In vitro and in vivo antiproliferative activity of organo-nickel SCS-pincer complexes on estrogen responsive MCF7 and MC4L2 breast cancer cells. Effects of amine fragment substitutions on BSA binding and cytotoxicity

Article abstract of DOI:10.1039/c8dt03079k

A family of organonickel complexes has been prepared, fully characterized, and tested for their antiproliferative activity against estrogen-responsive human breast cancer cells (MCF7). The three SCS-type pincer ligands HL1, HL2, and HL3 and their corresponding Ni(ii) complexes NiL1, NiL2, and NiL3 have been synthesized and fully characterized, including by single crystal diffraction studies for the complexes. The complexes possess square planar geometry with two symmetrical 5-membered nickellacycles. Fluorescence spectroscopy, circular dichroism measurements, molecular modeling, colorimetric based assay and tumor transplantation studies were used to evaluate the protein binding and antiproliferative activities of these organometallic complexes both in vitro and in vivo. Fluorescence quenching was used to investigate bovine serum albumin (BSA) interaction at different temperatures (293, 303 and 313 K), and the results were analyzed using the classical Stern-Volmer equation, allowing us to propose a dynamic quenching mechanism. Studies in vitro on the antiproliferative activity of the three organonickel complexes against estrogen-responsive human breast cancer cells (MCF7) showed promising antitumor activity for NiL1 containing pyrrolidine fragments. In vivo administration of this compound significantly inhibits tumor growth in estrogen-dependent MC4L2 cancer cells in female BALB/c mice.

Full text of DOI:10.1039/c8dt03079k

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
In vitro and in vivo antiproliferative activity of
organo-nickel SCS-pincer complexes on estrogen
responsive MCF7 and MC4L2 breast cancer cells.
Effects of amine fragment substitutions on BSA
binding and cytotoxicity†
Cite this: Dalton Trans., 2018, 47,
16944
Mahboubeh Hosseini-Kharat,
*
a,b Davit Zargarian, *a Ali Mohammad Alizadeh,*b,c
Kazem Karami, d Maryam Saeidifar,e Solmaz Khalighfard,f Laurent Dubrulle,a
Mostafa Zakariazadeh,g Jean-Philippe Cloutiera and Zahra Sohrabijame
A family of organonickel complexes has been prepared, fully characterized, and tested for their antiproli-
ferative activity against estrogen-responsive human breast cancer cells (MCF7). The three SCS-type
pincer ligands HL1, HL2, and HL3 and their corresponding Ni(II) complexes NiL1, NiL2, and NiL3
have been synthesized and fully characterized, including by single crystal diffraction studies for the com-
plexes. The complexes possess square planar geometry with two symmetrical 5-membered nickella-
cycles. Fluorescence spectroscopy, circular dichroism measurements, molecular modeling, colorimetric
based assay and tumor transplantation studies were used to evaluate the protein binding and
antiproliferative activities of these organometallic complexes both in vitro and in vivo. Fluorescence
quenching was used to investigate bovine serum albumin (BSA) interaction at different temperatures (293,
303 and 313 K), and the results were analyzed using the classical Stern–Volmer equation, allowing us to
propose a dynamic quenching mechanism. Studies in vitro on the antiproliferative activity of the three
organonickel complexes against estrogen-responsive human breast cancer cells (MCF7) showed promis-
ing antitumor activity for NiL1 containing pyrrolidine fragments. In vivo administration of this compound
significantly inhibits tumor growth in estrogen-dependent MC4L2 cancer cells in female BALB/c mice.
Received 27th July 2018,
Accepted 25th October 2018
DOI: 10.1039/c8dt03079k
rsc.li/dalton
1. Introduction
Thioamide derivatives, R–C(vS)–NR1R2(H), have shown sig-
nificant antituberculosis, antitumor, anthelmintic and thyro-
toxic activities.1–7 For instance, a platinum–pyridine thioamide
aDépartement de Chimie, Université de Montréal, Montréal, Québec H3C 3J7,
Canada. E-mail: m.hosseinikharat@gmail.com, zargarian.davit@umontreal.ca
bCancer Research Center, Tehran University of Medical Sciences, Tehran, Iran.
E-mail: aalizadeh@sina.tums.ac.ir
complex has been patented for clinical use in cancer treat-
ment,8 and fungicidal, insecticidal, and acaricidal activities
have also been reported for these compounds.9 Significantly,
compared to the biological activities of uncomplexed thio-
amides, their metal complexes show even greater activities.10,11
For this reason, considerable effort is currently being directed
towards the synthesis of metal complexes of aromatic thioa-
mides due to their remarkable chemical and biological
properties.12–15
In general, organometallic complexes consisting of organic
ligands and one or more transition metals constitute a new
class of compounds possessing promising pharmacological
properties. A number of organometallic complexes have thus
found application in the treatment of cancer, including ferroci-
fen, an iron-based derivative of the anticancer drug
cBreast Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran
dDepartment of Chemistry, Isfahan University of Technology, Isfahan 84156-83111,
Iran
eDepartment of Nanotechnology and Advanced Materials, Materials and Energy
Research Center, Karaj, Iran
fDepartment of Biology, Science and Research Branch, Islamic Azad University,
Tehran, Iran
gResearch Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz,
Iran
†Electronic supplementary information (ESI) available: Figures, tables, and pro-
cedures for data collection and the Experimental section of biological evalu-
ation. CCDC 1813281 (NiL3), 1813282 (NiL2) and 1813283 (NiL1). For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/
c8dt03079k
16944 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
tamoxifen.16–18 Indeed, ferrocifen and its family of compounds cal activities of organonickel complexes. As initial targets, we
are organometallic species classified as an original category of have selected the SCS-type pincer ligands HL1–HL3 featuring
ferrocifen-type breast cancer drugs.
various amine fragments and their Ni(II) complexes
Another Earth-abundant metal, nickel, is present at the (Scheme 1) for bioevaluation against the estrogen-responsive
active sites of several important classes of metalloproteinases, human breast cancer cell lines MCF7 (in vitro) and MC4L2
such as 2-mercaptoethanol-inhibited urease and Ni/Fe hydroge- (in vivo). To the best of our knowledge, this is the first report
nases.19,20 Several hydrogenases and carbon monoxide dehydro- describing the in vivo anticancer activity of organonickel com-
genases contain nickel at their active site.21 Complementing plexes. In addition, spectrophotometric methods and mole-
nickel’s natural biochemical functions, complexes of nickel also cular docking techniques have been employed to study the
display pharmacological potential.22 More specifically, nickel interaction of the nickel–SCS–pincer complexes with BSA, the
complexes with varied biological activities have been reported latter being a model protein thanks to its structural resem-
in the literature with antibacterial, antifungal and antiprolifera- blance to human serum albumin (HSA) and its low-cost and
tive/anticancer properties.23–27 For instance, a glycosyl sacchar- availability.
ide nickel complex has shown promising cytotoxic activity and
inhibited Ehrlich Ascites Carcinoma (EAC) induced cancer cell
lines in Swiss albino mice at LC50 = 2.89 × 10−8 M.28
Furthermore, Ni(II) thiosemicarbazone complexes exhibited sig-
2. Experimental
nificant degrees of cytotoxicity against the human lung adeno-
carcinoma (A549) cell line. Studies have shown that these com-
plexes induce apoptosis in A549 cells and inhibit lung cancer
2.1. Materials
The starting materials for the synthesis of the thioamide-
based compounds were purchased from Sigma-Aldrich (USA)
and used as received. BSA, warfarin, ibuprofen, tris(hydroxy-
methyl)aminomethane and sodium chloride were obtained
from Merck, Germany. High purity solvents used in the prepa-
ration and physical measurements were supplied by Merck,
Germany. HL1 and HL2 were synthesized by the Willgerodt–
Kindler reaction according to a previously described pro-
cedure.32,33 The synthesis of HL3 is given below.
metastasis through
a
mitochondrial-mediated pathway.29
Evidently, Ni(II) complexes featuring nitrogen and sulfur donor
ligands are highly interesting for their desirable biological
properties.
