ProChimia/HS(CH2)11EGnOCH2CONH2/TH 009n60.5/500 mg

Inorg. Chem.All Publications/WebsiteOR SEARCH CITATIONS Recently ViewedYou have not visited any articles yet, Please visit some articles to see contents here. Received5 August 2011Published online27 October 2011Published inissue 21 November 2011https://doi.org/10.1021/ic201704uCopyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoiceArticle Views1565Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Get e-AlertsAbstractHigh Resolution ImageDownload MS PowerPoint SlideSix 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 (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.SynopsisThree organic compounds L1−L3, known as potential Cdk inhibitors, and six novel complexes of the general formula [MIICl(η6-p-cymene)(L)]Cl, where M = Ru (11a, 12a, 13a) or Os (11b, 12b, 13b) and L = L1−L3, correspondingly, have been synthesized and comprehensively characterized by elemental analysis, ESI mass-spectrometry, spectroscopic and X-ray diffraction methods. Structure−activity relationships, with regard to cytotoxicity and cell cycle effects, in human cancer cells, as well as Cdk inhibitory activity, are reported.IntroductionARTICLE SECTIONSJump ToTumor-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 identified as Cdk modulators, are currently in preclinical or clinical development.(1-5) 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.(7-9) 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,(10-13) metal-based indolo-[3,2-d]benzazepines (paullones; Ga, Cu, Ru, and Os),(14-19) and indolo-[3,2-c]quinolines (Ru, Os).(20)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 recently as potent Cdk1 inhibitors with antiproliferative activity in HeLa (cervical carcinoma), HCT116 (colon carcinoma), and A375 (melanoma) human cancer cell lines.(21-23) Comparison of 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 properties.(24-26) 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 structure–activity relationships revealed that a methoxymethyl group in position 7b (4b′) 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.Chart 1Chart 1. Compounds Reported in This Work with Atom Numbering Schemes for NMR Signal AssignmentaHigh Resolution ImageDownload MS PowerPoint SlideChart aUnderlined compounds have been characterized by X-ray diffraction (XRD).Our previous experience with metal-based indolo-[3,2-d]benzazepines 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 compounds [M(η6-arene)(YZ)X]n (where M = Ru, Os) exhibit promising anticancer activity and are the focus of attention for several groups.(27-32) These compounds have shown activity toward classic (DNA) and nonclassic (e.g., Cdks) targets in anticancer chemotherapy.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 (L1–L3) (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 4b′-L3 in a metal-free state; and (v) cell cycle effects, as well as the antiproliferative and Cdk inhibitory activities of both metal-free ligands and organometallic complexes.Experimental SectionARTICLE SECTIONSJump To Starting Materials3-Acetyl-2-chloropyridine and 3-methyl-1H-pyrazolo[3,4-b]pyridine were prepared according to literature protocols.(33-35) 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), tetrahydrofuran (THF), diethyl ether (Et2O)] were dried using standard procedures. [RuIICl(μ-Cl)(η6-p-cymene)]2 and [OsIICl(μ-Cl)(η6-p-cymene)]2 were synthesized as described previously.(37, 38) 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, and the resulting mixture was stirred at room temperature for 2–2.5 h and then at 110–115 °C for 2.5–3 h. Afterward, water (300–350 mL) was added and the mixture was stirred at room temperature. The formed light yellow precipitate was filtered off and dried in vacuo at 40–50 °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 [M–H]−. 1H 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 (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 sodium 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+2Na–H]+; (negative): m/z 241 [M–H]−. 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. 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 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 fraction, a red-orange oil crystallized to form a red solid at 4 °C; yield: 3.68 g, 65%). Mr (C8H10N2O3) = 182.18 g/mol. 1H 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 18–24 h. The catalyst was removed by filtration through GF-3-filter under argon and washed with dry EtOH (50–70 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), 6.42–6.36 (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,N′-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 20–24 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 (3–5 mL), and dried in a sublimator in vacuo at 60 °C, to remove the imidazole as a contaminant. Yield: 1.13 g; 25–29%, 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 [M+Na]+; (negative): m/z 212 [M–H]−. 1H 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,N′-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 20–24 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 (10–15 mL), and dried in a sublimator in vacuo at 60 °C, to remove the imidazole as a contaminant. Yield: 7.27 g, 13–16%, 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 [M–H]−. 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 (10–12 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 [M–H]−. 