Synthesis and antiproliferatory activity of ruthenium complexes containing N-heterocyclic carboxylates

Article abstract of DOI:10.1016/j.inoche.2017.02.011

Solvates of the complexes Ru₂Cl(pic)₄(EtOH) (2), Ru(Im-CO₂)₃ (3), and Ru(Im-CO₂)(Im-CO₂H)Cl₂ (4), were synthesized from reaction of RuCl₃·3H₂O or K₃[RuCl₆] with the N-heterocyclic carboxylic acids pyridine-2-carboxylic acid (picolinic acid, Hpic), and imidazole-4-carboxylic acid (Im-CO₂H). Crystals of 2–4 could not be grown and hence characterization was done by elemental analysis, NMR, IR, and conductivity data; 2 and 4 were tested for antiproliferatory activity in vitro against a human breast cancer cell line, but were less active than, for example, Ru complexes containing bis-imidazole or 4,4′-biimidazole that we studied previously [see Can. J. Chem. 89 (2011) 948]. Preliminary work with a third potential ligand, 3-nitro-1,2,4-triazole-5-carboxylic acid (abbreviated HCANT), and other nitro heterocyclic compounds is also presented.

Full text of DOI:10.1016/j.inoche.2017.02.011

Accepted Manuscript
Synthesis and antiproliferatory activity of ruthenium complexes
containing N-heterocyclic carboxylates
David C. Kennedy, Brian R. James
PII:
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doi: 10.1016/j.inoche.2017.02.011
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ACCEPTED MANUSCRIPT
Synthesis and antiproliferatory activity of ruthenium complexes containing N-
heterocyclic carboxylates
David C. Kennedy, Brian R. James
Department of Chemistry, The University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Received …….2016; accepted ……..
Published online:
Abstract
Solvates of the complexes Ru Cl(pic) (EtOH)(2), Ru(Im-CO ) (3), and Ru(Im-CO )(Im-CO H)Cl (4),
2
4
2 3
2
2
2
were synthesized from reaction of RuCl ∙3H O or K [RuCl ] with the N-heterocyclic carboxylic acids pyridine-2-
3
2
3
6
carboxylic acid (picolinic acid, Hpic), and imidazole-4-carboxylic acid (Im-CO H). Crystals of 2-4 could not be
2
grown and hence characterization was done by elemental analysis, NMR, IR, and conductivity data; 2 and 4 were
tested for antiproliferatory activity in vitro against a human breast cancer cell line, but were less active than, for
example, Ru complexes containing bis-imidazole or 4,4'-biimidazole that we studied previously [see Can. J. Chem.
8
9 (2011) 948]. Preliminary work with a third potential ligand, 3-nitro-1,2,4-triazole-5-carboxylic acid (abbreviated
HCANT), and other nitro heterocyclic compounds is also presented.
Keywords: Ruthenium; N-heterocyclics; Carboxylates; Antiproliferatory activity
Our group first published work on the use of ruthenium complexes as radiosensitizers in 1986,
reporting on a fluorinated nitroimidazole ligand system [1], and have since continued interest in
Ru-imidazole and -4,4'-biimidazole complexes [2] and their ‘biological properties’, particularly
antiproliferatory activity against cancer cells. Ruthenium species we have studied include co-
ligands such as sulfoxides [3], and acac and the related maltolato and pyridonate anions were
also studied as ligands or co-ligands [4]. Meantime, we have naturally followed the great interest
III
in development through clinical trials of the anticancer agents [HIm][trans-Ru Cl (Im)(S-
4
III
DMSO)] (Im = imidazole) and [HInd][trans-Ru Cl (Ind) ] (Ind = indole), labelled NAMI and
4
2
KP1019, respectively [5]. We reported, in 2011, the exponential general interest in
bioinorganic’ Ru-systems [4d], and noted that ‘many synthetic-type coordination and
‘
organometallic researchers in the Ru chemistry area are now testing their new complexes for
antiproliferatory activity!’ This trend, including studies of other biological properties, has
continued and, for example, around 400 publications are listed in SciFind for 2014, 2015 and the
first 6 months of 2016; as well as ref. 5(a-c), 13 other arbitrary 2016 references are listed [6(a-
m)].
1

