Organometallic 3-(1 H -benzimidazol-2-yl)-1 H -pyrazolo[3,4- b ]pyridines as potential anticancer agents

Article abstract of DOI:10.1021/ic201704u

Six organometallic complexes of the general formula [M^IICl(η ⁶-p-cymene)(L)]Cl, where M = Ru (11a, 12a, 13a) or Os (11b, 12b, 13b) and L = 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1-L3) have been synthesized. The latter are known as potential cyclin-dependent kinase (Cdk) inhibitors. All compounds have been comprehensively characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, UV-vis spectroscopy, ESI mass spectrometry, and X-ray crystallography (11b and 12b). The multistep synthesis of 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1-L3), which was reported by other researchers, has been modified by us essentially (e.g., the synthesis of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3) via 5-bromo-3-methyl-1H-pyrazolo[3,4-b]pyridine (2); the synthesis of 1-methoxymethyl-2,3-diaminobenzene (5) by avoiding the use of unstable 2,3-diaminobenzyl alcohol; and the activation of 1H-pyrazolo[3,4-b]pyridine-3- carboxylic acids (1, 3) through the use of an inexpensive coupling reagent, N,N′-carbonyldiimidazole (CDI)). Stabilization of the 7b tautomer of methoxymethyl-substituted L3 by coordination to a metal(II) center, as well as the NMR spectroscopic characterization of two tautomers 7b-L3 and 4b′-L3 in a metal-free state are described. Structure-activity relationships with regard to cytotoxicity and cell cycle effects in human cancer cells, as well as Cdk inhibitory activity, are also reported.

Full text of DOI:10.1021/ic201704u

ARTICLE
pubs.acs.org/IC
Organometallic 3-(1H-Benzimidazol-2-yl)-1H-pyrazolo-
[3,4-b]pyridines as Potential Anticancer Agents
Iryna N. Stepanenko, Maria S. Novak, Gerhard M€uhlgassner, Alexander Roller, Michaela Hejl,
Vladimir B. Arion,* Michael A. Jakupec, and Bernhard K. Keppler*
University of Vienna, Institute of Inorganic Chemistry, W€ahringer Strasse 42, A-1090 Vienna, Austria
S Supporting Information
b
ABSTRACT: Six organometallic complexes of the general formula
[MIICl(η6-p-cymene)(L)]Cl, where M = Ru (11a, 12a, 13a) or Os
(11b, 12b, 13b) and L = 3-(1H-benzimidazol-2-yl)-1H-pyrazolo-
[3,4-b]pyridines (L1ꢀL3) have been synthesized. The latter are
known as potential cyclin-dependent kinase (Cdk) inhibitors.
All compounds have been comprehensively characterized by
elemental analysis, one- and two-dimensional NMR spectroscopy,
UVꢀvis spectroscopy, ESI mass spectrometry, and X-ray crystal-
lography (11b and 12b). The multistep synthesis of 3-(1H-benzi-
midazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1ꢀL3), which was
reported by other researchers, has been modified by us essentially
(e.g., the synthesis of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3) via 5-bromo-3-methyl-1H-pyrazolo[3,4-
b]pyridine (2); the synthesis of 1-methoxymethyl-2,3-diaminobenzene (5) by avoiding the use of unstable 2,3-diaminobenzyl
alcohol; and the activation of 1H-pyrazolo[3,4-b]pyridine-3-carboxylic acids (1, 3) through the use of an inexpensive coupling
reagent, N,N0-carbonyldiimidazole (CDI)). Stabilization of the 7b tautomer of methoxymethyl-substituted L3 by coordination to a
metal(II) center, as well as the NMR spectroscopic characterization of two tautomers 7b-L3 and 4b0-L3 in a metal-free state are
described. Structureꢀactivity relationships with regard to cytotoxicity and cell cycle effects in human cancer cells, as well as Cdk
inhibitory activity, are also reported.
’ INTRODUCTION
Cdk1 inhibitory activity with the inhibiting activity in four other
protein kinases (VEGF-R2, HER2, Aurora-A, and RET) revealed
selectivity for Cdk1. Structural modifications consisting of a
replacement of both bicyclic rings in 3-(1H-benzimidazol-2-yl)-
1H-pyrazolo[3,4-b]pyridines by monocycles while retaining the
imidazolyl pyrazole core have been proposed in order to obtain
inhibitors with improved pharmacokinetic and solubility pro-
perties.24ꢀ26 The most promising were suggested to be 3-(1H-
benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (see Chart 1).
The inspection of substitution patterns on the benzimidazole
moiety and structureꢀactivity relationships revealed that a meth-
oxymethyl group in position 7b (4b0) is favorable for Cdk1
inhibiting potency. The role of the pyrazole NH group is also of
note, since its methylation led to a significant reduction of Cdk1
activity. The effect of various heteroaryl groups in position 5a was
also remarkable for the development of more-effective Cdk
inhibitors and antiproliferative agents.
Tumor-associated cell cycle defects, manifesting in unscheduled
proliferation, and the associated genomic and chromosomal
instabilities are mediated by misregulation of cyclin-dependent
kinases (Cdks).1 Because of the main role in the division cycle,
Cdks have been recognized as targets for anticancer therapy.
Many small-molecule organic compounds, which have been identi-
fied as Cdk modulators, are currently in preclinical or clinical
development.1ꢀ5 However, no Cdk inhibitors have gained marketing
approval, despite 20 years of scientific investigation.1
Several studies have shown synergism when Cdk inhibitors
were combined with organic (e.g., doxorubicin, paclitaxel)2,6
and inorganic (e.g., cisplatin, carboplatin) cytotoxic drugs.7ꢀ9
The reported effects inspired the design of metal complexes
with biologically active ligands. The first publications have
appeared recently and include Fe, Cu, and Pt complexes with
Cdk inhibitors derived from 6-benzylaminopurine,10ꢀ13 metal-
based indolo-[3,2-d]benzazepines (paullones; Ga, Cu, Ru, and
Os),14ꢀ19 and indolo-[3,2-c]quinolines (Ru, Os).20
Our previousexperiencewithmetal-basedindolo-[3,2-d]benza-
zepines prompted the use of the half-sandwich metal-arene moiety
as a suitable scaffold to which 3-(1H-benzimidazol-2-yl)-1H-
pyrazolo[3,4-b]pyridines may be attached. Organometallic com-
pounds [M(η6-arene)(YZ)X]n (where M = Ru, Os) exhibit
Another class of compounds potentially suitable for targeted
metal-based chemotherapy is that of 3-(1H-benzimidazol-2-yl)-
1H-pyrazolo[3,4-b]pyridines. These have been documented re-
cently as potent Cdk1 inhibitors with antiproliferative activity in
HeLa (cervical carcinoma), HCT116 (colon carcinoma), and
A375 (melanoma) human cancer cell lines.21ꢀ23 Comparison of
Received: August 5, 2011
Published: October 27, 2011
r
2011 American Chemical Society
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Chart 1. Compounds Reported in This Work with Atom Numbering Schemes for NMR Signal Assignmenta
aUnderlined compounds have been characterized by X-ray diffraction (XRD).
promising anticancer activity and are the focus of attention for
several groups.27ꢀ32 These compounds have shown activity toward
classic (DNA) and nonclassic (e.g., Cdks) targets in anticancer
chemotherapy.
and the resulting mixture was stirred at room temperature for 2ꢀ2.5 h
and then at 110ꢀ115 °C for 2.5ꢀ3 h. Afterward, water (300ꢀ350 mL)
was added and the mixture was stirred at room temperature. The
formed light yellow precipitate was filtered off and dried in vacuo at
40ꢀ50 °C. Yield: 17 g. The raw product was used without further
purification in the next step. Purification by column chromatography
afforded a white powder (SiO2, EtOAc, Rf = 0.79; 12.5 g, 72.6% yield).
Mr (C7H6BrN3) = 212.05 g/mol. ESI-MS in MeOH (positive): m/z
213 [M+H]+, 235 [M+Na]+, 253 [M+K]+; (negative): m/z 211
Herein, we report (i) the modified synthetic approach to
3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (recall
Chart 1: L1, X = H, Y = H; L2, X = Br; Y = H; L3, X = Br;
Y = CH2OCH3); (ii) the synthesis and characterization of a new
family of organoruthenium(II) (11a, 12a, 13a) and osmium(II)
(11b, 12b, 13b) complexes of the general formula [MCl(η6-p-
cymene)L]Cl, where L = 3-(1H-benzimidazol-2-yl)-1H-pyrazolo-
[3,4-b]pyridines (L1ꢀL3) (Chart 1); (iii) stabilization of the 7b
tautomer of methoxymethyl-substituted L3 by metal coordination
as well as (iv) NMR spectroscopic characterization of two
tautomers 7b-L3 and 4b0-L3 in a metal-free state; and (v) cell
cycle effects, as well as the antiproliferative and Cdk inhibi-
tory activities of both metal-free ligands and organometallic
complexes.
1
[MꢀH]ꢀ. H NMR (500.32 MHz, MeOH-d4): δ 8.55 (d, 1H, J =
2.16 Hz, CH), 8.43 (d, 1H, J = 2.15 Hz, CH), 2.56 (s, 3H, CH3) ppm.
1H NMR (500.32 MHz, DMSO-d6): δ 13.42 (brs, 1H, NH), 8.54 (d,
1H, J = 2.19 Hz, CH), 8.53 (d, 1H, J = 2.18 Hz, CH), 2.49 (s, 3H, CH3)
ppm. Colorless crystals of 2 0.5H2O suitable for X-ray diffraction
3
(XRD) study were grown in EtOAc (see Figure S1 in the Supporting
Information).
5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3). To so-
dium hydroxide (9.54 g, 0.24 mol) in water (150 mL) was added the raw
product 2 (7.1 g, 0.03 mol). After a dropwise addition of KMnO4 (16.98 g,
0.11 mol) in water (300 mL) at 100 °C over 2 h, the reaction mixture was
further heated for 1 h. MnO2 was filtered off from the hot reaction
mixture and washed with hot water. The filtrates were combined, the
water was evaporated to ca. 400 mL, and the yellow solution was
acidified to pH ∼2, using concentrated HCl. The yellow precipitate was
filtered off and dried in vacuo at 60 °C. Yield: 2.28 g. The crude
hydrochloride of 3 was used without further purification in the next step.
