Half-sandwich rhodium(III) transfer hydrogenation catalysts: Reduction of NAD+ and pyruvate, and antiproliferative activity

Article abstract of DOI:10.1016/j.jinorgbio.2015.10.008

Organometallic complexes have the potential to behave as catalytic drugs. We investigate here Rh(III) complexes of general formula [(Cp^x)Rh(N,N′)(Cl)], where N,N′ is ethylenediamine (en), 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen) or N-(

Full text of DOI:10.1016/j.jinorgbio.2015.10.008

Half-sandwich rhodium(III) transfer hydrogenation catalysts: Reduction of
+
NAD and pyruvate, and antiproliferative activity
Joan J. Soldevila-Barreda, Abraha Habtemariam, Isolda Romero-Canel o´ n,
Peter J. Sadler
PII:
DOI:
Reference:
S0162-0134(15)30097-0
doi: 10.1016/j.jinorgbio.2015.10.008
JIB 9823
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
7 August 2015
5 October 2015
7 October 2015
Please cite this article as: Joan J. Soldevila-Barreda, Abraha Habtemariam, Isolda
Romero-Canel ´o n, Peter J. Sadler, Half-sandwich rhodium(III) transfer hydrogenation
+
catalysts: Reduction of NAD and pyruvate, and antiproliferative activity, Journal of
Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.10.008
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ACCEPTED MANUSCRIPT
For submission to J. Inorg. Biochem. (Organometallic/Medicinal)
JIB 15-0807Rev
Half-sandwich rhodium(III) transfer hydrogenation
+
catalysts: reduction of NAD and pyruvate, and
antiproliferative activity
†
†
†
Joan J. Soldevila-Barreda , Abraha Habtemariam , Isolda Romero-Canelón and Peter J.
†
Sadler*
†
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL,
UK
+
KEYWORDS: NADH, NAD , pyruvate, transfer-hydrogenation, anticancer, organo-
rhodium.
1

ACCEPTED MANUSCRIPT
ABSTRACT: Organometallic complexes have the potential to behave as catalytic drugs. We
x
investigate here Rh(III) complexes of general formula [(Cp )Rh(N,N′)(Cl)], where N,N′ is
ethylenediamine (en), 2,2'-bipyridine (bpy), phenanthroline (phen) or N-(2-aminoethyl)-4-
x
(
trifluoromethyl)benzenesulfonamide (TfEn), and Cp is pentamethylcyclopentadienyl (Cp*),
xPh
1
-phenyl-2,3,4,5-tetramethylcyclopentadienyl (Cp ) or 1-biphenyl-2,3,4,5-tetramethyl
xPhPh
+
cyclopentadienyl (Cp
). These complexes can reduce NAD to NADH using formate as
an hydride source under biologically-relevant conditions. The catalytic activity decreased in
5
the order of N,N-chelated ligand bpy > phen > en with Cp* as the η -donor. The en
X
complexes (1-3) became more active with extension to the Cp ring, whereas the activity of
+
the phen (7-9) and bpy (4-6) compounds decreased. Cp*Rh(bpy)Cl] (4) showed the highest
-1
catalytic activity, with a TOF of 37.4 ± 2 h . Fast hydrolysis of the chlorido complexes 1-10
1
was observed by H NMR (< 10 min at 310 K). The pK * values for the aqua adducts were
a
determined to be ca. 8-10. Complexes 1-9 also catalysed the reduction of pyruvate to lactate
using formate as the hydride donor. The efficiency of the transfer hydrogenation reactions
x
was highly dependent on the nature of the chelating ligand and the Cp ring. Competition
+
reactions between NAD and pyruvate for reduction by formate catalysed by 4 showed a
+
preference for reduction of NAD . The antiproliferative activity of complex 3 towards A2780
human ovarian cancer cells increased by up to 50% when administered in combination with
non-toxic doses of formate, suggesting that transfer hydrogenation can induce reductive stress
in cancer cells.
2

ACCEPTED MANUSCRIPT

Graphical abstract (synopsis)
We report for the first time half-sandwich Rh(III) complexes as catalytic anticancer
+
agents capable of reducing nicotinamide adenine dinucleotide (NAD ) and pyruvate in the
presence of a hydride source (formate). The complexes display low-moderate
antiproliferative activity towards cancer cells. However, a significant increase in activity
upon coadministration with formate can be achieved.
Graphical abstract (figure attached)
3

ACCEPTED MANUSCRIPT
1
. Introduction
Transfer hydrogenation reactions for the reduction of ketones, imines or C=C double bonds
have been intensively studied in recent years [1-4]. Precious metal complexes containing Ru,
Rh or Ir are often effective as transfer hydrogenation catalysts, and half-sandwich
organometallic complexes in particular have achieved high conversions, turnover frequencies
(
TOF) and enantio-selectivities [1-4] Ruthenium(II) catalysts are often the most efficient,
.
partly due to their slower rate of ligand exchange compared to Rh(III) or Ir(III), which, in
many cases, provides higher chemo- and enantio-selectivity [1-3,5-7]. Although rhodium
catalysts can be more active, this is often accompanied by a loss of chemo- and enantio-
selectivity. Nevertheless, catalytic activity and enantio-selectivity depend on the choice of
appropriate chiral ligands, on the substrate, the hydride donor, and on the reaction conditions
[
1-3,5-7].
There is current interest in metal-based complexes capable of catalysing reactions in cells [8-
1]. Metallodrugs with catalytic properties can potentially be administered in smaller doses
1
consequently leading to lower toxicity [8-11]. Transfer hydrogenation reactions have
attracted much attention for the reduction of molecules inside cells [8-11]. Organo-Ru(II) and
-
Ir(III) complexes can catalyse hydride transfer reactions under biologically-relevant
6
conditions (pH 7.2, 310 K, aqueous media) [12-17]. For example, [(η -
+
x
+
X
arene)Ru(bipyrimidine)Cl]
or
[(Cp Ir(phenanthroline)Cl]
where
Cp
is
xPh
pentamethylcyclopentadienyl (Cp*), 1-phenyl-2,3,4,5-tetramethylcyclopentadienyl (Cp ) or
xPhPh
1
-biphenyl-2,3,4,5-tetramethylcyclopentadienyl (Cp
) can utilise nicotinamide adenine
dinucleotide reduced (NADH) to transfer hydride and reduce biomolecules such as pyruvate
+
[
13,14]. The reverse transfer of hydride to nicotinamide adenine dinucleotide (NAD ) can
6
+
also be achieved using formate as an hydride source, [(η -arene)Ru(ethylendiamine)Cl] and
+
[
(arene)Ru(N-(2-aminoethyl)-sulfonamide)Cl] where arene = p-cymene (p-cym), benzene
4

ACCEPTED MANUSCRIPT
bn) or hexamethyl benzene (hmb)and biphenyl (bip) can regenerate NADH from NAD
12,15,16].
+
(
[
Our recent work suggests that transfer hydrogenation catalysed by organometallic complexes
can be achieved inside cells [12,13]. For example, the Ir(III) complex
xphph
+
[
(Cp
)Ir(phenylpyrdine)py] , where py is pyridine, can utilize NADH as a biological
hydride donor, to generate an iridium-hydride complex. The hydrido complex is able to
transfer hydride to molecular oxygen, increasing the levels of hydrogen peroxide and reactive
oxygen species in cancer cells [13]. Also, the Ru(II) complex [(p-cym)Ru(TsEn)Cl] where
+
TsEn is N-(2-aminoethyl)-4-toluene sulfonamide reduces the levels of NAD in A2780
ovarian cancer cells when co-administered with sodium formate, potentiating the anti-cancer
activity of the complex [12].
The aim of the present work was to synthesise a series of Rh(III) Cp* catalysts which can
carry out transfer hydrogenation reactions in cells. Half-sandwich complexes of the type
X
n+
[
(Cp Rh(N,N’)Cl] , where N,N’ = ethylenediamine (en), 2,2'-bipyridine (bpy), 2,2'-
dimethylbipyridine (mbpy) and phenanthroline (phen) or were synthesised and fully
+
characterized (Figure 1). The activity of these Rh(III) complexes for the reduction of NAD
6
in the presence of formate was compared with their Ru(II) analogues [(η -arene)Ru(en)Cl]
6
and [(η -arene)Ru(TsEn)Cl] We also studied the reduction of pyruvate to lactate via hydride
transfer from formate, a process carried out naturally in vivo by the enzyme lactate
dehydrogenase and the coenzyme NADH. Finally, the antiproliferative activity of the
complexes towards cancer cells in the presence of excess formate was also investigated.
5

