Effect of sulfonamidoethylenediamine substituents in RuII arene anticancer catalysts on transfer hydrogenation of coenzyme NAD+ by formate

Article abstract of DOI:10.1039/c8dt00438b

A series of neutral pseudo-octahedral Ru^II sulfonamidoethylenediamine complexes [(η⁶-p-cym)Ru(N,N′)Cl] where N,N′ is N-(2-(R¹,R²-amino)ethyl)-4-toluenesulfonamide (TsEn(R¹,R²)) R¹,R² = Me,H (1); Me,Me (2); Et,H (3); benzyl,H (Bz, 4); 4-fluorobenzyl,H (4-F-Bz, 5) or naphthalen-2-ylmethyl,H (Naph, 6), were synthesised and characterised including the X-ray crystal structure of 3. These complexes catalyse the reduction of NAD⁺ regioselectively to 1,4-NADH by using formate as the hydride source. The catalytic efficiency depends markedly on the steric and electronic effects of the N-substitutent, with turnover frequencies (TOFs) increasing in the order: 1 < 2 < 3, 6 < 4, 5, achieving a TOF of 7.7 h^-1 for 4 with a 95% yield of 1,4-NADH. The reduction rate was highest between pH? (deuterated solvent) 6 and 7.5 and improved with an increase in formate concentration (TOF of 18.8 h^-1, 140 mM formate). The calculations suggested initial substitution of an aqua ligand by formate, followed by hydride transfer to Ru^II and then to NAD⁺, and indicated specific interactions between the aqua complex and both NAD⁺ and NADH, the former allowing a preorganisation involving interaction between the aqua ligand, formate anion and the pyridine ring of NAD⁺. The complexes exhibited antiproliferative activity towards A2780 human ovarian cancer cells with IC₅₀ values ranging from 1 to 31 μM, the most potent complex, [(η⁶-p-cym)Ru(TsEn(Bz,H))Cl] (4, IC₅₀ = 1.0 ± 0.1 μM), having a potency similar to the anticancer drug cisplatin. Co-administration with sodium formate (2 mM), increased the potency of all complexes towards A2780 cells by 20-36%, with the greatest effect seen for complex 6.

Full text of DOI:10.1039/c8dt00438b

Dalton
Transactions
View Article Online
View Journal
PAPER
Effect of sulfonamidoethylenediamine substituents
in RuII arene anticancer catalysts on transfer
hydrogenation of coenzyme NAD+ by formate†
Cite this: DOI: 10.1039/c8dt00438b
a,b
Feng Chen, a Joan J. Soldevila-Barreda, a Isolda Romero-Canelón,
a
James P. C. Coverdale, a Ji-Inn Song, a Guy J. Clarkson,
c
Jana Kasparkova, c Abraha Habtemariam, a Viktor Brabec,
Juliusz A. Wolny, d Volker Schünemann d and Peter J. Sadler
*
a
A series of neutral pseudo-octahedral RuII sulfonamidoethylenediamine complexes [(η6-p-cym)Ru(N,N’)
Cl] where N,N’ is N-(2-(R1,R2-amino)ethyl)-4-toluenesulfonamide (TsEn(R1,R2)) R1,R2 = Me,H (1); Me,Me
(2); Et,H (3); benzyl,H (Bz, 4); 4-fluorobenzyl,H (4-F-Bz, 5) or naphthalen-2-ylmethyl,H (Naph, 6), were
synthesised and characterised including the X-ray crystal structure of 3. These complexes catalyse the
reduction of NAD+ regioselectively to 1,4-NADH by using formate as the hydride source. The catalytic
efficiency depends markedly on the steric and electronic effects of the N-substitutent, with turnover fre-
quencies (TOFs) increasing in the order: 1 < 2 < 3, 6 < 4, 5, achieving a TOF of 7.7 h−1 for 4 with a 95%
yield of 1,4-NADH. The reduction rate was highest between pH* (deuterated solvent) 6 and 7.5 and
improved with an increase in formate concentration (TOF of 18.8 h−1, 140 mM formate). The calculations
suggested initial substitution of an aqua ligand by formate, followed by hydride transfer to RuII and then
to NAD+, and indicated specific interactions between the aqua complex and both NAD+ and NADH, the
former allowing a preorganisation involving interaction between the aqua ligand, formate anion and the
pyridine ring of NAD+. The complexes exhibited antiproliferative activity towards A2780 human ovarian
cancer cells with IC50 values ranging from 1 to 31 μM, the most potent complex, [(η6-p-cym)Ru(TsEn(Bz,
H))Cl] (4, IC50 = 1.0 0.1 μM), having a potency similar to the anticancer drug cisplatin. Co-administration
with sodium formate (2 mM), increased the potency of all complexes towards A2780 cells by 20–36%,
with the greatest effect seen for complex 6.
Received 1st February 2018,
Accepted 5th March 2018
DOI: 10.1039/c8dt00438b
rsc.li/dalton
pounds (e.g. NADH) is of critical importance for energy storage
1. Introduction
and release in cell metabolism.3,4 Transition metal-mediated
Nicotinamide adenine dinucleotide (NAD+) and its reduced catalytic reduction of NAD+ to NADH, using hydrogen,5 2-pro-
form (NADH), as well as their phosphorylated derivatives, panol,6,7 glycerol8 and sodium formate as hydride donors, has
NADP+ and NADPH, play a vital role in biological systems as been intensively studied for the last three decades.9–13
redox coenzymes.1 More than 400 enzymatic redox reactions Compared to reduction with H2 (hydrogenation), transfer
rely on the action of nicotinamide enzymes, in which the hydrogenation (TH) reactions have the advantage of being
transformation of NAD(P)+ to NAD(P)H is involved.2–4 The simpler, without the need for any high external pressure and
reduction of pyridinium salts (e.g. NAD+) to dihydropyridine com- use readily available, safer-to-handle, hydride sources.14 Also,
TH reduction of NAD(P)+ artificially has attracted wide interest
as an in vitro mimic for enzymatic reactions performed under
aDepartment of Chemistry, University of Warwick, Gibbet Hill Road,
biologically relevant conditions.15
Coventry CV4 7AL, UK. E-mail: P.J.Sadler@warwick.ac.uk
bSchool of Pharmacy, University of Birmingham, Birmingham B15 2TT, UK
cDepartment of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12,
CZ-77146 Olomouc, Czech Republic
dDepartment of Physics, University of Kaiserslautern, Erwin-Schrödinger-Str. 46,
67663 Kaiserslautern, Germany
The pathways of hydride transfer between pyridinium salts
and dihydropyridine compounds are also of interest. The first
mechanistic study of the TH reduction of BNA+ (1-benzylnicoti-
namide), as a model for NAD+ was reported by Steckhan
and Fish et al. using [(η5-Cp*)Rh(bipy)Cl] as the catalyst and
sodium formate as a hydride source in aqueous media in the
1990s.10–12,16,17 They proposed a catalytic cycle involving a
†Electronic supplementary information (ESI) available. CCDC 1571331. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c8dt00438b
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Table 1 RuII complexes studied in this work
ring-slippage η4-Cp* intermediate with Rh coordinated to the
amide of the pyridine ring.18 Knör et al. reported a Rh-
coordinated poly(arylene-ethynylene)-alt-poly(arylene-vinylene)
polymer as photocatalyst for the reduction of NAD+; involving
a possible photoexcited polymer chain being quenched and
transferring an electron to the RhIII active centre.19 More
recently, Yoon et al. described a mechanism involving hydride
transfer to Cp* and formation of the RhI intermediate
[(η4-Cp*-H)Rh((CH2OH)2-bipy)]+ followed by hydride transfer
from the endo orientation of the C–H bond to maintain the
1,4-regioselectivity of NADH.20
Complex
R1
R2
1
2
3
4
Me
Me
Et
H
Me
H
Bz
H
The half-sandwich ruthenium complex [(η6-p-cym)Ru
(TsDPEN)Cl] (TsDPEN: N-((1S,2S)-2-amino-1,2-diphenylethyl)-
4-methylbenzenesulfonamide) was first reported by Noyori and
coworkers in 1995.21,22 Potent catalytic activity has been shown
in asymmetric TH reduction of aromatic ketones. Most
recently, the 16-electron Os analogues [(η6-arene)Os(TsDPEN)]
of Noyori type complexes were reported to reduce pyruvate
enantioselectively to (D- or L-) lactate via asymmetric transfer
hydrogenation in human cancer cells.23 Nonetheless, the
hydrophobic nature of the two phenyl groups on the ethylene
backbone limits its application as a possible catalyst for TH
reduction of NAD+ under biologically relevant conditions.