Mr(C7H4BrN3O2) = 242.03 g/mol. ESI-MS in MeOH (positive): m/z
243 [M+H]+, 265 [M+Na]+, 287 [M+2NaꢀH]+; (negative): m/z 241
[MꢀH]ꢀ. 1H NMR (500.32 MHz, MeOH-d4): δ 8.69 (d, 1H, J = 2.22
Hz, CH), 8.68 (d, 1H, J = 2.22 Hz, CH) ppm. 1H NMR (500.32 MHz,
DMSO-d6): δ 14.65 (s, 1H, NH), 13.45 (brs, 1H, NH), 8.72 (d, 1H, J =
2.23 Hz, CH), 8.58 (d, 1H, J = 2.25 Hz, CH) ppm.
’ EXPERIMENTAL SECTION
Starting Materials. 3-Acetyl-2-chloropyridine and 3-methyl-1H-
pyrazolo[3,4-b]pyridine were prepared according to literature protocols.33ꢀ35
1H-Pyrazolo[3,4-b]pyridine-3-carboxylic acid (1) was obtained via the
oxidation of 3-methyl-1H-pyrazolo[3,4-b]pyridine by KMnO4 in the
presence of a base,35 followed by acidification with 37% HCl. 2-Amino-
3-nitrobenzyl alcohol was obtained as reported in the literature.36 1,
2-Diaminobenzene and dry dimethylformamide (DMF) were purchased
from Acros Organics. Solvents [toluene, ethanol (EtOH), tetrahydro-
furan (THF), diethyl ether (Et2O)] were dried using standard proce-
dures. [RuIICl(μ-Cl)(η6-p-cymene)]2 and [OsIICl(μ-Cl)(η6-p-cymene)]2
were synthesized as described previously.37,38
1-Methoxymethyl-2-amino-3-nitrobenzene (4). NaH (1.75 g, 0.07 mol)
was suspended in dry THF (200 mL). A solution of 2-amino-3-nitrobenzyl
alcohol (5.23 g, 0.03 mol) in dry THF (100 mL) was added dropwise at 0 °C
and the mixture was stirred at the same temperature for 15 min. After a
dropwise addition of MeI (11.5 g, 0.08 mol), stirring was continued at room
Synthesis of Ligands. 5-Bromo-3-methyl-1H-pyrazolo[3,4-b]-
pyridine (2). 3-Methyl-1H-pyrazolo[3,4-b]pyridine (10.9 g, 0.08 mol)
and anhydrous sodium acetate (10.21 g, 0.13 mol) were suspended in
glacial acetic acid (42 mL). Bromine (20.42 g, 0.13 mol) was added,
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ARTICLE
temperature for 3 h. A saturated aqueous solution of NaHCO3
(300 mL) and MeOH (300 mL) then were added. The mixture was
filtered, and the white precipitate was washed with EtOAc. The filtrates
were combined, and organic solvents were evaporated under reduced
pressure. The remaining aqueous solution was extracted with EtOAc
(2 ꢁ 300 mL). The organic phase was dried over Na2SO4, filtered, and
evaporated to yield a red oil (4.85 g). The raw product was purified by
column chromatography (SiO2, EtOAc, or EtOAc/hexane 1/1, first frac-
tion, a red-orange oil crystallized to form a red solid at 4 °C; yield: 3.68 g,
(dd, 1H, J = 1.2 Hz, J = 7.89 Hz, CHbz), 6.64 (td, 1H, J = 1.2 Hz, J =
7.67 Hz, CHbz), 4.93 (s, 2H, NH2) ppm.
N-(2-Aminophenyl)-5-bromo-1H-pyrazolo[3,4-b]pyridine-3-car-
boxamide (9). The solution of 1,2-diaminobenzene (0.96 g, 8.88
mmol) in dry DMF (10 mL) was added to the suspension of 7 (2.28 g,
7.8 mmol) in dry DMF (10 mL), and this mixture was heated under
argon at 85 °C for 7 h. DMF then was evaporated in vacuo at 50 °C and
water (20mL) was added. The yellow precipitate was filtered off and dried
in vacuo at 50 °C. Yield: ∼2.2 g. The raw product was used without
purification in the next step. Mr(C13H10BrN5O) = 332.16 g/mol.
ESI-MS in methanol (positive): m/z 333 [M+H]+, 355 [M+Na]+;
(negative): m/z 331 [MꢀH]ꢀ. 1H NMR (500.32 MHz, DMSO-d6):
δ 14.54 (brs, 1H, NHpz), 9.80 (brs, 1H, CONH), 8.73 (d, 1H, J = 2.26
Hz, CHpy), 8.69 (d, 1H, J = 2.22 Hz, CHpy), 7.34 (dd, 1H, J = 0.98 Hz,
J = 7.88 Hz, CHbz), 6.99 (td, 1H, J = 1.43 Hz, J = 8.03 Hz, CHbz), 6.82
(dd, 1H, J = 1.22 Hz, J = 7.96 Hz, CHbz), 6.64 (td, 1H, J = 1.19 Hz, J =
7.73 Hz, CHbz), 4.94 (s, 2H, NH2) ppm.
N-[2-Amino-3-(methoxymethyl)phenyl]-5-bromo-1H-pyrazolo-
[3,4-b]pyridine-3-carboxamide (10). A mixture of 5 (1.17 g, 7.69 mmol)
and 7 (2.1 g, 7.2 mmol) in dry DMF (45 mL) was heated under argon at
85 °C for 20 h. DMF then was evaporated in vacuo at 50 °C and water
(20 mL) was added. The brown precipitate formed was filtered off and
dried in vacuo at 50 °C. Yield: ∼2.3 g. The product was used without
further purification in the next step. Mr(C15H14BrN5O2) = 376.21 g/mol.
ESI-MS in methanol (positive): m/z 399 [M+Na]+; (negative): m/z 375
[MꢀH]ꢀ. 1H NMR (500.32 MHz, DMSO-d6): δ 14.54 (brs, 1H, NHpz),
9.85 (brs, 1H, CONH), 8.73 (d, 1H, J = 2.13 Hz, CHpy), 8.68 (d, 1H, J =
2.05 Hz, CHpy), 7.29 (d, 1H, J = 7.56 Hz, CHbz), 7.03 (d, 1H, J = 7.29 Hz,
CHbz), 6.65 (t, 1H, J = 7.79 Hz, CHbz), 4.78 (brs, 2H, NH2), 4.42 (s, 2H,
CH2), 3.29 (s, 3H, CH3) ppm.
1
65%). Mr (C8H10N2O3) = 182.18 g/mol. H NMR (500.32 MHz,
DMSO-d6): δ 8.00 (d, 1H, J = 8.71 Hz, C6H3), 7.48 (d, 1H, J = 7.1 Hz,
C6H3), 7.09 (br, 2H, NH2), 6.67 (t, 1H, J = 8.45 Hz, C6H3), 4.48
(s, 2H, CH2), 3.31 (s, 3H, CH3) ppm.
1-Methoxymethyl-2,3-diaminobenzene (5). A mixture of 4 (1.4 g,
0.008 mol) and 10% Pd/C (0.18 g) in dry EtOH (55 mL) was stirred
under hydrogen atmosphere at room temperature for 18ꢀ24 h. The
catalyst was removed by filtration through GF-3-filter under argon and
washed with dry EtOH (50ꢀ70 mL). The filtrate was evaporated in
vacuo to give a light-orange solid (1.17 g, 100% yield), which was used
immediately in the next step. Mr(C8H12N2O) = 152.19 g/mol. 1H NMR
(500.32 MHz, DMSO-d6): δ 6.52 (dd, 1H, J = 1.87 Hz, J = 7.25, C6H3),
6.42ꢀ6.36 (m, 2H, C6H3), 4.48 (br, 2H, NH2), 4.31 (br, 4H, NH2+CH2),
3.24 (s, 3H, CH3) ppm.
(1H-Imidazol-1-yl)(1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (6).
N,N0-Carbonyldiimidazole (CDI, 4.16 g, 0.026 mol) was added in small
portions to 1 (2.34 g) in dry DMF (12 mL). The mixture was stirred at
room temperature for 20ꢀ24 h. Water (5 mL) then was added and the
suspension was stirred until all CO2 was ceased. The white precipitate
was filtered off, washed with water (3ꢀ5 mL), and dried in a sublimator
in vacuo at 60 °C, to remove the imidazole as a contaminant. Yield: 1.13 g;
25ꢀ29%, based on 3-methyl-1H-pyrazolo[3,4-b]pyridine. Mr (C10H7N5O) =
213.19 g/mol. ESI-MS in methanol (positive): m/z 214 [M+H]+, 237
3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridine (11, L1). The
raw product 8 (0.86 g) was heated in a glacial acetic acid (10 mL) at
125 °C for 2.5 h. The solvent was evaporated under reduced pressure
and the residue was dried in vacuo at 50 °C, then resuspended in
CH2Cl2/MeOH (4/1, 25 mL), filtered off, and dried in vacuo to give L1
as a white powder (0.4 g). The filtrate was evaporated and the residue
was purified by column chromatography (SiO2, EtOAc, Rf = 0.58) to
give an additional amount of L1 (0.22 g). Yield: 0.62 g, ∼60% based on
6. Mr (C13H9N5) = 235.24 g/mol. Anal. Calcd for 11 0.15H2O 0.1
1
[M+Na]+; (negative): m/z 212 [MꢀH]ꢀ. H NMR (500.32 MHz,
DMSO-d6): δ 14.95 (brs, 1H, NH), 8.91 (s, 1H, CHim), 8.74 (dd, 1H, J =
1.61 Hz, J = 4.51 Hz, CHpy), 8.63 (dd, 1H, J = 1.59 Hz,
J = 8.12 Hz, CHpy), 8.14 (t, 1H, J = 1.23 Hz, CHim), 7.51 (dd, 1H, J =
4.46 Hz, J = 8.06 Hz, CHpy), 7.20 (s, 1H, CHim) ppm.
(5-Bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)(1H-imidazol-1-yl)methanone
(7). N,N0-Carbonyldiimidazole (CDI, 16.2 g, 0.1 mol) was added in small
portions to crude 3 (11.58 g) in dry DMF (70 mL). The mixture was
stirred at room temperature for 20ꢀ24 h. Water (10 mL) then was added
and the suspension was stirred until all CO2 was ceased. The white
precipitate was filtered off, washed with water (10ꢀ15 mL), and dried in
a sublimator in vacuo at 60 °C, to remove the imidazole as a contaminant.
Yield: 7.27 g, 13ꢀ16%, based on 3-methyl-1H-pyrazolo[3,4-b]-
pyridine. Mr(C10H6BrN5O) = 292.09 g/mol. ESI-MS in methanol
(positive): m/z 315 [M+Na]+; (negative): m/z 291 [MꢀH]ꢀ. 1H NMR
(500.32 MHz, DMSO-d6): δ 14.95 (brs, 1H, NH), 8.89 (s, 1H, CHim),
8.83 (d, 1H, J = 2.12 Hz, CHpy), 8.75 (d, 1H, J = 2.07 Hz, CHpy), 8.13
(s, 1H, CHim), 7.20 (s, 1H, CHim) ppm.
N-(2-Aminophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide
(8). The solution of 1,2-diaminobenzene (0.5 g, 4.67 mmol) in dry
DMF (2 mL) was added to the suspension of 6 (0.94 g, 4.41 mmol) in
dry DMF (13 mL), and this reaction mixture was heated under argon
at 85 °C for 5 h. DMF then was evaporated in vacuo at 50 °C and
water (10ꢀ12 mL) was added. The light-yellow precipitate was
filtered off and dried in vacuo at 50 °C. Yield: 0.86 g. The raw
product was used without purification in the next step. Mr-
(C13H11N5O) = 253.26 g/mol. ESI-MS in methanol (positive):
m/z 255 [M+H]+, 277 [M+Na]+; (negative): m/z 253 [MꢀH]ꢀ.
1H NMR (500.32 MHz, DMSO-d6): δ 14.30 (brs, 1H, NHpz), 9.73
(brs, 1H, CONH), 8.64 (dd, 1H, J = 1.59 Hz, J = 4.44 Hz, CHpy), 8.56
(dd, 1H, J = 1.53 Hz, J = 8.02 Hz, CHpy), 7.39ꢀ7.36 (m, 2H,
CHpy+CHbz), 6.97 (td, 1H, J = 1.31 Hz, J = 7.73 Hz, CHbz), 6.82
3
3
EtOAc (Mr = 246.76 g/mol): C, 65.22; H, 4.13; N, 28.38. Found: C,
65.56; H, 3.90; N, 28.39. ESI-MS in methanol (positive): m/z 237
[M+H]+, 259 [M+Na]+; (negative): m/z 235 [MꢀH]ꢀ. UVꢀvis
(methanol), λmax, nm (ε, Mꢀ1 cmꢀ1): 232 (25618), 275 (15198),
1
324 (22333). H NMR (500.32 MHz, DMSO-d6): δ 14.19 (brs, 1H,
H1a), 13.11 (brs, 1H, H1b), 8.85 (dd, 1H, J = 1.5 Hz, J = 8.1 Hz, H4a),
8.66 (dd, 1H, J = 1.5 Hz, J = 4.5 Hz, H6a), 7.75 (d, 1H, J = 7.3 Hz, H4b or
H7b), 7.54 (d, 1H, J = 7.7 Hz, H4b or H7b), 7.39 (dd, 1H, J = 4.5 Hz, J =
8.0 Hz, H5a), 7.24 (m, 2H, H5b+H6b) ppm. 13C NMR (125.81 MHz,
DMSO-d6): δ 153.13 (C8a), 150.22 (C6a), 146.99 (C2b), 144.34 (C8b or
C9b), 136.06 (C3a), 134.68 (C8b or C9b), 131.82 (C4a), 123.35 (C5b or
C6b), 122.11 (C5b or C6b), 119.44 (C4b or C7b), 118.65 (C5a), 113.33
(C9a), 112.01 (C4b or C7b) ppm. 15N NMR (50.70 MHz, DMSO-d6):
δ 166.2 (N1a), 121.3 (N1b) ppm.