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2
. Experimental
2
.1 Materials.
Rhodium(III) trichloride hydrate was purchased from Precious Metals Online (PMO Pty Ltd.)
and used as received. Ethylenendiamine (en) was purchased from Sigma-Aldrich and freshly
XPh
distilled prior to use. The ligands 3-phenyl-1,2,4,5-tetramethyl-1,3-cyclopentadienyl (Cp
)
xPhPh
and 3-biphenyl-1,2,4,5-tetramethyl-1,3-cyclopentadienyl (Cp
)
were synthesised
following the methods in the literature [18,19]. The Rh(III)-arene precursor dimers
x
[
(Cp )RhCl ] were prepared following literature methods [20], as was the ligand N-(2-
2
2
aminoethyl)-4-(trifluoromethyl)benzenesulfonamide (TfEnH) [15]. 2,2'-Bipyridine, 4,4'-
dimethyl-2,2’-dipyridine, 1,10-phenanthroline, 4-bromo-biphenyl, 4-bromo-biphenyl, 1.6 M
n-butyllithium in hexane, phenyllithium in ether (1.8 mM), 2,4-pentamethylcyclopentadiene,
2
,3,4,5-tetramethyl-2-cyclopentanone were obtained from Sigma-Aldrich. Magnesium
sulphate, ammonium hexafluorophosphate, silver nitrate, potassium hydroxide, sodium
chloride, hydrochloric acid were obtained from Fisher Scientific. Sodium formate, perchloric
+
acid, β-nicotinamide adenine dinucleotide hydrate (NAD ), β-nicotinamide adenine
dinucleotide reduced disodium salt (NADH) and sodium pyruvate were purchased from
Sigma-Aldrich. DMSO-d , MeOD-d , D O, (CD ) CO-d and CDCl for NMR spectroscopy
6
4
2
3 2
6
3
were purchased from Sigma-Aldrich and Cambridge Isotope Labs Inc. Non-dried solvents
used in syntheses were obtained from Fisher Scientific and Prolabo.
2
.2 NMR Spectroscopy.
1
H NMR spectra were acquired in 5 mm NMR tubes at 298 K or 310 K on Bruker AV-400 or
Bruker AV III 600 spectrometers. Data processing was carried out using XWIN-NMR
1
version 3.6 (Bruker U.K. Ltd). H NMR chemical shifts were internally referenced to TMS
via 1,4-dioxane in D O (δ = 3.75), residual DMSO (δ = 2.52 ppm) or CHCl (δ = 7.26 ppm).
2
3
1
D spectra were recorded using standard pulse sequences. Typically, data were acquired with
6

ACCEPTED MANUSCRIPT
1
6 transients into 32 k data points over a spectral width of 14 ppm and for the kinetic
experiments, 32 transient into 32 k data points over a spectral width of 30 ppm using a
relaxation delay of 2 s.
2
.3 pH* measurements.
pH* values (pH meter reading without correction for the effect of deuterium on glass
electrode) were measured at ambient temperature using a minilab IQ125 pH meter equipped
with a ISFET silicon chip pH sensor and referenced in KCl gel. The electrode was calibrated
with Aldrich buffer solutions of pH 4, 7 and 10. pH* values were adjusted with KOH or
HClO solutions in D O.
4
2
2
.4 Elemental analysis.
Elemental analyses were performed by Warwick Analytical Service using an Exeter
Analytical elemental analyzer (CE440).
2
.5 Electrospray ionization mass spectrometry (ESI-MS).
Positive ion electrospray mass spectra were obtained on a Bruker Daltonics Esquire 2000 ion
trap mass spectrometer. All samples were prepared in methanol (100%). Data were processed
using Data-Analysis version 3.3 (Bruker Daltonics).
2
.6 Synthesis
[
(Cp*)RhCl ] Hydrated rhodium(III) trichloride (500 mg, 2.12 mmol) was reacted with 2,4-
2
2.
pentamethylcyclopentadiene (302 mg, 2.22 mmol) dissolved in dry methanol (50 mL) and
heated to reflux (343 K) under a nitrogen atmosphere for 48 h. The dark red precipitate was
filtered off and washed with ether (3 x 20 mL) to give a dark red powder. The crude product
1
was then recrystallised from methanol. Yield: 410.6 mg (62.7 %). H NMR (400 MHz,
DMSO-d ): δ 1.627 (s, 10H).
6
H
xPh
xPh
[
(Cp )RhCl ] [(Cp )RhCl ] was synthesised following the same procedure described for
2 2. 2 2
*
xPh
[
(Cp )RhCl ] using hydrated Rh(III)Cl (500.7 mg, 1.05 mmol) and HCp (532.5 mg, 2.68
2 2 3
7

ACCEPTED MANUSCRIPT
1
mmol). The red precipitate was recrystallised from methanol. Yield: 592.7 mg (75.47 %). H
NMR (400 MHz, DMSO-d ): δ 7.67 (m, 4H), 7.43 (m, 6H), 1.71 (s, 4H), 1.67 (m, 4H).
6
H
xPhPh
xPhPh
[(Cp
)RhCl ] [(Cp
)RhCl ] was synthesised following the procedure described for
2
2.
2 2
*
xPhPh
[
(Cp )RhCl ] using hydrated Rh(III)Cl (992.6 mg, 2.1 mmol) and HCp
(960.5 mg, 4.4
2
2
3
mmol). The red-orange precipitate obtained was recrystallised from methanol. Yield: 613.4
1
mg (33.2 %). H NMR (400 MHz, DMSO-d ): δ 7.75 (m, 12H), 7.49 (t, 4H, J = 7.4 Hz),
6
H
7
.40 (t, 2H, J = 7.4 Hz), 1.73 (s, 24H).
General Synthesis of complexes 1-9. The Rh(III) dimer was placed in a round-bottom flask
to which dry dichloromethane was added. Upon addition of the corresponding ligand (2.5
mol. equiv.), the reaction was stirred overnight at ambient temperature, after which the
solvent was removed on the rotary evaporator to afford a crude powder. The crude product
was re-dissolved in methanol and filtered. Excess ammonium hexafluorophosphate was then
added and the solution stored in the freezer. The resulting product was collected by filtration
and recrystallised from acetone or methanol.
[
(Cp*)Rh(en)Cl]PF (1). [(Cp*)RhCl ] (51.3 mg, 83.0 μmol), ethylenendiamine (9.88 mg,
6 2 2
1
0
.16 mmol, 11 µl). Bright yellow crystals were collected. Yield: 42.3 mg (78.3 %). H NMR
(
400 MHz, CDCl ): δ 5.91 (s, 2H), 3.05 (s, 2H), 2.88 (s, 2H), 2.74 (s, 2H), 1.90 (s, 15H).
3 H
Anal: Calc for C H ClF N PRh C: 27.91, H: 4.61, N: 6.21; Found C: 28.11, H: 4.81, N:
1
2
23
6
2
+
5
.95. ESI-MS: Calc for C H ClN O Rh (M) 333.0 m/z found 333.0 m/z.
1
2
23
2
2
xPh
xPh
[
(Cp )Rh(en)Cl]PF (2). [(Cp )RhCl ] (50.5 mg, 68.0 μmol), ethylenendiamine (8.99
6 2 2
mg, 0.15 mmol, 10 µl). Recrystallisation from methanol resulted in bright yellow crystals.
1
Yield: 31.7 mg (59.0 %). H NMR (400 MHz, CDCl ): δ 7.49 (m, 5H), 5.59 (s, 2H), 3.11 (s,
3
H
2
H), 3.02 (s, 2H), 2.76 (s, 2H), 2.05 (s, 6H), 1.91 (s, 6H). Anal: Calc for C H ClF N PRh
17 25 6 2
C: 37.76, H: 4.66, N: 5.18; Found C: 37.02, H: 4.67, N: 5.26. ESI-MS: Calc for
+
C H ClN Rh (M) 395.0 m/z found 395.0 m/z.
17
25
2
8