Complexes with chelating diamine ligands such as complex 7
in Fig. 1, display good aqueous solubility but poor catalytic
activity in TH reduction of NAD+.24 However, p-cymene (p-cym)
complexes with functional sulfonyl substituents such as [(η6-
p-cym)Ru(TsEn)Cl] (e.g. complex 8 in Fig. 1),25 exhibit good
solubility in water and improved catalysis for NAD+ reduction
to NADH in aqueous media. Moreover, co-administration of
5
6
4-F-Bz
Naph
H
H
(DFT) calculations. We also explored the effect of co-adminis-
tration of formate on the antiproliferative activity of these com-
plexes against A2780 human ovarian cancer cells.
2. Experimental
2.1 Materials
[(η6-p-cym)Ru(TsEn)Cl] with low non-cytotoxic doses of sodium Ruthenium(III) trichloride hydrate was purchased from
formate led to an enhancement of the antiproliferative activity Precious Metals Online (PMO Pty Ltd) and used as received.
against A2780 human ovarian cancer cells by up to 50×.15,26
Toluenesulfonyl
chloride,
sodium
formate
and
Here we investigate the effect on catalytic reduction of β-nicotinamide adenine dinucleotide hydrate (NAD+) were
NAD+ using formate as a hydride source upon variation of sub- obtained from Sigma-Aldrich. Magnesium sulfate, potassium
stituents on the amino group of the N,N-chelating TsEn ligand hydroxide, sodium chloride, and hydrochloric acid were
in RuII complexes [(η6-p-cym)Ru(N,N′)Cl] where N,N′ is N-(2- obtained from Fisher Scientific. α-Phellandrene was pur-
(methylamino)ethyl)-4-toluenesulfonamide (TsEn(Me,H), 1), chased from SAFC. The RuII precursor dimer [(η6-p-cym)
N-(2-(dimethylamino)ethyl)-4-toluenesulfonamide (TsEn(Me,Me), RuCl2]2 was prepared following literature methods,27 as
2), N-(2-(ethylamino)ethyl)-4-toluene sulfonamide (TsEn(Et,H), were the ligands 4-methyl-N-(2-(methylamino)ethyl)benzene
3), N-(2-(benzylamino)ethyl)-4-toluenesulfonamide (TsEn(Bz, sulphonamide (TsEn(Me,H))28 and N-(2-(dimethylamino)ethyl)-
H), 4), N-(2-((4-fluorobenzyl)amino) ethyl)-4-toluenesulfona- 4-methylbenzenesulfonamide (TsEn(Me,Me)).29 The solvents
mide (TsEn(4-F-Bz,H), 5) and N-(2-((naphthalen-2-ylmethyl) used for NMR spectroscopy were purchased from Sigma-
amino) ethyl)-4-toluenesulfonamide (TsEn(Naph,H), 6) Aldrich and Cambridge Isotope Laboratories Inc. Non-dried
(Table 1). In addition, the catalytic mechanism was investi- solvents used in synthesis were obtained from Fisher
gated both experimentally and by density functional theory Scientific. Solvents were used as received, except in the case of
ethanol, 2-propanol, and methanol, which were degassed prior
to use by bubbling with nitrogen.
A2780 human ovarian carcinoma cells were obtained from
the European Collection of Cell Cultures. The cell line was
grown in Roswell Park Memorial Institute medium
(RPMI-1640) supplemented with 10% of foetal calf serum, 1%
v/v of 2 mM glutamine and 1% v/v penicillin/streptomycin
(10 000 units). All cells were grown as adherent monolayers at
Fig. 1 Organometallic RuII complexes [(η6-biph)Ru(en)Cl]PF6 (7) and
[(η6-p-cym)Ru(TsEn) Cl] (8).
310 K in a 5% CO2-humidified atmosphere and passaged at ca.
70–80% confluency.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
2.2 Instruments
pH values were measured using a Minilab IQ125 pH meter
equipped with a ISFET silicon chip pH sensor and referenced
in KCl gel. pH* values (pH meter reading without correction
for the effect of deuterium on the sensor) of NMR samples in
D2O were measured at 310 K. Relative hydrophobicity measure-
ments were performed utilising the Agilent 1200 HPLC system
with a VWD and 50 µL loop. The column was an Agilent
Zorbax 300SB C18, 150 × 4.6 mm with a 5 µm pore size. The
mobile phase was H2O (50 mM NaCl)/H2O/CH3CN 1 : 1
(50 mM NaCl), with a flow of 1 mL min−1. The detection wave-
length was set at 254 nm with the reference wavelength at
360 nm.
NMR spectra were acquired on Bruker HD-300, HD-400,
HD-500, and AV III 600 spectrometers. 1H NMR chemical shifts
were internally referenced to TMS via 1,4-dioxane in D2O (δ =
3.75 ppm) or residual protiated d4-MeOD (δ = 3.31 ppm), or
CDCl3 (δ = 7.26 ppm). 1D spectra were recorded using standard
pulse sequences. Typically, data were acquired with 16 transi-
ents into 32k data points over a spectral width of 14 ppm and,
for the kinetic experiment, 32 transients into 32k data points
over a spectral width of 30 ppm using a relaxation delay of 2 s.
Elemental analysis were performed by Warwick Analytical
using an Exeter Analytical elemental analyzer (CE440).
Positive ion electrospray mass spectra were obtained on an
Agilent 6130B ion mass spectrometer. High resolution mass
spectrometry data were obtained on a Bruker Maxis Plus
Q-TOF instrument.
2.3 Turnover frequency determination
UV-vis spectroscopy. In a typical experiment, 330 μL of each
solution (84 µM complex in MeOH/H2O 1 : 9 v/v, 102 mM
sodium formate and 510 µM NAD+ in H2O) was added to a
1 mL cuvette, and the pH adjusted to 7.2, bringing the total
volume to 1 mL (final concentrations: Ru complex 28 µM;
NAD+ 170 µM; NaHCO2 34 mM; molar ratio 1 : 6 : 1200). UV
spectra were recorded and the absorbance at 340 nm was
monitored every 5 min until completion of the reaction.
NMR spectroscopy. Complexes were dissolved in d4-MeOD/
D2O (1 : 4, v/v) (1.4 mM) in a glass vial. Solutions of sodium
formate (35 mM) and NAD+ (5.6 mM) in D2O were also pre-
pared and then incubated at 310 K, pH* 7.2 0.1. An aliquot
of 200 μL from each solution was added to a 5 mm NMR tube,
giving a final volume of 0.64 mL (Ru complex 0.44 mM; NAD+
X-ray crystallographic diffraction data were collected on an
Oxford Diffraction Gemini four-circle system with a Ruby CCD
area detector. The structure was refined by full-matrix least-
squares against F2 using SHELXL 9735 and solved by direct
methods using SHELXS36 (TREF) with additional light atoms
found by Fourier methods. The atoms from the sulfonamide
nitrogen to the end of the chain (C10 C11 N12 C13) were mod-
elled as disordered over two positions related by a small ruffle
in the chain. The occupancy of the two positions was linked to
a free variable which refined to 86 : 14. The minor component
was refined isotropically. X-ray crystallographic data for
1
complex
3
has been deposited in the Cambridge
1.75 mM; NaHCO2 10.94 mM; molar ratio 1 : 4 : 25). A H NMR
Crystallographic Data Center (CCDC) under the accession
number CCDC 1571331.†
spectrum was recorded at 310 K every 162 s until the com-
pletion of the reaction. Further experiments under similar con-
ditions using different concentrations of sodium formate
(complex 4, NAD+ and sodium formate in ratio of 1: 4: X,
where X = 10, 25, 50, and 100 mol equiv.) and different concen-
trations of NAD+ (complex 4, NAD+ and sodium formate in
ratio of 1 : Y : 25, where Y = 2, 4, 6 and 10) were also studied.
