Luminescent anticancer ruthenium(II)-: P-cymene complexes of extended imidazophenanthroline ligands: Synthesis, structure, reactivity, biomolecular interactions and live cell imaging

Article abstract of DOI:10.1039/c9dt00921c

Of late, cancer has become a terrible disease affecting people throughout the world. Keeping this in mind, we tried to design drugs that are more lipophilic, target-specific, water-soluble, cytoselective and fluorescent. In this regard, we reported novel

Full text of DOI:10.1039/c9dt00921c

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Luminescent anticancer ruthenium(II)-p-cymene
complexes of extended imidazophenanthroline
ligands: synthesis, structure, reactivity,
Cite this: DOI: 10.1039/c9dt00921c
biomolecular interactions and live cell imaging†
a
Bidisha Sarkar, ‡a Ashaparna Mondal, ‡a Yukti Madaan, a Nilmadhab Roy,
a
Anbalagan Moorthy, b Yung-Chih Kuo c and Priyankar Paira
*
Of late, cancer has become a terrible disease affecting people throughout the world. Keeping this in
mind, we tried to design drugs that are more lipophilic, target-specific, water-soluble, cytoselective and
fluorescent. In this regard, we reported novel ruthenium(II)-p-cymene imidazophenanthroline scaffolds as
effective DNA targeting agents. The planarity of imidazophenanthroline ligands caused the Ru(II) complex
to be a good intercalator. An extended π-electronic conjugation was introduced in the imidazophenan-
throline moieties through the Suzuki and Sonogashira coupling reactions. Here, we synthesized nine Ru(II)
complexes (16a–b, 17a–d, and 19a–c). Among these, [(η6-p-cymene)RuCl(K2-N,N-2-(4’-methyl-[1,1’-
BIphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline)]·PF6 (16b) exhibited the best potency and selecti-
vity with excellent cellular uptake; [(η6-p-cymene)RuCl(K2-N,N-2-(4-(phenylethynyl)phenyl)-1H-imidazo
[4,5-f][1,10]phenanthroline)]·PF6 (17a) acted as a cytoselective probe for live cell imaging.
Received 1st March 2019,
Accepted 8th July 2019
DOI: 10.1039/c9dt00921c
rsc.li/dalton
brain cancers.10–12 Various other second-generation platinum
drugs such as carboplatin and oxaloplatin are also used.
Introduction
Cancer is the second most life-threatening disease around the However, the use of these platinum drugs towards cancer treat-
world after heart disease, and it is mainly caused by the ment is being reduced because of their poor aqueous solubi-
mutation in genes, which leads to abnormal and unrestricted lity, toxicity to normal cells and drug-resistance problems.13–24
cell growth.1 Chemotherapy is the most prevalent traditional Ruthenium complexes have displayed promising results as the
cancer therapy, but it is inefficient in terms of tumor speci- next possible anticancer therapeutics due to their high rate of
ficity, hence rendering normal cells at risk. Targeted therapy ligand exchange, a wide range of accessible oxidation states,
can only disrupt the process of carcinogenesis. Metal com- and exceptional aqueous solubility and stability in a biological
plexes have played an important role in medicine for around environment.25 In general, ruthenium shows anticancer
5000 years because of their physico-chemical properties, vari- activity in its +2 oxidation state when reduced from the +3
able oxidation states, hydrophobicity and lipophilicity, high state in vivo as ruthenium(III) complexes are relatively unreac-
aqueous solubility and positively charged nature.2–9 Cisplatin, tive due to higher electrostatic pull from the nucleus. The
the first FDA (Food Drug Administration)-approved anticancer reduction from ruthenium(III) to ruthenium(II) is most likely
platinum(II) drug, is used for the treatment of a large variety of to occur in a cancer cellular environment as it is acidic, has
cancers such as ovarian, lung, oesophageal, neck, cervical and
a lower oxygen content and has a higher glutathione
concentration.26–28 Hence ruthenium(III) complexes can selec-
tively treat cancer cells without disturbing the growth of
normal cells as they are inert to normal cells. NAMI-A and
KP1019 are two ruthenium(III) drugs that have reached clinical
trials so far. There are studies that support the binding of
NAMI-A with DNA and histidine of serum albumins (HSA) in
biological systems.29–31 NAMI-A showed low efficacy against
the progress of the disease in phase I of the clinical trial,
which restricted its use for further clinical trials.32
Consequently, Keppler et al. developed KP1019, which has
aDepartment of Chemistry, School of Advanced Sciences, VIT, Vellore-632014,
Tamil Nadu, India. E-mail: priyankar.paira@vit.ac.in; Fax: +91-416-2243092;
Tel: +91-416-2243091
bDepartment of Biotechnology, School of Bioscience & Technology,
Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India
cDepartment of Chemical Engineering, National Chung Cheng University, 168,
University Rd., Chia-Yi, Min-Hsiung, Taiwan 62102
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9dt00921c
‡Equal contribution.
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Fig. 1 Structures of NAMI-A, KP1019 and KP1339.
since moved into the clinical investigation stage.33,34 Also, as a
result of its solubility problem, its sodium salt KP1339 is
undergoing clinical investigations35 (Fig. 1).
The hydrophobic character of the arene ligand attached to
ruthenium facilitates the passive diffusion of the complex
through the cell membrane and enhances its cellular accumu-
lation.36 To support this phenomenon, Sadler et al. reported a
family of piano-stool ruthenium(II)-arene pyridyl complexes of
the type [(η6-arene)Ru(N,N′)(L)][PF6]2 for photoactivation and
ruthenium(II) arene complexes containing N,N-, N,O- and O,O-
chelating ligands for anticancer activity.37–39
Fig. 2 Structures of some Ru(II) polypyridyl-type complexes.
DNA and proteins have distinct structures and functions
and they both play acute roles in regulating cellular
functions.40,41 Hence, close investigation of the interactions of
ruthenium complexes with DNA and proteins is mandatory to
understand the anticancer mechanism and to design an
efficient target-specific drug. A variety of ruthenium complexes
have been reported to bind with DNA either in covalent mode
or in non-covalent mode.42–46 For example, the ruthenium
complex [Ru(η6-biphenyl)(en)Cl]+[PF6]− specifically binds to
guanine in aqueous environment, unlike adenine, thymine
and cytosine.47–49 Thus this kind of covalent binding deforms
the DNA backbone, which spoils DNA transcription and repli-
cation. The non-covalent binding is reversible in nature and it
can be further divided into various types, like groove binding,
electrostatic binding and intercalation. Ruthenium(II) com-
plexes can undergo intercalation when they have planar aro-
matic ligands and the complex gets inserted in between adja-
cent base pairs of DNA.50 Liu et al. reported two Ru(II) polypyri-
dyl complexes, namely [Ru(2,2′-bpy)2(MCMIP)]2+ (1) and
[Ru(phen)2(MCMIP)]2+ (2), for cancer therapy. These two com-
plexes bind to DNA through intercalation in between base
pairs.51 Tan et al. developed two novel Ru(II) polypyridyl com-
plexes, namely [Ru(bpy)2(BPIP)]2+ (3) and [Ru(phen)2(BPIP)]2+
Fig. 3 Design of Ru(II)-imidazophenanthroline complexes.
having some crucial units, like: (i) a labile chlorine ligand for
facilitating biomolecular interactions;55 (ii) a η6-p-cymene
moiety for stabilizing the oxidation state of ruthenium and to
increase the potency of drug transportation into the cell
membrane;56,57 (iii) d6 transition metal ruthenium for potency
and selectivity; and (iv) extended π-conjugated ligands for cel-
lular imaging and DNA intercalation. Herein for reporting, we
selected a variety of imidazophenanthroline analogues as
ligands along with ruthenium(II)-p-cymene precursors for the
synthesis of bioactive metal complexes (Fig. 3).
Results and discussion
Synthesis and characterization
(4), which also bind to CT-DNA via intercalation.52 Kumar et al. At the outset, 2-(4-bromophenyl)-1H-imidazo[4,5-f][1,10]phen-
reported three Ru(II) complexes, namely [Ru(bipy)2PRIP]2+ (5), anthroline (10) was prepared in a conventional way by the con-
[Ru(dmb)2PRIP]2+ (6) and [Ru(phen)2PRIP]2+ (7), which were densation of 1,10-phenanthroline-5,6-dione (8) and 4-bromo-
found to intercalate in between DNA base pairs through the benzaldehyde (9), taken in a 1 : 1 proportion in the presence of
aromatic ligand PRIP53 (Fig. 2). However, the development NH4OAc and glacial acetic acid (Scheme 1). The product (10)
of novel ruthenium(II)-arene complexes with extended was fully characterized by 1H, 13C NMR, IR and mass spec-
π-conjugation is still a challenge for target-oriented cancer troscopy. All 10 aromatic protons of compound 10 appeared in
therapy and cellular imaging.
the region of δ 7.78–9.02 ppm in the 1H NMR spectra. The
In continuation of our ongoing work on anticancer organo- protons adjacent to the nitrogen atom of the phenanthroline
rutheniums,54 in the present study, we designed complexes ring appeared as a doublet in the most downfield region of δ
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Scheme 1 Synthetic route of Ru(II)-arene imidazophenanthroline complexes.
9.01–9.02 ppm. In the 13C NMR, peaks of six quaternary the C–C coupled product. The LCMS peak at m/z: 373.1 [M + H]+
carbons and five tertiary carbons were also observed. The C–Br confirmed the formation of ligand 12a. The scope of this chem-
stretching frequency was obtained at 619 cm−1 in the istry was also extended in the other Suzuki product (12b). A
IR spectra, which indicated the presence of C–Br bonds in Sonogashira coupling reaction was also performed with com-
compound 10. The LCMS peak at m/z: 375.2 [M + H]+ con- pound 10 and phenylacetylene (13a) in the presence of
firmed the formation of compound 10.
10 mol% (PPh3)4Pd, NaOH in DMF solvent under microwave
Suzuki coupling was performed with the above compound irradiation (450, 150 °C, 15 min). The ligand 2-(4-(phenylethy-
(10) and benzene boronic acid (11a) in the presence of 10 mol% nyl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (14a) was
(PPh3)4Pd and NaOH in DMF under microwave irradiation (450 isolated in a high yield. This ligand was fully characterized by
W, 150 °C, 15 min). The desired ligand 2-([1,1′-biphenyl]-4-yl)- 1H and 13C NMR, IR and mass spectroscopy. All 15 aromatic
1H-imidazo[4,5-f][1,10]phenanthroline (12a) was isolated in a protons of ligand 14a appeared in the region of
high yield. The ligand 12a was fully characterized by 1H, 13C 7.45–9.01 ppm in the 1H NMR spectra.
δ
NMR, IR and mass spectroscopy. All 15 aromatic protons of
The four protons of the phenanthroline ring appeared as a
compound 12a appeared in the region of δ 7.43–9.02 ppm in multiplet in the most downfield region of δ 8.95–9.01 ppm.