On the other hand, there is interest in the analysis of drug–
protein interactions that greatly influence the absorption, dis-
tribution, metabolism, and excretion properties of drugs.30
Bovine serum albumin (BSA) has been frequently selected as
the protein model primarily due to its unusual binding pro-
perties and similarity to human serum albumin.31 Thus, a
precise understanding of the features that determine the
binding of metal complexes to proteins would be valuable in
both theory and application.
It is worth noting that less attention has been paid to the
biological properties of organonickel complexes in comparison
with their catalytic properties. In addition, relatively few
reports have been published involving the anticancer activity
of metal thioamide compounds, and among these the biologi-
cal evaluations of organometallic thioamide compounds are
rare.
2.2. Instruments used for physical measurements
Infrared spectra were recorded on a FT-IR JASCO 680 spectro-
photometer in the spectral range 4000–400 cm−1 using the KBr
pellet technique. NMR spectra were recorded on a Bruker
spectrometer at 400.13 MHz (1H) and 100.61 MHz (13C) using
standard pulse sequences at 298 K. Elemental analysis was per-
formed on a Leco, CHNS-932 apparatus. Electronic absorption
spectra were recorded on a PerkinElmer Lambda 25 UV/Vis
spectrophotometer and 1.0 cm quartz cells were used for
measurements. Fluorescence measurements were performed
on a Hitachi fluorescence spectrophotometer model MPF-4.
Circular dichroism spectra were obtained on an Aviv model
215 spectropolarimeter using a 1.0 mm cell. Mass spectra were
The above considerations have prompted us to initiate a
research program focused on investigating the pharmacologi-
Scheme 1 Synthesis of SCS pincer ligands HL and their Ni(II) complexes.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16945

View Article Online
Paper
Dalton Transactions
recorded on an Agilent 5975C VL MSD with a Triple-Axis ppm): δ = 6.98–7.55 (m, 3H), 4.23 (s-br, 4H), 3.72 (t, 4H), 1.88
detector.
(s-br, 8Hb,b′), 1.40 (s-br, 4Hc). 13C{1H} NMR (CD3CN-d3, ppm):
δ = 207.0 (CvS), 147.3 (C-2), 129.2 (C-1,3), 127.5 (C-4,6), 120.0
(C-5), 54.8, 31.0, 27.4, 25.6, 23.5 (C-a,a′,b,b′,c). Anal. Calc. for:
C18H23IN2NiS2: C, 41.81; H, 4.48; N, 5.42; S, 12.40. Found: C,
41.80; H, 4.46; N, 5.41; S, 12.40%.
2.3. Synthesis of HL3
A mixture of isophthalaldehyde (0.134 g, 1 mmol), cyclohexyl-
amine (0.23 mL, 2 mmol), sulfur (0.064 mg, 2 mmol), and
acetic acid (0.06 mL, 1 mmol) was stirred in DMF (20 mL) at
100 °C for 5 h, and the resulting brown suspension was
poured onto cold water (200 mL) to give a brown precipitate.
Filtration of the mixture gave a wet brown solid, which was
dried in air. Extraction of the dried solid with hot EtOH
(30 mL) gave an insoluble solid (S8) and a brown solution.
Evaporation of the latter gave the crude product as a yellow
solid, which was recrystallized from Et2O to give a light-yellow
solid (280 mg, 80% yield).
NiL3: 225 mg, 83% yield.
IR (KBr, cm−1): ν(NH)
= 3266, ν(CH)aromatic = 3053,
ν(CH)aliphatic = 2927, 2851, ν(C–N) = 1547, ν(CvS) = 1146, 631.
1H-NMR (CD3CN-d3, ppm): δ = 8.57 (s-br, SH), 7.67–8.19 (m,
H
aromatic), 7.62 (d, 1H), 7.54 (d, 1H), 7.16 (t, 1H), 4.31 (s-br, NH),
3.73 (s-br, 4H), 3.65 (s-br, 4H), 1.30–2.16 (m, 12H-c,c′,d). 13C{1H}
NMR (CD3CN-d3, ppm): δ = 201.5 (C–SH), 187.9 (CvS), 152.2,
146.6, 137.9, 133.2, 130.7, 129.8, 127.2, 125.8, 125.3, 121.8
(Caromatic), 57.0 (C-a), 45.0, 31.0 (C-b,b′), 25.5, 24.7 (C-c,c′), 20.7
(C-d). Anal. Calc. for: C20H27IN2NiS2: C, 44.06; H, 4.99; N, 5.14;
S, 11.76. Found: C, 44.05; H, 4.97; N, 5.12; S, 11.75%.5.
IR (KBr, cm−1): ν(NH)
ν(CH)aliphatic = 2930, 2851, ν(C–N) = 1527, ν(CvS) = 1135, 700.
1H-NMR (CD3CN-d3, ppm): δ = 8.58 (s-br, SH), 7.20–7.96 (m,
= 3231, ν(CH)aromatic = 3035,
H
aromatic), 4.48 (s-br, NH), 3.54 (t, 4H-b′), 3.16 (t, 4H-b),
1.71–2.12 (m, 12H-c,c′,d). 13C{1H} NMR (CD3CN-d3, ppm): δ =
199.8 (CvS), 196.4 (C–SH), 143.7, 142.2, 129.1, 128.1, 128.0,
127.5, 126.7, 125.4, 124.3, 123.0 (Caromatic), 55.0 (C-a), 44.4,
42.9 (C-b,b′), 31.4, 25.3 (C-c,c′), 24.5 (C-d). Anal. Calc. for:
C20H28N2S2: C, 66.62; H, 7.83; N, 7.77; S, 17.78. Found: C,
66.62; H, 7.82; N, 7.76; S, 17.76%.
3. Results and discussion
3.1. Synthesis and description of structures
3.1.1. Synthesis and spectroscopic characterization. The
SCS thioamide pincer ligands HL were synthesized by the
Willgerodt–Kindler reaction, and their corresponding Ni(II)
complexes were prepared by stirring the ligands with NiI2 in
toluene at 110 °C (Scheme 1). The direct C–H nickellation reac-
2.4. Synthesis of NiL1, NiL2, and NiL3
NiI2 (0.156 g, 0.5 mmol) and HL1 (0.152 g, 0.5 mmol) or HL2 tions proceeded to give the target (SCS)NiI complexes NiL1,
(0.166 g, 0.5 mmol) or HL3 (0.180 g, 0.5 mmol) were dissolved NiL2, and NiL3 in 78–83% yields.
in toluene (5 mL) and stirred at 110 °C under N2 for 72 h. The
The pincer complexes NiL are stable in air and water and
precipitate was collected by filtration, washed with hexane and show good solubility in organic solvents such as CH2Cl2,
a small amount of EtOH, and dried in vacuo to afford the CHCl3, DMF, and DMSO. Full characterization of NiL was
target complex as a dark violet powder.
accomplished by 1H NMR, 13C{1H} NMR, IR, UV-Vis spec-
troscopy and elemental analysis. In the IR spectra, a higher fre-
NiL1: 190 mg, 78% yield.
IR (KBr, cm-1): ν(CH)aromatic = 2977, ν(CH)aliphatic = 2941, quency shift of ν(C–N) for the thioamide group is observed in
2864, ν(C–N) = 1508, ν(CvS) = 1170, 709. H-NMR (CD3CN-d3, the range of 1508–1547 cm−1 (Table 1), indicating the
1
ppm): δ = 7.42 (d, 2H, J = 8.2 Hz), 7.35 (t, 1H, J = 8.2 Hz), 3.90 S-coordination of the thioamide group and an increase in the
(t, 4H), 3.50 (t, 4H), 2.10 (m-br, 8H). 13C{1H} NMR (CD3CN-d3, double bond character of the C–N bond in NiL.32 Furthermore,
ppm): δ = 195.1 (CvS), 146.1 (C-2), 129.5 (C-1,3), 128.5 (C-4,6), the ν(CvS) vibrational frequencies assigned to the stretching
120.1 (C-5), 55.0, 54.2, 25.6, 23.0 (C-a,a′,b,b′). Anal. Calc. for: and bending modes of complexes were observed in the ranges
C16H19IN2NiS2: C, 39.29; H, 3.92; N, 5.73; S, 13.11. Found: C, of 1132–1174 cm−1 and 615–635 cm−1 34,35
.
