.08; H, 4.00; N, 2.85; S, 3.26; Cu, 6.46. Found: C, 60.91; H, 3.90; N, 2.49; S,
3.13; Cu, 6.31%.
2.3.3.12.
(trifluoromethyl)phenyl)thiourea)bis(triphenylphosphine)copper(I) (12). Quantities used were
0.0809 (0.22 mmol) of the ligand (Tu12) and 0.20
(0.22 mmol) of
Synthesis
of
the
complex
chloro(1-(2-chlorobenzoyl)-3-(3-
g
chlorotris(triphenylphosphine)copper(I). Physical state: light yellow solid; Yield 0.157 g (71%),
M.P.: 158 °C; FT-IR (ATR, υ in cm-1): 3198 (N-H), 3044.4 (C-H), 1663.7 (C=O), 1531.9 (C=C),
1
(300 MHz,
1434 (C-P), 1327 (C-F), 1279.6 (C=S), 1092.6 (C-N), 744 (TPP); H NMR
CDCl
,
3
δ in ppm 7.17-7.76 (m, 38H, Ar-H), 12.34 (s, NH proton), 12.64 (s, NH proton); 13C NMR
):
(75 MHz, CDCl δ in ppm): 122.03, 123.61, 126.89, 128.20, 128.36-128.48 (d, 3J=8.89 Hz, due
,
3
1
to splitting of P-C coupling, Ar-C), 129.35-137.49 (t, J=306.72 Hz, due to splitting of F-C
coupling), 129.48, 130.65, 130.98, 131.41, 131.41, 132.01, 132.18, 132.92, 133.38, 133.79-
31P
2
133.99 (d, J=14.78 Hz, due to splitting of P-C coupling, Ar-C), 137.49, 168.77, 179.07;
19
NMR (121.49 MHz, 83% aqueous H3PO4
): (s)
282.23 MHz,
CFCl δ
,
3
δ in ppm
-3.35; F NMR (
,
in ppm): (s) -63.98, Anal. Calcd for C51H40Cl2CuF3N2OP2S (982.34): C, 62.36; H, 4.10; N, 2.85;
S, 3.26; Cu, 6.47. Found: C, 62.10; H, 4.01; N, 2.59; S, 3.04; Cu, 6.25%.
2.4. Biological assays
2.4.1. Brine shrimp lethality assay. For the selected ligands and their complexes in a 96 well
plate the lethality test was performed against brine shrimp (Artemia salina) larvae as previously
reported [32] with slight changes. For 24-48 h A.salina eggs (Ocean star, USA) were incubated
under light at 30-32 °C in a specially designed two-compartment plastic tray in simulated sea
water (38 g/L supplemented with 6 mg/L dried yeast). With the help of Pasteur pipettes, ten
mature phototropic nauplii were harvested and transferred to each well of the plate. A
corresponding volume of each extract containing ≤ 1% DMSO in sea water at final
concentrations of 500 and 200 µg/mL was transferred to the wells containing sea water and
shrimp larvae. The final volume in each well was kept at 300 µL. Positive and negative control
wells included serial concentrations of doxorubicin and 1% DMSO, respectively. After 24 h

incubation, live shrimps were counted and percentage of deaths was determined. Median lethal
concentration (LC50) was calculated using TableCurve 2D v5.01 software.
2.4.2. Antibacterial assay. Susceptibility of extracts against bacterial species was tested for the
selected ligands and their complexes according to a formerly described procedure [33]. Three
Gram negative strains (Bordetella bronchiseptica, ATCC # 4617, Salmonella typhimurium,
ATCC # 14028 and Enterobacter aerogenes, ATCC # 13048) were tested. 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 inoculums (100 µL) were then swabbed onto Petri plates containing 20
mL of nutrient agar. Test samples (5 µL of 20 mg/mL DMSO; 100 µg/disc) were infused on
sterile filter paper discs (6 mm) and placed on seeded nutrient agar plates. Cefixime-USP at a
concentration of 20 µg/disc and DMSO impregnated discs were included as positive and
negative controls, respectively. After 24 h of incubation, clear zones of growth inhibition were
measured.
2.4.3. Antifungal assay. Antifungal activity of extracts for selected ligands and their complexes
was evaluated following the reported literature standard protocol [33]. The fungal strains
(Aspergillus fumigatus FCBP# 66, Fusarium solani FCBP# 0291 and Mucor specie FCBP#
0300) were purchased from the fungal culture bank of Pakistan and cultured on SDA. Prior to the
sensitivity determination, the spores were harvested in 0.02% Tween 20 solution and turbidity
was adjusted according to the McFarland 0.5 turbidity standard. 100 µL of each harvested spore
type 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 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 measured using a Vernier
caliper.
2.4.4. Antileishmanial assays. In vitro antileishmanial activity for selected ligands and their
complexes was performed with Leishmania tropica khw23 strain with the previously reported
standard protocol [33] with slight modification. The parasite was cultured in M199 media

