(η6-Arene) ruthenium(II) complexes with ferrocene-tethered salicylaldimine ligands: Synthesis, characterization and anti-cancer properties

Article abstract of DOI:10.1016/j.poly.2020.114829

A series of ruthenium(II) complexes (5a-5f) derived from ferrocenyl salicylaldimine ligands (3a-3f), coordinated in a chelating mode through the deprotonated phenolic oxygen atom and the imine nitrogen, were synthesized and fully characterised. The molecular structures of selected ligands and complexes were also confirmed by X-ray diffraction analysis. The heterobimetallic Ru-Fe complexes 5a-5f were evaluated for their in vitro anti-cancer activity against human liver cancer (HepG2) and human cervical cancer (HeLa) cell lines and showed better activities compared to the ferrocenyl salicylaldimine ligands. Among the heterobimetallic complexes, 5c and 5d showed enhanced cytotoxicity against HeLa cancer cells (IC₅₀ value of 9.34 μM compared to 31.32 μM for cisplatin), and HepG2 cancer cells (IC₅₀ value of 15.74 μM compared to 27.95 μM for cisplatin), respectively. Mechanistic studies indicated compound 5d induced cell cycle arrest in the S phase. Further, compound 5d treatment resulted in increased ROS generation and loss of mitochondrial membrane potential in HepG2 cells.

Full text of DOI:10.1016/j.poly.2020.114829

Journal Pre-proofs
( η 6-Arene) ruthenium(II) complexes with ferrocene-tethered salicylaldimine
ligands: synthesis, characterization and anti-cancer properties
Jakku Ranjith Kumar, Eda Rami Reddy, Nedaossadat Mirzadeh, T. Srinivasa
Reddy, Anil Kumar Vardhaman, L. Giribabu, Balasubramanian Sridhar, Deep
Pooja, Sistla Ramakrisna, Suresh Bhargava, Rajiv Trivedi
PII:
S0277-5387(20)30486-1
DOI:
Reference:
https://doi.org/10.1016/j.poly.2020.114829
POLY 114829
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
22 July 2020
30 September 2020
2 October 2020
Please cite this article as: J.R. Kumar, E.R. Reddy, N. Mirzadeh, T. Srinivasa Reddy, A. Kumar Vardhaman, L.
6
Giribabu, B. Sridhar, D. Pooja, S. Ramakrisna, S. Bhargava, R. Trivedi, ( η -Arene) ruthenium(II) complexes
with ferrocene-tethered salicylaldimine ligands: synthesis, characterization and anti-cancer properties,
Polyhedron (2020), doi: https://doi.org/10.1016/j.poly.2020.114829
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(6-Arene) ruthenium(II) complexes with ferrocene-
tethered salicylaldimine ligands: synthesis,
characterization and anti-cancer properties.
Jakku Ranjith Kumar,a,b,c Eda Rami Reddy,a Nedaossadat Mirzadeh,*b,c T. Srinivasa Reddy,c
Anil Kumar Vardhaman,d L. Giribabu,d Balasubramanian Sridhar,e Deep Pooja,f Sistla
Ramakrisna,f Suresh Bhargava,*b,c Rajiv Trivedi*a,b
a
Catalysis and Fine Chemicals Division, CSIR-Indian Institute of Chemical Technology,
Uppal Road, Tarnaka, Hyderabad-500007, India.
b
IICT-RMIT Centre, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka,
Hyderabad-500007, India.
c
Centre for Advanced Materials and Industrial Chemistry (CAMIC), School of Science,
RMIT University, GPO Box 2476, Melbourne, 3001, Australia.
d Polymer and Functional Materials Division, CSIR-Indian Institute of Chemical Technology,
Uppal Road, Tarnaka, Hyderabad-500007, India.
e
Analytical Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007,
India.
f
Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad
500007, India.
Correspondence to: suresh.bhargava@rmit.edu.au, nedaossadat.mirzadeh@rmit.edu.au,
trivedi@iict.res.in, trivedi.iict@gov.in
Abstract: A series of ruthenium(II) complexes (5a-5f) derived from ferrocenyl
salicylaldimine ligands (3a-3f), coordinated in a chelating mode through the deprotonated
phenolic oxygen atom and the imine nitrogen, were synthesized and fully characterised. The
molecular structures of selected ligands and complexes were also confirmed by X-ray
diffraction analysis. The heterobimetallic Ru-Fe complexes 5a-5f were evaluated for their in
vitro anti-cancer activity against human liver cancer (HepG2) and human cervical cancer
(HeLa) cell lines and showed better activities compared to the ferrocenyl salicylaldimine
ligands. Among the heterobimetallic complexes, 5c and 5d showed enhanced cytotoxicity
against HeLa cancer cells (IC50 value of 9.34 µM compared to 31.32 µM for cisplatin), and
HepG2 cancer cells (IC50 value of 15.74 µM compared to 27.95 µM for cisplatin),
respectively. Mechanistic studies indicated compound 5d induced cell cycle arrest in the S
phase. Further, compound 5d treatment resulted in increased ROS generation and loss of
mitochondrial membrane potential in HepG2 cells.
Keywords: Ferrocene, salicylaldimine ligands, Ruthenium complexes, Anti-cancer activity

1.0 Introduction
The anti-cancer properties of metal containing drugs is an extensively researched area.
Notably, the antiproliferative activity of the square planar platinum(II) complex cis-
diamminedichloroplatinum(II), [cis-Pt(NH3)2Cl2], discovered by Barnett Rosenberg in the
1960s1,2 has led to the treatment of several types of cancers, including testicular, head and
neck.3 However, the range of side effects, including oto-, neuro- and nephrotoxicities, are
severe limitations to its clinical use. In view of this aspect, other transition metal-based
drugs have been investigated as alternatives to platinum-based therapies and in recent years,
ruthenium-based drugs have shown great potential in chemotherapy. For instance,
(H2im)[RuCl4(DMSO)(Him)] (NAMI-A, Him = 1H-imidazole) and, Na[RuCl4(Hind)2]
(NKP-1339, Hind = 1H-indazole) are among the most prominent ruthenium complexes that
have entered various stages of clinical trials.4 Furthermore, the half-sandwich arene
ruthenium complexes, RAPTA5 and RAED6 compounds, have been the most extensively
investigated organoruthenium complexes (Figure 1). According to Sadler and co-workers,
the electronic properties of the Ru(II) complexes are significantly affected by the change of
donor ligand.7 This change in ligand had direct consequence on the nucleobase to be
targeted. Subsequent studies were performed to establish the structure-activity relationships
of Ru(II) complexes with various chelating N,N-(diamines and bipyridine), N,O-(amino
acids) and O,O-(acetylacetonate) ligands.8 It has been shown that the presence of two non-
leaving cis-coordinated amine ligands was crucial for the anti-cancer properties of platinum
complexes.9 Numerous metal complexes, including ruthenium compounds, containing
aromatic N-donor ligands (phenanthroline, pyridine, and imidazole) have exhibited
promising anti-cancer properties.10 Free N-H moieties in the ligand of Ru(II) complexes
often facilitates an effective and strong interaction with DNA via hydrogen bonding
interactions.11
Figure 1. Examples of ruthenium-based bioactive complexes.