ACCEPTED MANUSCRIPT
xPhPh
xPhPh
[(Cp
)Rh(en)Cl]PF (3). [(Cp
)RhCl ] (51.6 mg, 57.6 μmol), ethylenendiamine
2 2
6
(7.11 mg, 0.12 mmol, 8 µl). Recrystallisation from methanol resulted in bright yellow
1
crystals. Yield: 47.7 mg ( 54.2 %) H NMR (400 MHz, CDCl ): δ 7.69 (d, 2H, J = 8.4 Hz),
3
H
7
.60 (m, 4H), 5.49 (t, 2H, J = 7.5 Hz), 7.41 (t, 1H, J = 7.5), 5.59 (s, 2H), 3.14 (s, 2H), 3.03 (s,
H), 2.79 (s, 2H), 2.07 (s, 6H), 1.97 (s, 6H). Anal: Calc for C H ClF N PRh C: 44.79, H:
2
23
29
6
2
+
4
.74, N: 4.54; Found C: 43.98, H: 4.65, N: 4.54. ESI-MS: Calc for C H ClN Rh (M) 471.1
23 29 2
m/z, found 471.1 m/z.
[
(Cp*)Rh(bpy)Cl]PF (4). [(Cp*)RhCl ] (100.1 mg, 16.2 mmol), 2,2'-bipyridine (64.7 mg,
6 2 2
4
1.4 mmol) Recrystallization from acetone resulted in bright orange crystals. Yield: 126.6 mg
1
(
67.9 %). H NMR (400 MHz, Acetone-d ): δ 9.14 (d, 2H, J = 5.7 Hz), 8.67 (d, 2H, J = 8.1
6
H
Hz), 8.38 (t, 2H, J = 8.0 Hz),7.95 (dd, 2H, J = 8.0 Hz, J = 5.7 Hz), 1.82 (s, 15H). Anal: Calc
1
2
for C H ClF N PRh C: 41.8, H: 4.03, N: 4.87; Found C: 41.85, H: 3.97, N: 4.84. ESI-MS:
2
0
23
6
2
+
Calc for C H ClN Rh (M) 430.1 m/z found 430.0 m/z.
2
0
23
2
XPh
xPh
[
(Cp )Rh(bpy)Cl]PF (5). [(Cp )RhCl ] (101.3 mg, 13.6 mmol, 2,2’-bipyridine (56.0
6 2 2
mg, 35.9 mmol). Recrystallization from acetone resulted in bright orange crystals. Yield:
1
1
13.5 mg (65.2 %). H NMR (400 MHz, Acetone-d ): δ 8.85 (d, 2H,J = 5.5 Hz), 8.70 (d, 2H,
6
H
J = 8.1 Hz), 8.37 (t, 2H, J = 8.4 Hz), 7.85 (t, 2H, J = 6.4 Hz), 7.76 (d, 2H, J = 6.6 Hz), 7.60
(
m, 3H), 1.92 (s, 6H), 1.85 (s, 6H). Anal: Calc for C H ClF N PRh + acetone C: 48.40, H:
25 25 6 2
+
4
.50, N: 4.03; Found C: 49.89, H: 4.22, N: 3.93. ESI-MS: Calc for C H ClN Rh (M) 490.1
25 25 2
m/z found 490.0 m/z.
xPhPh
xPhPh
[(Cp
)Rh(bpy)Cl]PF (6). [(Cp
)RhCl ] (100.8 mg, 11.2 mmol) 2,2’-bipyridine
2 2
6
(45.2 mg, 28.9 mmol). Recrystallization from acetone resulted in bright orange crystals.
1
Yield: 87.2 mg ( 54.3 %) H NMR (400 MHz, Acetone-d ): δ 8.91 (d, 2H, J = 8.2 Hz), 8.89
6
H
(d, 2H, J = 8.4 Hz), 8.35 (t, 2H, J = 8.0 Hz), 7.87 (m, 6H), 7.79 (d, 2H, J = 7.6 Hz), 7.54 (t,
2
H, J = 7.6 Hz), 7.45 (t, 1H, J = 7.6 Hz), 1.95 (s, 6H), 1.90 (s, 6H). Anal: Calc for
9

ACCEPTED MANUSCRIPT
C H ClF N PRh C: 52.23, H: 4.10, N: 3.93; Found C: 52.73, H: 4.31, N: 3.70. ESI-MS:
31
29
6
2
+
Calc for C H ClN Rh (M) 567.1 m/z, found 567.0 m/z.
3
1
29
2
[
(Cp*)Rh(phen)Cl]PF (7). [(Cp*)RhCl ] (100.2 mg, 16.2 mmol), 1,10-phenanthroline
6 2 2
(80.78 mg, 40.7 mmol). Recrystallization from acetone resulted in bright orange crystals.
1
Yield: 138.7 mg (71.4 %). H NMR (400 MHz, CDCl ): δ 9.43 (d, 2H, J = 5.2 Hz), 8.71 (d,
3
H
2
H, 8.1 Hz), 8.29 (dd, 2H, J = 8.2 Hz, J = 5.2 Hz), 8.1 (s, 2H), 1.89 (s, 15H). Anal: Calc for
1 2
C H ClF N PRh C: 44.13, H: 3.87, N: 4.68; Found C: 44.13, H: 3.79, N: 4.62. ESI-MS:
22
23
6
2
+
Calc for C H ClN Rh (M) 453.1 m/z found 453.0 m/z.
2
2
23
2
XPh
xPh
[
(Cp )Rh(phen)Cl]PF (8). [(Cp )RhCl ] (124.0 mg, 16.7 mmol), 1,10-phenanthroline
6 2 2
(83.6 mg, 42.1 mmol). Recrystallization from acetone resulted in bright red crystals. Yield:
1
1
8
30.3 mg (59.0 %). H NMR (400 MHz, CDCl ): δ 9.08 (d, 2H, J = 5.2 Hz), 8.69 (d, 2H, J =
3
H
.2 Hz), 8.12 (m, 4H), 7.78 (d, 2H, J = 7.6 Hz), 7.61 (m, 3H), 2.06 (s, 6H), 1.85 (s, 6H).
Anal: Calc for C H ClF N PRh + MeOH C: 48.54, H: 4.22, N: 4.04; Found C: 47.68, H:
2
7
25
6
2
+
4
.09, N: 4.17. ESI-MS: Calc for C H ClN Rh (M) 515.1 m/z found 515.0 m/z.
2
7
25
2
xPhPh
xPhPh
[(Cp
)Rh(phen)Cl]PF6 (9). [(Cp
)RhCl2]2 (100.7 mg, 11.2 mmol), 1,10-
1
phenanthroline (55.8 mg, 28.2 mmol). Yield: 109.8 mg (66.3 %) H NMR (400 MHz,
CDCl ): δ 9.11 (d, 2H, J = 5.2 Hz), 8.69 (d, 2H, J = 8.1 Hz), 8.13 (dd, 2H, J = 8.1 Hz, J =
3
H
1
2
5
.2 Hz), 8.12 (s, 2H), 7.86 (d, 2H, J = 8.2 Hz), 7.82 (d, 2H, J = 8.2 Hz), 7.70 (d, 2H J = 7.4
Hz), 7.53 (t, 2H, J = 7.4), 7.45 (t, 1H, J = 7.4 Hz), 2.06 (s, 6H), 1.88 (s, 6H). Anal: Calc for
C H ClF N PRh + MeOH C: 53.11, H: 4.33, N: 3.64; Found C: 54.37, H: 4.34, N: 3.49.
23
29
6
2
+
ESI-MS: Calc for C H ClN Rh (M) 591.1 m/z, found 591.1 m/z.
2
3
29
2
[
(Cp*)Rh(TfEn)Cl] (10). [(Cp*)RhCl ] (49.7 mg, 80.4 μmol) and N-(2-aminoethyl)-4-
2 2
(trifluoromethyl)benzenesulfonamide (TfEnH) (43.5 mg, 162.2 µmol) were dissolved in
dichlormethane (25 mL) and triethylamine (45 µL, 320.5 µmol) was added. The reaction was
then stirred under nitrogen atmosphere overnight. The solution was placed in a separating
1
0