These shifted
39.25; H, 3. 90; N, 5.71; S, 13.10%.
stretching frequencies indicate that the sulfur atom of the
NiL2: 206 mg, 80% yield.
thiocarbonyl group is coordinated to the Ni center.
IR (KBr, cm-1): ν(CH)aromatic = 2937, ν(CH)aliphatic = 2918,
In the 1H NMR spectra of the three complexes, the signal
1
2854, ν(C–N) = 1531, ν(CvS) = 1132, 635. H-NMR (CD3CN-d3, due to H at the 2-position of the central benzene ring dis-
Table 1 Wavenumbers (cm−1) and band assignments for the recorded FT-IR spectra of the SCS pincer ligands (HL) and their organonickel com-
plexes (NiL)
Compound
ν(N–H)
ν(C–H)aromatic
ν(C–H)aliphatic
ν(C–N)
ν(CvS)
HL1
NiL1
HL2
NiL2
HL3
NiL3
—
—
—
—
3231
3266
3043
2977
3008
2937
3035
3053
2987, 2950, 2874
2941, 2864
2937, 2858
2918, 2854
2930, 2851
2927, 2851
1498
1508
1491
1531
1527
1547
1170, 709
1174, 615
1140, 704
1132, 635
1135, 700
1146, 631
16946 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
appears. Moreover, a downfield shift of the C-2 carbon reso-
nance (δ 146.1 ppm (NiL1), δ 147.3 ppm (NiL2), and δ
146.6 ppm (NiL3)) in the 13C{1H} NMR spectra is observed.
These C-2 carbon resonances are deshielded by 17.6, 18.5, and
18.6 ppm with respect to the free HL1 (δ 128.5 ppm), HL2 (δ
128.8 ppm), and HL3 (δ 128.0 ppm). These observations are
consistent with the cyclometallation at the C-2 position.33,36–38
In addition, the large shifts of the 13C{1H} NMR signals in the
aromatic region suggest the binding of the aryl moiety to Ni.32
Finally, the signals at δ 195.1, δ 207.0 and δ 187.9 ppm are
attributed to the (CvS) group of the thioamide units in NiL,
respectively.
The secondary thioamide group in HL3 and NiL3 is involved
in equilibrium with its amino–thione and imino–thiol tauto-
mers, as shown in Fig. 1. This phenomenon has also been
1
observed in the related SCS pincer Pt(II) complexes.39 In the H
NMR spectra of HL3 and NiL3, two broad singlet peaks appeared
around δ 4.5 ppm and δ 8.6 ppm, attributed to NH and SH
groups, respectively. Moreover, in both compounds the aromatic
protons in the 7–8 ppm region displayed fine structures. In
addition, in the 13C{1H} NMR spectra of HL3 and NiL3, two
different signals around the range of δ 187.9–201.5 ppm were
observed and assigned to the CvS and C–SH groups.
Furthermore, two distinguishable signals attributed to C1,3–CvS
or C1,3–C–SH groups related to the thione or thiol structure were
observed in the 13C{1H} NMR spectra of both compounds. The
mass spectra of the Ni(II) complexes (Fig. S1†), under EI con-
ditions, show peaks at m/z 489.5, 517.6 and 545.6, corresponding
to the molecular formula of NiL1, NiL2 and NiL3, respectively.
In addition, several small peaks in the molecular ion regions of
the three complexes appeared at m/z and the ranges of
490.5–492.5 (NiL1), 518.6–520.6 (NiL2), and 546.6–548.6 (NiL3).
These assignments are based on the atomic weights of Ni-58, Ni-
60, N-14 and N-15 isotopes. The molecular peaks at m/z 362.3
(Fig. S1, A†), 390.4 (Fig. S1, B†) and 418.5 (Fig. S1, C†) corres-
pond to the loss of iodine in each complex. These observations
confirm the monomeric nature of NiL.
Fig. 2 The molecular structures of Ni(II) SCS pincer complexes NiL1 (A),
NiL2 (B), and NiL3 (C) with displacement ellipsoids drawn at the 50%
probability level.
3.1.2. Molecular structures of Ni(II) pincer complexes.
Fig. 2 depicts the solid state structures of NiL, determined by
X-ray crystallography, and the crystallographic data and
selected bond lengths and angles of the complexes are sum-
marized in Table S1† and Table 2, respectively.
All three complexes have tridentate SCS nickel(II) structures
containing two symmetrical 5-membered rings. They adopt a
nearly ideal square planar molecular geometry as reflected in
the sum of the bond angles around the Ni(II) center: 360.31°
(NiL1), 359.88° (NiL2), and 360.14° (NiL3), respectively.
Nevertheless, the coordination plane is bent out slightly in all
cases, as indicated by the dihedral angle between the SCS
plane and the benzene ring: 4.49° (NiL1), 19.19° (NiL2), and
5.08° (NiL3) in the three complexes. The Ni1–I1 bond lengths
in the three complexes are in the range of 2.5325 (16)–2.5733
(9) Å. In the packing diagrams of NiL (Fig. S2†), adjacent mole-
cules are connected to each other via CH–π interactions. The
Ni1–C1 bond lengths in these nickel SCS pincer complexes are
1.874 (5) Å (NiL1), 1.888 (6) Å (NiL2), and 1.877 (11) Å (NiL3),
Fig. 1 Tautomeric equilibrium in HL3.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16947

View Article Online
Paper
Dalton Transactions
Table 2 Crystallographic data and structure refinement of NiL1–NiL3
NiL1
NiL2
NiL3
Empirical formula
Formula weight
Temperature (K)
Radiation type
Wavelength (Å)
Crystal system
Space group
a (Å)
C16H19IN2NiS2
489.06
273.15
Ga Kα
1.34139
Orthorhombic
Pbca
12.5312(3)
12.1900(3)
22.2460(6)
90
C18H23IN2NiS2
517.11
273.15
C20H27IN2NiS2
545.16
273.15
Ga Kα
1.34139
Monoclinic
P21/c
12.5103(10)
13.1427(8)
14.0322(10)
90
Ga Kα
1.34139
Triclinic
ˉ
P1
9.0954(12)
10.4357(13)
11.0814(14)
64.612(4)
b (Å)
c (Å)
α (°)
β (°)
90
90
89.389(6)
87.803(6)
949.5(2)
2
1.809
15.61
516
98.902(5)
90
2279.4(3)
4
1.589
13.03
γ (°)
Volume (Å3)
Z
3398.20(15)
8
2.309
26.958
2263.0
Dcalcd. (Mg m−3
)
μ (mm−1
F(000)
)
1096
Crystal size (mm)
Theta range (°)
0.3 × 0.1 × 0.1
3.1–53.9
0.12 × 0.06 × 0.04
3.8–60.6
0.12 × 0.04 × 0.04
3.1–54.4
Reflections collected/unique
Data/restraints/parameters
Goodness-of-fit
Final R indices [I > 2σ(I)]
R indices (all data)
10 813/2976
2976/0/199
1.054
R1 = 0.044, wR2 = 0.1153
R1 = 0.049, wR2 = 0.1218
1.24 and −1.80 e Å−3
21 575/4348
4348/0/217
1.112
R1 = 0.066, wR2 = 0.1677
R1 = 0.079, wR2 = 0.1810
2.27 and −0.76 e Å−3
13 331/4181
4181/0/235
0.970
R1 = 0.085, wR2 = 0.1696
R1 = 0.101, wR2 = 0.2054
2.16 and −0.89 e Å−3
Largest diff. peak and hole
all of which are in the range of previously reported benzene- the quencher and c(Q) is the total concentration of the
centered nickel PCP pincer and C,P cyclonickellated complexes quencher.
