Biological activity and photocatalytic properties of a naphthyl-imidazo phenanthroline (HNAIP) ligand and its [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl complexes

Article abstract of DOI:10.1016/j.jinorgbio.2019.110885

The synthesized 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline (HNAIP) ligand and its new iridium ([Ir(ppy)₂(HNAIP)]Cl) and rhodium ([Rh(ppy)₂(HNAIP)]Cl) complexes, being ppy = 2-phenylpiridinate, show cytotoxic effects in SW480 (colon adenocarcinoma) and A549 (epithelial lung adenocarcinoma) cells. They all are cytotoxic in the tested cell lines. HNAIP and [Rh(ppy)₂(HNAIP)]⁺ are the most cytotoxic, whereas [Ir(ppy)₂(HNAIP)]⁺ displays negligible cytotoxicity towards A549 cells and moderate activity towards SW480. The interaction of all three compounds with Bovine Serum Albumin (BSA), L-glutathione reduced (GSH), nicotinamide adenine dinucleotide (NADH) and DNA was studied to explain the differences found in terms of cytotoxicity. None of them are able to interact with BSA, thus excluding bioavailability due to plasma protein interaction as the possible differentiating factor in their biological activity. By contrast, small differences have been observed regarding DNA interaction. In addition, taking advantage of the emission properties of these molecules, they have been visualized in the cytoplasmic region of A549 cells. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show, in turn, that the internalization ability follow the sequence [Rh(ppy)₂(HNAIP)]⁺ > [Ir(ppy)₂(HNAIP)]⁺ > cisplatin. Therefore, it seems clear that the cellular uptake by tumour cells is the key factor affecting the different cytotoxicity of the metal complexes and that this cellular uptake is influenced by the hydrophobicity of the studied complexes. On the other hand, preliminary catalytic experiments performed on the photo-oxidation of GSH and some amino acids such as L-methionine (Met), L-cysteine (Cys) and L-tryptophan (Trp) provide evidence for the photocatalytic activity of the Ir(III) complex in this type of reactions.

Full text of DOI:10.1016/j.jinorgbio.2019.110885

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Biological activity and photocatalytic properties of a naphthyl-
imidazo
phenanthroline
(HNAIP)
ligand
and
its
[Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl complexes
Ana R. Rubio, Jairo Fidalgo, Judit Martin-Vargas, Cristina Pérez-
Arnaiz, Sara R. Alonso-Torre, Tarita Biver, Gustavo Espino,
Natalia Busto, Begoña García
PII:
S0162-0134(19)30493-3
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110885
JIB 110885
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
26 July 2019
7 October 2019
7 October 2019
Please cite this article as: A.R. Rubio, J. Fidalgo, J. Martin-Vargas, et al., Biological
activity and photocatalytic properties of a naphthyl-imidazo phenanthroline (HNAIP)
ligand and its [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl complexes, Journal of
Inorganic Biochemistry (2019), https://doi.org/10.1016/j.jinorgbio.2019.110885
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Biological Activity and Photocatalytic Properties of a
naphthyl-imidazo phenanthroline (HNAIP) ligand and its
[Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl complexes
Ana R. Rubio,a,& Jairo Fidalgo,a,& Judit Martin-Vargas,a,& Cristina Pérez-Arnaiz,a Sara R. Alonso-
Torre,b Tarita Biver,c Gustavo Espino, a* Natalia Busto,a * Begoña García a *
aDepartamento de Química, Universidad de Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos,
Spain. BG E-mail: begar@ubu.es
bDepartamento de Biotecnología y Ciencia de los Alimentos, Universidad de Burgos, Plaza Misael
Bañuelos s/n, 09001 Burgos, Spain.
cDipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Moruzzi 13, 56124 Pisa, Italy.
&JMV, JF and ARR contributed equally to this work.
In memoriam of Professor Juan Manuel Salas Peregrín and his inspiring contribution to the development of
Bioinorganic Chemistry
Abstract
The synthesized 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenantroline (HNAIP) ligand and its
new iridium ([Ir(ppy)2(HNAIP)]Cl) and rhodium ([Rh(ppy)2(HNAIP)]Cl) complexes, being ppy 2-
phenylpiridinate, show cytotoxic effects in SW480 (colon adenocarcinoma) and A549 (epithelial
lung adenocarcinoma) cells. They all are cytotoxic in the tested cell lines; HNAIP and
[Rh(ppy)2(HNAIP)]+ are most cytotoxic, whereas [Ir(ppy)2(HNAIP)]+ displays negligible cytotoxicity
towards A549 cells and moderate activity towards SW480. The interaction of all three compounds
with Bovine Serum Albumin (BSA), L-glutathione reduced (GSH), Nicotinamide adenine
dinucleotide (NADH) and DNA was studied to explain the differences found in terms of
cytotoxicity. None of them are able to interact with BSA, thus excluding bioavailability due to
plasma protein interaction as the possible differentiating factor in their biological activity. By
contrast, small differences have been observed regarding DNA interaction. In addition, taking
advantage of the emission properties of these molecules, they have been visualized in the
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cytoplasmic region of A549 cells. Inductively coupled plasma mass spectrometry (ICP-MS)
experiments show, in turn, that the internalization ability follow the sequence [Rh(ppy)2(HNAIP)]+
> [Ir(ppy)2(HNAIP)]+ > cisplatin. Therefore, it seems clear that the cellular uptake by tumour cells is
the key factor affecting the different cytotoxicity of the metal complexes and that this cellular
uptake is influenced by the hydrophobicity of the studied complexes. On the other hand,
preliminary catalytic experiments performed on the photo-oxidation of GSH and some amino acids
such as L-methionine (Met), L-cysteine (Cys) and L-tryptophan (Trp) provide evidence for the
photocatalytic activity of the Ir(III) complex in this type of reactions.
Keywords: 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline; Iridium; Rhodium; cellular
test; DNA; photo-catalysis.
Introduction
The intense work on metal complexes aimed at solving the shortcomings of current cisplatinum-
like drugs and similar chemotherapeutic compounds is key to current medicinal chemistry [1–3].
Recent relevant studies are based on testing transition metal complexes other than platinum(II).
As a notable instance of second generation metal-based anticancer agents, Ru(II) metal complexes
such as (H2im)[RuIIICl4(DMSO)(Him)] (Himꢀ=ꢀ1H-imidazole) (NAMI-A) or trans-[tetrachlorobis(1H-
indazole)ruthenate(III)] (KP1019) have reached different phases of clinical trial [4–6]. To date,
other less explored transition metal ions such as gold, rhodium, iridium, iron, osmium, palladium,
silver, antimony, bismuth, copper and tin have aroused increasing attention [1,7–12]. Until very
recently, Rh(III) and particularly Ir(III) complexes were generally regarded as unsuitable candidates
as anticancer agents owing to the typical kinetic inertness of their transition metal centres.