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), 7.39–7.36 (m, 2H, CHpy+CHbz), 6.97 (td, 1H, J = 1.31 Hz, J = 7.73 Hz, CHbz), 6.82 (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-carboxamide (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 (20 mL) 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 [M–H]−. 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 [M–H]−. 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. 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·0.1EtOAc (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 [M–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 232 (25618), 275 (15198), 324 (22333). 1H 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 (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]+; (negative): m/z 313 [M–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 234 (28154), 282 (17628), 335 (17198). 1H NMR (500.32 MHz, DMSO-d6): δ 14.43 (brs, 1H, H1a), 13.19 (brs, 1H, H1b), 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 washing with CH2Cl2 (30 mL), CH2Cl2/MeOH (2/1, 5–7 mL) the gray 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 [M–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 235 (29776), 282 (17544), 337 (16958).NMR characterization of 7b-L3 and 4b′-L3 tautomers (1/1.3) in DMSO-d6: 1H 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, H4a+H4a′), 8.75 (d, (1 + 1.3)H, J = 2.3 Hz, H6a+H6a′), 7.72 (dd, 1H, J = 1.8 Hz, J = 6.8 Hz, H4b), 7.28–7.21 (m, (2 + 2.6)H, H5a, H6a+H5a′, H6a′), 4.80 (s, 2H, H10b), 3.37 (s, 3H, H11b) ppm. 1H NMR (500.32 MHz, DMSO-d6), 4b′-L3: δ 14.43 (brs, 1.3H, H1a′), 13.22 (brs, 1.3H, H1b′), 8.99 or 8.98 (d+d, (1 + 1.3)H, J = 2.3 Hz, H4a+H4a′), 8.75 (d, (1 + 1.3)H, J = 2.3 Hz, H6a+H6a′), 7.47 (dd, 1.3H, J = 1.2 Hz, J = 7.7 Hz, H7b′), 7.28–7.21 (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), 7b-L3: δ 151.51 (C8a+C8a′), 150.69 (C6a or C6a′), 150.65 (C6a or C6a′), 146.70 (C2b), 144.46 (C9b), 135.62 (C3a+C3a′), 133.45 (C4a or C4a′), 133.42 (C4a or C4a′), 133.29 (C8b), 123.38 (C6b; C5b or C6b′), 123.36 (C6b; C5b or C6b′), 122.95 (C7b), 122.16 (C5b or C6b′), 118.97 (C4b), 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. 13C NMR (125.81 MHz, DMSO-d6), 4b′-L3: δ 151.51 (C8a+C8a′), 150.69 (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′), 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 C9a′), 114.92 (C5a or C9a or C5a′ or C9a′), 113.49 (C5a or C9a or C5a′ or C9a′), 111.17 (C7b′), 69.90 (C10b′), 58.35 (C11b′) ppm. 15N NMR (50.70 MHz, DMSO-d6): δ 167.6 (N1a+N1a′), 121.4 (N1b+N1b′) ppm. Synthesis of Organometallic Complexes (η6-p-Cymene){3-(1H-benzimidazol-κN-2-yl)-1H-pyrazolo-κN-[3,4-b]pyridine}chloridoruthenium(II) chloride, [RuIICl(η6-p-cymene)(L1)]Cl, (11a)A mixture 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 50 °C. Yield: 90–95 mg, 70–74% as 11a·H2O. Mr(C23H23Cl2N5Ru) = 541.44 g/mol. Anal. Calcd for 11a·H2O (Mr = 559.45 g/mol): C, 49.38; 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 [M–HCl–Cl]+, 507 [M–Cl]+, 528 [M–HCl+Na]+; (negative): m/z 468 [M–2HCl–H]−, 505 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 251 (16335), 299 (26663), sh 347 (16012). UV–vis (H2O), λmax, nm (ε, M–1 cm–1): 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. (η6-p-Cymene){3-(1H-benzimidazol-κN-2-yl)-1H-pyrazolo-κN-[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 11b·H2O (Mr = 648.61 g/mol): C, 42.59; H, 3.88; N, 10.79. Found: C, 42.56; H, 3.57; N, 10.97. ESI-MS in methanol (positive): m/z [M–HCl–Cl]+, 596 [M–Cl]+, 618 [M–HCl+Na]+; (negative): m/z 595 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 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), 7.64–7.59 (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 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. (η6-p-Cymene){3-(1H-benzimidazol-κN-2-yl)-5-bromo-1H-pyrazolo-κN-[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: 106 mg, 73% as 12a·H2O. Mr(C23H22BrCl2N5Ru) = 620.33 g/mol. Anal. Calcd for 12a·H2O (Mr = 638.35 g/mol): C, 43.28; H, 3.79; N, 10.97. Found: C, 43.42; H, 3.54; N, 11.18. ESI-MS in methanol (positive): m/z 548 [M–HCl–Cl]+, 608 [M–HCl+Na]+; (negative): m/z 549 [M–2HCl–H]−, 582 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 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, 3H, J = 6.8 Hz, H8c or H9c), 0.89 (d, 3H, J = 6.9 Hz, H8c or H9c) ppm. 13C 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), 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. (η6-p-Cymene){3-(1H-benzimidazol-κN-2-yl)-5-bromo-1H-pyrazolo-κN-[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, 79% as 12b·H2O. Mr(C23H22BrCl2N5Os) = 709.49 g/mol. Anal. Calcd for 12b·H2O (Mr = 727.51 g/mol): C, 37.97; H, 3.33; N, 9.63. Found: C, 37.82; H, 3.02; N, 9.33. ESI-MS in methanol (positive): m/z 638 [M–HCl–Cl]+, 696 [M–HCl+Na]+; (negative): m/z 672 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 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, H8c 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 for XRD study have been obtained from a solution of 12b in methanol. (η6-p-Cymene){5-bromo-3-(7-methoxymethyl-1H-benzimidazol-κN-2-yl)-1H-pyrazolo-κN-[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 50 °C. Yield: 95.8 mg, 85% as 13a·1.5H2O. Mr(C25H26BrCl2N5ORu) = 664.39 g/mol. Anal. Calcd for 13a·1.5H2O (Mr = 691.41 g/mol): C, 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 [M–HCl–Cl]+, 650 [M–HCl+Na]+; (negative): m/z 591 [M–2HCl–H]−, 628 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1cm–1): 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 (C7c), 22.22 (C8c or C9c), 22.09 (C8c or C9c), 19.13 (C10c) ppm. 15N NMR (50.70 MHz, DMSO-d6): δ 122.9 (N1b) ppm. The red XRD-quality crystals of [RuIICl(η6-p-cymene)(L3–H)] (13c) were obtained from EtOH/Et2O and an EtOH solution of 13a (long crystallization). The yellow crystals of [RuIICl(η6-p-cymene)(L3)]Cl·[RuIICl(η6-p-cymene)(L3–H)]·CH3OH (13d·CH3OH) suitable for XRD study have been obtained from methanolic solution of 13a (short crystallization). 