(1.880(2)–1.931(2)).40–42 On the other hand, these Ni1–C1 bond
lengths are longer than those bonds in the recently published
F0
F
¼ 1 þ KSV½Lꢀ ¼ 1 þ Kqτ0cðQÞ
ð1Þ
nickel NCN pincer complexes (1.822(2)–1.825(5).43
Fluorescence quenching can occur by different mecha-
nisms, which are usually classified as dynamic quenching and
3.2. Interaction of NiL with bovine serum albumin (BSA)
3.2.1 Analysis of fluorescence quenching of BSA by pincer static quenching. The dynamic and static quenching can be
nickel(II) complexes. It is worth pointing out that the nickel distinguished by their differing dependence on temperature.
complexes under study showed no fluorescence emission in In the case of dynamic quenching, higher temperature results
buffer solutions, and that the fluorescence properties of BSA in faster diffusion and, consequently, greater quenching rate
arise from the intrinsic characteristics of the proteins, mainly constants. In contrast, in the case of static quenching, increas-
due to the presence of tryptophan and tyrosine residues. BSA ing temperature will result in decreasing complex stability and
has two tryptophan residues, Trp-134 and Trp-212. The effect hence in lowering of the value of the static quenching con-
of NiL1–NiL3 on the fluorescence intensity of BSA is shown in stant.45,46 In many cases the fluorophore can be quenched by a
Fig. 3, which shows the decrease in the fluorescence intensity combination of dynamic and static mechanisms.47
of BSA with the increase in the concentration of nickel com-
Fig. S4† depicts the curves of F0/F versus c(Q) for {Ni–BSA}
plexes at 293 K, for the three studied compounds. This trend systems at different temperatures, according to eqn (1). These
was also observed at the other temperatures we probed (303 results point to a good linearity at the three different tempera-
and 313 K) (Fig. S3†). It is interesting to note that the subtle tures. The binding constants calculated using the Stern–
differences in the structure of the pincer complexes, due Volmer (KSV) equation are presented in Table 3. In all cases,
mainly to the different amine fragments (pyrrolidine, piper- the results for the interaction between NiL and BSA show that
idine and cyclohexylamine), cause significant differences in the Stern–Volmer quenching constant KSV is correlated with
the binding affinity to albumin.
temperature, indicating that the probable quenching mecha-
Commonly, fluorescence quenching can be described by nism of a Ni–BSA binding reaction is a dynamic collision
the Stern–Volmer relationship,44 eqn (1), wherein F0 and F rep- rather than a complex formation. In addition, the values for Kq
resent the fluorescence intensities in the absence and presence are higher than the maximum diffusion collision quenching
of the quencher, respectively. Kq is the quenching rate constant rate constant (2.0 × 1010 L mol−1 s−1), which implies that some
of the biomolecule, KSV is a linear Stern–Volmer quenching static quenching effect may also exist in the {Ni–BSA}
constant, τ0 is the average life-time of the biomolecule without systems.47
16948 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Fig. 3 Fluorescence emission spectra of the NiL2-BSA system at different temperatures. ([BSA] = 4.2 μM, [NiL2] = 0–0.86 mM at Tris-HCl buffer
(0.02 M and 0.02 M NaCl), pH 7.4 at 298 K).
Table 3 Stern–Volmer quenching constants and values of Ka, n and R2 for the {Ni–BSA} system at different temperatures
Complex
T (K)
KSV × 103 (M−1
)
Kq × 1011 (M−1 s−1
)
R2
Ka × 103 (M−1
)
n
R2
NiL1
293
303
313
293
303
313
293
303
313
2.55 0.1
2.75 0.3
3.02 0.1
1.22 0.2
1.30 0.3
1.60 0.1
5.00 0.4
5.75 0.2
6.66 0.1
2.55 0.1
2.75 0.3
3.02 0.1
1.22 0.2
1.30 0.3
1.60 0.1
5.00 0.4
5.75 0.2
6.66 0.1
0.99
0.99
0.99
0.99
0.99
0.98
0.99
0.99
0.99
2.75 0.3
2.84 0.2
2.97 0.2
1.23 0.2
1.31 0.1
1.49 0.3
4.64 0.3
5.44 0.2
7.59 0.2
1.33
0.88
0.77
0.99
0.76
0.99
0.90
1.04
1.24
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
NiL2
NiL3
3.2.2 Binding constants and the number of binding sites. values of Ka, n and correlation coefficients R are listed in
Assuming that there are independent binding sites in the Table 3. These results show that the association constants Ka
protein and that small molecules can bind independently to a increased with the temperature, which may indicate the for-
set of equivalent sites, the equilibrium between free and mation of stable adducts. The values of n were approximately
bound molecules can be described by using eqn (2) wherein Ka equal to 1, thus indicating the existence of a single binding
is the association constant for a site and n is the number of site in BSA for the three nickel complexes. Thus, it can be con-
binding sites in the protein.48
cluded that NiL binds to BSA by forming 1 : 1 adducts.
3.2.3 Thermodynamic parameters and the nature of
binding forces. The interaction forces between a drug and a
protein may include multiple hydrogen bonds, van der Waals
ðF0 ꢁ FÞ
log
¼ log Ka þ n log cðQÞ
ð2Þ
F
Fig. S5† shows the binding equilibrium plots for the fluo- forces, electrostatic forces and hydrophobic interactions.49 The
rescence quenching of BSA by NiL at 293, 303 and 313 K; the thermodynamic parameters, enthalpy change (ΔH°), entropy
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16949

View Article Online
Paper
Dalton Transactions
change (ΔS°) and free energy change (ΔG°) are the main para- The negative values found for Gibbs free energy show that the
meters for identifying the binding mode. If the enthalpy interaction processes are more favorable. Positive values for
change (ΔH°) does not vary significantly in the studied temp- both ΔH° and ΔS° indicate typical hydrophobic interactions,
erature range, both the enthalpy (ΔH°) and entropy (ΔS°) vari- while negative values point out to van der Waals and hydrogen
ations can be evaluated from the van’t Hoff eqn (3) wherein R bonding.
is the universal gas constant and Kq is the bimolecular quench-
3.2.4 Characteristics of synchronous fluorescence spectra.
ing constants as mentioned in Table 3 at the corresponding Synchronous fluorescence spectroscopy is a useful tool for
temperature (T).
studying protein conformations. In synchronous fluorescence
experiments, the change in polarity around a chromophore
microenvironment induces a decrease and/or shifts in emis-
sion intensity. A blue shift in the maximum emission intensity
indicates that amino acid residues are in a hydrophobic
environment.50 On the other hand, a red shift is due to the
exposure of amino acid residues to the polar solvent environ-
ment. As Δλ = 15 nm, synchronous fluorescence offers charac-
teristics of tyrosine residues, while when Δλ = 60 nm, it pro-
vides the characteristic information of tryptophan residues.
The synchronous experiments were performed using a fixed
concentration of BSA with the increasing concentration of
nickel complexes. Our studies revealed that a decrease in emis-
sion intensity was more in Δλ = 60 nm rather than in Δλ =
15 nm (Fig. S6†). These results indicate that NiL binds mainly
to the tryptophan site, thereby causing changes in the micro-
environment around the tryptophan residue.51
ΔH°
RT
ΔS°
R
ln Kq ¼
ꢁ
þ
ð3Þ
The chosen temperatures were 293, 303 and 313 K, at which
BSA did not undergo any structural damage or degradation.
Table 4 shows the values of ΔH° and ΔS° obtained from the
slop and ordinate of the van’t Hoff relationship and lists the
corresponding values of the Gibbs free energy change (ΔG°).
Table 4 Thermodynamic parameters for the {Ni–BSA} system at
different temperatures
ΔG°
ΔH°
TΔS°
Complex
T (K)
(kJ mol−1
)
(kJ mol−1
)
(kJ mol−1
)
R2
NiL1
293
303
313
293
303
313
293
303
313
−2.46
−2.63
−2.83
−0.50
−0.68
−1.01
−3.74
−4.27
−5.28
5.36
5.54
5.72
7.41
7.66
7.82
8.17
8.56
7.91
8.35
0.9511
3.2.5 Three-dimensional (3D) fluorescence studies. Three-
dimensional (3D) fluorescence spectroscopy is another power-
ful tool to analyze the microenvironment and conformational
changes about BSA.52 The 3D fluorescence spectra of BSA and
BSA in the presence of nickel complexes are depicted in Fig. 4
and the resulting data are collected in Table S2.† It exhibited
NiL2
NiL3
0.9892
0.9681
7.92
8.93
22.34
23.10
23.86
26.08
27.37
29.14
Fig. 4 The three-dimensional fluorescence spectra of the BSA and Ni–BSA system (BSA (4.2 μM) (A), NiL1 (0.1 mM) (B), NiL2 (0.1 mM) (C), NiL3
(0.1 mM) (D)).