By introducing a second metal into potential anti-cancer agents, a new variant of the
metallodrugs can be examined.12 One of the most abundant metals, iron, as its organometallic
complex ferrocene, has gained much attention in the field of medicinal chemistry.13
Heterobimetallic complexes containing a ferrocenyl ligand exhibit different electrochemical
and biological properties compared to those observed for the free ligand. Furthermore, there
are examples of ferrocenyl motifs incorporated into heterobimetallic assemblies in which the
ferrocene fragment, a chemically robust moiety having low toxicity, acts as a one-electron
reservoir.14 The ferrocene moiety is stable in both aqueous and aerobic media, and its
favorable electrochemical properties make it a prominent molecule for use in biological
applications.13 The chemotherapeutic application of the ferrocene moiety is associated with its
electrochemical activation wherein the oxidation of iron atom produces the ferrocenium (Fc+)
species, which can assist in producing reactive oxygen species (ROS).15 Similar redox
processes are known to play pivotal roles in many metal-containing therapeutics.16 In an
attempt to design and develop heterobimetallic cytotoxic anti-cancer agents, different metal
complexes containing gold, silver, palladium, rhodium and iridium17 have been coordinated
with ferrocenyl-containing ligands to achieve a synergistic effect between the two active
metals. In addition, examples where ruthenium and iron have been combined within the same
molecule and their anti-cancer activity investigated have been reported (Figure 2).14
Figure 2. Structures of ferrocenyl-ruthenium anti-cancer complexes.
Recent work by Štěpnička and co-workers have shown that di- and tri-nuclear
heterobimetallic complexes containing ferrocenyl and arene-ruthenium fragments exhibited
moderate cytotoxic activities against cisplatin-sensitive (A2780) and cisplatin-resistant
(A2780cisR) human ovarian cancer cell lines,18 and Ramadevi and co-workers have
investigated ruthenium-arene complexes containing N-ferrocenyl amino acids against
MCF7 cell line.19 There are also a number of reports on ferrocene-free Schiff base
complexes containing the (p-cymene)Ru(II) moiety that exhibit a range of in vitro
antiproliferative activities against cell lines such as, human metastatic breast carcinoma
MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, hepatocellular carcinoma Hep
G2 and human lung cancer A549.20 As an extension of this fascinating chemistry to deliver
novel Ru-based drugs, we have designed and prepared the (6-arene)ruthenium(II) half-
sandwich complexes 5a-5f. Synthesized from the reaction of ferrocenyl salicylaldimine
ligands with [Ru(p-cymene)Cl2]2, these heterobimetallic complexes adopt "piano-stool"
geometry which offers structural stability and the opportunity for tuning the electronic
properties at the ruthenium center. Herein, we report the synthesis and characterization of a
series of novel Ru(II) complexes bearing bidentate N,O-donor Schiff-base ligands, and the

in vitro anti-cancer activity against two different human cancer cell lines [HepG2 (human
liver) and HeLa (human cervical)].
2.0 Experimental
2.1 Materials and Methods
Ferrocene, 4-nitroaniline, toluene, dichloro(p-cymene)ruthenium(II) dimer, and substituted
salicylaldehydes were purchased from Sigma Aldrich and used as supplied. HepG2 human
liver carcinoma cells and HeLa human cervical cancer cells were purchased from the
American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s modified
eagle medium (DMEM), trypsin–EDTA, antibiotic/anti-mycotic solution, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Foetal bovine serum
was purchased from Gibco, USA. Cell culture plasticware was purchased from Tarson Ltd
13
(Mumbai, India). 1H and C NMR spectra were recorded at room temperature on a Bruker
Avance 400 or Varian Inova 500 spectrometer as CDCl3 solutions. Chemical shifts (δ) for
protons are reported in ppm downfield from TMS as an internal standard and the carbon
13
chemical shifts are referenced to the C signal of CDCl3 at 77.0 ppm. Coupling constants
(J) are expressed in Hz. MALDI-TOF mass spectra were recorded on a Shimadzu Biotech
Axima spectrometer. For MALDI-TOF and ESI mass spectra, m/z values are expressed in
atomic mass units. FT-IR spectra were recorded on a Thermo Nicolet Nexus 670
spectrometer as KBr discs. Melting points were determined using a Toshniwal apparatus
and are uncorrected. The UV-vis. spectra were recorded on a UV 3600 Shimadzu
spectrophotometer over the range of 200-550 nm as CH3CN solution. Cyclic voltammetry
(CV) experiments were performed using a conventional three-electrode configuration
consisting of a glassy carbon working electrode, a platinum wire auxiliary electrode, and a
saturated calomel (SCE) reference electrode. The cyclic voltammograms were recorded on a
CHI620 model electrochemical analyzer in the presence of 0.1 M tetrabutylammonium
perchlorate (TBAP) supporting electrolyte at a scan rate of 0.1 Vs-1. Elemental analyses
were performed with an Elementary Vario MICRO analyzer. All reactions were carried out
in the absence of air using standard Schlenk techniques unless stated otherwise. Solvents
were deoxygenated, purified and dried prior to use.
2.2 General procedure for the synthesis of ligands 3a-3f
4-Ferrocenyl aniline (1) was synthesized in two steps by a reported method.23 Under a
nitrogen atmosphere, a two-neck round bottom flask equipped with magnetic stirrer was
charged with the appropriate salicylaldehyde 2a-2f (1 mmol) followed by a solution of 4-
ferrocenyl aniline 4-nitrophenyl ferrocene (277 mg, 1 mmol) in dry ethanol (10 mL). p-
Toluene sulfonic acid (3-4 mg) was added then the reaction mixture to stir for 4 hours at
room temperature and the precipitated solid was collected by suction filtration, washed with
cold dichloromethane and dried under vacuum. The product was purified by
recrystallization from dichloromethane/hexane.
Ligand 3a
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and 4-nitrosalicylaldehyde (2a) (167.12
1
mg, 1 mmol) gave 3a as an orange red crystalline solid, yield: 80%. H NMR (500 MHz,
CDCl3): δ (ppm)= 14.63 (s, 1H, -OH), 8.77 (s, 1H, CH=N), 8.40 (d, J = 2.7 Hz, 1H, ArH),
8.30 – 8.24 (m, 1H, ArH), 7.58 – 7.53 (m, 2H, ArH), 7.30 – 7.27 (m, 2H, ArH), 7.10 (d, J =
9.2 Hz, 1H, ArH), 4.71 (s, 2H, C5H4), 4.40 (s, 2H, C5H4), 4.11 (s, 5H, C5H5). 13C NMR (101
MHz, CDCl3): δ (ppm) = 161.59, 157.21, 147.54, 134.03, 128.08, 126.96, 121.65, 118.21,
115.61, 84.38, 69.73, 69.31, 66.58, 18.36. ESI: m/z 427 [M+H ]+

