Ruthenium(II)-arene complexes with naphthalimide-tagged N,O- and N,N-chelating ligands: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ica.2015.05.025

A new family of ruthenium(II)-arene complexes with naphthalimide functionalized N,O- and N,N-chelating ligands of the general formula [Ru(η⁶-p-cymene)Cl(L)] (2b-4b) (where: L = 4-[N-(2-((2-hydroxy-5-Br-phenyl)methyl imino)ethyl)]-N-butyl-1,8-na

Full text of DOI:10.1016/j.ica.2015.05.025

Accepted Manuscript
Ruthenium(II)-Arene Complexes with Naphthalimide-Tagged N,O-and N, N-
Chelating Ligands: Synthesis and Biological Evaluation
Kesete Ghebreyessus, Ashley Peralta, Meena Katdare, Krishnan Prabhakaran,
Shanthi Paranawithana
PII:
S0020-1693(15)00287-X
http://dx.doi.org/10.1016/j.ica.2015.05.025
ICA 16555
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
24 January 2015
26 May 2015
28 May 2015
Please cite this article as: K. Ghebreyessus, A. Peralta, M. Katdare, K. Prabhakaran, S. Paranawithana,
Ruthenium(II)-Arene Complexes with Naphthalimide-Tagged N,O-and N, N-Chelating Ligands: Synthesis and
Biological Evaluation, Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.2015.05.025
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Ruthenium(II)-Arene Complexes with Naphthalimide-Tagged N,O-and N, N-
Chelating Ligands: Synthesis and Biological Evaluation
Kesete Ghebreyessusa* Ashley Peraltaa#, Meena Katdareb Krishnan Prabhakaranb#,
Shanthi Paranawithanaa
aDepartment of Chemistry and b Skin of Color Research Institute, Hampton University,
Hampton, VA 23668
a* Corresponding Author: Kesete Ghebreyessus, Department of Chemistry, Hampton
University, Hampton, VA 23666, USA. E-mail: Kesete.Ghebreyessus@hamptonu.edu.
Telephone: +1-757-727-5475, Fax: +1-757-727-5604.
ABSTRACT
A new family of ruthenium(II)-arene complexes with naphthalimide
functionalized N,O- and N,N-chelating ligands of the general formula [Ru(η6-p-
cymene)Cl(L)] (2b-4b) (where:
L
=
4-[N-(2-((2-hydroxy-5-Br-phenyl)methyl
imino)ethyl)]-N-butyl-1,8-naphthalimide (2a), 4-[N-(2-((2-hydroxy-5-Cl-phenyl)methyl
imino)ethyl)]-N-butyl-1,8-naphthalimide (3a), and N-butyl-4-[N-(2-((2-hydroxy-5-NO2-
phenyl)methylimino)ethyl)]-N-butyl-1,8-naphthalimide (4a), and [Ru(η6-p-cymene)
Cl(L’)]Cl (8b-9b) (where L’ = N-(2,2'-dipyridylaminoethyl)-1,8-naphthalimide (8a) and
N-(2,2'-dipyridylaminopropyl)-1,8-naphthalimide (9a) have been synthesized and
characterized. The in vitro cytotoxic activities of the ligands (2a, 9a) and the complexes
(2b-4b, and 8b-9b) have been evaluated against the human melanoma skin cancer
(CRL7687) and normal noncancerous (CA-M75) cell lines. All the compounds exhibit
potent cytotoxic activities with IC50 values of ~ 1µM or less but displayed variable
selectivity. The compounds with N, O- ligands were found to be less selective than those
containing N, N-chelating ligands. Notably, complex 9b displayed the highest selectivity
towards cancer cells over health cells. The interactions of the compounds with calf
thymus DNA (CT-DNA) have also been investigated by UV-vis and fluorescence
spectra, ethidium bromide displacement assay and gel electrophoretic studies, which
revealed that the compounds bind to CT-DNA moderately presumably through an
intercalative mode.
1

Key Words: Ruthenium(II)-arene complexes; antitumor activity; DNA-binding;
Naphthalimide-tagged chelating ligands
1.
INTRODUCTION
Ruthenium based coordination and organometallic complexes are increasingly
gaining in importance as promising candidates for the design of new and more effective
metal-based anticancer agents [1-6]. This interest is stimulated by the successful entry of
two ruthenium(III) compounds, NAMI-A ([H2im][trans-RuCl4(S-dmso)-(Him)]; Him =
imidazole, dmso = dimethyl sulfoxide) and KP1019 ([H2ind][trans-RuCl4(Hind)2]; Hind
= indazole), into clinical trials [7-10]. More recently, promising result in clinical studies
of NKP-1339 ([Na][trans-RuCl4(Hind)2]; Hind = indazole), the sodium analogue of
KP1019 has also illuminated further interest for the design and development of
ruthenium(III) based coordination compounds as anticancer agents [11-12]. The most
attractive profile of NKP-1339 is its high water solubility compared to KP1019. It has
been established that ruthenium compounds exhibit low toxicities while maintaining high
selectivity toward cancer cells in vitro and high efficacy against platinum-drug-resistant
tumors [9-13]. The more selective activity of ruthenium compounds is believed to be due
to their preferential accumulation in cancer cells and the ability of ruthenium to mimic
iron in binding to biomolecules [3, 7, 9, 11-13]. Ruthenium compounds are also believed
to have a biological mode of action that is significantly different from those of platinum-
based drugs [9, 13-15]. Furthermore, the rich synthetic chemistry, diverse coordination
geometries, redox accessible oxidation states, and favorable ligand substitution reactions
of ruthenium complexes have been advantageously considered for the design of new
anticancer agents [3, 16].
2

More recently, the family of half-sandwich ruthenium(II)-arene organometallic
complexes are also being actively investigated and evaluated as a potential source of new
and effective metal-based anticancer agents. A favorable property of this type of
complexes is the versatile pseudo-tetrahedral coordination geometry conferred by the
metal center. Such a characteristic provides considerable possibilities for creating new
compounds with interesting biological properties through rational ligand design and
functionalization. In this context, several promising families of ruthenium(II)-arene based
complexes with diverse ligand frameworks have been synthesized, and evaluated for their
antitumor activity against a broad spectrum of cancer cell lines. For example,
ruthenium(II)-arene complexes of the type [(η6-arene)Ru(en)Cl]+ (en = 1,2-
ethylenediamine) developed by Sadler and co-workers have shown to exhibit high
antitumor activities in various cancer cell-lines [16-21]. Related compounds of the type
[(η6-arene)RuCl2(pta)] (pta = 1,3,5-triaza-7-phosphaadmantane) reported by the Dyson
group have also been shown to exhibit antimetastatic and antitumor activities [1, 10, 22-
25]. The antitumor activity of many ruthenium(II)-arene complexes is generally related to
their enhanced DNA binding affinity, which involves covalent coordination and/or
simultaneous intercalation of extended aromatic groups and specific hydrogen bonding
depending on the particular type of ligands used. In this regard, diverse ligand types are
increasingly being developed and combined with the ruthenium(II)-arene moiety to
enhance their DNA-binding properties, so as to achieve different biological functions and
to maximize their effectiveness as therapeutic agents [21-26].
In recent years, research on targeted and multifunctional ruthenium(II)-arene
complexes tethered to biologically active ligands have received increasing attention,
3

mainly due to the potential synergism that could be achieved by combining a metal ion
and a bioactive ligand [1, 27-30]. It has been noted that tethering of biologically active
ligands to the metal ion increases the biological potency of the complexes, through a
combination of increased solubility, altered mechanisms of action, increased uptake, and
improved cancer targeting properties. Dyson and co-workers have recently prepared new
half-sandwich ruthenium(II)-arene complexes that incorporate the bioactive 1,8-
naphthalimde-tagged arene and imidazole based ligands and,-which exhibited
significantly higher anticancer activities compared to the prototype [(η6-
arene)RuCl2(pta)] (pta = 1,3,5-triaza-7-phosphaadmantane) complexes. The higher
cytotoxic activity of these new complexes is attributed to the incorporation of the 1,8-
naphthalimide moiety onto the ruthenium(II)-arene unit, which provides multi-targeting
properties including strong DNA-binding and interaction with proteins [29].
In light of these promising results, we initiated the investigations on the design
and synthesis of new ruthenium(II)-arene complexes consisting of N,O- and N, N- based
chelating ligands conjugated with the bioactive 1,8-naphthalimide moiety as potential
anticancer agents. 1,8-naphthalimides are heterocyclic pharmacophores that are known to
readily interact with DNA through intercalation, and also to act as potent topoisomerase
II inhibitors [31-36]. As a result, 1,8-naphthalimide and its derivatives have been
extensively investigated for their potential use as anticancer drugs, and two of these
compounds (mitonafide, and amonafide) have reached clinical trials [32-34]. In addition
to their broad spectrum of biological activities, 1,8-naphthalimides have also been
described as strongly fluorescent agents, a property that could be useful for probing the
interaction of these compounds with biomolecules [37-40].
4