16950 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
four characteristic peaks; the extreme left one has been represent the typical α-helix structure of the protein. CD
assigned to the first order Rayleigh scattering peak whose measurements performed in the presence of different concen-
emission wavelength matches well with the excitation wave- trations of NiL showed that the binding of three nickel pincer
length (λem = λex), whereas the peak on the extreme right has complexes to BSA caused a decrease in both bands (Fig. S8†).
been allocated as the second order Rayleigh scattering peak
The CD results were expressed in terms of mean residue
(λem = 2λex) marked as “a” and “b”, respectively. Moreover, ellipticity (MRE) in deg cm2 dmol−1, according to eqn (4)
there are two additional peaks denoted as “peak 1” and “peak wherein Cp is the molar concentration of the protein, n is the
2” that describe the spectral behavior of tyrosine and trypto- number of amino acid residues of the protein and l is the path
phan residues (peak 1) and the fluorescence behavior of the length.
polypeptide backbone and secondary structure of the protein
(peak 2).53,54 These results showed that peak 1 did not exhibit
θobsðmdegÞ
MRE ¼
ð4Þ
ð10nlCpÞ
any significant shift, but its intensity decreased significantly,
thus indicating a decrease in the polarities of both residues as
well as implying that the new amino residues of BSA get
buried into the hydrophobic pocket. Furthermore, a decrease
in the fluorescence intensity of peak 2 suggested extension of
the polypeptide backbone to a greater extent in addition to
BSA conformational changes. Variation in the fluorescence
intensity for both peaks revealed that interaction between BSA
and nickel complexes leads to changes in both tyrosine and
tryptophan residues, microenvironment polarity of amino resi-
dues, and conformation of BSA.
3.2.6. Site probe studies. The binding sites of drugs are
determined by studying their ability to displace the site-
specific probes. The two primary regions in drugs bound to
BSA are usually located in hydrophobic cavities in subdomains
IIA and IIIA; the binding cavities associated with these sub-
domains are also referred to as Sudlow’s sites I and II.55 Site I
of BSA has an affinity for warfarin, phenylbutazone, eosin Y,
etc. and site II has an affinity for ibuprofen, flufenamic acid,
etc. BSA has two tryptophan residues (Trp-134 and Trp-213)
located in subdomains IA and IIA, respectively.56,57 In order to
determine the preferable binding sites of compounds to BSA,
competitive binding experiments were performed. Warfarin
was used as a Sudlow site I probe, and ibuprofen as a Sudlow
site II probe. BSA-site probe complexes were titrated with
nickel pincer complexes and fluorescence emission spectra
were recorded. Results are shown in Fig. S7† and in Table 5. As
can be seen from the experimental results, three nickel(II) com-
plexes bind to ibuprofen Sudlow’s site II of BSA.
The α-helix contents of free and combined BSA were calcu-
lated from mean residue ellipticity values at 222 nm using eqn
(5).58,59
ꢀ
ꢁ
ðMRE222 ꢁ 2340Þ
α‐Helix ð%Þ¼
ꢂ 100
ð5Þ
30 300
At the molar ratio of compound/protein (c/p) = 1.19, the
decrease of α-helical content is about 18.8%, 19.4% and 15.7%
for BSA and NiL1, NiL2 and NiL3, respectively (Table 6). The
decrease of the CD signal indicated that the binding of these
complexes to BSA induced some conformational changes, but
the secondary structure of BSA remains predominantly α-helix.
3.2.8 Molecular docking result with BSA. Serum albumin
(here BSA) is chosen for the current studies because it is the
most abundant protein in the serum, and it binds and trans-
ports hormones, fatty acids, and a multitude of metal ions.
Sudlow’s sites I and II are the two major sites on BSA that are
well known for binding of various compounds.60 In addition,
BSA has two well distinguished metal binding sites, the
N-terminal site (NTS), which is an important metal binding
site, especially for Ni2+ and Cu2+ ions,61 and the multi-metal
binding site (MMBS) that is at the interface of domain I and
domain II. These four binding sites due to the interaction of
BSA with nickel complexes are shown in Fig. 5.
MMBS binds Cu(II), Ni(II), Cd(II) and Zn(II) and is suggested
to consist of His105, His147, and His246 in BSA.62–64 Herein,
the binding affinity of three nickel complexes for the above-
3.2.7 Circular dichroism spectra. Circular dichroism (CD)
spectroscopy is widely used to monitor the conformation of
the protein in solution. The CD spectrum of BSA exhibits two
negative bands in the far UV region at 208 and 222 nm, which
Table 6 Conformation changes of the secondary structure of BSA with
and without NiL1–NiL3
B turn +
antiparallel +
parallel %
Random
coil %
Table 5 Effect of warfarin and ibuprofen on the Ka value of the Ni–BSA
system
Compound
α-Helix %
Only BSA (4.2 μM)
42.5
18.8
17.0
14.4
19.4
17.3
14.9
15.7
14.1
12.5
28.7
36.1
37.2
39.4
36.3
37.1
38.8
35.3
36.5
37.4
28.8
45.1
45.8
46.2
44.3
45.6
46.3
49.0
49.8
50.1
Ka × 103
BSA (4.2 μM) + NiL1 (5 μM)
BSA (4.2 μM) + NiL1 (10 μM)
BSA (4.2 μM) + NiL1 (20 μM)
Compound
(M−1
)
R2
BSA (4.2 μM) + NiL1 (0.1 mM) + Ibu (0.02 mM)
BSA (4.2 μM) + NiL1 (0.1 mM) + War (0.03 mM)
BSA (4.2 μM) + NiL2 (0.1 mM) + Ibu (0.02 mM)
BSA (4.2 μM) + NiL2 (0.1 mM) + War (0.03 mM)
BSA (4.2 μM) + NiL3 (0.1 mM) + Ibu (0.02 mM)
BSA (4.2 μM) + NiL3 (0.1 mM) + War (0.03 mM)
1.47
1.78
1.78
2.49
2.07
2.53
0.99 BSA (4.2 μM) + NiL2 (5 μM)
0.99 BSA (4.2 μM) + NiL2 (10 μM)
0.99 BSA (4.2 μM) + NiL2 (20 μM)
0.99 BSA (4.2 μM) + NiL3 (5 μM)
0.99 BSA (4.2 μM) + NiL3 (10 μM)
0.99 BSA (4.2 μM) + NiL3 (20 μM)
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16951

View Article Online
Paper
Dalton Transactions
Tyr149, Glu152, Tyr156, Ser191, Arg194, Arg198, Trp213,
Arg217, Leu218, Leu237, Arg256, His287, Ile289 and Ala290. It
is worth emphasizing that among the complexes studied, the
interaction of NiL3 with Sudlow’s site I resulted in the lowest
free binding energy (ΔG°) of −7.42 kcal mol−1. The amino acid
residues participating in this interaction are Tyr149, Glu152,
Arg198, Trp213, Arg217, Leu218, Lys221, Leu237, Leu259,
Ala260 and Ser286. Moreover in this case, hydrogen bonds
between Tyr149, Arg198 and Arg217 residues and NiL3 are
formed. As the Trp213 residue is located at Sudlow’s site I of
BSA, we obtained the distances between the NiL and the
Trp213 residue. These are <7.2 Å for the three compounds,
indicating that the energy transfer from BSA to the nickel com-
plexes occurred with high probability according to Förster’s
non-radioactive resonance energy transfer theory.65
At Sudlow’s site II of BSA, NiL1 interacts with amino acid
residues Leu386, Gln389, Asn390, Gln393, Ala405, Val408,
Arg409, Arg412 and Lys413 as depicted in Fig. S10 (A, B).†
Furthermore, Fig. S10 (C, D, E, F)† shows the amino acid resi-
dues involved in the interactions with NiL2 and NiL3, respect-
ively. Similar to the former case, NiL3 forms hydrogen bonds
with amino acid residues Gln389, Tyr410 and Ser488 through
the NH group in its structure. Overall, at Sudlow’s site I and II,
polar residues of BSA are the main amino acids that interact
with the three nickel complexes.