ACCEPTED MANUSCRIPT
funnel and washed with brine, the organic layer separated and dried over MgSO and filtered.
4
The solution was concentrated in vacuo and the product obtained recrystallised from
1
methanol to afford an orange powder. Yield: 40.3 mg (46.4 %). H NMR (400 MHz, CDCl ):
3
δH 8.00 (d, 2H, J = 8.1 Hz), 7.54 (d, 2H, J = 8.1 Hz), 3.19 (s, 2H), 2.60 (s, 3H), 1.71 (s, 15H).
Anal: Calc for C H ClF N O RhS C: 42.20, H: 4.66, N: 5.18; Found C: 41.92, H: 4.36, N:
1
9
25
3
2
2
+
5
.01. ESI-MS: Calc. for C H ClF N O RhS (M-Cl) 505.0 m/z, found 505.0 m/z.
19 25 3 2 2
2
.7 Aqueous solution chemistry
1
Solutions of complexes 1-10 (1.4 mM, in 20 % MeOD/ 80 % D O) were prepared and H
2
NMR spectra at 310 K were recorded at time 0 (<10 min) and 24 h. The samples were then
incubated at 310 K.
Aqueous solutions of the chlorido complexes 1-10 (1.4 mM, in 20 % MeOD/ 80 % D O)
2
were treated with silver nitrate (0.95 mol. equiv.) and stirred overnight at room temperature.
1
H NMR spectra were recorded after filtration of the samples through celite to remove the
silver chloride formed.
2
.8 pK determination for aqua species
a
Solutions of complexes 1-4, 7 and 10 (1.4 mM, D O) were prepared and treated with 0.95
2
mol equiv. of silver nitrate. The reaction mixture was then filtered through celite to obtain the
x
corresponding aqua adducts. Changes in the chemical shifts of the methyl protons of the Cp
ligand in the aqua adducts with the pH* (pH meter reading) over a range from 2 to 12 were
1
followed by H NMR spectroscopy. Solutions of KOH or HClO in D O were used to adjust
4
2
1
the pH*. H-NMR spectra were recorded at 298 K on a Bruker AV III 600 spectrometer. The
data were fitted to the Henderson–Hasselbalch equation using Origin 7.5.
1
1

ACCEPTED MANUSCRIPT
+
2
.9 Reduction of pyruvate and NAD
Complexes 1-3 and 10 were dissolved in D O (1.4 mM, 4 mL) in a glass vial. Complexes 1-9
2
were prepared in MeOD/D O (3:2 v/v) (1.4 mM, 4 mL) in a glass vial and treated with 0.95
2
mol. equiv. of silver nitrate.
Aqueous solutions of sodium formate (25 mol equiv.) and substrate (2-9 mol equiv, substrate
+
=
NAD or pyruvate) in D O were also prepared and incubated at 310 K. In a typical
2
experiment, an aliquot of 200 µL of the complex, formate and substrate solutions were added
to a 5 mm NMR tube. The pH* of the solution mixture was adjusted to 7.2 ± 0.2 bringing the
1
total volume to 0.635 mL H NMR spectra were recorded at 310 K every 162 s until the
completion of the reaction.
+
Molar ratios of substrate and product were determined by integrating the peaks for NAD ,
pyruvate, lactate and 1,4-NADH. The turnover number (TON) for the reaction was calculated
as follows:
ꢂꢃ ꢀꢁꢂꢃꢁꢄꢅꢆꢄꢇꢈ0
ꢂꢃ ꢄꢂ ꢀCaꢅalꢆsꢅꢈ
ꢀ
Oꢁ =
where I is the integral of the signal at n ppm. x = 6.96 (NADH) or 1.32 ppm (lactate). y =
n
+
+
9
.33 (NAD ) or 2.36 ppm (pyruvate). [substrate] is the concentration of NAD or pyruvate at
0
the start of the reaction.
2
.10 Mechanistic studies
A set of four experiments was performed to compare the characteristics of the catalytic cycle
of Rh(III) complexes 1 and 10 (1.4 mM, D O) with their Ru(II) analogues.
2
+
The turnover frequencies for the reduction reaction of NAD using complexes 1 or 10 and
formate (25 mol equiv.) were determined following the procedure described above. The
+
reaction was studied using different concentrations of NAD (1, 2 and 5 mol equiv. for
complex 10; 5, 7.5 and 10 mol equiv. for complex 1).
1
2

ACCEPTED MANUSCRIPT
A second series of experiments using different concentrations of sodium formate (5, 10, 25,
5
0 and 100 mol equiv. for complex 1; 10, 25, 50, 100 and 500 mol equiv. for complex 10)
+
and a constant concentration of NAD (5 mol equiv.) was also performed.
The optimum pH range for the catalytic process was studied in a series of experiments. Each
experiment was performed at a different pH* over a range from 6 to 10. The pH* of the
reaction was adjusted using solutions of KOH or HClO in D O.
4
2
Transfer hydrogenation reactions using 1,4-NADH (400 μL, 2.8 mM) as a hꢆdride source
1
(
400 μL, 2.8 mM) in D O by complex 1 and 10 were studied by H NMR for a period of 10 h.
2
The pH* of the reaction mixture was adjusted to 7.2 ± 0.2, and the experiment performed at
3
10 K.
2
.11 Competition studies.
Complexes 1 (1.4 mM) was dissolved in CD OD/D O (3:2 v/v ,4 mL) in a glass vial.
3
2
+
Aqueous solutions of sodium formate (100 mol equiv.), NAD (4.5 mol equiv.) and pyruvate
(
4.5 mol equiv.) in D O were also prepared and incubated at 310 K. In a typical experiment,
2
+
an aliquot of 200 µL of complex, 50 µL of formate and 150 µL of pyruvate and NAD were
added to a 5 mm NMR tube. The pH* of the solution mixture was adjusted to 7.2 ± 0.2
1
bringing the total volume to 0.635 mL H NMR spectra were recorded at 310 K every 162 s
until the completion of the reaction.
2
.12 Antiproliferative activity and cell viability modulation assays.
The antiproliferative activities of complexes 1-10 in A2780 ovarian cancer cells were
determined. Briefly, 96-well plates were used to seed 5000 cells per well. The plates were
pre-incubated in drug-free media at 310 K for 48 h before addition of various concentrations
of the compounds. The drug exposure period was 24 h, after which, the supernatant was
removed by suction and each well washed with PBS. A further 48 h was allowed for the cells
to recover in drug-free medium at 310 K. The sulforhodamine B (SRB) colorimetric assay
1
3

ACCEPTED MANUSCRIPT
was used to determine cell viability [21]. IC values, as the concentration which causes 50%
5
0
growth inhibition, were determined as duplicates of triplicates in two independent sets of
experiments and their standard deviations were calculated.
The data were analysed using Origin 8.5. IC50 values were obtained from plots of the
percentage survival of cells versus the logarithm of the concentration expressed in millimolar
units and fitted to a sigmoidal curve. IC values for cisplatin were determined in each well-
5
0
plate as a validation.
Cell viability modulation assays were carried out in A2780 ovarian cancer cells. These
experiments were performed as described above for IC50 determinations with the following
experimental modifications. A fixed concentration of complexes was used, 150 μM. Co-
administration of the complex with three different concentrations of sodium formate (0.5, 1
and 2 mM) was studied. Both solutions (complex and sodium formate) were added to each
well independently, but within 5 min of each other. Cell viability percentages were
determined as duplicates of triplicates in two independent sets of experiments and their
standard deviations were calculated.
Stock solutions of the Rh(III) complexes were freshly prepared for every experiment in 5%
DMSO and a mixture 0.9% saline : medium (1:1 v/v). The stock solution was further diluted
using RPMI-1640 to achieve working concentrations. The final concentration of DMSO was
between 0.5 and 0.1 % % v/v. Metal concentrations were determined by ICP-MS.
1
4