1
the H NMR spectra. The proton adjacent to the nitrogen atom There was an absence of C–Br stretching at 619 cm−1 in the
of the phenanthroline ring appeared as a broad singlet in the IR spectrum, which indicated the formation of the C–C
most downfield region of δ 9.02 ppm. In the 13C NMR spectra, coupled product. The scope of this chemistry was also
10 quaternary carbons peaks and 8 CH peaks were also extended to the other Sonogashira analogues (14b–d). The
observed. There was an absence of the C–Br stretching peak at ligand 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzene-
619 cm−1 in the IR spectra, which indicated the formation of 1,3-diol (18a) was prepared in a conventional way by the con-
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densation of 1,10-phenanthroline-5,6-dione (8) and 2,4-dihy- firmed the formation of complex 16a. The characteristic split-
droxybenzaldehyde (17), taken in a 1 : 1 proportion in the pres- ting pattern of the isotopes of ruthenium were also observed in
ence of NH4OAc and glacial acetic acid. The ligand 18a was the mass spectra. The same procedure was followed for the syn-
fully characterized by 1H and 13C NMR, IR and mass spec- thesis of complexes 16b, 17a–d and 19a–c using various
troscopy. All nine aromatic protons of the ligand appeared in ligands. In complex 17a, the ligand peaks were observed in the
the region of 6.46–9.00 ppm in the 1H NMR spectra. The ranges of δ 7.47–9.84 ppm. The methyl group of the p-cymene
protons adjacent to the nitrogen atom of the phenanthroline ring appeared as a singlet at 2.2 ppm, the six methyl protons as
ring appeared as a doublet in the most downfield region of δ a doublet at 0.89–0.91 ppm and the CH proton as a multiplet at
9.02–9.03 ppm and the meta proton of the 2,4-dihydroxyben- 2.60–2.63 ppm. The four aromatic protons of the p-cymene ring
zaldehyde ring appeared as a singlet at δ 6.46 ppm. The O–H appeared as distinct doublets at 6.10–6.12 ppm and
bending at 1404 cm−1 and C–O stretching at 1066 cm−1 con- 6.33–6.35 ppm. The LCMS peak at m/z: 667.2 [M]+ confirmed
firmed the formation of ligand 18a. The scope of this chem- the formation of complex 17a. The complexes 19a–c were also
istry was further extended to 18b–c.
characterized in a similar fashion.
The Ru(II)-p-cymene complex (16a) of ligand 12a was pre-
pared by dissolving the dichloro(p-cymene)ruthenium(II)
dimer (15) in an adequate amount of methanol and adding 2.1
equivalents of ligand 12a to the reaction mixture followed by
magnetic stirring for 2 h. After a change in colour from deep
yellow to deep orange, 2.5 equivalents of NH4PF6 was added
and stirred for another 1 h (Scheme 1). The complex 16a was
fully characterized by 1H and 13C NMR, IR and mass spec-
troscopy. Due to the formation of a metal complex, the 1H
NMR splitting pattern were changed slightly and all the peaks
were shifted towards the downfield region, i.e. the ligand peaks
in the region of 7.43–9.02 ppm were shifted to 7.43–9.85 ppm
in the complex. The characteristic methyl protons of the
p-cymene ring appeared as a singlet at 2.2 ppm, while the six
methyl protons appeared as a doublet at 0.88–0.90 ppm and
the CH proton as a multiplet at 2.58–2.62 ppm. The four aro-
matic protons of the p-cymene ring appeared as respective
doublets at 6.08–6.10 ppm and 6.32–6.34 ppm. All 19 aromatic
protons were observed in the region of δ 6.08–9.85 ppm in the
1H NMR spectra. The characteristic peaks of phosphorous were
observed in the region of δ −153.02 to −135.46 ppm in the 31P
NMR spectra; whereas specific fluorine peaks of PF6 were
found at δ −71.03 and −69.14 ppm. The P–F stretching at
831 cm−1, sp3 C–H stretching at 2924 cm−1 and sp3 C–H
bending at 1460 cm−1 in the IR spectra indicated the formation
of a Ru(II) complex. The LCMS peak at m/z: 643.3 [M]+ con-
UV-VIS and fluorescence study
To emphasize the cellular imaging properties of the syn-
thesized Ru(II)-arene complexes (16a–b, 17a–d, 19a–c), UV and
fluorescent studies were conducted in 5% DMSO in PBS at
room temperature (Fig. S1†). We observed the λmax values of
these complexes were in the region of 250–450 nm in the UV
spectra. Most of the complexes exhibited strong absorption
peak at 280–300 nm due to π → π* transition and a lower
energy broad absorption band at 350–450 nm region due to
metal-to-ligand charge transfer (MLCT). In order to know the
quantum yields (Φf) of these complexes, the molecules were
excited at λmax (300–360 nm) and the fluorescence was also
recorded (Table 1). Φf was calculated by using eqn (ii). Here,
we used quinine sulphate as a standard for calculating the
emission of the quantum yield (Table 1). From Table 1, it is
evident that all the compounds were moderate to highly fluo-
rescent, and among them, compound 17a showed the highest
quantum yield (Φf) of 0.003.
Solubility, lipophilicity and conductivity study
Equilibrium between the hydrophilicity and lipophilicity is
highly necessary to maintain the tumour-inhibiting potential
of metal complexes. These Ru(II)–arene complexes were highly
soluble in DMSO, DMF and have moderate to good solubility
in H2O, MeOH, EtOH and CH3CN and poor solubility in hydro-
Table 1 Selected physico-chemical data of the synthesized organoruthenium complexes (16a–b, 17a–d and 19a–c)
h
ΛM
λmax
λfb
Stoke’s
εd
a
Samples
(nm) (nm) shift
Peak area ODc
(M−1 cm−1
)
(ϕf)e
Solubilityf (M) log Pg
DMSO 10% aq DMSO
16a
16b
17a
17b
17c
17d
19a
19b
19c
336
304
354
356
360
340
342
344
344
432
438
440
442
442
434
442
436
440
452
96
134
86
86
82
94
100
92
96
102
434.182
920.323
1544.612
1137.83
664.172
831.862
545.378
369.054
685.346
56 001
0.70
0.569 18 966
0.44
0.27
0.263 8766
0.21 7000
0.165 5500
0.405 13 500
0.267 8900
23 333
0.0005 0.026
0.0013 0.024
0.89 0.03 65
162
160
92
94
95
96
84
86
86
1.10 0.09 60
0.92 0.06 25
0.94 0.07 27
0.88 0.08 25
0.82 0.02 28
0.49 0.06 28
0.79 0.03 32
0.78 0.08 30
14 666
9000
0.003
0.003
0.002
0.003
0.003
0.016
0.018
0.012
0.019
0.029
0.0007 0.026
0.002
0.564
0.025
—
Qninine sulphate 350
0.07
—
—
—
a Absorption maxima. b Emission wavelength. c Optical density. d Extinction coefficient. e Quantum yield. f DMSO-10% DMEM medium (1 : 99 v/v,
comparable to cell media). g n-Octanol/water partition coefficient. h Conductance in DMSO and 10% aqueous DMSO.
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carbon solvents. These complexes were soluble in the range of most widely using method to identify this type of interaction.
5–10 mg per ml of DMSO-10% DMEM medium (1 : 99 v/v, com- Here, we selected three different types of Ru(II)–arene com-
parable to cell media) at 25 °C, which is essential for delivering plexes (16b, 17a and 19b) for measuring the CT-DNA binding
the drug into the target. The lipophilicity of these complexes strength, which was performed by electronic absorption titra-
was measured by an n-octanol/water partition coefficient tion and ethidium bromide displacement assay.
(log P) study.58 To compare the lipophilic properties of the
The λmax values of the complexes 16b, 17a and 19b were
ruthenium complexes (16a–b, 17a–d, 19a–c), we estimated the observed at 291, 292 and 288 nm respectively, while the λmax of
n-octanol/water partition coefficient (log P) using the shake CT-DNA alone appeared at 258 nm. A decrease in wavelength
flask method (Table 1). The experimental log P values of the (i.e., hypsochromic shift) was observed (Fig. S3†) upon the first
complexes were obtained in the range 0.49–1.1. Complex 16b addition of CT-DNA to the complex solution. We also observed
exhibited the highest order of log P due to the lipophilic an increase in absorbance (i.e. hyperchromism) upon increas-
nature of its methyl group. The lowest order of log P value was ing CT-DNA addition to the metal complex. The binding con-
observed in compound 19a due to presence of hydrophilic stant (Kb) values calculated from eqn (i) were 4.4 × 103, 3.49 ×
–OH groups. The ruthenium complexes 17a–d and 19a–c 103 and 5.5 × 103 M−1 for 16b, 17a and 19b, respectively
exhibited the molar conductance values of ∼25–30 S m2 M−1 (Fig. S4,† and Table 2). These high binding constant values
in pure DMSO. While, complexes 16a–b displayed a greater and significant hyperchromism in absorbance and the hypso-
conductance (∼60–65 S m2 M−1) in DMSO. The molar conduc- chromic shift in wavelength after each addition of CT-DNA
tance of all these complexes were drastically increased in 10% indicated an electrostatic mode of binding for these complexes
aqueous DMSO (∼80–160 S m2 M−1, Table 1), suggesting their with DNA.
1 : 1 electrolytic nature in both media due to the charged
Ethidium bromide (EtBr) binding study. The addition of
ruthenium ions.59 The significant change in conductance in Ru(II) complexes 16b, 17a and 19b in the EtBr-CT-DNA adduct
aqueous and non-aqueous media suggests a high rate of dis- caused a decrease in fluorescence intensity (Fig. S5†) as the
sociation of chloride ligands from the ruthenium(II) complex. complexes displaced EtBr from the CT-DNA grooves so that the
Moreover, the conductance value increased gradually with complexes themselves bound to the DNA base pairs. The exci-
time (Table S1†). It is noteworthy that at lower pH, the conduc- tation wavelengths for the EtBr-bound DNA and complexes
tance values of the complexes steadily increased (Table S2†). 16b, 17a and 19b were at 485 nm and the recorded emission
Similar results were also observed in higher DNA and GSH wavelengths were at 600 nm. The concentrations of DNA and
concentrations (Tables S3 and S4†). All these results EtBr were 120 and 8 μM, respectively. The calculated value of
sufficiently supported the DNA covalent interaction through Kapp for the complexes were observed as Kapp = 2.6 × 106 M−1
aqua complex formation in a cancerous environment (low PH, (eqn (iii), Table 2). The value of KEtBr found from literature was
high GSH).
