[RuIII(medtra)(H2O)] (medtra = N-methylethylenediaminetriacetate) complex - A highly efficient NO inhibitor with low toxicity

Article abstract of DOI:10.1016/j.ica.2005.12.081

Stopped-flow kinetic measurements were used to compare the reactivities of [Ru(medtra)(H₂O)] (medtra^3- = N-methylethylenediaminetriacetate) (1) and [Ru(hedtra)(H₂O)] (2) (hedtra^3- = N-hydroxyethylethylenediaminetriacetate) with NO in aqueous solution at 15 °C, pH 7.2 (phosphate buffer). The measured second-order rate constants (3 × 10³ and 6 × 10⁴ M^-1 s^-1 for 1 and 2, respectively) are three to four order of magnitudes lower than that for the reaction between [Ru^III(edta)(H₂O)]^- (3) with NO. However, NO scavenging studies of complexes 1-3, conducted by measuring the difference in nitrite production between treated and untreated murine macrophage cells, revealed that despite being less kinetically reactive toward NO, the [Ru(medtra)(H₂O)] complex exhibited the highest NO scavenging ability and lowest toxicity of compounds 1-3.

Full text of DOI:10.1016/j.ica.2005.12.081

Inorganica Chimica Acta 359 (2006) 2285–2290
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[RuIII(medtra)(H2O)] (medtra = N-methylethylenediaminetriacetate)
complex – A highly efficient NO inhibitor with low toxicity
a,
a
a
b
b
Debabrata Chatterjee *, Anannya Mitra , Ayon Sengupta , Piu Saha , Mitali Chatterjee
a
Chemistry Group, Central Mechanical Engineering Research Institute, MG Avenue, Durgapur 713209, West Bengal, India
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, 244 B, AJC Bose Road, Kolkata 700020, West Bengal, India
b
Received 7 September 2005; received in revised form 19 December 2005; accepted 29 December 2005
Available online 23 February 2006
Abstract
Stopped-flow kinetic measurements were used to compare the reactivities of [Ru(medtra)(H2O)] (medtra3ꢀ = N-methylethylenediam-
inetriacetate) (1) and [Ru(hedtra)(H2O)] (2) (hedtra3ꢀ = N-hydroxyethylethylenediaminetriacetate) with NO in aqueous solution at
15 ꢀC, pH 7.2 (phosphate buffer). The measured second-order rate constants (3 · 103 and 6 · 104 Mꢀ1 sꢀ1 for 1 and 2, respectively)
are three to four order of magnitudes lower than that for the reaction between [RuIII(edta)(H2O)]ꢀ (3) with NO. However, NO scaveng-
ing studies of complexes 1–3, conducted by measuring the difference in nitrite production between treated and untreated murine mac-
rophage cells, revealed that despite being less kinetically reactive toward NO, the [Ru(medtra)(H2O)] complex exhibited the highest
NO scavenging ability and lowest toxicity of compounds 1–3.
ꢁ 2006 Elsevier B.V. All rights reserved.
Keywords: NO scavenging; RuIII-pac complexes; Kinetics; NO binding; Murine macrophages
1. Introduction
We have undertaken this study to compare the NO scav-
enging ability of Ru(medtra)(H2O)] (1) (medtra3ꢀ = N-
methylethylenediaminetriacetate) and [Ru(hedtra)(H2O)]
(2) (hedtra3ꢀ = N-hydroxyethylethylenediaminetriacetate)
with that of [RuIII(edta)(H2O]ꢀ (3) (edta4ꢀ = ethylene-
diaminetetraacetate) under biological conditions (pH 7.2).
The lability of aquo-substituents on 1 and 2 differs from
that of 3 in the following order: [RuIII(edta)(H2O]ꢀ ꢁ
[Ru(hedtra)(H2O)] > [Ru(medtra)(H2O)] [2]. Studies with
murine macrophages, described herein, reveal that, despite
being least kinetically reactive toward NO, [Ru(med-
tra)(H2O)] is the most efficient and least toxic NO scaven-
ger under physiological conditions.
