Synthesis, X-ray structure of organometallic ruthenium (II) p-cymene complexes based on P- and N- donor ligands and their in?vitro antibacterial and anticancer studies

Article abstract of DOI:10.1016/j.jorganchem.2017.06.017

Two new arene compounds containing bis-diphosphinomethane (dppm) and tert-butylpyridine (tbp) ligands as important components in ruthenium(II) complexes were synthesized and characterized by X-ray crystallography, and spectroscopy of ¹H NMR,

Full text of DOI:10.1016/j.jorganchem.2017.06.017

Accepted Manuscript
Synthesis, X-ray structure of organometallic ruthenium (II) p-cymene complexes
based on P- and N- donor ligands and their in vitro antibacterial and anticancer
studies
Parichad Chuklin, Vachirawit Chalermpanapan, Thidarat Nookeaw, Saowanit
Saithong, Kittipong Chainok, Saowalak Phongpaichit, Adisorn Rattanaphan, Nararak
Leesakul
PII:
S0022-328X(17)30412-6
10.1016/j.jorganchem.2017.06.017
JOM 20004
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 24 June 2016
Revised Date: 10 June 2017
Accepted Date: 18 June 2017
Please cite this article as: P. Chuklin, V. Chalermpanapan, T. Nookeaw, S. Saithong, K. Chainok, S.
Phongpaichit, A. Rattanaphan, N. Leesakul, Synthesis, X-ray structure of organometallic ruthenium (II)
p-cymene complexes based on P- and N- donor ligands and their in vitro antibacterial and anticancer
studies, Journal of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2017.06.017.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, X-ray structure of organometallic ruthenium (II) p-cymene complexes based
on P- and N- donor ligands and their in vitro antibacterial and anticancer studies
Parichad Chuklina, Vachirawit Chalermpanapana, Thidarat Nookeawb, Saowanit Saithonga,
,
Kittipong Chainokc, Saowalak Phongpaichitd, Adisorn Rattanaphanb, Nararak Leesakula
*
aDepartment of Chemistry and Center for Innovation in Chemistry, Faculty of Science,
Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
b Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of
Songkla University, Hat Yai, Songkhla 90110, Thailand
cDepartment of Physics, Faculty of Science and Technology, Thammasat University, Klong
Luang, Pathumthani, 12120 Thailand
dNatural Products Research Center and Department of Microbiology, Faculty of Science,
Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
Abstract
Two new arene compounds containing bis-diphosphinomethane (dppm) and tert-butylpyridine
(tbp) ligands as important components in Ruthenium(II) complexes were synthesized and
1
characterized by X-ray crystallography, and spectroscopy of H-NMR, 13C-NMR, 2D-NMR,
FTIR and CHN analysis. The synthesized complexes were evaluated in vitro as anticancer agents
of human breast cancer cell lines, MCF-7 and HCC-1937, using the MTT assay. Both
complexes showed an interesting behavior especially the compound of [Ru(p-cymene)
(dppm)Cl2]. It exhibited anticancer activity against both tested cell lines with greater IC50 values
than cisplatin against all breast cancer cells. Both MCF-7 and HCC1937 cells exhibited 16-
fold sensitivity to the [Ru(p-cymene)(dppm)Cl2] compared to cisplatin. Furthermore, the [Ru(p-

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cymene)(dppm)Cl2] complex
significantly inhibited both Staphylococcus
aureus
ATCC25923 ,and MRSA = methicillin - resistant Staphylococcus aureus with MIC/MBC
g. mL-1 and 32/128 g. mL-1, respectively. In addition, it showed inhibition
values of 8/200
µ
µ
activity on Cryptococcus neoformans ATCC90113 flucytosine - resistant, CN90113, with an
g. mL-1
MIC/MBC value of 64/128
µ
.
Keywords : antimicrobial activity, anticancer activity, Ru(II)-arene
Corresponding author : Tel.: 066 74 288421 (N. Leesakul), Fax.: 066 74 558841
E-mail addresses : nararak.le@psu.ac.th