supplemented with 10% Fetal Bovine Serum at 24 °C. The culture (promastigotes) was
ingathered at a concentration of 1×106 cells/mL. Stock solutions of each test sample were
prepared in DMSO (10 mg/mL) and serially diluted in 96 well plates. The antileishmanial
activity of each sample was determined at concentrations ranging from 500, 250, 125 and 31.25
µg/mL. The cultured plates, inoculated with parasite and test samples, were incubated at 24 °C
for 72 h. Amphotericin-B was employed as positive control and media as a negative control.
Subsequent to the incubation, the live promastigotes were counted under a light microscope
using an improved Neubauer chamber. The data obtained was then statistically analyzed and
LC50 was estimated using TableCurve 2D Ver.4 software.
2.4.5. Antioxidant assays. The antioxidant activity of the phosphine thiourea copper(I) halide
complexes was conducted using the following assays.
DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity: Copper(I) complex
(400 µg L-1) up to 20 µL (from 400 µg L-1) was taken in 96 well-plates and 180 µL of DPPH
from stock was added. Ascorbic acid and DMSO were used as positive and negative control,
respectively. The plates were incubated for 1 h. Readings were taken at 515 nm using a
microplate ELISA reader [34]. DPPH free radical scavenging activity of the complexes was
calculated as a percent (%) of radical inhibition by the following equation:
Reducing power: The reducing power of the complexes was estimated using vitamin C as
standard reference. In the procedure, 40 μL of the sample and 50 μL of phosphate buffer
(0.2 molL-1; pH 6.6) were taken in a lmate tube followed by the addition of 50 μL of potassium
ferricyanide (1%). After incubation, at 50 °C for 20 minutes, 50 μL of trichloroacetic acid (10%)
was added to the mixture and it was centrifuged (3000 rpm) for 10 minutes. After layer
separation, the upper-layer 166.66 μL was transferred to 96 well microplates and 33.33 μL was
added to the isolate. The reducing power of the synthesized complex was calculated as ascorbic
acid equivalents; μg of AE per mg of complex.
Total antioxidant capacity: The phosphomolybdenum method was used to estimate the
total antioxidant capacity of the synthesized complexes. The synthesized complex sample (20 μL
-1
taken in separate tubes. This was
from 4 mgL ) and ascorbic acid (20 μL), as standard, were