Fig. 5 Four selected binding sites of BSA for interaction with nickel
complexes.
The interaction of NiL1 with the BSA binding site NTS
involves the amino acid residues His3, Lys4, Ser5, Glu6, His9,
Lys12 and Glu57 (Fig. 6(A and B)); the interaction with NiL2
also involves the same number and types of amino acid resi-
dues (Fig. 6(C and D)). In contrast, NiL3 interacts with the
main residues Asp1, Thr2, His3, Lys4, Glu6 and His9 when the
best conformer was selected (Fig. 6(E and F)). Hydrogen
bonding is also formed between amino acid residues Asp1,
Thr2, His3, Glu6 and Glu57 and NiL3 in the bond length
range of 1.8–2.2 Å.
Finally, at the multi-metal binding site (MMBS) of BSA,
NiL1 interacts with amino acid residues Glu100, Leu103,
Ser104, Ile202, Gln203, Lys242, Lys245 and His246 (Fig. S11 (A,
B)†). Two amino acid residues Gln203 and Cys245 form hydro-
gen bonds with NiL1 in the bond length range of 2–2.2 Å.
From Fig. S10 (C, D, E, F),† amino acid residues in MMBS
involved in the interaction with NiL2 and NiL3 are shown. As
can be seen, Glu100 and Gln203 are the two major amino acid
residues which form hydrogen bonds with NiL3 in the bond
length range of 1.9–2.2 Å. The highlights of our findings from
the molecular docking studies at four different sites of BSA are
listed below:
noted four binding sites is calculated theoretically and the
results are compared to each other.
The computed lowest free binding energy values (ΔG°) of
interaction between nickel complexes NiL and BSA at four
independent binding sites are represented in Table 7.
According to the data, NiL3 shows the highest interaction at
the four studied binding sites on BSA in comparison with the
other complexes. In the docking simulation studies, the non-
covalent interactions such as hydrogen bonds, π–π stacking,
cation–π interactions and possibility of the formation of metal-
coordinating bonds between NiL and BSA were analyzed for all
the obtained conformers at these four studied binding sites
(Table S3†).
The interaction of the nickel complexes at Sudlow’s site I of
BSA has been studied first. After the docking of the complexes
into the above site, the results were used for selecting the best
conformer for each compound. In Fig. S9,† two- and three-
dimensional representations of binding of NiL to Sudlow’s site
I of BSA are presented, showing that NiL1 has interaction with
amino acid residues Tyr156, Lys187, Arg194, Arg198, Trp213,
Arg217, His287 and Glu291. In addition, NiL2 interacts with
(1) Based on the results of Table 7, MMBS of BSA is the pre-
ferable NiL1 binding site, whereas NiL2 and NiL3 are located
favorably within the binding pocket of subdomain IIA
(Sudlow’s site I) of BSA.
Table 7 The lowest calculated free binding energy values (ΔG°) at the
four binding sites of BSA by molecular docking studies. The unit of
values is kcal mol−1
(2) NiL3 shows better binding affinity to BSA in both experi-
mental and computational studies. This might be due to the
presence of an NH group in its structure that can have signifi-
cant effects on hydrogen bond formation. It should be noted
that the occurrence of hydrogen bonding increases the hydro-
Compound
Sudlow’s site I
Sudlow’s site II
NTS
MMBS
NiL1
NiL2
NiL3
−5.04
−6.23
−7.48
−4.84
−5.38
−6.60
−4.41
−5.63
−5.06
−5.58
−5.84
−5.97
16952 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Fig. 6 Two- and three-dimensional representation of interactions of nickel pincer complexes with BSA at the NTS binding site. H-bonding and π–π
stacking interactions are shown by green spheres and mesh structures, respectively. NiL1-BSA (A, B), NiL2-BSA (C, D), and NiL3-BSA (E, F).
phobic interaction within the protein binding sites and lowers 0.02 and 0.04 mM within 48 and 72 h, respectively. Moreover,
the hydrophilicity, thereby leading to the stabilization of the the IC50 values of NiL3 for MCF7 cells were 0.2 and 0.02 mM
BSA complexes.66
within 48 and 72 h, respectively. Based on the results given in
(3) Non-covalent π–π stacking and cation–π interactions are Fig. 7, the IC50 values of NiL1 at 24 h were dramatically lower
formed because of the existence of a benzene moiety in the than those of NiL2 and NiL3 (P < 0.05), while NiL2 and NiL3
structure of nickel complexes. Moreover, there is a high ten- had non-significant changes in comparison with each other at
dency for formation of cation–π interactions and π–π stacking 24 h.
at Sudlow’s sites and the NTS binding site, respectively. This is
due to the increase in the number of amino acid residues argi- dine rings in the amine moieties at 24, 48, and 72 h. It seems
nine and histidine at the above-mentioned sites. that the low steric hindrance of the amine fragments causes
Higher cytotoxicity was observed for NiL1 featuring pyrroli-
(4) Binding affinities of nickel complexes towards two metal more cytotoxic activity in the molecule. As mentioned in the
binding sites of BSA (MMBS and NTS) are different. It seems Molecular structures of Ni(II) pincer complexes section, all
that the three complexes have a greater tendency to bind BSA three complexes have the planar molecular geometry that
through MMBS rather than NTS.
makes the molecules ideally suited for a potential intercala-
(5) There is no evidence for the formation of a metal-coordi- tion into dsDNA. From the crystal structures, the pyrrolidine
nating bond between NiL and the four studied binding sites rings in the NiL1 have a slight deviation from the main
on BSA.
square plane of the central atom, implying that this molecule
has more potential to intercalate between the planar bases of
DNA and possesses more cytotoxic effect. The in vitro cyto-
3.3. In vitro and in vivo antitumor activity
3.3.1. In vitro cytotoxic activity. To evaluate the cytotoxic toxic activity also shows that the IC50 value of NiL1 against
activity of NiL1 on the human breast adenocarcinoma cells, NIH-3T3 (non-cancerous cells) is above 0.2 mM (within 48 h),
MCF7 was incubated with different doses of the nickel pincer showing little damage to normal cells. Under the same con-
complexes (0.0002 to 0.2 mmol L−1). After 24, 48, and 72 hours ditions, cisplatin showed a higher toxicity (IC50 = 0.03 mM)
of incubation, the cell viability was determined by using the on NIH-3T3 normal cells within 48 h. The selectivity index
MTT assay. All three nickel complexes inhibited the pro- (SI) was calculated based on the IC50 ratio of the normal
liferation of the MCF7 cells in a dose- and time-dependent fibroblast NIH cells and the cancer cells MCF7. The SI value
manner (P < 0.05) (Fig. 7). The cytotoxic activity of cisplatin indicates the selectivity of the sample to the tested cell lines,
was used as a standard reference for evaluation and compari- samples with greater SI values than 2 being considered to
son purposes (Fig. S12 and Table S4†). The IC50 values of NiL1 have a high selectivity.67 The SI value of NiL3 was found to be
for MCF7 cells were 0.2, 0.04, and 0.02 mM within 24, 48, and 0.89 showing less selectivity of NiL3 on MCF7 cells than NiL1
72 h, respectively. The IC50 values of NiL2 for MCF7 cells were and NiL2.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16953

View Article Online
Paper
Dalton Transactions
Fig. 7 The anti-growth effect after the treatment with NiL for 24, 48, and 72 h against MCF7 breast cancer cells by using the MTT assay. *Significant
differences of the marker levels in relation to the untreated control (p < 0.05) are marked with asterisks.