ACCEPTED MANUSCRIPT
We decided to study as ligands imidazoles containing a carboxylic acid substituent because
this would likely allow for any derived Ru complexes to be soluble in aqueous media, depending
on pH  an important property for biological tests. This paper reports the syntheses and
characterization work of the complexes, as well as some preliminary data on their
antiproliferatory properties. A further N-heterocyclic carboxylic acid, pyridine-2-carboxylic acid
2
III
(
Hpic), was also studied because the tris( -N,O-picolinate) complex mer-Ru (pic) was known
3
[
7], and its characterization data were thought to be of possible value for our studies. Because of
the ability of nitroimidazoles to function as radiosensitizers and hypoxic markers [1, 8], studies
were also initiated on carboxylic acid derivatives of nitro-substituted N-heterocyclic compounds:
examples include synthesis of 3-nitro-1,2,4-triazole-5-carboxylic acid and its reactivity with Ru-
precursors, and attempts to synthesize 2-nitroimidazole-4-dicarboxylic acid and the reported 2-
nitroimidazole-4,5-dicarboxylic acid [9].
Some ligand syntheses required the following precursors that were purchased from Aldrich:
imidazole-4-carboxylic acid (Im-CO H), imidazole-4,5-dicarboxylic acid, 4-(hydroxymethyl)-
2
imidazole, and 3-amino-1,2,4-triazole-5-carboxylic acid; Hpic, trityl chloride, and other common
chemicals were also Aldrich products. RuCl ∙3H O (with 39% Ru) was donated by Colonial
3
2
Metals Inc., while K [RuCl ] [10] and Ru Cl(-O CMe) [11] were made by literature methods.
3
6
2
2
4
Some solvents (Fisher products) were dried and distilled prior to use: MeOH and EtOH (distilled
from Mg), THF (Na); other solvents were used as received. The deuterated solvents D O,
2
CD OD, CDCl , and DMSO-d (Cambridge Isotope Labs products) were also used as received.
3
3
6
Medical grade N was used as received from Praxair.
2
1
H NMR spectra ( s = singlet, t = triplet, q = quartet, br = broad, and m = multiplet) were
recorded at room temperature (~20 C) on a Bruker AV300 instrument, and referenced using
residual protonated solvent signals: 7.24 (CDCl ), 4.65 (D O), 3.30 (CD OD), and 2.49 (DMSO-
3
2
3
-1
d ). IR spectra (KBr pellets, s = strong, m = medium) were recorded as cm on a Bomem-
6
Michelson MV-100 FT-IR spectrometer. Mass spectral data were measured on a Bruker Esquire
electrospray (ESI ion-trap) spectrometer and a Micromass LCT electrospray time of flight (ESI
+
TOF); M is defined as the positive molecular ion of a given species and does not include the
mass of solvated molecules. Conductivity measurements, carried out on a RCM151B Serfass
conductivity bridge (A.H. Thomas Co.) with a 3403 cell (Yellow Springs Instrument Co.), were
-1
2
-1
calibrated using 0.0100 M aq. KCl solution ( = 141.3  cm mol at 25 C, cell constant =
M
2