Nowadays, due to their readily tuneable properties (attainable by changing the ancillary ligand),
amphiphilic nature and relative accessibility, Ir(III) and Rh(III) complexes have become promising
alternatives to platinum and ruthenium complexes [13–16]. The influence of the ligands in the
biological activity of metal complexes is highly relevant; in some cases the ligand is not cytotoxic,
whereas the metal complex is [14,17]. In other cases, the cytotoxicity is provided by the ligand,
because the free ligand is toxic by itself [18]. However, very often the toxicity of the free ligands is
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not studied in the literature [16,19,20] and the cytotoxicity is attributed to the complex. For this
reason, it is advisable to study the effect of the ligand on the cytotoxicity of the metal complex;
this comparison should be routine work.
Half-sandwich arene Ir(III) and Rh(III) metal complexes with diimine ligands display potent
anticancer properties related to DNA intercalation [16,21,22] or perturbation of the cellular redox
state [19,23,24].
Cyclometallated Ir(III) and Rh(III) complexes have been synthesized as potential diagnostic agents.
Octahedral iridium(III) complexes of the type fac-[IrCl3(DMSO)(N^N)] (N^N = bipyridine,
phenanthroline, dipyridoquinoxaline, dipyrido[3,2-a:2’,3’-c]phenazine or benzo[i]-dipyrido[3,2-
a:2’,3’-c]phenazine) are cytotoxic against lung and colon tumour cell lines, whose activity depends
on the aromatic ligand and the presence of the fac-isomer. In spite of the aromatic ligands, DNA
intercalation was not the biological mechanism of action of these complexes [25]. A similar
Rhodium compound showed higher antiproliferative activity toward MCF-7 breast cancer cells
compared to its Iridium analogue. Recently, studies on the properties of octahedral Rh(III)
complexes of general formula [(C^N)2Rh(dppz)]Cl (where C^N = 2-phenylpyridinate type ligands
and dppz = dipyrido[3,2-a:2′,3′-c]phenazine) have demonstrated that the activity depends not only
on the DNA intercalator dppz, but also on the electronic and steric features of the C,N-chelating
ligands [26]. -[Rh(bpy)2(chrysi)]3+ (chrysi =5,6-chrysenquinonediimine) is able to intercalate into a
specific region of DNA and recognize a single mismatch in a 2725 base pair plasmid [27].
It is known that the imidazole unit efficiently interacts with biomolecules and displays biological
activity. Imidazo-phenanthroline-based ligands derivatives exhibit excellent light emitting
efficiency [28] and attracted our attention because of their potential anticancer activity [29].
However, phenanthroimidazole derivatives exhibit poor solubility, which severely hindered their
further biological application. Coordination of this scaffold to iridium(III) and rhodium(III) would
lead to significant improvement in solubility.
Bearing all this in mind, the 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline organic
ligand (HNAIP)[30] was used to synthesize M(III) complexes denoted as [M(HNAIP)(ppy)2]+ where
ppy = 2-phenylpiridinate and M = Ir(III) and Rh(III) (Scheme 1). The ability of the ligand and the two
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transition metal complexes synthesized to induce cellular damage was tested and their possible
mode of action was studied through biosubstrates-binding tests.
Experimental
Starting materials. RhCl3·xH2O and IrCl3·xH2O were purchased from Johnson Mattey and used as
received. The starting dimers [Rh(ppy)2(μ-Cl)]2, and [Ir(ppy)2(μ-Cl)]2 (ppy = 2-phenylpyridinate)
were prepared according to literature procedures [31]. The HNAIP ligand was prepared according
to a literature protocol [32,33]. The other reactants were purchased as follows: Phenanthroline
(Alfa Aesar), glacial acetic acid (VWR), 2-hydroxy-1-naphthaldehyde (SigmaAldrich), ammonium
acetate (Panreac), L-glutathione reduced (SigmaAldrich) and NADH (SigmaAldrich). CD3SOCD3 was
obtained from Euriso-top.
Synthesis methods and characterization of complexes. All synthetic manipulations were carried
out under an atmosphere of dry, oxygen-free nitrogen using standard Schlenk techniques. The
solvents were dried and distilled under nitrogen atmosphere before use. Elemental analyses were
performed with a Thermo Fisher Scientific EA Flash 2000 Elemental Microanalyzer. The analytical
data for the new compounds were obtained from crystalline samples. The discrepancy between
the calculated and the obtained values for carbon was > 0.4 %; introduction of solvent molecules
in the molecular formulae improved the agreement. HR ESI (+) Mass spectra (position of the peaks
in Da) were recorded with an Agilent LC-MS system (1260 Infinity LC / Quadrupole – Time of Flight
Mass Spectrometry (6545 Q-TOF MS spectrometer). Dichloromethane (DCM) was used as the
sample solvent and aqueous 0.1 % HCOOH/MeOH as the mobile phase. NMR samples were
prepared under nitrogen atmosphere by dissolving a suitable amount of the corresponding
compound in 0.5 mL of the respective oxygen-free deuterated solvent and the spectra were
recorded at 298 K with a Varian Unity Inova-400 (399.94 MHz for 1H; 100.6 MHz for 13C). Typically,
1H NMR spectra were acquired with 32 scans into 32 k data points over a spectral width of 16
1
13
1
13
ppm. H and C{1H} chemical shifts were internally referenced to TMS via the residual H and C
signals of CD3SOCD3 (δ = 2.50 ppm and δ = 39.52 ppm) according to the values reported by Fulmer
et al.[34]. Chemical shift values (δ) are reported in ppm and coupling constants (J) in Hertz. The
1
splitting of proton resonances in the reported H NMR data is defined as s = singlet, d = doublet, t
= triplet, m = multiplet, bs = broad singlet. 2D NMR spectra such as Correlation Spectroscopy (1H-
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1H gCOSY), Nuclear Overhauser Effect Spectroscopy (1H-1H NOESY), were recorded using standard
pulse sequences. The probe temperature (1 K) was controlled by a standard unit calibrated with
methanol as a reference. All NMR data processing were carried out using MestReNova version
10.0.2. See atom labelling for NMR peaks in Figures 1SI and 2SI in the Supporting Information.
Synthesis of (2-(hydroxy-1-naphtyl)imidazo[4,5-f][1,10]fenanthroline) (HNAIP). A mixture of 2-
hydroxy-1-naphthaldehyde (0.287 g, 1.67 mmol), 1,10-phenanthroline-5,6–dione (0.25 g, 1.19
mmol), ammonium acetate (2.03 g, 26.30 mmol), and glacial acetic acid (10 mL) was refluxed for 3
h. Afterwards, the mixture was cooled down to room temperature and diluted with distilled water
(40 mL approximately). Dropwise addition of concentrated aqueous ammonia to neutralize gave a
yellow precipitate, which was collected and washed with water (310 mL). The crude product was
dissolved in the minimum amount of warm ethanol and purified by crystallization. Yield: 237 mg
(0.654 mmol, 55 %).