The [RuIII(pac)(H2O)] complexes (pac = polyamino-
carboxylate) by virtue of their ability to bind to a number
of biological molecules via a rapid aquo-substitution rate
command a reputable position as potential metallodrugs
[1]. The pac ligands form stable 1:1 (metal:ligand) com-
plexes with ruthenium and function as pentadentate
ligands (as represented in Fig. 1) in [RuIII(pac)(H2O)] com-
plexes [2]. The sixth coordination site of the ruthenium cen-
tre is occupied by a water molecule at low pH or by a
hydroxide ion at high pH [3–6]. Recent studies show that
the RuIII-pac complexes exhibited most of the characteris-
tics of an effective NO scavenger in biological systems
[7–14]. Until now, [Ru(edta)(H2O)]ꢀ (edta4ꢀ = ethylene-
diaminetetraacetate) appeared to be the most promising
[RuIII(pac)(H2O)] NO scavenger [9,11,13].
2. Experimental
2.1. Materials
The K2[RuCl5(OH2)] complex used as the precursor
complex for the preparation of Ru-pac complexes (1–3)
was prepared by the reported method [15]. The complexes
*
Corresponding author. Tel.: +91 3432554017; fax: +91 3432546745.
E-mail address: dchat57@hotmail.com (D. Chatterjee).
0020-1693/$ - see front matter ꢁ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2005.12.081

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D. Chatterjee et al. / Inorganica Chimica Acta 359 (2006) 2285–2290
O
an on-line data analyzer (KinetAsyst3). The solution tem-
perature during kinetic experiments was maintained within
0.1 ꢀC using a circulating water bath (JEIO TECH RW-
1025G). The substitution reaction was followed by moni-
toring changes in absorbance at 300 nm. Rate constant
data were measured under pseudo-first order conditions
of excess (10–50-fold) substituting ligands. The pH of the
solutions was measured with a Mettler Toledo MA 235
pH/Ion analyzer. A phosphate buffer was used to maintain
the pH of the solution for the kinetic studies. All the spec-
tral and kinetic measurements were performed under
anaerobic conditions. Dilution of a stock solution of NO
(1.5 · 10ꢀ3M) was carried out using degassed phosphate
buffer solution. The experimentally observed rate constant
data (kobs) represented as an average of several kinetic runs
(at least five to six) are reproducible within 4%.
O
O
O
N
N
Ru
OH
2
R
O
O
1
2
3
R = CH3
;
;
;
pac = medtra3- (N-methylethylenediaminetetriacetate)
pac = hedtra3- (N-hydroxyethylethylenediaminetetriacetate)
pac = edtra4- (ethylenediaminetetraacetate)
R = CH2CH2OH
R = CH2COO-
Fig. 1. Schematic representation of Ru-pac complexes.
K[RuIII(hedta)Cl], K[RuIII(hedtra)Cl] and K[RuIII(med-
tra)Cl] were prepared by treating K2[RuCl5(H2O)] with
the respective ligands as described in the literature [16].
The complexes were characterized by comparing the spec-
tral (UV–Vis and IR) and micro-analysis (C,H,N) data
obtained for complexes 1–3 with previously reported data
[3–6]. All the K[RuIII(pac)Cl] complexes are rapidly aqua-
ted when dissolved in water to form their corresponding
aquo-species [3–6]. Table 1 summarizes acid-dissociation
constants along with spectral and electrochemical data
for [RuIII(pac)(H2O)] complexes.
The NO solution was prepared by dropwise addition of
an aqueous solution of H2SO4 (2 M) to NaNO2 (solid)
under N2(g) in a Kipps apparatus as described in the liter-
ature [13]. The gas produced was purged sequentially
through NaOH solution (1 M), cold distilled water, and
finally a degassed phosphate buffer solution saturated with
NO (final concentration estimated [17,18] was
ꢂ1.5 · 10ꢀ3M at 25 ꢀC). All other chemicals used were of
A.R. grade. Doubly distilled H2O was used throughout
the experiments.
2.3. Murine macrophage assay for NO scavenging by Ru-pac
complex
Swiss albino mice (av. wt. 25–30 g) were used. All ani-
mals were housed under standard conditions of tempera-
ture (25 5 ꢀC). A 12 h light/dark cycle was maintained
and animals were provided with standard pellet diet and
water ad libitum. To elicit activated peritoneal macro-
phages, Swiss albino mice were injected i.p. with starch
(2% in autoclaved phosphate buffered saline (PBS),
0.02 M, pH 7.2, 2 ml/animal). After 48 h, the peritoneal
cells were lavaged with chilled, sterile PBS (10 ml) and cen-
trifuged at 2000 rpm for 10 min at 4 ꢀC. The resultant cell
pellet was washed in PBS and finally re-suspended in com-
plete RPMI-1640 medium supplemented with 10% heat
inactivated fetal calf serum (HIFCS). Macrophage viability
(>95%) was confirmed by Trypan Blue exclusion.