Ligand 3b
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and salicylaldehyde (2b) (122 mg, 1
mmol) gave (3b)as an orange red crystalline solid, yield: 82%.1H NMR (400 MHz, CDCl3):
δ 13.32 (s, 1H, -OH), 8.62 (s, 1H, CH=N), 7.47 – 7.44 (m, 2H, ArH), 7.32 (dd, J = 15.4, 8.1
Hz, 2H, ArH), 7.18 – 7.14 (m, 2H, ArH), 6.96 (d, J = 8.1 Hz, 1H, ArH), 6.87 (d, J = 7.5 Hz,
1H, ArH), 4.61 – 4.59 (m, 2H, C5H4), 4.29 – 4.27 (m, 2H, C5H4), 4.00 (s, 5H, C5H5).13C
NMR (101 MHz, CDCl3): δ 161.4, 161.2, 146.1, 138.7, 133.0, 132.2, 126.9, 121.3, 119.4,
119.1, 117.3, 84.5, 69.7, 69.2, 66.5. ESI-MS: m/z 382 [M+H]+
.
Ligand 3c
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and 4-diethylaminosalicylaldehyde (2c)
1
(193.34 mg, 1 mmol) gave 3c as an orange red crystalline solid, yield: 86%. H NMR (400
MHz, CDCl3): δ 13.92 (s, 1H, -OH), 8.45 (s, 1H, CH=N), 7.50 – 7.45 (m, 2H, Ar-H), 7.20 –
7.14 (m, 3H), 6.28 – 6.18 (m, 2H), 4.66 (s, 2H, C5H4), 4.33 (s, 2H, C5H4), 4.06 (s, 5H,
C5H5), 3.40 (q, J = 7.1 Hz, 4H, CH2), 1.21 (t, J = 7.1 Hz, 6H, CH3). 13C NMR (101 MHz,
CDCl3): δ 164.4, 159.4, 151.8, 146.4, 136.7, 133.7, 126.9, 120.8, 109.3, 103.8, 97.9, 85.1,
69.6, 69.0, 66.4, 44.6, 12.8. ESI-MS: m/z 453.3 [M+H]+
.
Ligand 3d
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and 4-methoxysalicylaldehyde (2d)
1
(152.15 mg, 1 mmol) gave 3d as an orange red crystalline solid, yield: 84%. H NMR (300
MHz, CDCl3): δ 13.92 (s, 1H, -OH), 8.58 (s, 1H, CH=N), 7.51 (d, J = 8.5 Hz, 2H, ArH),
7.30 (s, 1H, ArH), 7.21 (d, J = 8.4 Hz, 2H, ArH), 6.57 – 6.45 (m, 2H, ArH), 4.66 (s, J = 1.8
Hz, 2H, C5H4), 4.39 (s, 2H, C5H4), 4.06 (s, 5H, C5H5), 3.86 (s, 3H, OCH3). 13C NMR (126
MHz, CDCl3): δ 164.1, 163.9, 160.3, 145.9, 137.9, 133.4, 126.9, 121.1, 113.3, 107.2, 101.2,
84.7, 69.7, 69.1, 66.5, 55.5. ESI-MS: m/z 412 [M+H]+.
Ligand 3e
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and 5-bromosalicylaldehyde (2e)
1
(201.02 mg, 1 mmol) gave 3e as an orange red crystalline solid, yield: 81%. H NMR (400
MHz, CDCl3): δ 13.36 (s, 1H, -OH), 8.63 (s, 1H, CH=N), 7.53 (d, J = 7.5 Hz, 4H, ArH),
7.23 (d, J = 8.0 Hz, 2H, ArH), 6.89 (s, 1H, ArH), 4.68 (s, 2H, C5H4), 4.37 (s, 2H, C5H4),
4.07 (s, 5H, C5H5). 13C NMR (101 MHz, CDCl3): δ 160.2, 159.7, 145.3, 139.2, 135.5, 134.1,
127.0, 121.3, 120.8, 119.3, 110.5, 84.3, 69.7, 69.3, 66.6. ESI-MS: m/z 460 [M+H]+
.
Ligand 3f
Reaction of 4-ferrocenyl aniline (277 mg, 1 mmol) and 2-hydroxy-1-naphthaldehyde (2f)
1
(172.05 mg, 1 mmol) gave 3f as an orange red crystalline solid, yield: 87%. H NMR (400
MHz, CDCl3): δ 15.63 (s, 1H, -OH), 9.36 (d, J = 6.1 Hz, 2H), 8.11 (d, J = 8.4 Hz, 1H,
CH=N), 7.80 (d, J = 9.2 Hz, 1H, ArH), 7.72 (d, J = 8.0 Hz, 1H, ArH), 7.58 – 7.52 (m, 3H,
ArH), 7.37 – 7.29 (m, 3H, ArH), 7.09 (d, J = 9.2 Hz, 1H, ArH), 4.68 (s, 2H, C5H4), 4.39 (s,
2H, C5H4), 4.10 (s, 5H, C5H5). 13C NMR (101 MHz, CDCl3): δ 170.7, 153.5, 142.7, 138.2,
136.6, 133.3, 129.4, 128.1, 127.3, 127.2, 123.5, 122.5, 120.3, 118.9, 108.9, 84.4, 69.7, 69.3,
66.5. ESI-MS: m/z 432 [M+H]+
.
2.4 General procedure for the synthesis of complexes 5a-5f
Under a nitrogen atmosphere, a solution of 3a-3f in dry ethanol (25 mL) was added to a
two-neck round bottom flask equipped with magnetic stirrer and reflux condenser.
Triethylamine was added dropwise to the solution, which was stirred at room temperature
for 30 min. A solution of [Ru(p-cymene)Cl2]2 in dry ethanol (5 mL) was added to the
mixture and the reaction was heated to 70°C and stirred for 5 hours. The precipitated crude
product and was isolated by filtration and purified by recrystallization from
dichloromethane/hexane.
Complex 5a