3-(1H-Benzimidazol-2-yl)-5-bromo-1H-pyrazolo[3,4-b]pyridine (12, L2).
The raw product 9(2.2 g) was heated in a glacial acetic acid (30 mL) at 125 °C
for 2 h. The solvent was evaporated under reduced pressure, and the residue
was dried in vacuo at 50 °C, then resuspended in CH2Cl2/MeOH (7/1,
50 mL), filtered off and dried in vacuo to give L2 as a white powder (1.15 g).
The filtrate was evaporated and the residue was purified by column
chromatography (SiO2, EtOAc/hexane 2/1, Rf = 0.6) to give an additional
amount of the product (0.27 g). Yield: 1.42 g, ∼58% based on 7. Mr-
(C13H8BrN5) = 314.14 g/mol. Anal. Calcd for 12 0.25H2O 0.04EtOAc
3
3
(Mr = 322.17 g/mol): C, 49.06; H, 2.76; N, 21.74. Found: C, 49.44; H, 2.45;
N, 21.59. ESI-MS in methanol (positive): m/z 315 [M+H]+, 337 [M+Na]+;
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(negative): m/z 313 [MꢀH]ꢀ. UVꢀvis (methanol), λmax, nm
(ε, Mꢀ1 cmꢀ1): 234 (28154), 282 (17628), 335 (17198). 1H NMR
(500.32 MHz, DMSO-d6): δ 14.43 (brs, 1H, H1a), 13.19 (brs, 1H,
(ε, Mꢀ1 cmꢀ1): sh 245 (10728), 294 (18597), 360 (9666). 1H NMR
(500.32 MHz, DMSO-d6): δ 14.91 (brs, 1H, H1b), 9.17 (d, 1H, J = 7.7
Hz, H4a), 8.82 (d, 1H, J = 3.8 Hz, H6a), 8.11 (d, 1H, J = 7.1 Hz, H4b),
7.84 (d, 1H, J = 8.5 Hz, H7b), 7.58 (m, 3H, H5a+H5b+H6b), 6.43 (m,
2H, H2c+H6c), 6.31 (d, 1H, J = 5.3 Hz, H3c or H5c), 6.12 (d, 1H, J = 5.5
Hz, H3c or H5c), 2.54 (sep, 1H, H7c, under DMSO-d6 peak), 2.21 (s,
3H, H10c), 0.94 (d, 3H, J = 6.6 Hz, H8c or H9c), 0.91 (d, 3H, J = 6.6 Hz,
H8c or H9c) ppm. 13C NMR (125.81 MHz, DMSO-d6): δ 153.97
(C8a), 150.45 (C6a), 146.52 (C2b), 141.37 (C9b), 134.70 (C8b), 134.64
(C3a), 131.48 (C4a), 125.39 (C5b or C6b), 124.92 (C5b or C6b), 119.56
(C5a), 117.85 (C4b), 113.96 (C7b), 111.54 (C9a), 103.96 (C4c), 103.74
(C1c), 84.44 (C2c or C6c), 83.73 (C2c or C6c), 82.15 (C3c or C5c), 80.84
(C3c or C5c), 31.12 (C7c), 22.19 (C8c+C9c), 19.17 (C10c) ppm. 15N
NMR (50.70 MHz, DMSO-d6): δ 128.7 (N1b) ppm.
H
1b), 8.99 (d, 1H, J = 2.2 Hz, H4a), 8.75 (d, 1H, J = 2.3 Hz, H6a), 7.77
(d, 1H, J = 7.9 Hz, H4b or H7b), 7.54 (d, 1H, J = 7.9 Hz, H4b or H7b),
7.26 (m, 2H, H5b+H6b) ppm. 13C NMR (125.81 MHz, DMSO-d6): δ
151.49 (C8a), 150.66 (C6a), 146.42 (C2b), 144.24 (C8b or C9b),
135.61 (C3a), 134.67 (C8b or C9b), 133.35 (C4a), 123.54 (C5b or
C6b), 122.27 (C5b or C6b), 119.55 (C4b or C7b), 114.87 (C5a or C9a),
113.49 (C5a or C9a), 112.09 (C4b or C7b) ppm. 15N NMR (50.70
MHz, DMSO-d6): δ 167.5 (N1a), 121.3 (N1b) ppm.
5-Bromo-3-(4-methoxymethyl-1H-benzimidazol-2-yl)-1H-pyrazolo-
[3,4-b]pyridine (13, L3). The raw product 10 (2.3 g) was heated in a
glacial acetic acid (40 mL) at 125 °C for 2 h. The solvent was evaporated
under reduced pressure, and the residue dried in vacuo at 50 °C. After
washingwithCH2Cl2 (30 mL), CH2Cl2/MeOH (2/1, 5ꢀ7 mL) thegray
product was purified by column chromatography (SiO2, EtOAc, Rf =
0.68) to give a white powder (0.9 g). The filtrates were evaporated and
the remaining solid was purified by column chromatography to give an
additional amount of the product (0.3 g). Yield: 1.2 g, 47%, based on 7.
Mr(C15H12BrN5O) = 358.19 g/mol. Anal. Calcd for 13: C, 50.29; H,
3.38; N, 19.55. Found: C, 50.03; H, 3.19; N, 19.19. ESI-MS in methanol
(positive): m/z 381 [M+Na]+; (negative): m/z 357 [MꢀH]ꢀ. UVꢀvis
(methanol), λmax, nm (ε, Mꢀ1 cmꢀ1): 235 (29776), 282 (17544), 337
(16958).
(η6-p-Cymene){3-(1H-benzimidazol-kN-2-yl)-1H-pyrazolo-kN-
[3,4-b]pyridine}chloridoosmium(II) chloride, [OsIICl(η6-p-cymene)-
(L1)]Cl, (11b). A mixture of L1 (42.5 mg, 0.18 mmol) and [OsCl2-
(η6-p-cymene)]2 (70 mg, 0.09 mmol) in dry ethanol (25 mL) was stirred
at room temperature for 2 h. Ethanol then was removed under reduced
pressure up to ca. 3 mL and dry Et2O (40 mL) was added. The yellow
precipitate was filtered off and dried in vacuo at 50 °C. Yield: 93 mg, 81%
as 11b H2O. Mr(C23H23Cl2N5Os) = 630.59 g/mol. Anal. Calcd for
3
11b H2O (Mr = 648.61 g/mol): C, 42.59; H, 3.88; N, 10.79. Found: C,
3
42.56; H, 3.57; N, 10.97. ESI-MS in methanol (positive): m/z
[MꢀHClꢀCl]+, 596 [MꢀCl]+, 618 [MꢀHCl+Na]+; (negative): m/z
595 [MꢀHClꢀH]ꢀ. UVꢀvis (methanol), λmax, nm (ε, Mꢀ1 cmꢀ1): sh
252 (17048), 299 (20228), 343 (19879). 1H NMR (500.32 MHz,
MeOH-d4): δ 8.99 (dd, 1H, J = 1.3 Hz, J = 8.1 Hz, H4a), 8.77 (dd, 1H, J =
1.4 Hz, J = 4.8 Hz, H6a), 7.96 (dd, 1H, J = 2.0 Hz, J = 6.4 Hz, H4b), 7.80
(dd, 1H, J = 1.9 Hz, J = 6.1 Hz, H7b), 7.64ꢀ7.59 (m, 3H, H5a+H5b+H6b),
6.66 (d, 1H, J = 5.6 Hz, H2c or H6c), 6.59 (d, 1H, J = 5.7 Hz, H2c or H6c),
6.43 (d, 1H, J = 5.6 Hz, H3c or H5c), 6.21 (d, 1H, J = 5.7 Hz, H3c or H5c),
2.43 (sep, 1H, J = 6.9 Hz, H7c), 2.38 (s, 3H, H10c), 0.96 (d, 3H, J = 6.9
Hz, H8c or H9c), 0.92 (d, 3H, J = 6.9 Hz, H8c or H9c) ppm. 13C NMR
(125.81 MHz, MeOH-d4): δ 153.25 (C8a), 148.91 (C2b), 147.42 (C6a),
140.49 (C9b), 135.15 (C3a), 134.25 (C8b), 132.07 (C4a), 125.47 (C5b or
C6b), 124.81 (C5b or C6b), 118.48 (C5a), 116.89 (C4b), 112.98 (C7b),
112.92 (C9a), 97.71 (C4c), 94.88 (C1c), 76.44 (C2c or C6c), 74.99 (C2c or
C6c), 71.93 (C3c or C5c), 70.44 (C2c or C6c), 31.37 (C7c), 21.35 (C8c or
NMR characterization of 7b-L3 and 4b0-L3 tautomers (1/1.3) in
1
DMSO-d6: H NMR (500.32 MHz, DMSO-d6), 7b-L3: δ 14.47 (brs,
1H, H1a), 13.25 (brs, 1H, H1b), 8.99 or 8.98 (d+d, (1 + 1.3)H, J = 2.3 Hz,
0
0
H4a+H4a ), 8.75 (d, (1 + 1.3)H, J = 2.3 Hz, H6a+H6a ), 7.72 (dd, 1H, J =
0
1.8 Hz, J = 6.8 Hz, H4b), 7.28ꢀ7.21 (m, (2 + 2.6)H, H5a, H6a+H5a
,
1
0
H6a ), 4.80 (s, 2H, H10b), 3.37 (s, 3H, H11b) ppm. H NMR (500.32
MHz, DMSO-d6), 4b0-L3: δ 14.43 (brs, 1.3H, H1a ), 13.22 (brs, 1.3H,
0
0
0
H1b ), 8.99 or 8.98 (d+d, (1 + 1.3)H, J = 2.3 Hz, H4a+H4a ), 8.75 (d, (1 +
0
1.3)H, J = 2.3 Hz, H6a+H6a ), 7.47 (dd, 1.3H, J = 1.2 Hz, J = 7.7 Hz,
0
0
0
H7b ), 7.28ꢀ7.21 (m, (2 + 2.6)H, H5a, H6a+H5a , H6a ), 4.96 (s, 2.6H,
H10b ), 3.44 (s, 3.9H, H11b ) ppm. 13C NMR (125.81 MHz, DMSO-d6),
0
0
0
0
0
7b-L3: δ 151.51 (C8a+C8a ), 150.69 (C6a or C6a ), 150.65 (C6a or C6a ),
0
0
146.70 (C2b), 144.46 (C9b), 135.62 (C3a+C3a ), 133.45 (C4a or C4a ),
0
0
133.42 (C4a or C4a ), 133.29 (C8b), 123.38 (C6b; C5b or C6b ), 123.36
0
0
(C6b; C5b or C6b ), 122.95 (C7b), 122.16 (C5b or C6b ), 118.97 (C4b),
C9c), 21.09 (C8c or C9c), 17.84 (C10c) ppm. Crystals of 11b 4H2O
suitable for XRD study have been obtained from a solution of 11b in
ethanol.