ACCEPTED MANUSCRIPT
3
. Results
Rh(III) complexes 1-9 were synthesised (Figure 1) using a similar procedure. Typically, the
ligand (2 - 2.5 mol equiv), was added to a dichloromethane solution of the rhodium dimer,
x
[
(Cp )RhCl ] and the reaction mixture stirred at ambient temperature. The details for
2
2
individual reactions are described in the experimental section. Complex 10 was synthesised
by reacting the dimer with the TfEnH ligand in dichloromethane, and in the presence of
triethylamine (4 mol equiv) at ambient temperature overnight.
The complexes were characterised by elemental analysis, NMR spectroscopy and mass
spectrometry. The x-ray crystal structures of complexes 3-6, 8 and 9 were determined and
will be reported elsewhere [22].
3
.1 Aqueous solution chemistry.
1
Aquation of complexes 1-10 (1.4 mM in D O) at 310 K was followed by H NMR over a
2
period of 24 h. For complexes 1-3 and 10, only one set of peaks (aqua adducts) was observed.
For complex 4-9, two sets of peaks were observed and assigned as the chlorido and the aqua
1
species. Peaks for aqua species were assigned by comparison of the H spectra of 1-10 in
D O and the products of reactions between 1-10 and silver nitrate. After 24 h incubation (310
2
1
K), no apparent changes were observed by H NMR. Compounds 6 and 9 gave rise to
1
precipitates after 24 h and H NMR spectra could not be recorded. The extent of hydrolysis is
shown in 0.
1
Changes in the H NMR chemical shifts of the methyl groups of cyclopentadienyl protons
from the aqua adducts of complexes 1-4, 7 and 10 were followed over the pH* range from 2
to 12. The data were fitted to the Henderson–Hasselbalch equation. The pK * values for
a
complexes 1-4, 7 and 10 are shown in 0 (Figure 2).
1
5

ACCEPTED MANUSCRIPT
+
3
.2 Transfer hydrogenation reactions with NAD
+
Catalytic conversion of NAD to NADH using complexes 1-10 and sodium formate (hydride
1
source) was followed by H NMR spectroscopy. In a typical experiment, 200 µL of complex
+
(
1.4 mM, D O or MeOD/D O 3:2 v/v), sodium formate (35 mM, D O) and NAD (2.8-12.6
2
2
2
mM, D O) were mixed in a 5 mm NMR tube, final ratio 1:25:2-9, complex : sodium formate :
2
+
1
NAD . The pH* of the reaction mixture was adjusted to 7.2 ± 0.2. H NMR spectra at 310 K
were recorded every 162 s until completion of the reaction. The turnover frequency (TOF) for
the reactions were determined as described in the experimental section (0).
xPh
The turnover frequencies for complexes 1-3 in D O, increased in the order Cp *< Cp
<
2
xPhPh
xPhPh
+
-1
Cp
(0). The most active complex was [(Cp
)Rh(en)Cl] (3), with a TOF of 24.19 h .
The reaction was regioselective giving 1,4-NADH exclusively. On changing the
ethylendiamine (en) chelating ligand (complex 1) to N-(2-aminoethyl)-4-
(trifluoromethyl)benzenesulfonamide (TfEn) (complex 10), the catalytic activity decreased
(0). Furthermore, a decrease in the regioselectivity of the reaction was also observed with
7
.5% of 1,6-NADH being produced.
+
Complex 1 was less active than complex 2 in the reduction of NAD in 20% MeOD/ 80%
D O, following the same trend as observed for the reaction in D O. However, the catalytic
2
2
activity of the complexes 1 and 2 in 20% MeOD was 2x times higher than that in D O.
2
+
Complexes 4-9 were also studied for the reduction of NAD in 20% MeOD / 80% D O.
2
xPh
Complexes containing Cp* (4 and 7) were more active than those containing Cp (5 and 7),
xPhPh
which, in turn, were more active than Cp
complexes (6 and 8), (0). The reactions of
complexes 4-9 were largely regioselective, giving only 1 to 10% of 1,6-NADH.
+
3
.2.1 Effect of NAD and sodium formate concentration on the catalysis rate.
Transfer hydrogenation reactions using complex 1, sodium formate and varying
+
concentrations of NAD were studied (ratio of 1:25:X, X = 5, 7.5 and 10 mol equiv of
1
6

ACCEPTED MANUSCRIPT
+
-1
NAD ). The turnover frequency (7.5 h ) remained the same with increasing concentrations of
+
-1
+
NAD . For complex 10, the TOF (4.12 h ) was unaffected by the concentration of NAD
X= 1, 2 or 5 mol equiv).
(
+
A second series of experiments on the reduction of NAD , varying the concentration of
+
sodium formate, was performed (complex 1, sodium formate and NAD in the ratio 1:X:5, X
=
5, 10, 25, 50 or 100 mol equiv). A notable increase in the catalytic activity was observed
with increase of the hydride source (sodium formate), (Figure 3). From the plot of TOF vs
formate concentration, typical Michaelis-Menten behaviour can be observed. From the
reciprocal of the TOF vs formate concentration, a maximum turnover frequency (TOFmax) of
-1
4
1.49 h and a Michaelis constant (K ) of 54.16 mM were calculated.
M
-1
The TOFmax (37.24 h ) and K (108.37 mM) for complex [(Cp*)Rh(TfEn)Cl] (10) were also
M
determined by performing a series of experiments with different concentrations of sodium
formate (10, 25, 50, 100 or 500 mol equiv.), Figure 3.
3
.2.2 pH dependence
The pH* dependence of the catalytic reaction (from pH* 4 to 10) was also investigated via a
+
series of experiments using complex 1, sodium formate and NAD (ratio 1:25:6, respectively)
-1
in D O. A dependence on pH* was observed. The highest TOF (7.5 h ) was achieved at pH*
2
7
.3 ± 0.1, however the turnover frequencies between pH* 6 - 8 are similar. A decrease in the
catalytic activity was observed when the pH* is higher than 9 (Figure 4).
3
.2.3 Transfer hydrogenation reactions using NADH as a hydride donor
NADH has previously been shown to be able to act as a hydride donor [14]. As a
consequence, experiments in which complex 1-3 and 10 were reacted with NADH were
performed, however, no hydride transfer from NADH was observed over a period of 10 h.
1
7

ACCEPTED MANUSCRIPT
3
.3 Transfer hydrogenation reactions with pyruvate
Lactate formation via hydride transfer from formate to pyruvate catalysed by complexes 1-9
1
was followed by H NMR. In a typical experiment, 200 µL of complex (1.4 mM, D O or
2
MeOD/D O 3:2 v/v), formate (35 mM, D O) and pyruvate (7 mM, D O) were mixed in a 5
2
2
2
1
mm NMR tube, final ratio 1:25:5, complex : formate : pyruvate. H NMR spectra at 310 K
were recorded every 162 s until completion of the reaction.
Molar ratios of pyruvate and lactate were determined by integrating the signals of pyruvate
(3H, singlet, 2.36 ppm) and of lactate (3H, singlet, 1.32 ppm), Figure 5. The activity of the
complexes towards reduction of pyruvate to lactate is dependent on the nature of both the
X
XPhPh
Cp ring and the N,N’ chelating ligand. Extended Cp
rings result in an increase in the
catalytic activity for complexes 1 and 2. However, there was a decrease in the TOF when the
N,N’-chelated ligand is 2,2'-bipyridine (bpy) or phenanthroline (phen) (Figure 6).
+
3
.4 Competition for the reduction between NAD and pyruvate.
+
Transfer hydrogenation reactions with complex [(Cp*)Rh(bpy)Cl] (4), sodium formate, and
+
both NAD and pyruvate as hydride acceptors were performed. For these experiments, 4.5
+
mol equiv of NAD and pyruvate were reduced using 25 mol equiv. of formate.
+
The reduction of both NAD and pyruvate by complex 4 was observed, but the rate of
reduction was slower for both substrates (Figure 7). In addition, pyruvate was reduced only
+
when the reduction of NAD was almost complete.
3
.5 Cancer cell growth inhibition.
The IC50 values for Rh(III) complexes 1- 10 in A2780 human ovarian cancer cells were
determined (0). Complexes 1-3 and 10 were inactive up to the maximum concentration tested
(
100 μM). Complexes 4-9 were moderately active with IC values between 14 and 65 μM.
50
1
8