Triethylamine (0.038 mL, 0.352 mmol) and ligand 3a (150 mg, 0.352 mmol) were reacted
with [Ru(p-cymene)Cl2]2 (107.7 mg, 0.176 mmol). The product 5a was isolated as a black
1
crystalline solid, yield: 78%. H NMR (400 MHz, CDCl3): δ 8.10 – 8.03 (m, 6H), 7.89 (s,
1H, CH=N), 7.58 – 7.50 (m, 12H), 6.95 (d, J = 9.4 Hz, 3H), 5.41 (d, J = 5.7 Hz, 3H), 5.33
(d, J = 5.5 Hz, 3H), 5.08 (d, J = 5.5 Hz, 3H), 4.73 – 4.69 (m, 6H), 4.42 – 4.39 (m, 6H), 4.31
(d, J = 5.9 Hz, 3H), 4.10 – 4.03 (s, 5H, Cp-H), 2.71 – 2.61 (m, 1H, 30-CH(η6-p-cymene)),
2.17 (s, 3H, -CH3 (η6-p-cymene)), 1.19 (dd, J = 19.0, 6.9 Hz, 6H, CMe2 (η6-p-cymene) ). 13C
NMR (101 MHz, CDCl3): δ 170.3, 163.5, 155.7, 139.4, 135.9, 133.5, 129.6, 126.3, 123.2,
117.6, 102.4, 98.5, 86.6, 84.1, 83.6, 83.4, 81.3, 69.8, 69.6, 66.7, 66.5, 30.5, 22.8, 21.7, 18.6.
Anal. Calcd. for C33H31ClFeN2O3Ru (696.04): C 56.95, H 4.49, N 4.03%. Found: C 56.76,
H 4.22, N 3.88%. MALDI-TOF MS: m/z 696.22 [M]+
.
Complex 5b
Triethylamine (0.056 mL, 0.524 mmol) and 3b (200 mg, 0.524 mmol) were reacted with
[Ru(p-cymene)Cl2]2 (160 mg, 0.262 mmol). The product was isolated as an orange
crystalline solid, yield: 83%. H NMR (400 MHz, CDCl3): δ 7.80 (s, 2H), 7.56 (dt, J = 18.9,
5.3 Hz, 8H), 7.26 – 7.19 (m, 2H), 7.02 – 6.95 (m, 4H), 6.46 – 6.41 (m, 2H), 5.35 (d, J = 6.1
Hz, 2H), 5.26 (d, J = 6.1 Hz, 2H), 5.04 (d, J = 5.7 Hz, 2H), 4.73 – 4.68 (m, 4H), 4.40 – 4.36
(m, 4H), 4.31 (d, J = 5.5 Hz, 2H), 4.09 (s, 5H), 2.64 (dq, J = 13.2, 6.6 Hz, 2H), 2.14 (s, 6H),
1.16 (dd, J = 19.8, 6.9 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 165.1, 163.9, 156.5, 138.4,
135.5, 135.3, 126.1, 123.8, 122.7, 118.3, 114.3, 101.5, 98.0, 86.4, 84.2, 83.6, 83.5, 80.6,
69.7, 69.4, 66.6, 66.5, 30.4, 22.9, 21.7, 18.6. Anal. Calcd. for C33H32ClFeNORu (650.08): C
60.95, H 4.95, N 2.15%. Found: C 60.31, H 4.93, N 2.71%. MALDI-TOF MS: m/z 616.26
[M-Cl]+
.
Complex 5c
Triethylamine (0.035 mL, 0.331 mmol) and 3c (150 mg, 0.331 mmol) were reacted with
[Ru(p-cymene)Cl2]2 (101.57 mg, 0.165 mmol). The product was isolated as an orange red
crystalline solid, yield: 85%. 1H NMR (400 MHz, CDCl3): δ 7.58 – 7.46 (m, 16H), 6.78 (d,
J = 8.9 Hz, 3H), 6.22 (d, J = 2.1 Hz, 3H), 5.96 (dd, J = 8.9, 2.3 Hz, 3H), 5.31 (d, J = 6.0 Hz,
3H), 5.22 (d, J = 6.0 Hz, 3H), 5.01 (d, J = 5.4 Hz, 3H), 4.71 – 4.67 (m, 6H), 4.38 – 4.34 (m,
6H), 4.29 (d, J = 5.6 Hz, 3H), 4.10 – 4.05 (m, 14H), 3.39 – 3.29 (m, 12H), 2.66 (dt, J =
13.8, 6.9 Hz, 3H), 2.16 (s, 9H), 1.62 (s, 30H). 13C NMR (101 MHz, CDCl3): δ 160.7, 136.8,
126.0, 124.4, 102.4, 100.6, 100.3, 97.6, 86.3, 82.9, 80.6, 69.7, 69.2, 66.5, 44.4, 30.4, 22.9,
21.9, 18.6, 13.0. Anal. Calcd. for C37H41ClFeNO2Ru (722.13): C 61.54, H 5.72, N 3.88%.
Found: C 60.85, H 5.52, N 3.50%. MALDI-TOF MS: m/z 687.32 [M-Cl]+
.
Complex 5d
Triethylamine (0.052 mL, 0.486 mmol) and 3d (200 mg, 0.486 mmol) were reacted with
[Ru(p-cymene)Cl2]2 (148.8 mg, 0.243 mmol). The product was isolated as an orange
1
crystalline solid, yield: 83%. H NMR (400 MHz, CDCl3): δ 7.67 (s, 2H), 7.58 – 7.52 (m,
7H), 6.86 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 2.0 Hz, 2H), 6.09 (dd, J = 8.7, 2.2 Hz, 2H), 5.29
(dd, J = 30.6, 6.1 Hz, 4H), 5.01 (d, J = 5.7 Hz, 2H), 4.70 (s, 4H), 4.37 (s, 4H), 4.28 (d, J =
5.6 Hz, 2H), 4.07 (s, 9H), 3.78 (s, 6H), 3.09 (q, J = 7.3 Hz, 4H), 2.66 (dt, J = 13.8, 6.8 Hz,
2H), 2.16 (s, 6H), 1.17 (dd, J = 17.3, 6.9 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 167.1,
166.1, 162.1, 156.8, 137.9, 136.5, 126.1, 124.0, 112.7, 105.8, 103.1, 101.0, 98.0, 86.5, 84.3,
83.3, 83.2, 80.8, 69.7, 69.3, 66.5, 66.4, 55.2, 45.9, 30.4, 22.9, 21.7, 18.6, 8.7. Anal. Calcd.
for C34H34ClFeNO2Ru (681.01): C 59.96, H 5.03, N 2.06%. Found: C 59.47, H 5.24, N
2.17%. MALDI-TOF MS: m/z 646.22 [M-Cl]+
.
Complex 5e
Triethylamine (0.035 mL, 0.325 mmol) and 3e (150 mg, 0.325 mmol) were reacted with
[Ru(p-cymene)Cl2]2 (99.73 mg, 0.162 mmol). The product was isolated as an orange-red
crystalline solid, yield: 79%. 1H NMR (500 MHz, CDCl3): δ 7.72 (s, 1H), 7.54 (s, 4H), 7.26

