DNA interaction and anticancer evaluation of new palladium(II), platinum(II) and silver(I) complexes based on (Δ)- and (Λ)-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol enantiomers

Article abstract of DOI:10.1016/j.poly.2018.07.020

The synthesis of some new palladium(II), platinum(II) and silver(I) complexes based on (Δ)- and (Λ)-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol (Δ-H₂bie and Λ-H₂bie) enantiomers in the absence and presence of the N,N-chelates 2,2′-bipyridyl and 9,10-phenanthroline, and triphenylphosphine is reported. The molecular structures of the new complexes are discussed on the basis of their IR, Raman, UV–Vis, NMR (¹H, ¹³C and ³¹P) and mass spectra, elemental analyses, molar conductivities and TGA properties. Both Δ-H₂bie and Λ-H₂bie coordinate to the metal ions in a neutral bidentate manner through the azomethine-N and protonated hydroxy-O atoms, while in basic media, Δ-Hbie^? and Λ-Hbie^? are bound to the metal ions through the azomethine-N and deprotonated-O atoms as mono-negative bidentate chelating ligands. The in vitro anticancer activity of the complexes has been evaluated against the human breast cancer (MDA-MB231) and ovarian cancer (OVCAR-8) cell lines. The Δ-H₂bie complexes are more active against the studied cell lines. The IC₅₀ values for cell growth proliferation of [Ag(PPh₃)(Λ-H₂bie)]ClO₄ and [Ag(PPh₃)(Δ-Hbie)] are 0.443 and 1.277 μM, and 0.427 and 1.437 μM for the MDAMB231 and OVCAR-8 cell lines, respectively. The corresponding IC₅₀ values for cisplatin were 3.20 (MDA-MB231) and 2.28 (OVCAR-8) μM. The DNA-binding properties of some of the complexes have been studied using circular dichroism (CD) spectroscopy. The results indicate that the complexes may have intercalative CT-DNA binding capabilities. The intercalation of the Δ-enantiomers appears to be greater than is that for the Λ-enantiomers. Insertion of the complexes into adjacent base pairs prevents neighboring base pairs from close stacking.

Full text of DOI:10.1016/j.poly.2018.07.020

Accepted Manuscript
DNA Interaction and Anticancer Evaluation of New Palladium(II), Platinum(II)
and Silver(I) Complexes Based on (Δ)- and (Λ)-1,2–Bis-(1H-benzimidazol-2-
yl)-1,2-ethanediol Enantiomers
Ahmed A. Shabana, Ian S. Butler, Annie Castonguay, Mohsen Mostafa,
Bertrand J. Jean-Claude, Sahar.I. Mostafa
PII:
S0277-5387(18)30407-8
https://doi.org/10.1016/j.poly.2018.07.020
POLY 13285
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
10 March 2018
2 July 2018
3 July 2018
Please cite this article as: A.A. Shabana, I.S. Butler, A. Castonguay, M. Mostafa, B.J. Jean-Claude, Sahar.I. Mostafa,
DNA Interaction and Anticancer Evaluation of New Palladium(II), Platinum(II) and Silver(I) Complexes Based on
(
Δ)- and (Λ)-1,2–Bis-(1H-benzimidazol-2-yl)-1,2-ethanediol Enantiomers, Polyhedron (2018), doi: https://doi.org/
1
0.1016/j.poly.2018.07.020
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DNA Interaction and Anticancer Evaluation of New Palladium(II), Platinum(II) and
Silver(I) Complexes Based on (∆
)- and (Λ
)-1,2–Bis-(1H-benzimidazol-2-yl)-1,2-ethanediol
Enantiomers
1
2,*
3
1
Ahmed A. Shabana , Ian S. Butler , Annie Castonguay , Mohsen Mostafa , Bertrand J.
4
Jean-Claude , Sahar. I. Mostafa
1,*
1
Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
2
Department of Chemistry, McGill University, Montreal QC, Canada H3A 2K6
3
INRS-Institut Armand-Frappier, Institut National de la Recherche Scientifique, Université du
Québec, Laval, Canada
4
McGill University, Royal Victoria Hospital, Department of Medicine, Montreal, QC H3A 1A1,
Canada
E-mail: ian.butler@mcgill.ca & sihmostafa@gmail.com
Tel: 002-01008502625
Fax: 0020502246781
Abstract
The synthesis of some new palladium(II), platinum(II) and silver(I) complexes based on (∆)- and
(
2 2
Λ)-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol (∆-H bie and Λ-H bie) enantiomers in the
absence and presence of the N,N-chelates 2,2’-bipyridyl and 9,10-phenanthroline, and
triphenylphosphine is reported. The molecular structures of the new complexes are discussed on
1
13
31
the basis of their IR, Raman, UV–Vis, NMR ( H, C and P) and mass spectra, elemental
analyses, molar conductivities and TGA properties. Both ∆-H bie and Λ-H bie coordinate to the
2
2
metal ions in a neutral bidentate manner through the azomethine-N and protonated hydroxy-O
1

-
-
atoms, while in basic media, ∆-Hbie and Λ-Hbie are bound to the metal ions through the
azomethine-N and deprotonated-O atoms as mono-negative bidentate chelating ligands. The in
vitro anticancer activity of the complexes has been evaluated against the human breast cancer
(
MDA-MB231) and ovarian cancer (OVCAR-8) cell lines. The ∆-H
active against the studied cell lines. The IC50 values for cell growth proliferation of [Ag(PPh
bie)]ClO and [Ag(PPh )(∆-Hbie)] are 0.443 and 1.277 µM, and 0.427 and 1.437 µM for the
2
bie complexes are more
3
)(Λ-
H
2
4
3
MDAMB231 and OVCAR-8 cell lines, respectively. The corresponding IC50 values for cisplatin
were 3.20 (MDA-MB231) and 2.28 (OVCAR-8) µM. The DNA-binding properties of some of
the complexes have been studied using circular dichroism (CD) spectroscopy. The results
indicate that the complexes may have intercalative CT-DNA binding capabilities. The
intercalation of the ∆-enantiomers appears to be greater than is that for the Λ- enantiomers.
Insertion of the complexes into adjacent base pairs prevents neighboring base pairs from close
stacking.
2 2
Keywords: ∆-H bie and Λ-H bie, palladium, platinum, silver, spectra, anticancer, DNA.
1
. Introduction
Benzimidazoles constitute a class of heterocyclic aromatic organic compounds that contain a
benzene ring fused to an imidazole ring. The most prominent benzimidazole compound found in
nature is N-ribosyldimethylbenzimidazole, which serves as an axial ligand for Co(III) ions in
vitamin B12 [1]. Benzimidazole and its derivatives are involved in various biological processes
[
2-5]. Substituted benzimidazoles are of interest since substitution at the 1, 2 and 5 positions has
been shown to be related to their pharmacological properties [6]. In addition, the benzimidazole
2

scaffold is considered to be a useful structural motif in the development of therapeutic agents
that display a broad spectrum of pharmacological activity. To date, many DNA minor groove
binders containing one or more imidazole heterocycles have been reported that have promising
anticancer and antiparasitic activities [7].
The synthesis and in vitro anticancer activities of bis-benzimidazole Zn(II) [7,8], Co(II) [8] and
Cu(II) [9,10] derivatives have been reported. In addition, monomeric and polymeric mixed-ligand
benzimidazole and malonic acid Co(II) and Cu(II) complexes has been described [11]. The
.
preparation of the complexes [Cr(2gb)
3
]Cl[ZnCl
4
]·CH
3
OH, [Cr(2gb)
3
]Cl
3
4H
2
O and [Cr
2
(2gb)
4
(µ-
.
OH)
2
](ClO
4
)
4
5H
2
O (2gb = 2-guanidinobenzimidazole) has been published [12], and the X-ray
crystal structure of the latter binuclear complex revealed the presence of two 2gb moieties and a
symmetric (µ-OH) bridge.
2
Tridentate facial chiral ligands deserve attention as they offer different types of binding sites with
a view to enantioselective catalysis, so the introduction of chirality in the ligands is an attractive
prospect. The synthesis and coordination chemistry of 1,2-bis(1H-benzimidazol-2-yl)-1,2-
ethanediol and its methylated derivative indicate that they are versatile chiral, tridentate facially
coordinating ligands (Scheme 1) [13]. The related 1,2-bis(2-benzimidazolyl) ethanol ligand, which
is formed using malic acid instead of tartaric acid, can act as a facially coordinating tridentate
ligand, with the -OH group coordinating to a Ni(II) ion in an octahedral geometry [14]. 1,2-
Bis(1H-benzimidazol-2-yl)-1,2-ethanediol and its methylated derivative act as chiral, facially
coordinating tridentate ligands, forming [Cu(SS-1)
2
](ClO
4
)
2
and [Cu(SS-2)
2
4 2
](ClO ) [14]. The fully
2
-
2-
protonated structures were determined for the [Ni(RR-1)
2
] and [Ni(SS-1)
2
] complexes, which
were formed from a racemate of the ligand and show homochirality [14]. On the other hand, the
2
-
mono-deprotonated complex with a dimeric structure, [Ni(RR-2)
2
Ni(RR-2-H) ] , shows the
2
3