1 × 107 M−1. The Stern–Volmer quenching constant (KSV
values calculated from eqn (iv) for complexes 16b, 17a and 19b
were 0.04 × 105, 0.01 × 105 and 0.1 × 105 −1, respectively
)
Stability study of the complexes by UV-Vis spectroscopy
M
The stabilities of the three complexes, namely 16b, 17a and (Table 2).
19b, were tested in 1% DMSO-PBS medium in order to assess Viscosity measurements. In order to find out the binding
them as a potent therapeutic agent, as the complexes need to mode of drugs with DNA, a hydrodynamic method, like a vis-
be stable in the biological environment of cell. These com- cosity study, needed to be conducted. Binding via intercalation
plexes were found to be fairly stable in DMSO-buffer system requires the adjacent base pairs separation by allowing the
from 0 h to the 48th h. There was no significant change in the drug molecules to enter into the DNA double helix, which
spectral pattern, as shown in Fig. S2,† which suggests the leads to an increase in the length of DNA and their viscosity.
stability of the complexes in MTT solvent and indicates they The interaction of a drug via groove binding or electrostatic
can be used for various cellular activities.
interaction does not change the relative viscosity of DNA, as
the molecule does not change the length of DNA upon
DNA binding study
Electronic absorption spectral studies. DNA is one of the
most crucial pharmacological targets for various FDA-approved
anticancer metallodrugs, like carboplatin, cisplatin, oxliplatin
and organic drugs (doxorubicin, gemcitabine, 5-fluorouracil,
etc.).60 Various photophysical properties and structural diversi-
ties make these metal complexes good for use as DNA foot-
printing agents, molecular light-switches, sensors, and charge
transfer agents, as well as useful for the site-specific reco-
gnition of DNA, and as diagnostic probes and for use in
therapeutics.61,62 Therefore, the interaction of metal complexes
with DNA is an effective strategy for designing effective che-
motherapeutic drugs. Electronic absorption titration is the
Table 2 Binding parameters for the interaction of complexes 16b, 17a
and 19b with CT-DNA
Complex
Kb a (M−1
)
KSV b (M−1
)
Kapp c (M−1
)
16b
17a
19b
4.4 × 103
0.04 × 105
0.01 × 105
0.1 × 105
2.7 × 106
2.4 × 106
2.0 × 106
3.49 × 103
5.5 × 103
a Kb, intrinsic DNA binding constant from UV-visible absorption titra-
b
c
tion. KSV, Stern–Volmer quenching constant. Kapp, apparent DNA
binding constant from competitive displacement from fluorescence
spectroscopy.
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Table 3 Binding parameters for the interaction of complexes 16b, 17a
and 19b with BSA
binding. A partial or non-classical intercalation of the com-
pounds can bend or kink DNA, resulting in a decrease in its
effective length, with a concomitant decrease in its viscosity.
When a molecule binds covalently to DNA, it results in an
unwinding and bending of the DNA double helix. This results
in a decrease in the effective length of DNA, which ultimately
decreases the relative viscosity of DNA solution. The viscosity
study of our prepared complex (16b) showed that initially, up
to 10 µM concentration, it exhibited a strong intercalative
capability towards DNA, which was evident from the steady
increase of viscosity from 0–10 µM concentration, and after
that it showed a decent intercalation with DNA molecules up
to 50 µM concentration, marked by the slow increase of vis-
cosity with the increase in complex concentration (Fig. S6†).
However, with a gradual increase in concentration after 50 µM
onwards, this drug dramatically modified itself as a good
electrostatic or groove binder to DNA which was apparent from
the line parallel to the X-axis in the viscosity graph. Again, if
we cast our vision beyond a 10 µM concentration, it is clear
that the drug binds to the DNA base pair covalently with the
gradual decrease in complex concentration, which could be
confirmed by the steady decrease in viscosity, which was also
consistent with the study of the conductance in the presence
of DNA, whereby the conductance increases with the gradual
decrease in ri value, indicating the covalent binding nature of
the complex. From that point of view, we can say that our justi-
fication perfectly fitted with all the experimental results, ensur-
ing the possibility of the binding ability of all our metal com-
plexes with DNA through different modes.
Complex
KBSAa (M−1
)
Kqb (M−1 s−1
)
Kc (M−1
)
nd
16b
17a
19b
0.58 × 106
0.26 × 106
2.01 × 106
0.58 × 1014
0.26 × 1014
2.01 × 1014
8.74 × 103
4.39 × 103
32.7 × 103
0.90
0.82
0.698
a KBSA, Stern–Volmer quenching constant. b Kq, quenching rate con-
stant. c K, binding constant with BSA. d n, number of binding sites.
Table 4 MTT cytotoxicity screening of Ru(II)-arene complexes (16a–b,
17a–d, 19a–c) at 24 h of drug exposure
IC50 (µM)a
SFb
Cell lines
Compounds
HeLac
Caco-2c
HEK-293
HeLa
Caco-2
16a
16b
17a
17b
17c
17d
19a
19b
6.7 0.7
1.2 0.9
3.4 0.8
17.6 1.4
3.2 0.5
15.3 1.6
>100
4.2 0.8
3.1 1.2
—
11.8 0.6
9.0 0.4
9.1 0.8
13.4 0.9
9.1 0.6
26.8 1.2
29.2 1.1
12.8 0.9
11.1 1.1
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
32.2 1.8
55.2 1.2
>14.9
>83.3
>29.4
>5.7
>31.2
>6.5
—
>23.8
>32.2
—
>8.4
>11.1
>10.9
>7.4
>11.1
>3.7
8.74
>7.8
>9.0
—
19c
DMSO
CisPlatin
RAPTA-C
20 0.8
16.2 0.5
11 0.6
11.9 0.8
1.61
3.40
2.93
5.17
a IC50 is the concentration at which 50% of cells undergo cytotoxic cell
death due to organoruthenium or cisplatin treatment. b SF (selectivity
factor) = ratio of IC50 for HEK-293 and IC50 for all the cancer cell lines.
HEK-293 fibroblasts are generally selected as the model for healthy
cells in the evaluation of chemotherapeutic drug selectivity. c 24 h
incubation time for the HeLa and Caco-2 cell lines.
BSA binding study. The gradual decrease in fluorescence
intensity (Fig. S7†) with the subsequent increase in concen-
trations of 16b, 17a and 19b confirmed the binding of the
complexes with BSA. The excitation wavelength used for this
titration study was 295 nm, while the emission was observed at
350 nm. The Stern–Volmer quenching constant for BSA fluo-
rescence (KBSA), calculated using eqn (v), was found to be 0.58 cate. The cells were treated with these ruthenium complexes
× 106, 0.26 × 106 and 2.0 × 106 M−1 for 16b, 17a and 19b, along with cisplatin and RAPTA-C as the standard positive
respectively (Fig. S8†). The highest value of KBSA of compound control with variable concentrations (0–200 μM) for 24 h. Most
19b among the other two is due to the presence of polar of the ruthenium complexes exhibited higher potency in HeLa
groups, like OH, which aid its binding with the polar amino and Caco-2 cell lines than cisplatin and RAPTA-C (Table 4). It
acids of BSA. Scatchard plot analysis gave binding affinity (K) was noteworthy that the majority of the compounds were 3–83-
values for 16b, 17a and 19b of 8.74 × 103, 4.39 × 103 and 32.7 × fold more selective with cancer cells (HeLa, Caco-2) than with
103 M−1, respectively (eqn (vi), Fig. S9†). These high binding normal human embryonic kidney cells (HEK-293).
constant values confirm the occurrence of strong interactions Nevertheless, cisplatin and RAPTA-C exhibited insignificant
among BSA and ruthenium complexes, which is essential for cytoselectivity with those cancer cell lines. Among the syn-
transport of the complexes across the biological system. The thesized ruthenium complexes, compound 16b presented the
number of sites (n) that BSA can make available to bind com- greatest potency and selectivity with both cell lines.
plexes 16b, 17a and 19b was found to be 0.9, 0.82 and 0.698,
Structure–activity relationship (SAR) study. Ru(II)-p-cymene
respectively (Table 3).
imidazophenanthroline complexes displayed excellent potency
Cytotoxic activity. Cytotoxicity study of all the synthesized in both cancer cell lines with high selectivity (Fig. 4).
Ru(II)-arene complexes (16a–b, 17a–d, 19a–c) was performed
These types of ruthenium complexes having planner imida-
via a typical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- zophenanthroline ligands induce cancer cell apoptosis via an
lium bromide (MTT) assay protocol together with a panel of effective DNA intercalation. The methyl group attached to the
cancer cell lines, i.e. colorectal adenocarcinoma cells (Caco-2), para position of biphenyl imidazophenanthroline ligands
human epitheloid cervix carcinoma (HeLa), and one normal enhanced the selective cellular uptake of complex 16b in
cell line, i.e. human embryonic kidney cells (HEK-293) in tripli- cancer cells, which induced apoptosis via DNA intercalation.
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(50 μM). After 2 h of incubation, the complex-treated cells were
excited through a 450–500 nm excitation filter for fluorescence
microscopy analysis. Live cells were tracked by the red fluo-
rescence of the compounds under a fluorescence microscope
(Fig. 5). These imaging results represent clear evidence of the
strong cellular uptake of complex 17a at 2 h incubation in the
HeLa cell line compared to a lesser time of incubation.
Experimental section
Materials and methods
All the reagents and solvents used had the highest commercial
quality. Organic solvents used for the chemical synthesis and
for chromatography were acquired from E. Merck (India) and
were of analytical grade. Dichloro-p-cymene ruthenium(II)
chloride, 1,10-phenanthroline-5,6-dione, 4-bromobenzalde-
hyde, organoboronic acids, phenyl acetylene and its deriva-
Fig. 4 SAR of Ru(II)-arene imidazophenanthroline complexes.
However, the ruthenium complexes having ((ethynyl)phenyl)-
1H-imidazo[4,5-f][1,10]phenanthroline ligands (17a–d) dis-
played lower efficacy than complex 16b. This is because of the
slightly non-planner nature of their ligands. Among these four
scaffolds, complexes 17a and 17c showed comparable efficacy
with complex 16b. After introducing benzene 1,3 diol at the
2-position of the imidazophenanthroline ligand, the potency
of complex 19a was drastically reduced because of the presence
of two hydrophilic –OH groups. However, compounds 19b and
19c showed significant cytoselectivity with both cancer cells
more than with normal cells. The lipophilic methyl and
methoxy groups of these complexes increase the cellular
accumulation. The presence of one –OH group also facilitates
the interaction with BSA followed by drug transportation.