– 7.21 (m, 1H), 7.08 (d, J = 2.6 Hz, 1H), 6.89 (d, J = 9.2 Hz, 1H), 5.35 (d, J = 6.1 Hz, 1H),
5.27 (d, J = 6.1 Hz, 1H), 5.02 (d, J = 5.7 Hz, 1H), 4.72 – 4.68 (m, 2H), 4.41 – 4.37 (m, 2H),
4.27 (d, J = 5.7 Hz, 1H), 4.07 (d, J = 6.8 Hz, 5H), 3.10 (d, J = 7.1 Hz, 1H), 2.64 (sept, J =
6.9 Hz, 1H), 2.15 (s, 3H), 1.17 (dd, J = 24.9, 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3): δ
164.2, 162.8, 156.2, 138.8, 138.0, 136.5, 126.1, 124.7, 123.5, 119.8, 104.9, 101.6, 98.2,
86.6, 88.0, 83.7, 83.5, 80.8, 69.8, 69.5, 66.6, 66.5, 30.4, 29.7, 22.9, 21.7, 18.6. Anal. Calcd.
for C33H31BrClFeNORu (728.97): C 54.30, H 4.28, N 1.92%. Found: C 54.35, H 4.63, N
2.00%. MALDI-TOF MS: m/z 694.13[M-Cl]+
.
Complex 5f
Triethylamine (0.040 mL, 0.371 mmol) and 3f (160 mg, 0.371 mmol) were reacted with
[Ru(p-cymene)Cl2]2 (113.6 mg, 0.185 mmol). The product was isolated as a maroon
crystalline solid, yield: 80%. 1H NMR (500 MHz, CDCl3): δ 8.64 (s, 1H), 7.75 (d, J = 8.2
Hz, 1H), 7.58 (dd, J = 25.4, 7.4 Hz, 6H), 7.33 (t, J = 7.2 Hz, 1H), 7.21 – 7.11 (m, 2H), 5.35
(d, J = 38.2 Hz, 2H), 5.03 (d, J = 4.8 Hz, 1H), 4.73 (s, 2H), 4.40 (s, 2H), 4.32 (d, J = 4.7 Hz,
1H), 4.09 (d, J = 16.3 Hz, 5H), 3.72 (s, 1H), 2.71 – 2.60 (m, 1H), 2.17 (s, 3H), 1.16 (dd, J =
33.1, 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 166.1, 157.5 (d, J = 25.8 Hz), 138.0,
135.8, 134.9, 128.9, 127.3, 126.7, 126.2, 125.5, 124.3, 121.9, 118.7, 108.3, 101.4, 98.0,
86.5, 84.3, 83.8, 80.8, 69.8, 69.6 (d, J = 41.4 Hz), 66.6, 30.5, 22.9, 21.7, 18.6. Anal. Calcd.
for C37H34ClFeNORu (701.07): C 63.39, H 4.89, N 2.00%. Found: 62.94, H 4.14, N 1.82%.
MALDI-TOF MS: m/z 666.22 [M-Cl]+
.
2.5. Electrochemistry
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed using
CH instruments model 620C series, using a conventional three-electrode cell consisting of a
glassy carbon working electrode, a platinum wire auxiliary electrode and a standard calomel
electrode (SCE) as the reference electrode with a scan rate of 100 mV s-1. All the experiments
were carried out by dissolving the ligands and complexes in CH3CN (1.0 mM) containing 0.1
M [n-Bu4N]ClO4 as supporting electrolyte. All potentials are relative to the
ferrocene/ferrocenium couple. Deoxygenation of all solutions was achieved by purging with
nitrogen gas for 5 minutes prior to analysis.
2.6 X- Ray diffraction studies
Orange crystals of 3e were obtained from the slow evaporation of a dichloromethane-hexane
(9:1) solution and maroon colored crystals of 5f were obtained by the slow evaporation of a
dichloromethane-methanol (9:1) solution. Data for compounds 3e (at room temperature) and
5f (at 100 K) were collected on a Bruker D8 QUEST diffractometer with a 1 μS Mo
microsource (λ = 0.7107 Å) and a PHOTON-100 detector. The raw data frames were reduced
and corrected for absorption effects using the Bruker Apex 3 software suite of programs. The
structures were solved using the intrinsic phasing method and further refined with the
SHELXL21 program and expanded using Fourier techniques. Anisotropic displacement
parameters were included for all non-hydrogen atoms. All C bound H atoms were positioned
geometrically and treated as riding on their parent C atoms [C-H = 0.93-0.97 Å and Uiso(H) =
1.5Ueq(C) for methyl H or 1.2Ueq(C) for other H atoms]. The dichloromethane solvent
molecule of 5f could not be resolved due to extensive disorder and its assumed presence was
removed from the overall scattering by the PLATON SQUEEZE procedure. The computer
program OLEX2 was used to generate the X-ray crystal structure images.22