1H NMR (13c, 500.32 MHz, DMSO-d6): δ 13.43 (s, 1H), 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){5-bromo-3-(7-methoxymethyl-1H-benzimidazol-κN-2-yl)-1H-pyrazolo-κN-[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 [M–HCl–Cl]+, 704 [M–2HCl+Na]+; (negative): m/z 680 [M–2HCl–H]−, 716 [M–HCl–H]−. UV–vis (methanol), λmax, nm (ε, M–1 cm–1): 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), 7.64–7.58 (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)(L3–H)]·0.75 CH3OH·0.25H2O (13e·0.75CH3OH·0.25H2O) suitable for XRD study have been obtained from a methanolic solution of 13b. Physical MeasurementsElemental analyses (C, H, N, Cl) were performed by the Microanalytical Service of the Institute of Physical Chemistry, University of Vienna. Electrospray ionization mass spectrometry (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. UV–vis spectra were recorded on a Lambda 20 UV–vis spectrophotometer (Perkin–Elmer), using samples dissolved in methanol (L1–L3, 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 HMBC, 1H,1H COSY, 1H,1H TOCSY, 1H,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 (L1–L3, 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 DeterminationXRD measurements were performed on a Bruker Model X8 APEXII CCD diffractometer. 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·2H2O, 13c, 13d·CH3OH, and 13e·0.75CH3OH·0.25H2O, correspondingly. 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 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 complex in 13d·CH3OH or in both crystallographically independent complexes in 13e·0.75CH3OH·0.25H2O is disordered with sof values of 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)Table 1. Crystal Data and Details of Data Collection for 2·0.5H2O, 11b·4H2O, 12b, 12b·2CH3OH·2H2O, 13c, 13d·CH3OH, and 13e·0.75CH3OH·0.25H2O 2·0.5H2O11b·4H2O12b12b·2CH3OH·2H2O13c13d·CH3OH13e·0.75CH3OH·0.25H2Oempirical formulaC7H7BrN3O0.5C23H31Cl2N5O4OsC23H22BrCl2N5OsC25H34BrCl2N5O4OsC25H25BrClN5ORuC51H55Br2Cl3N10O3Ru2C50.75H54.5Br2Cl3N10O3Os2formula wt, Fw221.06702.63709.47809.58627.931324.361499.11space groupP1̅P21/cP21/nP1̅P21/cP1̅P1̅a [Å]7.0878(3)19.2075(13)8.5092(10)8.4014(8)16.7948(13)9.9942(9)10.0042(5)b [Å]7.5561(3)7.7890(5)19.402(3)13.1678(14)12.4936(10)15.4370(13)15.3936(8)c [Å]16.5281(8)17.0807(11)14.497(3)13.7997(16)12.3028(8)17.8076(17)17.8050(9)α [°]98.267(3)  81.446(4) 104.380(5)104.233(2)β [°]97.046(3)99.334(4)91.531(5)81.191(5)108.744(5)93.139(5)93.029(2)γ [°]107.894(3)  76.887(5) 98.859(4)98.683(3)V [Å3]820.42(6)2521.6(3)2392.5(6)1458.9(3)2444.6(3)2617.1(4)2615.7(2)Z4442422λ [Å]0.710730.710730.710730.710730.710730.710730.71073ρcalcd[g cm–3]1.7901.8511.9701.8431.7061.6811.903crystal size [mm3]0.20 × 0.15 × 0.050.20 × 0.10 × 0.020.20 × 0.04 × 0.040.20 × 0.04 × 0.040.20 × 0.05 × 0.020.20 × 0.20 × 0.200.25 × 0.17 × 0.05temp, T [K]120(2)100(2)100(2)100(2)296(2)100(2)100(2)μ[mm–1]4.9545.3097.2455.9622.4142.3106.587R1a0.04340.05310.05600.03100.05470.05120.0442wR2b0.11810.10720.12210.07960.13550.14110.1055goodness of fit, GOFc1.0921.0110.9391.0870.9861.0481.054aR1 = ∑||Fo| – |Fc||/∑|Fo|.bwR2 = {∑[w(Fo2 – Fc2)2]/∑[w(Fo2)2]}1/2.cGOF = {∑[w(Fo2 – Fc2)2]/(n – p)}1/2, where n is the number of reflections and p is the total number of parameters refined. Cell Lines and Culture ConditionsA549 (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 Sigma–Aldrich. 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 100× 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 GrowthAntiproliferative 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/MTT mixture was removed, and the 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 concentration–effect curves. Calculations are based on at least three independent experiments with triplicates for each concentration level. Cell Cycle AnalysesTo 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 compounds 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 5 μg/mL propidium iodide overnight. Their fluorescence was measured with a FACS Calibur instrument (Becton Dickinson), and the obtained histograms were analyzed with Cell Quest Pro software (Becton Dickinson). At least two independent experiments were performed for each setting, and 2.5 or 3.0 × 104 cells were measured per sample. Kinase AssayThe 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 (Perkin–Elmer Tri-Carb 2800TR; Quanta Smart software). Results were obtained in duplicate in at least two independent experiments, and IC50 values were calculated by interpolation.Results and DiscussionARTICLE SECTIONSJump To Synthesis of Ligands and ComplexesSeveral 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 unsubstituted 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-diaminobenzene.