Hence, in this article, we report the synthesis and characterization of a series of
naphthalimide-tethered chelating ligands and their corresponding ruthenium(II)-arene
complexes. The cytotoxic activity of the complexes toward the human skin melanoma
cancer cell line (CRL7687) and normal skin melanocyte (CA-M75) was investigated by
using the methylthiazolyldiphenyltetrazolium bromide (MTT) assay. The DNA-binding
properties of the compounds were explored by UV-Vis and fluorescence spectroscopy,
and gel electrophoretic mobility studies. The results of our investigation revealed that
conjugation of the naphthalimide moiety to the ruthenium(II) center have no distinct
advantages on the cytotoxic activities of the ruthenium(II)-arene complexes on the cell
lines tested. The details are presented herein.
2. Experimental Section
2.1. Materials and Methods
All synthetic procedures were performed under nitrogen. All chemicals and
solvents were purchased from commercial sources and used as received. Double stranded
calf thymus DNA (CT-DNA) (Sodium salt, highly polymerized type) and supercoiled
pUC18 plasmid DNA were purchased from Sigma Aldrich. [(η6-cymene)RuCl2]2 was
1
prepared according literature methods [41]. H and 13C{1H} NMR spectra were recorded
on a JEOL Eclipse2-400 MHz spectrometer using solvent resonances as internal
references. Electrospray ionization (ESI) mass spectra were recorded on an Agilent
(Varian) MS-500 series and analyzed using MS-Varian 6.9.3 software. UV-vis absorption
spectra were recorded on a Varian Cary 50 BIO spectrometer and emission spectra on a
Cary Eclipse fluorospectrometer. Milli-Q H20 (18.2 mΩ) was used as a solvent for all
UV-vis, fluorescence, and gel electrophoresis studies.
5

2.2. Synthesis and characterization of the Ligands (2a-4a)
2.2.1. Synthesis of 4-[(2'-aminoethyl)amino]-N-butyl-1,8-naphthalimide (1)
The precursor compound, 4-[(2'-aminoethyl)amino]-N-butyl-1,8-naphthalimide
(1), was synthesized following a procedures reported in the literature [42-43].
2.2.2. Synthesis of N-(2-((2-hydroxy-5-Br-phenyl)methylimino)ethyl)-N-butyl-1,8-
naphthalimide (2a).
A stirred solution of the precursor compound 4-[(2'-aminoethyl)amino]-N-butyl-
1,8-naphthalimide (1), (0.50 g) and 5-Bromo-salicylaldehyde in ethanol (50 mL) was
heated to reflux for 24 h under a nitrogen atmosphere. The volume of the reaction
mixture was reduced to ~ 5 mL by rotary evaporation. The solution was then cooled to
room temperature and placed in an ice bath to precipitate the product. The resulting
yellow precipitate was isolated by filtration and washed with diethyl ether to give the
pure product as a yellow microcrystalline solid (yield: 0.56 g, 71%).
Anal. Calcd. for C25H25N3O3Br: C, 60.61; H, 5.09; N, 8.48.Found: C, 60.43; H, 4.91; N,
8.50. ESI-MS (CH3CN): m/z = 494.7 [2a + H]+. 1H NMR (CDCl3, 400 MHz) δ 8.58 (dd,
J = 7.3 Hz, J = 0.9 Hz, 1H, Ar-H), 8.48 (d, J = 8.7 Hz,1H, Ar-H), 8.31 (s, CH=N), 8.03
(d, J = 7.8 Hz,1H, Ar-H), 7.62 (d, J = 7.3 Hz, 1H, Ar-H), 7.40 (dd, J = 6.4 Hz, J = 2.8 Hz,
1H, Ar-H), 7.32 (d, J = 2.3 Hz, 1H, Ar-H), 6.87 (d, J = 8.7 Hz, 1H, Ar-H), 6.81 (d, J = 8.2
Hz, 1H, Ar-H), 5.47 (t, J = 6.0 Hz, 1H, NH), 4.16 (t, J = 7.8 Hz, 2H, NCH2CH2-, butyl),
4.00 (t, J = 5.5 Hz, 2H,NCH2-), 3.83 (q, J = 6.0 Hz, 2H, -NCH2CH2), 1.71 (m, 2H, -
CH2CH2CH3, butyl), 1.44 (m, 2H, -CH2CH2CH2-, butyl), 0.92 (t, J = 7.4 Hz, 3H, CH3,
13
butyl). C (CDCl3, 100 MHz) δ 166.0 (C=O), 164.6 (C=O), 164.1 (N=CH), 159.9 (Ar-
C), 148.5 (Ar-C), 135.5 (Ar-C), 134.1 (Ar-C), 133.7 (Ar-C), 131.2 (Ar-C), 129.8 (Ar-C),
6

125.5 (Ar-C, Ph), 125.2 (Ar-C, Ph), 123.4 (Ar-C, Ph), 120.4 (Ar-C), 119.8 (Ar-C), 119.1
(Ar-C),111.4 (Ar-C), 110.4 (Ar-C), 104.6 (Ar-C), 57.9 (NCH2-), 43.9 (NCH2, butyl), 40.0
(CH2NH), 30.3 (NCH2CH2-, butyl), 20.4 (-CH2CH2CH2-), 13.9 (CH3, butyl).
2.2.2. Synthesis of N-(2-((2-hydroxy-5-Cl-phenyl)methylimino)ethyl)-N-butyl-1,8-
naphthalimide (3a).
The same procedure as in 2a using 5-Chloro-salicylaldehyde to give 3a (yield:
0.60 g, 83%). Anal. Calcd. for C25H25N3O3Cl: C, 66.59; H, 5.59; N, 9.32. Found: C,
1
66.40; 5.38; N, 9.20. ). ESI-MS (CH3CN): m/z = 450.4 [3a]+. H NMR (CDCl3, 400
MHz) δ 8.58 (d, J = 7.3 Hz, 1H, Ar-H), 8.48 (d, J = 8.2 Hz, 1H, Ar-H), 8.31 (s, 1H,
N=CH), 8.04 (d, J = 8.3 Hz, 1H, Ar-H), 7.61 (d, J = 7.3 Hz, 1H, Ar-H, Ph), 7.28 (d, J =
2.8 Hz, 1H, Ar-H), 7.17 (d, J = 2.8 Hz, 1H, Ar-H), 6.92 (d, J = 8.7 Hz, 1H, Ar-H, Ph),
6.81 (d, J = 8.3 Hz, 1H, Ar-H, Ph), 5.50 (t, J = 6.0 Hz, 1H, NH), 4.16 (t, J = 7.3 Hz, 2H, -
NCH2CH2-, butyl), 4.00 (t, J = 6.0 Hz, 2H, -CH2N), 3.83 (q, J = 5.5 Hz, 2H, NHCH2),
1.71 (m, 2H, -CH2CH2CH2-, butyl), 1.44 (m, 2H, -CH2CH2CH3, butyl), 0.97 (t, J = 7.3
Hz, 3H, CH3, butyl). 13C (CDCl3, 100 MHz) δ 166.0 (C=O), 164.6 (C=O), 164.1
(C=NH), 159.9 (C-O), 148.5 (C-NH), 134.1 (Ar-C), 132.7 (Ar-C), 131.2 (Ar-C), 130.6
(Ar-C), 129.8 (Ar-C, Ph), 125.5 (Ar-C, Ph), 125.1 (Ar-C, Ph), 123.6 (Ar-C, Ph), 123.4
(Ar-C), 120.4 (Ar-C), 119.2 (Ar-C), 118.6 (Ar-C), 111.4 (Ar-C), 104.5 (Ar-C, Ph), 57.9
(NCH2-), 43.9 (-NCH2-, butyl), 40.0 (-CH2NH-), 30.3 (-CH2CH2-, butyl), 20.4 (-CH2CH3,
butyl), 13.9 (CH3, butyl).
7