ACCEPTED MANUSCRIPT
-
1
1
1
.016 cm ), and data are given in  units. Elemental analyses were done on a Carlo Erba, EA
M
106 analyzer in this department.
All reagents, cell handling techniques, and details for a so-called MTT colourimetric biological
assay, which examines antiproliferatory activity of compounds over large concentration ranges
MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium], have been described in detail in
earlier publication [3, 4c]. The MTT assay gives IC values (the drug concentration at which
[
5
0
5
0% of the cells are viable relative to a control) that are determined from plots of % cell viability
vs. drug concentration (logarithmic scale). The Ru complexes were examined on human breast
cancer cells (MDA-MB-435S), purchased from ATCC (American Type Culture Collection).
Modified literature methods [7] were used to synthesize mer-Ru(pic) ∙H O (1). (a) Hpic (0.14
3
2
g, 1.2 mmol) was added to a red solution of K [RuCl ] (0.17 g, 0.39 mmol) in 5 mL H O, and the
3
6
2
o
solution then heated at 60 C for 5 h. The yellow precipitate that formed was filtered off, washed
o
with MeOH (2 x 5 mL) and H O (1 x 5 mL), and then dried in vacuo at ~80 C for 48 h; yield:
2
0
.11 g (58 %). (b) Hpic (0.096 g, 0.78 mmol) was added to a brown solution of Ru Cl(-
2
O CMe) (0.062 g, 0.13 mmol) in water /MeOH, 1:1 (10 mL), and the mixture was refluxed for
2
4
8
h to give the yellow precipitate that was isolated by filtration, washed with H O (2 x 5 mL),
2
and dried as above. Yield: 0.049 g (77 %). IR: 3443 (O-H, s), 1661 (C=Oasym, s), 1565 (m), 1315
+
(
C=O , m), 1280 (s), 1057(m), in agreement with literature data [7]. ESI-MS: 468 (M ). Anal.
sym
Calcd. for C H N O Ru∙H O: C, 44.54; H, 2.91; N, 8.66. Found: C, 44.5; H, 2.7; N, 8.3.
1
8
12
3
6
2
For synthesis of Ru Cl(pic) (EtOH)•H O (2), Hpic (0.072 g, 0.59 mmol) was added to a
2
4
2
brown solution of RuCl ∙3H O (0.051 g, 0.19 mmol) in 10 mL EtOH, and the solution was then
3
2
refluxed for 6 h, with the colour becoming orange; after evaporation to a volume of ~1 mL,
addition of Et O (10 mL) gave an orange precipitate that was collected, washed with Et O (2 x 5
2
2
o
1
mL), and then dried in vacuo at ~80 C for 24 h. Yield: 0.066 g (86 %). H NMR (D O):  8.76-
2
7
.92 (m, pic), 4.53 (br q, coordinated HOCH CH ), 3.55 (q, free OHCH CH ), 1.45 (br t,
2 3 2 3
coordinated HOCH CH ), 1.12 (free HOCH CH ). IR: 3468 (O-H, s), 3124 (N-H, m), 1672
2
3
2
3
(C=Oasym, s), 1639 (C=Oasym, s), 1600 (m), 1456 (m), 1396 (C=Osym, m), 1316 (C=Osym, m), 1282
+
+
(
m), 1147 (m), 856 (m), 759 (s), 691 (m). ESI-MS: 727 (M EtOH), 382 (Ru(pic) Cl ), 346
2
+
(
Ru(pic) ).  (MeOH) = 112. Anal. Calcd. for C H N O ClRu ∙H O: C, 39.52; H, 3.06; N,
2
M
26 22
4
9
2
2
7
.09. Found: C, 39.4; H, 2.9; N, 6.9.
3

ACCEPTED MANUSCRIPT
Ru(Im-CO ) ∙2H O (3) was synthesized by two methods: (a) Im-CO H) (0.094 g, 0.84 mmol)
2
3
2
2
was added to a solution of K [RuCl ] (0.12 g, 0.28 mmol) in H O (10 mL). Refluxing for 6 h
3
6
2
afforded a yellow precipitate that was collected at r.t., washed with MeOH (2 x 5 mL), and dried
o
in vacuo at 78 C for 48 h; yield: 0.074 g (57 %). (b) Im-CO H (0.092 g, 0.82 mmol) was added
2
to a solution of Ru Cl(-O CMe) (0.065 g, 0.14 mmol) in 1:1water/MeOH (10 mL) that was
2
2
4
1
then refluxed for 8 h to give the yellow precipitate, isolated as in (a). Yield: 0.043 g (67 %). H
NMR (DMSO-d ):  0.31, 0.09, 0.09 (s, H -Im), 2.67, 13.55, 17.87 (s, H -Im). IR: 3423 (O-
6
5
2
H, s), 3109 (N-H, s), 1642 (C=Oasym, s), 1324 (C=Osym, s), 1201 (m), 1090 (m), 1024 (m), 930
+
(
m), 829 (m). ESI-MS: 435 (M ). Anal. Calcd. for C H N O Ru∙2H O: C, 30.64; H, 2.79; N,
12
9
6
6
2
1
7.87. Found: C, 30.9; H, 2.6; N, 17.5.
For synthesis of RuCl (Im-CO )(Im-CO H) ∙EtOH∙H O (4), Im-CO H (0.084 g, 0.75 mmol)
2
2
2
2
2
2
was added to a solution of RuCl ∙3H O (0.065 g, 0.25 mmol) in EtOH (10 mL); on being
3
2
refluxed for 4 h, the solution became yellow. The volume was then reduced to ~2 mL, when
addition of Et O (15 mL) afforded a yellow precipitate that was collected, washed with Et O (3 x
2
2
1
5
mL), and dried as for 3. Yield: 0.11 g (76 %). H NMR (CD OD):  3.45 (q, HOCH CH ), 1.12
3
2
3
(
t, HOCH CH ), 4.49, 7.20, 10.16 (s, H -Im), 14.83, 16.75, 23.92 (br s, H -Im). IR: 3467
2
3
5
2
(O-H, s), 3217 (O-H, m), 3149 (N-H, m), 1718 (C=Oasym, s), 1706 (C=Oasym, s), 1570 (C=Oasym,
s), 1384 (C=Osym, m), 1350 (C=Osym, m), 1313 (C=Osym, m), 1188 (m), 1089 (m). ESI-MS: 508
+
+
+
+
(
M ), 472 (M Cl), 396 (M ImCO H), 360 (M ImCO H–Cl).  (MeOH) = 19. Anal.
2
2
M
Calcd. for C H N O Cl Ru∙EtOH∙H O: C, 29.43; H, 3.35; N, 14.71. Found: C, 29.4; H, 3.2; N,
1
2
11
6
6
2
2
1
4.4.
-Nitro-1,2,4-triazole-5-carboxylic acid (HCANT) was made by a modified literature method
3
for conversion of amino-triazoles to the nitro derivative [12]. After addition of NaNO (1.10 g,
2
o
1
1
5.9 mmol) to 7 mL of H SO at –5 C, glacial CH CO H (15 mL) and finely ground 3-amino-
2
4
3
2
,2,4-triazole-5-carboxylic acid (2.00 g, 15.6 mmol) were added. The mixture was then stirred
for 10 min to dissolve most of the triazole; H O (25 mL) was then added at a temperature of
2
around zero. The resulting yellow solution was then added dropwise to a nitrite solution (200 g
o
NaNO in 200 mL H O) at 50 C (a rapid addition generates a hot foam of diazonium salts, with
2
2
o
contents erupting from the flask). The green product solution on heating for 2 h at 50 C becomes
colourless, and is then extracted with EtOAc (4 x 50 mL); evaporation of the extracts gives
4