(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 substituted 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 i–iv): 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-carboxylic 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 ester in the presence of NaOH, followed by acidification with HCl.(23) The overall yield of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3) was 18%.Scheme 1Scheme 1. Synthesis of 5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3)aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (i) KMnO4, NaOH, 3 h, 100 °C, H2SO4;(35) (ii) methanol, H2SO4, reflux, 6–8 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, 2.5–3 h; and (vi) KMnO4, NaOH, 3 h, 100 °C, HCl, 24–35% (v + vi).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 24–35%. 5-Bromo-3-methyl-1H-pyrazolo[3,4-b]pyridine (2) is a known compound, the synthesis of which is well-documented.(43-45)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 65–75% yield (see Scheme 2, steps ii and iii).Scheme 2Scheme 2. Synthesis of 1-Methoxymethyl-2,3-diaminobenzene (5)aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents 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, chromatographic purification, 65–75% yield; (iii) 10% Pd/C, ethanol, H2, room temperature, 18–24 h, 100% yield.Patented benzimidazole ring formation by cyclization of the 3-carboxyl group of 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic 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,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate) was used as a coupling reagent.(21, 23)Three 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (L1–L3) (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-bromo-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)For the synthesis of L1–L3, we used N,N′-carbonyldiimidazole (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: 25–29% (6) and 13–16% (7). In the second step, monoacylation of phenylenediamines (1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene (5)) using acyl-imidazolides was performed in DMF at 80–85 °C.Scheme 3Scheme 3. Synthesis of 3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines L1–L3aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (i) CDI, dry DMF, room temperature, 20–24 h; (ii) 1,2-diaminobenzene or 5, dry DMF, 80–85 °C; 5–20 h; and (iii) glacial AcOH, 120–125 °C, 2.5–4.5 h, chromatographic purification.Amides 8–10 were used without further purification in ring closure reactions in a glacial acetic acid at 120–125 °C and afforded the desired 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines: L1 (55–60%), L2 (58–61%), and L3 (44–51%), 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 L1–L3 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 (11a, 11b, 12a, 12b, 13a, 13b) in quantitative yields. Crystallization of [RuIICl(η6-p-cymene)(L3)]Cl (13a) in EtOH or EtOH/Et2O resulted in XRD-quality crystals of [RuIICl(η6-p-cymene)(L3–H)] (13c), while the crystallization of 13a in methanol afforded crystals of composition [RuIICl(η6-p-cymene)(L3)]Cl·[RuIICl(η6-p-cymene)(L3–H)]·CH3OH (13d·CH3OH). The osmium(II) analogue 13e·0.75CH3OH·0.25H2O was obtained via the crystallization of 13b in methanol. NMR Evidence of Ligand CoordinationThe full assignment of proton, carbon, and nitrogen resonances for L1–L3, 11a, 11b, 12a, 12b, 13a, and 13b is quoted in Tables S1–S3 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 pyrazolopyridine moiety, two doublets for H4b, H7b (7.54 and 7.75 ppm), the overlapped H5b, H6b proton signals in one multiplet (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 benzimidazole moiety spectrum remains almost unchanged. Two singlets 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 benzimidazole 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. 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 4b′-L3 tautomers (see Chart 1). The signals of pyrazolopyridine moieties are partially overlapped (e.g., H1a (H1a′) at 14.47 and 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 proton at 13.22 ppm gives a cross-peak with H7b′ at 7.47 ppm and belongs to 4b′-L3 tautomer (Figure 1).Figure 1Figure 1. Part of the 1H,1H ROESY plot of L3.High Resolution ImageDownload MS PowerPoint SlideTautomers 7b-L3 and 4b′-L3 are present in solution in 1:1.3 molar ratio and give, for the substituted benzimidazole moiety, 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 (H5b, H6b, H5b′, H6b′ at 7.28–7.21 ppm), two CH2 (4.80 (H10b), 4.96 (H10b′) ppm) and two CH3 singlets at 3.37 (H11b) and 3.44 (H11b′) ppm. The absence of cross-peaks between H7b′ and H4b in the 1H,1H COSY plot supports their relation to the two different molecules.The binding site in 4b′-L3 tautomer is sterically shielded by a methoxymethyl group. Consistent with this NMR spectra display, 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).Scheme 4Scheme 4. Coordination of L3 (7b-L3 (left) and 4b′-L3 (right) Tautomers)High Resolution ImageDownload MS PowerPoint SlideThe 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 coordinated ligand and metal-free 7b-L3 tautomer are very similar (see Table S2 in the Supporting Information). The nitrogen resonance 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 pyrazolopyridine proton H1a resonance was not detected.According to the 1H,1H ROESY plots of 11a, 12a, 