ACCEPTED MANUSCRIPT
The percentage cell survival of A2780 cells after incubation with complexes 1-10 and
varying concentrations of sodium formate (0, 0.5, 1, 2 mM) was determined (Figure 8). A
significant increase of the antiproliferative activity of the Rh(III) complexes upon addition of
xPhPh
+
formate is evident. The largest increase was observed for complex [(Cp
with a decrease in cell survival of up to 50%.
)Rh(en)Cl] (3),
4
. Discussion
Ruthenium(II), rhodium (III) and Iridium (III) half sandwich complexes have previously been
+
reported to catalyse the reduction of NAD via transfer hydrogenation using sodium formate
as a hydride source [22,23]. In the previous reports, Rh(III) complexes generally displayed
higher turnover frequencies (TOF) than Ir(III), while Ru(II) complexes have the lowest
catalytic activity [16,24-27]. However, the reported catalytic reactions were usually carried
out under non-physiological conditions: high temperatures, very high concentrations of
formate, pH ≠ 7.4 or with high concentrations of non-aqueous solvents [16,24-27].
+
The complexes selected for the present work have all been studied for the reduction of NAD
+
under non physiological conditions: [(Cp*)Rh(bpy)Cl] (4), developed by Steckhan and Fish
+
+
[
27,28], [(Cp*)Rh(phen)Cl] (7), published by Süss-Fink [26], [(Cp*)Rh(en)Cl] (1), rhodium
+
+
analogue of [(p-cym)Ru(en)Cl] studied in our group [16], and [(Cp*)Rh(TfEn)Cl] (10).
Noyori-type catalysts such as [(Cp*)Rh(TfEn)Cl] (10) are some of the more successful
+
catalysts for transfer hydrogenation reactions [3] . Reduction of NAD using catalysts has
been recently studied successfully by several groups [24,29,30].
In our previous work, we observed that the nature of arene and Cp rings can have a
significant effect on the catalytic properties of half-sandwich metal complexes. For example,
x
iridium complexes containing Cp with extended aromatic substituents had improved
1
9

ACCEPTED MANUSCRIPT
+
catalytic activity for the regeneration of NAD [14]. With this in mind, complexes containing
xPh
xPhPh
Cp and Cp
rings were studied in the present work.
4
.1 Hydrolysis and acidity of aqua adducts
e-
Previous studies with related compounds have shown that the 16 species or the aqua adducts
of the complexes are the active catalysts in transfer hydrogenation reactions [2,31]. However,
in aqueous media the presence of water and pH has been shown to play a critical role [2,31].
1
Aquation of the complexes 1-10 in D O (1.4 mM) was confirmed by comparing the H NMR
2
spectra of D O solutions of the complexes and solutions obtained after removal of the
2
chloride ligand by reaction with AgNO to precipitate AgCl. Complexes 1-10 hydrolysed
3
1
rapidly at 310 K, reaching equilibrium by the time the first H NMR spectrum was recorded
(< 10 min). Consequently the turnover frequency for transfer hydrogenation is not affected by
the use of the chlorido complex instead of the aqua adduct.
Complete conversion of the chlorido complexes 1-3 and 10 to their aqua adducts was
observed. However, complexes 4-9 reached equilibrium with 30-60% formation of the aqua
species (0). This lower conversion would not be expected to affect the catalytic activity since
the hydrolysis is fast, and the formation of hydrido species will shift the equilibria towards
aqua adduct formation.
pH* titrations to determine the pK of the aqua adducts of complexes 1-4, 7 and 10 were
a
carried out so that the nature of the complexes at pH 7.2 (aqua or hydroxido complexes)
could be determined. The formation of hydroxido adduct would be expected to hamper the
catalytic reaction since hydroxido ligands bind more tightly than the aqua ligands. The pKa*
values of the aqua adducts determined by NMR pH* titrations, are all > 9 (0). These
complexes will therefore exist largely as the aqua complexes at pH values close to 7.4.
2
0

ACCEPTED MANUSCRIPT
+
4
4
.2 Transfer hydrogenation reactions with NAD
.2.1 Comparison of Ru(II) and Rh(III)
+
+
First, we compared the catalytic activity for the reduction of NAD by [(Cp*)Rh(en)Cl] (1)
and [(Cp*)Rh(TfEn)Cl] (13) with the arene-Ru(II) analogues we studied previously
+
[
(hmb)Ru(en)Cl] , [(p-cym)Ru(TfEn)Cl] and [(bn)Ru(TfEn)Cl], (0) [16,24].
The catalytic activity of the Rh(III) complexes was expected to be higher than that of their
Ru(II) analogues based on the literature. [16,24-27] Complex 1 was up to 8.5x times more
+
active than [(hmb)Ru(en)Cl] , (0). However, complex 10 had TOF values in the same range
6
-1
-1
as those of ꢅhe Ru analogues [(η -arene)Ru(TfEn)Cl] (TOFp-cym = 3.1 h , TOF = 6.6 h )
bn
[
24].
The TOF of complex 10 was 2x times lower than complex 1 (0). This decrease in catalytic
activity could be due to steric hindrance generated by the sulpfonamide group. Despite the
fact that half-sandwich complexes containing N-(2-aminoethyl)-sulfonamides are known to
display very high catalytic activity for hydride transfer reactions [32,33] [3,34,35], our
results are perhaps not surprising, since low catalytic activities have been previously reported
+
with Noyori-type catalysts for the reduction of NAD compared with other Rh(III) catalysts
[
30].
x
Next a series of ethylenediamine-Rh(III) complexes containing extended Cp ligands was
x
studied. A trend was observed in which the presence of a more electron-withdrawing Cp ring
xPhPh
+
gives higher catalytic activity (0). Accordingly, the complex [(Cp
)Rh(en)Cl] shows the
xPh
+
highest activity followed by [(Cp )Rh(en)Cl] and, in turn, both are more active than
+
[
(Cp*)Rh(en)Cl] . With 3, an improvement in the TOF of 24x was achieved, compared with
+
the ruthenium analogue [(hmb)Ru(en)Cl] . This increase in activity can be attributed to the
effect of a less electron-rich Cp ring. The more acidic metal centre may facilitate the
2
1

ACCEPTED MANUSCRIPT
coordination of negatively-charged formate, which can then undergo β-elimination to
generate Rh-H and CO2.
In order to compare the reactivity of Cp-Rh(III) with their arene-Ru(II) analogues, a set of
experiments was performed using complexes 1 and 10: dependence of the reaction rate on the
+
NAD and formate concentrations, reaction with NADH, and optimum pH* of the reaction.
+
For reactions of [(Cp*)Rh(en)Cl] (1) and [(Cp*)Rh(TfEn)Cl] (10) and varying
+
concentrations of NAD , no significant alterations in the reaction rate were observed (TOF =
-1
7
.5 and 4.1 h , respectively). The unchanged turnover frequency implies that the reaction rate
+
does not depend on the NAD concentration. However, when the experiments were
performed with increasing concentrations of the hydride source (sodium formate), the
reaction rate increased, suggesting that formate is involved in the rate-determining step
+
(
Figure 3). This behaviour is similar to that of [(Cp*)Rh(bpy)Cl] previously observed by
Fish et al. [36], with the immobilised tethered Cp*Rh complex (Rh(III)-TsDPEN) where
TsDPEN is (N-[(R,2R)-2-amino-1,2-diphenylethyl]-4-toluene sulfonamide) studied by
6
+
6
Hollmann et al. [30] and wiꢅh [[(η -arene)Ru(en)Cl] and [[(η -arene)Ru(TfEn)Cl] studied in
our laboratory [16,24]. Plotting the turnover frequency against formate concentration shows a
typical Michaelis-Menten behaviour. The maximum turnover frequency calculated from the
-1
double reciprocal of TON vs formate concentration for complex 1 is TOFmax = 41.49 h , ca.
+
-1
2
8 x higher than that of [(hmb)Ru(en)Cl] (TOF =1.46 h ) [16].
max
-1
The TOFmax for complex 10 was 37.2 h , only ca. 5x higher than that of [(p-
-1
cym)Ru(TsEn)Cl] (TOFmax=6.9 h ) [24]. At high concentrations of hydride (Sodium
formate), the complexes containing TfEn ligands are 5x times more active than their
ruthenium analogues. However, complex [(Cp*)Rh(TsEn)Cl] (10) is still less active than the
+
ethylenediamine complex [(Cp*)Rh(en)Cl] (1).
The Michaelis constant for complex 10 (K = 108.4 mM) indicates a weaker affinity for
M
+
+
formate compared to complexes [(Cp*)Rh(en)Cl] (K = 41.5 mM), [(hmb)Ru(en)Cl] (K =
M
M
2
2