coordinated -OH groups of one complex molecule are hydrogen bonded to the coordinated
alkoxide groups of the another complex molecule.
The mainstay of cancer treatment is chemotherapy [15] and the chief objective is to attain the
maximum therapeutic damage to cancer cells with a minimal concentration of drugs [16,17].
Some metal ions are essential for cellular processes and their redox activity, reactivity and
complexation ability towards organic substrates, and intracellular availability is tightly regulated.
Even though they may be associated with various pathological disorders, including cancer, they
3 2 2
are potential candidates as anticancer agents [18]. Cisplatin, cis-[Pt(NH ) Cl ], is one of the most
potent chemotherapy drugs and is widely used to treat many cancers [18-21]. However, cisplatin
and some other platinum-based anticancer drugs (carboplatin and oxaliplatin) have several
disadvantages, including side effects such as injury to renal tubular epithelial cells [20-22],
nausea, vomiting [23], gastrointestinal toxicity [24], nephrotoxicity, elevated blood pressure and
others [18,22,25,26], and some tumors are resistant to cisplatin [27,28]. Moreover, cisplatin has
limited solubility in aqueous media [27,29,30] and responds to only a restricted spectrum of
cancers [31,32]. Thus, there is scope to improve the toxicity [33,34] and effectiveness, along
with a broader spectrum of action, elimination of side effects and increased solubility in future
drugs [23]. Other strategies have already encompassed the development of water-soluble Pt(II)
and multinuclear Pt(II) complexes, and complexes with bioactive donor molecules as secondary
ligands [35-37] and the design the new anticancer agents with other central metal ions
[
27,38,39]. In particular, Pd(II) complexes have readily been chosen owing to their structural
similarity to those containing Pt(II) ions [32,33,40,41]. These Pd(II) complexes were expected to
exhibit lower kidney toxicity than cisplatin owing to the inability of the proteins in the kidney
tubules to replace the tightly bound Pd(II) chelates with sulfhydryl groups [22,41,42]. In fact,
4

4
5
Pd(II) compounds exchange ligands 10 -10 times faster than their Pt(II) analogs, i.e., Pt(II)
complexes are thermodynamically and kinetically more stable than Pd(II) complexes [32,43].
The Pd(II) derivatives do not maintain their structural integrity in biological fluids for a long
enough time to reach their pharmacological targets [26,32,44,45]. Nevertheless, several Pd(II)
complexes containing N-, N,N- and N,S- aromatic ligands have shown promising anticancer
activities [32,45,46,47].
Although it is generally accepted that DNA is the primary intracellular target of metal-based
anticancer drugs, DNA-binding is chelate dependent. Changes in the structure of DNA upon
binding with Pd(II) complexes have been reported, but a definite correlation between the
structure and the mode of interaction has not been established [32,43,47]. Our research reported
here deals with the synthesis of new Pd(II), Pt(II) and Ag(I) complexes based on (∆)- and (Λ)-
1
,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol (∆-H
2
bie and Λ-H
2
bie) enantiomers in both the
. The structures of the
absence and presence of N,N-chelating ligands (bpy and phen) and PPh
3
new complexes obtained are discussed in terms of their spectroscopic and other physical
properties. In addition , we have examined their in vitro anticancer activity against human breast
cancer (MDA-MB231) and ovarian cancer (OVCAR-8) cell lines as compared to cisplatin.
Finally, we have used circular dichroism (CD) spectroscopy to study the DNA binding properties
of the new complexes.
2
. Experimental
2
.1. Materials
5

All reagents and solvents were purchased from Alfa/Aesar and were used without further
purification. All manipulations were performed under aerobic conditions. DMSO-d was used for
the NMR measurements, referenced against TMS. The [M(L)Cl ] [M(II) = Pd, Pt; L = bpy, phen,
PPh ] complexes were prepared by the literature methods [48].
The human (MDA-MB231) cell line was obtained from the American Type Culture Collection
ATCC catalog number), while the human ovarian cancer (OVCAR-8) cell line was generously
6
2
3
(
donated by Dr. Marie-Claude Beauchamp (MUHC, Lady Davis Institute, Jewish General
Hospital, Montreal, QC, Canada). Cells were maintained in RPMI media from Wisent Bio
Products, which was supplemented with Fetal Bovine Serum (FBS; 10%), HEPES (12 mL), L-
glutamine (5 mL), gentamicin sulfate (500 µL), fungisone (250 µL) and ciprofloxacin (170 µL).
All the bio-products used in the preparation of the media were purchased from Wisent Inc. The
cells were grown in Corning cell cultured treated polystyrene flasks, which were placed in an
2
incubator at 37 °C and at a CO level of 5%. The media of each flask were changed when
necessary and cell passaging was done between 85 and 95% confluence.
2
.2. Measurements
Elemental analyses (C, H, N, Cl) were performed in the Microanalytical Unit, Department of
Chemistry, Cairo University. Palladium and silver contents were determined by the reported
methods [36]. Infrared spectra were recorded on a Nicolet 6700 Diamond ATR spectrometer in
-1
the 4000-200 cm range. NMR spectra were measured on Varian Mercury 200, 300 and 500
MHz spectrometers, in DMSO d using TMS as a reference. Mass spectra (ESI-MS) were
6
recorded using LCQ Duo and double focusing MS25RFA instruments, respectively. Electronic
spectra were obtained in DMF using a Hewlett-Packard 8453 spectrophotometer. Thermal
6

o
o
-1
analysis measurements were made in the 20-1000 C range at a heating rate of 20 C min using
Ni and NiCo as references, on a on a TA instrument TGA model Q500 Analyzer TGA-50. Molar
conductivity measurements were carried out at room temperature on a YSI Model 32
conductivity bridge. SEM analysis was performed using JEOL JSM 6510 lv.
2
.3. Syntheses
)- and (Λ)-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol (∆
(∆
-H
2
2
bie and Λ-H bie)
A mixture of 1,2-phenylenediamine (10.00 g, 92.5 mmol) and D(-)- or L(+)-tartaric acid (6.94 g,
6.3 mmol) was dissolved in 4M HCl (200 mL). The reaction mixture was heated under reflux
4
for 24 h. Upon cooling, green crystals of the chloride salt of the protonated ligand were obtained.
These were dissolved in water (200 mL) and mixed with activated carbon under reflux for 2 h.
4
The reaction mixture was treated with NH OH until neutralized and a white pprecipitate was
obtained, which was filtered off and recrystallized from water-ethanol (1:1; V/V) to yield white
needles or plates.
For ∆
-H
2
14 4 2
bie: Yield (25%). Elemental anal. Calcd. for (C16H N O ) C, 65.31; H, 4.76; N,
1
9.05%. Found: C, 65.30; H, 4.78; N, 19.01%.
For Λ
2 14 4 2
-H bie: Yield (28%). Elemental anal. Calcd. for (C16H N O ) C, 65.31; H, 4.76; N,
1
9.05%. Found: C, 65.33; H, 4.75; N, 19.00%.
[
Pd(L)
2
]Cl
2
.nH
2
O (L = ∆
-H
2
2
bie, n = 3; L = Λ-H bie, n = 0)
2 4 2 2
An aqueous solution of K [PdCl ] (0.16 g, 0.5 mmol, 3 mL) was added to ∆-H bie or Λ-H bie
(
0.147 g, 0.5 mmol) in EtOH (15 mL). The suspension was stirred at 45 °C for 2 h and the
7