13a, and 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. Crystal StructuresThe results of the XRD studies of [OsIICl(η6-p-cymene)(L1)]Cl·4H2O (11b·4H2O), [OsIICl(η6-p-cymene)(L2)]Cl (12b), [OsIICl(η6-p-cymene)(L2)]Cl·2CH3OH·2H2O (12b·2CH3OH·2H2O), [RuIICl(η6-p-cymene)(L3–H)] (13c), [RuIICl(η6-p-cymene)(L3)]Cl·[RuIICl(η6-p-cymene)(L3–H)]·CH3OH (13d·CH3OH) and [OsIICl(η6-p-cymene)(L3)]Cl·[OsIICl(η6-p-cymene)(L3–H)]·0.75CH3OH·0.25H2O (13e·0.75CH3OH·0.25H2O) are shown in Figures 2, Figure S2 in the Supporting Information, and Figures 3–6, 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 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.Figure 2Figure 2. Fragment of the crystal structure of 11b·4H2O showing nitrogen atoms N3 and N4, which act as proton donors in intermolecular hydrogen bonding interactions N3–H···Cl2 [N3···Cl2 3.067(7) Å, N3–H···Cl2 177.3°] and N4–H···O3 [N4···O3 2.671(9) Å, N4–H···O3 170.9°]; thermal ellipsoids have been drawn at 50% probability level. Selected bond lengths and angles: Os–Cl1, 2.421(2) Å; Os–N1, 2.072(7) Å; Os–N5, 2.096(7) Å; Os–C(arene)av, 2.198(32) Å; C1–N2, 1.349(11) Å; N2–N1, 1.366(10) Å; N1–C6, 1.357(11) Å; C6–C7, 1.428(12) Å; C7–N5, 1.345(11) Å; N5–C13, 1.391(11) Å; N1–Os–N5, 75.6(3)°; N1–Os–Cl1, 85.5(2)°; N5–Os–Cl1, 83.4(2)°.High Resolution ImageDownload MS PowerPoint SlideThe bidentate 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines, which can have different substituents in positions 5a and 7b, reveal different acid–base properties. It can act as a neutral organic ligand, with nitrogen atoms N2 and N4 as proton donors involved in intermolecular hydrogen bonding interactions N4–H···O1i(−x + 1, −y + 1, −z + 2) [N4···O1i, 2.730(4) Å; N4–H···O1i, 176.1°] and N2–H···O3ii(−x + 2, −y + 1, −z + 1 [N2···O3ii, 2.697(4) Å; N4–H···O1i, 172.3°] with one methanol and one water molecule, respectively, as is the case for 12b·2CH3OH·2H2O (Figure 3) or N2–H···Cl2(Cl2X) and N4–H···Cl1i(−x, −y, −z + 2) [N4···Cl1i, 3.213(9) Å; N4–H···O1i, 162.91°] in 12b (see Figure S2 in the Supporting Information), correspondingly. The ligand can be protonated at N3 (N3–H···Cl2) and deprotonated at N2 [N2···H–O4i (−x + 1, −y + 1, −z + 1) with N2···O4i 2.923(9) Å, N2···H–O4i 138.7°] (overall charge zero) with atom N4 as a proton donor to one of the four co-crystallized water molecules N4–H···O3, as occurs in 11b·4H2O (see Figure 2).Figure 3Figure 3. ORTEP plot of the structure of the cation [OsIICl(η6-p-cymene)(L2)]+ in 12b·2CH3OH·2H2O. Thermal ellipsoids have been drawn at the 50% probability level. Selected bond lengths and angles: Os–Cl1, 2.4043(1) Å; Os–N1, 2.083(3) Å; Os–N5, 2.097(3) Å; Os–C(arene)av, 2.197(26) Å; C1–N2, 1.361(5) Å; N2–N1, 1.344(4) Å; N1–C6, 1.336(5) Å; C6–C7, 1.447(6) Å; C7–N5, 1.335(5) Å; N5–C13, 1.380(5) Å; N1–Os–N5, 74.73(13)°, N1–Os–Cl1, 84.29(10)°; and N5–Os–Cl1, 83.25(10)°.High Resolution ImageDownload MS PowerPoint SlideIn 13c, the ligand was found to be deprotonated at N2, acting as a proton acceptor in the intermolecular hydrogen bonding interaction N2···H–N4i (i denotes symmetry transformations x, −y + 1.5, z + 0.5) used to generate equivalent atoms) [N2···N4i, 2.896(7) Å; N2···H–N4i, 155.6°] (see Figure 4).Figure 4Figure 4. ORTEP plot of the complex [RuIICl(η6-p-cymene)(L3–H)] (13c). Thermal ellipsoids have been drawn at 30% probability level. Selected bond lengths and angles: Ru–Cl, 2.399(2) Å; Ru–N1, 2.067(6) Å; Ru–N5, 2.089(5) Å; Ru–C(arene)av, 2.186(31) Å; C1–N2, 1.366(8) Å; N2–N1, 1.353(7) Å; N1–C6, 1.362(7) Å; C6–C7, 1.426(9) Å; C7–N5, 1.335(8) Å; N5–C13, 1.398(8) Å; N1–Ru–N5, 75.9(2)°; N1–Ru–Cl, 87.35(17)°; and N5–Ru–Cl, 85.73(17)°.High Resolution ImageDownload MS PowerPoint SlideComplexes 13d·CH3OH and 13e·0.75CH3OH·0.25H2O crystallized both in the centrosymmetric triclinic space group P1̅. The asymmetric unit in both consists of a neutral complex [MIICl(η6-p-cymene)(L3–H)] and a complex cation [MIICl(η6-p-cymene)(L3)]+ (M = Ru or Os), a chloride counteranion and co-crystallized solvent (methanol or methanol/water). Deprotonation of the organic ligand in [MIICl(η6-p-cymene)(L3–H)] is corroborated by the presence of hydrogen bonding of the type N2a···H–N2bi (−x + 1, −y + 1, −z + 1) in the crystal structure of the ruthenium complex 13d·CH3OH (Figure 5) and a similar interaction N2b···H–N2ai (x, y + 1, z) [N2b···N2ai, 2.810(9) Å; N2b···H–N2ai, 170.1°] in the crystal of the osmium analogue 13e·0.75CH3OH·0.25H2O. In Figure 6 the structure of [OsIICl(η6-p-cymene)(L3–H)] is shown. In both structures, the atoms N4a and N4b act as proton donors in strong hydrogen bonding interactions to the chloride counteranion, namely, N4a···H–Cl2ii (−x + 1, −y + 2, −z + 1) [N4···Cl2ii, 3.275(6) Å; N4a···H–Cl2ii, 176.9°], N4b···H–Cl2 [N4b···Cl2, 3.211(5) Å; N4b···H–Cl2, 176.0°] (13d·CH3OH) and N4a···H–Cl2ii (−x + 1, −y + 1, −z + 1) [N4a···Cl2ii, 3.273(8) Å; N4a···H–Cl2ii, 177.0°], N4b···H–Cl2ii [N4b···Cl2, 3.204(7) Å; N4b···H–Cl2, 177.0°] (13e·0.75CH3OH·0.25H2O).Figure 5Figure 5. Fragment of the crystal structure of [RuIICl(η6-p-cymene)(L3)]Cl·[RuIICl(η6-p-cymene)(L3–H)]·CH3OH (13d·CH3OH) showing the intermolecular hydrogen bonding N2a–H···N2b [N2a···N2bi (−x + 1, –y + 1, –z + 1), 2.814(7) Å; N2a–H···N2bi, 163.3°]. Selected bond lengths and angles: Ru1a–Cl1a, 2.3952(17) Å; Ru1a–N1a, 2.078(5) Å; Ru1a–N5a, 2.099(5) Å; Ru1a–C(arene)av, 2.194(33) Å; C1a–N2a, 1.358(8) Å; N2a–N1a, 