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1. Introduction
At the present time, some platinum drugs like cis-platin, carbo-platin and oxaliplatin are
commonly used in the treatment of numerous types of cancer cells [1]. Nevertheless, these
kinds of drugs can cause side effects including dehydration, risk of infection, kidney toxicity
and many other abnormalities [2]. Half sandwich ruthenium(II)-arene complexes have been
widely investigated and challenged to develop their pharmaceutical potential as anti-cancer
agents with lower toxicity to normal cells than platinum(II) complexes. Half-sandwich
metallocenes are effectively used for medicinal applications. Various half sandwich
organometallic (η6 -arene)-ruthenium (II) complexes with p-cymene ligands show promising
anticancer behavior [3-4]. A distorted psuedo-tetrahedral structure coordinated with p-
cymene to the ruthenium(II) center, like the typical “piano-stool” geometry, is of extensive
interest. Other coordinated bonds normally occur with functional and chloro ligands. There
exist several types of functional ligands with Nitrogen [5], Oxygen [6], Sulfur [7] and
Phosphorus [8] donors. Most structures are designed to be ionic complexes soluble in water
[9-10]. On the other hand, many neutral complexes exhibit promise as anticancer drugs [11-
12] because they are kinetically stable, relatively lipophilic, and their metal atoms are in
states of low oxidation [13]. Notable examples of anticancer compounds are ruthenium
complexes consisting of diphosphine derivative ligands like 1,1-bis(diphenylphosphino)
methane (dppm)
and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos).
Recently reported by Rodríguez-Bárzano and coworkers [14], they exhibited excellent IC50
values in nanomolar normoxic A2780 (human ovarian carcinoma) and HT-29 (human colon
carcinoma) cell lines. Their reported complexes are both in the form of neutral and ionic
structures of chelating bidentate diphosphine ligands. Nevertheless, no study has been carried
out of the monodentate bonding of this kind of ligand.

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Here we present the synthesis and structure determination, by single crystal X-ray diffraction
and spectroscopic techniques, of half sandwich neutral complexes of organometallic Ru(II)-
p-cymene with two different kinds of P and N-donor ligands of 1,1-bis(diphenyl
phosphino)methane (dppm) and tert-butylpyridine (tbp), respectively. The complexes have
general structures of [Ru(p-cymene)(L)Cl2], where (L) = dppm and tbp and (p-cymene) = η6-
p-cymene. As a consequence of their particular chemical structure, dichloro ligands are
believed to display similar activity to the cis-dichloro motif of the well-established anticancer
drug cisplatin. We investigated the ability of these two complexes to inhibit the growth of
the breast cancer cell lines MCF-7 and HCC-1937, and also their anti-bacterial and anti-
fungal activities . The [Ru(p-cymene)(dppm)Cl2] complex is more encouraging than pyridine
ligand and cis-Platin for the treatment of breast cancer.
2. Experimental Section
2.1 Materials
The chemicals of [RuCl(η6-p-cymene)(µ-Cl)]2 were purchased from Merck, tert-
butylpyridine (tbp) was obtained from Sigma-Aldrich. The tetrahydrofuran, diethyl ether and
acetonitrile solvents were reagent grades from RCI Labscan and used as received without any
further purification.
2.2 Instrumentation
o
The melting points were determined using a Thomas HOOVER, Unimelt 0-360 C
apparatus. FTIR spectra (KBr disk, 4000–400 cm−1) were recorded with a BX PerkinElmer
FTIR spectrophotometer. 1H NMR data were measured using a CDCl3 solvent with a Bruker

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300 MHz NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard. The
orange single crystal of [Ru(p-cymene)(dppm)Cl2] was obtained by recrystallization and the
diffraction collected with a Bruker APEX-II CCD diffractometer with graphite-
monochromated Mo Kα radiation (λ= 0.71073 Å), 33925 reflections. The diffraction data
were obtained by SMART, SAINT v8.34A and SADABS [15]. The structure was solved by
ShelXS [16]. The anisotropic thermal parameters were refined to all non-hydrogen. All
hydrogen atoms were placed in calculated, ideal positions and refined using a riding model.
The Olex2 [17], WinGXv2014.1 [18] and Mercury3.8 [19] programs were used to prepare the
materials and molecular graphics for publication. Crystallographic data of [Ru(p-
cymene)(dppm)Cl2] has been deposited at Cambridge Crystallographic Data Center via
http://www.ccdc.cam.ac.uk/ data_request/cif (or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB21EZ, U.K.; fax: +44 1223 336 033 or email
deposit@ccdc.cam.ac.uk) with the CCDC1486230 and can be received upon request. The X-
ray data are reported as supplementary crystallographic data.
2.3 Synthesis pathway
The complexes of [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2] were synthesized
in the same procedure. The starting material of [RuCl((p-cymene))(µ-Cl)]2 dimer (0.1837 g,
0.3 mmol) was dissolved in warm THF (15 mL) and stirred continuously for 1 hour. The
dppm (0.192 g, 0.5 mmol) or tbp (0.2 mL, 1.2 mmol) ligands were slowly added to the warm
(40oC) Ru(II) dimer solutions. Diethyl ether (5 mL) was added for precipitation. The solution
was kept at room temperature for over one week. Orange precipitates of [Ru(p-
cymene)(dppm)Cl2] and brownish-orange precipitates of [Ru(p-cymene)(tbp)Cl2]) were
obtained. The products were filtered and washed twice with diethyl ether and the synthesized
complexes crystallized in a mixture of THF:ethylacetate (2:1 ratio) after a week. The