ACCEPTED MANUSCRIPT
HCANT (1.41 g, 57 %). IR: 3416 (N-H, s), 3257 (O-H, m), 1710 (C=O, s), 1574 (NO , m), 1383
2
+
(
NO , m), 1268 (m), 720 (m). ESI-MS: 159 (M ). Anal. Calcd. for C H N O : C, 22.78; H, 1.28;
2
3
2
4
4
N, 35.44. Found: C, 22.5; H, 1.4; N, 35.4.
For synthesis of methyl imidazole-4-carboxylate (5), conc. H SO (1.5 mL) was added to a
2
4
suspension of Im-CO H (1.00 g, 8.92 mmol) in MeOH (20 mL), and the mixture was refluxed
2
o
for 24 h to give a solution that was then cooled to 0 C and neutralized to pH 8 using 5 M NaOH.
Evaporation in vacuo left a white residue that was re-dissolved in a minimal volume of boiling
water; cooling the solution deposited white crystals of the ester that were collected, washed with
1
cold H O (1 x 5 mL), and dried in vacuo at r.t. for 24 h. Yield: 0.87 g (77 %). H NMR (DMSO-
2
d ):  7.74 (s, 1H, H -Im), 7.65 (s, 1H, H -Im), 2.51 (s, 3H, CH -Im). IR: 3105 (N-H, s), 2976,
6
5
2
3
+
2
846 (C-H, m), 1619 (C=O, m), 1363 (s), 1156 (m), 864 (m). ESI-MS: 127 (M ). Anal. Calcd.
for C H N O : C, 47.62; H, 4.80; N, 22.21. Found: C, 47.5; H, 4.85; N, 21.7.
5
6
2
2
For methyl 1-tritylimidazole-4-carboxylate (6), trityl chloride (1.77 g, 6.35 mmol) was first
stirred into a solution of 5 (0.80 g, 6.3 mmol) in DMF (20 mL) under N . NEt (0.98 mL, 7.0
2
3
mmol) was then added, and the mixture stirred for 16 h at r.t. before being poured over ice; the
cold mixture was then filtered and the isolated solid was washed with H O (2 x 5 mL), and then
2
1
dried in vacuo at r.t. for 24 h. Yield: 2.01 g (86 %). H NMR (CDCl ):  7.65 (s, 1H, H -Im),
3
5
+
7
.52 (s, 1H, H -Im), 7.03-7.36 (m, 15H, Ph C), 2.42 (s, 3H, CH -Im). ESI-MS: 369 (M ), 243
2 3 3
(
CPh ). Anal. Calcd. for C H N O : C, 78.24; H, 5.47; N, 7.60. Found: C, 78.35; H, 5.6; N, 7.4.
3 24 20 2 2
4
-Hydroxymethyl-1-tritylimidazole (7) was made by two methods: (a) a modified, reported
method was first used [9]. A hexanes solution (5.5 mL) of 1.0 M diisobutylaluminum hydride
(
Dibal-H) was added to a THF solution (15 mL) of 6 (1.02 g, 2.72 mmol) under N , and the
2
o
mixture was stirred for 1 h, and then cooled to 0 C. The following were then slowly added in
sequence: H O (0.8 mL), 15 % aq. NaOH (1.0 mL), and H O (0.8 mL). The mixture was then
2
2
filtered, and the precipitate washed with THF (3 x 10 mL), the combined filtrate and washings
then being evaporated in vacuo. The residue was dissolved in CH Cl and the solution, after
2
2
being washed with H O to remove any remaining inorganic salts, was evaporated in vacuo to
2
give a white product that was further dried for 24 h at r.t. in vacuo. Yield: 0.41 g (44 %). (b) To a
stirred solution of 4-(hydroxymethyl)imidazole (0.52 g, 5.3 mmol) in DMF (10 mL) under N2
was added trityl chloride (1.48 g, 5.30 mmol), and after ~10 min NEt (0.90 mL, 6.4 mmol) was
3
5