0.6 molL-1, Na3PO4; 28 mmolL-1,
followed by the addition of reagent solution (H2SO4;
(NH4)6Mo7O24; 4 mmolL-1). The tubes were properly capped and incubated at 95 °C in a thermal
block for 90 minutes. After heat incubation, the samples were cooled to room temperature and
transferred to 96-well microplates. The assurance of the solution was measured versus blank at
630 nm using a Microplate ELISA reader. The blank was prepared using the same procedures i.e.
having 200 μL of reagent solution and appropriate volume of the same solvent. Water soluble
antioxidant capacities were expressed as equivalents of ascorbic acid for the synthesized
complexes. Ascorbic acid equivalents were calculated using a standard graph of ascorbic acid.
The experiments were carried out in triplicate and values were expressed as ascorbic acid
equivalents (AAE) µgmg-1 of complex.
2.4.6. DNA Binding study by UV-Vis spectroscopy. For the DNA binding study, UV-Vis
spectroscopy was used to determine the DNA binding constant. For this purpose the spectrum of
the free complex without the addition of DNA was taken using the solvent in the reference cell.
The spectra were recorded by adding different concentrations of DNA to the solution of the
investigated complexes having constant concentration. The concentration and volume of the
compound were kept constant during evaluation while the concentration of the DNA was
changed [35].
The equilibrium constants (binding constant) were 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, and εG
and εH-G are the molar extinction coefficients of free compound and of the compound–DNA
complex, respectively.
2.4.7. Molecular docking method. Molecular Operating Environment (MOE) from Chemical
Computing Inc. 2015.08 was used to draw and optimize Cu complexes 1-12 on MOE window
using MOE builder. Optimized molecular structures were entered into the MOE database. The
X-ray crystallographic structure of the DNA with PDB ID 5JLT was obtained through the
protein data bank (PDB) and imported to the MOE window. 5JLT fragment was protonated with
their standard geometry using MOPAC 7.0. All water molecules were removed from the
complex with 11 base pairs running in 5’-3’ direction. The resulting DNA model was subjected

to systematic conformational search analysis at default parameters with RMS gradient of 0.01
kcal mol-1 using Site Finder. In order to perform docking simulations, a number of
conformational runs were carried out to get a final binding docking pose with the lowest energy
as accurate as possible. The best conformation was selected based on energetic ground and the
minimum Final Docking Energy (ΔG) [36, 37].
3. Results and discussion
3.1. Elemental analyses
For elemental analyses, CHNS analyzer was used to determine the percentages of carbon,
hydrogen, nitrogen and sulfur while an atomic absorption spectrophotometer (AAS) was used to
find the copper content in the synthesized complexes. The results are given in the Experimental
section and have close agreement with those of calculated values that confirmed the complexes
formation.
3.2. FT-IR Results
The FT-IR analysis of the synthesized complexes was carried out for structural elucidation and
functional group identification. All the significant recorded bands of the complexes are listed in
the Experimental section. The observed bands in all the complexes are at (ν in cm–1): 3150-3350
(N-H), 2910-3050 (Ph; C-H), 1660-1685 (C=O), 1475-1600 (Ph; C=C), 1433 and 1477 (C-P),
1320 and 1327 (C-F), 1220-1270 (C=S), 1146 (C-O), 1035-1090 (C-N), 690-695 (C-Cl), 320-
337 (Cu-S) and 263-297 (Cu-X). The FT-IR results clearly showed the addition of secondary
amine to C=N bond resulting in the formation of thiourea [22]. Band shifting for the NH-group
(3400 cm-1) and carbonyl C=O (1750 cm-1) to lower wavelength is attributed to hydrogen
bonding between H of one NH-group and the O of carbonyl on one side and between H of
another NH-group and halogen attached to Cu on another side, as can be seen in the data of
single crystal analysis. An IR band for SH (2500-2600 cm-1) has not been observed in the
complexes, confirming non-tautomeric behavior of the ligand [22, 38, 39].
Comparison between the FT-IR data of free ligands [40] and complexes have revealed
that ligand bonding to metal has not modified NH, CN, CO and CS stretching, probably due to
hydrogen bonding. All the FT-IR spectra of the complexes have characteristic bands of
phosphine: 1433 cm-1, 1477 cm-1, 1092 cm-1 and 741-747 cm-1 [29].