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resulting crystals of [Ru(p-cymene)(dppm)Cl2] were separated and dried under vacuum. The
obtained complexes are readily soluble in DMSO.
2.3.1 Synthesis of [Ru(p-cymene)(dppm)Cl2]
Yield : 69 %. Melting point: 178-180 oC. Anal Calcd for RuC35H36P2Cl2 (690.55): C,
60.87; H, 6.02. Found: C, 60.21; H, 6.22. IR: 2985 (νC-H), 1436 (νC=C), 1094 (νP-Ph), 800
(δC-H para disubstituted benzene), 708 (νP-C) cm-1. 1H NMR (300 MHz, CDCl3) 12 signals:
δ (ppm): 7.61 (dd, 4H, JHH = 6.3 Hz), 7.21 (t, 2H, JHH = 7.2 Hz), 7.10 (t, 4H, JHH = 6.6 Hz),
5.15 (d, 2H, JHH = 6.0 Hz), 4.90 (d, 2H, JHH = 5.4 Hz), 4.60 (d, 4H, JHH = 7.3 Hz), 3.75 (t,
4H, JHH = 6.5 Hz), 2.47 (m, 4H, JHH = 6.9 Hz), 1.91 (s, 3H), 1.85 (t, 2H, JHH = 6.5 Hz), 1.71
(s, 2H), 0.94 (d, 6H, JHH = 6.9 Hz).
2.3.2 Synthesis of [Ru(p-cymene)(tbp)Cl2]
o
Yield : 61 %. Melting point: 178-180 C. Anal Calcd for RuC19H26NCl2 (690.55): C,
51.70; H, 6.17; N, 3.17. Found: C, 51.48; H, 6.28; N, 3.13. IR: 3073 (νC-H, aromatic ring),
1
2958 (νC-H, alkyl) , 1617 (νC=C), 835 (δC-H para disubstituted benzene) cm-1. H NMR
(300 MHz, CDCl3) 8 signals: δ (ppm): 8.81 (d, 2H, JHH = 6.3 Hz), 7.23 (d, 2H, JHH = 7.2 Hz),
5.38 (d, 2H, JHH = 6.0 Hz), 5.20 (d, 2H, JHH = 5.4 Hz), 2.93 (m, 4H, JHH = 6.9 Hz), 2.06 (s,
13
3H), 1.24 (d, 2H, JHH = 6.5 Hz), 1.23 (s, 2H). C NMR (300 MHz, CDCl3): 163, 115, 122,
103, 97, 83, 77, 35, 30, 27, 18 ppm. The 13C NMR spectrum was assigned on the basis of the
proton-decoupled 13C and the HMQC, DEPT 135, DEPT 90 spectra (Supplementary data).
2.4 Antibacterial assay
All compounds were dissolved in dimethyl sulfoxide and tested against
Staphylococcus aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus

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(MRSA) SK1, and Escherichia coli ATCC25922 by a microdilution method involving a
modification of Clinical and Laboratory Standards Institute (CLSI) M07-A9 [20]. The MICs
are the lowest concentration of synthesized compounds with visible growth inhibition.
Synthesized compounds of higher concentrations than the MIC, and the MIC were streaked
onto a nutrient agar plate and incubated under appropriate conditions. The lowest
concentration of compounds showing no growth was recorded as the MBC. Vancomycin and
gentamicin were used as standard antibacterial agents for positive inhibitory controls.
2.5 Antifungal assay
The MICs of synthesized compounds were determined by a modification of the
microbroth dilution CLSI M27-A3 [21] against yeast (Cryptococcus neoformans
ATCC90113) and a modification of the microbroth dilution CLSI M38-A2 [22] against a
clinical isolate of Microsporum gypseum MU-SH4. Microtiter plates were incubated at 35oC
for 48 h for C. neoformans. The MFCs of the active compounds were determined by the
streaking method on Sabouraud’s dextrose agar. Amphotericin B was used as a positive
inhibitory control for the yeasts.
2.6 Cell culture
Human breast adenocarcinoma cell lines, including MCF-7 (BRCA1 wild type,
estrogen receptor (ER) positive) and HCC1937 (BRCA1 mutant, triple-negative breast cancer
(TNBC)) were purchased from the American Type Culture Collections (ATCC, Rockville,
MD). MCF-7 cells were grown in Dulbecco’s modified eagle’s medium (DMEM) without
phenol red, while HCC1937 cells were grown in Roswell Park Memorial Institute 1640
medium (RPMI 1640) without phenol red. Both media were supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin-streptomycin. All cell lines were cultured at a constant
temperature of 37°C in a 5% carbon dioxide (CO2) humidified atmosphere.

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2.7 In vitro cytotoxicity assay
The cytotoxic effect of both complexes on MCF-7 and HCC1937 cells was performed
by the tetrazolium salt reduction (MTT) assay. Ten thousand cells were plated in each well
of 96-well culture plates and grown at 37°C in 5% CO2. After 24 h of seeding cells, the
medium was removed and cells were treated with different concentrations of the two
complexes. [Ru(p-cymene)(dppm)Cl2] wasdissolved in 1% DMSO at final concentrations of
0.01, 1, 5, 10,50 and 100 µM and [Ru(p-cymene)(tbp)Cl2] was dissolved in 1% DMSO at
final concentrations of 100, 200, 500, 1000 and 2000 µM. The cells were then incubated at
37°C in 5% CO2 for 48 h, after which each well was washed twice with 100 µl of phosphate
buffered saline (PBS). Then 100 µl of 0.5 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5
diphenyltetrazolium bromide was added and the plates were further incubated at 37°C in 5%
CO2 for 4 h. Subsequently, the medium was removed and 200 µl of 100% DMSO was added
to dissolve the purple formazan crystal. The absorbance of each well was determined
spectrophotometrically at 570 nm. The percentage of cell viability was calculated as follows,
% cell viability = (absorbance of the ruthenium complex treated cells)/(absorbance of the
vehicle treated cells) x 100. The inhibiting concentration of each ruthenium complex that
reduced the number of viable cells to 50% (IC50) was derived by plotting log of the
percentage cell viability versus concentration. Results were derived from four independent
experiments each performed in at least triplicate.
Data are expressed as the standard error of the mean (±S.E.M.). Statistical analysis
comparisons of the significant differences between the mean values was performed using
one-way ANOVA. A probability of 0.01 or less was deemed statistically significant. The
following notation is used throughout the manuscript: *, p<0.01, relative to the control.

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3. Results and Discussion
3.1 Synthesis and characterization
Two novel ruthenium(II) complexes [Ru(p-cymene)(dppm)Cl2] and [Ru(p-
cymene)(tbp)Cl2] were synthesized by the reaction of [RuCl((p-cymene))(µ-Cl)]2 with the P
and N donors of 1,1-bis(diphenylphosphino) methane and tert-butyl pyridine in
tetrahydrofuran, respectively (scheme I).
<< Scheme I >>
The elemental analysis data of both complexes corresponded to the theoretically
calculated values. The differences of C, H and N (in [Ru(tbp)(p-cymene)Cl2] complex)
percentages between the calculated and experimental values deviate within 0.04-0.6 %. The
FTIR spectra displayed some characteristic peaks in the 1600-700 cm-1 region (see
supplementary data, S1). The [Ru(p-cymene)(dppm)Cl2] complex exhibited the stretching
modes of P-C(Phenyl) at 520 cm-1 and 490 cm-1 corresponding to the frequencies reported by
Jensen and Nielsen, 1963 [23]. P-C(alkyl) stretching frequencies appeared in the range of
700-1100 cm-1. The [Ru(p-cymene)(tbp)Cl2] complex showed the stretching modes of C=C
and C=N in the pyridine ring in the region of 1420-1620 cm-1. Importantly, these peaks do
not exist in the FTIR spectrum of the starting material, [RuCl(p-cymene)(µ-Cl)]2, which is
evidence that the functional ligand coordinated with the Ru(II) center. The vibrational
frequencies of C=C and C=N were compared with the free 4-tert-butylpyridine which are in
the range of 1500-1700 cm-1. A shift of ca. 100 cm-1 was observed. This red shifting
frequency may be a result of the decrease of C=C and C=N bond order caused by π-
backbonding from the d-orbital of Ru(II) to the π* orbital of the pyridine moiety. Likewise,
the C-P stretching mode of [Ru(p-cymene)(dppm)Cl2] is different from its free dppm ligand
(1000 cm-1 [24]) for almost 500 cm-1.