1H NMR (400 MHz, DMSO-d6, 25 C): δ 13.75 (bs, 1H), δ 10.82 (bs, 1H), 9.05 (dd, J = 4.2, 1.7 Hz,
2H), 8.95 – 8.88 (m, 2H), 8.03 – 7.90 (m, 3H), 7.83 (dd, J = 8.1, 4.3 Hz, 2H), 7.50 – 7.44 (m, 1H), 7.39
(dd, J = 8.3, 2.9 Hz, 2H).
Synthesis of [Ir(ppy)2(HNAIP)]Cl. In a 100 mL Schlenk flask, previously purged with nitrogen,
HNAIP (0.020 g, 0.055 mmol) was added to a solution of [Ir(ppy)2(μ-Cl)]2 (0.029 g, 0.027 mmol) in a
methanol/dichloromethane (21 mL, 4:3) mixture. The mixture was stirred overnight at 60 C under
a nitrogen atmosphere. The solution was concentrated under vacuum and a crude solid
precipitated with diethyl ether (10 mL) and n-hexane (10 mL). Lastly, the product was filtered,
washed with diethyl ether (5 mL) and dried under vacuum. Yield: yellow solid 0.030 g (0.033 mmol,
61 %).
Mr (C45H30IrN6OCl) = 898.43 g/mol. Analysis Calculated for C45H30ClIrN6O·(H2O)2: C = 57.84 %; H =
3.67 %; N = 8.99 %; Found: C = 57.53 %; H = 3.28 %; N = 8.83 %. 1H NMR (400 MHz, DMSO-d6, 25
C): δ 14.41 (bs, 1H, NH), δ 10.75 (bs, 1H, OH), δ 9.23 (d, J = 7.2 Hz, 2H, Hc), 8.28 (d, J = 8.2 Hz, 2H,
H3), 8.16 (d, J = 4.0 Hz, 2H, Ha), 8.08 (dd, J = 8.3, 5.1 Hz, 2H, Hb), 8.03 (d, J = 9.1 Hz, 1H, Hd/e), 7.99 –
7.86 (m, 6H, H9, H4, Hg/i, Hf/j), 7.55 (d, J = 5.8 Hz, 2H, H6), 7.50-7.41 (m, 2H, He/d, Hf/j), 7.38 (t, J = 7.4
Hz, 1H, Hg/i), 7.07 (t, J = 7.1 Hz, 2H, H10), 6.99 (m, 4H, H5, H11), 6.33 (d, J = 7.2 Hz, 2H, H12). 13C{1H}
NMR (101 MHz, DMSO-d6, 25 C): δ 166.87, 154.93, 150.51, 149.19, 148.10, 144.05, 143.93,
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138.64, 133.22, 132.28, 131.70, 131.25, 130.23, 127.67, 127.22, 127.00, 125.06, 124.05, 123.84,
122.32, 119.96, 118.24, 109.85. HR ESI(+) MS (DCM): m/z (%) = 863.2108 (100) Da (m/zcalcd
[C45H30IrN6O]+ = 863.2111 Da). Molar Conductivity (CH3CN): 54.6 S·cm2·mol-1.
Synthesis of [Rh(ppy)2(HNAIP)]Cl. The synthetic protocol for the Rh complex is analogous to that
followed for [Ir(ppy)2(HNAIP)]Cl. Typical amounts are: HNAIP (0.020 g, 0.055 mmol), [Rh(ppy)2Cl]2
(0.024 g, 0.027 mmol). Yield: yellow solid 0.023 g (0.028 mmol, 53 %).
Mr (C45H30RhN6OCl) = 809.14 g/mol. Analysis Calculated for C45H30ClRhN6O·(H2O): C = 64.64 %; H =
3.98 %; N = 10.05 %; Found: C = 64.86 %; H = 3.55 %; N = 9.72 %. 1H NMR (400 MHz, DMSO-d6, 25
C): δ 14.40 (bs, 1H, NH), δ 10.61 (bs, 1H, OH), δ 9.23 (d, J = 8.1 Hz, 2H, Hc), 8.30 (d, J = 8.0 Hz, 2H,
H3), 8.20 (d, J = 3.5 Hz, 2H, Ha), 8.10 – 7.91 (m, 9H, 2Hb, Hd/e, 2H9, 2H4, Hg/i, Hf/j), 7.54 – 7.35 (m, 5H,
H6, He/d, Hf/j, Hg/i), 7.14 (t, J = 7.4 Hz, 2H, H10), 7.05 (dd, J = 7.4 Hz, 4H, H5, H11), 6.34 (d, J = 7.7 Hz,
13
2H, H12). C{1H} NMR (101 MHz, DMSO-d6, 25 C): δ 167.62 (d, J = 32.1 Hz), 164.21, 154.82,
149.11, 147.50, 143.89, 142.61, 138.78, 133.20, 132.38, 132.25, 129.96, 128.19, 127.69, 127.24,
126.63, 124.87, 124.05, 123.86, 123.26, 120.16, 118.19, 109.85. HR ESI(+) MS (DCM): m/z (%) =
773.1529 Da (m/zcalcd [C45H30RhN6O]+ = 773.1536 Da). Molar Conductivity (CH3CN): 55.5 S ·cm2·
mol-1.
Preparation of solutions for biological studies. For the 2-(hydroxy-1-naphtyl)imidazo-[4,5-
f][1,10]phenantroline (HNAIP) ligand and the [M(ppy)2(HNAIP)]Cl complexes, weighed amounts of
the purified solids were dissolved in DMSO as stock solutions and maintained at 4 °C protected
from light. They can be referred to as dyes/drugs and their molar concentration denoted as CD.
Calf thymus DNA (highly polymerized lyophilized sodium salt) from Sigma-Aldrich was dissolved in
water and sonicated, producing short polynucleotide fragments (ca. 1000 base pairs) according to
a known procedure. The molar DNA concentration (in base pairs, ε (260 nm) = 13200 M-1 cm-1)[35]
will be expressed as CP. Bovine Serum Albumin (BSA) is supplied as crystallized and lyophilized
powder (≥ 98 %, agarose gel electrophoresis and ≤0.005 % fatty acids); BSA concentration of the
stock solutions was spectrophotometrically determined (ε (278 nm) = 45000 M−1 cm−1) [36]. All
other reagents were of analytical grade. The solutions were prepared with doubly distilled water
from a Puranity TU System with UV lamp and ultrafilter (VWR). The aqueous solutions were
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prepared using sodium cacodylate (NaCaC) as a buffer to keep the pH constant at pH = 7.0 and
sodium chloride (NaCl) to adjust the ionic strength.