yellow-beige precipitate that formed was filtered off and washed with water, ethanol and air-
dried.
For [Pd(∆
2 2 2 2
-H bie) ]Cl .3H
O: Yield: 73%. Elemental anal. Calcd.: C, 46.86; H, 4.15; Cl, 8.66;
Pd); Found: C, 46.83; H, 4.08; Cl, 8.70; N, 13.54; Pd,
N, 13.67; Pd, 12.99 (C32Cl
2
34 8 7
H N O
-3
-1
2
−1
1
M
3.05%. Conductivity data (10 M in DMF): Λ = 189.2 ohm cm mol .
For [Pd(Λ
2 2 2
-H bie) ]Cl : Yield: 61%. Elemental anal. Calcd.: C, 50.17; H, 3.52; Cl, 9.28; N,
1
4.63; Pd, 13.90 (C32Cl H N O
2 28 8 4
Pd); Found: C, 50.22; H, 3.78; Cl, 9.24; N, 14.45; Pd, 13.94%.
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 188.4 ohm cm mol .
[
2 2 2
M(Λ-H bie)Cl(H O)]Cl.nH O (M = Pd, n = 2; M = Pt, n = 1)
A similar procedure to that for the [Pd(Λ-H
orange (Pd) or yellow (Pt) precipitate was isolated after stirring for 24 h at room temperature.
For [Pd(Λ-H bie)(H O)Cl]Cl.2H O: Yield: 61%. Elemental anal. Calcd.: C, 36.54; H, 3.81; Cl,
3.51; N, 10.66; Pd, 20.25 (C16 Pd); Found: C, 36.28; H, 3.86; Cl, 13.44; N, 10.48;
Pd, 20.56%. Conductivity data (10 M in DMF): Λ
For [Pt(Λ-H bie)(H O)Cl]Cl.H O: Yield: 69%. Elemental anal. Calcd.: C, 32.21; H, 3.02; Cl,
1.91; N, 9.40 (C16 Pt); Found: C, 32.14; H, 3.11; Cl, 12.03; N, 9.55%. Conductivity
2 2 2
bie) ]Cl analogue was employed. A bright yellow-
2
2
2
1
2 4 5
H20Cl N O
-3
-1
2
−1
M
= 88.0 ohm cm mol .
2
2
2
1
2 4 4
H18Cl N O
-3
-1
2
−1
M
data (10 M in DMF): Λ = 86.2 ohm cm mol .
[
2 2 6 2 2
Pt(∆-H bie) ](PF ) .2H O
8

K
2
[PtCl
MeOH (10 mL), and the resulting solution was stirred at 40 °C for 24 h. A yellow precipitate
was obtained upon adding saturated aqueous solution of NH PF (2 mL). This precipitate was
filtered off, washed with water, MeOH and air-dried. Yield: 83%. Elemental anal. Calcd.: C,
4 2
] (0.05 g, 0.125 mmol) in water (3 mL) was added to ∆-H bie (0.0733 g, 0.25 mmol) in
4
6
3
4.63; H, 2.89; N, 10.1 (C32
H
32
F
12
N
8
O
6
P
2
Pt); Found: C, 34.53; H, 2.80; N, 10.02%.
-3
-1
2
−1
Conductivity data (10 M in DMF): Λ
M
= 65.0 ohm cm mol .
[
M(L)(L’)]Cl.nH
2
O {M(II) = Pd, Pt; L = ∆
-Hbie, Λ-Hbie; L’ = bpy, phen}
2 2
To a stirred suspension of [Pd(L’)Cl ] (L’ = bpy, phen; 1 mmol) in MeOH (20 mL), ∆-H bie or
Λ-H bie (0.294 g, 1 mmol) in MeOH containing KOH (0.056 g, 1 mmol; 7 mL) was added
2
o
dropwise with stirring over 30 min. The reaction mixture was warmed to 40 C and allowed to
stand for 36 h. After slow evaporation, a yellow-to-orange precipitate was isolated, which was
filtered off, washed with water, MeOH and dried in vacuo.
For [Pd(bpy)(∆
-Hbie)]Cl.H
2
O: Yield: 77%. Elemental anal. Calcd.: C, 51.24; H, 3.78; Cl, 5.83;
N, 13.80; Pd, 17.47 (C26
H
23ClN O Pd); Found: C, 51.40; H, 3.84; Cl, 5.80; N, 13.66; Pd,
6 3
-3
-1
2
−1
1
M
7.45%. Conductivity data (10 M in DMF): Λ = 84.0 ohm cm mol .
For [Pd(bpy)(Λ
-Hbie)]Cl: Yield: 70%. Elemental anal. Calcd.: C, 52.80; H, 3.55; Cl, 6.00; N,
1
6 2
4.22; Pd, 18.00 (C26ClH21N O Pd); Found: C, 52.61; H, 3.78; Cl, 5.83; N, 14.11; Pd, 18.11%.
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 79.0 ohm cm mol .
9

For [Pt(bpy)(∆-Hbie)]Cl: Yield: 77%. Elemental anal. Calcd.: C, 45.92; H, 3.09; Cl, 5.22; N,
-3
1
6 2
2.36 (C26ClH21N O Pt); Found: C, 45.78; H, 3.22; Cl, 5.01; N, 12.33%. Conductivity data (10
-1
2
−1
M in DMF): Λ
M
= 89.0 ohm cm mol .
For [Pt(bpy)(Λ
-Hbie)]Cl: 70%. Elemental anal. Calcd.: C, 45.92; H, 3.09; Cl, 5.22; N, 12.36
-3
6 2
(C26ClH21N O Pt); Found: C, 45.93; H, 3.10; Cl, 4.89; N, 12.21%. Conductivity data (10 M in
-1
2
−1
M
DMF): Λ = 80.0 ohm cm mol .
For [Pd(phen)(∆
-Hbie)]Cl: Yield: 69%. Elemental anal. Calcd.: C, 54.64; H, 3.42; Cl, 5.77; N,
Pd); Found: C, 54.52; H, 3.56; Cl, 5.74; N, 13.60; Pd, 17.32%.
1
3.66; Pd, 17.30 (C28ClH21N O
6 2
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 89.0 ohm cm mol .
For [Pd(phen)(Λ
-Hbie)]Cl: Yield: 64%. Elemental anal. Calcd.: C, 54.64; H, 3.42; Cl, 5.77; N,
1
1
3.66; Pd, 17.30 (C28ClH21 Pd); Found: C, 54.63; H, 3.27; Cl, 5.76; N, 13.52; Pd,
6 2
N O
-
3
-1
2
−1
M
7.24%.Conductivity data (10 M in DMF): Λ = 81.0 ohm cm mol .
For [Pt(phen)(∆-Hbie)]Cl: Yield: 65%. Elemental anal. Calcd.: C, 47.76; H, 3.00; Cl, 5.04; N,
-3
1
6 2
1.94 (C28ClH21N O Pt); Found: C, 47.74; H, 3.01; Cl, 5.12; N, 11.83%. Conductivity data (10
-1
2
−1
M
M in DMF): Λ = 88.0 ohm cm mol .
For [Pt(phen)(Λ-Hbie)]Cl: Yield: 60%. Elemental anal. Calcd.: C, 47.76; H, 3.00; Cl, 5.04; N,
-3
1
6 2
1.94 (C28ClH21N O Pt); Found: C, 47.56; H, 3.09; Cl, 5.22; N, 11.56%. Conductivity data (10
-1
2
−1
M
M in DMF): Λ = 81.0 ohm cm mol .
1
0

[
M(PPh
.5)
3
)(L)Cl].nH
2
O (M(II) = Pd, L = ∆
-Hbie, Λ
-Hbie, n = 0; M(II) = Pt, L = ∆-Hbie, n =
1
A suspension of [Pd(PPh
bie or Λ-H bie (0.037 g, 0.125 mmol) in MeOH containing KOH (0.007 g, 0.125 mmol; 4
mL). The reaction mixture was heated under reflux for 48 h. A yellow complex was obtained,
which was filtered off, washed with CH Cl , CH OH and dried in vacuo.
3 2 2 2 2
) Cl ] (0.0877 g, 0.125 mmol) in CH Cl (10 mL) was mixed with ∆-
H
2
2
2
2
3
For [Pd(∆
-Hbie)(PPh
3
)Cl]: Yield: 80%. Elemental anal. Calcd. C, 58.54; H, 4.02; Cl, 5.09; N,
PPd); Found: C, 58.50; H, 4.15; Cl, 5.00; N, 8.07; Pd, 15.24 %.
8
.04; Pd, 15.27 (C34ClH28N O
4 2
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 8.3 ohm cm mol .
For [Pt(∆
-Hbie)(PPh
3
)Cl].1.5H
2
O: Yield: 76%. Elemental anal. Calcd. C, 50.22; H, 3.81; Cl,
4
.37; N, 6.89 (C34ClH31N O
4
3.5PPt); Found: C, 50.14; H, 3.89; Cl, 4.26; N, 6.92%. Conductivity
-3
-1
2
−1
M
data (10 M in DMF): Λ = 7.5 ohm cm mol .
For [Pd(PPh
3
)(Λ
-H
2
bie)Cl]: Yield: 75%. Elemental anal. Calcd.: C, 58.54; H, 4.02; Cl, 5.09; N,
PPd); Found: C, 58.44; H, 4.00; Cl, 4.75; N, 8.04; Pd, 15.23 %.
8
.04; Pd, 15.27 (C34ClH28N O
4 2
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 6.8 ohm cm mol .
[
Ag(L)
2
]ClO
4
(L = ∆
-H
2
2
bie, Λ-H bie)
4 2 2
An aqueous solution of AgClO (0.1036 g, 0.5 mmol; 2 mL) was added to ∆-H bie or Λ-H bie
(
0.1471 g, 0.5 mmol) in MeOH containing KOH (0.028 g, 0.5 mmol; 10 mL). The reaction
1
1