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1
For H-NMR spectra (see supplementary data, S2) of both complexes measured in
CDCl3, the prospective resonances are detected for the (p-cymene) and the functional ligands
of dppm and tbp. In consequence of the coordination of the functional ligands, downfield
shifts of 0.15-0.25 ppm of the ligand ring protons are noticed in comparison with the free
ligands. Likewise, downfield shifts were also found for the coordinated (p-cymene) in both
complexes compared to the p-cymene ligand in RuCl(p-cymene)(µ-Cl)]2 complex. The
chemical shifts are presented in the experimental section. The 13C-NMR spectra (see
supplementary data, S3) of [Ru(p-cymene)(tbp)Cl2] are in good agreement with the resonance
signals of its structure.
The structure of the [Ru(p-cymene)(dppm)Cl2] complex was determined by single
crystal x-ray diffraction. Its molecular structure with atom numbering is displayed in Figure
1, selected bond lengths and angles are given in Table 1. The crystal structure of [Ru(p-
cymene)(dppm)Cl2] is a triclinic system with a P-1 space group. The mononuclear complex
of Ru(II) is in four coordinations with π conjugated carbons in cymene, and in dppm through
one of the phosphorus atoms and two choro ligands show the piano-stool distorted psuedo-
tetrahedral geometry. The Ru-C(p-cymene) lengths are between 2.161(4)and 2.235(3) Å; the
average distance between Ru(II) and the centroid of the p-cymene ring is 1.6941(16) Å: and
the average length of Ru-Cl is 2.4095(9) Å. All these measurements are similar to the
relevant complexes in [25-26]. The length of Ru-P is 2.350(8) Å which is also close to the
other compounds [27-28]. The bond angles around Ru(II) are in the range of 82.45(3)o to
160.65(10)o. The largest angle can be observed in the C(2)-Ru-P(1). It is probably due to the
steric bulk of the phosphinomethane groups. In the molecular structure, intramolecular π-π
stacking is observed of the two opposed phenyl rings in the dppm ligand. The centroid-
centroid distance is 3.955(3) Å as shown in Figure 2. In addition, there is intermolecular π-π
stacking of two dppm phenyl rings and π-π stacking between the cymene and dppm phenyl

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rings of two alternate adjacent molecules (Figure 2). This stabilizes the crystal packing with a
centroid-centroid (Cg5---Cg5) distance of 4.328(3) Å. The π-π stacking between the cymene
ring (Cg1) and the phenyl ring (Cg2) of dppm, Cg1----Cg2 stabilizes at 4.460(2) Å.
In addition, the intermolecular contacts in the packing were studied by Hirshfeld
surface analysis. The Crystal Explorer program (Wolff et al., 2012) [31] was used to generate
Hirshfeld surfaces mapped over dnorm. The mapping of dnorm was used to analyze the
intermolecular contact distances, di and de , from the Hirshfeld surfaces between the nearest
atom inside and outside molecules, respectively. Hirshfeld surfaces mapped over dnorm
,
shown in Figure 3, reveal a pair of hydrogen-bonds representing acceptors on the surfaces
and they are shown as bright-red spots at Cl1 of C2-H2---Cl1(#1) and at Cl2 of C3-H3---
Cl2(#1) with distances of 3.725(4) and 3.529(4) Å, respectively (for symmetry operation #1 :
-x,1-y,1-z). Two-dimensional fingerprint plots (Rohl et al., 2008) [32] are shown in Figure 4
as the combination of de and di and provide a summary of intermolecular contacts in the
crystal. The overall two-dimensional fingerprint plot is depicted in Fig. 4a, and those for the
contacts of H---H , H---Cl/Cl---H, C---H/H---C are shown in Fig 4b-d. The greatest
contribution to the overall Hirshfeld surface, i.e. 72.8%, is provided by H---H contacts in
crystal packing. The contribution of 9.9 % from the H---Cl/Cl---H contacts corresponds to the
C—H---Cl interactions, which are represented by a pair of asymmetric spikes at de + di ca 3.2
A ° (Fig. 4c). The asymmetrical peaks of the delineated finger print plot of Figure 4d,
indicate C---H/H---C contacts with 14.3%, de + di ca 3.6 Å, representing π-π stacking
interactions in crystal packing.
<< Table 1 >>
<<Figure 1>>
<<Figure 2>>