3
0
0
0
0
115.04 (C5a or C9a or C5a or C9a ), 114.92 (C5a or C9a or C5a or C9a ),
113.49 (C5a or C9a or C5a or C9a ), 70.49 (C10b), 57.95 (C11b) ppm. 13
C
0
0
(η6-p-Cymene){3-(1H-benzimidazol-kN-2-yl)-5-bromo-1H-pyrazo-
lo-kN-[3,4-b]pyridine}chloridoruthenium(II) chloride, [RuIICl(η6-p-
cymene)(L2)]Cl, (12a). A mixture of L2 (73.2 mg, 0.23 mmol) and
[RuCl2(η6-p-cymene)]2 (70 mg, 0.11 mmol) in dry ethanol (25 mL)
was stirred at room temperature for 1 h. Ethanol then was removed
under reduced pressure up to ca. 2 mL and dry Et2O (40 mL) was added.
The yellow precipitate was filtered off and dried in vacuo at 40 °C. Yield:
NMR (125.81 MHz, DMSO-d6), 4b0-L3: δ 151.51 (C8a+C8a ), 150.69
0
0
0
0
0
(C6a or C6a ), 150.65 (C6a or C6a ), 146.13 (C2b ), 142.50 (C9b ), 135.62
(C3a+C3a ), 134.42 (C8b ), 133.45 (C4a or C4a ), 133.42 (C4a or C4a ),
0
0
0
0
0
0
0
129.38 (C4b ), 123.38 (C6b; C5b or C6b ), 123.36 (C6b; C5b or C6b ),
122.16 (C5b or C6b ), 120.81 (C5b ), 115.04 (C5a or C9a or C5a or C9a0),
0
0
0
114.92 (C5a or C9a or C5a or C9a ), 113.49 (C5a or C9a or C5a or C9a0),
0
0
0
111.17 (C7b ), 69.90 (C10b ), 58.35 (C11b ) ppm. 15N NMR (50.70 MHz,
0
0
0
0
0
DMSO-d6): δ 167.6 (N1a+N1a ), 121.4 (N1b+N1b ) ppm.
106 mg, 73% as 12a H2O. Mr(C23H22BrCl2N5Ru) = 620.33 g/mol.
3
Synthesis of Organometallic Complexes. (η6-p-Cymene){3-
(1H-benzimidazol-kN-2-yl)-1H-pyrazolo-kN-[3,4-b]pyridine}chlorid-
oruthenium(II) chloride, [RuIICl(η6-p-cymene)(L1)]Cl, (11a). A mix-
ture of L1 (54.7 mg, 0.23 mmol) and [RuCl2(η6-p-cymene)]2 (70 mg,
0.11 mmol) in dry ethanol (25 mL) was stirred at room temperature for
1 h. Ethanol then was evaporated up to ca. 3 mL and dry Et2O (40 mL)
was added. The yellow precipitate was filtered off and dried in vacuo at
Anal. Calcd for 12a H2O (Mr = 638.35 g/mol): C, 43.28; H, 3.79; N,
3
10.97. Found: C, 43.42; H, 3.54; N, 11.18. ESI-MS in methanol
(positive): m/z 548 [MꢀHClꢀCl]+, 608 [MꢀHCl+Na]+; (negative):
m/z 549 [Mꢀ2HClꢀH]ꢀ, 582 [MꢀHClꢀH]ꢀ. UVꢀvis (methanol),
λmax, nm (ε, Mꢀ1 cmꢀ1): sh 254 (17778), 303 (29953), 351 (17387). 1H
NMR (500.32 MHz, DMSO-d6): δ 14.34 (brs, 1H, H1b), 9.21 (s, 1H,
H4a), 8.73 (s, 1H, H6a), 8.06 (d, 1H, J = 7.6 Hz, H4b), 7.79 (d, 1H, J = 8.5
Hz, H7b), 7.52 (m, 2H, H5b+H6b), 6.32 (d, 2H, J = 5.8 Hz, H2c+H6c),
6.22 (d, 1H, J = 6.2 Hz, H3c or H5c), 6.03 (d, 1H, J = 6.1 Hz, H3c or H5c),
2.52 (sep, 1H, H7c, under DMSO-d6 peak), 2.18 (s, 3H, H10c), 0.94 (d,
50 °C. Yield: 90ꢀ95 mg, 70ꢀ74% as 11a H2O. Mr(C23H23Cl2N5Ru) =
3
541.44 g/mol. Anal. Calcd for 11a H2O (Mr = 559.45 g/mol): C, 49.38;
3
H, 4.50; N, 12.52; Cl, 12.67. Found: C, 49.68; H, 4.25; N, 12.11; Cl,
12.26. ESI-MS in methanol (positive): m/z 470 [MꢀHClꢀCl]+, 507
[MꢀCl]+, 528 [MꢀHCl+Na]+; (negative): m/z 468 [Mꢀ2HClꢀH]ꢀ,
505 [MꢀHClꢀH]ꢀ. UVꢀvis (methanol), λmax, nm (ε, Mꢀ1 cmꢀ1):
251 (16335), 299 (26663), sh 347 (16012). UVꢀvis (H2O), λmax, nm
3H, J = 6.8 Hz, H8c or H9c), 0.89 (d, 3H, J = 6.9 Hz, H8c or H9c) ppm. 13
C
NMR (125.81 MHz, DMSO-d6): δ 154.04 (C8a), 151.24 (C6a), 146.57
(C2b), 141.37 (C9b), 134.63 (C8b), 133.43 (C3a), 131.82 (C4a), 125.33
(C5b or C6b), 124.88 (C5b or C6b), 117.78 (C4b), 114.72 (C5a or C9a),
11718
dx.doi.org/10.1021/ic201704u |Inorg. Chem. 2011, 50, 11715–11728

Inorganic Chemistry
ARTICLE
113.85 (C7b), 112.48 (C5a or C9a), 103.96 (C4c), 103.74 (C1c), 84.44
(C2c or C6c), 83.69 (C2c or C6c), 82.16 (C3c or C5c), 80.76 (C3c or C5c),
31.09 (C7c), 22.20 (C8c or C9c), 22.17 (C8c or C9c), 19.16 (C10c) ppm.
15N NMR (50.70 MHz, DMSO-d6): δ 127.3 (N1b) ppm.
9.18 (d, 1H, J = 2.1 Hz), 8.48 (d, 1H, J = 2.2 Hz), 7.94 (d, 1H, J = 8.2 Hz),
7.45 (t, 1H, J = 7.8 Hz), 7.39 (d, 1H, J = 7.2 Hz), 6.16 (d, 1H, J = 6.0 Hz),
6.13 (d, 1H, J = 6.1 Hz), 6.07 (d, 1H, J = 5.9 Hz), 5.89 (d, 1H, J = 6.4 Hz),
4.85 (s, 2H), 4.04 (s, 3H), 2.14 (s, 3H), 0.93 (d, 3H, J = 6.8 Hz), 0.86 (d,
3H, J = 6.9 Hz) ppm.
(η6-p-Cymene){3-(1H-benzimidazol-kN-2-yl)-5-bromo-1H-pyrazolo-
kN-[3,4-b]pyridine}chloridoosmium(II) chloride, [OsIICl(η6-p-cymene)-
(L2)]Cl, (12b). A mixture of L2 (56.4 mg, 0.18 mmol) and [OsCl2(η6-p-
cymene)]2 (70 mg, 0.09 mmol) in dry ethanol (25 mL) was stirred at
room temperature for 2 h. Ethanol then was removed under reduced
pressure up to ca. 3 mL and dry Et2O (40 mL) was added. The yellow
precipitate was filtered off and dried in vacuo at 50 °C. Yield: 102 mg,
(η6-p-Cymene){5-bromo-3-(7-methoxymethyl-1H-benzimidazol-
kN-2-yl)-1H-pyrazolo-kN-[3,4-b]pyridine}chloridoosmium(II) chloride,
[OsIICl(η6-p-cymene)(L3)]Cl, (13b). A mixture of L3 (59 mg, 0.17
mmol) and [OsCl2(η6-p-cymene)]2 (60 mg, 0.08 mmol) in dry ethanol
(25 mL) was stirred at room temperature for 3 h. Ethanol was
evaporated up to ca. 3 mL and dry Et2O (40 mL) was added. The
yellow precipitate was filtered off and dried in vacuo at 50 °C. Yield: 80
mg, 70%. Mr(C25H26BrCl2N5OOs) = 753.55 g/mol. Anal. Calcd for
13b: C, 39.85; H, 3.48; N, 9.29. Found: C, 39.60; H, 3.32; N, 9.20. ESI-
MS in methanol (positive): m/z 682 [MꢀHClꢀCl]+, 704 [Mꢀ2HCl
+Na]+; (negative): m/z 680 [Mꢀ2HClꢀH]ꢀ, 716 [MꢀHClꢀH]ꢀ.
UVꢀvis (methanol), λmax, nm (ε, Mꢀ1 cmꢀ1): sh 256 (19825), 303
(24634), 357 (18850). 1H NMR (500.32 MHz, MeOH-d4): δ 9.33 (d,
1H, J = 2.1 Hz, H4a), 8.84 (d, 1H, J = 2.2 Hz, H6a), 7.91 (dd, 1H, J = 1.2
Hz, J = 7.8 Hz, H4b), 7.64ꢀ7.58 (m, 2H, H5b+H6b), 6.69 (d, 1H, J = 5.6
Hz, H2c or H6c), 6.62 (d, 1H, J = 5.7 Hz, H2c or H6c), 6.47 (d, 1H, J = 5.5
Hz, H3c or H5c), 6.21 (d, 1H, J = 5.6 Hz, H3c or H5c), 4.94 (q, 2H, J = 12.4
Hz, H10b), 3.49 (s, 3H, H11b), 2.41 (sep, 1H, J = 6.9 Hz, H7c), 2.39 (s,
3H, H10c), 0.94 (d, 3H, J = 6.9 Hz, H8c or H9c), 0.89 (d, 3H, J = 6.9 Hz,
H8c or H9c) ppm. 13C NMR (125.81 MHz, MeOH-d4): δ 153.18 (C8a),
152.16 (C6a), 148.36 (C2b), 140.82 (C9b), 134.35 (C3a), 132.68 (C8b),
131.68 (C4a), 125.63 (C5b or C6b), 124.85 (C5b or C6b), 124.51 (C7b),
116.72 (C4b), 115.24 (C5a or C9a), 112.26 (C5a or C9a), 98.75 (C4c),
95.50 (C1c), 75.98 (C2c or C6c), 75.34 (C2c or C6c), 71.82 (C3c or C5c),
70.29 (C10b), 70.04 (C3c or C5c), 57.17 (C11b), 31.39 (C7c), 21.34 (C8c
or C9c), 21.16 (C8c or C9c), 17.85 (C10c) ppm. The yellow crystals of
[OsIICl(η6-p-cymene)(L3)]Cl [OsIICl(η6-p-cymene)(L3ꢀH)] 0.75
79% as 12b H2O. Mr(C23H22BrCl2N5Os) = 709.49 g/mol. Anal.
3
Calcd for 12b H2O (Mr = 727.51 g/mol): C, 37.97; H, 3.33; N, 9.63.