2.4. Measurement of nitric oxide (NO) production in
macrophages
2.2. Instrumentation
The electronic absorption spectra were measured with a
Lambda 35 (Perkin–Elmer) UV–Vis spectrophotometer.
The cell compartment was equipped with a PTP-1 Peltier
System for temperature control of the test solution. All
kinetic measurements were carried out with a SF-61SX2
(HI-TECH) stopped-flow spectrophotometer coupled with
Macrophages obtained by peritoneal lavage, as
described above, were seeded (2 · 105 cells/200 ll/ well) in
RPMI-1640 (Phenol red free) medium supplemented with
10% HIFCS in 96 well tissue culture plates and incubated
at 37 ꢀC, 5% CO2 for 3–4 h. Ru-pac complexes (1–3) were
co-incubated with lipopolysaccharide (LPS, 10 lg/ml) for
an additional 48 h at 37 ꢀC, 5% CO2. At the end of 48 h,
100 ll of the supernatant was withdrawn from each well
and nitrite accumulation as an indicator of NO production
was measured using the Griess reagent [8]. Briefly, 100 ll of
Griess reagent (1:1 of naphthyl ethylene diamine dihydro-
chloride (NED), 0.1% in water) and sulfanilamide (1% in
5% orthophosphoric acid) was added to 100 ll of superna-
tant and incubated in dark at room temperature (25–30 ꢀC
for 10 min). Absorbances at 550 nm were measured in a
microplate ELISA reader (BioRad model 680). A standard
curve using sodium nitrite (0–100 lM) was used to
calculate the concentrations of nitrite. To demonstrate
specificity, macrophages were incubated in the presence
Table 1
Spectral, electrochemical and acid-dissociation constant data for [RuIII
(pac)(H2O)] complexes
-
Complex
kmax (nm)
E1/2
(V vs. NHE)
pK1
pK2
(emax/Mꢀ1 cmꢀ1
in water
)
[RuIII(medtra)(H2O)]
[RuIII(hedtra)(H2O)]
[RuIII(hedta)(H2O)]
290 (2400 30)
380 (920 60)
285 (1950 20)
350 (850 20)
280 (2800 50)
350 (680 30)
ꢀ0.10
ꢀ0.07
ꢀ0.04
6.3
4.9
7.6
2.4

D. Chatterjee et al. / Inorganica Chimica Acta 359 (2006) 2285–2290
2287
of N-monomethyl arginine (L-NMMA, 0.1 mM), an estab-
lished inhibitor of NO production. A standard curve was
generated using sodium nitrite. The % inhibition of NO
production by the RuIII-pac complexes was calculated as
follows: ([NaNO2]control macrophages ꢀ [NaNO2]test macrophages)/
[NaNO2]control macrophages · 100.
mented with 10% HIFCS in 96-well tissue culture plates
and incubated at 37 ꢀC, 5% CO2 for 3–4 h. The Ru-pac
complexes (1–3) along with LPS (10 lg/ml) were incubated
at 37 ꢀC, 5% CO2 for 48 h. At the end of exposure of
the compounds (1–3), the MTS assay was performed.
MTS {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner
salt,
2.5. Cytotoxicity assay
2 mg/ml} was prepared in PBS and stored in the dark at
ꢀ20 ꢀC. A stock solution of PMS (phenazine methosulfate,
0.92 mg/ml in PBS) was similarly prepared. After MTS and
PMS were mixed in a 5:1 ratio, each well was treated with
MTS/PMS mixture (20 ll). The plates were then incubated
To determine viability of macrophages in the presence of
the compounds, macrophages were seeded (2 · 105 cells/
200 ll/well) in RPMI-1640 phenol red free medium supple-
Fig. 2. Spectral changes associated with the reaction of NO with (A) 1 and (B). Inset: Kinetic traces for the reaction of NO with (A) 1 and (B) 2 at 15 ꢀC,
pH 7.2 (phosphate buffer), [RuIII] = 2 · 10ꢀ5 M, [NO] = 1.5 · 10ꢀ3 M.