Live-cell-imaging study. We performed cellular imaging
experiments using the HeLa cell line. The cells were incubated
for different times (30 min, 1 h, 2 h) at 37 °C with complex 17a
tives,
2,4-dihydroxybenzaldehyde,
2-hydroxy-4-methyl-
benzaldehyde, 2-hydroxy-4-methoxybenzaldehyde and tetrakis
(triphenylphosphine)palladium(0) were purchased from Sigma
Aldrich Chemical Ltd, Merk and Spectrochem. Microwave reac-
tions were performed in catalytic systems. HeLa and HEK-293
cell lines were purchased from NCCS, Pune. The Caco-2 cell
line was procured from ATCC. CT-DNA and BSA were pur-
1
chased from Sigma Aldrich Chemical Ltd. H NMR, 13C NMR,
19F NMR and 31P NMR spectra were recorded on a Bruker DPX
spectrometer at 400 MHz with tetramethylsilane as the
internal standard and the chemical shifts has been reported
herein in ppm units. The melting points of the complexes
were measured on an Elchem Microprocessor DT apparatus
using an open capillary tube and are uncorrected herein. The
synthesized compounds were also characterized using
a
Schimadzu LCMS-4000 LC-MS instrument, having 4000 triple
quadraupole MS, using methanol as the solvent. TLC was per-
formed on pre-coated silica gel 60 F254 aluminium sheets
(E. Merck, Germany) using methanol/ethyl acetate mixture as
the solvent system. UV-Visible spectra were recorded on a
JASCO V-730 spectrometer and fluorescence measurements
were carried out using an HITACHI fluorescence spectrophoto-
meter equipped with a xenon lamp. An Elisa reader and
96-well plates were used for the MTT assay. The fluorescence
imaging study was performed using an Olympus model
CKX41 microscope.
Synthetic procedure of 2-(4-bromophenyl)-1H-Imidazo[4,5-f]
[1,10]phenanthroline (10)
First, 500 mg (2.380 mmol) of 1,10-phenanthroline-5,6-dione
(8) was taken in a 50 ml round-bottom flask followed by the
addition of 484 mg (2.617 mmol, 1.1 equiv.) of 4-bromobenzal-
dehyde (9) and 1.46 g (19.03 mmol, 8 equiv.) of ammonium
acetate. All the reagents were dissolved in 10 ml of glacial
acetic acid and the mixture was kept in reflux for 24 h. TLC
was monitored to observe the change in the reaction mixture.
As soon as the reaction was over, the reaction mixture was
poured into ice cold water and concentrated ammonia was
Fig. 5 Fluorescence images of live HeLa cells with compound 17a (a)
and control HeLa cell without drug in bright field; (b) 17a + cell under
green filter [50 µM in PBS buffer; incubation time 30 min] (c) 17a + cell
in green filter [50 µM in PBS buffer; incubation time 1 h]; (d) 17a + cell in
green filter [50 µM in PBS buffer; incubation time 2 h] Scale bar 200 µm.
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added to neutralize the solution. Then, the above-neutralized bending (1571), Arm CvC stretching (1477), sp3 C–H bending
solution was allowed to settle for 30 min and was then filtered. (1448), C–N stretching (1352), C–H bending (736).
The yellowish–orange product was dried and recrystallized
Synthesis of ((phenylethynyl)phenyl)imidazo[4,5-f][1,10]
Phenanthroline analogues (14a–d)
from ethyl acetate in a high yield (∼96%).
2-(4-Bromophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (10).
Yield: 96%; m.p: 250–252 °C; Rf: 0.54 (ethyl acetate : methanol First, 50 mg (0.133 mmol) of compound 2-(4-bromophenyl)-
in 3 : 1); 1H NMR (DMSO-d6, 400 MHz): δ 7.78–7.83 (m, 4H, 1H-imidazo[4,5-f][1,10]phenanthroline (10), 2 equivalent of
ArH), 8.23 (d, 2H, J = 8 Hz, ArH), 8.91 (d, 2H, J = 8 Hz, ArH), phenylacetylene derivatives (13a–d), 0.5 mol% of (PPh3)4Pd,
9.01 (d, 2H, J = 4 Hz, ArH); 13C (DMSO-d6, 100 MHz): δ 122.0 20 mol% of NaOH and 4 ml of DMF were taken in a microwave
(CH), 123.3 (C), 123.8 (C), 128.6 (C), 129.8 (CH), 130.2 (CH), flask. Microwave-assisted reaction was performed for 15 min,
131.7 (CH), 132.4 (C), 143.9 (CH), 148.2 (C), 150.2 (C); at 450 W and a temperature of 150 °C. The completion of the
IR (cm−1): ν sp2 C–H stretching (3176), Arm C–H stretching reaction was marked by a colour change of the reaction
(3026), N–H bending (1664), Arm CvC stretching (1400), C–N mixture from yellow to dark brown. TLC was monitored to
stretching (1352), C–H bending (719), C–Br stretching (619); confirm the completion of the reaction. The mixture was then
LCMS (MeOH): m/z: 375.2 [M + H]+.
poured into a crushed-ice medium followed by extraction with
ethyl acetate using a separating funnel. After 4–5 times
washing, all the organic layers were collected and dried with
anhydrous sodium sulphate and the solvent was evaporated
Synthesis of biphenyl imidazo[4,5-f][1,10]phenanthroline
(12a–b)
First, 50 mg (0.133 mmol) of compound 2-(4-bromophenyl)- using a rotary evaporator. The dried product was washed with
1H-imidazo[4,5-f][1,10]phenanthroline (10), 1.3 equivalent of hexane followed by recrystallization from ethyl acetate. Finally,
aryl boronic acid (11a–b), 0.5 mol% of (PPh3)4Pd, 20 mol% of dark yellow needle-like crystals of compounds 14a–d were
NaOH in water and 4 mL of DMF were taken in a microwave obtained in high yields (∼90–95%).
flask. Microwave-assisted reaction was performed for 15 min,
2-(4-(Phenylethynyl)phenyl)-1H-imidazo[4,5-f][1,10]phenan-
at 450 W and a temperature of 150 °C. The completion of the throline (14a). Yield: 90%; m.p: 200–220 °C; Rf: 0.59 (ethyl
reaction was marked by a colour change of the reaction acetate : methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz):
mixture from orange to dark brown. TLC was monitored to δ 7.46 (d, 2H, J = 8 Hz, ArH), 7.58 (m, 4H, ArH), 7.70 (d, 1H, J =
confirm the completion of the reaction. The mixture was then 8 Hz, ArH), 7.80 (t, 2H, J = 4 Hz, ArH), 8.42 (d, 1H, J = 8 Hz,
poured into a crushed-ice medium followed by extraction with ArH), 8.58 (s, 1H, ArH), 8.98 (m, 4H, ArH); 13C (DMSO-d6,
ethyl acetate using a separating funnel. Then, the organic layer 400 MHz): δ 123.5 (CH), 126.8 (C), 129.2 (C), 129.3 (CH), 130.3
(i.e. ethyl acetate) was separated and the water layer was (CH), 131.9 (C), 131.9 (CH), 132.0 (C), 132.2 (C); IR (cm−1): ν sp
drained off. After 4–5 times washing, the organic layers were C–H stretching (3298), N–H bending (1583), Arm CvC stretch-
collected and dried with anhydrous sodium sulphate and the ing (1479), C–N stretching (1348), C–H bending (738).
solvent was evaporated using a rotary evaporator. The dried
2-(4-(p-Tolylethynyl)phenyl)-1H-imidazo[4,5-f][1,10]phenan-
product was washed with hexane followed by recrystallization throline (14b). Yield: 92%; m.p: 185–187 °C; Rf: 0.52 (ethyl
from ethyl acetate. Finally, brown needle-shaped crystals of acetate : methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz):
compounds 12a–b were obtained in high yields (∼85–90%).
δ 2.29 (s, 3H, CH3), 7.22 (d, 2H, J = 8 Hz, ArH), 7.33 (d, 2H, J =
2-([1,1′-Biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenanthro- 8 Hz, ArH), 7.72 (m, 4H, ArH), 8.23 (d, 1H, J = 8 Hz, ArH), 8.36
line (12a). Yield: 85%; m.p: 175–177 °C; Rf: 0.7 (ethyl acetate : (d, 1H, J = 8 Hz, ArH), 8.89 (d, 4H, J = 4 Hz, ArH); 13C (DMSO-
1
methanol = 1 : 1); H NMR (DMSO-d6, 400 MHz): δ 7.43 (s, 1H, d6, 100 MHz): δ 123.3 (CH), 129.0 (C), 129.2 (C), 129.3 (CH),
ArH), 7.50 (t, 2H, J = 8 Hz, ArH), 7.78 (d, 2H, J = 8 Hz, ArH), 129.4 (CH), 130.1 (C), 131.9 (CH), 132.0 (C); IR (cm−1): ν N–H
7.84 (t, 2H, J = 4 Hz, ArH), 7.91 (d, 2H, J = 8 Hz, ArH), 8.38 (d, stretching (3317), sp3 C–H stretching (2922), N–H bending
2H, J = 8 Hz, ArH), 8.96 (d, 2H, J = 8 Hz, ArH), 9.02 (brs, 2H, (1658), Arm CvC stretching (1438), C–N stretching (1352),
ArH); 13C (DMSO-d6, 100 MHz): δ 123.8 (CH), 127.1 (C), 127.3 C–H bending (696).
(C), 127.6 (C), 127.6 (C), 127.8 (CH), 128.4 (CH), 129.1 (CH),
2-(4-((4-Methoxyphenyl)ethynyl)phenyl)-1H-imidazo[4,5-f]
129.4 (CH), 129.5 (CH), 129.6 (CH), 130.2 (C), 130.6 (C), 139.7 [1,10]phenanthroline (14c). Yield: 93%; m.p: 180–182 °C; Rf:
(C), 141.5 (C), 143.9 (CH), 148.3 (C), 150.9 (C); IR (cm−1): ν Arm 0.64 (ethyl acetate : methanol in 1 : 1); 1H NMR (DMSO-d6,
C–H stretching (3032), N–H bending (1664), Arm CvC stretch- 400 MHz): δ 3.78 (s, 3H, OCH3), 7.23 (d, 1H, J = 8 Hz, ArH),
ing (1400), C–N stretching (1072), C–H bending (732); LCMS 7.43 (d, 1H, J = 8 Hz, ArH), 7.59 (m, 4H, ArH), 7.83 (t, 2H, J =
(MeOH): m/z: 373.1 [M + H]+.