Another series of experiments using different pH* values of
the reaction solutions (5, 6, 7, 8 and 9) were also performed.
Molar ratios of NAD+ and NADH were determined by integrat-
ing 1H NMR peaks corresponding to NAD+ (9.33 ppm) and 1,4-
NADH (6.96 ppm). The turnover number (TON) for the reaction
was calculated as follows:
ICP-OES analysis were carried out on a PerkinElmer Optima
5300 DV series Optical Emission Spectrophotometer. The water
used for ICP-OES analysis was doubly deionized (DDW) using a
Millipore Milli-Q water purification system and a USF Elga
UHQ water deionizer. The ruthenium Specupure plasma stan-
dard (ruthenium chloride, 1004 5 µg mL−1 in 10% v/v hydro-
chloric acid) was diluted with 3.6% v/v HNO3 to freshly prepare
calibrants at concentrations of 50–700 ppb. Calibration stan-
dards were adjusted to match the sample matrix by standard
addition of sodium chloride (TraceSELECT®). Total dissolved
solids did not exceed 0.2% w/v. Data were acquired and pro-
cessed using WinLab32 V3.4.1 for Windows.
ICP-MS analysis were carried out on an Agilent
Technologies 7500 series ICP-MS instrument. The water used
for ICP-MS analysis was double-deionized (DDW) using a
Millipore Milli-Q water purification system and a USF Elga
UHQ water deionizer. The Ruthenium Specpure plasma stan-
dard (ruthenium chloride, 1004 5 µg mL−1 in 10% v/v hydro-
chloric acid) was diluted with 3.6% v/v HNO3 to prepare cali-
brants freshly at concentrations of 0.1–1000 ppb. The ICP-MS
instrument was set to detect 101Ru in no gas mode. Total dis-
solved solids did not exceed 0.1% w/v. An internal standard of
166Er (50 ppb) was used. Data were acquired using ICP-MS-TOP
and proceeded using Offline Data Analysis (ChemStation
version B.03.05, Agilent Technologies, Inc.).
I6:96
½NADþꢀ
TON ¼
I6:96 þ I9:93 ½Catalystꢀ
where In is the integral of the signal at n ppm and [NAD+] is
the concentration of NAD+ at the start of the reaction.
2.4 Cell growth inhibition assays
The antiproliferative activity of complexes 1–6 was determined
in A2780 ovarian cancer cells. Briefly, 96-well plates were used
to seed 5000 cells per well. Cells were incubated in drug-free
medium at 310 K for 48 h before addition of tested com-
pounds (prepared by serial dilution in culture medium con-
taining 5% DMSO, typically 6 concentrations in the range:
0.01–100 μM). Exact Ru concentrations were determined by
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
ICP-OES and the maximum concentration of DMSO to which Sciences) according to the manufacturer’s instructions. Briefly,
cells were exposed never exceeded 0.5% v/v. A drug exposure 1.0 × 106 A2780 cells per well were seeded in a six-well plate.
period of 24 h was allowed. After this, supernatants were Cells were preincubated in drug-free media at 310 K for 24 h in
removed by suction and each well was washed with PBS. A a 5% CO2 humidified atmosphere, and then drugs were added
further 72 h were allowed for the cells to recover in drug-free to triplicates wells at IC50 concentration. After 24 h of drug
medium at 310 K. The sulforhodamine B (SRB) assay was used exposure, supernatants were removed by suction and cells were
to determine cell viability.30 IC50 values, as the concentration washed with PBS and harvested. Staining was achieved by re-
that causes 50% cell death, were determined as duplicates of suspending the cell pellets in buffer containing the orange/
triplicates in two independent sets of experiments and their green fluorescent reagents. Cells were analysed in a Becton
standard deviation were calculated. Data were processed using Dickinson FACScan flow cytometer using FL1 channel Ex/Em:
Microsoft Excel and sigmoidal curves fitted using Origin 9.1.
490/525 nm for the oxidative stress and FL2 channel Ex/Em:
550/620 nm for superoxide detection. Data were gated using
positive-stained (pyocyanin positive control), untreated-stained
2.5 Co-administration of Ru complexes with formate
Cell viability assays were carried out with complexes 1–6 with and untreated-unstained control samples, acquired as instru-
co-administration of sodium formate in A2780 ovarian cancer mental triplicates, and were processed using FlowJo V10 for
cells. These experiments were carried out as above (in vitro Windows. At all times, samples were kept under dark con-
growth inhabitation assay) with the following modifications: a ditions to avoid light-induced ROS production.
fixed equipotent concentration of each Ru complex equal to
2.8 DFT calculations
1/3 × IC50 in that cell line was used in coadministration with
three different concentrations of sodium formate (0.5, 1.0 and The DFT calculations of electronic energy levels of the catalytic
2.0 mM). Drug stock solutions (ca. 100 µM) were prepared and cycle were based on the crystal structure of complex 3. The
they were further diluted using media until working concen- method of the calculation was functional CAM-B3LYP31 with
trations were achieved. Separately, a stock solution of sodium basis set CEP-31G,32–34 using Gaussian 16 software.35 Ultrafine
formate was prepared in saline. The complex and formate solu- grid of integration was used in each case. The starting geometry
tions were added to each well independently, but within was taken from X-ray data for 3, with an appropriate change of
5 minutes of each other. All other experiment conditions were substituents for other systems. All given energy values are the
kept unchanged (drug exposure and cell recovery time, as well result of the full geometry optimisation with subsequent fre-
as, end point assay used).
quency calculations. Optimisations were performed with mod-
elling of water as solvent, within the continuous polarisation
model with integral equation formalism variant (IEFPCM
2.6 Cellular accumulation
The accumulation studies for Ru complexes 1–6 were per- keyword of Gaussian). Grimme empirical corrections for dis-
formed on A2780 ovarian cancer cells. 1.5 × 106 cells were persion were applied (keyword GD3). The optimisations were
seeded on a six-well plate using 2 mL of cell culture medium. performed using the solvent-accessible surface option and the
After 24 h of pre-incubation in drug-free medium at 310 K, final energy was calculated with using solvent-excluding
cells were exposed to complexes at equipotent IC50 concen- surface options (keywords surface = sas and ses, respectively).
trations for 24 h (prepared by serial dilution of a ca. 100 μM NAD+ was modelled with an effective charge of −1, with two
stock solution, prepared using culture medium containing 5% deprotonated phosphate groups; the same protonation of
DMSO. This solution was analysed by ICP-OES to determine phosphate was used for NADH, giving an effective charge of
Ru concentration before treatment of cells with Ru complex). −2.