3
Found: C, 37.82; H, 3.02; N, 9.33. ESI-MS in methanol (positive):
m/z 638 [MꢀHClꢀCl]+, 696 [MꢀHCl+Na]+; (negative): m/z 672
[MꢀHClꢀH]ꢀ. UVꢀvis (methanol), λmax, nm (ε, Mꢀ1 cmꢀ1): sh 253
(21638), 302 (30505), 357 (21813). 1H NMR (500.32 MHz, MeOH-
d4): δ 9.08 (d, 1H, J = 2.1 Hz, H4a), 8.84 (d, 1H, J = 2.1 Hz, H6a), 7.96
(dd, 1H, J = 2.1 Hz, J = 6 Hz, H4b), 7.80 (dd, 1H, J = 2.1 Hz, J = 6.1 Hz,
H7b), 7.63 (m, 2H, H5b+H6b), 6.69 (d, 1H, J = 5.7 Hz, H2c or H6c), 6.63
(d, 1H, J = 5.6 Hz, H2c or H6c), 6.48 (d, 1H, J = 5.6 Hz, H3c or H5c), 6.22
(d, 1H, J = 5.7 Hz, H3c or H5c), 2.42 (sep, 1H, J = 6.9 Hz, H7c), 2.39 (s,
3H, H10c), 0.95 (d, 3H, J = 6.9 Hz, H8c or H9c), 0.91 (d, 3H, J = 6.9 Hz,
H
8c or H9c) ppm. 13C NMR (125.81 MHz, MeOH-d4): δ 153.04 (C8a),
152.49 (C6a), 148.15 (C2b), 140.47 (C9b), 134.58 (C3a), 134.19 (C8b),
131.13 (C4a), 125.79 (C5b or C6b), 125.05 (C5b or C6b), 116.99 (C4b),
115.39 (C5a or C9a), 113.09 (C7b), 112.11 (C5a or C9a), 98.54 (C4c),
95.52 (C1c), 75.73 (C2c or C6c), 75.25 (C2c or C6c), 71.87 (C3c or C5c),
69.99 (C3c or C5c), 31.41 (C7c), 21.34 (C8c or C9c), 21.16 (C8c or C9c),
17.84 (C10c) ppm. Crystals of 12b and 12b 2CH3OH 2H2O suitable
3
3
for XRD study have been obtained from a solution of 12b in methanol.
(η6-p-Cymene){5-bromo-3-(7-methoxymethyl-1H-benzimidazol-
kN-2-yl)-1H-pyrazolo-kN-[3,4-b]pyridine}chloridoruthenium(II) chloride,
[RuIICl(η6-p-cymene)(L3)]Cl, (13a). A mixture of L3 (64 mg, 0.18
mmol) and [RuCl2(η6-p-cymene)]2 (50 mg, 0.08 mmol) in dry ethanol
(25 mL) was stirred at room temperature for 1 h. Ethanol then was
removed under reduced pressure up to ca. 3 mL and dry Et2O (40 mL)
was added. The yellow precipitate was filtered off and dried in vacuo at
3
3
CH3OH 0.25H2O (13e 0.75CH3OH 0.25H2O) suitable for XRD
3
3
3
study have been obtained from a methanolic solution of 13b.
Physical Measurements. Elemental analyses (C, H, N, Cl) were
performed by the Microanalytical Service of the Institute of Physical
Chemistry, University of Vienna. Electrospray ionization mass spectro-
metry (ESI-MS) was carried out with an Esquire 3000 instrument
(Bruker Daltonics, Bremen, Germany), using solutions of compounds in
methanol. The expected and measured isotope distributions were
compared. UVꢀvis spectra were recorded on a Lambda 20 UVꢀvis
spectrophotometer (PerkinꢀElmer), using samples dissolved in metha-
nol (L1ꢀL3, 11a, 12a, 13a, 11b, 12b, 13b) and water (11a) over 24
or 48 h, correspondingly. The one-dimensional (1H, 13C, 15N) and
two-dimensional spectra (15N,1H HSQC, 13C,1H HSQC, 13C,1H
50 °C. Yield: 95.8 mg, 85% as 13a 1.5H2O. Mr(C25H26BrCl2N5ORu) =
3
664.39 g/mol. Anal. Calcd for 13a 1.5H2O (Mr = 691.41 g/mol): C,
3
43.43; H, 4.23; N, 10.13; Cl, 10.26. Found: C, 43.81; H, 4.24; N, 10.11;
Cl, 10.57. ESI-MS in methanol (positive): m/z 592 [MꢀHClꢀCl]+,
650 [MꢀHCl+Na]+; (negative): m/z 591 [Mꢀ2HClꢀH]ꢀ, 628
[MꢀHClꢀH]ꢀ. UVꢀvis (methanol), λmax, nm (ε, Mꢀ1cmꢀ1): sh 252
(18744), 306 (28529), 354 (16889). 1H NMR (500.32 MHz, DMSO-
d6): δ 13.88 (brs, 1H, H1b), 9.41 (s, 1H, H4a), 8.68 (s, 1H, H6a), 7.99 (d,
1H, J = 7.7 Hz, H4b), 7.49 (m, 2H, H5b+H6b), 6.29 (m, 2H, H2c+H6c),
6.20 (d, 1H, J = 5.5 Hz, H3c or H5c), 6.00 (d, 1H, J = 5.9 Hz, H3c or H5c),
4.88 (s, 2H, H10b), 3.39 (s, 3H, H11b), 2.48 (sep, 1H, H7c, under DMSO-
d6 peak), 2.17 (s, 3H, H10c), 0.93 (d, 3H, J = 6.9 Hz, H8c or H9c), 0.87 (d,
3H, J = 6.8 Hz, H8c or H9c) ppm. 13C NMR (125.81 MHz, DMSO-d6): δ
155.35 (C8a), 150.27 (C6a), 147.43 (C2b), 141.64 (C9b), 133.07 (C8b),
132.68 (C3a), 131.68 (C4a), 124.87 (C5b or C6b), 124.59 (C5b or C6b),
124.53 (C7b), 117.18 (C4b), 114.23 (C5a or C9a), 112.78 (C5a or C9a),
103.74 (C4c), 103.09 (C1c), 85.38 (C2c or C6c), 83.68 (C2c or C6c),
82.13 (C3c or C5c), 81.05 (C3c or C5c), 70.19 (C10b), 58.03 (C11b), 31.04
1
1
1
HMBC, H,1H COSY, H,1H TOCSY, H,1H NOESY (L3, 12a),
1H,1H ROESY (L3, 11a, 12a, 13a, 13b)) were recorded with a Bruker
Model DPX500 (Ultrashield Magnet) system in DMSO-d6 (L1ꢀL3,
11a, 12a, 13a), MeOH-d4 (11b, 12b, 13b), D2O (1H NMR, 11a), and
D2O/DMSO-d6 (1H NMR, 12a), using standard pulse programs at
500.32 (1H), 125.81 (13C) and 50.70 (15N) MHz. 1H signals are
referenced relative to the solvent signals (DMSO-d6 at 2.51, MeOH-d4
at 3.33 ppm).
Crystallographic Structure Determination. XRD measure-
ments were performed on a Bruker Model X8 APEXII CCD diffract-
ometer. Single crystals were positioned at distances of 35, 40, 40, 40, 40,
40, and 35 mm from the detector, and 1556, 1131, 518, 1911, 1176,
1978, and 1039 frames were measured, each for 30, 60, 30, 20, 70, 60, and
30 s over 1° scan width for 2 0.5H2O, 11b 4H2O, 12b, 12b 2CH3OH
3
(C7c), 22.22 (C8c or C9c), 22.09 (C8c or C9c), 19.13 (C10c) ppm. 15
N
NMR (50.70 MHz, DMSO-d6): δ 122.9 (N1b) ppm. The red XRD-
quality crystals of [RuIICl(η6-p-cymene)(L3ꢀH)] (13c) were obtained
from EtOH/Et2O and an EtOH solution of 13a (long crystallization).
3
3
3
2H2O, 13c, 13d CH3OH, and 13e 0.75CH3OH 0.25H2O, corre-
3
3
3
spondingly. The data were processed using SAINT software.39 Crystal
data, data collection parameters, and structure refinement details are
given in Table 1. The structures were solved by direct methods and
refined by full-matrix least-squares techniques. Non-H atoms were
The yellow crystals of [RuIICl(η6-p-cymene)(L3)]Cl [RuIICl(η6-p-
3
cymene)(L3ꢀH)] CH3OH (13d CH3OH) suitable for XRD study
3
3
have been obtained from methanolic solution of 13a (short crystal-
lization). 1H NMR (13c, 500.32 MHz, DMSO-d6): δ 13.43 (s, 1H),
11719
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Inorganic Chemistry
ARTICLE
refined with anisotropic displacement parameters. H atoms were inserted
in calculated positions and refined with a riding model. Refinement of the
structure of 12b revealed that the chloride counteranion occupies two
statistically disordered positions with site occupation factor (sof) values of
0.57:0.43, whereas, in 13c, the methoxymethylene group was found to be
disordered over two positions with sof values of 0.80:0.20. Similarly, the
methoxymethylene group of one crystallographically independent com-
plex in 13d CH3OH or in both crystallographically independent com-
3
plexes in 13e 0.75CH3OH 0.25H2O is disordered with sof values of
3
3
0.35:0.65 and 0.60:0.40, 0.80:0.20, correspondingly. The disorder was
resolved with constrained anisotropic displacement parameters and
restrained bond distances using EADP and SADI instructions of
SHELX97, respectively. Structure solution was achieved with SHELXS-97
and refinement with SHELXL-97,40 and graphics were produced with
ORTEP-3.41
Cell Lines and Culture Conditions. A549 (non-small cell lung
carcinoma, human) and SW480 (colon carcinoma, human) cells were
kindly provided by Brigitte Marian (Institute of Cancer Research,
Department of Medicine I, Medical University of Vienna, Austria).
CH1 cells (ovarian cancer, human) were a gift from Lloyd R. Kelland
(CRC Centre for Cancer Therapeutics, Institute of Cancer Research,
Sutton, U.K.). All cell culture media and supplements were purchased
from SigmaꢀAldrich. Cells were grown in 75-cm2 culture flasks (Iwaki)
in a complete medium (i.e., Minimum Essential Medium supplemented
with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate,
4 mM L-glutamine, and 1% nonessential amino acids from 100ꢁ stock)
as adherent monolayer cultures. Cultures were grown at 37 °C under a
humidified atmosphere containing 5% CO2 and 95% air.
Inhibition of Cancer Cell Growth. Antiproliferative activity in
vitro was determined by the colorimetric MTT assay (MTT = 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Fluka). For
this purpose, cells were harvested from culture flasks by the use of
trypsin and seeded in complete medium (100 μL/well) into 96-well
plates (Iwaki) in densities of 4 ꢁ 103 (A549), 1.5 ꢁ 103 (CH1), and
2.5 ꢁ 103 (SW480) viable cells per well. Cells were allowed for 24 h to
settle and resume proliferation. Test compounds were dissolved in
DMSO first, appropriately diluted in complete medium, and instantly
added to the plates (100 μL/well), where the DMSO content did not
exceed 0.4% and 1% for the ligands and complexes, respectively. After
exposure for 96 h, the medium was replaced with 100 μL/well RPMI
1640 medium (supplemented with 10% heat-inactivated fetal bovine
serum and 4 mM L-glutamine) plus 20 μL/well MTT solution in
phosphate-buffered saline (5 mg/mL), followed by incubation for 4 h.
Subsequently, the medium/MTTmixture wasremoved, andthe formazan
product formed by viable cells was dissolved in DMSO (150 μL/well).
Optical densities were measured with a microplate reader (Tecan Spectra
Classic) at 550 nm (and a reference wavelength of 690 nm) to yield
relative quantities of viable cells as percentages of untreated controls, and
50% inhibitory concentrations (IC50) were interpolated from concentra-
tionꢀeffect curves. Calculations are based on at least three independent
experiments with triplicates for each concentration level.
Cell Cycle Analyses. To study the effects on the cell cycle of
exponentially growing CH1 cells by flow cytometric analysis of their
relative DNA content, cells were harvested from culture flasks, seeded in
complete medium into 90-mm Petri dishes (1 ꢁ 106 cells/dish) and,
after recovery for 24 h, exposed to various concentrations of the test com-
pounds for 24 h. For this purpose, test compounds were diluted from
DMSO stocks with complete medium (see above) such that the effective
DMSO content did not exceed 0.5%. After exposure, treated and control
cells were collected by scratching, washed with PBS, and stained with
5μg/mL propidium iodide overnight. Their fluorescence was measured with
a FACS Calibur instrument (Becton Dickinson), and the obtained histo-
grams were analyzed with Cell Quest Pro software (Becton Dickinson).