Crystal Data for 3e C23H18NOFeBr (M = 460.15 g/mol): monoclinic, space group P21/c (no.
14), a = 22.6635(4) Å, b = 7.3233(9) Å, c = 11.8142(2) Å, β = 102.8624(5)°, V = 1911.6(2)
Å3, Z = 4, T = 293.15 K, μ(Mo Kα) = 2.891 mm-1, Dcalc = 1.5987 g/cm3, 38785 reflections
measured (5.54 ≤ 2Θ ≤ 55.1), 4400 unique (Rint = 0.0277, Rsigma = 0.0176) which were used
in all calculations. The final R1 was 0.0449 ( 2u(I)) and wR2 was 0.1111 (all data). CCDC
1818439 contains the supplementary crystallographic data for the structure.
Crystal Data for 5f
C38H36Cl2FeNORu (M = 750.50 g/mol): monoclinic, space group P21/n (no. 14), a =
16.0789(2) Å, b = 9.75390(10) Å, c = 20.1984(2) Å, β = 101.0720(5), V = 3108.79(6) Å3, Z =
4, T = 100.09 K, μ(MoKα) = 1.158 mm-1, Dcalc = 1.603 g/cm3, 67265 reflections measured
(4.654 ≤ 2Θ ≤ 56.694 ), 7728 unique (Rint = 0.0383, Rsigma = 0.0227) which were used in all
calculations. The final R1 was 0.0332 (I > 2σ(I)) and wR2 was 0.0830 (all data). CCDC
1818440 contains the supplementary crystallographic data for the structure. These data can be
obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
2.7 In vitro anti-cancer activity
HepG2 human liver cancer cells and HeLa human cervical cancer cells were cultured in
DMEM medium supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL) and
streptomycin (0.1 mg/mL) in a CO2 incubator (5% CO2, 37 °C). The anti-cancer activity of
the compounds were assessed using the MTT assay. Stock solutions of the compounds were
prepared in DMSO (10 mM). Cells (5 × 103) were seeded in 96-well culture plates and
allowed to adhere overnight at 37 °C. The cells were then incubated with varying
concentrations (0.01-100 µM) of the compounds. The concentration of DMSO did not exceed
1% in the treatment wells. After 48 h, the media was replaced with fresh media (containing
0.5 mg/mL MTT) and the cells were further incubated for 4 h. The media was removed and
150 μL of DMSO was added to dissolve the formazan crystals. The absorbance of the
solution was measured at 570 nm using a micro-plate reader (Synergy-4, Biotek, USA).
Untreated cells were used as a negative control (100% cell viability). The percent cell
viability was calculated using the formula
% Cell viability = As/Ac × 100%,
where, As = absorbance of the sample and Ac = absorbance of the control or untreated cells,
incubated under identical conditions. The half-maximal inhibitory concentration (IC50) values
were determined using the probit analysis software package of MS-excel. Data are expressed
as the average ± the standard deviation (n = 4).
Cell Cycle analysis
HepG2 cells were seeded in a 6 well plate at cell densities of 1  105 cells/well and incubated
overnight. The cells were treated with various concentrations (10, 15 and 20 µM) of
compound 5d for 48 h. After the treatment, the cells were harvested using 0.05% trypsin-
EDTA and washed with phosphate buffered saline. Then, the cells were fixed with 70% cold
ethanol and incubated at 4 °C for 30 min. The fixed cells were washed with PBS and stained
with propidium iodide staining buffer [PI (20 µg/mL), 0.01 % Triton X] containing 200
µg/mL DNase free RNase A and incubated in the dark for 30 min. The DNA content of the
cells in each phase of the cell cycle was analyzed from 10000 events in each sample using a
BDC6-Accuri flow cytometer.
Assessment of mitochondrial membrane potential
HepG2 cells seeded in a 24 well plate were incubated with various concentrations (10, 15 and
20 µM) of compound 5d for 48 h. Rhodamine 123 solution was added to cells and incubated

for 30 min. The medium containing the drug and dye was removed and the cells were washed
with PBS. The cells were harvested and washed with PBS and the fluorescence intensity was
determined using a plate reader.
Reactive oxygen species
HepG2 cells were plated in a 24 well plate at a density of 3  104 cells/mL and incubated
overnight. The cells were then treated with different concentrations (10, 15 and 20 µM) of
compound 5d and incubated for 48 h. Carboxy-DCFHDA (5 mM) solution was added to the
cells to give a final concentration of 10 µM in each well 30 min prior to the termination of the
experiment. At the end of the experiment, the medium was replaced with fresh complete
medium and the cells were incubated for a further 30 min in a humidified atmosphere at 37
°C. Images were captured using a BIORAD microscope.
3.0 Results
3.1 Synthesis and characterization
p-Ferrocenyl aniline 1 was synthesized according to a literature method by the diazotization
of 4-nitroaniline followed by reaction with ferrocene and subsequent reduction of the nitro
group using Sn/HCl.23 Through a typical Schiff-base condensation reaction, 1 was reacted
with the aldehyde 2a-2f to afford the corresponding ferrocenyl phenyl salicylaldimine
ligands 3a-3f (Scheme 1), which were subsequently treated with [Ru(p-cymene)Cl2]2 in the
presence of triethylamine to give the desired Ru-Fe complexes 5a-5f (Scheme 2).
Scheme 1. Synthesis of ferrocenyl Schiff base ligands 3a-3f.
Scheme 2. Synthesis of hetero-bimetallic Ru-Fe complexes 5a-5f.

1
The H NMR spectra for the ligands 3a-3f and complexes 5a-5f revealed the expected
resonances for the ferrocenyl moiety. The proton signals for the unsubstituted
cyclopentadienyl ring appeared as a singlet at δ 4.28 ppm. The resonances for the protons of
the substituted cyclopentadienyl ring appeared as broadened singlets at δ 4.55 ppm and 4.78
ppm. Typically, the protons on the substituted ring of the ferrocenyl moieties that contain a
monosubstituted Cp ring appear as a doublet or triplet.24 The resonances for the aromatic
protons on the phenyl ring attached to the ferrocenyl moiety appeared around δ 6.5-7.5 ppm.
In the ligands 3a-3f, the –OH protons appeared in the range δ 13.3-15.6 and the presence of
a signal at around δ 8.23-8.86 ppm was assigned to the imine proton (Supporting
Information). The formation of the ruthenium complexes 5a-5f was indicated by the
disappearance of the phenolic –OH resonance and an upfield shift of the imine proton
13
resonance to about δ 7.31-7.74 ppm (Supporting Information). In the C NMR spectra of
the ligands and complexes, the characteristic peaks associated with the substituted and
unsubstituted cyclopentadienyl rings of the ferrocenyl moiety were observed in the range δ
69.53-71.58 ppm and at δ 69.07 ppm, respectively. The imine carbon resonances appeared
around  162-166. In the infrared spectrum, the imine stretching bands were typically
observed in the range 1580-1650 cm-1. For the complexes, the imine vibration of the
salicylaldimine moiety was observed around 1619 cm-1 while the band for the ferrocenyl
imine moiety appeared around 1585 cm-1 (Supporting Information, Figures S37-S46). In the
ESI mass spectra of 3a-3f, peaks corresponding to the [M+H]+ ions were observed. The
MALDI-TOF mass spectra of complexes 5b-5f showed peaks corresponding to the [M–Cl]+
fragments, whereas 5a showed the [M]+ parent ion, confirming the formation of the
expected ruthenium(II) complexes.
The molecular structures of the ligand 3e and the complex 5f were determined by X-ray
diffraction and are shown in Figures 3 and 4, respectively. Selected bond angles and bond
lengths for 3e and 5f are summarized in Table S4 (Supporting Information). The compounds
3e and 5f crystallized in the monoclinic P21/c and P21/n space groups, respectively. The
molecular structure of 5f reveals the orientation of the ferrocene group and ruthenium η6-p-
cymene with respect to the salicylaldimine moiety. The ruthenium atom is coordinated by
the imine nitrogen atom (N1), phenolic oxygen atom (O1), chlorine atom (Cl1) and the p-
cymene ring to adopt the typical distorted piano-stool geometry.25 The Ru-O [2.0566(16) Å]
and Ru-N [2.0767(18) Å] bond lengths are typical for Ru(II) half-sandwich complexes
containing N,O ligands.26
C13
C8
H10
C14
O1
N1
Fe1
C3
C11
C1
C9
C20
C7
C6
Br1
Figure 3. Molecular structure of 3e. Ellipsoids show 50% probability levels. The intramolecular
hydrogen bond is shown as a dashed line.