mixture was stirred in the dark at 50 °C for 4 h. The off-white precipitate that formed was
filtered off, washed with warmed MeOH and dried in vacuo.
For [Ag(∆
2 2 4
-H bie) ]ClO
: Yield: 73%. Elemental anal. Calcd.: C, 48.27; H, 3.52; Ag, 13.58; N,
); Found: C, 48.21; H, 3.78; Ag, 13.44; N, 14.21; Cl, 4.40%.
1
4.08; Cl, 4.46 (AgC32ClH28N O
8 8
-3
-1
2
−1
M
Conductivity data (10 M in DMF): Λ = 89.0 ohm cm mol .
For [Ag(Λ
-H
2
bie)
2
4
]ClO : Yield: 73%. Elemental anal. Calcd.: C, 48.27; H, 3.52; N, 14.08; Cl,
-3
4
.46 (AgC32ClH28N O ); Found: C, 48.04; H, 3.51; N, 14.33; Cl, 4.34%. Conductivity data (10
8 8
-1
2
−1
M
M in DMF): Λ = 80.4 ohm cm mol .
[
3
Ag(L)(∆-Hbie)] (L = phen, PPh )
A mixture of AgClO
MeOH (10 mL) was added to ∆-H
.5 mmol; 5 mL). The reaction mixture was stirred in the dark for 3 h at room temperature. The
4
3
(0.101 g, 0.5 mmol) in water (1 mL) and phen or PPh (0.5 mmol) in
2
bie (0.147 g, 0.5 mmol) in MeOH containing KOH (0.028 g,
0
pale-yellow solid that formed was filtered off, washed with water, MeOH, Et
vacuo.
2
O and dried in
For [Ag(phen)(∆
-Hbie)]: Yield: 73%. Elemental anal. Calcd.: C, 52.01; H, 3.41; N, 13.00
-3
(
C
28
H22AgN
6
O
6
); Found: C, 51.82; H, 3.24; N, 12.83%. Conductivity data (10 M in DMF): Λ
M
-1
2
−1
=
7.0 ohm cm mol .
1
2

For [Ag(PPh
3
)(∆
-Hbie)]: Yield: 66%. Elemental anal. Calcd. C, 61.38; H, 4.22; N, 8.44
-3
(
C
34
H28AgN
4
O
2
P); Found: C, 61.48; H, 4.30; N, 8.44%. Conductivity data (10 M in DMF): Λ
M
-1
2
−1
=
5.0 ohm cm mol .
[
2 4 3
Ag(L)(Λ-H bie)]ClO (L = bpy, PPh )
A mixture of AgClO
MeOH (10 mL) was added to Λ-H
mixture was stirred in the dark for 3 h at room temperature. The resulting pale beige-yellow solid
was filtered off, washed with water, MeOH, Et O and dried in vacuo.
4
(0.05 g, 0.25 mmol) in water (0.5 mL) and bpy or PPh
3
(0.5 mmol) in
2
bie (0.147 g, 0.5 mmol) in MeOH (10 mL). The reaction
2
For [Ag(bpy)(Λ
-H
2
bie)]ClO
4
: Yield: 73%. Elemental anal. Calcd.: C, 47.45; H, 3.34; Cl, 5.40;
N, 12.77 (C26
H
22AgClN O ); Found: C, 47.41; H, 3.29; Cl, 5.30; N, 12.74%. Conductivity data
6 6
-3
-1
2
−1
(
M
10 M in DMF): Λ = 99.0 ohm cm mol .
For [Ag(PPh
N, 5.27 (C34
3
)(Λ
-H
2
bie)]ClO
4
: Yield: 66%. Elemental anal. Calcd. C, 60.58; H, 4.31; Cl, 4.6;
H
29AgClN O P); Found: C, 60.50; H, 4.18; Cl, 4.61; N, 5.13%. Conductivity data
4 6
-3
-1
2
−1
(
M
10 M in DMF): Λ = 92.0 ohm cm mol .
2
2
.4. Biological applications
.4.1. Anticancer assays
1
3

-2
Drug treatment: In all assays, drug stocks of 0.3125 x 10 to 100 µM were prepared by
dissolution in DMSO.
In vitro growth inhibition essay: To evaluate the biological activity of the drugs on cancer cells,
the growth inhibiting effects on human breast cancer (MDAMB231) and human ovarian cancer
(
OVCAR-8) cell lines were examined in vitro and the results were compared against negative
controls.
The human breast cancer (MDA-MB231) and human ovarian cancer (OVCAR-8) cell lines were
grown to 80% confluence and then incubated in RPMI media onto 96-well plates (Corning Inc.)
at a density of 3000-5000 cells per well (100 µL medium/well). They were allowed to attach for
2
4 h and then treated with a wide range of drugs, free ligands and their complexes, at
-2
concentrations ranging from 0.3125 x 10 to 100 µM. The treatment was designed to be carried
out in triplicate over 5 days in the incubator. After treatment with the drugs, the cells were fixed
with cold trichloroacetic acid (TCA) and dried as much as possible before staining with
Sulforhodamine B (SRB; 50 µL, 0.4 g/100 mL) for at least 1 h at room temperature.
Subsequently, the SRB well plates were rinsed with acetic acid (1%) and allowed to air dry
overnight. Finally, the dye was dissolved in Tris-base (10 mM, pH 10–10.5; 200 µL) for 2-5 min
on a shaking platform. The optical density for each well was recorded using a microplate reader
(
model 2550; Bio-Rad) at 492 nm [49]. Each drug concentration was run in triplicate in at least
two independent experiments. The readings were analyzed using the GraphPad Prism program
GraphPad software, Inc.) and a sigmoidal dose-response curve was used to determine the 50%
inhibitory concentration (IC50) [49].
(
1
4

2
.4.2. DNA binding
The circular dichroism spectral technique is useful in monitoring the conformational variations
of DNA during complex-DNA interactions. The interactions of the new complexes with DNA
were examined in 5 mM phosphate buffer – 50 mM NaCl (pH 7.2). The circulating tumor DNA
(
CT-DNA) shows a ratio of absorbance at 260 and 280 nm of about >1.86, indicating that the
DNA was sufficiently free from protein [50]. The concentration of CT-DNA was determined
-1 -1
-1
from the absorption intensity at 260 nm, having a value of 6600 (mol L ) cm . A stock solution
was stored at 4 ºC and was used within one day. In addition, a stock solution (5 mM) of each
complex was freshly prepared in water prior to use. Aliquots were added to a solution of CT-
DNA (200 µM) freshly prepared in 5 mM phosphate buffer – 50 mM NaCl (pH 7.2) to achieve
molar ratios of 0, 0.1, 0.21, 0.42, 0.63, 0.84 [complex]/[DNA] while keeping a total volume of 2
mL. The samples were incubated at 37 ºC for 24 h in the dark. All CD spectra of DNA and the
DNA-complexes were recorded at 25 ºC in the 220–320 nm range. The absorption of free CT-
DNA was canceled by adding equimolar CT-DNA to a pure buffer solution in the reference
compartment and the resulting spectra were assumed to arise from the metal complexes and the
DNA-metal complex aggregates. All solutions were mixed thoroughly and allowed to equilibrate
for 60 min before data collection. While measuring the absorption spectra, an equal amount of
DNA was added to both the complexes and the reference solutions to eliminate the CD
contribution by CT-DNA, and the phosphate buffer – 50 mM NaCl (pH 7.2) was subtracted
through base line correction.
3
. Results and discussion
1
5

The syntheses of new complexes of ∆- and Λ-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol
bie) enantiomers with Pd(II), Pt(II) and Ag(I) ions are described in the Experimental section.
The elemental analyses are in agreement with the assigned formulae. The molar conductivities
(H
2
(
[
Ʌ
M
) in DMF at room temperature suggest that the complexes are 1:1 electrolytes, except for
Pd(L) ]Cl , (L = ∆-H bie, Λ-H bie) and [Pt(∆-H bie) ](PF , which are 1:2 electrolytes [51,52].
2
2
2
2
2
2
6 2
)
3
.1. Vibrational spectra
2
Isele et. al. [53] have reported the potentiometric titrations of Λ-H bie. The first deprotonation
takes place, for the alcoholic -OH hydrogen, at neutral pH (6–8), while the second is not
observed below pH 10, indicating stabilization of the second –OH hydrogen atom, which may
probably be attributed to hydrogen bond formation [53]. The IR spectra of the free ligands (∆-
–
1
2 2
H bie and Λ-H bie), show intramolecular H-bonding in the 2600–3450 cm region as broad
bands, which are weakened in the complexes, probably due to the participation of one of the two
hydroxyl-O atoms in coordination. Both alcoholic groups become more acidic upon
complexation. Some more intense hydrogen bonding is evidenced in the IR and Raman spectra
of the complexes than in the free ligands owing to intermolecular H-bonding between the OH
and NH groups [54].
–
1
The IR spectra of ∆-H
2 2
bie and Λ-H bie show a medium band near 3055 cm due to the ν(NH)
–
1
stretch [54], while the bands near 3430 cm belong to the ν(OH) stretches [54,55]. The medium-
–
1
-1
intense bands near 1597, 1317 and 1273 cm (∼1594, 1310 and 1277 cm in the Raman spectra)
in the free ligands are attributed to the ν(C=N), ν(C–N) and ν(C–O) stretches, respectively.
Upon complexation, these bands are shifted to lower wavenumbers [54,55]. The out-of-plane C–
1
6