3.3.2. Tumor incidence and size. As NiL1 showed promis- control group, the average body weight changed from 18.0 g
ing antitumor activity in vitro, it was chosen for further eluci- on day 7 to 20.5 g on day 19. No behavioral changes were
dation as an antitumor agent in vivo. To test the efficacy of observed in the animals during the course of administration,
NiL1 to inhibit tumor growth in vivo, an experiment was or in the ensuing follow up period. At the end of the second
carried out on MC4L2 breast cancer cells. NiL1 (2 and 10 week of tumor implant, the tumor take rate was 100% in both
mg kg−1 d−1) was injected intraperitoneally (ip) into one group control and treatment groups. Interestingly, the mice that
of female BALB/c mice for 14 days, and only normal saline was received the NiL1 complex showed a tumor growth inhibition
injected into another group of female mice as a control. As over time, whereas the mice that received normal saline
shown in Fig. 8, the animals’ weight in the control and treated showed an exponential increase in the tumor growth (Fig. 9).
groups increased slightly. Body weight rose steadily to reach a The mice treated at a dose of 10 mg kg−1 NiL1 had a smaller
plateau after about 2 weeks in all mice, but showed a slight fall tumor volume in comparison with those mice treated at a dose
in the treated group at a dose of 2 mg kg−1 on day 19. In the of 2 mg kg−1 NiL1 (Fig. 10). The average tumor volume in the
Fig. 8 Effects of NiL1 on the body weight (gr) in a mouse model of
Fig. 9 Effects of NiL1 on the tumor volume (mm3) in a mouse model of
breast cancer MC4L2.
breast cancer MC4L2.
16954 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
formation of H-bonding with amino acid residues and hence
higher binding constant values. Moreover, the thermodynamic
parameters indicated that the studied interactions are more
favorable (ΔG° < 0), with the hydrophobic interactions being
the major forces between NiL and BSA (ΔH > 0 and ΔS° > 0).
Molecular docking studies showed that MMBS of BSA is the
preferable NiL1 binding site, whereas NiL2 and NiL3 favor
Sudlow’s site I. In addition, the circular dichroism spectra
reveal that the α-helix content of BSA decreases with the
addition of nickel complexes.
The three nickel complexes were screened for their antipro-
liferative activity against estrogen-responsive human breast
cancer cells MCF7 and the normal fibroblast NIH-3T3 cell line
in vitro. It is to be noted that a change in amine fragments in
the nickel complexes (pyrrolidine, piperidine and cyclohexyl-
amine) has a major effect on their cytotoxic activity. The result
of the MTT assay reveals that NiL1 containing pyrrolidine
groups is more active compared to NiL2 and NiL3. In addition,
the SI (selectivity index) values of NiL1 and NiL2 were found to
be higher than 2 in the MCF7 cell line. Finally, the higher cyto-
toxicity of NiL1 in vitro prompted us to examine its in vivo
activity in female BALB/c mice. In mice with mammary carci-
noma (MC4L2 cancer cells), NiL1 treated mice display augmen-
ted tumor regression compared to the control group.
Fig. 10 Mice treated with the NiL1 complex (A) and control group (B)
on different days.
treated group was significantly less than the control group on
days 19, 24 and 29 after the tumor implantation (P < 0.05). The
mean final tumor volume reached approximately 3.6 0.7, 2
0.5, and 1.2 0.4 mm3 in the control and treated (2 and 10 mg
kg−1) groups, respectively, whereas the treated group that
received NiL1 at a dose of 10 mg kg−1 resulted in the lowest
tumor size.
Conflicts of interest
There are no conflicts to declare.
3.3.3 Metastases. Routine surgery was performed on all
mice at the end of the study. A thorough necropsy was made
and the vital organs including the liver, spleen, lung, brain,
and bone were examined for lesions and metastatic deposits.
There were no signs of metastasis in the major organs of both
the control and treated groups.
Acknowledgements
The authors are grateful to the Department of Chemistry,
Université de Montreal, Montreal, Canada, for providing the
facilities necessary for the execution of this work. M. H. K also
acknowledges the financial support provided by the Iran
National Science Foundation (INSF) through the Visiting
Researcher Program at Université de Montreal, Montreal,
Canada.
4. Conclusion
Three thioamide based pincer ligands and their organonickel
complexes have been synthesized and fully characterized. X-ray
crystallography studies indicate that all three complexes NiL
have pincer structures containing two symmetrical 5-mem-
bered rings and fairly planar geometries in the coordination
plane. Interaction between NiL and bovine serum albumin
(BSA) has been studied by fluorescence spectroscopy, circular
dichroism and molecular docking. All three complexes have
the ability to quench the intrinsic fluorescence of BSA through
the dynamic quenching mechanism. The analysis of the
binding equilibria shows that the number of binding sites
between NiL and BSA was about 1. The result of binding con-
stant values indicates that the complexes bind to BSA in the
order of NiL3 > NiL1 > NiL2. The pincer complex NiL3 pos-
sesses one cyclohexylamine group and one H at each amine
site. It can be concluded that the presence of an NH group in
NiL3 enhances the protein binding interactions through the
Notes and references
1 X. Hanoulle, J. M. Wieruszeski, P. Rousselot-Pailley,
I. Landrieu, A. R. Baulard and G. Lippens, Biochem.
Biophys. Res. Commun., 2005, 331, 452–458.
2 C. Vilchèze, T. R. Weisbrod, B. Chen, L. Kremer,
M. H. Hazbón, F. Wang, D. Alland, J. C. Sacchettini and
W. R. Jacobs Jr., Antimicrob. Agents Chemother., 2005, 49,
708–720.
3 K. L. Yu, A. F. Torri, G. Luo, C. Cianci, K. Grant-Young,
S. Danetz, L. Tiley, M. Krystal and N. A. Meanwell, Bioorg.
Med. Chem. Lett., 2002, 12, 3379–3382.
4 Y. Hitotsuyanagi, T. Hasuda, Y. Matsumoto, K. Yamaguchi,
H. Itokawa and K. Takeya, Chem. Commun., 2000, 1633–
1634.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16955

View Article Online
Paper
Dalton Transactions
5 Y. Nakagawa, K. Irie, H. Ohigashi, H. Hayashi and 29 P. Kalaivani, S. Saranya, P. Poornima, R. Prabhakaran,
P. A. Wender, Bioorg. Med. Chem. Lett., 2000, 10, 2087–
2090.
F. Dallemer, V. Vijaya Padma and K. Natarajan, Eur. J. Med.
Chem., 2014, 82, 584–599.
6 M. P. Wentland, X. Sun, Y. Bu, R. Lou, D. J. Cohen and 30 J. Flarakos, K. L. Morand and P. Vouros, Anal. Chem., 2005,
J. M. Bidlack, Bioorg. Med. Chem. Lett., 2005, 15, 2547–
2551.
7 Q. L. Wei, S. S. Zhang, J. Gao, W. H. Li, L. Z. Xu and
G. Z. Yu, Bioorg. Med. Chem., 2006, 14, 7146–7153.
8 E. S. Raper, Coord. Chem. Rev., 1996, 153, 199–256.
77, 1345–1353.
31 X. M. He and D. C. Carter, Nature, 1992, 358,
209–215.
32 Y. Nojima, M. Nonoyama and K. Nakajima, Polyhedron,
1996, 15, 3795–3809.
9 S. Sinha, A. K. Srivastava, C. M. Tripathi, O. P. Pandey and 33 M. Akaiwa, T. Kanbara, H. Fukumoto and T. Yamamoto,
S. K. Sengupta, Bioinorg. Chem. Appl., 2007, 87918, 1–9. J. Organomet. Chem., 2005, 690, 4192–4196.
10 S. Miranda, E. Vergara, F. Mohr, D. de Vos, E. Cerrada, 34 E. Pahontu, V. Fala, A. Gulea, D. Poirier, V. Tapcov and
A. Mendía and M. Laguna, Inorg. Chem., 2008, 47, 5641–
5648.
11 R. Raj Kumar, M. K. Mohamed Subarkhan and R. Ramesh,
RSC Adv., 2015, 5, 46760–46773.