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D. Chatterjee et al. / Inorganica Chimica Acta 359 (2006) 2285–2290
at 37 ꢀC for 3 h and absorbances measured at 490 nm using
the inset of Fig. 2. The kinetics of the interaction of
[RuIII(edta)(H2O)]ꢀ (3) with NO in phosphate buffer was
reported earlier [9]. Similar, but comparatively much
slower kinetic behavior was exhibited by complexes 1 and
2 while reacting with NO under the specified conditions.
Kinetic traces (decay at 300 nm) typical of the reaction of
1 and 2 with NO shown in Fig. 2 are single exponential
a microplate ELISA reader (BioRad model 680). The %
viability was calculated as follows: (490Atreated macrophages
/
490
490
490
A ) · 100, where
control
A
and
A
control
treated macrophages
are the specific absorbance of treated macrophages and
control macrophages, respectively, at 490 nm. The specific
absorbance was calculated by subtracting absorbance of
the medium from the total absorbance.
in nature. The values of the observed rate constant (kobs
)
obtained by performing single exponential analysis of the
kinetic traces increased linearly with [NO] with a negligible
intercept (Fig. 3), indicating that neither a reverse aquation
of the substituted product nor parallel reaction contributes
significantly. Considering that [RuIII(pac)(OH)]ꢀ would be
the predominating species at pH 7.2 [3–6], the kinetic
behavior of the present system can be rationalized in terms
of the following reaction (1):
3. Results and discussion
3.1. Kinetics of the interaction of RuIII-pac complexes with
NO
The representative example of the spectral changes that
occurred upon addition of NO dissolved in degassed phos-
phate buffer to an aqueous solution of 1 and 2 is shown in
40
30
20
10
0
(b)
100
(B)
(a)
(b)
80
60
40
20
(b)
(a)
(a)
(A)
0.0016
0
0.0004
0.0006
0.0008
0.0010
[NO]
0.0012
0.0014
0
1
2
3
Control
Fig. 3. Plots of kobs vs. [NO] for the reaction of NO with (A) 1 and (B) 2 at
15 ꢀC, pH 7.2 (phosphate buffer), [RuIII] = 2 · 10ꢀ5 M, [NO] = 1.5 ·
10ꢀ3 M.
Fig. 4. Inhibition of NO production in murine peritoneal macrophages by
RuIII-pac complexes (1–3, 100 lM) in absence of (a) or presence (b) of
LPS (10 lg/ml).
Table 2
Value of second-order rate constant (k) for the formation of [RuIII(pac)(Nu)] in the reaction of [RuIII(pac)(H2O)] complexes with some selected
nucleophiles at 25 ꢀC
RuIII-pac complex
k (Mꢀ1 sꢀ1
)
Thiourea
Thiocyanate
Azide
Pyridine
Cysteine
Glutathione
[RuIII(medtra)(H2O)]
[RuIII(hedtra)(H2O)]
[RuIII(edta)(H2O)]ꢀ
[RuIII(medtra)(OH)]ꢀ
[RuIII(hedtra)(OH)]ꢀ
[RuIII(edta)(OH)]2ꢀ
2.34a
22.6c
2970f
0.15a
2.8c
0.28a
6.8c
NR
18.5c
1885f
NR
2.6c
75e
NR
18d
6300g
NR
NR
NR
0.3b
2.59e
170i
0.35
0.6e
0.37b
1.97e
260i
0.41
0.8e
520h
270g
0.04a
1.1c
NR
32f
425h
a
Ref. [6].
Unpublished data.
Ref. [5].
Ref. [19].
Ref. [20].
Ref. [4].
Ref. [3].
Ref. [20].
b
c
d
e
f
g
h
i
At pH 7.2, Ref. [21]; NR, not reported.

D. Chatterjee et al. / Inorganica Chimica Acta 359 (2006) 2285–2290
2289
Table 3
Effect of RuIII-pac complexes on NO production in macrophagesa
RuIII-pac complexes
LPS (ꢀve)
LPS (+ve)
NaNO2 (lM)c
% Inhibitiond
% Viabilitye
NaNO2 (lM)c
% Inhibitiond
% Viabilitye
Control
7.65
4.9
6.21
6.72
100
8.1
5.3
5.44
6.35
100
100
68.07
59.58
1b
2b
3b
a
35.95
18.82
12.16
90.45
76.86
59.95
39.57
32.84
21.60
See experimental for details.