ACCEPTED MANUSCRIPT
added. This mixture was then stirred for 16 h, and poured onto ice; the resulting precipitate was
1
collected, washed with H O (2 x 10 mL), and dried in vacuo for 24 h. Yield: 1.53 g (85 %). H
2
NMR (CDCl ):  7.39 (s, 1H, H -Im), 7.27-6.97 (m, 15H, CPh ), 6.80 (s, 1H, H -Im), 4.62 (s,
3
2
3
5
+
2
8
H, CH OH). ESI-MS: 341 (M ), 243 (CPh ). Anal. Calcd. for C H N O: C, 81.15; H, 5.92; N,
2 3 23 20 2
1
.23. Found: C, 81.2; H, 5.9; N, 7.9. The H NMR data agree with reported values [9].
4
,5-Dihydroxymethyl-1-tritylimidazole was made by a modified, reported method [13]. A
hexanes solution (10.5 mL) of 1.0 M Dibal-H solution was added to a THF solution (20 mL) of
dimethyl 1-tritylimidazole-4,5-dicarboxylate (1.03 g, 2.41 mmol) (see below) under N , and the
2
o
mixture was stirred for 4 h, and then cooled to 0 C; the following were then added: H O (1.5
2
mL), 15 % aq. NaOH (2 mL), and H O (1.5 mL). The mixture was then filtered, and the
2
precipitate washed with warm THF (3 x 10 mL), the combined filtrate and washings then being
evaporated in vacuo. The residue was dissolved in CH Cl , the solution filtered through Celite
2
2
(~10 g), and the filtrate was evaporated. The resulting white solid was dried in vacuo at r.t. for
1
2
4
4 h. Yield: 0.42 g (44 %). H NMR (CDCl )  7.67 (s, 1H, H -Im), 7.41-7.19 (m, 15H, CPh ),
3
2
3
+
.83 (s, 2H, CH OH), 3.89 (s, 2H, CH OH). ESI-MS: 371 (M ), 243 (CPh ). Anal. Calcd. for
2
2
3
C H N O : C, 77.81; H, 5.99; N, 7.56. Found: C, 77.7; H, 5.9; N, 7.4.
24
22
2
4
The following compounds were made by the indicated literature methods: 4-hydroxymethyl-
-nitroimidazole (8), but in only 3% yield from 4-hydroxymethyl-1-tritylimidazole [9]; dimethyl
2
imidazole-4,5-dicarboxylate, in 85% yield, from imidazole-4,5-dicarboxylic acid [13]; and
dimethyl 1-tritylimidazole-4,5-dicarboxylate, in 71% yield, from dimethyl imidazole-4,5-
dicarboxylate [13].
III
II
First, it is worth noting that formation of Ru or Ru complexes via the mixed-valence
Ru Cl(-O CMe) is common [7b], but details of the redox chemistry remain unclear. The
2
2
4
III
structurally characterized Ru -picolate yellow complex, mer-Ru(pic) ∙H O (1), was synthesized
3
2
from reaction of Hpic with K [RuCl ] and with Ru Cl(-O CMe) by modified literature
3
6
2
2
4
methods [7] in 58 and 77% yields, respectively; however, 1 was too insoluble in the medium
required for the IC measurements [3, 4c]. A reported reaction [14] giving 1 in 62% yield using
5
0
RuCl ∙3H O, and Na CO to deprotonate the Hpic, was also tested but generated only a black
3
2
2
3
solid; it should be noted that commercially available ‘RuCl ∙3H O’ contains variable amounts of
3
2
IV
III
Ru impurities [15], and irreproducibility of some Ru chemistry using the trichloride precursor
6