2.2.3. Synthesis of N-(2-((2-hydroxy-5-NO2-phenyl)methylimino)ethyl)-N-butyl-1,8-
naphthalimide (4a).
The same procedure as in 2a using 5-nitro-salicyladehyde to give 4a (yield: 0.52
g, 70%). Anal. Calcd. for C25H25N4O5: C, 65.08; H, 5.46; 12.14. Found: C, 64.92; H,
5.42; N, 19.93. ESI-MS (CH3CN): m/z = 461.4 [4a]+. 1H NMR (CDCl3, 400 MHz) δ 8.65
(s, 1H, N=CH), 8.61 (d, J = 8.7 Hz, 1H, Ar-H), 8.41 (d, J = 6.8 Hz, 1H, Ar-H), 8.28 (dd, J
= 8.7 Hz, J = 2.7 Hz, 1H, Ar-H), 8.14 (m, 1H, Ar-H, Ph), 8.01 (dd, J = 9.2 Hz, J = 2.8 Hz,
1H, Ar-H), 7.81 (t, J = 7.8 Hz, 1H, N-H), 7.61 (t, J = 7.8 Hz, 1H, Ar-H), 6.86 (d, J = 8.7
Hz, 1H, Ar-H, Ph), 6.67 (d, J = 9.6 Hz, 1H, Ar-H, Ph), 4.00 (m, 4H, NCH2, ethyl; NCH2-,
butyl), 3.77 (q, J = 5.5 Hz, 2H, -CH2NH), 1.58 (m, 2H, NCH2CH2-, butyl), 1.35 (m, 2H, -
CH2CH2CH3, butyl), 0.92 (t, J = 7.3 Hz, 3H, CH3, butyl), 13C (CDCl3, 100 MHz) δ 167.2
(C=O), 165.8 (C=O), 164.5 (N=CH), 164.0 (C-O), 148.4 (Ar-C, Ph), 139.5 (Ar-C), 134.0
(Ar-C), 131.2 (Ar-C), 129.7 (Ar-C), 128.2 (Ar-C), 127.9 (Ar-C), 125.6 (Ar-C, Ph), 125.2
(Ar-C, Ph), 123.3 (Ar-C, Ph), 120.4 (Ar-C), 118.3 (Ar-C), 117.2 (Ar-C), 111.4 (Ar-C),
104.4 (Ar-C, Ph), 57.2 (-NCH2), 43.7 (NCH2CH2CH2-, butyl), 40.0 (-CH2N), 30.3 (-
CH2CH2N-, butyl), 20.4 (-CH2CH2CH2-, butyl), 13.9 (CH3, butyl).
3. Synthesis of the 2,2'-dipyridylamine containing ligands (8a, 9a)
3.1. Synthesis of N-(2,2'-dipyridylaminoethyl)-1,8-naphthalimide (8a)
The second series of ligands were synthesized using the following general
procedure [44]. Under nitrogen, solutions of N-(1-bromoethyl)-1,8-naphthalimide (7)
(0.305 g, 1.0 mmol) and 2,2’-dipyridylamine (0.20 g, 1.1 mmol) were suspended in
acetonitrile. To this suspension, Cs2CO3 (0.36 g, 1.1 mmol) was added with stirring. The
resulting mixture was refluxed for 24 h under nitrogen. The solution was cooled to room
8

temperature and the solvent was removed under reduced pressure. The resulting residue
was dissolved in CH2Cl2 and layered with diethyl ether to precipitate the product. The
precipitate was collected by filtration as a pale yellow solid: (yield: 0.35 g, 88%). Anal.
Calcd. for C24H18N4O2: C, 73.09; H, 4.60; N, 14.21. Found: C, 73.49; H, 4.75; N, 14.09.
ESI-MS (CH3CN): m/z = 395.1 [8a + H]+. 1H NMR (CDCl3, 400 MHz) δ 8.60 (d, J = 7.3
Hz, 2H, Ar-H, Py), 8.26 (d, J = 5.0 Hz, 2H, Ar-H, Py), 8.22 (d, J = 8.2 Hz, 2H, Ar-H),
7.75 (t, J = 7.8 Hz, 2H, Ar-H), 7.59 (m, 4H, Ar-H, Py), 6.84 (t, J = 6.0 Hz, 2H, Ar-H, Py),
4.47 (t, J = 5.0 Hz, 2H, -NCH2), 4.00 (t, J = 5.5 Hz, 2H, -CH2N-).13C NMR (CDCl3, 100
MHz) δ 165.1 (C=O), 153.9 (Ar-C, Py), 147.7 (Ar-C), 137.7 (Ar-C), 134.2 (Ar-C), 131.5
(Ar-C), 128.2 (Ar-C), 126.9 (Ar-C), 122.4 (Ar-C), 116.4 (Ar-C), 111.6 (Ar-C), 61.7 (-
NCH2), 42.8 (-CH2N-).
3.2. Synthesis of N-(2,2'-dipyridylaminopropyl)-1,8-naphthalimide (9a)
The same experimental procedure as reported for 8a (yield: 0.40 g, 85%). Anal.
Calcd. for C25H20N4O2 408.4: C, 73.51; H, 4.93; N, 13.62. Found: C, 73.30; H, 5.03; N,
1
13.54. ESI-MS (CH3CN): m/z = 408.3 [9a]+. H NMR (CDCl3, 400 MHz) δ 8.60 (d, J =
7.3 Hz, 2H, Ar-H, Py), 8.26 (d, J = 5.0 Hz, 2H, Ar-H), 8.21 (d, J = 8.2 Hz, 2H, Ar-H),
7.75 (t, J = 7.8 Hz, 2H, Ar-H), 7.56 (m, 4H, Ar-H, Py), 6.84 (t, J = 6.0 Hz, 2H, Ar-H, Py),
4.33 (t, J = 7.3 Hz, 2H,-NCH2CH2), 3.50 (t, J = 6.9 Hz, 2H, -CH2CH2N), 2.33 (m, 2H, -
CH2CH2CH2-).13C (CDCl3, 100 MHz) δ 164.2 (C=O), 154.0 (Ar-C, Py), 147.8 (Ar-C),
137.8 (Ar-C), 134.1 (Ar-C), 131.6 (Ar-C), 131.4 (Ar-C), 128.2 (Ar-C), 127.0 (Ar-C),
122.5 (Ar-C), 116.4 (Ar-C), 111.6 (Ar-C), 39.3 (NCH2CH2-), 31.4 (-CH2CH2N-), 30.6
(-CH2CH2).
9

4. Synthesis and Characterization of the (η6-p-cymene)ruthenium(II) complexes
(2b-4b)
4.1. Synthesis of (η6-p-cymene)(N-(2-((2-hydroxy-5-Br-phenyl)methylimino)ethyl)-N-
butyl-1,8-naphthalimide)chlororuthenium(II) (2b)
The ruthenium(II)-cymene complexes were synthesized following a similar
procedure described in the literature, but with a slight modification [45]. The N-(2-((2-
hydroxy-5-Br-phenyl) methylimino)ethyl)-N-butyl-1,8-naphthalimide ligand (2a) (0.25 g,
0.56 mmol) was dissolved in 25 mL of dry CH2Cl2, and K-OtBu (0.15 g, 0.56 mmol) was
added at room temperature. The mixture was stirred for 30 min at room temperature.
After the suspension was cooled in an ice bath, [(η6-p-cymene) RuCl2]2 (0.15 g, 0.25
mmol) was added. The solution was stirred for 24 h in an ice bath. The mixture was
filtered through a short pad of Celite to remove insoluble materials. The volume of the
filtrate was reduced to ~5 mL by rotary evaporation and the product was precipitated by
the addition of hexane. The precipitate was isolated via filtration, washed with diethyl
ether and air dried to give the products as a dark red solid: (yield: 0.50 g, 82%). Anal.
Calcld. for C35H37N3O3ClBrRu: C, 55.02; H, 4.88, N, 5.50. Found: C, 54.94; H, 4.72; N,
5.40. ESI-MS (CH3CN): m/z = 728.4 [(η6-p-cymene)Ru(2a)–Cl]+. 1H NMR (CDCl3, 400
MHz) δ 8.50 (d, J = 7.3 Hz, 1H, Ar-H), 8.45 (d, J = 8.2 Hz, 1H, Ar-H), 8.06 (d, J = 8.7
Hz, 1H, Ar-H), 7.41 (d, J = 7.8 Hz, 1H, Ar-H), 7.15 (d, J = 2.8 Hz, 1H, Ar-H), 7.13 (d, J
= 2.8 Hz, 1H, Ar-H), 7.07 (s, 1H, HC=N), 6.78 (d, J = 9.2 Hz, 1H, Ar-H), 6.71 (d, J = 8.2
Hz, 1H, Ph), 5.57 (d, J = 6.4 Hz, 1H, p-cymene), 5.52 (d, J = 6.4 Hz, 1H, p-cymene), 5.43
(d, J = 5.5 Hz, 1H, p-cymene), 5.01 (d, J = 5.5 Hz, 1H, p-cymene), 4.51 (m, 1H, NCH2),
4.26 (m, 2H, -CH2NH), 4.16 (t, J = 7.3 Hz, 2H, -NCH2CH2-, butyl), 3.94 (m, 1H, NCH2),
2.74 (sept, J = 6.9 Hz, 1H, CH(CH3)2, p-cymene), 2.30 (s, 3H, CH3, p-cymene), 1.71 (m,
10