3.3. NMR Results
NMR spectroscopy was conducted for the structural elucidation of the synthesized copper(I)
complexes. The 1H NMR data of the complexes have peaks for the aromatic protons in the range
of 7.14-8.20 ppm [41, 42]. The other peaks at around 10 ppm and 12 ppm are assigned to the N-
1
H protons of CONH and CSNH, respectively. Two separate peaks for the NH protons in the H
NMR spectra confirm that none of the N is bonded with copper metal.
All the 13C NMR signals, aromatic ring, of the synthesized complexes are in close
agreement to the reported values [43]. The carbon signals at 160-168 ppm and 173-183 ppm
were assigned to the carbon of CONH and CSNH, respectively. The 31P NMR data of the
complexes have singlet peaks in the range of -1.36 to -3.75 ppm. The 19F NMR of complexes 1,
4 and 12 give singlet peaks in the range of -62.61 to -63.99 ppm.
3.4. Single crystal X-ray diffraction study
Recrystallizations of copper(I) complexes of thiourea were carried out in chloroform and pure
yellow crystals were collected for solid state X-ray studies after 24 h. Suitable crystals of
complexes 1, 2, and 12 were analyzed on a Bruker Smart diffractometer equipped with an
APEX II CCD Detector, an Incoatec IMuS source and a Quazar MX mirror. The crystal-to-
detector distance was 4.0 cm, and the data collection was carried out in 512 × 512 pixel mode.
The initial unit cell parameters were determined by a least-squares fit of the angular
setting of strong reflections, collected by a 180.0 degree scan in 180 frames over three different
parts of the reciprocal space. Suitable crystals of small sizes were selected and mounted on a
Bruker Smart APEX diffractometer. The crystals were kept at 100 K during data collection. The
Cu Kα radiations with wavelength 1.54178 Å were used. For each reflection, integrated intensity
information was obtained by reduction of data frames using APEX2 [44]. Using Olex2 [45], the
structure was solved with the SHELXT [46] structure solution program using intrinsic Phasing
and refined with the XL [47] refinement package using full matrix least squares minimization on
F2 and using all the unique data [45, 46]. Atoms other than hydrogen were refined with
anisotropic thermal parameters and all hydrogens were at calculated positions and were refined
isotropically. The fluorine atoms in CF3 group of complex 12 were disordered and had the
occupancies F1A 0.52(13), F1B 0.48(13), F2A 0.52(13), F2B 0.48(13), F3A 0.52(13), F3B

0.48(13). The additional systematic absences and thermal voids were verified with Platon [48].
For the absorption corrections SADABS was used [49]. The crystallographic data and the results
of the refinements are summarized in table 1.
The complex ORTEP structures are given in figures 2-4, crystal data and refinement
parameters in table 1, selected bond lengths and angles given in table S1, and hydrogen-bond
lengths in table S2. The X-ray analysis of complexes 1, 2 and 12 (figures 2-4) show the
coordination of triphenylphosphine, thiourea and chloride with copper(I) in a distorted
tetrahedral-fashion. Complex 1 has space group P-1 with Z = 2 in the triclinic crystal system,
while complexes 2 and 12 have space group P21/c in the monoclinic crystal system with Z = 4, as
shown in table 1. The 3D crystal packing diagrams of complexes 1, 2 and 12 are given in figures
S1-S3, which confirm the Z values. The Cu–S bond lengths lie in the range of 2.3425(4) Å to
2.4243(6) Å, the CuP bond lengths lie in the range of 2.2456(6) to 2.2762(4) Å, and the Cu–Cl
bond lengths lie in the range of 2.3166(6) to 2.3415(4) Å (table S1). The average value of bond
angles of Cl–Cu–S lies in the range of 104.729(13) to 109.628(15)°, P–Cu–Cl bond angles lie in
the range of 105.87(8) to 114.52(2)°, P–Cu–S bond angles lie in the range of 102.85(2) to
109.972(14)°, and P–Cu–P bond angles lie in the range of 117.15(2) to 124.616(15)°, which are
nearly close to the perfect tetrahedral geometry (109.5 Å), table S1. The values of the torsion
angles showed that the carbonyl groups and thiocarbonyl of the thiourea ligand are coplanar. The
structural analysis further confirmed that there are two types of intramolecular hydrogen-
bonding, between O of C=O and H of –NH group (O1···HN2) and between Cl and H of –NH
group (Cl1··· HN1) presenting the trans-configuration of thiourea N protons (table S2).
3.5. Biological activity results
3.5.1. Antibacterial activity. In antibacterial tests, clear zones of growth inhibition were
measured after 24 h incubation. From the data in table S3, it is clear that complex 11 is more
active against E. Aerogens while against B. Bronchiseptaca it is complex 8 and against S.
Typhimurium it is complexes 2 and 3 with ZOI values comparable to the reference drugs. From
the data it is clear that the ligands and precursor are also active while complex activities are
higher than precursor and the ligands which mean complex formation further enhances activity.