4 Hz, ArH), 8.20 (d, 1H, J = 8 Hz, ArH), 8.32 (d, 1H, J = 8 Hz,
2-(4′-Methyl-[1,1′-biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phe- ArH), 8.98 (m, 4H, ArH); 13C (DMSO-d6, 400 MHz): δ 55.6
nanthroline (12b). Yield: 90%; m.p: 192–194 °C; Rf: 0.62 (ethyl (OCH3), 114.3 (CH), 123.8 (C), 126.7 (CH), 126.8 (C), 129.2 (C),
acetate : methanol = 1 : 1); 1H NMR (DMSO-d6, 400 MHz): 129.3 (C), 129.4 (CH), 130.4 (CH), 130.4 (CH), 131.8 (C), 131.9
δ 2.37 (s, 3H, CH3), 7.33 (d, 2H, J = 8 Hz, ArH), 7.69 (d, 2H, J = (CH), 132.6 (C), 143.7 (C), 148.2 (C); IR (cm−1): ν N–H stretch-
8 Hz, ArH), 7.85 (m, 2H, ArH), 7.91 (d, 2H, J = 8 Hz, ArH), 8.37 ing (3280), sp3 C–H stretching (2931), N–H bending (1600),
(d, 2H, J = 8 Hz, ArH), 8.96 (d, 2H, J = 8 Hz, ArH), 9.05 (d, 2H, Arm CvC stretching (1444), C–N stretching (1350), C–O
J = 4 Hz, ArH); IR (cm−1): ν Arm C–H stretching (3061), N–H stretching (1247), C–H bending (736).
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2-(4-(Pyridin-2-ylethynyl)phenyl)-1H-imidazo[4,5-f][1,10]phe- 123.7 (C), 124.2 (C), 127.5 (CH), 130.1 (CH), 143.8 (CH), 148.2
nanthroline (14d). Yield: 95%; m.p: 168–170 °C; Rf: 0.42 (ethyl (C), 151.9 (C), 159.6 (C), 162.2 (C); IR (cm−1): ν O–H stretching
acetate : methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz): (3180), Arm C–H stretching (3061), sp3 C–H stretching (2933),
δ 7.62–7.65 (m, 2H, ArH), 7.71 (d, 1H, J = 8 Hz, ArH), 7.87 (d, N–H bending (1631), Arm CvC stretching (1589), O–H
4H, J = 8 Hz, ArH), 8.38 (d, 2H, J = 8 Hz, ArH), 8.65 (d, 1H, J = 4 bending (1481), C–N stretching (1386), C–O stretching (1261),
Hz, ArH), 8.95 (d, 2H, J = 8 Hz, ArH), 9.05 (d, 2H, J = 4 Hz, C–H bending (734).
ArH); 13C (DMSO-d6, 400 MHz): δ 126.9 (CH), 127.9 (CH), 129.2
Synthesis of [(η6-p-cymene)RuCl(K2-N,N-2-(biphenyl)-1H-Imidazo
[4,5-f][1,10]phenanthroline)]·PF6 analogues (16a–b)
(CH), 129.3 (C), 131.9 (C), 132.0 (CH), 132.5 (C), 132.9 (C);
IR (cm−1): ν N–H stretching (3346), Arm C–H stretching (3059),
CuC stretching (2216), N–H bending (1581), Arm CvC First, 20 mg (0.033 mmol) of dichloro(p-cymene)ruthenium(II)
stretching (1431), C–N stretching (1348), C–H bending (738).
dimer (15) was dissolved in 7 ml of methanol in a 50 ml
round-bottom flask and stirred for 10 min to dissolve the com-
pound in methanol. Thereafter, 2.1 equivalent of the pre-
viously prepared ligand 12a,b (12a: 25.8 mg, 12b: 26.78 mg)
Synthesis of (imidazo[4,5-f][1,10]phenanthrolin-2-yl) phenol
analogues (18a–c)
First, 50 mg (0.238 mmol) of 1,10-phenanthroline-5,6-dione (8) was added to the reaction mixture followed by magnetic stir-
was taken in a 25 ml round-bottom flask and 1.1 equivalent of ring for another 2 h. A change in colour occurred from deep
2-hydroxybenzaldehyde derivatives (17a–c) was added followed yellow to deep orange. Then, 13 mg (0.082 mmol, 2.5 equiv.) of
by 147 mg (1.903 mmol, 8 equiv.) of ammonium acetate. The NH4PF6 was added to the reaction mixture and stirred for
reagents were dissolved in 5 ml of glacial acetic acid and kept another 1 h. The reaction was monitored by TLC using 100%
in reflux for 24 h. TLC was monitored to observe the change in methanol as the solvent system. After completion of the reac-
the reaction. As soon as the reaction was completed, the reac- tion, the methanol was evaporated off using a rotary evapor-
tion mixture was poured in ice-cold water and concentrated ator and the crude product was further recrystallized from a
ammonia was added in drops to neutralize the solution. Then, diethyl ether/methanol mixture. Orange fine crystals of 16a–b
the above neutralized solution was allowed to settle for were isolated in high yields (∼96–97%).
30 min. The precipitate formed was then filtered. The products
18a–c were dried and recrystallized from ethyl acetate in high imidazo[4,5-f][1,10]phenanthroline)]·PF6 (16a). Yield: 49.9 mg
yields (∼96–97%).
(0.0634 mmol, 97%). Mr (C35H30N4ClF6PRu) = 788.13 g mol−1
[(η6-p-Cymene)RuCl(K2-N,N-2-([1,1′-biphenyl]-4-yl)-1H-
.
4-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzene-1,3-diol Anal. Calcd for C35H30N4ClF6PRu: C 53.34, H 3.84, N 7.11;
(18a). Yield: 96%; m.p: 224–226 °C; Rf: 0.35 (ethyl acetate : found: C 53.06; H 3.44; N 7.40; m.p: 164–166 °C; Rf (100%
methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz): δ 6.46 (s, 1H, methanol): 0.52; 1H NMR (DMSO-d6, 400 MHz): δ 0.89 (d, 6H, J
ArH), 6.50 (d, 1H, J = 8 Hz, ArH), 7.82 (m, 2H, ArH), 7.99 (d, = 8 Hz, cymene isopropyl-CH3), 2.2 (s, 3H, cymene CH3),
1H, J = 8 Hz, ArH), 8.88 (d, 2H, J = 8 Hz, ArH), 9.02 (d, 2H, J = 2.58–2.64 (m, 1H, cymene CH), 6.09 (d, 2H, J = 8 Hz, cymene
4 Hz, ArH); 13C (DMSO-d6, 400 MHz): solubility problem; ArH), 6.33 (d, 2H, J = 8 Hz, cymene ArH), 7.48 (t, 1H, ArH),
IR (cm−1): ν sp2 C–H stretching (3062), Arm C–H stretching 7.53 (d, 2H, J = 8 Hz, ArH), 7.80 (d, 2H, J = 8 Hz, ArH), 7.95 (d,
(2978), N–H bending (1627), Arm CvC stretching (1477), O–H 2H, J = 8 Hz, ArH), 8.20 (m, 2H, ArH), 8.43 (d, 2H, J = 8 Hz,
bending (1404), C–N stretching (1253), C–O stretching (1066), ArH), 9.30 (d, 2H, J = 8 Hz, ArH), 9.84 (d, 2H, J = 4 Hz, ArH);
C–H bending (736).
13C NMR (DMSO-d6, 100 MHz): δ 18.3 (Me, p-cymene), 21.9
2-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)-5-methylphenol (Me, p-cymene), 22.1 (Me, p-cymene), 30.5 (CH, p-cymene),
(18b). Yield: 97%; m.p: 190–192 °C; Rf: 0.85 (ethyl acetate : 84.4 (ArCH, p-cymene), 85.9 (ArCH, p-cymene), 86.7 (ArCH,
methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz): δ 2.31 (s, 3H, p-cymene), 86.8 (ArCH, p-cymene), 100.6 (ArC, p-cymene),
CH3), 6.79 (d, 2H, J = 16 Hz, ArH), 7.77 (d, 2H, J = 4 Hz, ArH), 103.6 (ArC, p-cymene), 106.9, 126.8, 127.2, 129.1, 129.7, 132.7,
8.08 (d, 1H, J = 8 Hz, ArH), 8.87 (d, 2H, J = 8 Hz, ArH), 8.98 (d, 133.0, 143.6, 154.4; 19F NMR (DMSO-d6, 376 MHz): δ −71.03
2H, J = 4 Hz, ArH), 9.10 (s, 1H, OH); 13C (DMSO-d6, 100 MHz): (PF6), −69.14 (PF6); 31P NMR (DMSO-d6, 162 MHz): δ −153.02
δ 21.6 (CH3), 117.6 (CH), 123.6 (CH), 124.4 (CH), 126.3 (CH), (PF6), −148.64 (PF6), −144.24 (PF6), −139.85 (PF6), −135.46
130.1 (C), 131.0 (C), 147.7 (C); IR (cm−1): ν Arm C–H stretching (PF6); IR (cm−1): ν N–H stretching (3369), Arm C–H stretching
(2920), sp3 C–H stretching (2849), O–H stretching (2743), N–H (3124), sp3 C–H stretching (2924), N–H bending (1604), sp3
bending (1635), Arm CvC stretching (1550), O–H bending C–H bending (1460), Arm CvC stretching (1406), C–N stretch-
(1400), C–N stretching (1355), C–O stretching (1066), C–H ing (1199), P–F stretching (831), C–H bending (769); LCMS
bending (734).
(MeOH): m/z: 643.3 [M]+.
2-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)-5-methoxyphe-
[(η6-p-Cymene)RuCl(K2-N,N-2-(4′-methyl-[1,1′-biphenyl]-4-yl)-
nol (18c). Yield: 96%; m.p: 182–184 °C; Rf: 0.5 (ethyl acetate : 1H-Imidazo[4,5-f][1,10]phenanthroline)]·PF6 (16b). 50.3 mg
methanol in 1 : 1); 1H NMR (DMSO-d6, 400 MHz): δ 3.84 (s, 3H, (0.0627 mmol, 96%); Mr (C36H32N4ClF6PRu) = 802.15 g mol−1
;
OCH3), 6.59 (s, 1H, ArH), 6.65 (d, 1H, J = 4 Hz, ArH), 7.78 (m, Anal. Calcd for C36H32N4ClF6PRu: C 53.90, H 4.02, N 6.98;
2H, ArH), 8.07 (d, 1H, J = 8 Hz, ArH), 8.84 (d, 2H, J = 12 Hz, Found: C 53.56; H 3.84; N 7.34; m.p: 170–172 °C; Rf (100%
ArH), 8.99 (d, 2H, J = 4 Hz, ArH), 9.09 (s, 1H, OH); 13C (DMSO- methanol): 0.67; 1H NMR (DMSO-d6, 400 MHz): δ 0.90–0.92 (d,
d6, 400 MHz): δ 55.8 (OCH3), 101.9 (CH), 106.8 (C), 107.0 (CH), 6H, J = 8 Hz, cymene isopropyl-CH3), 2.21 (s, 3H, cymene CH3),
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2.38 (s, 1H, CH3, ligand), 2.58–2.64 (m, 1H, cymene CH), 6.11 154.4; 19F NMR (DMSO-d6, 376 MHz): δ −71.08 (PF6), −69.19
(d, 2H, J = 8 Hz, cymene ArH), 6.34 (d, 2H, J = 8 Hz, cymene (PF6); 31P NMR (DMSO-d6, 162 MHz): δ −152.99 (PF6), −148.60
ArH), 7.34 (d, 2H, ArH), 7.71 (d, 2H, J = 8 Hz, ArH), 7.86 (d, 1H, (PF6), −144.21 (PF6), −139.82 (PF6), −135.43 (PF6); IR (cm−1): ν
J = 8 Hz, ArH), 7.95 (d, 1H, J = 8 Hz, ArH), 8.20–8.24 (m, 2H, N–H stretching (3616, 3379), sp C–H stretching (3336), Arm
ArH), 8.29–8.31 (d, 1H, J = 8 Hz, ArH), 8.42 (d, 1H, J = 8 Hz, C–H stretching (3145), sp3 C–H stretching (2964), N–H
ArH), 9.30 (d, 2H, J = 4 Hz, ArH), 9.86 (d, 2H, J = 4 Hz, ArH); bending (1606), sp3 C–H bending (1458), Arm CvC stretching
13C NMR (DMSO-d6, 100 MHz): δ 18.8 (Me, p-cymene), 21.9 (1367), C–N stretching (1199), P–F stretching (831), C–H
(Me, p-cymene), 22.0 (Me, p-cymene), 24.4 (Me, ligand), 30.9 bending (692); LCMS (MeOH): m/z: 667.2 [M]+.