2H, CH2, butyl), 1.44 (m, 2H, CH2, butyl), 1.25 (d, J = 6.9 Hz, 3H, CH(CH3), p-cymene),
13
1.15 (d, J = 6.9 Hz, 3H, CH(CH3), p-cymene), 0.96 (t, J = 7.3 Hz, 3H, CH3, butyl). C
(CDCl3, 100 MHz) δ 164.7 (C=O), 164.3 (C=O), 164.0 (N=CH), 149.0 (C-O), 137.9 (Ar-
C), 135.9 (Ar-C), 134.0 (Ar-C), 131.6 (Ar-C), 130.0 (Ar-C), 127.8 (Ar-C), 125.2 (Ar-C,
Ph), 124.0 (Ar-C, Ph), 122.6 (Ar-C, Ph), 120.5 (Ar-C), 119.2 (Ar-C), 110.5 (Ar-C), 104.8
(Ar-C), 103.4 (Ar-C, Ph), 100.8 (Ar-C, Ph), 99.2 (Ar-C, p-cymene), 88.7 (Ar-C, p-
cymene), 83.3 (Ar-C, p-cymene), 80.7 (Ar-C, p-cymene), 80.5 (Ar-C, p-cymene), 66.7
(NCH2), 42.2 (-CH2NH), 39.9 (N-CH2CH2, butyl), 30.7 (CHCH3, p-cymene), 30.3
(CH2CH2, butyl), 23.0 (CH3, p-cymene), 21.7 (CH2CH2, butyl), 20.5 (CH3, p-cymene),
18.9 (CH3, p-cymene), 13.9 (CH3, butyl).
4.2. Synthesis of (η6-p-cymene)(N-(2-((2-hydroxy-5-Cl-phenyl)methylimino)ethyl)-N-
butyl-1,8-naphthalimide)chlororuthenium(II) (3b)
The same experimental procedure as reported for 2b, was employed using ligand
3a to give 3b as a red solid (yield: 0.50 g, 75%). Calcd. for C35H37N3O3Cl2Ru: C, 58.41;
H, 5.18; N, 5.84. Found: C, 58.11; H, 5.10; N, 5.54. ESI-MS (CH3CN): m/z = 684.4 [(η6-
1
p-cymene)Ru(3a)–Cl]+. H NMR (CDCl3, 400 MHz) δ 8.51 (d, J = 7.4 Hz, 1H, Ar-H),
8.46 (d, J = 8.2 Hz, 1H, Ar-H), 8.08 (d, J = 8.7 Hz, 1H, Ar-H), 7.42 (t, J = 7.8 Hz, 1H,
Ar-H), 7.10 (s, 1H, N=CH), 7.02 (dd, J = 9.2 Hz, J = 2.8 Hz, 1H, Ar-H), 6.84 (d, J = 9.2
Hz, 1H, Ar-H, Ph), 6.76 (m, 1H, N-H), 6.72 (d, J = 8.3 Hz, 1H, Ar-H, Ph), 6.20 (d, J =
2.8 Hz, 1H, Ar-H, Ph), 5.57 (d, J = 6.4 Hz, 1H, p-cymene), 5.52 (d, J = 6.0 Hz, 1H, p-
cymene), 5.43 (d, J = 6.6 Hz, 1H, p-cymene), 5.00 (d, J = 5.5 Hz, 1H, p-cymene), 4.52
(m, 1H, -NCH2), 4.27 (m, 2H, -CH2NH), 4.16 (t, J = 7.4 Hz, 2H, -NCH2-, butyl), 3.95
(m, 1H, NCH2-), 2.75 (sept, J = 6.8 Hz, 1H, CH(CH3)2, p-cymene), 2.32 (s, 3H, CH3, p-
11

cymene), 1.71 (m, 2H, CH2, butyl), 1.44 (m, 2H, CH2, butyl), 1.25 (d, J = 7.3 Hz, 3H,
CH(CH3), p-cymene) 1.15 (d, J = 6.9 Hz, 3H, CH(CH3), p-cymene), 0.97 (t, J = 7.4 Hz,
13
3H, CH3, butyl). C (CDCl3, 100 MHz) δ 164.7 (C=O), 164.3 (C=O), 163.7 (HC=N),
148.9 (C-O), 135.4 (Ar-C), 134.0 (Ar-C), 132.7 (Ar-C), 131.5 (Ar-C, p-cymene), 130.0
(Ar-C), 127.8 (Ar-C), 125.2 (Ar-C, Ph), 123.5 (Ar-C, Ph), 122.6 (Ar-C, Ph), 120.5 (Ar-
C), 118.5 (Ar-C), 118.3 (Ar-C), 110.4 (Ar-C), 103.3 (Ar-C, Ph), 100.7 (Ar-C), 99.2 (Ru-
C, p-cymene), 88.9 (Ru-C, p-cymene), 83.3 (Ru-C, p-cymene), 80.7 (Ru-C, p-cymene),
80.4 (Ru-C, p-cymene), 66.9 (-N-CH2), 42.2 (-CH2-NH), 40.0 (N-CH2CH2), 30.6
(CH(CH3)2, p-cymene), 30.3 (-CH2CH2, butyl), 23.0 (CH3, p-cymene), 21.6 (CH2CH2,
butyl), 20.5 (CH(CH3), p-cymene), 18.9 (CH(CH3), p-cymene), 13.9 (CH3, butyl).
4.3. Synthesis of (η6-p-cymene)(N-(2-((2-hydroxy-5-NO2-phenyl)methylimino)ethyl)-N-
buthyl-1,8-naphthalimide)chlororuthenium(II) (4b)
The same experimental procedure as reported for 3b, using ligand 4a to give 4b as
an red-orange solid (yield: 0.50 g, 70%). Anal. Calcd. for Calcd for C35H37N4O5ClRu: C,
57.57; H, 5.11; N, 7.67. Found: C, 57.89; H, 4.95; N, 7.55. ESI-MS (CH3CN): m/z =
695.2 [(η6-p-cymene)Ru(4a)–Cl]+. 1H NMR (CDCl3, 400 MHz) δ 8.46 (t, J = 7.8 Hz, 1H,
Ar-H), 8.03 (d, J = 8.2 Hz, 1H, Ar-H), 7.95 (dd, J = 9.6 Hz, J = 2.7 Hz, 1H, Ar-H), 7.40
(t, J = 7.3 Hz, 1H, Ar-H), 7.32 (s, 1H, N=CH), 6.86 (d, J = 9.2 Hz, 1H, Ar-H), 6.71 (d, J
= 8.2 Hz, 1H, Ar-H), 6.65 (m, 1H, Ar-H), 5.63 (d, J = 6.4 Hz, 1H, p-cymene), 5.59 (d, J =
6.4 Hz, 1H, p-cymene), 5.52 (d, J = 5.5 Hz, 1H, p-cymene), 5.06 (d, J = 6.0 Hz, 1H, p-
cymene), 4.55 (m, 1H, -NCH2), 4.29 (m, 2H, -CH2NH-), 4.14 (t, J = 7.4 Hz, 1H,
NCH2CH2-, butyl), 4.00 (m, 1H, -NCH2), 2.76 (sept, J = 6.9 Hz, 1H, CH(CH3)2), p-
cymene), 2.33 (s, CH3, 3H, p-cymene), 1.71 (m, 2H, -CH2CH2CH2-, butyl), 1.43 (m, 2H,
12