After this time, drug solutions were removed by suction, cells
were washed with PBS and then treated with trypsin–EDTA. A
2.9 DNA binding
suspension of single cells was counted, and cell pellets were The reactions of complex 4 (ca. 2 mM) with nucleobases (9-EtG
collected. Each pellet was digested overnight in freshly-dis- and 5′-AMP) were studied typically by addition of an aqueous
tilled concentrated nitric acid (200 μL, 72% v/v) at 353 K; the solution of nucleobase (3 mM, 1.5 mol equiv.) in 10% of d4-
1
resulting solutions were diluted with double-distilled water to MeOD and 90% of D2O, pH* 7.2 0.1, and monitored by H
a final concentration of 3.6% v/v HNO3, and the amount of Ru NMR at 310 K. Solutions of double-helical calf thymus DNA
in A2780 ovarian cells was determined by ICP-MS. These (ct-DNA) at a concentration of 32 µg mL−1 were incubated with
experiments did not include any cell recovery time in drug-free complex 4 at ri value of 0.1 in 10 mM NaClO4 at 310 K (ri is
media; they were carried out in triplicate, and the standard defined as the molar ratio of free ruthenium complex to
deviations were calculated. Data were processed using nucleotide phosphates at the onset of incubation with DNA).
Microsoft Excel and reported as ng Ru × 106 cells.
The concentration of ruthenium associated with DNA in these
samples was determined by flameless atomic absorption spec-
trometry (FAAS). The concentrations of DNA were determined
2.7 ROS determination
Flow cytometry analysis of ROS/superoxide induction in A2780 by absorption spectrophotometry. Plasmid DNA pBR322 (28
cells caused by exposure to complexes 1 and 4 was carried out µg mL−1) and complex 4 in various molar ratios (ri = 0.05–1)
using the Total ROS/Superoxide detection kit (Enzo-Life were incubated in 0.01 M NaClO4 at 310 K for 24 h in the dark.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Then the samples were directly mixed with the loading buffer
and loaded onto a 1% agarose gel running at 298 K in the dark
with Tris-acetate-EDTA (TAE) buffer and the voltage set at 25
V. No separation step was included before loading the samples
into the gel to allow detection of potential noncovalent
binding (if any). The gels were then stained with EtBr, followed
by photography with a transilluminator.
3. Results and discussion
3.1 Synthesis and characterisation
RuII complexes 1–6 were synthesised using a similar procedure
to that reported for related complexes (Scheme 1).25 Typically,
triethylamine (4 mol equiv.) and ligands (ca. 2 mol equiv.)
were added to a solution of [(η6-p-cym)RuCl2]2 in degassed iso-
propanol, and the reaction was stirred under a N2 atmosphere
at 365 K for 12 h. All synthesised complexes were characterised
Fig. 2 ORTEP diagrams for complex 3. Ellipsoids are shown at the 50%
probability level. All hydrogen atoms have been omitted for clarity.
2.126(9)) is within the expected range of 2.11–2.14 Å,37 but the
Ru–N12 length (2.1702(11) Å) is longer than the neutral
analogue [(η6-biph)Ru(TsEn)Cl] (2.122(3) Å),25 suggesting that
the presence of N-ethyl substituent causes a slight weakening
of this Ru–N bond. The remaining bond length and angles
show no significant difference.
1
by H and 13C NMR spectroscopy, mass spectrometry (ESI-MS)
and elemental analysis (CHN). A crystal of complex 3 suitable
for X-ray analysis was obtained by diffusion of diethyl ether
into a solution of the complex in methanol at ambient temp-
erature. Selected bond lengths and angles for complex 3 are
listed in Table 2. Crystallographic data are presented in
Table S1,† and the structure of complex 3 is shown in Fig. 2.
Complex 3 adopts a pseudo-octahedral geometry with the η6-
bonded aromatic ring occupying 3 coordination sites. The che-
lating ligand is deprotonated and bonded as a monoanionic
bidentate ligand. The CH2CH2N-Et atoms from N,N′ chelated
ligand (C10 C11 N12 C13) were modelled as disordered over
two positions whose occupancy refined to 86 : 14. Compared to
reported ruthenium ethylenediamine complexes (either
neutral or +1 charge),25,36,37 the Ru–N− bond length (N9,
3.2 Hydrolysis and pKa* determination
The hydrolysis of complex 4 was studied by dissolving the RuII
complex in d4-MeOD/D2O (1.4 mM, 1 : 9 (v/v)). The 1H NMR
spectrum remained unchanged after 24 h and the hydrolysis
was assumed to be rapid since the peaks could be assigned to
the aqua RuII species (4a) by comparison to those from the
aqua species generated in a reaction with silver nitrate in D2O
(1 mol equiv.). The pKa* (pKa value determined in deuterated
solvent) of complex 4a was determined by a pH* (meter
reading) titration ranging from 2 to 12 by addition of NaOD or
DNO3 solutions as appropriate. Changes in the chemical shift
1
of a tosyl H NMR resonance were followed and the data were
fitted to the Henderson–Hasselbalch equation, giving a pKa*
value of 9.73 0.06 (Fig. 3).
3.3 Kinetics of transfer hydrogenation reactions
The ratio of coenzyme NAD+/NADH greatly influences the
intracellular potential and can drive many reactions in vivo.38
The reduction of coenzyme nicotinamide adenine dinucleotide
(NAD+) to NADH was investigated in an aqueous medium
using complexes 1–6 as catalysts and sodium formate as the
hydride source. Initially, the TH reactions were studied by UV-
visible spectroscopy under conditions of pH 7.2 0.1, 310 K
and MeOH/H2O (1 : 9, v/v, Table 3); in all the cases, an increase
in intensity of the band at 340 nm was observed, which is
assignable to formation of NADH (Fig. S1, ESI†). The kinetics
of conversion were also monitored by 1H NMR at 310 K and
Scheme 1 Synthetic routes for diamine ligands and RuII complexes
1–6.
Table 2 Selected bond lengths (Å) and angles (°) for complex 3
Bonds
Length/angle
Ru1–N9
Ru1–N12
Ru1–N12A
Ru1–Cl1
Ru1–arene (centroid)
N9–Ru1–N12
N9–Ru1–N12A
N9–Ru1–Cl1
N12–Ru1–Cl1
2.1256(9)
2.1702(11)
2.157(8)
2.4173(3)
1.664
78.74(4)
76.1(2)
pH* 7.2
0.1. The reactions were performed in a mixed
solvent d4-MeOD/D2O (1 : 4, v/v), due to the poor aqueous solu-
bility of complexes 5 and 6, although the presence of methanol
in such reactions is known to enhance the reaction rate.25
89.47(3)
87.55(4)
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
The dependence of the rate of catalysis on pH was deter-
mined. Six pH* values ranging from 5 to 9 were studied for
complex 4 at a mol ratio complex 4 : NAD+ : formate of 1 : 4 : 25,
in the same mixed solvent at 310 K (Fig. S2, ESI†). The TOF
was relatively insensitive to pH* over the range pH* 6–8
(ca. 7.5 h−1), but decreased slightly at lower and higher pH*
(5.6 h−1 at pH* 5, 6.6 h−1 at pH* 9).
The dependence of turnover frequency on the concen-
trations of sodium formate and NAD+ was also investigated for
complex 4 in d4-MeOD/D2O (1 : 4) at 310 K. The mol ratio of
complex 4 : NAD+ : formate was 1 : 4 : X, where X = 5, 10, 25, 50
and 100 (Fig. S3, ESI†). The TOF increased steadily from
2.2 h−1 to 18.8 h−1 as the concentration of formate was
increased from 7 mM to 140 mM. Next the dependence of TOF
on the NAD+ concentration was studied for mol ratio complex
4 : NAD+ : formate = 1 : Y : 25, where Y = 2, 6 and 10. The TOF
was found to be independent of NAD+ concentration (7.7
0.5 h−1).
Fig. 3 Dependence of the 1H NMR chemical shift of a tosyl proton (red)
on pH* of aqua complex 4a. The red curve is the best fit to the
Henderson–Hasselbalch equation corresponding to a pKa* of 9.73
0.06.