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<<Figure 3>>
<<Figure 4>>
3.2 Absorption
The absorption spectra of the [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2]
complexes (Figure 5) in chloroform were measured in the range of 200-800 nm. The
absorption bands of [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2] complexes in the
visible region appear at the maximum wavelengths of absorption at 397 nm and 420 nm,
respectively, providing low molar extinction coefficients (< 1,700 M-1cm-1) which are
ascribed to d-d transition of Ru(II). In contrast, π-π* transition with high molar extinction
coefficients (> 10,000 M-1cm-1) is to be found in the non-visible UV region.
<<Figure 5>>
3.3 Antimicrobial activity of the [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2]
complexes
Using the agar microdilution method, we tested the antimicrobial activity of the two
studied compounds against three types of bacteria, namely S. aureus (SA), methicillin -
resistant S. aureus (MRSA) and E. coli ATCC25922 (EC). Growth inhibition was compared
with that of the antibacterial drugs, vancomycin and gentamicin. In addition, we measured the
antifungal activity of the complexes against one type of yeast (C. neoformans ATCC 90113).
A comparison of these results with those produced by the standard antifungal drug
amphotericin B is presented in Table 2. No activity was found from checking against each
tested organism.

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The [Ru(p-cymene)(dppm)Cl2] complex shows antibacterial activities at
concentrations < 32
µ
g. mL-1. However, the [Ru(p-cymene)(tbp)Cl2] complex does not
exhibit such activities in the studied system. The results imply that the dppm ligand may have
a strong influence on the bacterial growth inhibition mechanism not shown by the free
ligand. The [Ru(p-cymene)(dppm)Cl2] complex significantly inhibited Staphylococcus
aureus ATCC25923, MRSA = methicillin - resistant Staphylococcus aureus with MIC/MBC
values of 8/200 and 32/128 µ
g. mL-1, respectively. The variation in the antimicrobial activity
of the free ligand and the different metal complexes against the different microorganisms is
due either to the differences in the ribosomes in the microbial cells or the impermeability of
the microbe cells. It is worth noting that chelation is able to increase the ability of the
complexes to permeate the microorganism cell membranes by decreasing the polarizability of
the metal, as explained by Tweedy’s chelation theory [23]. The [Ru(p-cymene)(dppm)Cl2]
has a more lipophilic structure than that of the [Ru(p-cymene)(tbp)Cl2] due to the extra
phenyl rings in the diphosphinomethane group. Penetration through the cell walls of bacteria
is, therefore, much more possible than it is with the [Ru(p-cymene)(tbp)Cl2] complex,
leading to greater inhibition of bacterial growth.
The data obtained from the experiments suggested that the [Ru(p-cymene)(dppm)Cl2]
compound exhibits mild to good antifungal activity. Interestingly, the compound was more
effective against bacteria than against fungi.
<< Table 2 >>
3.4 Anticancer activity
The antiproliferative property of the new ruthenium(II)arene complexes, [Ru(p-
cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2] were tested in two different human breast
cancer cells using the MTT assay. The percentage of cell viability was assessed as shown in