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Inorganic Chemistry
ARTICLE
Scheme 1. Synthesis of 5-Bromo-1H-pyrazolo[3,4-b]-
Scheme 2. Synthesis of 1-Methoxymethyl-2,3-diamino-
pyridine-3-carboxylic acid (3)a
benzene (5)a
a Reagents and conditions: (i) KMnO4, NaOH, 3 h, 100 °C, H2SO4;35
(ii) methanol, H2SO4, reflux, 6ꢀ8 h, NaHCO3, 42% (i + ii);35 (iii) Br2,
AcOH, AcONa, 115 °C, overnight, chromatographic purification,
43%;23 (iv) NaOH, MeOH, reflux, 4 h, HCl, 100%;23 (v) Br2, AcOH,
AcONa, 115 °C, 2.5ꢀ3 h; and (vi) KMnO4, NaOH, 3 h, 100 °C, HCl,
24ꢀ35% (v + vi).
a Reagents and conditions: (i) NaH, MeI, dry THF, 0 °C, 30 min, room
temperature, overnight, chromatographic purification, 34% yield;23 (ii)
NaH, MeI, dry THF, 0 °C, 15 min, room temperature, 3 h, chromato-
graphic purification, 65ꢀ75% yield; (iii) 10% Pd/C, ethanol, H2, room
temperature, 18ꢀ24 h, 100% yield.
At least two independent experiments were performed for each setting,
and 2.5 or 3.0 ꢁ 104 cells were measured per sample.
The overall yield of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-car-
boxylic acid (3) was 18%.
Kinase Assay. The Cdk-inhibitory capacities of test compounds
were studied by a radiochemical assay using recombinant Cdk1/cyclin B
and Cdk2/cyclin E isolated from Sf21 insect cells and histone H1 as the
substrate for phosphorylation, as described by Marko et al.42 Briefly,
MOPS-buffered assay mixtures containing the test compound (with a
maximum of 1% DMSO), the respective kinase/cyclin complex, histone
H1, and 0.4 μCi (γ-32P)ATP per sample were incubated for 10 min at
30 °C. Aliquots of the solution were spotted onto phosphocellulose
squares, which had been washed three times with 0.75% phosphoric acid,
followed by acetone. The dried squares were measured in scintillation
vials by β-counting (PerkinꢀElmer Tri-Carb 2800TR; Quanta Smart
software). Results were obtained in duplicate in at least two independent
experiments, and IC50 values were calculated by interpolation.
We performed the synthesis of 3 in two steps (see Scheme 1,
steps v and vi): bromination of 3-methyl-1H-pyrazolo[3,4-b]-
pyridine in AcOH/AcONa mixture and oxidation of crude
5-bromo-3-methyl-1H-pyrazolo[3,4-b]pyridine (2) by KMnO4
in a basic medium, followed by acidification with 37% HCl, with
an overall yield of 24ꢀ35%. 5-Bromo-3-methyl-1H-pyrazolo-
[3,4-b]pyridine (2) is a known compound, the synthesis of which
is well-documented.43ꢀ45
For the benzimidazole ring formation, 1,2-diaminobenzene
and 1-methoxymethyl-2,3-diaminobenzene have been used. The
reported synthesis of 1-methoxymethyl-2,3-diaminobenzene (5)
from 2,3-diaminobenzyl alcohol afforded the desired product in
34% yield (see Scheme 2, step i).23 However, the instability and
low yield of diamines, as well as the necessity to purify the desired
ether using column chromatography, stimulated the search for a
more-convenient procedure that was subsequently proposed:
etherification of 2-amino-3-nitrobenzyl alcohol, followed by the
reduction of 1-methoxymethyl-2-amino-3-nitrobenzene (4) with
10% Pd/C in ethanol under a hydrogen atmosphere afforded 5 in
65ꢀ75% yield (see Scheme 2, steps ii and iii).
’ RESULTS AND DISCUSSION
Synthesis of Ligands and Complexes. Several routes to 3-
(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines have been
proposed by Johnson & Johnson Pharmaceutical Research &
Development L.L.C. The first one was developed for 3-(1H-
benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines with an un-
substituted benzimidazole moiety and involved sulfur-induced
benzimidazole ring formation via the treatment of 5-bromo-1H-
pyrazolo[3,4-b]pyridine-3-carboxaldehyde with 1,2-diamino-
benzene.22 The poor reproducibility of this synthesis prompted the
exploration of an alternative way, via 5-bromo-1H-pyrazolo[3,4-
b]pyridine-3-carboxylic acid, which was used for the preparation of
3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines with a sub-
stituted benzimidazole moiety.
The patented route to 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-
carboxylic acid (3) consists of four steps (see Scheme 1, steps
iꢀiv): oxidation of 3-methyl-1H-pyrazolo[3,4-b]pyridine by
KMnO4 in the presence of a base with subsequent acidification
with H2SO4,35 esterification of 1H-pyrazolo[3,4-b]pyridine-3-car-
boxylic acid (1) in the presence of H2SO4 in methanol,35
bromination of 1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid
methyl ester in an AcOH/AcONa mixture,23 and hydrolysis of
5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl es-
ter in the presence of NaOH, followed by acidification with HCl.23
Patented benzimidazole ring formation by cyclization of the
3-carboxyl group of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-car-
boxylic acid (3) with substituted 1,2-diaminobenzenes was
realized via amide formation using coupling reagents, followed
by treatment with glacial acetic acid.21 HATU (N,N,N0,N0-
tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluoro-
phosphate) was used as a coupling reagent.21,23
Three 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines
(L1ꢀL3) (where X = H, Br; and Y = H, CH2OCH3) have been
synthesized in this work (see Chart 1): 3-(1H-benzimidazol-2-yl)-
1H-pyrazolo[3,4-b]pyridine (L1) is a new compound that we have
prepared as a model ligand for coordination to metals; 3-(1H-
benzimidazol-2-yl)-5-bromo-1H-pyrazolo[3,4-b]pyridine (L2) was
previously known as BOC-protected compound prepared via
5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxaldehyde;23 5-bro-
mo-3-(4-methoxymethyl-1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]-
pyridine (L3) was synthesized via 5-bromo-1H-pyrazolo[3,4-b]-
pyridine-3-carboxylic acid (3) and patented as a potential Cdk
inhibitor and antiproliferative agent.23
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Inorganic Chemistry
ARTICLE
Scheme 3. Synthesis of 3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines L1ꢀL3a
a Reagents and conditions: (i) CDI, dry DMF, room temperature, 20ꢀ24 h; (ii) 1,2-diaminobenzene or 5, dry DMF, 80ꢀ85 °C; 5ꢀ20 h; and (iii) glacial
AcOH, 120ꢀ125 °C, 2.5ꢀ4.5 h, chromatographic purification.
(11a, 11b, 12a, 12b, 13a, 13b) in quantitative yields. Crystal-
lization of [RuIICl(η6-p-cymene)(L3)]Cl (13a) in EtOH or
EtOH/Et2O resulted in XRD-quality crystals of [RuIICl(η6-p-
cymene)(L3ꢀH)] (13c), while the crystallization of 13a in methanol
afforded crystals of composition [RuIICl(η6-p-cymene)(L3)]Cl
3
[RuIICl(η6-p-cymene)(L3ꢀH)] CH3OH (13d CH3OH). The
3
3
osmium(II) analogue 13e 0.75CH3OH 0.25H2O was obtained
3
3
via the crystallization of 13b in methanol.
NMR Evidence of Ligand Coordination. The full assignment
of proton, carbon, and nitrogen resonances for L1ꢀL3, 11a, 11b,
12a, 12b, 13a, and 13b is quoted in Tables S1ꢀS3 in the
Supporting Information.
3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines with an
unsubstituted benzimidazole moiety (L1, L2) display one set of
signals. L1 is characterized by three doublets of doublets for H4a,
H5a, H6a (8.85, 7.39, 8.66 ppm, correspondingly) of pyrazolopyr-
idine moiety, two doublets for H4b, H7b (7.54 and 7.75 ppm), the
overlapped H5b, H6b proton signals in onemultiplet (7.24 ppm) for
a benzimidazole moiety and two singlet resonances for H1a and H1b
(for atom numbering, see Chart 1). The substitution of H5a by
bromine (L2) results in reduced multiplicity for the H4a and H6a
signals (two doublets at 8.99 and 8.75 ppm), whereas the benzi-
midazole moiety spectrum remains almost unchanged. Two sing-
lets were attributed to NH protons and related to pyrazolopyridine
(H1a, at 14.19 (L1), 14.43 (L2) ppm) and benzimidazole (H1b, at
13.11 (L1), 13.19 (L2) ppm) moieties.
NMR spectra for 11a, 11b, 12a, and 12b, where L1 and L2
coordinate as bidentate ligands via N2a and N3b with the
formation of a stable five-membered chelate cycle, show one
set of signals. There was no evidence for monodentate or
tridentate coordination of ligands with the formation of dinuclear
or polynuclear complexes.
Coordination of L1 and L2 to ruthenium(II) made it possible
to assign the two doublets to proton resonances of H4b and H7b
of the benzimidazole moiety. In the 1H, 1H ROESY plots, one of
them has cross-peaks with a CH cymene ring and the nearest to
them is H4b (e.g., at 8.11 (11a), 8.06 (12a) ppm). A singlet at
14.91 (11a), 14.34 (12a) ppm was assigned to NH proton and
showed no couplings with other atoms. The nitrogen resonance
shift at 128.7 (11a), 127.3 (12a) ppm is closer to the benzimi-
dazole NH chemical shift in metal-free ligands (121.3 (L1, L2)
ppm) and, therefore, was assigned as N1b (see Table S3 in the
Supporting Information). The H1b resonance shows a significant
shift by 1.8 and 1.15 ppm for 11a and 12a, respectively, upon
ligand coordination (L1 and L2) to the metal(II)-arene moiety.
Figure 1. Part of the 1H,1H ROESY plot of L3.
For the synthesis of L1ꢀL3, we used N,N0-carbonyldiimida-
zole (CDI) as an amide-coupling reagent, because it is relatively
inexpensive and the side products, carbon dioxide and imidazole,
could be easily removed from the reaction mixture.
CDI-mediated amidation of acids 1 and 3 was performed as
shown in Scheme 3 (steps i and ii). In the first step, the acyl-
imidazolides 6 and 7 were obtained in dry DMF at room
temperature and isolated as white solids. The yields based on
3-methyl-1H-pyrazolo[3,4-b]pyridine are: 25ꢀ29% (6) and
13ꢀ16% (7). In the second step, monoacylation of phenylene-
diamines (1,2-diaminobenzene and 1-methoxymethyl-2,3-diami-
nobenzene (5)) using acyl-imidazolides was performed in DMF
at 80ꢀ85 °C.
Amides 8ꢀ10 were used without further purification in ring
closure reactions in a glacial acetic acid at 120ꢀ125 °C and
afforded the desired 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]-
pyridines: L1 (55ꢀ60%), L2 (58ꢀ61%), and L3 (44ꢀ51%), based
on 6 and 7 (see Scheme 3, step iii). The reported synthesis of L3 is a
one-pot approach for amide formation using HATU with 57% yield,
followed by cyclization under acidic conditions (acetic acid) with
87% yield.
Thus, the ligands L1, L2, and L3 have been prepared in 7, 8,
and 11 steps, correspondingly.
Finally, the ligands L1ꢀL3 were reacted with [MIICl2(η6-p-
cymene)]2 (where M = Ru, Os) in a 2:1 molar ratio in dry ethanol
at room temperature to give [MCl(η6-arene)(L)]Cl complexes
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Inorganic Chemistry
ARTICLE
Scheme 4. Coordination of L3 (7b-L3 (left) and 4b0-L3
(right) Tautomers)
The nearest to the metal binding site pyrazolopyridine proton
H1a was not detected for 11a, 11b, 12a, and 12b, and the proton
resonance of H1b was also not seen for 11b or 12b.