The Michaelis–Menten kinetic behaviour is apparent from
a plot of turnover frequency versus formate concentration. A
reciprocal plot of turnover frequency versus formate concen-
tration gave a Michaelis constant of KM = 0.086 mM (Fig. S3
and S4, ESI†). The maximum turnover frequency TOFmax for
complex 4 (30.3 h−1) is ca. 5× higher than for [(η6-p-cym)Ru
Table 3 Turnover frequencies for transfer hydrogenation reactions
using Ru complexes 1–6 as catalysts
Complex
R1,R2
TOFa (h−1
)
TOFb (h−1
)
1
2
3
4
5
6
Me,H
Me,Me
Et,H
Bz,H
4-F-Bz,H
Naph,H
2.97 0.04
3.9 0.1
4.3 0.1
7.4 0.1
7.1 0.1
6.1 0.9
4.0 0.3
4.1 0.1
5.9 0.2
7.7 0.3
6.5 0.4
4.9 0.5
(TsEn)Cl] (complex 8, TOFmax = 6.4 h−1 25
and 20× higher than
)
for the complex [(η6-hmb)Ru(en)Cl]PF6 (TOFmax = 1.46 h−1).24
The much lower Michaelis–Menten constant (KM = 0.086 mM)
for the N-benzyl complex 4 indicates a stronger affinity of the
complex for formate compared to [(η6-p-cym)Ru(TsEn)Cl] (KM
=
a By UV-vis spectroscopy. b By NMR spectroscopy.
27.8 mM)25 and [(η6-hmb)Ru(en)Cl]PF6 (KM = 58 mM).24
The maximum turnover frequency was observed at pH* 6
(TOFmax = 7.7 h−1) (Fig. S2, ESI†). The TOF for complex 4 gradu-
In general, the introduction of substituents on the terminal ally decreased when the pH* was raised above 6. Transfer
nitrogen improved the catalytic activity. The bulkier the substi- hydrogenation was halted below pH* 4 because of the
tuents on the terminal nitrogen, the higher the TH reaction decomposition of the complex.
rate becomes. The turnover frequency reaches a maximum (ca.
7.54 h−1) when the substituent on the terminal N is benzyl
3.4 Antiproliferative activity and anticancer activity with
formate
(complex 4), making it as efficient as the RhIII complex
[(η5-Cp*)Rh(bipy)Cl]PF6.16 Interestingly, the TOF decreases when Ruthenium complexes have shown promise for their activity
the substituent is para-fluoro-benzyl (complex 5) or naphtha- against various types of cancer cells.42 The antiproliferative
lene (complex 6), probably, because these ligands hamper the activity of complexes 1–6 towards A2780 human ovarian cancer
approach of NAD+ to the Ru centre. Compared to the en cells was determined and compared with the clinically
complex with unsubstituted nitrogens [(η6-biph)Ru(en)Cl]PF6, approved drug cisplatin, Fig. 4. The IC50 values (50% inhi-
the turnover frequency of complex 4 is 41× higher,24 and 2.7× bition of cell growth) range from 1 to 6.5 μM for complexes
higher compared to [(η6-p-cym)Ru(TsEn)Cl].25
containing aromatic R substituents (4–6), whereas those con-
The NH proton of the chelated diamine ligand appears to taining aliphatic R substituents were less potent with IC50
be essential for the TH reduction of ketones to alcohols;39 nor- values of 12–31 µM. The complex [(η6-p-cym)Ru(TsEn(Bz,H))Cl]
mally, RuII catalysts for TH of ketones form 16-e intermedi- (4) (IC50, 1.0 μM) has a potency similar to cisplatin in this cell
ates.40,41 It has been reported that a RuII complex with two line (CDDP, 1.20 0.02 µM). It is apparent that the presence of
N-alkyl groups (R,R)-[(η6-benzene)Ru(TsDPEN-Me2)Cl] exhibi- aromatic substituents on the chelated ligands of complexes
ted poor catalytic reactivity in TH reaction of ketones.40 4–6 give rise to more potent cytotoxicity than aliphatic substi-
However, complex 2 [(η6-p-cym)Ru(TsEn(Me,Me))Cl] exhibited tuents in complexes 1–3, most probably due to their higher
good catalytic activity towards the TH reduction of NAD+ to lipophilicity.
NADH (TOF = 4.1 h−1, Table 3), despite not having an NH
Combination treatment with formate can greatly increase
proton, which suggests, as expected, that an N–H is not essen- the antiproliferative activity of RuII arene sulfonyl diamine
tial in the transfer reduction of NAD+ to NADH.
complexes, which offers a potential new strategy for cancer
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Interestingly for complex 6, a 28% decrease in cell viability was
observed with only 0.5 mM formate present (Fig. 5, for percen-
tage of viability decrease see Table S3, ESI†). The largest
decrease of cell survival was 31% for complex 6 in the presence
of 2 mM sodium formate, followed by 29% and 32% for the
other two complexes with aromatic substituents, complexes 4
and 5, respectively. Complexes 1–3 with aliphatic functional
groups showed an increase in potency of 18%, 21% and 22%,
respectively.
3.5 Cell accumulation and relative hydrophobicity
Hydrophobicity and cellular accumulation are often important
factors that play key roles in the potency of organometallic and
other anticancer drugs.43 The cellular accumulation, as an
equilibrium between uptake and efflux, of ruthenium in
A2780 human ovarian cancer cells after exposure to complexes
1–6 at their IC50 equipotent concentrations was determined by
inductively coupled plasma mass spectrometry (ICP-MS) and is
shown in Fig. 6.
Complex 4 gave the lowest cellular accumulation (0.52
0.08 ng of Ru per 106 cells), while complex 6 with moderate
anticancer activity, exhibited the highest extent of cell accumu-
lation with 4.5 0.2 ng of Ru per 106 cells at IC50 concen-
tration, 8.6× higher than complex 4. Complexes 1–3 and 5,
gave rise to similar cell uptake 2.4 0.3 ng, 1.2 0.2 ng, 3.0
0.2 ng and 1.3 0.2 ng per 106 cells, respectively, following the
order: 4 < 2, 5 < 1 < 3 < 6.
The relative hydrophobicity of complexes 1–6 was deter-
mined by RP-HPLC. The more hydrophobic complexes have
longer retention times on a reverse-phase C18 column.44 To
ensure solubility of the RuII complexes in water, methanol was
used as co-solvent (MeOH/H2O, 1 : 9 v/v) together with NaCl
(50 mM) to suppress hydrolysis of the complexes. The HPLC
solvents were also prepared with 50 mM NaCl (measurements
see Fig. S5, ESI†). The resulting retention times are shown in
Table 4, and follow the order: 1, 2, 3 < 4, 5 < 6. Complex 3
shows the shortest retention time (least hydrophobic) of
Fig. 4 Antiproliferative activity of RuII complexes 1–6 and cisplatin
towards A2780 human ovarian cancer cells.
treatment.15 In this work, the antiproliferative activity of RuII
complexes in A2780 human ovarian cancer cells in the pres-
ence of sodium formate was studied (Fig. 5). Firstly, the cyto-
toxicity of sodium formate alone towards A2780 human
ovarian cancer cells was investigated. No significant toxicity
was found up to formate concentrations of 2 mM which is in
agreement with the previous report.15 Then, A2780 human
ovarian cancer cells were coincubated with equipotent concen-
trations of complexes 1–6 (1/3 × IC50) and three different con-
centrations of sodium formate (0.5, 1 and 2 mM) in order to
observe the formate-concentration dependence of the cell via-
bility. The antiproliferative activity of complexes 1–6 increased
significantly upon coincubation with 2 mM formate. The
formate-induced decrease in viability of A2780 cells ranged
from 20% to 36% in the presence of complexes 1–6.
Fig. 5 Percentage of cell survival when equipotent concentrations of
complexes 1–6 (1/3 × IC50) were co-administered with different con-
centrations of sodium formate, p-values were calculated after a t-test
against the negative control data (without sodium formate), *p < 0.05,
**p < 0.01.
Fig. 6 IC50 values (µM) for complexes 1–6 against A2780 human
ovarian cancer cells (orange bars) and cellular accumulation of Ru in
A2780 cancer cells at equipotent IC50 concentrations in the absence of
sodium formate (in green).