ACCEPTED MANUSCRIPT
5
8
mM), [16] and [(p-cym)Ru(TsEn)Cl] (42.1 mM) [24]. Compared with
2
+
[
(Cp*)Rh(bpy)H O] (K 140 mM) [36], complex 10 show stronger affinity for formate.
2 M
This implies that the low catalytic activity when using 25 mol equiv. of formate is due to
reduced affinity for formate, but the catalytic activity increases markedly in formate-saturated
solutions.
+
Some
iridium
and
ruthenium
complexes
such
as
([Cp*Ir(phen)Cl] ,
([Cp*Ir(phenylpyridine)Cl] and[(p-cym)Ru(bipyrimidine)Cl]) have been shown to oxidise
NADH through transfer hydrogenation [13,14,17,37]. In such processes, the NADH acts as a
natural hydride donor, and the metal catalyses hydride transfer from NADH to other
substrates such as quinones, pyruvate or oxygen [13,14,37]. In this study, we investigated the
possibility of oxidising NADH using the rhodium(III) complexes 1 and 10. No oxidation of
NADH occurred after 12 h at 310 K and pH 7.2 ± 0.2. In contrast to the above mentioned
compounds, the lack of reactivity with NADH is similar to the ruthenium complex [(p-
cym)Ru(TfEn)Cl]. These experiments emphasise the critical influence of the ligands in half-
sandwich complexes on the reactivity of the complex.
The last experiment performed was to determine the optimum pH* for the reduction of
+
+
NAD . In line with our aim of applying the catalytic reduction of NAD in biological
systems, we worked at pH 7.2. However, it is interesting to note the effect of pH on the
reaction rate. Surprisingly, the maximum activity for complex 1 was observed at pH* 7.3
(Figure 3) which is close to physiological pH. Furthermore, slight variations were observed
-
over the pH range 5 to 9. At pH* > 9, the concentration of OH inhibits the reaction due to the
formation of the more inert Rh(III) hydroxido adduct. This effect of pH was also observed for
the ruthenium systems [2,24].
+
4
.2.2 Reduction of NAD
In order to select the best catalyst candidate to work with, 3 well-known half sandwich
+
Rh(III) compounds were studied for their catalytic activity towards the reduction of NAD .
2
3

ACCEPTED MANUSCRIPT
X
We also studied the effect of extended phenylation of Cp rings on their TOF. These
reactions were performed in 20% methanol and 80% water to aid solubility.
As we observed previously, there is an increase on the TOF for ethylendiamine-containing-
complexes when using extended rings. Interestingly, there is a 2-fold increase in the TOF due
to the effect of 20% methanol. This effect was observed previously for the ruthenium(II)
complexes [(arene)Ru(TfEn)Cl]] [24].
+
xPh
xPhPh
The TOF of complexes 4-9 for the reduction of NAD decreases when using Cp or Cp
as ligands (0). Those results are surprising, since the trend is opposite to that for complexes 1-
. These results may be attributable to steric factors due to the extended Cp ring and the size
3
of the chelating ligand (bipyridine and phenanthroline compared to ethylenediamine).
+
The catalytic activity for the reduction of NAD is also dependent on the N,N’ chelating
ligand. For complexes containing Cp*, the turnover frequencies increase in the order en <
XPh
XPhPh
phen < bpy, whereas for complexes containing Cp
or Cp
, the TOFs increase in the
-1
order phen < bpy < en (0). Higher activity (TOF = 37.4 ± 2 h ) was obtained with
+
+
[
Cp*Rh(bpy)Cl] (4). However, the catalytic activity for the reduction of NAD
XPh
+
ethylendiamine complex [(Cp )Rh(en)Cl] (2) is in the same range as that obtained with
+
complex [Cp*Rh(bpy)Cl] (4).
4
.3 Reactions with pyruvate
+
We initially studied the reduction of NAD , but it is well known that this type of catalyst can
also be used for the reduction of other molecules. Previously, the possibility of reducing
+
biomolecules such as pyruvate was reported, using [(Cp*)Ir(phen)Cl] as a catalyst [14].
Therefore, we studied the catalytic reduction of pyruvate using complexes 1-9.
Complexes 1 and 2 reduce pyruvate in the presence of sodium formate, and complete
conversion to lactate was achieved. Complexes containing the Cp* ring were less active than
those containing an extended cyclopentadienyl ring (Figure 6). Similar to the reduction of
2
4

ACCEPTED MANUSCRIPT
+
NAD , when using bipyridine (4-6) and phenanthroline (7-9) as chelating ligands, the
catalytic activity decreases with extended Cp rings. Interestingly, the catalytic activity of
compounds 6 and 9 was extremely low, and after 8 h less than 30% conversion was achieved.
XPhPh
The complexes containing Cp
were not able to reduce pyruvate effectively before
decomposition, and the reactions cannot be considered as catalytic (Figure 6).
The low catalytic activity of complexes 1-9 for the reduction of pyruvate was expected since
both, pyruvate and formate, contain negatively-charged carboxylate groups which can
compete with hydride for binding to Rh(III).
+
4
.4 Competition for the reduction of NAD and pyruvate.
+
Competition experiments were performed using complex [(Cp*)Rh(bpy)Cl] (4). For these
+
experiments, 4.5 mol equiv. of NAD and pyruvate were reduced using 25 mol equiv. of
+
formate. The experiment showed a clear preference of complex 4 for the reduction of NAD .
+
NAD was reduced completely but at a slight lower rate than when the reaction was
performed without pyruvate (Figure 7), attributable to competitive binding of formate and
pyruvate.
+
Pyruvate was reduced only after the levels of NAD became very low. The reduction of
+
pyruvate in the presence of NAD was extremely slow compared with the reduction of
pyruvate on its own, with 50% reduction of pyruvate to lactate after 2 h 15 min.
4
.5 Cancer cell growth inhibition
The antiproliferative activity of complexes 1-10 in A2780 human ovarian cancer cells was
studied (0). IC values for complexes 1-3 and 10 were higher than 100 µM (inactive), while
5
0
complexes 4-9 showed moderate activity, with IC values between 14 and 65 µM. Higher
50
activities were obtained with the phenanthroline series (IC (7) = 17.8 ± 0.6 µM, (9) = 14.68
5
0
±
0.08 µM). Interestingly, in all cases lower IC50 values were obtained when using the
xPhPh
Cp
capping ligand, perhaps due to an increase in hydrophobicity and increased
2
5