C37
C28
C11
Fe1
C21
N1
C22
O1
Ru1
Cl1
Figure 4. Molecular structure of 5f. Ellipsoids show 50% probability levels. Hydrogen atoms and
disordered CH2Cl2 solvent of crystallization have been omitted for clarity.
3.2 UV-vis. spectroscopy
The UV-vis. absorption spectra (Figure 5) of the ligands and complexes in acetonitrile
displayed bands corresponding to n–π* and π–π* transitions. The stronger and higher energy
bands were attributed to the π–π* transition of the Schiff base in 3a-3f and 5a-5f due to
symmetry allowed transitions of the phenyl rings while the less energetic bands were
assigned to n–π* transitions.27 The bands at 295 and 445 nm are typical of Fe(e2g) → Cp(e1g)
charge transfer and symmetry forbidden Fe(a1g) → Fe(e1g) transitions, respectively. The
ligands 3b, 3d and 3e exhibited absorption maxima at around 355 nm, while ligand 3a
showed a shoulder peak at 285 nm due to the strong electron withdrawing effect of the nitro
group. For ligands 3c and 3f, the absorption maxima appeared at 400 nm. The complexes 5a,
5b, 5d-5f exhibited two absorption maxima at around 295 and 445 nm. In contrast, the
spectrum for 5c showed a band at 365 nm due to the presence of the strongly electron
donating diethylamino group (Supporting Information, Figures S47-S48).
a)
b)
Figure 5. UV-vis. spectra of 0.6 10-5 M solutions of a) ligands 3a-3f and b) complexes 5a-5f in
acetonitrile at 25 °C.
3.3 Electrochemical studies

The electrochemical properties of 3a-3f and 5a-5f were studied in acetonitrile by cyclic
voltammetry (CV) and differential pulse voltammetry (DPV). The cyclic and differential
pulse voltammograms of the compounds are shown in the Supporting Information (Figures
S49 and S50, respectively). In the CV, a reversible oxidation wave at 0.4 V was observed
for 3a-3f and 5a-5f due to the [Fe(II) → Fe(III)] ferrocenyl oxidation wave, as shown in
Figure 6 for selected examples. An irreversible oxidation wave for complexes 5a-5f due to
the [Ru(II) → Ru(III)] oxidation of the ruthenium center appeared in the region of 0.75 to
0.9 V. Similarly, based on the electron donating/withdrawing substituents on the ligands,
other irreversible oxidation potentials were observed, as shown in Tables S1 and S2
(Supporting Information). According to existing literature, facile oxidation of the ferrocenyl
moiety in the complexes can produce reactive oxygen species that disrupt lipid membranes
and in turn influence the anti-tumor activity of the complexes.10 This phenomenon is in
agreement with the results found for complexes 5a-5f.
a)
b)
c)
Figure 6. Cyclic voltammograms of a) 3b, 5b, b) 3e, 5e and c) 3f, 5f in acetonitrile solution at 25 °C
with 0.1 M tetrabutylammonium perchlorate as a supporting electrolyte using a SCE reference electrode,
glassy carbon working electrode and platinum wire counter electrode.
3.4 In vitro anti-cancer activity
The antiproliferative activities of 3a-3f and 5a-5f were evaluated against two different
cancer cell lines, human cervical carcinoma (HeLa) and human liver carcinoma (HepG2)
using the MTT assay.28 The cell viability after treatment with the compounds at different
concentrations for both cell lines are shown in Figure 7 and compared to cisplatin as a
standard. The calculated IC50 are shown in Table 1. All the complexes exhibited moderate to
high cytotoxicity towards both HeLa and HepG2 cancer cell lines with low IC50 values.
With the exception of 5c, all the tested compounds showed greater sensitivity towards
HepG2 cells than HeLa cells. Enhanced cytotoxicity was observed against both cell lines for
the ruthenium-containing complexes 5a-5f compared to the ferrocenyl compounds 3a-3f.
Exceptionally, the bromo-substituted ferrocenyl salicylaldimine 3e exhibited significant
cytotoxicity (IC50, 18.29±1.47 µM) against HepG2 cells. For the HepG2 cell line,
compound 5d showed the greatest cytotoxicity (IC50, 15.74±2.63 µM) among all the
complexes, including the standard (cisplatin, IC50, 27.93±3.15 µM). Interestingly, complex
5c exhibited the most greatest cytotoxicity against HeLa cells with IC50 9.34±1.23 µM and
was 3.3 times more cytotoxic than cisplatin (IC50, 31.32±3.47 µM).

Table 1 IC50 values of different complexes against HeLa and HepG2 cell lines determined using the
MTT assay (n = 4, ± SD).
Test
Metal
IC50 (µM)a
Test
Metal
IC50 (µM)
compound
compound
HeLa
96.73±7.52
84.84±7.22
488.55±14.63
252.44±11.87
50.28±4.74
127.22 ±14.23
31.32 ±3.47
HepG2
HeLa
137.74± 8.72
29.91± 2.56
9.34 ±1.23
21.08± 1.98
25.36 ±3.89
137.81± 7.56
31.32 ±3.47
HepG2
Fe
Fe
Fe
Fe
Fe
Fe
Pt
67.73±5.32
37.73±5.33
52.08±3.65
64.96±4.85
18.29±1.47
30.58± 2.85
27.95± 3.15
Ru-Fe
Ru-Fe
Ru-Fe
Ru-Fe
Ru-Fe
Ru-Fe
Pt
43.97 ±4.42
17.88± 3.56
38.37± 4.12
15.74 ±2.63
21.90 ±2.85
30.73 ±3.96
27.95± 3.15
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
Cisplatin
Cisplatin
Figure 7. Percentage cell viability of HepG2 human liver carcinoma cells (a and b) and HeLa human
cervical cancer cells (c and d) after treatment with varying concentration of ligands 3a-3f (left) and
complexes 5a-5f (right) (mean ± SD, n = 4). Untreated cells were taken as control.
3.5 Cell cycle analysis
Considering the cytotoxicity results of complexes 5a-5f towards cervical and liver cancer
cells, compound 5d was selected to further investigate the mechanism of cell growth
inhibition in liver cancer cells. In order to investigate whether compound 5d induces cell
cycle arrest in HepG2 cells, flow cytometric analysis was carried out after 48 h of treatment
with various concentrations of the compound.29 As shown in Figure 8, compound treatment