ACCEPTED MANUSCRIPT
is not surprising. Fortunately, when the Na CO was omitted, a bright orange complex
2
3
formulated as Ru Cl(pic) (EtOH)•H O (2) was obtained in 86% yield, and was readily water-
2
4
2
soluble. Elemental analysis (EA) strongly supports this formulation, and the MS data (in MeOH)
+
show a Ru splitting pattern with a parent peak for [M EtOH] at 727 (Fig. S1); this pattern is
2
distinctly different from that of a mononuclear species (Fig. S2). The IR data, specifically the
difference in the asymmetric and symmetric carboxylate stretching frequencies [16], which is
very similar to that of 1 that contains solely N,O-bonded picolinate (asym = 1661; sym = 1315
-1
cm ), implies the absence of a bridging carboxylate; for -carboxylates, the difference is
-1
-1
typically < 150 cm for a Ru species [16]. The band at 3468 cm is assigned to  of the H O,
2
OH
2
-1
the value being close to one of 3460 cm in 1, in which the H O is H-bonded to carboxylate O-
2
atoms, this resulting in chains within the crystal [7b]. Coordinated
1
H2
EtOH is identified by H NMR resonances (in D O) at  1.45 (the Me-
2
O
III
EtOH
N
N
O
O
N
Cl
II
Ru
Ru
triplet) and  4.53 (the CH quartet) that are downfield from those seen
2
N
O
O
also for free EtOH ( 1.12 and 3.55, respectively); the relative
intensities of the two sets of signals (Fig. S3) imply ~40% of 2 is
2
converted to a species with coordinated D O. Conductivity for 2 in MeOH is in the 1:1
2
-
-
electrolyte range [17], consistent with dissociation of a terminal Cl ; loss of a bridged Cl would
likely decompose the bimetallic unit, but the ESI-MS data in MeOH show this remains intact. In
their synthesis of 1, Barral et al. [7b] isolated also a co-product Ru (pic) , with IR data for the
2
4
carboxylate being again consistent with coordination via N- and O-atoms; possible coordination
of the keto group of a carboxylate at an axial position of a further Ru (pic) unit to give a
2
4
polymeric structure was also suggested to rationalize low solubility of the complex. An
II
III
‘
imaginative’ non-polymeric Ru -Ru solid state structure for 2 containing chelated bridging
and non-bridging N,O-pic ligands is shown above; an alternative structure with four bridging pic
1
ligands is also feasible. The H NMR spectrum (Fig. S3) supports only qualitatively this type of
structure, because the partial loss of coordinated EtOH and likely loss of chloride means that
1
several species are present; meaningful integration of the H signals was not possible. The
related mixed-valence complex, Ru Cl(hp) (Hhp) where Hhp is 2-hydroxypyridine, similarly
2
4
contains bridging and non-bridging N,O-ligands [18].
III
The yellow complex Ru (Im-CO )
•
2H O (3), like 1, was synthesized in good yield using
2
3
2
reaction of either K [RuCl ] or Ru Cl(-O CMe) with Im-CO H in H O or MeOH, respectively,
3
6
2
2
4
2
2
7