(CH, p-cymene), 84.3 (ArCH, p-cymene), 85.9 (ArCH,
p-cymene), 86.7 (ArCH, p-cymene), 86.8 (ArCH, p-cymene), Imidazo[4,5-f][1,10]Phenanthroline)]·PF6 (17b). 52.9 mg
100.7 (ArC, p-cymene), 104.3 (ArC, p-cymene), 107.1, 126.6, (0.0640 mmol, 98%); Mr (C38H32N4ClF6PRu) = 826.15 g mol−1
[(η6-p-Cymene)RuCl(K2-N,N-2-(4-(P-tolylethynyl)Phenyl)-1H-
;
126.8, 127.0, 127.5, 127.7, 129.3, 129.7, 130.2, 132.9, 143.6, Anal. Calcd for C38H32N4ClF6PRu: C 55.24, H 3.90, N 6.78;
154.2; 19F NMR (DMSO-d6, 376 MHz): δ −71.07 (PF6), −69.19 found: C 55.56; H 3.54; N 7.10; m.p: 146–148 °C; Rf (100%
1
(PF6); 31P NMR (DMSO-d6, 162 MHz): δ −157.38 (PF6), −152.98 methanol): 0.71; H NMR (DMSO-d6, 400 MHz): δ 0.90 (d, 6H,
(PF6), −148.60 (PF6), −144.20 (PF6), −139.81 (PF6), −135.43 J = 8 Hz, cymene isopropyl-CH3), 2.2 (s, 3H, cymene CH3), 2.29
(PF6), −131.03 (PF6); IR (cm−1): ν sp2 C–H stretching (3142), (s, 3H, CH3), 2.60–2.68 (m, 1H, cymene CH), 6.11 (d, 2H, J =
Arm C–H stretching (3049), sp3 C–H stretching (2968), N–H 8 Hz, cymene ArH), 6.33 (d, 2H, J = 8 Hz, cymene ArH), 7.46 (d,
bending (1606), sp3 C–H bending (1450), Arm CvC stretching 2H, J = 8 Hz, ArH), 7.57–7.65 (m, 4H, ArH), 8.21 (s, 2H, ArH),
(1406), C–N stretching (1116), P–F stretching (831), C–H 8.26 (d, 1H, J = 8 Hz, ArH), 8.38 (d, 1H, J = 8 Hz, ArH), 9.33 (s,
bending (721); LCMS (MeOH): m/z: 657.8 [M]+.
2H, ArH), 9.86 (s, 2H, ArH); 13C NMR (DMSO-d6, 100 MHz):
18.3 (Me, p-cymene), 21.9 (Me, p-cymene), 22.1 (Me,
p-cymene), 24.4 (Me, ligand), 30.5 (CH, p-cymene), 82.3 (ArCH,
p-cymene), 84.4 (ArCH, p-cymene), 85.9 (ArCH, p-cymene), 86.8
δ
Synthesis of [(η6-p-cymene)RuCl(K2-N,N-2-(ethynyl)phenyl)-1H-
Imidazo[4,5-f][1,10]phenanthroline]·PF6 analogues (17a–d)
First, 20 mg (0.033 mmol) of dichloro(p-cymene)ruthenium(II) (ArCH, p-cymene), 100.6 (ArC, p-cymene), 102.2 (ArC,
dimer (15) was dissolved in 7 ml of methanol in a 50 ml p-cymene), 107.0, 129.5, 126.5, 126.8, 127.0, 129.0, 129.2,
round-bottom flask and stirred for 10 min to dissolve the com- 129.2, 129.3, 129.5, 129.7, 131.9, 132.0, 132.9, 143.6, 154.3; 19F
pound in the methanol. Thereafter, 2.1 equivalent of the pre- NMR (DMSO-d6, 376 MHz): δ −71.07 (PF6), −69.18 (PF6); 31P
viously prepared ligand 14a–d (14a: 27.5 mg, 14b: 28.5 mg, NMR (DMSO-d6, 162 MHz): δ −152.98 (PF6), −148.60 (PF6),
14c: 29.5 mg, 14d: 27.5 mg) was added to the reaction mixture −144.20 (PF6), −139.82 (PF6), −135.43 (PF6); IR (cm−1): ν N–H
followed by magnetic stirring for another 2 h. A change in stretching (3317), Arm C–H stretching (3161), N–H bending
colour occurred from deep yellow to brown. Then, 13 mg (1606), Arm CvC stretching (1409), C–N stretching (1116), P–F
(0.082 mmol, 2.5 equiv.) of NH4PF6 was added to the reaction stretching (831), C–H bending (696); LCMS (MeOH): m/z:
mixture and stirred for another 1 h. The reaction was moni- 681.4 [M]+.
tored by TLC using 100% methanol as the solvent system.
After completion of the reaction, the methanol was evaporated phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline)]·PF6
[(η6-p-Cymene)RuCl(K2-N,N-2-(4-((4-methoxyphenyl)ethynyl)
(17c).
off using a rotary evaporator and the crude product was 52.8 mg (0.0627 mmol, 96%); Mr (C38H32N4OClF6PRu) =
further recrystallized from a diethyl ether/methanol mixture. 842.17 g mol−1; Anal. Calcd for C38H32N4OClF6PRu: C 54.19, H
Orange fine crystals of 17a–d were obtained in high yields 3.83, N 6.65; found: C 54.46; H 3.64; N 6.90; m.p: 138–140 °C;
(∼96–98%).
Rf (100% methanol): 0.67; 1H NMR (DMSO-d6, 400 MHz): δ
[(η6-p-Cymene)RuCl(K2-N,N-2-(4-(phenylethynyl)phenyl)- 0.91 (d, 6H, J = 8 Hz, cymene isopropyl-CH3), 2.2 (s, 3H,
1H-imidazo[4,5-f][1,10]phenanthroline)]·PF6 (17a). 51.5 mg cymene CH3), 2.60–2.68 (m, 1H, cymene CH), 3.75 (s, 3H,
(0.0634 mmol, 97%); Mr (C37H30N4ClF6PRu) = 812.15 g mol−1
;
OCH3), 6.10 (d, 2H, J = 8 Hz, cymene ArH), 6.34 (d, 2H, J =
Anal. Calcd for C37H30N4ClF6PRu: C 54.72, H 3.72, N 6.90; 8 Hz, cymene ArH), 7.58 (s, 2H, ArH), 7.60–7.65 (m, 4H, ArH),
found: C 54.36; H 3.44; N 7.28; m.p: 172–174 °C; Rf (100% 8.21 (t, 2H, J = 4 Hz, ArH), 8.25 (d, 1H, J = 8 Hz, ArH), 8.37 (d,
1
methanol): 0.81; H NMR (DMSO-d6, 400 MHz): δ 0.89 (d, 6H, 1H, J = 8 Hz, ArH), 9.32 (d, 2H, J = 8 Hz, ArH), 9.87 (d, 2H, J = 4
J = 8 Hz, cymene isopropyl-CH3), 2.2 (s, 3H, cymene CH3), Hz, ArH); 13C NMR (DMSO-d6, 100 MHz): δ 18.8 (Me,
2.60–2.63 (m, 1H, cymene CH), 6.11 (d, 2H, J = 8 Hz, cymene p-cymene), 21.9 (Me, p-cymene), 22.0 (Me, p-cymene), 30.9
ArH), 6.34 (d, 2H, J = 8 Hz, cymene ArH), 7.47 (d, 2H, J = 4 Hz, (CH, p-cymene), 55.5 (OMe, ligand), 84.2 (ArCH, p-cymene),
ArH), 7.62 (d, 4H, J = 4 Hz, ArH), 7.79 (d, 1H, J = 8 Hz, ArH), 85.9 (ArCH, p-cymene), 86.7 (ArCH, p-cymene), 86.8 (ArCH,
8.19 (d, 2H, J = 4 Hz, ArH), 8.44 (d, 2H, J = 8 Hz, ArH), 9.29 (m, p-cymene), 100.7 (ArC, p-cymene), 103.9 (ArC, p-cymene),
2H, ArH), 9.82 (d, 2H, J = 8 Hz, ArH); 13C NMR (DMSO-d6, 107.1, 114.3, 126.5, 126.8, 127.1, 129.2, 129.3, 129.7, 130.4,
100 MHz): δ 18.3 (Me, p-cymene), 21.9 (Me, p-cymene), 22.1 131.8, 131.9, 132.9, 143.6, 154.2; 19F NMR (DMSO-d6,
(Me, p-cymene), 30.5 (CH, p-cymene), 82.3 (ArCH, p-cymene), 376 MHz): δ −71.08 (PF6), −69.19 (PF6); 31P NMR (DMSO-d6,
84.4 (ArCH, p-cymene), 85.9 (ArCH, p-cymene), 86.8 (ArCH, 162 MHz): δ −152.98 (PF6), −148.60 (PF6), −144.20 (PF6),
p-cymene), 96.9, 100.6 (ArC, p-cymene), 104.4 (ArC, p-cymene), −139.82 (PF6), −135.43 (PF6); IR (cm−1): ν Arm C–H stretching
107.0, 126.8, 127.5, 129.3, 130.3, 131.0, 131.9, 133.0, 143.8, (3147), sp3 C–H stretching (2962), N–H bending (1602), Arm
Dalton Trans.