resulted in a dose dependent increase in apoptotic cells (Sub G1 cells). The percentage of
cells undergoing apoptosis increased remarkably at 15 and 20 µM (10.4 and 15.6%)
compared to untreated cells (1.3%). Similarly, the number of cells in the S phase increased
significantly in a concentration dependent manner from 13.2 to 35.5%. These results
indicate that compound 5d induces cell cycle arrest in the Sub G1 and S phases in HepG2
cells.
Figure 8. Cell cycle analysis of HepG2 cells. A) The cells were treated with various concentrations (10,
15 and 20 µM) of the compound 5d and were stained with propidium iodide. The samples were analyzed
using flow cytometry. B) Histogram showing the percentage of cells in each phase of the cell cycle. The
results are expressed as the meanꢀ±ꢀstandard deviation of three independent experiments.
3.6 Mitochondrial membrane potential
The depolarization of mitochondrial membrane potential (ΔΨ m) is considered an early event
that occurs during apoptosis.30 Therefore, we measured the changes in mitochondrial
membrane potential (Ψm) to determine the possible mechanism of apoptosis induction by
compound 5d using rhodamine-123 (Rh123), which is a fluorescent probe specific for the
detection of alterations in ΔΨm in living cells.31 HepG2 cells incubated with various
concentrations of compound 5d were stained with Rh123 and the fluorescence intensity of
Rh123 in mitochondria was analyzed by spectrophotometry. The results, shown in Figure 9,
demonstrated that compound 5d treatment resulted in a 13.4 to 31.6% reduction in
fluorescence intensity, corresponding to a decrease in mitochondrial membrane potential. The
loss of ΔΨm was found to be 13.4, 22.5 and 31.6% at concentrations of 10, 15 and 20 µM,
respectively. These results suggest that compound 5d could induce apoptosis through
disruption of mitochondrial membrane potential in HepG2 cells.

Figure 9. Effect of compound 5d on intracellular mitochondrial membrane potential. The image shows
graphically the decrease in fluorescence intensity of mitochondrial specific dye Rh 123 with increasing
concentration of 5d compared to control. The results are expressed as the meanꢀ±ꢀstandard deviation of
three independent experiments.
3.7 Reactive oxygen species (ROS)
It has been reported that disruption of mitochondrial membrane potential leads to increased
ROS production.32 Therefore, to investigate whether this event occurred in HepG2 cells
treated with compound 5d, we used the oxidation-sensitive membrane permeable probe 2′,7′-
dichlorofluorescein diacetate (DCFH-DA).33 As shown in Figure 10, the incubation of HepG2
cells with various concentrations of compound 5d significantly increased ROS levels, as
indicated by the increased DCF fluorescence. For instance, 6- and 16-fold increased ROS
accumulation relative to the control was observed after treatment with 15 and 20 μM of
compound 5d for 48 h. These results illustrate that compound 5d treatment led to increased
intracellular ROS levels in a dose dependent manner.
Figure 10. Impact of compound 5d on intracellular ROS in HepG2 cells. A) HepG2 cells were treated
with different concentrations (10, 15 and 20 µM) of compound 5d and stained with carboxy-DCFH-DA.
The images were captured with a BIORAD microscope. B) Quantification of the green fluorescence was
carried out using software imageJ.
4.0 Conclusion
The synthesis and characterization of half-sandwich ruthenium η6-p-cymene complexes
containing ferrocenyl salicylaldimine ligands are described. All the compounds exhibited

good to moderate cytotoxicity towards HeLa and HepG2 cells. Complexes containing
strong donor groups on the salicylaldehyde ligands, such as diethylamino (NEt2) and
methoxy (OMe), displayed cell-specific activity. In particular, compounds 5c and 5d
showed promising anti-cancer activity against HeLa cancer cells (IC50 values of 9.34 and
21.08 μM, respectively) and HepG2 cancer cells (IC50 values of 38.37 and 15.74 μM,
respectively) which were found to be among the best IC50 values reported for ferrocenyl-
ruthenium complexes. Mechanistic studies indicated that compound 5d arrests the HepG2
cells in the S phase of the cell cycle. Compound 5d also induces apoptosis through
mitochondrial membrane disruption and increased ROS. These results indicate the
importance of the salicylaldehyde derived scaffolds in designing ferrocenyl-ruthenium
complexes, hence the present work describes a simple approach towards the development of
ruthenium-based imines containing ferrocenyl derivatives. Further experiments involving
the study of structural modifications and other biological studies to determine the role of
these compounds on the apoptotic and proliferative pathways in tumor lines are in progress.
Acknowledgements
This work was financially supported by the CSIR-IICT (in-house project MLP-0007) and
DST-(EMR/2016/006410). ERR acknowledges DST-SERB, India for the SERB‐NPDF
(PDF/2016/003495) and AKV acknowledges SERB-NPDF (PDF/2016/001158). JRK is
grateful to the IICT-RMIT Centre for the award of a research fellowship. We also thank the
Director, CSIR-IICT (Ms. No. 11CT/Pubs/2020/103).
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Mixed metal ruthenium-ferrocene complexes with anti-cancer properties

Highlights
Six novel and fully characterized heterobimetallic Ru-Fe complexes are reported and
their in vitro activities towards liver (HepG2) and human cervical (HeLa) cancer cell
lines are reported.
Among the prepared complexes, 5c and 5d showed enhanced cytotoxicity towards
HeLa and HepG2, with IC50 values of 9.34 and 15.74, respectively, compared to
cisplatin (31.32 and 27.95, respectively).
Complex 5d induced cell cycle arrest in the S phase and apoptosis through collapse of
mitochondrial membrane potential and ROS.

CRediT authorship contribution statement
Ranjith kumar Jakku: Conceptualization, Methodology, Validation, Formal analysis,
Investigation, Writing - original draft, Data curation. Eda Rami Reddy: Validation, Formal
analysis. Nedaossadat Mirzadeh: Conceptualization, Methodology, Writing - review &
editing, Supervision. T. Srinivasa Reddy: Data curation, software, investigation. Anil
Kumar Vardhaman: Data curation, Software. L. Giribabu: Formal analysis, Validation.
Balasubramanian Sridhar: Resources, formal analysis. Deep Pooja: Validation, Data
curation. Sistla Ramakrisna: Resources, investigation. Suresh Bhargava: Supervision,
Project administration, Funding acquisition. Rajiv Trivedi: Supervision, Formal analysis,
Methodology.