–
1
H bending appears at ∼750 cm as a strong band in the free ligands, while the weak bands in the
–
1
2
815–2891 cm range in the spectra of both the ligands and the complexes are assigned to the
aliphatic ν(CH) vibrational stretches [54,55].
Mono-deprotonated complex formation can be achieved after addition of one equivalent of base
-
-
(
NaOH or KOH) per metal [53,54]. Thus, in these complexes, both ∆-Hbie and Λ-Hbie function
as mono-negative bidentate ligands, coordinating the metal ions through the deprotonated
hydroxyl-O and azomethine-N centers, forming five-membered chelate rings (Fig. 2). The ν(OH)
−
1
stretch is shifted to a lower wavenumber (∼3380 cm ), revealing the participation of one
deprotonated hydroxyl-O atom in complexation, while the other remains uncoordinated [53-57].
-1
The ν(C=N) stretches observed near 1600 and 1550 cm (in the Raman spectra near 1590 and
−
1
1
545 cm ) indicate the involvement of one azomethine-N atom in complexation, while the other
plays no role in coordination [58,59]. The spectra of the ligands display bands near 3055 and
−
1
1
280 cm that are assigned to the imidazole ring NH stretch [60] and these remain more or less
in the same position in the complexes, indicating no participation in coordination.
In the complexes, [Pd(L) ]Cl (L = ∆-H bie, Λ-H bie; Fig. 3), [Pt(∆-H bie)
bie)Cl(H O)]Cl (M(II) = Pd, Pt), [Ag(∆-H bie) ]ClO and [Ag(L’)(Λ-H bie)]ClO
PPh ), the pH values of ∆-H bie or Λ-H bie were initially 7.5, but these were lowered to 5.5–6.1
upon addition of the corresponding metal salt, reflecting the inability of deprotonating the
2
2
2
2
2
2
](PF
6
)
2
, [M(Λ-
H
2
2
2
2
4
2
4
(L’ = bpy,
3
2
2
−
1
alcoholic -OH group [53,56,58,59]. The two ν(OH) modes, near 3200 and 3390 cm , and the
-1
−1
two ν(C=N) modes {near 1590 and 1540 cm (∼ 1595 and 1545 cm in the Raman spectra)}
indicate the involvement of one protonated hydroxy-OH and one azomethine-N atom in
coordination, while the other groups do not participate in complex formation [54,56]. The ν(NH)
1
7

stretch remains more or less in the same position as in the free ligand, indicating no participation
in coordination. The presence of the coordinated PPh groups in [Pd(L-H)(PPh )Cl], [Ag(L-
H)(PPh )] (L = ∆-H bie) and [Ag(Λ-H bie)(PPh )]ClO is confirmed by the strong IR bands near
095 and 750 cm , which are attributed to the ν(P–Cph) and δ(C–CH) vibrations, respectively
61].
The IR spectra of the [M(L)(L’)]Cl (M(II) = Pd, Pt; L = ∆-Hbie or Λ-Hbie; L’ = bpy, phen),
Ag(phen)(∆-Hbie)] and [Ag(bpy)(Λ-H bie)]ClO complexes display bands near 854, 841, 750
3
3
3
2
2
3
4
-1
1
[
[
2
4
-1
and 725 (in the case of bpy) and 1580, 1515, 1495 and 1420 cm (in the case of phen), which are
assigned to the γ(CH) vibrations of the coordinated phen or bpy ligands [62]. These bands are
observed at higher wavenumbers compared with those for the free phen or bpy ligands,
indicating their participation in complex formation [62]. Furthermore, the IR spectra of the
complexes, [Ag(Λ-H
2
bie)(L’)]ClO
4
and [Ag(L)
2
]ClO
4
(L = ∆-H
2
bie, Λ-H
2
bie; L’ = bpy, PPh
) and ν (T ) modes of
bie) ](PF
3
),
-1
exhibit bands near 1105 (strong) and 625 (medium) cm due to the ν
3
(T
2
4
2
-
the uncoordinated T
d
symmetry ClO
4
ion, respectively [63]. The complex [Pt(∆-H
2
2
6 2
)
-1
-
displays a strong IR band at 865 cm arising from the O
h
symmetry [PF
6
] ion [64]. The Raman
-1
and IR spectra of the reported complexes show several bands in the 550–200 cm region that are
attributed to the ν(M-O), ν(M-N), ν(M-P) and ν(M-Cl) stretches [65,66].
3
.2. NMR spectra
1
13
Assignments of the H and C NMR chemical shifts were obtained from the analysis of gCOSY
1
1
1
1
13
1
13
1
(
H), NOESY ( H), ROESY ( H), gHMBC ( H- C) and gHSQC ( H- C) spectra. In the H
NMR spectra of the free ligands, ∆-H bie and Λ-H bie, the aliphatic protons (H , H1’) appear as
doublets near δ 5.25 ppm, which change to singlets with considerable downfield shifts (δ 5.35-
2
2
1
1
8

5
.85 ppm) in the complexes (see Fig.1 for the numbering scheme). The protons of the aliphatic
OH and imidazole NH groups appear as doublets near δ 5.99 ppm and broad singlets at δ 12.38
ppm, respectively. The broad NH singlet indicates its fluxional behavior due to resonance
…
.
….
through the N
and consequently, the NH proton appears as a sharp signal in comparison to that for the free
ligand [54,55]. The aromatic protons appear as doublets (H , H7’) and triplets (H
, H6’) at δ 7.15 and 7.50 ppm, respectively (Fig. 4, Table 1).
C
N system [54,55,58,59], which is changed to HN-C=N in the complexes,
4
, H4’, H
7
5
, H5’,
H
6
In the spectra of the complexes prepared in the presence of base (1:1; M: KOH), the resonance of
the hydroxy protons at δ 5.99 ppm is absent , while a new singlet appears near δ 6.07 ppm,
indicating deprotonation of one –OH group upon coordination to the metal ion, while the second
–
OH group remains uncoordinated [67]. The intensity of the new –OH singlet is less than that of
the doublet in the free ligand, which may be attributed to the involvement of the uncoordinated –
OH group in H-bonding with the solvent [56]. The signals for the imidazole NH proton (δ 12.40–
1
3.8 ppm), the aromatic protons (δ 7.17–8.0 ppm) and aliphatic protons are all shifted downfield
62]. This feature may be attributed to a decrease in the electron density in the imidazole ring
upon complexation through the azomethine nitrogen atom [68,69]. In addition, two doublets
near δ 7.80, 8.00 ppm) and two triplets (near δ 7.15, 7.5 ppm) are observed for the aromatic
[
(
protons, indicating coordination of the metal ion via a deprotonated hydroxyl-O atom and a close
azomethine-N atom in a mono-negative bidentate manner, while the other side remains
uncoordinated (Fig. 4c) [53-55].
In the spectra of the complexes, [Pd(L)
bie)Cl(H O)]Cl (M = Pd, Pt), [Ag(∆-H
PPh ), two doublets and two triplets for the aromatic protons, and two singlets for the hydroxy
2
]Cl
2
(L = ∆-H
2
bie, Λ-H
2
bie), [Pt(∆-H
2
bie)
2
6 2
](PF ) , [M(Λ-
H
2
2
2
bie) ]ClO
2
4
and [Ag(L’)(Λ-H
2
bie)]ClO
4
(L’ = bpy,
3
1
9