ACCEPTED MANUSCRIPT
and characterized by methods used for 1. The EA is consistent with a dihydrate, and the presence
-1
of H O is shown by  at 3423 cm , and an increase in intensity of the residual H O signal in
2
OH
2
1
the H NMR spectrum (in DMSO-d ) compared with that seen in the absence of the complex; 3
6
is only sparingly soluble in DMSO (and insoluble in other solvents) and the NMR spectrum
recorded over 6 h (~20,000 scans) shows 6 weak signals for the bound imidazoles at  0.31, 0.09,
and 0.09 for H(5) and  2.67, 13.55, and 17.87 for H(2), the assignments being based on
III
those observed earlier for monodentate imidazole ligands at Ru [4d]. The asym sym value of
-1
3
18 cm for the carboxylates in the IR spectrum is consistent with a chelated structure with
binding via the imidazole-N(3) and one carboxylate-O atom as in 1.
Unlike the reaction with RuCl ∙3H O with Hpic, reaction with Im-CO H does not form a
3
2
2
III
bimetallic complex. A reflux reaction in EtOH yields a product of formulation Ru Cl (Im-
2
CO )(Im-CO H)
•
EtOH H O (4) that satisfies the EA; one possible isomer consistent with the
•
2
2
2
2
data is shown below in (a). The IR data show two types of carboxylates with asym = 1718, 1706,
-1
II
and 1570 cm . The reported complex Ru (PPh ) (L-H) , where L-H = imidazole-4,5-
3
2
2
2
dicarboxylic acid, has two coordinated carboxylates and two non-coordinated carboxylic acid
-1
groups [see (b)] with respective asym values of 1627 and 1720 cm [19]. This suggests that for 4,
-1
-
the stretches at 1570 and 1718/1706 cm are for the bound CO and CO H groups,
2
2
-1
respectively; a strong 3467 cm band likely results from the H O solvate. ESI-MS data show a
2
+
parent M peak at 508 (Fig S4) with the same isotope splitting pattern as a calculated spectrum
(Fig. S2). In MeOH, the conductivity of 3, consistent with a neutral species, remained unchanged
1
over 3h. The H NMR spectrum in CD OD shows three signals for both the H(2) and H(5)
3

protons of the Im-CO H and ImCO ligands, consistent with structure (a), where the two Im-
2
2
CO H ligands are in different environments; resonances for the free solvate EtOH are also seen.
2
H
N
H
N
CO H
2
N Cl
Ru
N
O
N
HO C
O
2
CO H
N
O
2
O
Ru
O
Cl
CO H
O
PPh3
PPh3
N
2
(
b)
(
a)
N
H
8

ACCEPTED MANUSCRIPT
To our knowledge, there are no previous reports on Ru complexes containing Im-CO H or
2

ImCO . Indeed, complexes with the imidazole-4-carboxylate ligand are surprisingly rare; one
2
type is M(CO) (H O)(Im-CO ), where M = Re, Tc [20]. There is a RuH(CO)(MeIm-CO )(PPh )
3 2
3
2
2
2
complex, but this contains an N-methylated imidazole -2-carboxylate ligand [21].
HCANT was synthesized by modifying a reported Sandmeyer reaction
with the 3-amino precursor that was stated to be soluble in acetic acid
NO2
4
N
O
3
5
N
[
12]. However, this is incorrect, and we used a higher H SO :MeCO H
2 4 2
2
HO
NH
HCANT 1
ratio to solubilize the amino-triazole; addition of the diazonium salt
solution to the NaNO solution needs to be dropwise with rapid stirring and gentle heating to
2
prevent foam erupting from the flask. The isolated solid was well characterized. Attempts to
form Ru complexes from HCANT via methods analogous to those described with Hpic and
ImCO H in were unsuccessful. Reaction of K [RuCl ] with 3 equiv. of HCANT in H O gave a
2
3
6
2
purple solution from which a purple solid was isolated, the MS showing a parent peak consistent
III
with the formulation Ru Cl (CANT)(HCANT) analogous to 4. There are IR bands at 1677,
2
2
-1
-1
-1
1
665, 1652 cm (
of C=O), and 1542 cm (NO2), but an intense 1912 cm band may be due
asym
to a coordinated CO or NO (see below); also, for the above formulation, EA data were variable
but always low in C (e.g. 11.5 vs. 16.74) and N (21.6 vs. 26.05), and high in H (3.0 vs. 0.78)
suggesting the presence of considerable water (consistent with an observed, intense broad IR
-1
band at ~3400 cm ). A purple solid was also isolated with use of Ru Cl(-O CMe) as a
2
2
4
-1
precursor. The IR data are very similar to those noted above, except that the 1912 cm band is
even more intense. ESI-MS (negative, MeOH) showed a single peak at 444 with a Ru isotope
-1
splitting factor consistent with the formulation Ru(CANT) (NO), and the 1912 cm band is in
2
II
the range for a Ru (NO) species [22]; a variable EA was again always low in C and N, and high
in H for this formulation. Further studies are needed to characterize these purple solids.
Synthesis of 2-nitroimidazole-4-carboxylic acid was attempted using intermediates 58.
Treatment of Im-CO H with MeOH in conc. H SO generated the methyl ester (5), the procedure
2
2
4
CH OH
2
CO Me
CO Me
CH OH
2
CO H
2
2
2
4
5
N
N-CPh3
N
NH
NO2
N-CPh3
N
N
NH
N
NH
3
1
2
7
8
Im-CO H
5
6
2
9