-CH2CH2CH3), 1.26 (d, J = 6.8 Hz, 3H, CH(CH3)2, p-cymene), 1.17 (d, J = 6.9 Hz, 3H,
CH(CH3)2, p-cymene), 0.95 (t, J = 7.3 Hz, 3H, -CH2CH3, butyl). 13C (CDCl3, 100 MHz) δ
170.1 (C=O), 165.1 (C=O), 164.5 (N=CH), 164.2 (C-O),148.6 (Ar-C), 135.7 (Ar-C),
134.0 (Ar-C), 132.7 (Ar-C), 131.5 (Ar-C), 130.0 (Ar-C), 129.6 (Ar-C),127.2 (Ar-C),
125.2 (Ar-C, Ph), 122.8 (Ar-C, Ph), 122.6 (Ar-C, Ph), 120.4 (Ar-C), 117.0 (Ar-C), 110.7
(Ar-C), 103.2 (Ar-C), 101.7 (Ar-C, Ph), 99.6 (Ar-C, Ph), 88.8 (Ar-C, p-cymene), 83.7
(Ar-C, p-cymene), 80.9 (Ar-C, p-cymene), 80.4 (Ar-C, p-cymene), 67.4 (-NCH2), 42.1
(-CH2NH), 39.9 (N-CH2CH2, butyl), 30.7 (CHCH3, p-cymene), 30.3 (-CH2CH2, butyl)
23.0 (CH3, p-cymene), 21.6 (-CH2CH2, butyl), 20.4 (CH3, p-cymene), 18.9 (CH3, p-
cymene), 13.9 (CH3, butyl).
4.4. Synthesis of (η6-p-cymene)(N-(2,2'-dipyridylaminoethyl)-1,8-naphthalimide)chloro
ruthenium(II) chloride (8b).
The cationic ruthenium(II)-cymene complexes were synthesized using the same
procedure as follows [46]. To a solution of [(η6-cym)RuCl2]2) (0.15 g , 0.30 mmol, 1.0
equiv) in CH2Cl2 (20 mL) was added N-(2,2'-dipyridylaminoethyl)-1,8-naphthalimide
(8a) (0.25 g, 0.50 mmol, 2.10 equiv) and stirred at room temperature for 24 h. The
solution was filtered through a pad of Celite to remove insoluble materials. The volume
of the solution was reduced to ~5 mL and layered with diethyl ether to precipitate the
product. The precipitate was isolated via filtration, washed with diethyl ether and air
dried to give the pure product (8b) as an orange solid: (yield: 0.35 g, 80%). Anal. Calcd.
for C34H32N4O2Cl2Ru: C, 58.29; H, 4.60; 8.00. Found: C, 58.57; H, 4.50; N, 7.52. ESI-
1
MS (CH3CN): m/z = 632.5 [(η6-p-cymene)RuCl(8a)–HCl–3H + Li]-. H NMR (CDCl3,
400 MHz) δ 8.60 (dd, J = 7.4 Hz, J = 0.9 Hz, 2H, Ar-H), 8.41 (dd, J = 6.0 Hz, J = 1.4 Hz,
13

2H, Ar-H, Py), 8.22 (dd, J = 8.2 Hz, J = 0.9 Hz, 2H, Ar-H), 8.11 (d, J = 8.2 Hz, 2H, Ar-
H), 7.74 (4H, m, Ar-H, Py), 7.0 (t, J= 7.3 Hz, 2H, Ar-H, Py), 5.42 (d, J = 6.4 Hz, 2H, Ar-
H, p-cymene), 5.27 (d, J = 6.0 Hz, 2H, Ar-H, p-cymene), 4.45 (t, J = 5.0 Hz, 2H,-NCH2),
3.97 (t, J = 5.5 Hz, 2H, -CH2N), 2.73 (sept, J = 6.9 Hz, 1H, -CH(CH3)2, p-cymene), 1.94
13
(s, 3H,CH3, p-cymene), 1.22 (d, J = 6.9 Hz, 6H, CH(CH3)2, p-cymene). C (CDCl3, 100
MHz) δ 165.0 (C=O), 154.3 (Ar-C, Py), 152.4 (Ar-C), 139.7 (Ar-C), 134.3 (Ar-C), 131.5
(Ar-C, p-cymene), 128.1 (Ar-C), 126.9 (Ar-C), 122.3 (Ar-C), 119.2 (Ar-C), 116.1 (Ar-
C), 106.4 (Ar-C), 99.4 (Ar-C, p-cymene), 84.4 (Ar-C, p-cymene), 61.7 (N-CH2), 42.7
(-CH2N), 30.6 (CH(CH3)2, p-cymene), 22.2 (CH3, p-cymene), 18.1 (CH(CH3)2, p-
cymene).
4.5. Synthesis of [(η6-p-cymene)(N-(2,2'-dipyridylaminepropyl)-1,8-naphthalimide)
chlororuthenium(II) chloride (9b)
The same experimental procedure as reported for 8b, was used with ligand 9a to
give 9b as an orange solid (yield: 0.33 g, 77%). Anal. Calcd. for C35H34N4O2Cl2Ru: C,
58.82; H, 4.79; N, 7.84. Found: C, 59.15; H, 4.53; N, 7.66. ESI-MS (CH3CN): m/z =
646.7 [(η6-p-cymene)RuCl(9a)–HCl–3H + Li]-. 1H NMR (CDCl3, 400 MHz) δ 8.60 (d, J
= 7.3 Hz, 2H, Ar-H), 8.41 (d, J = 5.0 Hz, 2H, Ar-H, 2-Py), 8.22 (d, J = 7.8 Hz, 2H, Ar-
H), 8.15 (d, J = 7.8 Hz, 2H, Ar-H), 7.76 (m, 4H, Ar-H, Py), 7.03 (t, J = 7.8 Hz, 2H, Ar-H,
Py), 5.42 (d, J = 5.0 Hz, 2H, p-cymene), 5.27 (d, J = 5.0 Hz, 2H, p-cymene), 4.32 (t, J =
6.9 Hz, 2H,-NCH2CH2), 3.49 (t, J = 6.9 Hz, 2H, -CH2CH2N), 2.74 (sept, J = 7.0 Hz, 1H, -
CH(CH3)2, p-cymene), 2.31 (t, J = 6.9 Hz, 2H, -CH2CH2CH2-), 1.95 (s, 3H, CH3, p-
cymene), 1.23 (d, J = 6.9 Hz, 6H, CH(CH3)2, p-cymene).13C (CDCl3, 100 MHz) δ 164.2
(C=O), 154.3 (Ar-C), 152.4 (Ar-C), 139.7 (Ar-C), 134.1 (Ar-C), 131.5 (Ar-C, p-cymene),
14