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Figure 4 and 5. As can be seen in Figure 6, for each type of breast cancer cell, the observed
cell growth inhibitory effect of [Ru(p-cymene)(dppm)Cl2] varied at similar concentrations.
The same results for the [Ru(p-cymene)(tbp)Cl2] complex show clear differences at
concentrations from 100 µM to 1000 µM, and no variation at concentrations from 1000 µM
to 2000 µM. Representative results showed that the percentage cell viability of both breast
cancer cells decreased as concentrations of
[Ru(p-cymene)(dppm)Cl2] and [Ru(p-
cymene)(tbp)Cl2] increased. The cytotoxic activities of the ruthenium complexes, compared
to cisplatin, were determined as the IC50 values and are summarized in Table 3. Both
ruthenium complexes can inhibit breast cancer cell growth, but with different cellular
responses. Interestingly, [Ru(p-cymene)(dppm)Cl2] exhibited significantly greater
cytotoxicity than [Ru(p-cymene)(tbp)Cl2] against cells of both cisplatin-resistant MCF-7 and
cisplatin-sensitive, BRCA1-defective HCC1937 .
A feature of the antiproliferative activity studies was tested as chemotherapeutic agents
candidates for both cisplatin-resistant, BRCA1-competent MCF-7 and cisplatin-sensitive,
BRCA1-deficient, triple-negative HCC1937 cells by the two ruthenium(II) arene complexes
with different ligands, [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(tbp)Cl2] as shown in
Figure 6, 7 and Table 3. Both ruthenium complexes exerted cytotoxicity against both breast
cancer cells in a concentration-dependent manner. It was of interest that the cytotoxicity of
[Ru(p-cymene)(dppm)Cl2]
was clearly greater than that of cisplatin or [Ru(p-
cymene)(tbp)Cl2] against all breast cancer cells. Both MCF-7 and HCC1937 cells were
16times more sensitive to the [Ru(p-cymene)(dppm)Cl2] than to cisplatin. Compared to
cisplatin, [Ru(p-cymene)(tbp)Cl2] was less cytotoxic to the same cells by factors of 15 and 16
respectively. Compared to [Ru(p-cymene)(dppm)Cl2], it was respectively 247 and 275times
less cytotoxic. The greater cytotoxicity of [Ru(p-cymene)(dppm)Cl2] may be attributed to the
larger size and surface area of its structure and the effect that has on the activity of the

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coordinated diphenylphosphino ligand. The hydrophobicity of the complex, and its π-
extended system could also be associated with its superior uptake into breast cancer cells
[24.] These results agree very well with a previous study which showed that ruthenium(II)
complexes containing 1,1′-bis(diphenylphosphino) ferrocene (dppf) exerted an enhanced
anticancer activity against S-180 murine ascetic sarcoma 180, DU145 human prostate
carcinoma, K562 chronic myeloid leukemia and A549 human lung carcinoma [25]. It was
also interesting that HCC1937, known to be a BRCA1-defective (5382insC mutation) cell
line lacking an estrogen receptor (ER), was significantly more sensitive than the BRCA1-
competent MCF-7 cell line. Ruthenium sensitivity in the BRCA1-mutated cells might be
related to dysfunctional BRCA1 that is unable to repair DNA damage induced by [Ru(p-
cymene)(dppm)Cl2] treatment, ultimately leading to breast cancer cell death [26-29].
However, the precise molecular mechanisms of action of this ruthenium(II)-arene complex
remain largely unexplored and are of great interest for further investigation. Our results are
the first evidence of the anticancer activity of [Ru(p-cymene)(dppm)Cl2] against both
cisplatin-resistant and BRCA1-defective breast cancer cells. Therefore, the ruthenium(II)
arene complex containing diphenylphosphino ligand could be a promising therapeutic
ruthenium-based agent for breast cancers.
<< Table 3 >>
<<Figure 6>>
<<Figure 7>>