3.5.2. Antifungal activity. Fungal zone of inhibition values of the selected ligands and their
complexes are given in table S4 and it has been found that almost all the complexes showed
higher activities against the tested species than the ligands and precursor.
3.5.3. Brine shrimp assay. Live shrimps were counted and percentage of deaths was determined
after 24 h incubation. Median lethal concentration (LC50) was calculated using TableCurve 2D
v5.01 software. It is clear from table S5 that the tested compounds are active in damaging the
cells. The values are in the range of 52.6 to 88.6 µg/mL for the complexes while the ligands and
the precursor are less active in damaging the cells as compared to the complexes.
3.5.4. Antileishmanial activity. The data in table S6 shows that the precursor and the ligands are
active, but showed low activities compared to their copper(I) complexes. From the data it is clear
that complex 2 shows high activity with LC50 value of 21.25 as compared to the standard drug
while complexes 1 and 8 show moderate activities and complex 5 shows low activity.
3.5.5. Antioxidant activity. In antioxidant activities, it was observed that all the complexes
showed higher activity than the precursor and ligands toward DPPH. The antioxidant activities
for the complexes were found in the range of 54.96 to 62.67%. This high activity can be
attributed to the high electron-withdrawing power of chlorine which facilitates the hydrogen
release and can reduce the DPPH [50]. Likewise all the tested complexes have shown
significantly high reducing power toward Fe3+ to Fe2 (table S7) and significantly high total
antioxidant activity compared to the precursor and the ligands, which means the complex
formation further increase the activity.
3.5.6. DNA Interaction studies by UV-visible spectroscopy and molecular docking. The
tested complexes showed strong hypochromicity which was observed in the spectral order by the
addition of DNA concentration (figure 5). All the complexes showed DNA interactions which
can be attributed to the presence of chloride and phenyl rings in their structures. It has been
reported previously that copper(I) tetrahedral complexes show DNA binding either by partial
interaction or by the phenyl ring binding to one of the grooves [51, 52]. The plot of 1/[DNA] vs.
Ao/A-Ao also supports the groove binding of the complexes with DNA (figure 6). Complexes of

current studies have demonstrated groove binding with DNA, which is in close agreement with
molecular docking studies.
The optimized molecular geometries of Cu complexes 1-12 were fully optimized with the
PM3 semi-empirical quantum mechanical method and the two representative optimized
geometries of complexes 1 and 2 with electronic charge distribution are presented in figure 7.
Optimized structures show that all Cu-complexes have puckered conformations with crescent
shaped geometry, which is the characteristic property of groove binders [53]. In order to have an
understanding of the molecular mechanism of interaction between Cu compounds and double
stranded DNA, different results of docking poses were obtained. Lowest energy docked poses of
Cu compounds with double stranded DNA showed that all compounds interact with DNA
through mixed mode of interaction for groove binding with the major groove of DNA, except for
complexes 1, 2, 4 and 5, which interact with the major groove of DNA via groove binding.
Ligplots of 1, 2, 4 and 5 (figure 7 left) show that these compounds develop weak van der Waal’s
forces of attractions with the sugar phosphate backbone in the groove and fewer interactions with
DNA base pairs.
The most important parameter obtained from docking simulations is the binding constant
(Kb) calculated by scoring function free energy using the equation ∆G = -RTlnKb, where Kb is
measure of binding strength of a ligand with receptor. The highest value of Kb was found to be
1.08×105 for “complex 8” (table S8) which is attributed to the intrastrand cross linking of
benzo[d]thiazol-2-ylmoiety of complex 8 with DA17 and DT6 base pairs of DNA in addition to
groove binding interactions. Following “complex 8”, 6 and 7 are the other complexes having
higher values of binding strength, which is also attributed to their mixed mode of interactions
with DNA. The lowest value of Kb was found for “4” and “5” (table S8). This fact is also
manifested in figures of molecular docked complexes and the ligplot DNA base pairs with
complexes 4 and 5 that these complexes are significantly distant from DNA and not cohesively
attached to backbone. Negative values of free energies (∆G) in the case of all Cu-complexes with
DNA showed that they interact with DNA spontaneously.
A number of steric descriptors were calculated for the comprehension of macroscopic
interactions. Electronic descriptors cannot be calculated because of the presence of Cu -- a heavy
metal. An important steric descriptor is molar refractivity (MR) which is an extent of the total
polarizability of a mole of a substance. As evident from figure 8 direct correlation of the binding