protons are observed together with clear downfield shifts of the aliphatic, imidazole NH and
aromatic proton resonances. These observations support coordination of ∆-H bie and Λ-H bie in
2
2
a neutral bidentate manner, through one protonated hydroxyl-OH group and a nearby
azomethine-N atom [56].
2 2 2 4 2 2 2 2 6 2
For the complexes [Pd(L) ]Cl , [Ag(L) ]ClO (L = ∆-H bie, Λ-H bie) and [Pt(∆-H bie) ](PF ) ,
1
cis and/or trans isomers are possible. Their H NMR spectra reveal the presence of only one
resonance for each of the two equivalent protons, suggesting the presence of a trans
2 2 6 2 2 2 2
configuration, except for [Pt(∆-H bie) ](PF ) and [Pd(Λ-H bie) ]Cl (Figs. 2 and 5) which have
two equivalent signals for each proton, confirming their presence in a cis-configuration [70].
1
3
In the C NMR spectra of free ∆-H
56.29, 134.73, 122.18, 119.04, 112.09, 121.6 and 122.20 ppm, corresponding to C(1), C(2),
C(4), C(5), C(6), C(7) and C(8), respectively (Table 2). In the complexes, [Pd(∆-H bie) ]Cl
Pt(∆-H bie) ](PF and [Pd(Λ-H bie)Cl(H O)]Cl, the carbon atoms adjacent to the coordination
2 2
bie and Λ-H bie, there are eight resonances near δ 71.6,
1
2
2
2
,
[
2
2
6
)
2
2
2
centers C(1), C(2) and C(8) are shifted to ∼ δ 69.80, 153.75 and 132.10 ppm, respectively,
indicating the coordination of both protonated hydroxyl-O and azomethine-N atoms in a neutral
2 2 2 2 2 6 2
bidentate manner [71]. In addition, the spectra of [Pd(Λ-H bie) ]Cl and [Pt(∆-H bie) ](PF )
show two resonances for each carbon atom, supporting the cis-configuration that was concluded
1
from their H NMR spectra [72].
1
3
Upon comparing the C NMR spectra of the complexes [M(phen)(∆-Hbie)]Cl (M = Pd, Pt) and
[
Pd(phen)(Λ-Hbie)]Cl to those of ligands, there are clear shifts in the C(1), C(2) and C(8)
resonances, together with only small shifts for C(3) and C(7) being observed, indicating the
participation of one deprotonated -OH group and the nearest azomethine nitrogen atom in
coordination [68].
2
0

3
1
Finally, the P NMR spectra of [Pd(PPh
3
)(∆-Hbie)Cl], [Pd(PPh
3
)(Λ-Hbie)Cl] and [Ag(PPh
group in these
2 2 6 2
complexes [73]. The P NMR spectrum of [Pt(∆-H bie) ](PF ) has one signal at δ 28.7 ppm,
3
)(∆-
Hbie)] exhibit one signal near δ 52 ppm, indicating the presence of one PPh
3
3
1
-
revealing the presence of the uncoordinated [PF
6
] ion [74].
3
.3. Electronic spectra
The electronic spectra of the complexes were recorded in DMSO in the 200-800 nm range. The
spectra of ∆-H bie and Λ-H bie in DMSO show absorption bands near 240 and 300 nm due to n
π* and π → π * transitions, respectively [65]. The electronic spectra of the diamagnetic Pd(II)
2
2
→
1
1
1
and Pt(II) complexes display bands near 470 and 320 nm attributable to A1g → B1g and A1g
→
1
E
1g transitions associated with the square-planar configuration [52,54]. The absorption band
near 375 nm in the [M(L)(∆-Hbie)]Cl and [M(L)(∆-Hbie)]Cl (M(II) = Pd, Pt; L = bpy, phen)
complexes is assigned to a mixture of charge-transfer transitions from a M(II) d-orbital to the π*-
orbital of bpy or phen [49].
3
.4. Mass spectra
The mass spectra of the complexes exhibit fragmentation patterns that reflect the stepwise loss of
the ligands. The mass spectra of the complexes, [Pd(∆-H bie) ]Cl and [Pd(Λ-H bie) ]Cl (Fig
), display signals at m/z 693.99 (Calcd. 694.4) and 1388.9 (Calcd. 1388.4) with 10 and 12%
2
2
2
2
2
2
6
+
2 2 2
abundance, in agreement with the molecular ions of the complexes, [Pd(∆-H bie) ] and [Pd (Λ-
+
H
2
bie)
4
] , respectively [75]. The fragmentation pattern of the former complex indicates the
+
formation of a [Pd(∆-H
2
bie-C
6
H
4
)
2
] fragment at m/z 542.7 (Calcd. 542.4), while that of the latter
indicates the loss of Λ-H
2
bie, Λ-H
2
bie, Pd, C and C fragments at m/z 1095.2 (Calcd.
6
H
4
6 6 2
H N
2
1

+
+
1
{
{
094.8) {[Pd
2
(Λ-H
2
bie)
3
] }, 801.3 (Calcd. 800.8) {[Pd
2
(Λ-H
2
2
bie) ] }, 695.3 (Calcd. 694.4)
+
+
[Pd(Λ-H
2
2
bie)
2
] }, 618.8 (Calcd. 618.4) {[Pd(Λ-H
2
bie-C
6
H
4
)
2
] } and 512.5 (Calcd. 512.4)
+
[Pd(Λ-H
bie-C
6
6 2 2 2 6 2
H N) ] } [68]. The mass spectrum of [Pt(∆-H bie) ](PF ) , shows the first
molecular ion peak at m/z 783.6 (Calcd. 783.0) with 81% abundance, corresponding to [Pt(∆-
+
H
2
bie)
2
] . The peaks at m/z 707.3 (Calcd. 707.1) and 489.3 (Calcd. 489.0) represent the
+
+
fragments, [Pt(∆-H
2
bie)(∆-H
2
bie-C
6
H
4
)] and [Pt(∆-H
2
bie)] , respectively [71,76]. The spectra of
the complexes [Pd(Λ-H
2
bie)Cl(H
2
O)]Cl and [Pt(Λ-H
2
bie)Cl(H O)]Cl shows ion peaks at m/z
2
4
00.8 (Calcd. 400.4) and 490.0 (Calcd. 489.0) with 11 and 12.3% abundance, representing the
+
+
molecular ions [Pd(Λ-H
2
bie)] and [Pt(Λ-H
2
bie)] , respectively. The former one shows one more
+
peak at m/z 294.8 (Calcd. 294.4), corresponding to the [Pd(Λ-H
2
bie-C
6
H
6
N
2
)] fragment.
The mass spectra of the complexes, [Pd(bpy)(∆-Hbie)]Cl, [Pd(bpy)(Λ-Hbie)], [Pd(phen)(∆-
Hbie)] and [Pd(phen)(Λ-Hbie)] exhibit molecular ion peaks at m/z 555.1 (Calcd. 555.4), 554.99
(
Calcd. 555.4), 579.4 (Calcd. 579.4) and 579.3 (Calcd. 579.4) with 100, 42, 6.7 and 12.8%
+
+
abundance, in agreement with the molecular ions, [Pd(bpy)(∆-Hbie)] , [Pd(bpy)(Λ-Hbie)] ,
+
+
[
Pd(phen)(∆-Hbie)] and [Pd(phen)(Λ-Hbie)] , respectively. The fragmentation patterns indicate
+
the loss of a C
6
H
5
N fragment from {[Pd(bpy)(∆-Hbie-C
6
H
5
N)] at m/z 464.2 (Calcd. 464.4)},
while those of the phen complex show the loss of C
6
H
4
fragments from {[Pd(phen)(∆-Hbie-
+
+
C
6
H
4
)] at m/z 503.6 (Calcd. 503.4) and [Pd(phen)(Λ-Hbie-C
6
H
4
)] 503.5 (Calcd. 503.4),
respectively}. On the other hand, the mass spectra of the complexes, [Pt(bpy)(∆-Hbie)]Cl,
Pt(bpy)(Λ-Hbie)], [Pt(phen)(∆-Hbie)] and [Pt(phen)(Λ-Hbie)] (Fig. 6), show signals at m/z
[
6
6
44.3 (Calcd. 644.0), 645.0 (Calcd. 644.0), 668.3 (Calcd. 668.0) and 668.6 (Calcd. 668.0) with
+
6, 5.4, 26 and 10% abundance, in agreement with the molecular ions, [Pt(bpy)(∆-Hbie)] ,
2
2