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4. Conclusion
The structures of half sandwich [Ru(p-cymene)(dppm)Cl2] and [Ru(p-cymene)(dppm)Cl2]
complexes are pseudo-tetrahedral distorted geometry. The [Ru(p-cymene)(dppm)Cl2]
complex presents as a promising powerful anticancer against MCF-7 and HCC1937 breast
cell lines with a lower IC50 than that of cisplatin, while [Ru(p-cymene)(tbp)Cl2] shows much
lower activity. It is favored by the diphosphine ligand more than the pyridine moiety.
Although the mechanism of the inhibition of growth in cancer cells is not yet well
understood, the binding of synthesized ruthenium complexes to DNA cancer cells is the main
reason for their anticancer effect. Chloro ligands are labile which can cause further hydrolysis
and allow Ru(II) to attach to base pairs of DNA cancer cells. The [Ru(p-cymene)(dppm)Cl2]
exhibits moderate to good activity against SA and MRSA bacteria but only weak inhibition of
CN yeast growth.
Acknowledgement
NL acknowledges financial support from the Prince of Songkla University under contract
number (SCI560355S) as well as Center for Innovation in Chemistry (PERCH–CIC), the
Commission on Higher Education and the Ministry of Education and Faculty of Science,
PSU. AR thanks the National Research Council of Thailand and Prince of Songkla University
(PHA570058S, PHA580500S and PHA580926S) for financial support. We would also like to
thank Mr.Thomas Duncan Coyne for assistance with the English.
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Table 1 Selected bond lengths (Å) and bond angles (o) of [Ru(p-cymene)(dppm)Cl2] complex
Ru(1)-P(1)
2.3500(8)
1.6941(16)
2.4150(9)
2.4040(9)
1.825(3)
1.823(4)
87.85(3)
88.78(3)
Ru(1)-ring centroid
Ru(1)-Cl(1)
Ru(1)-Cl(2)
P(1)-C(11)
P(1)-C(17)
P(1)-Ru(1)-Cl(1)
P(1)-Ru(1)-Cl(2)
Table 2. Antimicrobial activity levels of [Ru(p-cymene)(dppm)Cl2] and [Ru(p-
cymene)(tbp)Cl2] complexes and starting materials in dmso.
Bacteria (µg/mL)
Yeast (µg/mL)
Compounds
SA
MBC
MRSA
EC
MBC
CN90113
MIC
MIC
MBC
MIC
MIC
MFC
[Ru(dppm)(pcymene)Cl2]
[Ru(tbp)(p-cymene)Cl2]
[[RuCl((p-cymene))(µ-
Cl)]2]
8
200
NA
NA
32
128
NA
NA
NA
NA
NA
NA
NA
NA
64
128
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.5
-
NA
NA
1
NA
NA
1
NA
NA
2
NA
NA
-
NA
NA
NA
-
NA
NA
-
dppm
NA
tbp
-
-
-
Vancomycin
-
1
1
-
0.5
-
-
-
Gentamicin
-
-
-
0.25
0.5
Amphotericin B
SA = Staphylococcus aureus ATCC25923 , MRSA = methicillin - resistant Staphylococcus aureus, Escherichia coli
ATCC25922, CN90113 = Cryptococcus neoformans ATCC90113 flucytosine - resistant MIC = minimum inhibitory
concentration (µg/mL) , MBC= minimum bactericidal concentration (µg/mL) , MFC= minimum fungicidal concentration
(µg/mL), NA = non active

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Table 3. IC50 mean values (µM) for [Ru(p-cymene)(dppm)Cl2], [Ru(p-cymene)(tbp)Cl2], and
cisplatin against MCF-7 and HCC1937 cells after 48 h of treatment. (All data are the mean
and standard errors obtained from four independent experiments, each performed in at least
triplicate)
IC50 (µM)
Metal complexes
MCF-7
HCC1937
23.4 ± 7 *,**
1.4 ± 0.3 *,**
385.1 ± 5.3 *,**
Cisplatin[29]
42.2 ± 8 *,**
2.6 ± 0.2 *,**
[Ru(p-cymene)(dppm)Cl2]
[Ru(p-cymene)(tbp)Cl2]
642.6 ± 6.6 *,**
*
Statistical significance differences are indicated by p<0.01, compared to the IC50 values of
the same complex on cell lines, and **p<0.001, compared to the IC50 values of the complexes
on each cell line.

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[RuCl(η6-p-cymene)(µ-Cl)]2
-L = bis-phenyldiphosphinomethane (dppm)
THF
-
or tert-butylpyridine (tbp)
[Ru(dppm)(p-cymene)Cl2]
[Ru(tbp)(p-cymene)Cl2]
Scheme I. Synthesis of the [Ru(dppm)(p-cymene)Cl2] and [Ru(tbp)(p-cymene)Cl2]
complexes.

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Figure 1. An ORTEP structure of [Ru(p-cymene)(dppm)Cl2] complex with atom numbering

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Figure 2. The packing interactions of [Ru(p-cymene)(dppm)Cl2] complex

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Figure 3. Hirshfeld surface analysis mapped for [Ru(p-cymene)(dppm)Cl2] complex over
d norms showing hydrogen bonds of C-H---Cl with neighboring molecules.

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Figure 4. Two-dimensional fingerprint plots of [Ru(p-cymene)(dppm)Cl2] complex:
(a) overall interactions and pictured into contributions from different contacts, (b) H---H ,
(c) H---Cl/Cl---H and (d) C---H / H---C, respectively.

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Figure 5 Normalized absorption spectra of [Ru(p-cymene)(dppm)Cl2] and
[Ru(p-cymene)(tbp)Cl2]