12 R. A. Begum, D. Powell and K. Bowman-James, Inorg.
Chem., 2006, 45, 964–966.
13 T. Koizumi, T. Teratani and K. Okamoto, Inorg. Chim. Acta,
2010, 363, 2474–2480.
T. Rosu, Molecules, 2013, 18, 8812–8836.
35 R. K. Agarwal, L. Singh and D. K. Sharma, Bioinorg. Chem.
Appl., 2005, 2006, 1–10.
36 F. Neve, A. Crispini, C. D. Pietro and S. Campagna,
Organometallics, 2002, 21, 3511–3518.
37 M. Ghedini, I. Aiello, M. L. Deda and A. Grisolia, Chem.
Commun., 2003, 2198–2199.
38 C. S. Consorti, G. Ebeling, F. Rodembusch, V. Stefani,
P. R. Livotto, F. Rominger, F. H. Quina, C. Yihwa and
J. Dupont, Inorg. Chem., 2004, 43, 530–536.
14 S. Kagaya, H. Miyazaki, M. Ito, K. Tohda and T. Kanbara,
J. Hazard. Mater., 2010, 175, 1113–1115.
15 A. Kanchanadevi, R. Ramesh and D. Semeril, J. Organomet. 39 K. Okamoto, T. Yamamoto, M. Akita, A. Wada and
Chem., 2016, 808, 68–77. T. Kanbara, Organometallics, 2009, 28, 3307–3310.
16 U. Schatzschneider and N. Metzler-Nolte, Angew. Chem., 40 V. Pandarus and D. Zargarian, Chem. Commun., 2007, 978–
Int. Ed., 2006, 45, 1504–1507. 980.
17 G. Jaouen, S. Top, A. Vessieres, G. Leclercq and 41 V. Pandarus and D. Zargarian, Organometallics, 2007, 26,
M. J. McGlinchey, Curr. Med. Chem., 2004, 11, 2505–
2517.
18 G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2011,
54, 3–25.
19 A. Volbeda, M. H. Charon, E. C. Piras, M. Frey and
J. C. F. Camps, Nature, 1995, 373, 580–587.
20 S. Wang, M. H. Lee, R. P. Hausinger, P. A. Clark,
4321–4334.
42 L. P. Mangin and D. Zargarian, Dalton Trans., 2017, 46,
16159–16170.
43 J. P. Cloutier, B. Vabre, B. Moungang-Soume and
D. Zargarian, Organometallics, 2015, 34, 133–145.
44 J. R. Lakowicz and G. Weber, Biochemistry, 1973, 12, 4161–
4170.
D. E. Wilcox and R. A. Scott, Inorg. Chem., 1994, 33, 1589– 45 M. R. Eftink and C. A. Ghiron, J. Phys. Chem., 1976, 80,
1593. 486–493.
21 R. R. Kumar, M. K. M. Subarkhan and R. Ramesh, RSC 46 X. F. Zhao, Y. Ouyang, Y. Z. Liu, Q. J. Su, H. Tian, C. Z. Xie
Adv., 2015, 5, 46760–46773. and J. Y. Xu, New J. Chem., 2014, 38, 955–965.
22 J. Haribabu, K. Jeyalakshmi, Y. Arun, N. S. P. Bhuvanesh, 47 T. S. Morais, F. C. Santos, L. Corte-Real and M. Helena
P. T. Perumal and R. Karvembu, RSC Adv., 2015, 5, 46031–
46049.
23 R. Kurtaran, L. T. Yıldırım, A. D. Azaz, H. Namli and
O. Atakol, J. Inorg. Biochem., 2005, 99, 1937–1944.
24 W. Luo, X. Meng, X. Sun, F. Xiao, J. Shen, Y. Zhou,
Garcia, J. Inorg. Biochem., 2013, 129, 94–101.
48 F. Ding, J. X. Diao, X. L. Yang and Y. Sun, J. Lumin., 2011,
131, 2244–2251.
49 D. Leckband, Annu. Rev. Biophys. Biophys. Chem., 2000, 29,
1–26.
G. Cheng and Z. Ji, Inorg. Chem. Commun., 2007, 10, 1351– 50 J. R. Lakowicz, Principles of fluorescence spectroscopy,
1354. Springer, New York, 2006.
25 Z. Afrasiabi, E. Sinn, W. Lin, Y. Ma, C. Campana and 51 D. Ray, B. K. Paul and N. Guchhait, Phys. Chem. Chem.
S. Padhye, J. Inorg. Biochem., 2005, 99, 1526–1531. Phys., 2012, 14, 12182–12192.
26 A. Buschini, S. Pinelli, C. Pellacani, F. Giordani, 52 P. V. Bernhardt, P. C. Sharpe, M. Islam, D. B. Lovejoy,
M. B. Ferrari, F. Bisceglie, M. Giannetto, G. Pelosi and
P. Tarasconi, J. Inorg. Biochem., 2009, 103, 666–677.
D. S. Kalinowski and D. R. Richardson, J. Med. Chem.,
2009, 52, 407–415.
27 P. Raj, A. Singh, A. Singh and N. Singh, ACS Sustainable 53 X. Han, P. Mei, Y. Liu, Q. Xiao, F. Jiang and R. Li,
Chem. Eng., 2017, 5, 6070–6080. Spectrochim. Acta, Part A, 2009, 74, 781–787.
28 M. P. Sathisha, S. Budagumpi, N. V. Kulkarni, 54 J. Q. Tong, F. F. Tian, Q. Li, L. L. Li, C. Xiang, Y. Liu, J. Dai
G. S. Kurdekar, V. K. Revankar and K. S. R. Pai, Eur. J. Med.
Chem., 2010, 45, 106–113.
and F. L. Jiang, Photochem. Photobiol. Sci., 2012, 11, 1868–
1879.
16956 | Dalton Trans., 2018, 47, 16944–16957
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
55 G. Sudlow, D. J. Birkett and D. N. Wade, Mol. Pharm., 1975, 61 C. Harford and B. Sarkar, Acc. Chem. Res., 1997, 123–130.
11, 824–832.
62 W. Bal, J. Christodoulou, P. J. Sadler and A. Tucker, J. Inorg.
Biochem., 1998, 70, 33–39.
56 A. M. Merlot, S. Sahni, D. J. R. Lane, A. M. Fordham,
N. Pantarat, D. E. Hibbs, V. Richardson, M. R. Doddareddy, 63 P. J. Sadler and J. H. Viles, Inorg. Chem., 1996, 35, 4490–
J. A. Ong, M. L. H. Huang, D. R. Richardson and
D. S. Kalinowski, Oncotarget, 2015, 6, 10374–10398.
57 D. Li, M. Zhu, C. Xu and B. Ji, Eur. J. Med. Chem., 2011, 46,
588–599.
4496.
64 C. A. Blindauer, I. Harvey, K. E. Bunyan, A. J. Stewart,
D. Sleep, D. J. Harrison, S. Berezenko and P. J. Sadler,
J. Biol. Chem., 2009, 284, 23116–23224.
58 A. Varlan and M. Hillebrand, Molecules, 2010, 15, 3905– 65 F. L. Cui, J. Fan, D. L. Ma, M. C. Liu, X. G. Chen and Z. Hu,
3919. Anal. Lett., 2003, 36, 2151–2166.
59 M. Hosseini-Kharat, K. Karami, M. Saeidifar, C. Rizzoli, 66 R. K. Gupta, G. Sharma, R. Pandey, A. Kumar, B. Koch,
R. Zahedi-Nasab, Z. Sohrabijam and T. Sharifi, New
J. Chem., 2017, 41, 9897–9907.
P. Z. Li, Q. Xu and D. S. Pandey, Inorg. Chem., 2013, 52,
13984–13996.
60 G. Sudlow, D. J. Birkett and D. N. Wade, Mol. Pharmacol., 67 A. Koch, P. Tamez, J. Pezzuto and D. Soejarto,
1976, 12(6), 1052–1061.
J. Ethnopharmacol., 2005, 101, 95–99.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans., 2018, 47, 16944–16957 | 16957