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Table 4 Retention times (tR) of RuII complexes 1–6 by RP-HPLC and
cellular accumulation (at equipotent of IC50 concentrations) in A2780
cells
Complex
tR (min)
Cellular-Ru (ng per 106 cells)
1
2
3
4
5
6
15.4 0.9
14.5 0.3
14.0 0.3
17.4 0.2
17.27 0.08
2.4 0.3
1.2 0.2
3.0 0.2
0.52 0.08
1.3 0.2
4.5 0.2
20
1
Fig. 7 ROS in A2780 cells induced by complexes 1 and 4, FL1 channel
detects total oxidative stress, and FL2 channel detects superoxide pro-
duction. (A) Induction of ROS by complexes 1 and 4. (B) Four different
populations induced by complexes 1 and 4 at equipotent IC50 concen-
trations. p-Values were calculated after a t-test against the negative
control data, **p < 0.01, ***p < 0.001.
14.0 min, while complex 6 shows the longest retention time
(most hydrophobic), 20.9 min.
It is evident from Table 4 that the RuII complexes with aro-
matic substituents (complexes 4–6) exhibit higher hydrophobi-
city than complexes with aliphatic substituents (complexes
1–3). The most hydrophobic complex (6) shows the highest cell
accumulation. Nonetheless, there is no linear correlation
between the hydrophobicity of complexes 1–6 and their cellu-
lar accumulation. This has been observed before.45 In these
cases, the chemistry and the mechanism of action of each par-
ticular complex has a higher impact on the compound’s anti-
cancer activity than cellular accumulation per se. However,
complex 4 has the lowest extent of cell uptake, but the most
potent antiproliferative activity, suggesting that it is the chemi-
cal properties of the intracellular drug that are more important
for activity than the total amount of Ru entering the cell. In
general, a high hydrophobicity could facilitate interaction
between the organometallic complex and cell membranes, and
also correlate with the potency of the complex, but that is not
always the case.43,45
ROS are induced in the majority, if not in all, of the cell popu-
lation. These ROS may play a major role in killing the cancer
cells (Table S4, ESI†).
3.7 DNA related binding for complex 4
The interaction of complex 4 with DNA nucleobase models:
9-ethylguanine (9-EtG) and adenosine 5′-monophosphate
1
(5′-AMP) was studied by H NMR spectroscopy (Fig. S6, ESI†).
The reactions were performed by adding nucleobase solution
(3 mM in D2O) to RuII complex solution (2 mM in 10% d4-
MeOD/90% D2O) at 310 K, to give a final 1.5 : 1 mol ratio. The
formation of adduct 4-9-EtG was confirmed by following the
new set of peaks, and up to 90% yield of adduct was obtained
when 1.5 mol equiv. nucleobase solution was added. However,
no adduct was found when 1.5 mol equiv. of 5′-AMP was
added to complex 4, even after 24 h incubation at 310 K.
Reactions of double-helical calf thymus DNA (ct-DNA, 32
µg mL−1) and plasmid DNA pBR322 (28 µg mL−1) with
complex 4 in various molar ratios (ri = 0.05–1, ri = the molar
ratio of free Ru complex to nucleotide phosphates at the onset
of incubation with DNA) were studied. Very low amounts of
ruthenium (5–7% of initial Ru) were found in the samples of
DNA treated with complex 4 for 24 h. No significant changes
in the mobilities of supercoiled (sc) or open circular (oc) form
of plasmid DNA were observed even when incubated with high
concentration of complex 4 (ri = 1, Fig. S7, ESI†). DNA is
3.6 ROS induction
Reactive oxygen species (ROS) are metabolic byproducts of
aerobic respiration and are responsible for maintaining redox
homeostasis in cells.46 ROS also play a significant role in the
mechanism of action of anticancer agents.47,48 Some organo-
metallic complexes, e.g. Ir and Os,49–51 can generate high ROS
levels or bursts of superoxide in cancer cells to induce cell
apoptosis,49 but by comparison, other complexes are known to
induce cell death by reductive stress.15 The levels of reactive
oxygen species (ROS) were determined in A2780 human
ovarian cancer cells for complexes 1 and 4 at IC50 concen-
trations by flow cytometry fluorescence analysis (Fig. 7). This
included the monitoring of H2O2, peroxy and hydroxyl radicals
using a green probe, and superoxide levels using the orange
channel. Induction of total ROS and superoxide were deter-
mined in A2780 cells after 24 h exposure to complexes 1 and 4
when compared to the negative untreated control. The popu-
lations of cells that show high fluorescence in both FL-1 and
thought to be a cellular target for the en complex
7
(Fig. 1).52,53 However, for the substituted-en complex studied
here, no obvious unwinding of DNA was observed after coincu-
bation of ct-DNA with complex 4, suggesting that binding is
weak, nor changes in the ratio of sc and oc forms of plasmid
DNA, suggesting that complex 4 does not cleave DNA.
3.8 DFT calculations
FL-2 channels (both high total ROS and high superoxide gene- We modelled the catalytic cycle by considering seven states of
ration) for complexes 1 and 4 are 16.5 1.0% and 31.3 0.3%, the reaction: (1) the initial aqua complex [(η6-p-cym)Ru(TsEn
respectively, which indicates a higher induction of superoxide (R1,R2))(OH2)]+ and formate (with isolated NAD+); (2) [(η6-p-
by complex 4. Remarkably, the total increase of the population cym)Ru(TsEn(R1,R2))(OH2)]+ interacting intermolecularly with
in the high FL-1 green channel shows that the levels of total NAD+,
and
formate;
(3)
[(η6-p-cym)Ru(TsEn(R1,R2))
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Fig. 8 (Top) Reduction cycle for conversion of NAD+ to 1,4-NADH via transfer hydrogenation with formate as the hydride donor. (Bottom) DFT
energy profile for the formation of Ru formate species, Ru hydride complex and hydride transfer from ruthenium; brown line, complex 1; red line,
complex 2; blue line, complex 3; green line, complex 4; purple line, complex 5; black line, complex 6. Sets of calculated structures of states 1–7 are
supplied in the ESI and illustrated graphically in Fig. S8† for complex 2. To calculate the energy of states 1 and 7, the energies of the states rep-
resented in pdb files 1 and 7 were added to the energies calculated for NAD+ and NADH.
(HCOO−)]·NAD+ and water; (4) [(η6-p-cym)Ru(TsEn(R1,R2))H] were found ca. 100 kJ mol−1 higher in energy. It is notable that
·NAD+ and water and CO2; (5) [(η2-p-cym)Ru(TsEn(R1,R2))(OH2) the Ru atoms of all complexes in state 5 are coordinated to the
NADH] and CO2; (6) [(η6-p-cym)Ru(TsEn(R1,R2))(OH2)]NADH amide oxygen atom of NADH, while only weakly bound to the
and CO2; (7) [(η6-p-cym)Ru(TsEn(R1,R2))(OH2)]+, water and CO2, (hydridic) CH2 of NADH, giving a Ru–H distance of 3.11–3.12 Å
and isolated NADH (Fig. 8).
for R1,R2 = Me,H (1); Et,H (3); Naph,H (6) and 3.06–3.07 Å for
For state 5, with ring-slipped coordinated η2-p-cymene, the R1,R2 = Bz,H (4) and 4-F-Bz,H (5). For R1,R2 = Me,Me (2) the
introduction of water into coordination sphere was necessary, calculations revealed a true bonding of the (hydridic) CH2,
while highly distorted complexes without coordinated water with a Ru–H distance of 1.99 Å. The results obtained are
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
shown in Fig. 8. Four general conclusions can be drawn from potent, the benzyl complex 4 being as potent as the anticancer
these data: (a) there is a strong interaction between the drug cisplatin (Fig. 6). In general, the most hydrophobic com-
[(η6-p-cym)Ru(TsEn(R1,R2))(OH2)]+ cation and NAD+ and NADH plexes were found to be the most biologically active. However,
molecules, leading to a stabilisation of the cationic form by the activity does not correlate closely with total cell accumu-
60–70 kJ mol−1 for NAD+ (41 kJ mol−1 for R1,R2 = 4-F-Bz,H (5)) lation of Ru or with hydrophobicity (Table 3). Although DNA
and 130–150 kJ mol−1 for NADH; (b) depending on the can be a target for related arene RuII diamine complexes, it
N-substituent, the species of the lowest energy is either [(η6-p- does not appear to be a target for these sulfonyl-en RuII cata-
cym)Ru(TsEn(R1,R2))(HCOO−)]·NAD+ (R1,R2 = Me,H (1); Bz,H lysts since we observe very weak binding to both calf thymus
(4); 4-F-Bz,H (5) and Naph,H (6)) or [(η6-p-cym)Ru(TsEn(R1,R2)) and plasmid DNA (Fig. S7, ESI†).