+
+
+
[
Pt(bpy)(Λ-Hbie)] , [Pt(phen)(∆-Hbie)]
and [Pt(phen)(Λ-Hbie)] , respectively. The
fragmentation patterns indicate the loss of C
6
4 6 5
H and C H N fragments from the second and
+
fourth complexes, i.e., [Pt(bpy)(Λ-Hbie-C
6
H
4
)] at m/z 568.7 (Calcd. 568.0) and [Pt(phen)(Λ-
+
Hbie-C
6
H
5
N)] at m/z 485.9 (Calcd. 486.0), respectively.
The mass spectra of the complexes [Pd(PPh
3
3
)(∆-Hbie)Cl], [Pd(PPh )(Λ-Hbie)Cl] and
[
(
[
Pt(PPh
Calcd. 750.0) with ∼10% abundance due to the molecular ions, [Pd(PPh
3
)(∆-Hbie)Cl] show signals at m/z 661.8 (Calcd. 661.4), 663.3 (Calcd. 661.4) and 750.3
+
3
)(∆-Hbie)] ,
+
+
Pd(PPh
3
)(Λ-Hbie)] and [Pt(PPh
3
)(∆-Hbie)] . The spectrum of the former complex exhibits one
+
3 6 4
more peak at m/z 584.3 (Calcd. 585.4), corresponding to the [Pd(PPh )(∆-Hbie-C H )]
fragment.
2 2 4 2 2 4
The mass spectra of [Ag(∆-H bie) ]ClO and [Ag(Λ-H bie) ]ClO show signals at m/z 696.4
(
Calcd. 696.0) and 697.1 (Calcd. 696.0) with 21 and 15.3% abundance, corresponding to [Ag(∆-
+
+
H
2
bie)
2
] and [Ag(Λ-H
2
bie)
2
] , respectively. The former complex show two more signals at m/z
5
14.3 (Calcd. 514.0) and 402.2 (Calcd. 402.0), corresponding to the [Ag(∆-H
2
bie)(∆-H bie-
2
+
+
C
6
H
5
N)] and [Ag(∆-H
2
bie)] fragments, respectively, while the latter shows one peak at m/z
+
4
02.9 (Calcd. 402) due to the [Ag(Λ-H
2
bie)] fragment.
The mass spectrum of [Ag(PPh
3
)(∆-Hbie)] (Fig. 6) exhibits the first signal at m/z 664.1 (Calcd.
+
6
4
63.0), corresponding to [Ag(PPh
3
)(∆-Hbie)] . A stepwise ligand loss reveals signals at m/z
+
00.8 (Calcd.400.0) and 369.8 (Calcd. 370.0), corresponding to [Ag(PPh
3
)(∆-Hbie-C16
)] fragments, respectively [68]. The mass spectrum of [Ag(bpy)(Λ-H
Fig. 3.2.4.11), exhibits a signal at m/z 559.1 (Calcd. 558.0) with 65.5% abundance,
H
14
N
3
O)]
+
and [Ag(PPh
3
2
bie)]ClO
4
(
+
2 3 2 4
corresponding to [Ag(bpy)(Λ-H bie)] , while that of [Ag(PPh )(Λ-H bie)]ClO shows three
2
3

peaks at m/z 665.2 (Calcd. 664.0), 634.6 (Calcd. 634.0) and 371.2 (Calcd. 370.0), due to
+
+
+
[
[
Ag(PPh
3 2 3 2 3
)(Λ-H bie)] , [Ag(PPh )(Λ-H bie-2NH)] and [Ag(PPh )] fragments, respectively
68,72].
3
.5. Thermal measurements
According to the TGA curves, the observed mass-loss stages arise from the elimination of
hydrated water, coordinated water and/or Cl , and elimination of PPh followed by
bie, ∆-Hbie or Λ-Hbie moiety {or decomposition of ∆-
2
3
-
-
decomposition of the ∆-H
2
bie, Λ-H
2
-
-
H
2
bie, Λ-H
2
bie, ∆-Hbie or Λ-Hbie followed by loss of phen or bpy}. The decomposition of the
-
-
∆
-H
2
2
bie, Λ-H bie, ∆-Hbie or Λ-Hbie moiety takes place through the breakdown of the weak
C
6
H
4
-N, C
6
4
H -NH, C(1)-N, C(1’)-N, C(1)-O or C(1’)-O hetero bonds [61,77-79]. All the
o
complexes show thermal stability up to 200 C. The complexes of both ∆-H
2
bie and Λ-H
2
bie or
-
-
∆
-Hbie and Λ-Hbie enantiomers display the same thermal behavior with the same metal ion
and/or the second chelating agent (bpy, phen or PPh ). The thermograms of the complexes,
Ag(∆-Hbie)(PPh )] and [Ag(Λ-H bie)(PPh )}ClO , do not reflect the same behavior, based on
the difference in the chelation modes of ∆-Hbie and Λ-H
3
[
3
2
3
4
-
2
bie, respectively. The weight loss
observed below ∼ 130 ºC is due to dehydration as the colors of the complexes changed from pale
to a deeper one [62,79].
2 2 2 2
The thermogram of [Pd(∆-H bie) ]Cl .3H O shows the first weight loss between 50 and 175 ºC,
arising from the release of three hydrated water molecules (Calcd. 6.6, Found 6.7%). The second
endothermic decomposition step occurs between 176 and 415 ºC, and is attributed to the loss of
2
4

two C
inflections (416-620 and 621-910 ºC) may arise from the elimination of two C
9.3, Found 39.3%) and two C 1/2 (Calcd. 15.1, Found 15.3%) fragments, respectively,
leaving 14.9% for the PdO residue [80,81].
6
H
4
and Cl
2
species (Calcd. 27.2, Found 27.0%), while the third and fourth endothermic
8
7 3
H N
O (Calcd.
3
2 2 2
H N O
The TGA thermogram of [Pt(∆-H
80, 181–389 and 390-700 ºC regions that are due to the release of hydrated water (Calcd. 3.3,
Found 3.3%), 2PF , 4C , N and H species (Calcd. 55.0, Found 55.4%) and {two C
Calcd. 21.3, Found 21.3%) fragments, respectively, leaving a PtO residue (18.1%) [80,81].
2 2 6 2 2
bie) ](PF ) .2H O is characterized by three steps in the 50–
1
6
6
H
4
2
2
4 4 3 2
H N O }
(
The complex [Pd(Λ-H
2
bie)
2
]Cl
2
(Fig. 7) shows weight losses in the 55– 450 and 451–590 ºC
, N and 4C N (Cacld. 60.5, Found 60.3%)
regions that may correspond to the release of Cl
2
2
6 5
H
and 2C
3
H
4
NO and O
2
(Calcd. 20.4, Found 20.6%) species, respectively, leaving a mixed Pd(II)
o
oxide and carbide residue at 650 C (19.8%). The thermogram of [Pd(Λ-H
2
bie)(H
2
O)Cl]Cl.2H
2
O
consists of three steps in the 62–117, 200–322 and 324-526 ºC regions. These are assigned to the
elimination of {½ Cl and hydrated water} (Calcd. 13.6, Found 13.7%), {coordinated water, ½
Cl , N and H } (Calcd. 15.9, Found 16.1%) and 2C (Calcd. 28.9, Found 29.0) fragments,
respectively, leaving a palladium nitride, carbide and oxide residue (26.4%). On the other hand,
the thermogram of [Pt(Λ-H bie)(H O)Cl]Cl.H O shows four decomposition steps in the 45-180,
81-500, 501-812 and 813-900 C regions, which may be attributed to the elimination of
hydrated water (Calcd. 3.0, Found 3.2%), coordinated water and Cl (Calcd. 14.9, Found 14.5%),
(Calcd. 12.8, Found 12.6%) and C N (Calcd. 15.3, Found 15.5%) fragments,
respectively, leaving a Pt(II) residue (45.5%) [72].
2
2
2
2
6 4
H
2
2
2
o
1
2
C
6
H
4
6 5
H
2
The thermogram of [Pd(bpy)(∆-Hbie)]Cl.H O (Fig. 7) indicates the first weight loss between 38
2
5

o
and 145 C due to the release of a hydrated water molecule (Calcd. 3.0, Found 3.2%). There are
five more endothermic TG inflections in the 146-225, 226-325, 326-562, 563-615 and 616–970
ºC regions, which arise from the elimination of ½ Cl
2
(Calcd. 5.8, Found 5.7%), 2C
7.4, Found 27.0%), ½ bpy (Calcd. 12.8, Found 12.7%), {½ bpy, N , ½ O and 1.5H
0.5, Found 20.2%) and C N (Calcd. 10.5, Found 10.6%) species, respectively, leaving a PdO
6
H
5
N (Calcd.
2
2
2
2
2
} (Calcd.
4
H
2
residue (20.1%) [82]. The thermogram of [Pd(bpy)(Λ-Hbie)]Cl is characterized by three steps in
the 280–333, 334-428 and 742–790 ºC regions. These are assigned to the elimination of {½ Cl
bpy, C N} (Calcd. 34.6, Found 34.9%), {½ bpy, C N} (Calcd. 28.6, Found 28.2%) and ½
(Calcd. 2.4, Found 2.3%) fragments, respectively, leaving a residue (27.7%). The thermogram
of [Pd(phen)(∆-Hbie)]Cl shows three TG inflections in the 55–210, 211–512 and 513-790 ºC
regions, which are attributed to the elimination of ½ Cl (Calcd. 5.8, Found 5.4%), {phen, 2C
and ½ H species} (Calcd. 56.4, Found 55.8%) and a C O fragment (Calcd. 17.9,
Found 18.3%), leaving a PdO residue (17.3%) [72].
2
,
½
6
H
5
6 5
H
N
2
2
6 4
H ,
½
N
2
2
4 4 3
H N
The TG of [Pd(phen)(Λ-Hbie)]Cl has two endothermic decomposition steps in the 296–320 and
3
1
40–444 ºC regions, assigned to the loss of {½ Cl
2 6 4
and C H fragments} (Calcd. 18.1, Found
8.3%) and {phen, C , ½ N and ½ H species} (Calcd. 48.8, Found 49.1%), respectively,
6
H
5
N
2
2
2
leaving a residue (35.4%) [77].
3 2
The thermogram of [Pd(∆-Hbie)(PPh ) Cl] shows the first step weight loss between 218 and 400
o
C that corresponds to the release of ½ Cl
2
and PPh
3
and 2C
6
H
4
fragments (Calcd. 64.5, Found
o
6
5.0%). The second decomposition occurs between 401 and 828 C due to the elimination of a
C
3
H
4
N
3
O fragment (Calcd. 14.1, Found 13.8%) [53], leaving a PdO residue (21.6%). The
thermogram of [Pt(∆-Hbie)(PPh Cl]1.5H O shows TG inflections in the 45-180, 181-230, 231-
3
)
2
2
2
6