Cellular Assays
Cellular Visualization. To study the cellular uptake, A549 cells were seeded at a density of 5×103
cells per well, incubated for 24 h at 37 °C and 5 % CO2 and then exposed to different
concentrations of the studied compounds and 1 µM of Hoechst33258. After 30 minutes of
incubation, the cells were visualized in a Cytation 5 Cell Imaging Multi-Mode Reader (Biotek
Instruments, USA) in bright field, blue and orange fluorescence emission with a 20× objective.
MTT Assay. 1104 SW480 and 5103 A549 cells were cultured in 200 μL culture medium per well
Dulbecco’s Modified Eagle’s Medium (DMEM medium), supplemented with 10 % newborn calf
serum and 1 % amphotericin-penicillin-streptomycin solution in 96-well plates and incubated
under 5 % CO2 atmosphere at 37 C. The cells were grown for 24 h and then exposed to the
studied compounds. To determine cytotoxicity, the MTT (thiazolyl blue tetrazolium bromide) assay
was performed. Briefly, cells were incubated for 24 h in the presence of different concentrations
of the tested drugs (dissolved in DMSO). Cisplatin was used as a positive control. Then, 100 μL of
500 μg/mL of MTT was added to each well and incubated for 4 h. At the end of the incubation, the
precipitated formazan product was dissolved by adding 100 μL of solubilizing solution (10 % SDS in
0.01 M HCl). The absorbance at 590 nm was read in a microplate reader (Biotek Instruments, USA).
Four replicates per dose were included. The IC50 values calculated using the GraphPadPrism
Software Inc. (v.6.01) were averaged out from at least two independent experiments.
Cellular Uptake by ICP-MS. To properly quantify the metal accumulation inside the cells, SW480
cells were seeded in culture flasks (2.5 × 106 cells in 6 mL DMEM culture medium) and incubated
for 24 h at 37 C and 5 % CO2. After that, cells were treated for 24 h with the indicated complex
concentrations, then they were harvested with trypsin and resuspended in phosphate buffered
saline. The samples were digested for 24 h with 65 % HNO3 at room temperature and then diluted
with Milli-Q water to obtain 2 % HNO3 solutions to be analyzed by inductively coupled plasma-
mass spectrometry (ICP-MS).
Biophysical methods
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pH measurements. To ensure near physiological conditions, the pH of all aqueous solutions was
measured at 25 C with a pH-meter fitted out with a combined glass electrode, with 3 M KCl as a
liquid junction.
UV-vis absorbance and NMR spectra. Absorbance measurements were performed, to study the
stability and possible aggregation processes of all three HNAIP, [Ir(ppy)2(HNAIP)]+ and
[Rh(ppy)2(HNAIP)]+ under the working conditions; then, we evaluated the DNA binding and
thermal stabilization, L-glutathione reduced (GSH) binding and NADH interaction. All these
measurements were performed at 25 C on a Hewlett-Packard 8453A (Agilent Technologies, Palo
Alto, CA) photodiode array spectrophotometer fitted out with a Peltier temperature control
1
13
system and, for GSH, on a Varian Unity Inova-400 (399.94 MHz for H; 100.6 MHz for C). DNA
thermal denaturation was studied in 0.01 M buffer (NaCl and NaCaC) by heating DNA and DNA-
drug solutions, from 30 to 90 C at 0.3 C/min scan rate, with 1 min stabilization time prior to
measurements.
Photophysical measurements: Emission spectra, quantum yield and lifetime were recorded on a
FLS980 Edinburgh Instrument. Emission lifetime was determinated by time-correlated single
photon counting following excitation with a pulsed laser diode. Quantum yield was measured with
an integrating sphere. Samples of 10-5 M in dry and deoxygenated DMSO and DMSO/H2O (1:3)
were prepared in quartz cuvettes and in N2 atmosphere.
Hydrophobicity (log P). The hydrophobicity of [Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ was
measured by the “shake flask” method between octanol/water phase partitions [37]. Octanol-
saturated water and water-saturated octanol were prepared using analytical grade octanol (VWR)
and double distilled water by shaking both solvents for 24 h and then to let them stand long
enough to allow splitting of phases. First, the calibration curve was elaborated at 360 nm for both
complexes. Then, 10 µM solutions of iridium and rhodium complex were shaken for 0.5 h in equal
volume (50:50) at room temperature. After attaining equilibrium, the organic and aqueous phases
were separated. Finally, metal complex concentrations were determined by UV-vis measurements
at the wavelength mentioned. Partition coefficients were calculated using log P = log
[(complex)octanol/(complex)water]. Triplicate experiments were performed for each complex.
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Native protein electrophoresis. Native PAGE electrophoresis was carried out by incubating BSA
overnight at T = 25 C with different concentrations of the complexes in Tris HCl buffer (0.5 M,
pH = 6.8) for [complex]/[protein] concentration ratios of 1, 15 and 30. After that, 5 µL of buffer
sample 2 (0.01 % Bromophenol Blue and 20 % glycerol in Tris HCl buffer 0.5 M, pH 6.8) were
added to 5 µL of the sample solutions and loaded onto 10 % polyacrylamide gels. Gels were run in
native PAGE buffer (250 mM Tris Base, 1.92 M glycine, pH = 8.3) at 6.6 V/cm for 4 h at 4 C to
avoid thermal denaturation of the protein. Lastly, gels were stained with Coomassie brilliant blue
R-250 and visualized with a Gel Doc XR+ Imaging System (Bio-Rad).
Circular dichroism (CD). CD studies were performed by adding variable amounts of drug solution
to a cell containing DNA, in 0.01M buffer (NaCl and NaCaC). CD measurements were performed
with a MOS-450 biological spectrophotometer (Bio-Logic SAS, Claix, France) fitted out with a 1.0
cm path-length cell.
Differential scanning calorimetry (DSC). DSC samples were prepared by adding different amounts
of drug solution to different CD/CP (drug/DNA concentration) ratios maintaining the DMSO content
at 5 % and constant DNA concentration (410-4 M). After homogenization, the samples were
degassed for 30 min in a degassing station (TA Instruments, Newcastle, USA). DSC thermal
denaturation studies were performed with a nanoDSC (TA Instruments, Newcastle, USA),
equipped with 300 μL platinum capillary tube cells. Measurements were performed by heating the
DNA and DNA/drug systems, in 0.01M buffer (NaCl and NaCaC), from 20 to 100 C, at 1 C/min
scan rate and 3 atm pressure. The DSC curves were analyzed using the NanoAnalyze (TA
Instruments, Newcastle, USA) software.