131.3 (Ar-C), 128.1 (Ar-C, p-cymene), 127.0 (Ar-C), 122.4 (Ar-C), 119.2 (Ar-C), 116.2
(Ar-C), 106.5 (Ar-C), 99.4 (Ar-C), 84.4 (Ar-C, p-cymene), 84.3 (Ar-C, p-cymene), 39.2
(-NCH2CH2-), 31.4 (-CH2CH2N-), 30.7 (CH(CH3)2), p-cymene), 30.5 (-CH2CH2CH2-),
22.3 (CH3, p-cymene), 18.1 (CH(CH3)2, p-cymene).
5. Stability Studies
For stability studies, the UV-vis spectra of 30 µM of the ruthenium(II)-arene
complexes (2b-4b) and (8b-9b) in 1% DMSO at 37 °C in a phosphate buffer saline (PBS)
was monitored over 24 h.
6. DNA Binding Studies
Concentrated calf thymus DNA (CT-DNA) stock solutions for both absorption
and fluorescence studies were prepared in 5 mM Tris-HCl buffer (pH = 7.4) containing
50 mM NaCl. Milli-Q water (18.2 mΩ) was used in all experiments. Concentration of the
DNA solution was determined by UV absorbance at 260 nm. The molar absorption
coefficient was taken as 6600 M-1cm-1. Solutions of CT-DNA gave a ratio of UV
absorbance at 260 nm and 280 nm ≥ 1.8, indicating that the DNA solution was
sufficiently free of protein. All stock solutions were stored at 4 °C and were used within
one week.
6.1. Absorption titration studies.
The absorption titrations were performed using fixed concentration of the ligands
and the ruthenium(II)-cymene complexes (10 µM), while varying the concentration of
CT-DNA from 0-160 µM. After addition of DNA to the ligand and/or metal complex, the
resulting solutions were allowed to equilibrate 3-5 min at 25 °C, after which the
absorption readings were recorded. The intrinsic binding constants (Kb), of the ligands
15

and the ruthenium(II)-cymene complexes were determined from the spectroscopic
titration data using the following equation:[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/Kb(εb-εf)
[47].The 'apparent' extinction coefficient (εa) was obtained by calculating Aobsd/[Ru]. The
terms εf and εb correspond to the extinction coefficients of free (unbound) and the fully
bound complexes, respectively. The Kb values were calculated from the ratio of the slope
to the intercept of a plot of [DNA]/(εa-εf) vs. [DNA] [46].
6.2. Fluorescence titration studies
The fluorescence titrations were carried out in 5 mM Tris-HCl buffer (pH = 7.4)
containing 50 mM NaCl. Solutions of the ligands and their corresponding metal
complexes were titrated with varying concentrations of CT-DNA (0-160 µM). After each
addition of CT-DNA, the samples were allowed to equilibrate for 3-5 min prior to
recording of the spectra. The data was used to calculate the binding constant (Kb) of the
ligands and metal complexes.
6.3. Ethidium Bromide Fluorescence Displacement Experiments
The ethidium bromide (EB) displacement assay titration experiments were
performed on a Varian Cary Eclipse spectrofluorometer using 1 cm path length quartz
cell and 5 cm slits. The fluorescence measurements were carried out by successive
addition of 0-50 µM of each of the ligands (2a, 9a) or the ruthenium complexes (2b, 9b)
to a 2.5 mL of a solution that is 40 µM CT-DNA and 5 µM EB in Tris-HCl buffer
(pH = 7.4) at room temperature. After each addition of CT-DNA, the solution was stirred
for 5 min before measurement. The emission spectra of the EB-DNA system in the
presence and absence of solutions of the ligands and ruthenium complexes were
16

performed by exciting at 520 nm, and the emitted fluorescence was analyzed from 530 to
700 nm at room temperature.
6.4. Agarose Gel Electrophoresis Experiments
In the gel electrophoresis experiments supercoiled pUC18 plasmid DNA was
treated with freshly prepared solutions (40 µM) of the free ligands (2a-4a) and ruthenium
complexes (2b-4b) in Tris-HCl buffer (5 mM, pH 7.4, 50 mM NaCl) in Milli-Q (18.2
mΩ) water with 5-10% DMSO. The treated DNA samples were incubated at 37 °C for 2
h in the dark. Samples of the treated pUC18 plasmid DNA were loaded on 0.8% (w/v)
agarose gel in TAE buffer (Tris-acetate-EDTA buffer, pH = 8.2), and the gel was run for
2.5 h at 70 V. Afterwards, the gel was stained with ethidium bromide (0.5 µg/mL, in
TAE) for 5 min, distained in TAE buffer for 15-20 min, and the bands were visualized by
photographing the fluorescence of ethidium bromide using a UV illuminator. A sample of
pUC18 DNA in TAE buffer alone was used as a control.
7. Cell Culture and Inhibition of Cell Growth
7.1 Cell Culture
Human melanoma skin cancer cells CRL7687 were obtained from the American
Type Culture Collection (ATCC: Manassas, VA) and normal skin melanocytes GM22275
(CA-M75 code in our lab) were obtained from Coriel Cell Repositories (Campton, NJ).
The cells were routinely grown in a Dulbecco’s Modified Eagle’s Medium (DMEM)
containing 10% of fetal bovine serum (FBS) at 37 °C with antibiotics in a humidified
atmosphere of 5% CO2.
17

7.2 Cytotoxicity Assay
Cytotoxic activity of the compounds was determined using the MTT assay (MTT
= 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma Aldrich C).
For evaluation of growth inhibition tests, 1.0 x 104 cells per plate (100 µL) were seeded
in 96-well culture plates and grown for 24 h in complete medium prior to addition of the
drugs. The stock solutions of the compounds were prepared in DMSO in the
concentration ranges of 1.0 x 10-2 – 2.5 x 10-2 M. The drug solutions were then directly
diluted in the culture medium to concentration ranges of 0-40 µM with a final DMSO
concentration of 0.5%, and added to each well. After 72 h incubation at 37 °C, 20 µl of a
solution of MTT in PBS (5 mg/mL) was added to each cell, and the plates were incubated
for a further 3.5 h at 37 °C. All tests were carried out in triplicate. The culture medium
was then aspirated, and the formazan crystals were dissolved in 400 µL of isopropanol in
0.04 M HCl. The absorbance of the plates was read at a 590 nm using microplate reader
(Bio-Rad, Hercules, CA, USA) and compared to the values of the control cells incubated
without compounds. The IC50 values defined as the concentrations of the compounds at
which 50% cell growth inhibition is observed were obtained by a Sigma Plot software
version 11.0.
8. Results and Discussion
8.1 Synthesis and Characterization
In the present study, two series of naphthalimide functionalized ligands and their
corresponding ruthenium(II)-cymene complexes were synthesized. The first series of
ligands 2a-4a were synthesized via the typical condensation route from the reactions of
the 5-subtituted-salicylaldehydes with the corresponding 4-[(2'-aminoethyl)amino]-N-
18

butyl-1,8-naphthalimide precursor (1) in refluxing ethanol affording the products as
yellow microcrystalline solids in 70-85% yield (Scheme 1).
(Scheme 1 here)
Subsequently, reactions of the dimeric [(p-cymene)RuCl2]2 precursor with two
equiv of the N,O-donor ligands 2a-4a in the presence of potassium tert-butoxide (K-
OBut) in dichloromethane gave the neutral half-sandwich mononuclear ruthenimu(II)-
cymene complexes (2b-4b) as air stable orange or dark red solids in good yield (Scheme
2) [45]. Half-sandwich ruthenium(II)-arene complexes analogous to 2b-4b, based on
similar ligand structural motifs that coordinate to the ruthenium center in an N,O- fashion
through the imine nitrogen and the hydroxyl oxygen are known in the literature [45,48-
49]. In addition, reported studies by Schmid et al. showed that ligands with similar
structures can also coordinate to the ruthenium center in a bidentate N, N-mode through
the ethylenediamine moiety [50]. However, these reactions were conducted under base
free conditions. In our part, the reactions were conducted in the presence of a base which
make the ligands prone to coordinate in an N,O-chelating fashion. All the complexes are
soluble in most common organic solvents such as acetone, chloroform, dichloromethane,
dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and methanol, but insoluble in
water. The N,O-chelating ligands (2a-4a) and their corresponding ruthenium(II)-cymene
1
13
complexes (2b-4b) were characterized by means of H, C{1H} and electrospray mass
spectrometry (ESI-MS).
(Scheme 2 here)
19