strength with the MR was observed implying increase of binding strength with an increase in the
molar refractivity of the compound.
Another important steric descriptor calculated was hydrophobic surface area (Vsurf). It is
clear from figure 9 that a reasonably good inverse correlation of Kb with Vsurf was observed
depicting that compounds with higher Vsurf have greater ability to overlap electronic clouds of
DNA base pairs rendering a stronger association with DNA molecules.
4. Conclusion
Copper(I) complexes 1-12 of chlorine, triphenylphosphine (TPP) and different N,Nʹ-disubstituted
thioureas were synthesized for biological applications. The crystal structure investigation
revealed that the complexes were distorted tetrahedral with copper as central metal ion
coordinated with ligands. All the complexes were screened for different biological activities. In
antibacterial studies complex 11 was the most active against E. Aerogens, complex 8 was the
most active against B. Bronchiseptaca and complexes 2 and 3 were active against S.
Typhimurium with ZOI comparable to the reference drug. The antileishmanial activities showed
that complex 2 was the most active while complexes 3 and 8 were moderately active, and
complex 5 showed low activity. The antioxidant activities showed that all the complexes have
high total antioxidant activity and high reducing power toward Fe3+ to Fe2+. During DNA
interaction studies, through UV/Vis, a hypochromicity in the spectral order was observed with
increasing concentration of DNA which was attributed to the groove binding. This type of
interaction was further supported by molecular docking studies.
Supplementary data
Supplementary information is available containing data of bond lengths, bond angles, hydrogen
bonds, crystals packs and tables of biological activates. Author ORIC ID is 0000-0002-0377-
0356 and CCDC Nos. 1834952-1834953 and 1834957-1834958 contain the supplementary
crystallographic data for this article. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif on request.

Acknowledgements
S.I.K. is highly thankful to Higher Education Commission of Pakistan for Doctoral fellowship
under International Research Support Initiative Program (IRSIP) at University of Montreal
Canada. S.I.K. is also highly thankful to Dr. Michel Simard for training of single crystal XRD
machine in XRD lab at University of the Montreal, Montreal, Canada.
Conflicts of interest
The authors declare no conflict of interest.
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H3CO
H
CF3
O
S
O
S
H
C NH C N
C NH C N
Tu2
Tu1
Cl
Cl
O
S
O
S
H
H
C NH C N
F3C
Cl
C NH C N
Tu4
Tu3
Cl
Cl
Cl
O
S
O
S
H
H
C NH C N
Cl
C NH C N
Tu6
Tu5
Cl
Cl
O
S
S
O
S
H
H
C NH C N
C NH C N
N
Tu7
Tu8
Cl
O
S
O
S
H
H
C NH C N
C NH C N
Cl
Cl
Tu10
Tu9
CF3
Cl
Cl
H
H
S
O
H
H
S
O
C N C N
C N C N
Cl
Cl
Tu12
Tu11
Figure 1. The chemical structure of the ligands (Tu1 to Tu12) used in this work.

Figure 2. ORTEP drawing of complex 1 with atomic numbering scheme.

Figure 3. ORTEP drawing of complex 2 with atomic numbering scheme.

Figure 4. ORTEP drawing of complex 12 with atomic numbering scheme showing disorder in
the CF3 group.

Figure 5. DNA binding studies by UV/Vis graphs for complexes 1 (a), 2 (b), 3 (c), 4 (d), 5 (e)
and 6 (f).