(OH2)]·NAD+ (R1,R2 = Et,H (3) and Me,Me (2)), the difference
We showed that complexes 1 and 4 can generate high levels
between them being only 6–12 kJ mol−1; (c) the effective of ROS in A2780 human ovarian cancer cells, especially 4, the
NADH-hydride coordination for bulky R1,R2 = Me,Me (2) most potent complex. This is consistent with interference in
lowers the energy, relative to the state of lowest energy, of the cellular redox pathways and possible attack on NAD+ when
species with coordinating NADH by 30–40 kJ mol−1, compared sodium formate is present. The enhancement of anticancer
to other complexes; (d) the formation of the state with the Ru– activity by low non-toxic dose of formate might be clinically
H hydride bond, including the twist of formate and the elimin- useful since it introduces a new mechanism of activity which
ation of carbon dioxide, corresponds to the highest energy does not involve DNA attack, unlike the clinical drug cisplatin.
step. These four factors seem all to influence the turnover. The Such a regime might therefore avoid some unwanted side-
energy barriers and optimized structures for the seven states effects. Formate itself is a natural biochemical molecule
of complex 2 in the cycle with NAD+ are listed in Table S5 and enriched in some cancer cells.54 However, more work remains
illustrated graphically in Fig. S8.† The structure files for the to be done to investigate possible intracellular catalysis,
remaining complexes are supplied as ESI.†
especially since a range of metabolites might readily poison
these catalysts in cells.
4. Conclusions
Author contributions
In this work, we have synthesised a series of new RuII com-
plexes of the type [(η6-p-cym)Ru(N,N′)Cl] where N,N′ are mono-
sulfonamide chelating ligands derived from tosylethyl-
enediamine, with either alkyl (Me,H (1); Me,Me (2); Et,H (3)) or
aryl (Bz,H (4); 4-F-Bz,H (5); Naph,H (6)) substituents on the
terminal N. These substituents have a significant effect on the
rate of transfer hydrogenation of coenzyme NAD+ with formate
as hydride donor as determined by NMR and UV-vis spec-
troscopy. In general, the bulkier aromatic substituents gave
rise to faster hydrogenation rates (Table 3). DFT calculations
provided insight into the mechanism of hydride transfer form
formate to NAD+ involving initial coordination of formate fol-
lowed by transfer of hydride to ruthenium and then to NAD+
with release of CO2. The calculations suggested a preorganiza-
tion of the initial aqua complex, formate and NAD+ involving
T-shaped adenosine NH-tosyl stacking, H-bonding of the NH
of the chelated ligand and phosphate O of NAD+, H-bonding
between formate and water, and between formate and the pyri-
dine ring of NAD+. They also indicated strong interactions with
NADH involving T-shaped adenosine NH-tosyl stacking, as well
as H-bonds to phosphate and (hydridic) CH2-tosylate O
(Fig. S8, ESI†).
Feng Chen, Joan J. Soldevila-Barreda, Abraha Habtemariam
and Peter J. Sadler designed the experiments and interpreted
data.
Feng Chen carried out synthesis and characterisation of
ligands and complexes, investigated hydrolysis, determined
the pKa value, and TH turnover frequencies.
Isolda Romero-Canelón and Ji-Inn Song carried out the cell
antiproliferative screening and related biochemical assays.
Guy J. Clarkson carried out the X-ray crystallography.
Juliusz A. Wolny and Volker Schünemann carried out all
the DFT calculations.
Jana Kasparkova and Viktor Brabec carried out DNA
binding studies.
James P. C. Coverdale carried out metal analyses by
ICP-OES and ICP-MS, and related biological and biochemical
assays.
All authors contributed to the writing of the paper.
Conflicts of interest
The authors declare no conflicts of interest.
To investigate the possibility of achieving transfer hydro-
genation mediated by formate in cells, we investigated the
effect of formate on the antiproliferative activity of these com-
plexes towards human ovarian cancer cells. In each case a
Acknowledgements
dose-dependent increase in potency of the complexes We thank ERDF/AWM (Science City), NWO (Rubicon grant),
(20–36%) was observed with increasing formate concentration EPSRC (grant no. EP/F042159/1), and ERC (grant no. 247450)
over a range of non-toxic formate concentrations (0–2 mM). for support for this work, China Scholarship Council (CSC) for
The complexes with aromatic substituents were the most a scholarship for F. C., and Bruker Daltonics and Warwick
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Dalton Transactions
Paper
Collaborative Postgraduate Research Scholarship (WCPRS, 22 A. Fujii, S. Hashiguchi, N. Uematsu, T. Ikariya and
funding for J. P. C. C.); J. A. W. and V. S. acknowledge support R. Noyori, J. Am. Chem. Soc., 1996, 118, 2521–2522.
of the research initiative NANOKAT and the German Federal 23 J. P. C. Coverdale, I. Romero-Canelón, C. Sanchez-Cano,
Ministry of Education and Research (BMBF under 05K14UK1)
and are grateful to the Allianz für Hochleistungsrechnen
G. J. Clarkson, A. Habtemariam, M. Wills and P. J. Sadler,
Nat. Chem., 2018, 10, 347–354.
Rheinland-Pfalz (AHRP) for providing CPU-time within the 24 Y. K. Yan, M. Melchart, A. Habtemariam, A. F. A. Peacock
project TUK-SPINPLUSVIB. and P. J. Sadler, J. Biol. Inorg. Chem., 2006, 11, 483–488.
We also thank Dr Ivan Prokes, Dr Lijiang Song, and Mr 25 J. J. Soldevila-Barreda, P. C. A. Bruijnincx, A. Habtemariam,
Philip Aston (University of Warwick) for their excellent assist-
ance with the NMR and MS measurements.
G. J. Clarkson, R. J. Deeth and P. J. Sadler, Organometallics,
2012, 31, 5958–5967.
26 S. Betanzos-Lara, Z. Liu, A. Habtemariam, A. M. Pizarro,
B. Qamar and P. J. Sadler, Angew. Chem., Int. Ed., 2012, 51,
3897–3900.
27 A. Habtemariam, M. Melchart, R. Fernández, S. Parsons,
I. D. H. Oswald, A. Parkin, F. P. A. Fabbiani, J. E. Davidson,
A. Dawson, R. E. Aird, D. I. Jodrell and P. J. Sadler, J. Med.
Chem., 2006, 49, 6858–6868.
References
1 U. Eisner and J. Kuthan, Chem. Rev., 1972, 72, 1–42.
2 J. G¸ebicki, A. Marcinek and J. Zielonka, Acc. Chem. Res.,
2004, 37, 379–386.
3 A. McSkimming and S. B. Colbran, Chem. Soc. Rev., 2013, 28 J. E. D. Martins, G. J. Clarkson and M. Wills, Org. Lett.,
42, 5439–5488. 2009, 11, 847–850.