Viscosity. The viscosity assays were performed introducing 3 mL of 210-4 M DNA solution in 0.01
M buffer in a Micro-Ubbelohde viscometer (temperature control by an external thermostat – 25.0
± 0.1 C) and the efflux time was measured. Then, different amounts of drug solution, with DMSO
as a solvent, were added and efflux times of the mixture were measured. The experiments were
repeated for DNA-DMSO, and buffer-DMSO. The viscosity data were analyzed using η/η0 = (t −
t0)/(tDNA − t0), where t0, tDNA and t are the flow times for the buffer-DMSO, DNA-DMSO and DNA-
drug systems, respectively, expressed as the average of at least three independent readings.
Finally, relative elongation of the DNA double helix was calculated according to L/L0 = (η/η0)1/3.
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General procedure for the photocatalytic experiments. In a septum-capped tube we added 0.5
mL of a solution with the L-aminoacids or L-glutathione (10 mM, 0.005 mmol), the metal PC (0.01
mM – 1 % mol for L-Metionine (Met) and L-Cysteine (Cys), or 0.1 mM – 1 % mol for L-Tryptophan
(Trp) and L-Glutathione (GSH) in a mixture of DMSO-d6/D2O (1:3 v/v for Met and Trp and 1:10 v/v
for Cys) or in a mixture of MeOD/D2O (1:3 v/v for GSH). The system was purged with O2 until
atmosphere saturation and irradiated with Blue LED light (λ= 460 nm), 24W at room temperature
during 12 h. The conversion values have been calculated by 1H NMR and ESI(+) MS AR.
Results and discussion
SYNTHESIS AND CHARACTERIZATION
Synthesis of Ligand. The HNAIP ligand was synthesized following a protocol from the literature
which consists of reacting 2-hydroxy-1-naphthaldehyde with 1,10-phenanthroline-5,6–dione and
ammonium acetate, in acetic acid as the solvent (see Scheme 1) [32,33].
Synthesis of Complexes. The new complexes of general formula [M(ppy)2(N^N)]Cl (ppy = 2-
phenylpyridinate, N^N = HNAIP; M = Ir, [Ir(ppy)2(HNAIP)]Cl or M = Rh, [Rh(ppy)2(HNAIP)]Cl were
synthesized from the appropriate starting material [M(μ-Cl)(ppy)2]2 (M = Rh, or M = Ir) through a
bridge splitting reaction in the presence of HNAIP, using a solvent mixture of dichloromethane and
methanol [31,38]. The details, outlined in Scheme 1, are given in the experimental section. These
complexes were isolated as pale yellow (Rh) or yellow (Ir) solids, consisting of racemic mixtures of
Δ and Λ enantiomers with good yields and purities according to analytical and spectroscopic data.
Moreover, they are soluble in common organic solvents such as DMSO, acetone, methanol, CH3CN,
and CH2Cl2 and slightly soluble in methanol/water or DMSO/water solutions.
The new derivatives were comprehensively characterized by multinuclear NMR and mass
1
spectrometry and by elemental analysis. Assignment of the signals in the H NMR spectra was
carried out using 2D NMR experiments. (See Experimental part and Figures 1SI and 2SI for atom
labelling).
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Scheme 1. Synthesis of HNAIP and the Rh(III) and Ir(III) complexes.
1
13
Characterization of Complexes. The H and C{1H} NMR spectra of complexes [Ir(ppy)2(HNAIP)]+
and [Rh(ppy)2(HNAIP)]+ in CD3SOCD3 display distinctive signals for both types of ligands, i.e.,
HNAIP and ppy⁻ (Figures 1SI and 2SI, Supporting information). These resonances are shifted
compared to those of free HNAIP and the metal precursors with ppy⁻, which proves the formation
of the new derivatives. Moreover, the overall NMR patterns of both complexes revealed the
following significant features: (a) one single set of peaks for the two equivalent 2-phenylpyridinate
1
13
ligands in the H and C NMR spectra, consistent with the tautomeric equilibrium affecting the
imidazole ring and the C2-symmetry for the molecular structures of [Ir(ppy)2(HNAIP)]+ and
1
[Rh(ppy)2(HNAIP)]+ in solution; (b) two broad singlets around 14.5 and 10.6 ppm in the H NMR
spectra of both complexes, assigned to the N-H and O-H protons, respectively; (c) Besides, in the
13C{1H} NMR spectra of [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl we ascribed respectively, the
13
singlet at 166.9 ppm and the doublet at 167.6 ppm (1J103
-
Rh
≈ 32 Hz) to the metallated C atoms
C
of the C^N ligands.
The HR ESI MS spectra of [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl display peaks whose m/z
values and isotopic distribution are fully compatible with those calculated for the cationic metal
complexes of formula [M(ppy)2(HNAIP)]+ (M = Ir, Rh). Molar conductivity (ΛM) measurements
conducted with solutions of complexes [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl in
acetonitrile (10-3 M) were too low for 1:1 electrolytes, suggesting the presence of ion-pairs in
solution stabilized through N-H---Cl interactions [39].
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Stability of complexes. The stability of the compounds was also evaluated by means of
absorbance measurements as a function of time. All three HNAIP, [Ir(ppy)2(HNAIP)]+ and
[Rh(ppy)2(HNAIP)]+ are stable for weeks. The photostability of 10-2 M solutions of complexes in
1
DMSO-d6 and D2O/DMSO-d6 (3:1) was studied by H NMR spectroscopy over a period of 24 h
under irradiation (blue LED light and UV light) in an NMR tube exposed to atmospheric air. Both
metal complexes are stable and do not show decomposition.
HNAIP presents an absorbance band at 330 nm, spread up to 375 nm (Figure 1A). Both complexes,
[Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ display intense, broad absorption bands in the UV
region, attributed to spin-allowed (1IL) (1  *) intra ligand transitions in the cyclometallated
(N^C) and (N^N) ancillary ligands. In addition, the complexes exhibit a band at about 380 nm
attributed to a mixed charge transfer (CT).