L3 displays two sets of signals originated from 7b-L3 and 4b0-
L3 tautomers (see Chart 1). The signals of pyrazolopyridine
Figure 2. Fragment of the crystal structure of 11b 4H2O showing
nitrogen atoms N3 and N4, which act as proton donors in intermole-
3
cular hydrogen bonding interactions N3ꢀH Cl2 [N3 Cl2
3 3 3
3 3 3
0
moieties are partially overlapped (e.g., H1a (H1a ) at 14.47 and
3.067(7) Å, N3ꢀH Cl2 177.3°] and N4ꢀH O3 [N4 O3
3 3 3
3 3 3
3 3 3
0
0
14.43 ppm, H4a (H4a ) at 8.99 and 8.98 ppm, H6a (H6a ) at 8.75
ppm), whereas the signals of the benzimidazole moieties are
better resolved (see Table S1 in the Supporting Information).
According to the 1H,1H ROESY plot, one of the CH2 groups at
4.80 ppm couples with the NH proton at 13.25 ppm, indicating
their attribution to the 7b-L3 tautomer, whereas another NH
2.671(9) Å, N4ꢀH O3 170.9°]; thermal ellipsoids have been drawn
3 3 3
at 50% probability level. Selected bond lengths and angles: OsꢀCl1,
2.421(2) Å; OsꢀN1, 2.072(7) Å; OsꢀN5, 2.096(7) Å; OsꢀC(arene)av,
2.198(32) Å; C1ꢀN2, 1.349(11) Å; N2ꢀN1, 1.366(10) Å; N1ꢀC6,
1.357(11) Å; C6ꢀC7, 1.428(12) Å; C7ꢀN5, 1.345(11) Å; N5ꢀC13,
1.391(11) Å; N1ꢀOsꢀN5, 75.6(3)°; N1ꢀOsꢀCl1, 85.5(2)°;
N5ꢀOsꢀCl1, 83.4(2)°.
0
proton at 13.22 ppm gives a cross-peak with H7b at 7.47 ppm and
belongs to 4b0-L3 tautomer (Figure 1).
Tautomers 7b-L3 and 4b0-L3 are present in solution in 1:1.3
molar ratio and give, for the substituted benzimidazole moiety,
0
two singlets (H1b at 13.22 ppm, H1b at 13.25 ppm), two doublets
of doublets (H7b at 7.47 ppm, H4b at 7.72 ppm), one multiplet
0
0
0
(H5b, H6b, H5b , H6b at 7.28ꢀ7.21 ppm), two CH2 (4.80 (H10b),
0
4.96 (H10b ) ppm) and two CH3 singlets at 3.37 (H11b) and 3.44
0
0
(H11b ) ppm. The absence of cross-peaks between H7b and H4b
in the H,1H COSY plot supports their relation to the two
1
different molecules.
The binding site in 4b0-L3 tautomer is sterically shielded by a
methoxymethyl group. Consistent with this NMR spectra dis-
play, only one set of signals is shown for 13a and 13b with the
coordination of the 7b-L3 tautomer to ruthenium(II) and
osmium(II) via N2a and N3b (see Scheme 4).
The preference for coordination of the 7b-L3 tautomer is also
confirmed by 1H,1H ROESY plots: H4b (7.99 (13a), 7.91 (13b)
ppm) has couplings with CH protons of cymene ring. The cross-
peak between H10b at 4.88 ppm (13a) and NH at 13.88 ppm -
(13a) enabled the assignment of this singlet to a benzimidazole
moiety (H1b). In addition, the chemical shifts for the C atoms of
the benzimidazole moiety (C4b, C7b, C5b, C6b) of the coordi-
nated ligand and metal-free 7b-L3 tautomer are very similar (see
Table S2 in the Supporting Information). The nitrogen reso-
nance at 122.9 ppm (13a) compares well to the benzimidazole
NH chemical shift in metal-free L3 at 121.4 ppm. As for 11a, 11b,
12a, and 12b, the nearest to the coordination place pyrazolopyr-
idine proton H1a resonance was not detected.
Figure 3. ORTEP plot of the structure of the cation [OsIICl(η6-p-
cymene)(L2)]+ in 12b 2CH3OH 2H2O. Thermal ellipsoids have been
3
3
drawn at the 50% probability level. Selected bond lengths and angles:
OsꢀCl1, 2.4043(1) Å; OsꢀN1, 2.083(3) Å; OsꢀN5, 2.097(3) Å;
OsꢀC(arene)av, 2.197(26) Å; C1ꢀN2, 1.361(5) Å; N2ꢀN1,
1.344(4) Å; N1ꢀC6, 1.336(5) Å; C6ꢀC7, 1.447(6) Å; C7ꢀN5,
1.335(5) Å; N5ꢀC13, 1.380(5) Å; N1ꢀOsꢀN5, 74.73(13)°,
N1ꢀOsꢀCl1, 84.29(10)°; and N5ꢀOsꢀCl1, 83.25(10)°.
[RuIICl(η6-p-cymene)(L3)]Cl [RuIICl(η6-p-cymene)(L3ꢀH)]
1
According to the H,1H ROESY plots of 11a, 12a, 13a, and
3
3
3
CH3OH (13d CH3OH) and [OsIICl(η6-p-cymene)(L3)]Cl
13b only CH cymene ring protons have couplings with the
nearest H4b benzene ring proton, suggesting that the isopropyl or
methyl groups are further away from the H4b proton.
3
[OsIICl(η6-p-cymene)(L3ꢀH)] 0.75CH3OH 0.25H2O (13e 0.75-
3
3
3
CH3OH 0.25H2O) are shown in Figures 2, Figure S2 in the
3
Crystal Structures. The results of the XRD studies of [OsIICl(η6-
p-cymene)(L1)]Cl 4H2O (11b 4H2O), [OsIICl(η6-p-cymene)-
Supporting Information, and Figures 3ꢀ6, respectively. All complexes
have a typical “three-legged piano-stool” geometry of ruthenium(II)
and osmium(II) arene complexes, with an η6 π-bound p-cymene ring
forming the seat and three other donor atoms (two nitrogens N1 and
3
3
(L2)]Cl (12b), [OsIICl(η6-p-cymene)(L2)]Cl 2CH3OH 2H2O
3
3
(12b 2CH3OH 2H2O), [RuIICl(η6-p-cymene)(L3ꢀH)] (13c),
3
3
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Inorganic Chemistry
ARTICLE
Figure 5. Fragment of the crystal structure of [RuIICl(η6-p-cymene)-
(L3)]Cl [RuIICl(η6-p-cymene)(L3ꢀH)] CH3OH (13d CH3OH)
3
3
3
showing the intermolecular hydrogen bonding N2aꢀH N2b
3 3 3
3 3 3
[N2a N2bi (ꢀx + 1, ꢀy + 1, ꢀz + 1), 2.814(7) Å; N2aꢀH N2bi,
3 3 3
163.3°]. Selected bond lengths and angles: Ru1aꢀCl1a, 2.3952(17) Å;
Ru1aꢀN1a, 2.078(5) Å; Ru1aꢀN5a, 2.099(5) Å; Ru1aꢀC(arene)av,
2.194(33) Å; C1aꢀN2a, 1.358(8) Å; N2aꢀN1a, 1.354(7) Å; N1aꢀC6a,
1.350(8) Å; C6aꢀC7a, 1.441(9) Å; C7aꢀN5a, 1.335(8) Å; N5aꢀC13a,
1.381(8) Å; N1aꢀRu1aꢀN5a, 75.6(2)°; N1aꢀRu1aꢀCl1a, 84.45(15)°;
and N5aꢀRu1aꢀCl1a, 85.05(15)°.
Figure 4. ORTEP plot of the complex [RuIICl(η6-p-cymene)(L3ꢀH)]
(13c). Thermal ellipsoids have been drawn at 30% probability level.
Selected bond lengths and angles: RuꢀCl, 2.399(2) Å; RuꢀN1,
2.067(6) Å; RuꢀN5, 2.089(5) Å; RuꢀC(arene)av, 2.186(31) Å; C1ꢀ
N2, 1.366(8) Å; N2ꢀN1, 1.353(7) Å; N1ꢀC6, 1.362(7) Å; C6ꢀC7,
1.426(9) Å; C7ꢀN5, 1.335(8) Å; N5ꢀC13, 1.398(8) Å; N1ꢀRuꢀN5,
75.9(2)°; N1ꢀRuꢀCl, 87.35(17)°; and N5ꢀRuꢀCl, 85.73(17)°.
N5 of the corresponding 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-
b]pyridine and one chlorido ligand) as the legs of the stool. Selected
bond distances and angles are quoted in the figure captions. All
complexes crystallize as racemates, because of the presence of the
stereogenic metal center.
The bidentate 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]-
pyridines, which can have different substituents in positions 5a
and 7b, reveal different acidꢀbase properties. It can act as a
neutral organic ligand, with nitrogen atoms N2 and N4 as proton
donors involved in intermolecular hydrogen bonding interac-
tions N4ꢀH O1i(ꢀx + 1, ꢀy + 1, ꢀz + 2) [N4 O1i,
3 3 3
3 3 3
2.730(4) Å; N4ꢀH O1i, 176.1°] and N2ꢀH O3ii(ꢀx + 2,
3 3 3
3 3 3
3 3 3
3 3 3
ꢀy + 1, ꢀz + 1 [N2 O3ii, 2.697(4) Å; N4ꢀH O1i, 172.3°]
with one methanol and one water molecule, respectively, as is the
case for 12b 2CH3OH 2H2O (Figure 3) or N2ꢀH Cl2-
3
3
3 3 3
(Cl2X) and N4ꢀH Cl1i(ꢀx, ꢀy, ꢀz + 2) [N4 Cl1i,
3 3 3
3 3 3
3 3 3
3.213(9) Å; N4ꢀH O1i, 162.91°] in 12b (see Figure S2 in
the Supporting Information), correspondingly. The ligand can be
protonated at N3 (N3ꢀH Cl2) and deprotonated at N2
3 3 3
Figure 6. ORTEP plot of [OsIICl(η6-p-cymene)(L3ꢀH)] in [OsIICl-
(η6-p-cymene)(L3)]Cl [OsIICl(η6-p-cymene)(L3ꢀH)] 0.75CH3OH
[N2 HꢀO4i (ꢀx + 1, ꢀy + 1, ꢀz + 1) with N2 O4i
3 3 3
3 3 3
2.923(9) Å, N2 HꢀO4i 138.7°] (overall charge zero) with
3
3
3
3 3 3
0.25H2O (13e 0.75CH3OH 0.25H2O). Thermal ellipsoids have been
3
3
atom N4 as a proton donor to one of the four co-crystallized
drawn at the 40% probability level. Selected bond lengths and angles:
Os1aꢀCl1a, 2.402(2) Å; Os1aꢀN1a, 2.068(7) Å; Os1aꢀN5a, 2.086(7) Å;
Os1aꢀC(arene)av, 2.192(25) Å; C1aꢀN2a, 1.349(10) Å; N2aꢀN1a
1.369(9) Å; N1aꢀC6a, 1.361(10) Å; C6aꢀC7a, 1.431(11) Å; C7aꢀN5a,
1.339(10) Å; N5aꢀC13a, 1.398(11) Å; N1aꢀOs1aꢀN5a, 75.1(3)°;
N1aꢀOs1aꢀCl1a, 83.56(18)°; and N5aꢀOs1aꢀCl1a, 84.2(2)°.
water molecules N4ꢀH O3, as occurs in 11b 4H2O (see
3 3 3
3
Figure 2).