ACCEPTED MANUSCRIPT
following that reported for an analogous ethyl ester [9]. Then the trityl group was used to form 6
with a protected imidazole-N(1) position, as this group is easily removed later with dilute acid,
as shown in reported syntheses of 4,4'-biimidazole derivatives [23], and used by us previously
[
2a]. The protected ester 6 was then reduced at r.t. using Dibal-H to give 4-hydroxymethyl-1-
tritylimidazole (7) in 44% yield (an 86% yield was reported for LiAlH reduction of the ethyl
4
ester [9], but attempts to use this for the methyl ester were unsuccessful); 7 was also made by
treatment of 4-(hydroxymethyl)imidazole with trityl chloride in 85% yield. Nitration of 7 using
n
PrNO , on the planned route to the 2-nitro derivative (8), gave only a 3% yield, despite a
3
reported 29% [9]. We found that the same nitrate procedure to make 2-nitroimidazole from 1-
tritylimidazole gave a 39% yield (vs. the reported 35-50% [9]). The low yield of 4-
hydroxymethyl-2-nitroimidazole prevented attempts at oxidation to the 4-carboxylic acid
derivative.
In imidazole-4,5-dicarboxylic acid, there is only the C(2) position available for substitution in
n
the ring, and direct nitration using PrNO , HNO or HNO /H SO seemed plausible; that
3
3
3
2
4
nitration of 4-nitroimidazole to form 2,4-dintroimidazole has been reported [24] shows that
nitration at C(2) is possible. However, neither imidazole-4,5-dicarboxylic acid nor its dimethyl
n
ester was reactive. Treatment of 4,5-bis(hydroxymethyl)-1-tritylimidazole with PrNO also gave
3
no nitrated products.
IC values (+10%) against MDA-MB-435S cells for complexes 2 and 4 are 100 and 400 M,
5
0
respectively, with the values for the free associated ligands Hpic and Im-CO H being 950 and
2
4
000 M; an example of a cell viability vs. concentration plot is shown in Fig. S5 (for complex
2
). The IC values for some Ru-imidazole/biimidazole complexes (and cisplatin) are an order of
5
0
magnitude lower [2a, 4d]. Thus, the introduction of carboxylic acid groups into the ligand N-
heterocyclic rings would appear from limited data to decrease antiproliferatory activity of these
types of Ru complexes.
Conclusions
III
A mixed-valence, dinuclear Ru complex, and two Ru complexes containing N-heterocyclic
2
carboxylate ligands, have been synthesized, and tested via an MTT assay for antiproliferatory
activity in vitro against a human breast cancer cell line. Limited findings, compared with
literature data, suggest that inclusion of a carboxylic acid group into an imidazole ligand
1
0

ACCEPTED MANUSCRIPT
decreases such activity. Efforts to make nitro-substituted derivatives of carboxylated imidazoles,
and their Ru complexes, of potential interest as radiosensitizers, are also described.
Acknowledgements
We thank Drs. E. Polishchuk and M. Ezhova of this department for help, respectively, with
the MTT assay and NMR analysis, and the Natural Sciences and Engineering Research Council
of Canada (NSERC) for financial support.
Supplementary material
1
Supplementary data for this article (ESI-MS for 2 and 4, H NMR spectrum for 2, and cell
viability vs. [2] for IC measurment) can be found online at …………….
5
0
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1
3

ACCEPTED MANUSCRIPT
One of:
Ru2Cl( -O2CMe)4
RuIII(ImCO2)3
RuCl3 3H2O
+ L
RuIIICl2(ImCO2)(ImCO2H)*
Ru2II/IIICl(pic)4(EtOH)*
K3[RuCl6]
HO C
2
N
* * IC50 values measured
against breast cancer cells
CO H
L =
2
N
N
ImCO H
H
Hpic
2
Graphical abstract
1
4

ACCEPTED MANUSCRIPT
Highlights


Ru complexes containing carboxylated imidazole ligands
Antiproliferatory activity (IC values)
50
1
5