10
A
B
0.6
0.4
0.2
0.0
8
6
4
2
0
[Ir(ppy)2(HNAIP)]+
[Rh(ppy)2(HNAIP)]+
HNAIP
[Ir(ppy)2(HNAIP)]+
[Rh(ppy)2(HNAIP)]+
HNAIP
300
400
500
600
400
500
600
700
800
, nm
, nm
Figure 1. A) UV-Vis spectra of HNAIP, [Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ in buffer, I = 0.01 M, pH
7.0 and T =25 C. B) Emission spectra of HNAIP and complexes [Ir(ppy)2(HNAIP)]Cl (exc = 285 nm) and
[Rh(ppy)2(HNAIP)]Cl (exc = 375 nm) in deoxygenated water/DMSO (3:1) (10-5 M) at 25 ᵒC.
The absorbance values increased linearly with concentration (Figure 3SI); fulfillment of Lambert
Beer law is observed for HNAIP up to 3.5×10-5 M with 290 nm = 2258 cm-1M-1, for [Ir(ppy)2(HNAIP)]+
up to 2.0×10-5 M with 380 nm = 12758 cm-1M-1 and for [Rh(ppy)2(HNAIP)]+ up to 6.0×10-5 M with

380 nm = 20758 cm-1M-1. Beyond these concentrations, changes in the linear profile are observed
due to precipitation. Interestingly, the rhodium complex is the most soluble.
Regarding the photoluminescence properties, HNAIP displays moderate blue photoluminescence
and the Ir(III) complex exhibits moderate orange photoluminescence under UV irradiation,
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whereas the Rh(III) derivative shows very weak yellow photoluminescence in H2O/DSMO. The
emission spectra in deoxygenated water/DMSO (3:1) upon excitation at 285 nm for HNAIP and 375
nm for its metal complexes at room temperature are shown in Figure 1B. The emission band
observed in Ir(III) complex (λmax = 605 nm) is broad and unstructured, revealing a dominant
contribution of the CT transitions to the emitting excited state, T1. The photophysical data in the
water/DMSO (3:1) mixture and in deoxygenated DMSO are listed in Table 1, the photoluminescent
quantum yields (PL) for the [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl derivatives are 0.57 %
and <0.5 %, respectively, and the lifetimes for the excited state (τ1/2) are 35.6 and 6.7 ns. These
features are consistent with phosphorescence as the emission mechanism for the Ir derivative
[40].
Table 1. Photophysical data for 10-5 M of HNAIP ligand and its complexes [Ir(ppy)2(HNAIP)]Cl and
[Rh(ppy)2(HNAIP)]Cl at 25 ᵒC.
Compound
HNAIP

exc (nm)

em (nm)
PL
ns
470a
450b
605a
610b
510a
530b
< 0.5a
0.98b
0.57a
1.86b
< 0.5a
< 0.5b
4.3a
4.6b
35.6a
204.1b
6.7a
285
[Ir(ppy)2(HNAIP)]Cl
[Rh(ppy)2(HNAIP)]Cl
375
375
1.5b
ain deoxygenated water/DMSO (3:1) and bin deoxygenated DMSO
CELLULAR TESTS
Cellular visualization. Since these compounds are fluorescent molecules, their visualization in
A549 (epithelial lung adenocarcinoma) cells was investigated using a bioimaging microplate reader
in phase contrast and blue, orange or green fluorescence emission modes. The images of A549
cells treated with either HNAIP, [Ir(ppy)2(HNAIP)]+ or [Rh(ppy)2(HNAIP)]+ during 30 min of
incubation (Figure 2) show that all of them are successfully and quickly internalized by the cells.
The co-staining with Hoechst33258 confirms that the metal complexes are mainly localized in the
cytoplasm.
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Cytotoxic Activity. Once their ability to get into cells was confirmed, MTT tests on human colon
adenocarcinoma SW480 and epithelial lung adenocarcinoma A549 cells have been performed to
evaluate the cytotoxic activity of HNAIP, [Ir(ppy)2(HNAIP)]+and [Rh(ppy)2(HNAIP)]+. The IC50
values are shown in Table 2. Interestingly, the metal centre exhibits a strong influence on the
cytotoxicity of the synthesized complexes since the Ir(III) complex is less cytotoxic than the
isoelectronic and isostructural Rh(III) complex; this complex displays almost the same cytotoxic
activity than the ligand, both being more active than cisplatin. Therefore, we can conclude that not
only the ligand but also the metal are key for the biological activity.
Figure 2. A549 cells treated for 30 minutes with 25 μM of the ligand HNAIP (1st row), 1 μM Hoechst 33258
and 10 μM of [Ir(ppy)2(HNAIP)]+ (2nd row), 1 μM Hoechst 33258 and 25 μM of Rh(ppy)2(HNAIP)]+ (3rd row).
Bright Field (A, D and G), blue (B), orange (E and H) fluorescence emission, merged bright field and blue
emission (C) and orange fluorescence emission overlapped (F and I).
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Table 2. Calculated IC50 values of the studied compounds and the positive control, cisplatin, in SW480
(colon adenocarcinoma) and A549 (lung adenocarcinoma) cells after 24 h of exposure time.
IC50, µM
Compound
SW480
4 ± 1
A549
2 ± 1
HNAIP
[Rh(ppy)2(HNAIP)]+
[Ir(ppy)2(HNAIP)]+
Cisplatin
5 ± 1
9 ± 2
29 ± 2
39 ± 1
>100
34 ± 1
Due to the low cytotoxicity and moderate PL and 1/2 values of the Ir complex (Table 1), it follows
that this species could be adequate for its use in photodynamic therapy (PDT), as occurs with
other Ir(III) cyclometallated complexes [41,42]. In view that the absorption bands in both
complexes extend from the UV to the Vis region (Fig. 2A), these were irradiated with UV (= 365
nm) and Vis ( = 460 nm) light and the IC50 were evaluated. Unfortunately, neither
[Ir(ppy)2(HNAIP)]+ nor [Rh(ppy)2(HNAIP)]+ showed any IC50 variation upon irradiation.
Cellular uptake: Quantification by ICP-MS measurements. Since the cytotoxicity of the Rh(III)
derivative differs from that of its Ir(III) analogue, a plausible explanation is searched in terms of
their ability to get into cell and the cellular uptake of both complexes was quantified by ICP-MS.
SW480 cells were treated for 24 h with [Ir(ppy)2(HNAIP)]+, [Rh(ppy)2(HNAIP)]+ and cisplatin at
CD = 2 µM. The results show greater cell internalization of the Rh complex compared to the Ir
derivative and to the well-known anticancer agent cisplatin (Figure 3). Moreover, cellular uptake
and cytotoxicity usually correlate satisfactorily in other anticancer drugs described in the literature
[43]. In this work, the correlation is precise, since [Rh(ppy)2(HNAIP)]+ is approximately five-fold
more cytotoxic than [Ir(ppy)2(HNAIP)]+, and exhibits a five-fold increase in its accumulation ability
inside SW480 cells. The observed differences in the uptake of the complexes are related to the
ability to cross lipid bilayers and this is often related to the partition coefficients.