In 13c, the ligand was found to be deprotonated at N2, acting
as a proton acceptor in the intermolecular hydrogen bonding
interaction N2 HꢀN4i (i denotes symmetry transformations
3 3 3
x, ꢀy + 1.5, z + 0.5) used to generate equivalent atoms)
[N2 N4i, 2.896(7) Å; N2 HꢀN4i, 155.6°] (see Figure 4).
3 3 3
3 3 3
corroborated by the presence of hydrogen bonding of the type
Complexes 13d CH3OH and 13e 0.75CH3OH 0.25H2O
3
3
3
N2a HꢀN2bi (ꢀx + 1, ꢀy + 1, ꢀz + 1) in the crystal structure
crystallized both in the centrosymmetric triclinic space group
P1. The asymmetric unit in both consists of a neutral complex
[MIICl(η6-p-cymene)(L3ꢀH)] and a complex cation [MIICl(η6-
p-cymene)(L3)]+ (M = Ru or Os), a chloride counteranion and
co-crystallized solvent (methanol or methanol/water). Deproto-
nation of the organic ligand in [MIICl(η6-p-cymene)(L3ꢀH)] is
3 3 3
of the ruthenium complex 13d CH3OH (Figure 5) and a similar
3
interaction N2b HꢀN2ai (x, y + 1, z) [N2b N2ai, 2.810(9)
3 3 3
3 3 3
Å;N2b HꢀN2ai, 170.1°] in thecrystalof theosmium analogue
3 3 3
13e 0.75CH3OH 0.25H2O. In Figure 6 the structure of
3
3
[OsIICl(η6-p-cymene)(L3ꢀH)] is shown. In both structures,
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Inorganic Chemistry
ARTICLE
Table 2. Antiproliferative Activity of Metal-Free Ligands
(L1ꢀL3), and Their Ruthenium(II) (11aꢀ13a) and Osmium(II)
(11bꢀ13b) Arene Complexes, in Three Human Cancer Cell
Lines (CH1, SW480, and A549)a
IC50 [μM]b
compound
metal
CH1
SW480
A549
L1
11 ( 3
96 ( 18
64 ( 19
1.5 ( 0.6
21 ( 3
23 ( 6
>320
29 ( 7
525 ( 102
>640
11a
11b
L2
Ru
Os
223 ( 29
5.1 ( 1.0
70 ( 8
6.7 ( 0.3
268 ( 35
123 ( 21
5.2 ( 0.5
68 ( 12
89 ( 11
12a
12b
L3
Ru
Os
22 ( 3
29 ( 2
0.63 ( 0.09
11 ( 1
0.74 ( 0.26
11 ( 2
13a
Ru
Os
13b
7.9 ( 2.2
12 ( 2
a CH1 denotes ovarian cancer, human; SW480 denotes colon carcino-
ma, human; and A549 denotes non-small-cell lung carcinoma, human.
b 50% inhibitory concentrations (mean value ( standard deviation from
at least three independent experiments) obtained by the MTT assay
(96-h exposure).
the atoms N4a and N4b act as proton donors in strong hydrogen
bonding interactions to the chloride counteranion, namely, N4a
3 3 3
HꢀCl2ii (ꢀx + 1, ꢀy + 2, ꢀz + 1) [N4 Cl2ii, 3.275(6) Å;
3 3 3
N4a HꢀCl2ii, 176.9°], N4b HꢀCl2 [N4b Cl2, 3.211(5)
3 3 3
3 3 3
3
3 3 3
Å; N4b HꢀCl2, 176.0°] (13d CH3OH) and N4a HꢀCl2ii
3 3 3
3 3 3
(ꢀx + 1, ꢀy + 1, ꢀz + 1) [N4a Cl2ii, 3.273(8) Å; N4a
3 3 3
3 3 3
HꢀCl2ii, 177.0°], N4b HꢀCl2ii [N4b Cl2, 3.204(7) Å;
3 3 3
3 3 3
Figure 7. Concentrationꢀeffect curves of each ligand, compared to the
corresponding ruthenium and osmium complexes, in CH1 ovarian
cancer cells (MTT assay, 96 h exposure): (A) L1, 11a, 11b; (B) L2,
12a, 12b; and (C) L3, 13a, 13b. Both the presence of substituents in the
ligands and complexation result in a marked shift of antiproliferative
activity.
N4b HꢀCl2, 177.0°] (13e 0.75CH3OH 0.25H2O).
3 3 3
3
3
Antiproliferative Activity. Ligands L1ꢀL3, as well as the
corresponding ruthenium(II) (11aꢀ13a) and osmium(II)
(11bꢀ13b) arene complexes, were studied with regard to their
capacity of inhibiting cell growth in vitro in the human cancer cell
lines CH1 (ovarian carcinoma), SW480 (colon carcinoma), and
A549 (non-small cell lung cancer), yielding the IC50 values listed
in Table 2. All compounds show the strongest effect in the
generally chemosensitive CH1 cells, whereas the more chemore-
sistant A549 cells are also less affected by the compounds
investigated here. The antiproliferative activity of the metal-free
ligands decreases in the rank order L3 > L2 > L1, indicating that
bromination (L2, L3) and, even more so, the double substitution
(bromination and an additional replacement of H by a methox-
ymethyl group (L3)) are advantageous, with regard to activity.
These structureꢀactivity relationships are also reflected in the
rank orders of the corresponding ruthenium and osmium com-
plexes for the ruthenium complexes, 13a > 12a > 11a, and for the
osmium complexes, 13b > 12b > 11b.
However, the IC50 values are shifted to higher concentrations
(see Figure 7). The differences between the ruthenium and
osmium analogues are mostly small (IC50 values differ only up to
a factor of 2.4), with the osmium complexes being at least as
active as their ruthenium counterparts.
Cell Cycle Effects. Since 3-(1H-benzimidazol-2-yl)-1H-pyrazolo-
[3,4-b]pyridines have been reported to be potent Cdk inhibitors, we
expected the investigated ligands and complexes to induce cell cycle
perturbations. To confirm this assumption in a sensitive cell line,
exponentially growing CH1 cells were treated with the compounds
for 24 h, stained with propidium iodide, and analyzed for their DNA
content by fluorescence-activated cell sorting (FACS). Surprisingly,
the metal-free ligands L1ꢀL3 turned out to exert only modest
effects on cell cycle distribution, with a slight increase of the G2/M
fraction from 32% in the untreated control to 53% by 20 μM L2 as
the strongest effect observed in this setting (higher concentrations
of this compound led to disintegration of cells already after 24 h).
However, the less cytotoxic ruthenium complexes 12a and 13a, both
bearing substituted ligands (L2 and L3 correspondingly), cause a
more pronounced G2/M phase arrest, as reflected by an increase of
this cell fraction to 65% at 80 μM and 59% at 40 μM, respectively,
and a concomitant decrease of the G0/G1 fraction to 21% and 24%
(compared to 42% in controls), whereas ruthenium complex 11a
bearing an unsubstituted ligand L1 is devoid of activity on the cell
cycle. On the other hand, the osmium complexes do not generally
show stronger effects on the cell cycle than the metal-free ligands,
perhaps with the exception of 13b, which induces an increase of the
G2/M fraction up to 53% at 80 μM, accompanied by a decline of
the G0/G1 fraction to 31% (see Figure 8).
Cdk-Inhibitory Activity. Although the lack of generally
pronounced cell cycle effects does not argue for a strong role
of Cdk inhibition in the mechanism of action of the investigated
compounds, inhibitory potencies were studied in cell-free experi-
ments with two recombinant Cdk/cyclin complexes, by means of
the histone H1 kinase assay. Results reveal that all compounds
are capable of inhibiting kinase activities in a concentration-
dependent manner, being more effective on Cdk2/cyclin E than
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Inorganic Chemistry
ARTICLE
Figure 8. Concentration-dependent effects of metal-free ligands (top), and the corresponding ruthenium (middle) and osmium complexes (bottom),
on the cell cycle distribution of CH1 cells (( 2) G0/G1, (O) S, and (9) G2/M phase). Note the different concentration scales.
on Cdk1/cyclin B (see Figure 9). In contrast to the observed cell
cycle effects, but in accordance with antiproliferative activities,
Cdk-inhibitory potency of the metal-free ligands is consistently
higher than that of the metal complexes. In particular, only
L1ꢀL3 effectively inhibit Cdk2/cyclin E in low concentrations
(1 μM or 10 μM), whereas all complexes require higher
concentrations to exert >50% inhibitory effects. As in the
MTT assay, differences between the effects of corresponding
ruthenium and osmium complexes are minor, compared to the
differences from those of the metal-free ligands.
’ CONCLUSION
The known multistep synthesis of 3-(1H-benzimidazol-2-yl)-
1H-pyrazolo[3,4-b]pyridines, which we have modified, essen-
tially afforded three organic compounds (L1ꢀL3) that possess
Cdk-inhibiting properties. These were used as bidentate ligands,
and six novel organometallic complexes of the general formula
[MIICl(η6-p-cymene)(L)]Cl, where M = Ru (11a, 12a, 13a) or
Os (11b, 12b, 13b) and L = L1ꢀL3, have been synthesized and
comprehensively characterized using spectroscopic and X-ray
diffraction methods. Complexation of L1ꢀL3 with ruthenium or
osmium yielded compounds with increased solubility in the
biological medium, yet lowered antiproliferative activity in hu-
man cancer cell lines. Modulation of biological activities by
substitution at the ligands can be accomplished in the metal-free
molecules and their metal complexes in a similar way. The known
Cdk-inhibitory activity of the ligands could be confirmed in cell-
Figure 9. Concentration-dependent effects of metal-free ligands (L1ꢀL3)
as well as corresponding ruthenium (11aꢀ13a) and osmium complexes
(11bꢀ13b), on kinase activities of recombinant Cdk1/cyclin B (top) and
Cdk2/cyclin E (bottom).
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Inorganic Chemistry
ARTICLE
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stronger effects on Cdk activity parallel their higher capacity of
inhibiting cancer cell growth in vitro, compared to their metal
complexes. Nevertheless, the lack of pronounced effects on the
cell cycle of chemosensitive ovarian cancer cells argues against a
major role for inhibition of cell growth, at least in this setting.
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’ ASSOCIATED CONTENT
Supporting Information. Assigned NMR (1H, 13C, 15N)
S
b
signals for L1ꢀL3, 11aꢀ13a, 11bꢀ13b (Tables S1ꢀS3); OR-
TEP plot of 5-bromo-3-methyl-1H-pyrazolo[3,4-b]pyridine in
2 0.5H2O (Figure S1); ORTEP plot of [OsIICl(η6-p-cymene)-
3
(L2)]+ in 12b (Figure S2); stability of complexes in solution;
time-dependent UVꢀvis spectra of L1, 11a, and 11b in MeOH
(Figure S3); time-dependent UVꢀvis spectra of L3, 13a, and
13b in MeOH (Figure S4); time-dependent UVꢀvis spectra of
11a in H2O for 48 h (Figure S5); crystallographic data for
2 0.5H2O, 11b 4H2O, 12b, 12b 2CH3OH 2H2O, 13c, 13d
3
3
3
3
3
CH3OH, and 13e 0.75CH3OH 0.25H2O (in CIF format). This
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: vladimir.arion@univie.ac.at (V.B.A.); bernhard.
keppler@univie.ac.at (B.K.K.).
(24) Lin, R.; Chiu, G.; Yu, Y.; Connolly, P. J.; Li, S.; Lu, Y.; Adams,
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’ ACKNOWLEDGMENT
We thank Prof. Markus Galanski for the two-dimensional
(2D) NMR measurements. We are indebted to Prof. Verena
Dirsch and Daniel Schachner (Institute of Pharmacognosy,
University of Vienna, Austria) for providing FACS equipment
and technical assistance and to Prof. Georg Schmetterer
(Institute of Physical Chemistry, University of Vienna, Austria)
for providing radiochemical facilities. The research was funded
by the Austrian Science Fund (FWF): T 393-N19.
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