o
5
21, 522-800 and 800–900 C regions. These weight losses arise from the release of hydrate
water (Calcd. 3.3, Found 3.5%), {½ Cl , Ph} (Calcd. 13.8, Found 13.5%), {½ N , ½ H , PPh
Calcd. 24.6, Found 24.9%), {2C N, H , ½ O } (Calcd. 24.6, Found 24.7%) and C HN (Calcd.
.3, Found 6.6%) fragments, respectively, leaving a PtO residue (26.0%) [75].
The thermogram of [Pd(PPh (Λ-H bie)]Cl, is characterized by only one endothermic step in the
70–488 C region due to the elimination of ½ Cl , PPh and C fragments (Calcd. 57.8,
2
2
2
2
}
(
6
H
5
2
2
4
6
3
)
2
2
o
2
2
3
6 5 2
H N
o
Found 58.0%), leaving a Pd(II) residue at 650 C (36.4%) [75]. The thermogram of [Ag(∆-
o
H
2
bie)
These arise from the release of {½ Cl
2C O and ½ N } (Calcd. 23.1, found 23.4) fragments, respectively [83], leaving a residue
2
]ClO
4
shows two endothermic TG inflections in the 200–510 and 511–620 C regions.
2
2 6 5 6 4
, 2O , C H N and 3C H } (Calcd. 52.6, Found 52.9%) and
{
3
H
5
N
2
2
of silver carbide, oxide and azide (23.7%). On the other hand, the thermogram of [Ag(∆-
o
H
2
bie)
regions. These weight losses may arise from the release of {½ Cl
Found 24.0%), {C , N , H } (Calcd. 17.0, Found 17.1%) and 2C
9.5) fragments, respectively [80], leaving a residue of silver oxide (38.6%) [75].
The thermogram of [Ag(PPh )(∆-Hbie)] shows three TG inflections in the 205-315, 316-520 and
21-720 C ranges, indicating the release of {PPh and 2C } (Calcd. 62.4, Found 62.6%),
, ½ O , ½ N , H } (Calcd. 13.3, Found 13.8%) and {C N, ¼ O } (Calcd. 6.9, Found
.1%) species, respectively, leaving the Ag O residue (17.5%). The TG thermogram of
)(Λ-H bie)]ClO (Fig. 7) displays two TG weight losses. The first between 190 and 310
C may arise from the release of {½ Cl
2
]ClO
4
(Fig. 7), shows three TG inflections in the 200-295, 296-425 and 426-520 C
2
, 2O
2
, C
6
H
5
N} (Calcd. 23.9,
6
H
5
N
2
2
2
6 4
H (Calcd. 19.1, found
1
3
o
5
{
7
3
6 4
H
C
2
H
5
N
2
2
2
2
2
2
2
[
Ag(PPh
3
2
4
o
2
, 2O
2
, C H
6 5
N
2
, ½ N
2
, ½ H
2
, PPh
3
} (Calcd. 63.1, Found
o
6
1
3.0%), while the second (311-900 C) is assigned to the release of {C
6
4, 2 2
H C H O1.5} (Calcd.
6.5, Found 16.8%) fragments [80], leaving a residue of silver oxide.
2
7

Finally, the TG thermogram of [Ag(phen)(∆-Hbie)] shows the first endothermic weight loss step
o
between 196 and 310 C, corresponding to the release of C
6
H
5
N
2
, ½ N
2
, ½ H
2
(Calcd. 20.7,
o
Found 20.6%). The second decomposition step occurs between 311 and 545 C, which is
6 4
attributed the loss of phen and C H fragments (Calcd. 44.1, Found 43.6%). The third TG
o
inflection between 546 and 607 C is attributed to the loss of a C
2
H
2
NO fragment (Calcd. 9.6,
1/5 (Calcd. 5.5,
o
Found 10.2%). The last step between 608 and 800 C, is due to the release of C
2
O
2
Found 5.1%), leaving a Ag O residue (24.7%) [72,80].
3
.6. Biological applications
In the field of pharmacology, the most important advances have been the proven efficacy of
chiral or enantiopure drugs. Single enantiopure drugs have been shown to be better drugs, i.e.
safer and more potent, than racemate drugs [84]. Chiral drugs are assumed to be therapeutically
active as most of the bio-targets of drugs are chiral in nature. Therefore, considerable attention
has been paid to the design and construction of chiral molecules that are versatile in occupying
the active site, possess a metal-binding domain and have a bioactive organic functionality
(
pharmacophore), which pre-orients the molecule as a drug entity and reduces the toxicity
parameters [53]. The introduction of chirality enhances the pharmacological behavior of a metal
complex by adopting a specific conformation and a target selective binding affinity for DNA (as
DNA itself exists in nature only in one chiral form) [85]. The design of metal-based
pharmaceuticals depends on the donor framework, the choice of metal ion and its oxidation state
[
86]. The donors can significantly alter the biological properties by modifying reactivity or
substitution inertness. Tailored, multifunctional donors introduced into metal-based medicinal
agents facilitate metal ion redistribution, limit their adverse effects upon overloading and
2
8

inhibiting selected metallo-enzymes [87]. Therefore, chiral metal complexes have a promising
future as robust chemotherapeutic agents in medicinal inorganic chemistry.
3
.6.1. Anticancer studies
As stated earlier, the main objective of this research was to obtain new pairs of enantiomeric
chiral, ∆-1,2–bis-(1H-benzimidazol-2-yl)-1,2-ethanediol (∆-H bie) and Λ-1,2–bis-(1H-
benzimidazol-2-yl)-1,2-ethanediol (Λ-H bie) complexes, with low cytotoxicity and high efficacy
2
2
against the human breast (MDA-MB231) and ovarian cancer (OVCAR-8) cell lines, which
unfortunately have high incidence rates throughout the world. Chirality is an attractive prospect,
because of its ability to enhance the pharmacological uptake of the drug entity by adopting a
specific conformation and stereo-selective binding with the DNA molecular target [88].
To evaluate the anticancer efficacy of the pairs of enantiomeric ∆-H
the in vitro anticancer activities of free ∆-H bie and Λ-H bie and their complexes, [Pd(∆-
bie) ]Cl , [Pt(∆-H bie) ](PF , [Pd(bpy)(∆-Hbie)]Cl, [Pd(phen)(∆-Hbie)]Cl, [Pt(bpy)(∆-
Hbie)]Cl, [Pt(phen)(∆-Hbie)]Cl, [Pd(∆-Hbie)(PPh )Cl], [Pt(∆-Hbie)(PPh )Cl], [Ag(∆-
bie) ]ClO , [Ag(phen)(∆-Hbie)], [Ag(PPh )(∆-Hbie)], [Pd(bpy)(Λ-Hbie)]Cl, [Pd(phen)(Λ-
Hbie)]Cl, [Pt(bpy)(Λ-Hbie)]Cl, [Pt(phen)(Λ-Hbie)]Cl, [Pd(Λ-H bie) ]Cl , [Pd(Λ-Hbie)(PPh )Cl],
Pd(Λ-H bie)(H O)Cl]Cl, [Pt(Λ-H bie)(H O)Cl]Cl, [Ag(Λ-H bie) ]ClO [Ag(bpy)(Λ-
bie)]ClO and [Ag(PPh )(Λ-H bie)]ClO , were tested for their inhibitory effects on the cell
2 2
bie and Λ-H bie complexes,
2
2
H
2
2
2
2
2
6 2
)
3
3
H
2
2
4
3
2
2
2
3
[
2
2
2
2
2
2
4
,
H
2
4
3
2
4
growth proliferation of the human breast (MDA-MB231) and ovarian cancer (OVCAR-8) cell
lines using cisplatin as a reference compound. The cell growth proliferation was evaluated by
RPMI assays. The final IC50 values for cell growth proliferation after 24 h of incubation with
2
9