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1 .0
0 .5
0 .0
+
+
n
]
]
i
)
)
t
P
P
I
a
l
I
p
A
A
s
i
N
N
c
H
H
(
(
2
2
)
)
y
y
p
p
p
p
(
(
r
h
I
[
R
[
Figure 3. Metal accumulation expressed as nmoles of metal in 106 SW480 cells after 24 h of exposition at
CD = 2 µM.
Hydrophobicity (log P). In general, hydrophobicity is considered to play a crucial role in
structure−activity relationships and in determining the biological properties of the drug [44–47].
The lipophilicity of a drug strongly affects its cellular internalization and consequently, its cytotoxic
activity.
In the light of the differences observed in the cellular uptake and cytotoxicity of the complexes
under study, their octanol/water partition coefficients (log P) were determined. HNAIP was not
soluble in octanol, therefore its log P could not be obtained. The log P values for iridium and
rhodium complexes were -0.11 ± 0.03 and 2.58 ± 0.08, respectively indicating greater
hydrophobicity for the Rh(III) derivative compared to that of its iridium analogue. These results are
consistent with cytotoxicity of the metal complexes.
BIOSUBSTRATES INTERACTION TESTS
To shed some light into the possible mode of action responsible for the obtained cytotoxicity
results, the interaction of the ligand and the two organometallic complexes with several bio-
targets was studied.
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Interaction with BSA, GSH and NADH. Since albumin is the most abundant plasma protein, the
interaction of the studied compounds with this biomolecule can easily affect their bioavailability
[48]. Therefore, to verify if the differences reported above between cellular uptake of
[Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ could be attributed not only to their hydrophobicity
but also to their interaction with serum proteins such as albumin and, consequently, to a
reduction in the final drug concentration that reaches the cell, the interaction of BSA with the
compounds under study was tested. We have verified by native polyacrylamide gel
electrophoresis that neither the HNAIP ligand, nor the corresponding Ir(III) and Rh(III) complexes
do interact with the protein (Figure 4SI). Therefore, the differences in terms of cytotoxicity and
cellular uptake cannot be justified on the basis of the interaction with seroalbumin.
In addition, the reactivity of
[Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ towards GSH,
overexpressed in most cancer cells, and NADH, involved in numerous enzymatic processes, were
explored, since both biomolecules have been reported in the literature as adequate targets for
similar metal complexes [49,50]. Therefore, UV-Vis experiments were carried out. Unfortunately,
there is no binding of the metal complexes to GSH (Figure 5SI) or NADH (Figure 6SI).
Interaction with CT-DNA. The HNAIP ligand is endowed with a phenanthroline moiety. It is known
that phenanthrolines essentially behave as intercalating agents between DNA nucleobases, so that
they are able to cause severe structural distortions, leading to lengthening, stiffening and
unwinding of the double helix [18]. On the other side, the HNAIP molecule possesses one 2-
hydroxy-1-naphthyl group, which could bind to the groove by H-bonding. Transition-metal
complexes containing bipyridine or phenanthroline ligands have been shown to act as groove
binders or possible partial intercalators [51,52]. That is why we study here the possible interaction
of both the ligand and the metal complexes with DNA. The reaction of CT-DNA with HNAIP,
[Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ can be represented by eqn 1:
(1)
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where P stands for the polynucleotide CT-DNA, D for the drug (the ligand or metal complexes
under study) and PD represents the drug/DNA complex. Henceforth, CD and CP denote the
analytical drug and biomolecule concentration, respectively.
Circular Dichroism. CD titrations were performed by adding increasing amounts of the tested drug
directly to the DNA solution. The variation of the CD spectra is somewhat modest for the ligand,
but it was notable for the complexes, revealing that the metal complexes can modify the DNA
structure to a greater extent than the ligand (Figure 4). The molar ellipticity in the DNA intrinsic,
negative CD band (243 nm) increases as a function of the CD/CP ratio for all systems; this band is
related to changes in the helicity of the double helix [53]. Moreover, a new band at 315 nm
emerges upon increasing the concentration of the complexes, thus making evident the formation
of the drug/DNA complex. The observed behaviour of HNAIP is compatible with binding to the
groove (rather than intercalation), whereas the interaction of [Ir(ppy)2(HNAIP)]+ and
[Rh(ppy)2(HNAIP)]+ could be compatible with partial intercalation or binding to the groove.
20
15
10
5
15
10
5
B
A
0
0
-5
-5
-10
-15
-20
-25
-10
-15
-20
250
300
350
400
450
500
250
300
 nm
350
400
, nm
15
10
5
C
0
-5
-10
-15
-20
-25
250
300
350
400
450
500
, nm
Figure 4. Circular dichroism titrations for (A) HNAIP/DNA, (B) [Ir(ppy)2(HNAIP)]+/DNA and (C)
[Rh(ppy)2(HNAIP)]+/DNA systems; (A) CP = 40 μM, CD/CP = 0 - 0.9; (B and C) CP = 40 μM, CD/CP = 0 – 1.3; I =
0.01 M, pH 7.0 and 25 ˚C.
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Thermal stability: UV-vis and DSC. The thermal stabilization of the DNA double helix caused by the
ligand and its Ir(III) and Rh(III) complexes was studied by UV-Vis and differential scanning
calorimetry (DSC). These techniques show that neither HNAIP nor its Ir(II) and Rh(III) derivatives
stabilize the DNA double helix, since no significant variation of ΔTm (difference between melting
temperature, Tm , in the presence and in the absence of the studied compounds) was observed
(Figure 7SI). This outcome points to the absence of intercalation as the mode of binding with DNA.
Viscosity. Noticeable differences in the relative elongation of DNA have been found (Figure 5). The
behaviour of HNAIP is different (20 % contraction for CD/CP  0.2) from that of [Rh(ppy)2(HNAIP)]+
and [Ir(ppy)2(HNAIP)]+ (no significant effect). Higher CD/CP ratios could not be explored due to
precipitation phenomena.
1.05
1.00
0.95
0.90
0.85
0.80
0.00
0.05
0.10
0.15
0.20
CD/CP
Figure 5. Relative contour length of the HNAIP/DNA (▲), [Ir(ppy)2(HNAIP)]+/DNA () and
Rh(ppy)2(HNAIP)]+/DNA (▪) systems; CP = 2 × 10-4 M, I = 0.01 M, pH 7.0 and 25C.