4 S. Fukuzumi and T. Suenobu, Dalton Trans., 2013, 42, 18–28. 29 X. Li, L. Li, Y. Tang, L. Zhong, L. Cun, J. Zhu, J. Liao and
5 S. M. Barrett, C. L. Pitman, A. G. Walden and A. J. M. Miller,
J. Deng, J. Org. Chem., 2010, 75, 2981–2988.
J. Am. Chem. Soc., 2014, 136, 14718–14721.
30 V. Vichai and K. Kirtikara, Nat. Protoc., 2006, 1, 1112–1116.
6 M. C. Carrión, F. Sepúlveda, F. A. Jalón, B. R. Manzano and 31 D. T. T. Yanai and N. Handy, Chem. Phys. Lett., 2004, 393,
A. M. Rodríguez, Organometallics, 2009, 28, 3822–3833. 51–57.
7 Y. Maenaka, T. Suenobu and S. Fukuzumi, J. Am. Chem. 32 W. J. Stevens, H. Basch and M. Krauss, J. Phys. Chem., 1984,
Soc., 2012, 134, 9417–9427. 81, 6026–6033.
8 A. Wolfson, C. Dlugy, Y. Shotland and D. Tavor, Tetrahedron 33 W. J. Stevens, M. Krauss, H. Basch and P. G. Jasien,
Lett., 2009, 50, 5951–5953. Can. J. Chem., 1992, 70, 612–630.
9 R. Ruppert, S. Herrmann and E. Steckhan, Tetrahedron 34 T. R. S. Cundari, J. Chem. Phys., 1993, 98, 5555–5565.
Lett., 1987, 28, 6583–6586.
35 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato,
A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts,
B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov,
J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini,
F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson,
D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng,
W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery Jr.,
J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. J. Heyd,
E. N. Brothers, K. N. Kudin, N. Staroverov, T. A. Keith,
R. Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell,
J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,
J. M. Millam, M. Klene, C. Adamo, R. Cammi,
J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas,
J. B. Foresman and D. J. Fox, Gaussian 16, Revision A. 03,
Gaussian, Inc., Wallingford CT, 2016.
10 E. Steckhan, S. Herrmann, R. Ruppert, E. Dietz, M. Frede
and E. Spika, Organometallics, 1991, 10, 1568–1577.
11 E. Steckhan, S. Herrmann, R. Ruppert, J. Thömmes and
C. Wandrey, Angew. Chem., Int. Ed. Engl., 1990, 29, 388–390.
12 V. D. Westerhausen, S. Herrmann, W. Hummel and
E. Steckhan, Angew. Chem., Int. Ed. Engl., 1992, 31, 1529–
1531.
13 R. T. Hembre and S. McQueen, J. Am. Chem. Soc., 1994,
116, 2141–2142.
14 C. Wang, B. Villa-Marcos and J. Xiao, Chem. Commun.,
2011, 47, 9773–9785.
15 J. J. Soldevila-Barreda, I. Romero-Canelón, A. Habtemariam
and P. J. Sadler, Nat. Commun., 2015, 6, 6582.
16 H. C. Lo, O. Buriez, J. B. Kerr and R. H. Fish, Angew. Chem.,
Int. Ed., 1999, 38, 1429–1432.
17 H. C. Lo, C. Leiva, O. Buriez, J. B. Kerr, M. M. Olmstead
and R. H. Fish, Inorg. Chem., 2001, 40, 6705–6716.
18 J. M. O’Connor and C. P. Casey, Chem. Rev., 1987, 87, 307–
318.
36 R. E. Morris, R. E. Aird, M. P. Socorro, H. Chen,
J. Cummings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd,
D. I. Jodrell and P. J. Sadler, J. Med. Chem., 2001, 44, 3616–
3621.
19 K. T. Oppelt, J. Gasiorowski, D. A. M. Egbe, J. P. Kollender,
M. Himmelsbach, A. W. Hassel, N. S. Sariciftci and
G. Knör, J. Am. Chem. Soc., 2014, 136, 12721–12729.
20 V. Ganesan, D. Sivanesan and S. Yoon, Inorg. Chem., 2017, 37 A. F. A. Peacock, A. Habtemariam, R. Fernández,
56, 1366–1374.
V. Walland, F. P. A. Fabbiani, S. Parsons, R. E. Aird,
D. I. Jodrell and P. J. Sadler, J. Am. Chem. Soc., 2006, 128,
1739–1748.
21 S. Hashiguchi, A. Fujii, J. Takehara, T. Ikariya and
R. Noyori, J. Am. Chem. Soc., 1995, 117, 7562–7563.
This journal is © The Royal Society of Chemistry 2018
Dalton Trans.

View Article Online
Paper
Dalton Transactions
38 J. Zhang, A. Pierick, H. M. Rossum, R. M. Seifar, C. Ras, 46 A. T. Dharmaraja, J. Med. Chem., 2017, 60, 3221–3240.
J. Daran, J. J. Heijnen and S. A. Wahl, Sci. Rep., 2015, 5, 47 D. Trachootham, J. Alexandre and P. Huang, Nat. Rev. Drug
12846.
Discovery, 2009, 8, 579–591.
39 M. Yamakawa, H. Ito and R. Noyori, J. Am. Chem. Soc., 48 J. Watson, Open Biol., 2013, 3, 120144.
2000, 122, 1466–1478.
40 R. Soni, F. K. Cheung, G. C. Clarkson, J. E. D. Martins,
49 Z. Liu, I. Romero-Canelón, A. Habtemariam, G. J. Clarkson
and P. J. Sadler, Organometallics, 2014, 33, 5324–5333.
M. A. Graham and M. Wills, Org. Biomol. Chem., 2011, 9, 50 Z. Liu, I. Romero-Canelón, B. Qamar, J. M. Hearn,
3290–3294.
A. Habtemariam, N. P. E. Barry, A. M. Pizarro,
G. J. Clarkson and P. J. Sadler, Angew. Chem., Int. Ed., 2014,
53, 3941–3946.
41 X. Wu, X. Li, W. Hems, F. King and J. Xiao, Org. Biomol.
Chem., 2004, 2, 1818–1821.
42 Z. Adhireksan, G. E. Davey, P. Campomanes, M. Groessl, 51 Y. Fu, M. J. Romero, A. Habtemariam, M. E. Snowden,
C. M. Clavel, H. Yu, A. A. Nazarov, C. H. F. Yeo, W. H. Ang,
P. Droge, U. Rothlisberger, P. J. Dyson and C. A. Davey, Nat.
Commun., 2014, 5, 3462.
L. Song, G. J. Clarkson, B. Qamar, A. M. Pizarro,
P. R. Unwin and P. J. Sadler, Chem. Sci., 2012, 3, 2485–
2494.
43 A. J. Millett, A. Habtemariam, I. Romero-Canelón, 52 H. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall,
G. J. Clarkson and P. J. Sadler, Organometallics, 2015, 34,
2683–2694.
R. O. Gould and P. J. Sadler, J. Am. Chem. Soc., 2002, 124,
3064–3082.
44 T. Bugarcic, O. Nováková, A. Halámiková, L. Zerzánková, 53 G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2010,
O. Vrána, J. Kašpárková, A. Habtemariam, S. Parsons, 54, 3–25.
P. J. Sadler and V. Brabec, J. Med. Chem., 2008, 51, 5310– 54 P. M. Tedeschi, E. K. Markert, M. Gounder, H. Lin,
5319.
D. Dvorzhinski, S. C. Dolfi, L. L. Y. Chan, J. Qiu,
R. S. DiPaola, K. M. Hirshfield, L. G. Boros, J. R. Bertino,
Z. N. Oltvai and A. Vazquez, Cell Death Dis., 2013,
4, 877.
45 M. Hanif, A. A. Nazarov, C. G. Hartinger, W. Kandioller,
M. A. Jakupec, V. B. Arion, P. J. Dyson and B. K. Keppler,
Dalton Trans., 2010, 39, 7345–7352.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2018