The 1H NMR spectra of the ligands 2a-4a show characteristic peaks
corresponding to the imine (N=CH) linkage between 8.31-8.65 ppm. The signals for the
methylene protons of the aminoethyl moiety also appeared at 3.77-4.00 ppm. The
naphthalimide and phenyl ring resonances appear between 6.67-8.61 ppm. The remaining
signals in the 1H NMR spectra of the ligands 2a-4a show the expected resonances for the
aliphatic protons (see experimental part). Similarly, the C{1H} spectra of the ligands
13
2a-4a also display characteristic peaks between 166.0-167.2 ppm assigned for the
carbonyl carbons and peaks at 164.4-165.8 ppm corresponding for the imine carbon. The
structures of the ligands 2a-4a were further confirmed by ESI-MS. Acquired in the
positive ion mode, acetonitrile solutions of the N,O-chelating ligands (2a-4a) show
strong peaks corresponding to the molecular ions as [2a + H]+ for (2a); [3a]+ and [4a]+
for 3a and 4a.
The NMR and ESI-MS spectral data strongly support the proposed structures of
the neutral half sandwich ruthenium(II)-cymene complexes. This reaction also results in
the formation of chiral complexes with the ruthenium center being the stereogenic center
1
and the ligands acting as bidentate N,O-chelates. Hence, in the H NMR spectra the
complexes 2b-4b display four separate doublets for the aromatic protons of the p-cymene
(5.00-5.59 ppm) and two doublets each for the methyl protons (1.17-1.26 ppm) of the
isopropyl group. The nonequivalence of the protons in both the aromatic ring and the
isopropyl group indicate the absence of any symmetry element in the complexes,
reflecting the asymmetric coordination of the chelating N,O-donor ligands to the
ruthenium center. The coordination of the η6-cymene ring to the ruthenium(II) center is
1
also clearly indicated by the H NMR spectra, in which the resonances of the aromatic
20

protons (5-5.63 ppm) are considerably upfield shifted compared to those of the free
arene. The septet corresponding to the isopropyl proton was observed near the 2.74-2.76
ppm, and the singlet for the methyl protons at 2.30-2.33 ppm. Apart from these signals,
the 1H NMR spectra of 2b-4b display resonances at 7.07-7.31 ppm corresponding to the
imine HC=N proton, which are significantly upfield shifted compared to those of the free
ligands (8.31-8.65 ppm), suggesting coordination of the imine nitrogen to the
ruthenium(II) ion. However, signals attributed to the protons of the 1,8-naphthaimide
moiety do not show a noticeable shift upon coordination of the ligands to the
ruthenium(II) center, indicating that the naphthalimide does not significantly interact with
the ruthenium center. In addition, in the 13C{1H} NMR spectra of the complexes, six well
separated resonances were observed for the aromatic carbons of the p-cymene moiety
(see experimental part). The imine carbons (HC=N) exhibit peaks in the region 164.2-
1
13
166.5 ppm. The remaining H and C{1H} signals show chemical shifts typical of the
aliphatic region along with signals in the aromatic region due to both the 1,8-
naphthalimide and phenyl ring. Any attempts to obtain crystals of 2b-4b suitable for X-
ray structural analysis were unsuccessful.
Electrospray ionization mass spectra (ESI-MS) provided further evidence for the
formation of the ruthenium(II)-cymene complexes. The mass spectra of acetonitrile
solutions of the complexes 2b-4b show peaks corresponding to the [[(η6-p-
cymene)RuCl(2a-4a)–Cl]+ ion for 2b, 3b and 4b, respectively corroborating their
structures.
The second groups of ligands 8a-9a were also easily prepared in good yield by
reacting the naphthatlimide functionalized intermediate compounds (7) with 2,2’-
21

dipyridylamine in the presence of a slight excess of Cs2CO3 in acetonitrile at room
temperature (Scheme 3) [44].
(Scheme 3 here)
The synthesis of the cationic mononuclear ruthenium(II)-cymene complexes 8b
and 9b was achieved via the bridge cleavage of the [(η6-p-cymene)RuCl2]2, dimer with
2.1 equiv of the 1,8-naphthalimide–tethered 2,2’-dipyridylamine ligands (8a, 9a) in
CH2Cl2 at room temperature for 24 h in 77-80% yield (Scheme 4) [46]. Isolated as their
chloride salts, complexes 8b and 9b are air-stable red-orange solids, that dissolve in most
common organic solvents (acetone, acetonitrile, dichloromethane, DMSO, DMF and
methanol) and slightly soluble in water. The ligands and the new ruthenium(II)-cymene
complexes were characterized by 1H, 13C{1H} NMR and ESI-MS spectroscopy.
(Scheme 4 here)
1
The H NMR spectrum of 8a displays two well-separated triplet resonances at
3.99 and 4.46 ppm attributed to the methylene protons. The CH protons adjacent to the
nitrogen atom of the 2,2’-dipyridylamine unit also display doublet resonances at 8.26
1
13
ppm. The remaining H and C NMR spectra show all the expected resonances for the
1,8-naphthalimide and 2.2'-dipyridyl amine moieties. The main NMR spectroscopic
properties of 9a are similar to those of 8a. The ESI-MS spectra of acetonitrile solutions of
the ligands 8a and 9a show peaks corresponding to the [8a + H]+ and [9a]+ ions.
Both the 1H and 13C{1H}NMR spectra confirm coordination of the ligands 8a and
9a to the ruthenium center. The 1H NMR spectra of the ruthenium(II)-cymene complexes
8b and 9b contain resonances at ca. 8.41 ppm attributed to the CH groups (2H) adjacent
to the pyridine nitrogen, indicating the symmetrical coordination of the N,N-chelating
22

ligands to the ruthenium by the pyridine nitrogen atoms. These resonances are shifted
downfield relative to the free ligands from 8.26 ppm to 8.41 ppm indicating coordination
of the ruthenium(II) ion to the nitrogen atoms of the 2,2'-bipyridylamine ligands. In
addition to this the symmetrical nature of the compounds was evidenced by the
observation of two sets of doublets for the aromatic protons on the p-cymene ligand
1
(5.27- 5.43 ppm). The H NMR spectra of the complexes (8b, 9b) also exhibit sets of
doublet peaks for the methyl protons of the isopropyl group. Likewise, a septet at ca.
2.60-2.80 ppm is observed for the isopropyl proton. The C{1H} NMR spectrum also
13
exhibits appropriate signals. The methyl carbons display signals in the range 15 and 30
ppm. The methyl carbons of the isopropyl group appear around 29-34 ppm, while the
cymene carbons appear in the range 84.35-99.44 ppm for the C-H carbons. These
chemical shifts are similar to those reported for similar ruthenium arene compounds [44].
The carbons of the 1,8-naphthalimide and 2,2'-dipyridylamine groups give peaks in the
range 116-165 ppm. The remaining 1H and 13C{1H} signals show chemical shifts typical
of the aliphatic region along with signals in the aromatic region due to both the 1,8-
naphthalimide and pyridine moieties.
Electrospray ionization mass spectrometry (ESI-MS) provided further evidence
for the formation of the ruthenium(II)-cymene complexes. The ESI-MS of the complexes
8b and 9b measured in the negative ion mode in acetonitrile showed peaks corresponding
to the fragment ions [[(η6-p-cymene)RuCl(8a)–HCl–3H + Li]- for 8b and [[(η6-p-
cymene)RuCl(9a)–HCl–3H + Li]- for 9b.
23

9. Stability Studies
The stability of the ruthenium(II)-arene complexes (2b-4b) and (8b-9b) was
studied at 37 °C in PBS containing 1% DMSO over 24 h by UV-vis absorption
spectroscopy. As representative examples, the UV-vis spectral profiles of complexes 2b
and 9b are shown in Figure 1. The absorption spectra of 9b remains quite stable with no
Figure 1 here
significant shift in the λmax (345 nm) value in the aqueous buffer solution with 1% DMSO
(Fig.1b). Complex 8b also exhibits similar spectral profile as shown in Figure S1b. In
contrast, the UV-vis spectra of complex 2b display significant decrease in the maximum
absorbance at 440 nm in PBS (Fig.1a). Likewise, complexes 3b-4b show very similar
UV-vis absorption profiles to that of 2b (Fig. S1a and S2). On the other hand, as is also
found for complexes 8b-9b, no significant shift in the λmax values was observed for 2b-
4b. However, a slight cloudiness was observed in the aqueous and 1% DMSO solutions
of complexes 2b-4b after 24 h at 37 °C. Hence, the decrease in absorbance of complexes
2b-4b is probably due to the partial dissociation of the ruthenium(II) complexes, which
results in lowering of their concentrations in solution. Overall, the results of the UV-vis
studies indicate that the ruthenium(II) complexes 8b-9b are quite stable in the aqueous
PBS solution, while the complexes 2b-4b undergo hydrolysis to a certain extent.
However, despite the difference in stability of the complexes, there does not appear to be
a strong correlation between hydrolysis and cytotoxicity, both series of complexes (2b-
4b) and (8b-9b) essentially displaying similar cytotoxic profiles (Table 1).
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10. Biological Evaluations
10.1. In vitro cytotoxic activity
The in vitro cytotoxicity of the 1,8-naphthalimide-tagged free ligands (2a and 9a),
and the ruthenium(II)-cymene complexes (2b-4b and 8b-9b) were evaluated against the
human melanoma skin cancer cell line (CR7687) by means of the colorimetric MTT
assay. The results obtained from the UV-vis titration studies indicated that the ligands;
2a-4a, with bromo, chloro and nitro substituents at the para position of the phenyl ring,
respectively, bind to CT-DNA in a similar fashion (vide infra). Similarly, the second
series of ligands; 8a-9a, containing two different aliphatic linkers also showed
comparable binding ability to CT-DNA. Due to these similarities, the ligands 2a and 9a
were selected as representative members of the ligand systems explored in this study to
evaluate their in vitro cytotoxicity. The inhibitory percentage against growth of cancer
cells was determined after treatment of the cells with different concentrations of the free
ligands and the metal complexes for 72 h. In addition, in order to evaluate the selectivity
of the compounds for cancerous cells rather than healthy cells, the compounds were also
tested for their cytotoxic activity against the normal skin melanocytes (CA-M75). The
IC50 values of both the free ligands and their corresponding ruthenium(II)-cymene
complexes are summarized in Table 1.
(Table 1 here)
The following general conclusions can be drawn based on the results displayed in
Table 1: First, it is evident that both the naphthalimide based representative ligands (2a
and 9a), and the new ruthenium(II)-cymene complexes (2b-4b, and 8b-9b) exhibit
significant cytotoxic activities against the melanoma cell line tested, with IC50 values in
25