The observed decrease in the DNA relative contour length and the absence of thermal
denaturation are compatible with binding of HNAIP to the DNA groove by H-bonding. By contrast,
the absence of significant changes in the DNA relative viscosity and melting temperature suggest
external binding as the plausible binding mode for [Rh(ppy)2(HNAIP)]+ and [Ir(ppy)2(HNAIP)]+. The
external complex formed is favoured by the electrostatic interaction between the positive charge
of the metal complex and the DNA phosphate backbone. In principle, hydrogen bonding
interactions are also possible for the complexes. However, the geometry features of the complex
are not adequate for this type of interaction because the cyclometallated ring prevents
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accommodation of the complex in the groove and the subsequent access to the functional groups
of the DNA bases.
PHOTOCATALYTIC ACTIVITY IN THE OXIDATION OF AMINO ACIDS AND PEPTIDES
L- glutathione (GSH) is overexpressed in most cancer cells where it plays a remarkable role as an
antioxidant agent, preventing or mitigating oxidative stress. Proteins and enzymes, for its part, are
ubiquitous and play multiple essential roles. However, Glutathione and some amino acid residues
of proteins can be targets for photooxidative damage in the presence of suitable photosensitizers.
On the other hand, the catalytic behavior of biscyclometallated Ir(III) complexes with 2,2′‐
dipyridylamine-based ligands in the photooxidation of some sulfur-containing aminoacids have
been previously reported [54].
Therefore, we performed preliminary catalytic experiments on the photooxidation of L-
glutathione and some amino acids like a L-methionine (Met), L-cysteine (Cys) and L-tryptophan
(Trp) in the presence of [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl at room temperature under
an O2 atmosphere (balloon) with blue LED irradiation in DMSO-d6/D2O (Cys, Met, Trp) or
1
MeOD/D2O (GSH). Interestingly, we concluded by H NMR that complex [Ir(ppy)2(HNAIP)]Cl
promotes the photooxidation of Met, Cys, Trp and GSH in a very efficient and selective manner
after 12 h under the afore-mentioned conditions (entries 2, 5, 8, 11 in Table 3) (Figures 8SI to
10SI). The identity of the oxidized products was unambiguously established by ESI(+) MS Figures
11SI to 13SI), according to the structures shown in Figure 6. Nevertheless, the photocatlytic
activity of [Rh(ppy)2(HNAIP)]Cl in the same transformations depends strongly on the substrate, so
that it is modest for Met, quantitative for Cys and GSH and null for Trp (entries 3, 6, 12 and 9 in
Table 3). Control experiments in the absence of the metal photocatalysts (PC) were unproductive
(entries 1, 7 and 10 in Table 3), except for cysteine which is oxidized to cystine with a yield of 44 %
(entry 4 in Table 3).
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Figure 6. Reactions carried out in the presence and absence of the synthesized metal complexes
[Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl.
Table 3. Photocatalytic activity of complexes [M(ppy)2(HNAIP)]Cl (M = Rh, Ir) in the oxidation of L-
methionine, L-cysteine, L-Tryptophan and L-Glutathione under an O2 atmosphere and blue-LED irradiation.
% CONVERSION
12 H
ENTRY
SUBSTRATE
CONDITIONS
1
2
3
4
5
6
L-methionine
L-methionine
No Cat / Blue Light / O2
0
[Ir(ppy)2(HNAIP)]Cl / Blue Light / O2
100
15
L-methionine [Rh(ppy)2(HNAIP)]Cl / Blue Light / O2
L-cysteine
L-cysteine
L-cysteine
No Cat / Blue Light / O2
44
[Ir(ppy)2(HNAIP)]Cl / Blue Light / O2
[Rh(ppy)2(HNAIP)]Cl / Blue Light / O2
100
100
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7
8
L-Tryptophan
L-Tryptophan
No Cat / Blue Light / O2
0
100a
0
[Ir(ppy)2(HNAIP)]Cl / Blue Light / O2
9
L-Tryptophan [Rh(ppy)2(HNAIP)]Cl / Blue Light / O2
10
11
12
L-Glutathione
L-Glutathione
No Cat / Blue Light / O2
0
[Ir(ppy)2(HNAIP)]Cl / Blue Light / O2
100
100
L-Glutathione [Rh(ppy)2(HNAIP)]Cl / Blue Light / O2
aDetermined by ESI(+) MS.
Conclusions
HNAIP and the new complexes [Ir(ppy)2(HNAIP)]+ and [Rh(ppy)2(HNAIP)]+ have been synthesized
and fully characterized. The emission properties of the three compounds under study enable their
visualization in A549 epithelial lung adenocarcinoma cells after 30 minutes of incubation.
The cytotoxicity of HNAIP is really promising, showing the role played by the ligand in the
biological activity of the complexes. Together with the ligand, the metal center plays a key role, as
[Rh(ppy)2(HNAIP)]+ is 6-times more cytotoxic than [Ir(ppy)2(HNAIP)]+ complex in SW480 and more
than 10-times in A549 cancer cells. It has been verified by ICP-MS that the cellular uptake by
SW480 cells is much greater for the Rh complex than for the Ir analog due to their different
hydrophobicity, and that both complexes are internalized to a greater extent than cisplatin.
Moreover, the uptake values concur with the observed difference for the IC50 values in SW480
cells.
The interaction of all three compounds with BSA, GSH, NADH and DNA was studied. None of them
is able to react with BSA, GSH and NADH and HNAIP can interact with DNA by binding to the
groove, whereas the complexes can bind externally.
Regarding their behavior as photosensitizers, [Ir(ppy)2(HNAIP)]Cl is successfully used in the
aerobic catalytic photoxidation of GSH, Met, Cys and Trp.
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Acknowledgements
This work was supported by "la Caixa" Banking Foundation (LCF/PR/PR12/11070003), Ministerio
de Ciencia, Innovación y Universidades (RTI2018-102040-B-100), Junta de Castilla y León
(BU305P18, FEDER Funds), Spain.
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Graphical Abstract
IC 5 0
= 5 .1  M
1 .0
0 .5
0 .0
IC 5 0
= 2 9  M
cisp la tin
Ir
R h
2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline ligand (HNAIP) used to synthesize M(III)
complexes denoted as [M(HNAIP)(ppy)2]+ where ppy = 2-phenylpiridinate and M = Ir(III) and
Rh(III). Both complexes are more accumulated than cisplatin, being the cellular uptake of the Rh
complex higher than that of the Ir complex which could explain their different cytotoxic activity.
Highlights
1. An imidazophenanthroline Rh complex is six times more cytotoxic than its Ir analogue.
2. The metal complexes are internalized by cells and visualized mainly in the cytoplasm
3. The Rh complex is more accumulated inside tumor cells than the Ir complex
4. Ir complex is used in the catalytic photoxidation of Glutathione, Met, Cys and Trp.
27