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CvC stretching (1408), C–O stretching (1249), C–N stretching J = 4 Hz, ArH) 9.94 (d, 1H, OH), 9.97 (d, 1H, OH); 13C NMR
(1174), P–F stretching (831), C–H bending (721); LCMS (DMSO-d6, 100 MHz): δ 18.7 (Me, p-cymene), 21.8 (Me,
(MeOH): m/z: 693.3 [M]+.
p-cymene), 21.9 (Me, p-cymene), 31.1 (CH, p-cymene), 84.2
[(η6-p-Cymene)RuCl(K2-N,N-2-(4-(pyridin-2-yl-ethynyl)phenyl)- (ArCH, p-cymene), 85.9 (ArCH, p-cymene), 86.7 (ArCH,
1H-imidazo[4,5-f][1,10]phenanthroline)]·PF6 (17d). 51.5 mg p-cymene), 86.8 (ArCH, p-cymene), 100.8 (ArC, p-cymene),
(0.0634 mmol, 97%); Mr (C36H29N5ClF6PRu) = 813.13 g mol−1
;
103.6 (ArC, p-cymene), 104.3, 107.2, 126.7, 129.4, 132.9, 143.3,
Anal. Calcd for C36H29N5ClF6PRu: C 53.18, H 3.59, N 8.61; 154.2, 159.4, 161.3; 19F NMR (DMSO-d6, 376 MHz): δ −71.07
found: C 53.42; H 3.79; N 8.20; m.p: 134–136 °C; Rf (100% (PF6), −69.18 (PF6); 31P NMR (DMSO-d6, 162 MHz): δ −157.38
1
methanol): 0.62; H NMR (DMSO-d6, 3 MHz): δ 0.93 (d, 6H, J = (PF6), −152.98 (PF6), −148.60 (PF6), −144.20 (PF6), −139.81
8 Hz, cymene isopropyl-CH3), 2.2 (s, 3H, cymene CH3), (PF6), −135.43 (PF6) −131.03 (PF6); IR (cm−1): ν sp2 C–H
2.63–2.68 (m, 1H, cymene CH), 6.13 (d, 2H, J = 8 Hz, cymene stretching (3053), Arm C–H stretching (2968), N–H bending
ArH), 6.35 (d, 2H, J = 4 Hz, cymene ArH), 7.55–7.58 (m, 2H, (1606), sp3 C–H bending (1409), Arm CvC stretching (1325),
ArH), 7.62–7.67 (m, 4H, ArH), 8.22–8.25 (m, 2H, ArH), 8.40 (d, C–N stretching (1147), C–O stretching (1055), P–F stretching
2H, J = 8 Hz, ArH), 9.32 (d, 2H, J = 4 Hz, ArH), 9.88 (d, 2H, J = (833), C–H bending (721); LCMS (MeOH): m/z: 599.7 [M]+.
4 Hz, ArH); 13C NMR (DMSO-d6, 100 MHz): δ 18.7 (Me,
[(η6-p-Cymene)RuCl(K2-N,N-2-(1H-imidazolo[4,5-f][1,10]
p-cymene), 21.9 (Me, p-cymene), 22.0 (Me, p-cymene), 31.1 phenanthroline-2-yl)-5-methylphenol)]·PF6 (19b). 47.0 mg
(CH, p-cymene), 84.3 (ArCH, p-cymene), 84.7 (ArCH, (0.0634 mmol, 97%); Mr (C30H28N4OClF6PRu)
= 742.06
p-cymene), 85.9 (ArCH, p-cymene), 86.8 (ArCH, p-cymene), g mol−1; Anal. Calcd for C30H28N4OClF6PRu: C 48.56, H 3.80,
100.8 (ArC, p-cymene), 103.9 (ArC, p-cymene), 107.1, 126.5, N 7.55; found: C 48.26; H 3.44; N 7.70; m.p: 142–144 °C; Rf
1
126.7, 129.2, 129.3, 129.4, 131.8, 131.9, 132.7, 143.8, 154.2; 19F (100% methanol): 0.61; H NMR (DMSO-d6, 400 MHz): δ 0.86
NMR (DMSO-d6, 376 MHz): δ −71.07 (PF6), −69.18 (PF6); 31P (d, 6H, J = 8 Hz, cymene isopropyl-CH3), 2.18 (s, 3H, cymene
NMR (DMSO-d6, 162 MHz): δ −157.38 (PF6), −152.98 (PF6), CH3), 2.32 (s, 3H, CH3), 2.55–2.61 (m, 1H, cymene CH), 6.07
−148.60 (PF6), −144.20 (PF6), −139.81 (PF6), −135.43 (PF6); IR (d, 2H, J = 8 Hz, cymene ArH), 6.30 (d, 2H, J = 8 Hz, cymene
(cm−1): ν Arm C–H stretching (3048), N–H bending (1600), Arm ArH), 8.12 (t, 3H, J = 4 Hz, ArH), 8.20 (d, 1H, J = 8 Hz, ArH),
CvC stretching (1462), C–N stretching (1157), P–F stretching 8.27 (d, 1H, J = 8 Hz, ArH), 9.27 (d, 2H, J = 8 Hz, ArH), 9.77 (d,
(833), C–H bending (721). LCMS (MeOH): m/z: 668.1 [M]+.
1H, J = 4 Hz, ArH) 9.95 (d, 1H, J = 8 Hz, ArH); 13C NMR
(DMSO-d6, 100 MHz): δ 18.3 (Me, p-cymene), 21.9 (Me,
p-cymene), 22.1 (Me, p-cymene), 24.2 (Me, ligand), 31.1 (CH,
p-cymene), 84.3 (ArCH, p-cymene), 85.9 (ArCH, p-cymene), 86.7
Synthesis of [(η6-p-cymene)RuCl(K2-N,N-4-(1H-imidazo[4,5-f][1,10]
phenanthrolin-2-yl)phenol)]·PF6 analogues (19a–c)
First, 20 mg (0.033 mmol) of dichloro(p-cymene)ruthenium(II) (ArCH, p-cymene), 86.8 (ArCH, p-cymene), 100.7 (ArC,
dimer (15) was dissolved in 7 ml of methanol in a 50 ml p-cymene), 104.4 (ArC, p-cymene), 107.0, 118.0, 119.7, 121.0,
round-bottom flask and stirred for 10 min to dissolve the com- 133.0, 140.5, 143.5, 154.4, 157.5, 162.0; 19F NMR (DMSO-d6,
pound in the methanol. Thereafter, 2.1 equivalent of the pre- 376 MHz): δ −71.07 (PF6), −69.18 (PF6); 31P NMR (DMSO-d6,
viously prepared ligand 18a–c (18a: 22.8 mg, 18b: 22.6 mg, 18c: 162 MHz): δ −157.38 (PF6), −152.99 (PF6), −148.60 (PF6),
23.7 mg) was added to the reaction mixture followed by mag- −144.21 (PF6), −139.82 (PF6), −135.43 (PF6); IR (cm−1): ν Arm
netic stirring for another 2 h. A change in colour occurred C–H stretching (3140), sp3 C–H stretching (3047), N–H
from deep yellow to black. Then, 13 mg (0.082 mmol, bending (1606), sp3 C–H bending (1409), Arm CvC stretching
2.5 equiv.) of NH4PF6 was added to the reaction mixture and (1352), C–N stretching (1201), C–O stretching (1147), P–F
the mixture stirred for another 1 h. The reaction was moni- stretching (831), C–H bending (721); LCMS (MeOH): m/z:
tored by TLC using 100% methanol as the solvent system. 597.7 [M]+.
After completion of the reaction, the methanol was evaporated
off using a rotary evaporator and the crude product was nanthroline-2-Yl)-5-methoxyphenol)]·PF6 (19c). 48.1 mg
further recrystallized from a diethyl ether/methanol mixture. (0.0634 mmol, 97%); Mr (C30H29N4O2ClF6PRu) 759.06
Black fine crystals of complexes 19a–c were obtained in high g mol−1; Anal. Calcd for C30H29N4O2ClF6PRu: C 47.47, H 3.85,
N 7.38; found: C 47.86; H 3.64; N 7.60; m.p: 138–140 °C; Rf
[(η6-p-Cymene)RuCl(K2-N,N-2-(1H-imidazo[4,5-f][1,10]phe-
=
yields (∼95–97%).
1
[(η6-p-Cymene)RuCl(K2-N,N-4-(1H-imidazo[4,5-f][1,10]phe- (100% methanol): 0.53; H NMR (DMSO-d6, 400 MHz): δ 0.89
nanthrolin-2-yl)benzene 1,3 diol)]·PF6 (19a). 47.1 mg (d, 6H, J = 8 Hz, cymene isopropyl-CH3), 2.18 (s, 3H, cymene
(0.0634 mmol, 97%); Mr (C29H26N4O2ClF6PRu) = 744.03 g CH3), 2.58–2.67 (m, 1H, cymene CH), 3.84 (s, 3H, OCH3), 6.11
mol−1; Anal. Calcd for C29H26N4O2ClF6PRu: C 46.81, H 3.52, N (d, 2H, J = 8 Hz, cymene ArH), 6.34 (d, 2H, J = 8 Hz, cymene
7.53; Found: C 46.46; H 3.81; N 7.70; m.p: 178–180 °C; Rf ArH), 8.18 (m, 3H, ArH), 8.27 (d, 2H, J = 8 Hz, ArH), 9.37 (d,
1
(100% methanol): 0.47; H NMR (DMSO-d6, 400 MHz): δ 0.90 2H, J = 8 Hz, ArH), 9.84 (d, 2H, J = 8 Hz, ArH) 9.96 (m, 1H,
(d, 6H, J = 8 Hz, cymene isopropyl-CH3), 2.19 (s, 3H, cymene OH); 13C NMR (DMSO-d6, 100 MHz): δ 18.7 (Me, p-cymene),
CH3), 2.57–2.66 (m, 1H, cymene CH), 6.10 (d, 2H, J = 8 Hz, 21.9 (Me, p-cymene), 22.0 (Me, p-cymene), 31.1 (CH,
cymene ArH), 6.33 (d, 2H, J = 8 Hz, cymene ArH), 6.48 (s, 1H, p-cymene), 55.9 (OMe, ligand), 84.3 (ArCH, p-cymene), 85.9
ArH), 6.53 (d, 1H, J = 8 Hz, ArH), 8.12–8.17 (m, 1H, ArH), 8.19 (ArCH, p-cymene), 86.7 (ArCH, p-cymene), 86.8 (ArCH,
(d, 2H, J = 4 Hz, ArH), 9.35 (d, 2H, J = 8 Hz, ArH), 9.84 (d, 2H, p-cymene), 102.0 (ArC, p-cymene), 103.6 (ArC, p-cymene),
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Dalton Transactions
104.3, 107.3, 126.6, 128.5, 132.9, 143.4, 154.2, 159.4, 162.8; 19F sample was equilibrated with CT-DNA for about 5 min then
NMR (DMSO-d6, 376 MHz): δ −71.08 (PF6), −69.19 (PF6); 31P absorbance of the complex was measured. The intrinsic DNA
NMR (DMSO-d6, 162 MHz): δ −152.98 (PF6), −148.59 (PF6), binding constant (Kb) was calculated using the eqn (i):
−144.20 (PF6), −139.81 (PF6), −135.43 (PF6), −131.03 (PF6);
½DNAꢀ
½DNAꢀ
1
IR (cm−1): ν Arm C–H stretching (3053), sp3 C–H stretching
(2935), N–H bending (1627), Arm CvC stretching (1446), C–N
stretching (1261), C–O stretching (1151), P–F stretching (833),
C–H bending (721); LCMS (MeOH): m/z: 613.4 [M]+.