the submicromolar ranges (0.62-1.10 µM). In addition, in the first series of complexes,
the nature of substituent groups, chloro (2b), bromo (3b) and nitro (4b) in the para
position of the phenyl ring have no significant influence on the in vitro antitumor activity;
to within experimental errors, all the complexes showing almost the same cytotoxicity.
Likewise, in the second series of compounds 8b and 9b, the spacer length between the
naphthalimide moiety and 2,2’-dipyridylamine unit has minor influence on the cyctotoxic
activity of the complexes. Compound 8b with the ethyl linker exhibited slightly higher
anticancer activity compared to that of the ruthenium(II) compound with the propyl
linker, 9b. Second, in the first series of compounds both the free ligands and their
corresponding ruthenium(II)-cymene complexes showed comparable cytotoxicities
towards the human melanoma skin cancer cell line tested. This indicates that conjugation
of the ruthenium(II)-cymene fragment with the naphthalimide functionalized ligands
provides no additional advantages in improving the cytotoxicity of the complexes.
Naphthalimide and its derivatives are known bioactive molecules with potent cytotoxic
activities. In fact, two of its derivatives have been widely investigated and entered into
clinical trials as potential anticancer drugs [32-34]. Hence, the bioactive naphthalimide
moiety is probably mainly responsible for the antiproliferative activity of these new
ruthenium(II)-cymene complexes.
Furthermore, it is notable that the free ligand 2a and the ruthenium(II)-cymene
complexes (2b-4b) display similar cytotoxicities towards the cancerous and normal cells,
which indicates that these compounds are not selective. In contrast, in the second series
of compounds (8b-9b), coordination of the naphthalimide-functionalized N, N-chelating
ligands to the ruthenium(II)-cymene unit leads to a significant increase in selectivity of
26

the complexes for tumors rather than the normal cells. For example, the free ligand 9a
has an IC50 value of 0.620 µM against the melanoma skin cancer cell line (CR7686) and
an IC50 value of 0.613 µM against the normal skin melanocytes (CA-M75). In
comparison, complex 9b displays more toxicity towards the cancerous cells (IC50 of
1.103 µM), which much lower than that for the normal cells (IC50 of 19.70 µM).
The reason for the selectivity of complex 9b needs further investigation which is
currently undergoing in our lab. However, some preliminary assessments can be drawn
that correlate the selectivity of the compound; 9b to the spacer length between the
naphthalimide and the N,N-chelating ligands in the complex. The longer the alkyl chain
the more selective the compound. Thus, it may be likely that the presence of the cationic
charge and the longer propyl linker are influencing the biological activities and the
selectivity of the complex 9b.
10.2 DNA-Binding Studies
While the exact mode of action of the cytotoxic ruthenium(II)-arene complexes is
not clearly understood yet, DNA is considered to be one of their potential biological
targets [51-55]. Hence, to evaluate the mode of interactions of the new ruthenium(II)-
cymene complexes explored in the present study with DNA, UV-vis absorption and
fluorescence spectroscopy, and gels electrophoresis studies were carried out.
10.2.1 Electronic absorption titration studies
Electronic absorption titration studies of the ligands 2a-4a, along with their metal
complexes 2b-4b were carried out to evaluate their DNA binding properties. The
absorption spectra of the ligands show intense bands at λmax 440 nm (2a), 437 nm (3a),
and 408 nm (4a). Similarly, the corresponding metal complexes display intense bands at
27

λmax 444 nm (2b), 442 nm (3b), and 412 nm (4b) with a slight red shift (1-2 nm)
compared to those of the free ligands. The ligand 4a and its ruthenium(II) complex 4b
which have a nitro substituent at the 5-postion of the phenyl ring have an absorption
maximum slightly lower (408 nm for 4a) and (412 nm for 4b) compared to those of the
chloro- and bromo substituents. The difference in the maximum absorption peaks could
mainly be due to the strong electron withdrawing capacity of the nitro-substituent on the
phenyl ring. Otherwise, all the ruthenium(II)-cymene complexes have photophysical
properties similar to those observed in the free ligands, indicating that these bands are
mainly due to the 1,8-naphthalimide chromophore. Since the ligands and their metal
complexes have similar structural features, and displayed comparable cytotoxic activities,
the ligand 2a and its metal complex 2b are selected for discussions of their DNA binding
as typical examples.
The electronic absorption titration data for 2a and 2b in the absence and presence
of CT-DNA are given in Figures 2 and 3. Addition of increasing amounts of CT-DNA (0-
160 µM) to constant concentrations (10 µM) of 2a and/or 2b resulted in a significant
reduction in the absorption spectra along with a slight red shift (1-2 nm). For 2a the
absorption spectra at λmax 440 nm exhibited 28% hypochromism upon binding to CT-
DNA. Likewise, significant changes were also observed for the metal complex 2b, where
the absorption spectra showed 30% hypochromism at λmax 444 nm upon binding to CT-
DNA. In general, significant hypochromism and large red shifts of the absorption spectral
bands are characteristics of compounds that interact with DNA by a strong intercalative
mode [56-59]. The observed hypochromism in the spectra of CT-DNA upon addition of
2a and/or 2b can be partially evidenced as binding via intercalation through the
28

naphthalimide part; as DNA intercalators, in general, leads to a hypochromic effect [29,
36]. However, the observed low hypochromism with no red shift in the band position
could also suggest an electrostatic mode of DNA binding of the ligands and the new
metal complexes. Overall, the results suggested that the ligands and the new metal
complexes interact with CT-DNA, but the extent of interaction is weak. These
characteristic spectral changes are also very similar to those reported in the literature that
involves weak naphthalimide-DNA interactions [36].
((Figure 2 here)
(Figure 3 here)
The intrinsic binding constant Kb of the compounds 2a and 2b was also
determined by monitoring the changes in absorbance at the corresponding maximum
wavelengths. Obtained as the ratio of the slope to intercept from the plot of [DNA]/(εa-εf)
versus [DNA], the values of the binding constant showed that 2b have high DNA binding
propensity Kb = 2.0 x 104 M-1, while 2a gave Kb = 5.0 x 103 M-1, which is in agreement
with the observed hypochromism trends. The higher DNA binding affinity of the
ruthenium complex 2b relative to the corresponding free ligand 2a may suggest that the
1,8-naphthalimide pharmacophore and the ruthenium(II) ion may be interacting in a
cooperative manner through intercalation and covalent coordination. Similar binding
constant values were obtained for the ligands 3a-4a and the ruthenium(II) complexes 3b-
4b. The absorption spectra of the compounds (3a and 3b) and complexes (4a and 4b) are
given as supporting materials in Figures S3-S4.
The DNA binding properties of the ligand 8a and its ruthenium complexes 8b
were also investigated in a manner similar to those of 2a and 2b described above. The
29