ACCEPTED MANUSCRIPT
accumulation of the metal complex in cells [38,39], or due to the intercalation of the extended
aromatic unit between nucleobases of DNA [32,33].
Cell survival with complexes 1-10 upon addition of formate at concentrations of 0.5, 1 and 2
mM was also determined (Figure 8). The complexes show enhanced activity in combination
with formate. Formate alone had no effect on cell viability. It seems reasonable to propose
that co-administration of the complexes with formate gives rise to catalytic transfer
hydrogenation reactions in the A2780 human ovarian cancer cells, although perhaps with few
turnovers in view of the variety of nucleophiles in the cell which might terminate the
reactions.
In our previous work we have shown that transfer hydrogenation catalysts such as
+
[
(bip)Ru(en)Cl] or [(p-cym)Ru(TfEn)Cl] together with formate can reduce the levels of
+
+
nicotinamide adenine dinucleotide (NAD ) in cells [12]. NAD is an important co-enzyme
involved in maintaining the redox balance, the Kerb’s cꢆcle and other metabolic pathways
such as synthesis of ADP-ribose, ADP-ribose polymers and cyclic ADP-ribose which are
crucial for genome stability, DNA repair, and maintenance of calcium homeostasis [40-45].
Changes in the cellular redox status play an important role in cell death. In particular, cancer
cells, might be more sensitive to redox variations, since they are under constant oxidative
stress due to high production of reactive oxygen species (ROS) [40-45].
+
Despite the higher catalytic activity towards the reduction of NAD of the Rh(III) complexes
1
-10 compared with the Ru(II) complexes studied previously [16,24], the enhancement in
antiproliferative activity of the Rh(III) complexes is not as high as for their Ru(II) analogues
+
[
12]. The increase in catalytic activity for the reduction of NAD does not correlate with an
increase in antiproliferative activity when the drugs are co-administered with sodium formate
Figure 8b). The inconsistency between the catalytic activity and effects on cells might be
(
due to various factors. The poisoning of the catalyst [46], for example. Ward et al have
studied the possibility of synthesizing Ir(III) Noyori-type catalysts capable of reducing
2
6

ACCEPTED MANUSCRIPT
+
NAD , quinones and ketones [29,47]. In order to reduce poisoning due to sulfur-containing
molecules, such as glutathione, the compounds were conjugated to biotin-streptavidine [47].
The artificial metalloenzyme was able to carry out transfer hydrogenation reactions and
reduce, to a certain extent, ,the effect of poisoning due to glutathione [47].
The anticancer activity of the compounds may be due in part not only to the reduction of
+
NAD but also other biomolecules. For example, in this work we have demonstrated that
complexes 1-5 and 7-8 can reduce pyruvate, while compounds 6 and 9 cannot. Pyruvate is an
essential component of cellular energy pathways (Krebs’ cycle) and of special importance in
cancer cells. It has been previously observed that cancer cells consume high levels of glucose
and release lactate and carbon dioxide (Warburg effect) [48,49]. This behaviour is linked to
the malfunctioning of mitochondria. Cancer cells have a high rate of consumption of glucose
(glycolysis) due to the need to generate nutrients such as nucleotides and aminoacids.
+
However, a high rate of glycolysis, requires high levels of NAD . Cancer cells regenerate
+
NAD at a very fast rate by reducing pyruvate to lactate (Figure 9) [48,49].
In the presence of the Rh(III) catalysts 1-5 and 7-8, the pool of pyruvate in cells may be
+
reduced due to conversion to lactate. As a consequence, the regeneration of NAD and the
anaerobic glycolysis in cancer cells will be disrupted. The reduction of pyruvate, in
+
combination with the reduction of NAD will affect not only the redox regulatory system of
the cell, but also the generation of ATP (energy) and formation of various nutrients necessary
for cell growth and reproduction.
5
. Conclusions
During the last decade, there has been increasing interest in developing metal-based catalysts
which are effective in cells. Such technology might be useful in areas such as protein
labelling, imaging, and for the treatment of diseases. For example, Chen and Meggers have
3
shown that metal compounds such as Pd(dba) or [CpRu(QA)(η -allyl)]PF (QA = 2-
2
6
quinolinecarboxylate) can be used to remove protector groups from N-protected anticancer
2
7

ACCEPTED MANUSCRIPT
drugs [50,51]. Cowan et al. have shown that Cu(II)/Ni(II)-ATCUN compounds can
decompose RNA in hepatitis or HIV [52,53]. Also organo-ruthenium compounds can oxidise
glutathione catalytically [54].
In the current work, we have studied the possibility that the Rh(III) complexes
x
[
(Cp )Rh(N,N′)(Cl)] may be more active catalysts than their Ru(II) arene analogues. Four
+
series of compounds which can reduce NAD to NADH using formate as an hydride source,
have been investigated under biologically-relevant conditions (aqueous solution, pH = 7.2,
3
10 K). The catalytic activity decreased in the order of N,N-chelated ligand bpy > phen > en
5
with Cp* as the η -donor. However, while the ethylenediamine-containing compounds (1-3)
X
became more active with extension to the Cp ring, we observed a decrease in catalytic
activity for N,N-chelated phenanthroline- (7-9) and bipyridine– (4-6) compounds, perhaps
+
due to an increase in steric hindrance. The complex [Cp*Rh(bpy)Cl] (4) showed the highest
+
-1
catalytic activity towards the reduction of NAD , with a TOF of 37.4 ± 2 h .
x
+
-1
The catalytic activity of complexes [Cp Rh(en)Cl] (1-3) (7.50, 19.65 and 24.19 h ,
respectively) is up to two orders of magnitude higher than for their Ru(II) analogues (TOF
-1
[
(hmb)Ru(en)Cl]+ = 0.85 h ). Interestingly, Noyori type Rh(III) compounds such as 10 with
-1
a turnover frequency of 4.12 h , display no marked improvement compared to the Ru(II)
-1
arene analogues (c.f. 6.62 h for [(bn)Ru(TfEn)Cl]).
Mechanistic studies on these Rh(III) complexes were carried out in order to investigate the
1
catalytic cycle. Fast hydrolysis of the chlorido complexes 1-10 was observed by H NMR (<
1
0 min at 310 K). Compounds 1-3 and 10 gave quantitative conversion to aqua adducts,
while complexes 4-9 (at millimolar concentrations) reached equilibrium with a 30-60% of the
aqua species present. The pK * values determined for the aqua adducts of ca. 8-10 indicate
a
that the Rh(III) complexes are likely to be present in their aqua forms around pH 7 rather than
forming less reactive hydroxido species. Complexes 1 and 10 showed a dependence of the
2
8

ACCEPTED MANUSCRIPT
+
reaction rate on formate concentration, but not on NAD concentration. No reaction between
complexes 1-4 and 1,4-NADH was detected after 10 h. Optimum pH* for the reaction with
complex 1 was 7.3 ± 0.1, close to physiological pH. These results indicate that these Rh(III)
x
Cp compounds exhibit similar behaviour to their Ru(II) arene analogues.
-1
The maximum turnover frequencies of Rh(III) complexes 1 and 10 of 41.5 and 37.2 h ,
respectively, at high concentrations of formate are significant improvements in catalytic
-1
activity compared to the Ru(II) analogues (6.9 h ), even for complex [(Cp*)Rh(TfEn)Cl]
(10). This improvement in the maximum turnover frequency might be expected to translate in
significant enhancements in antiproliferative activity towards cancer cells when the
complexes are used in combination with high concentrations of formate.
We also demonstrated that some Rh(III) complexes, notably 1-5, 7 and 8, can catalyse the
reduction of pyruvate to lactate using formate as the hydride donor. Such reactions might
occur in cells. However complexes 6 and 9 with very low TOFs are not effective catalysts.
The transfer hydrogenation reactions were shown to be greatly affected by the chelating
x
ligand and the capping Cp ring. The catalytic activity of ethylenediamine compounds (1-3)
x
increases for extended Cp rings, while the bipyridine and phenanthroline compounds (4-9)
+
show the opposite trend. Studies of competition reactions between NAD and pyruvate for
reduction by formate catalysed by complex 4 suggested a clear preference for the reduction of
+
NAD , although, some lactate was still formed.
The antiproliferative activity of the Rh(III) complexes towards A2780 human ovarian cancer
cells increased by up to 50% (for complex 3) when administered in combination with
formate. However, the improvement in the activity of these Rh(III) complexes induced by
formate is much lower than in the case of Ru(II) complexes. It is possible that Rh(III) centres,
being more reactive than Ru(II), are more easily poisoned in the complicated mixture of
biomolecules present in culture media and in cells.
2
9