[RuIII] = 100 lM.
b
c
A standard curve using sodium nitrite (0–100 lM) was used to calculate concentrations of nitrite.
% Inhibition with RuIII-pac complexes was calculated as described in Section 2.
% Viability of macrophages treated with RuIII-pac complexes was measured as described in Section 2.
d
e
k
½RuIIIðpacÞðOHÞꢃ þ NO ! ½RuIIIðpacÞðNOÞꢃ þOHꢀ ð1Þ
a much faster rate than 1 and 2 (Table 2). The affinity that
3 exhibits for sulfur containing biomolecules may explain
its increased cytotoxicity. In contrast, the slower reactivity
toward cellular thio-molecules displayed by 1 and 2 may
explain why 2 is less toxic than 3 and, most importantly,
why 1 exhibits no toxicity at all. While evaluating com-
plexes for use as potential NO scavengers it is important
to consider the potential scavenger’s affinity for thio-con-
taining molecules to ensure that the decrease in NO pro-
duction is not due to enhanced cytotoxicity.
ðpac ¼ medtra3ꢀ; hedtra3ꢀÞ
for which the following rate expressions (Eq. (2)) is derived.
kobs ¼ kf ½NOꢃ
ð2Þ
The values of the second-order rate constant (k) for the
reaction of NO (15 ꢀC and pH 7.2) with 1 and 2 are
3 · 103 and 6 · 104 Mꢀ1 sꢀ1, respectively. It had been re-
ported earlier [9,12,13] that the rate of the aquo-substitu-
tion of [RuIII(edta)(H2O/OH)]ꢀ/2ꢀ with NO is very fast
(1.95–3.29 · 107 Mꢀ1 sꢀ1 at 7.3 ꢀC) in the studied pH range
6.5–8.0. Comparison of the rate constant values (k) ob-
tained in the present studies with that reported for the reac-
tion of Ru-edta complex with NO [9,12,13] revealed the
following reactivity order towards NO binding:
3 ꢁ 2 > 1. The [RuIII(edta)(H2O/OH)]ꢀ/2ꢀ complex binds
with NO at a much higher rate than 1 and 2, whereas 2
exhibited a higher reactivity than 1. Table 2 summarizes
the previously reported data for the reaction between 1
and 3 and some selected nucleophiles. The data in Table
2 substantiate our results and underscore the significant
difference in the rate constants for the aqua complexes as
compared to the hydroxo complexes. Within the series of
aqua/hydroxo complexes shown in Table 2, the rate
constants substantially decrease along the series edta ꢁ
hedtra > medtra (see Fig. 4).
4. Conclusion
Results of kinetic studies revealed that 1 and 2 react with
NO in a similar manner as reported for the corresponding
‘edta’ complex (3), but at much slower rates. The NO scav-
enging ability of 1 and 2 has been demonstrated. In both
(LPS treated and non-treated) cases, NO scavenging effi-
ciency of 1 and 2 is appreciably higher than that reported
for 3. It may be envisaged that the lower rate of deactiva-
tion through binding of cellular thio-molecules could be
one of the reasons. Further, the lower efficiency observed
in NO scavenging for 3 could be associated with its deacti-
vation by sulfur containing compounds, since 3 binds sul-
fur containing compounds more readily than does 1 or 2.
Acknowledgements
3.2. NO scavenging activity of [RuIII (pac)(H2O)]ꢀ
complexes
We thank Dr. G.P. Sinha, Director of this institute for
his encouragement with respect to this work. D.C. grate-
fully acknowledges financial support from the DST, Govt.
of India (Grant SP/S1/F35/99). A.M. is thankful to CSIR
for RA.
The results (Table 3) of the studies revealed that the
addition of 1–3 (100 lM) to macrophages reduced the
ꢀ
NO2 levels in the system both in the presence and absence
of LPS, indicating that all three Ru-pac complexes (1–3)
are capable of scavenging NO in biological systems. In
the present investigation, the NO scavenging efficiency of
3 was comparable to that reported earlier [13]. The most
significant findings were that both complexes 1 and 2,
despite being less reactive than 3 towards NO binding in
aqueous solution, lowered the nitrite levels more efficiently
than 3. Although due to its remarkable lability, the
[RuIII(edta)(H2O)]ꢀ complex (3) rapidly binds with NO
[9,12,13], it also binds sulfur containing bio-molecules at
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