¼
þ
ðiÞ
ðεa ꢁ εf Þ ðεb ꢁ εf Þ Kbðεa ꢁ εf Þ
where [DNA] is the concentration of DNA in the base pairs, εa
is the apparent extinction coefficient observed for the complex,
εf corresponds to the extinction coefficient of the complex in
its free form and εb refers to the extinction coefficient of the
complex when fully bound to DNA. Data were plotted using
Origin Lab, version 8.5 to obtain the [DNA]/(εa − εf) vs. [DNA]
linear plot. The ratio of the slope to the intercept from the
linear fit gives the value of the intrinsic binding constant (Kb).
In vitro cytotoxic study
In vitro cytotoxicity was evaluated using the standard MTT
assay protocol.63 The synthesized complexes (16a–b, 17a–d and
19a–c) were dissolved in 0.1% DMSO and then serially diluted
with DMEM medium. Two cancer cell lines, i.e. human epithe-
lioid cervix carcinoma (HeLa), human epithelial colorectal
adenocarcinoma cells (Caco-2), and one normal kidney cell
(HEK 293), were used in this assay. Approximately 1 × 104 cells
per well were cultured in 100 μl of a growth medium in 96-well
plates and incubated at 37 °C under a 5% CO2 atmosphere.
The cells were then treated with different concentrations of the
drugs (0–200 µM) in a volume of 100 µM per well. Cisplatin
and RAPTA-C were used as standard positive control drugs.
Cells in the control wells also acquired the same volume of
medium containing 0.1% DMSO. After 24 h, the medium was
superfluous and the cell cultures were incubated with 100 μl
MTT reagent (1 mg ml−1) for 5 h at 37 °C. Then, the suspen-
sion was placed on a micro vibrator for 10 min followed by
recording the absorbance at λ = 620 nm in an ELISA reader.
The experiment was also performed in triplicate. The data
were represented as the growth inhibition percentage, i.e. %
growth inhibition = 100 − [(AD × 100)/AB], where AD is the
measured absorbance in the wells containing the samples and
AB is the measured absorbance for the blank wells (cells with
a medium and a vehicle only).
UV and fluorescence study
UV and fluorescence studies of all these Ru(II)complexes, were
performed in 5% DMSO-buffer solution. The fluorescence
quantum yields (Φ) were calculated by using the comparative
William’s method, which involves the use of a well-character-
ized standard with a known quantum yield value using 5%
DMSO in PBS buffer solution.65 Quinine sulphate was used as
the standard. The quantum yield was calculated according to
the eqn (ii):
IS ODR ηS
ϕ ¼ ϕRꢂ
ꢂ
ꢂ
ðiiÞ
IR ODS ηR
where ϕ is the quantum yield, I is the peak area, OD is the
absorbance at λmax and η is the refractive index of the solvent
(s) and reference (R). Here, we used quinine sulphate as the
standard for calculating the quantum yield.
Ethidium bromide displacement assay
The ethidium bromide (EtBr) displacement assay was carried
out to explain the mode of binding between the potent com-
pounds with DNA.66 The apparent binding constants (Kapp) of
the complexes 16b, 17a and 19b to CT-DNA were calculated
using ethidium bromide (EtBr) as a spectral probe in 5 mM
Tris-HCl buffer (pH 7.4). Here, EtBr does not exhibit any fluo-
rescence in its free form as its fluorescence is quenched by the
solvent molecules, but its fluorescence intensity increases in
the presence of CT-DNA, which suggests the intercalative
mode of binding of EtBr with DNA grooves. The fluorescence
intensity was found to decrease with a further increase in con-
centration of the complexes. Thus, it can be said that the com-
plexes displace EtBr from the CT-DNA grooves and the com-
plexes themselves then get bound to the DNA base pairs. The
values of the apparent binding constant (Kapp) were obtained
by using eqn (iii):
Stability study
The stabilities of the three Ru(II) complexes (16b, 17a and 19b)
were tested in 1% DMSO in PBS buffer as the complexes were
dissolved in this solution during their cytotoxicity and imaging
studies.
DNA binding study
The binding of the complexes with calf-thymus DNA (CT-DNA)
was observed by their electronic spectra and through a com-
petitive binding assay using ethidium bromide (EtBr) as a
quencher in fluorescence spectroscopy.
UV-visible studies
A DNA binding assay was carried out by using complexes 4, 4′,
5 and 5′ in Tris-HCl buffer (5 mM Tris-HCl in water, pH 7.4) in
a water medium.64 The concentration of CT-DNA was calcu-
lated from its absorbance intensity at 260 nm and its known
Kapp ꢂ ½Complexꢀ50 ¼ KEtBr ꢂ ½EtBrꢀ
ðiiiÞ
molar absorption coefficient value 6600 M−1 cm−1. An equal where KEtBr is the EtBr binding constant (KEtBr = 1.0 × 107
amount of DNA was taken both in the sample and in the refer- M−1), and [EtBr] = 8 × 10−6 M. The Stern–Volmer equation has
ence in cuvettes. Titration was carried out by increasing the been employed for the quantitative determination of the
concentration of CT-DNA. Before each measurement, each Stern–Volmer quenching constant (KSV).67 Origin Lab (8.5)
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software was used to plot the fluorescence data to obtain a n-Octanol–water partition coefficient (log P)
linear plot of I0/I vs. [complex]. The value of KSV was calculated
The log P values of the ruthenium complexes were determined
via the shake flask method using the previously published pro-
cedure.71 A known amount of each complex (16a–b, 17a–d and
19a–b) was suspended in water (pre-saturated with n-octanol)
and shaken for 48 h on an orbital shaker. To allow the phase
separation, the solution was centrifuged for 10 min at 3000
rpm. Then, the amount of ruthenium present in the saturated
aqueous solution was measured by ICP-MS. To obtain the par-
tition coefficient, different ratios (0.5 : 1, 1 : 1 and 2 : 1) of the
saturated solutions were shaken with pre-saturated n-octanol
for 20 min on an orbital shaker and followed the same
procedure.
from the following equation.
I0=I ¼ 1 þ KSV½Qꢀ
ðivÞ
where I0 is the fluorescence intensity in the absence of a
complex and I is the fluorescence intensity in the presence of a
complex of concentration [Q].
Protein-binding studies
Serum albumin proteins are major components in blood
plasma proteins and play significant roles in drug transport
and metabolism.68 The interaction of the drug with bovine
serum albumin (BSA), a structural homologue with human
serum albumin (HSA), has been studied in tryptophan emis-
sion quenching experiments. Tryptophan emission quenching
experiment was thus performed here to detect the interaction
of the ruthenium complexes 16b, 17a and 19b with protein
BSA. Initially, BSA solution (2 × 10−6 M) was prepared in Tris-
HCl/NaCl buffer. Aqueous solutions of the complexes were
subsequently added to the BSA solution with increasing their
concentrations. After each addition, the solutions were shaken
slowly for 5 min before recording the fluorescence at a wave-
length of 295 nm (λex = 295 nm). A gradual decrease in fluo-
rescence intensity of BSA at λ = 340 nm was observed upon
increasing the concentration of the complex, which confirmed
that an interaction between the complex and BSA had
occurred. The Stern–Volmer equation was employed to quanti-
tatively determine the quenching constant (KBSA). Origin Lab,
version 8.5 was used to plot the emission spectral data to
obtain a linear plot of I0/I vs. [complex] using the following
eqn (v):
Conclusion
In summary, we synthesized and characterized some novel imi-
dazophenanthroline ligands and their ruthenium(II)-p-cymene
complexes. The ligands were prepared by following a novel pro-
cedure of microwave-assisted Suzuki and Sonogashira cross-
coupling reactions. The stability study of the three complexes
16b, 17a and 19b in DMSO-buffer solution indicated that all
three complexes were fairly stable in DMSO-buffer solution
conditions from 0 to 48 h. From the DNA binding studies, it
was observed that the intrinsic binding constant (Kb) values
were in the order of 19b > 16b > 17a, whereas the apparent
binding constant (Kapp) was observed in the order of 16b > 17a
> 19b, which could be correlated to the fact that complex 16b
intercalates DNA double strands more effectively than other
derivatives because of its planarity. The significant hyperchro-
mism in the absorbance spectra of the DNA binding curve,
high conductance in aqueous DMSO, high Kapp value along
with the results from the viscosity study indicated a covalent
and non-covalent mode of binding of the complexes with
DNA. The BSA binding study also revealed that complex 19b
had a many folds greater binding efficiency with BSA com-
pared to 16b and 17a. However, the number of binding sites of
BSA was more for 16b. The cytotoxicity study indicated that
complex, 16b displayed the best potency and selectivity with
both cancer cells. Live-cell imaging with the moderately fluo-
rescent compound 17a in HeLa cell suggested that it was
intensely consumed by the HeLa cell after 2 h.
I0=I ¼ 1 þ KBSA½Qꢀ ¼ 1 þ kqτ0½Qꢀ
ðvÞ
where I0 is the fluorescence intensity of BSA in the absence of
the complex and I indicates the fluorescence intensity of BSA
in the presence of the complex of concentration [Q], τ0 is the
lifetime of the tryptophan in BSA and was found to be 1 × 10−8
and kq is the quenching constant. The Scatchard eqn (vi) was
used to give the binding properties of the complexes,69 where
K is the binding constant and n is the number of binding sites.
logðI0 ꢁ I=IÞ ¼ log K þ n log½Qꢀ
ðviÞ
Conflicts of interest
There are no conflicts to declare.
Conductivity measurement
To confirm the interaction of the complexes with water,
DMSO, GSH and CT-DNA solutions, conductivity tests of the
prepared complexes were performed using a conductivity-TDS
Acknowledgements
meter-307 (Systronics, India) and a cell constant of 1.0 cm−1 70
.
Authors are grateful to Department of Science and Technology,
The rate of conductivity was also measured in different pH Government of India for supporting the work through the
media. Time-dependent conductivity measurements were also DST-EMR project grant (EMR/2017/000816) and DST young
performed.
scientist grant (YSS/2014/000842). The authors are thankful to
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VIT University for providing VIT SEED funding. Authors are 21 L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins,
also thankful to DST-VITFIST for NMR and GC-MS for research
facilities.
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