1.354(7) Å; N1a–C6a, 1.350(8) Å; C6a–C7a, 1.441(9) Å; C7a–N5a, 1.335(8) Å; N5a–C13a, 1.381(8) Å; N1a–Ru1a–N5a, 75.6(2)°; N1a–Ru1a–Cl1a, 84.45(15)°; and N5a–Ru1a–Cl1a, 85.05(15)°.High Resolution ImageDownload MS PowerPoint SlideFigure 6Figure 6. ORTEP plot of [OsIICl(η6-p-cymene)(L3–H)] in [OsIICl(η6-p-cymene)(L3)]Cl·[OsIICl(η6-p-cymene)(L3–H)]·0.75CH3OH·0.25H2O (13e·0.75CH3OH·0.25H2O). Thermal ellipsoids have been drawn at the 40% probability level. Selected bond lengths and angles: Os1a–Cl1a, 2.402(2) Å; Os1a–N1a, 2.068(7) Å; Os1a–N5a, 2.086(7) Å; Os1a–C(arene)av, 2.192(25) Å; C1a–N2a, 1.349(10) Å; N2a–N1a 1.369(9) Å; N1a–C6a, 1.361(10) Å; C6a–C7a, 1.431(11) Å; C7a–N5a, 1.339(10) Å; N5a–C13a, 1.398(11) Å; N1a–Os1a–N5a, 75.1(3)°; N1a–Os1a–Cl1a, 83.56(18)°; and N5a–Os1a–Cl1a, 84.2(2)°.High Resolution ImageDownload MS PowerPoint Slide Antiproliferative ActivityLigands L1–L3, as well as the corresponding ruthenium(II) (11a–13a) and osmium(II) (11b–13b) 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 chemoresistant 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 methoxymethyl group (L3)) are advantageous, with regard to activity. These structure–activity relationships are also reflected in the rank orders of the corresponding ruthenium and osmium complexes 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.Table 2. Antiproliferative Activity of Metal-Free Ligands (L1–L3), and Their Ruthenium(II) (11a–13a) and Osmium(II) (11b–13b) Arene Complexes, in Three Human Cancer Cell Lines (CH1, SW480, and A549)a  IC50 [μM]bcompoundmetalCH1SW480A549L1 11 ± 323 ± 629 ± 711aRu96 ± 18 320525 ± 10211bOs64 ± 19223 ± 29 640L2 1.5 ± 0.65.1 ± 1.06.7 ± 0.312aRu21 ± 370 ± 8268 ± 3512bOs22 ± 329 ± 2123 ± 21L3 0.63 ± 0.090.74 ± 0.265.2 ± 0.513aRu11 ± 111 ± 268 ± 1213bOs7.9 ± 2.212 ± 289 ± 11aCH1 denotes ovarian cancer, human; SW480 denotes colon carcinoma, human; and A549 denotes non-small-cell lung carcinoma, human.b50% inhibitory concentrations (mean value ± standard deviation from at least three independent experiments) obtained by the MTT assay (96-h exposure).Figure 7Figure 7. Concentration–effect 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.High Resolution ImageDownload MS PowerPoint Slide Cell Cycle EffectsSince 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 L1–L3 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).Figure 8Figure 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 (( ▲) G0/G1, (○) S, and (■) G2/M phase). Note the different concentration scales.High Resolution ImageDownload MS PowerPoint Slide Cdk-Inhibitory ActivityAlthough 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 experiments 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 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 L1–L3 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.Figure 9Figure 9. Concentration-dependent effects of metal-free ligands (L1–L3) as well as corresponding ruthenium (11a–13a) and osmium complexes (11b–13b), on kinase activities of recombinant Cdk1/cyclin B (top) and Cdk2/cyclin E (bottom).High Resolution ImageDownload MS PowerPoint SlideConclusionARTICLE SECTIONSJump ToThe known multistep synthesis of 3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines, which we have modified, essentially afforded three organic compounds (L1–L3) 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 = L1–L3, have been synthesized and comprehensively characterized using spectroscopic and X-ray diffraction methods. Complexation of L1–L3 with ruthenium or osmium yielded compounds with increased solubility in the biological medium, yet lowered antiproliferative activity in human 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-free experiments, in particular in Cdk2/cyclin E, and their 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.Supporting InformationARTICLE SECTIONSJump ToAssigned NMR (1H, 13C, 15N) signals for L1–L3, 11a–13a, 11b–13b (Tables S1–S3); ORTEP 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)(L2)]+ in 12b (Figure S2); stability of complexes in solution; time-dependent UV–vis spectra of L1, 11a, and 11b in MeOH (Figure S3); time-dependent UV–vis spectra of L3, 13a, and 13b in MeOH (Figure S4); time-dependent UV–vis spectra of 11a in H2O for 48 h (Figure S5); crystallographic data for 2·0.5H2O, 11b·4H2O, 12b, 12b·2CH3OH·2H2O, 13c, 13d·CH3OH, and 13e·0.75CH3OH·0.25H2O (in CIF format). This material is available free of charge via the Internet at http://pubs.acs.org.ic201704u_si_001.pdf (250.59 kb)ic201704u_si_002.cif (226.03 kb) Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html. Author InformationARTICLE SECTIONSJump ToCorresponding AuthorsVladimir B. Arion - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, Austria;  Email: [email protected] bernh[email protected]Bernhard K. Keppler - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, Austria;  Email: [email protected] [email protected]AuthorsIryna N. Stepanenko - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaMaria S. Novak - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaGerhard Mühlgassner - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaAlexander Roller - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaMichaela Hejl - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaMichael A. Jakupec - University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, AustriaAcknowledgmentARTICLE SECTIONSJump ToWe 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.ReferencesARTICLE SECTIONSJump To This article references 45 other publications. 1Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2009, 9, 153– 166[Crossref], [PubMed], [CAS], Google Scholar1Cell cycle, CDKs and cancer: a changing paradigmMalumbres, Marcos; Barbacid, MarianoNature Reviews Cancer (2009), (3), 153-166CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group) A review. Tumor-assocd. cell cycle defects are often mediated by alterations in cyclin-dependent kinase (CDK) activity. Misregulated CDKs induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, mammalian CDKs are essential for driving each cell cycle phase, so therapeutic strategies that block CDK activity are unlikely to selectively target tumor cells. However, recent genetic evidence has revealed that, whereas CDK1 is required for the cell cycle, interphase CDKs are only essential for proliferation of specialized cells. Emerging evidence suggests that tumor cells may also require specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide therapeutic benefit against certain human neoplasias. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXit1ans78%253D md5=2a7eb34b2381e288b3f43ec1bffc0aec2Senderowicz, A. M. Oncogene 2003, 22, 6609– 6620Google ScholarThere is no corresponding record for this reference.3Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770– 1783[Crossref], [PubMed], [CAS], Google Scholar3Cyclin-dependent kinase pathways as targets for cancer treatmentShapiro, Geoffrey I.Journal of Clinical Oncology (2006), (11), 1770-1783CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology) A review. Cyclin-dependent kinases (cdks) are crit. regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor-dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B-responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, esp. in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compds. potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD28XksVKgtbg%253D md5=420a31b47f6600625f7d499a4b8a5fc94Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101, 2511– 2526[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.5Malumbres, M.; Pevarello, P.; Barbacid, M.; Bischoff, J. R. Trends Pharmacol. Sci. 2008, 29, 16– 21Google ScholarThere is no corresponding record for this reference.6Bible, K. C.; Kaufmann, S. H. Cancer Res. 1997, 57, 3375– 3380Google ScholarThere is no corresponding record for this reference.7Bible, K. C.; Boerner, S. A.; Kirkland, K.; Anderl, K. L.; Bartelt, D., Jr.; Svingen, P. A.; Kottke, T. J.; Lee, Y. K.; Eckdahl, S.; Stalboerger, P. G.; Jenkins, R. B.; Kaufmann, S. H. Clin. Cancer Res. 2000, 6, 661– 670[PubMed], [CAS], Google Scholar7Characterization of an ovarian carcinoma cell line resistant to cisplatin and flavopiridolBible, Keith C.; Boerner, Scott A.; Kirkland, Kathryn; Anderl, Kari L.; Bartelt, Duane, Jr.; Svingen, Phyllis A.; Kottke, Timothy J.; Lee, Yean K.; Eckdahl, Steven; Stalboerger, Paul G.; Jenkins, Robert B.; Kaufmann, Scott H.Clinical Cancer Research (2000), (2), 661-670CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research) Flavopiridol, the first inhibitor of cyclin-dependent kinases to enter clin. trials, has shown promising antineoplastic activity and is currently undergoing Phase II testing. Little is known about mechanisms of resistance to this agent. In the present study, we have characterized an ovarian carcinoma cell line [OV202 high passage (hp)] that spontaneously developed drug resistance upon prolonged passage in tissue culture. Std. cytogenetic anal. and spectral karyotyping revealed that OV202 hp and the parental low passage line OV202 shared several marker chromosomes, confirming the relatedness of these cell lines. Immunoblotting demonstrated that OV202 and OV202 hp contained similar levels of a variety of polypeptides involved in cell cycle regulation, including cyclin-dependent kinases 2 and 4; cyclins A, D1, and E; and proliferating cell nuclear antigen. Despite these similarities, OV202 hp was resistant to flavopiridol and cisplatin, with increases of 5- and 3-fold, resp., in the mean drug concns. required to inhibit colony formation by 90%. In contrast, OV202 hp and OV202 displayed indistinguishable sensitivities to oxaliplatin, paclitaxel, topotecan, 1,3-bis(2-chloroethyl)-1-nitrosourea, etoposide, doxorubicin, vincristine, and 5-fluorouracil, suggesting that the spontaneously acquired resistance was not attributable to altered P-glycoprotein levels or a general failure to engage the cell death machinery. After incubation with cisplatin, whole cell platinum and platinum-DNA adducts measured using mass spectrometry were lower in OV202 hp cells than OV202 cells. Similarly, after flavopiridol exposure, whole cell flavopiridol concns. measured by a newly developed high performance liq. chromatog. assay were lower in OV202 hp cells. These data are consistent with the hypothesis that acquisition of spontaneous resistance to flavopiridol and cisplatin in OV202 hp cells is due, at least in part, to reduced accumulation of the resp. drugs. These observations not only provide the first characterization of a flavopiridol-resistant cell line but also raise the possibility that alterations in drug accumulation might be important in detg. sensitivity to this agent. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3cXhs1akt7s%253D md5=eab27239203f5f7c8b0229ec4d7d93c78Bible, K. C.; Lensing, J. L.; Nelson, S. A.; Lee, Y. K.; Reid, J. M.; Ames, M. M.; Isham, C. R.; Piens, J.; Rubin, S. L.; Rubin, J.; Kaufmann, S. H.; Atherton, P. J.; Sloan, J. A.; Daiss, M. K.; Adjei, A. A.; Erlichman, C. Clin. Cancer Res. 2005, 11, 5935– 5941[Crossref], [PubMed], [CAS], Google Scholar8Phase 1 Trial of Flavopiridol Combined with Cisplatin or Carboplatin in Patients with Advanced Malignancies with the Assessment of Pharmacokinetic and Pharmacodynamic End PointsBible, Keith C.; Lensing, Janet L.; Nelson, Sacha A.; Lee, Yean K.; Reid, Joel M.; Ames, Matthew M.; Isham, Crescent R.; Piens, Jill; Rubin, Stacie L.; Rubin, Joseph; Kaufmann, Scott H.; Atherton, Pamela J.; Sloan, Jeffrey A.; Daiss, Michelle K.; Adjei, Alex A.; Erlichman, CharlesClinical Cancer Research (2005), (16), 5935-5941CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research) Purpose: Flavopiridol, a cyclin-dependent kinase inhibitor, transcription inhibitor, and DNA-interacting agent, was combined with cisplatin or carboplatin to establish toxicities, evaluate pharmacokinetics, and examine its effects on patient cancers and levels of selected polypeptides in patient peripheral blood mononuclear cells (PBMC). Exptl. Design: Therapy was given every 3 wk. Stage I: cisplatin was fixed at 30 mg/m2 with escalating flavopiridol. Stage II: flavopiridol was fixed at the stage I max. tolerated dose (MTD) with escalation of cisplatin. Stage III: flavopiridol was fixed at the stage I MTD with escalation of carboplatin. Results: Thirty-nine patients were treated with 136 cycles of chemotherapy. Neutropenia was seen in only 11% of patients. Grade 3 flavopiridol/CDDP toxicities were nausea (30%), vomiting (19%), diarrhea (15%), dehydration (15%), and neutropenia (10%). Flavopiridol combined with carboplatin resulted in unexpectedly high toxicities and one treatment-related death. Stable disease ( 3 mo) was seen in 34% of treated patients, but there were no objective responses. The stage II MTD was 60 mg/m2 cisplatin and 100 mg/m2/24 h flavopiridol. As given, CDDP did not alter flavopiridol pharmacokinetics. Flavopiridol induced increased p53 and pSTAT3 levels in patient PBMCs but had no effects on cyclin D1, phosphoRNA polymerase II, or Mcl-1. Conclusions: Flavopiridol and cisplatin can be safely combined in the treatment of cancer patients. Unexpected toxicity in flavopiridol/carboplatin-treated patients attenuates enthusiasm for this alternative combination. Anal. of polypeptide levels in patient PBMCs suggests that flavopiridol may be affecting some, but not all, of its known in vitro mol. targets in vivo. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2MXos1Cisr4%253D md5=2a0cd103f2eead0b4720d4ab3ea600e89Coley, H. M.; Shotton, C. F.; Kokkinos, M. I.; Thomas, H. Gynecol. Oncol. 2007, 105, 462– 469[Crossref], [PubMed], [CAS], Google Scholar9The effects of the CDK inhibitor seliciclib alone or in combination with cisplatin in human uterine sarcoma cell linesColey, Helen M.; Shotton, Christine F.; Kokkinos, Maria I.; Thomas, HilaryGynecologic Oncology (2007), (2), 462-469CODEN: GYNOA3; ISSN:0090-8258. (Elsevier) Objectives: Inhibition of cyclin-dependent-kinases (CDKs) represents an interesting approach in cancer therapy. We have explored this in cell lines of human uterine sarcoma-tumors assocd. with poor survival, chemo-unresponsiveness and deregulation of cell cycle components. We studied the effects of the CDK inhibitor seliciclib (CYC202, R-roscovitine) when used alone or in combination with cisplatin. Methods: Cell lines used: SK-UT-1, SK-UT-1b and SK-LMS-1, the cytotoxicity of seliciclib and cisplatin was measured by the MTT assay. In combination with cisplatin the effects of seliciclib were examd. by isobologram anal. CDK2 levels were examd. at mRNA and protein level by immunoblotting and PCR. We also looked at the effects of seliciclib on p53-dependent response of cells to seliciclib using immunoblotting. The effects of combination treatment were analyzed using annexin V and PI staining by flow cytometric anal. Results: IC50 values for seliciclib were 10.5, 7.1 and 25.7 μM, for SK-UT-1, SK-UT-1b and SK-LMS-1 resp., P53 in the SK-UT-1b (wild-type) and SK-LMS-1 lines (mutant) showed a wild-type response with induction seen with seliciclib treatment for 24 and 48 h. Seliciclib caused a decrease in CDK2 mRNA and protein over 72 h. A combination of cisplatin and seliciclib was synergistic in all three cell lines. Effects of combination treatment were an enhancement in apoptosis as judged by the emergence of a sub-G1 population in cell cycle anal. and a sub-G1 population with PI staining. Conclusions: Our data demonstrate the effectiveness of seliciclib as a single agent and when used in combination with cisplatin where the effects are synergistic. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXksVOnu7k%253D md5=85796769ec1921ca2d149e82c26b77d010Travnicek, Z.; Popa, I.; Cajan, M.; Herchel, R.; Marek, J. Polyhedron 2007, 26, 5271– 5282Google ScholarThere is no corresponding record for this reference.11Malon, M.; Travnicek, Z.; Marysko, M.; Zboril, R.; Maslan, M.; Marek, J.; Dolezal, K.; Rolcik, J.; Krystof, V.; Strnad, M. Inorg. Chim. Acta 2001, 323, 119– 129Google ScholarThere is no corresponding record for this reference.12Dvorak, L.; Popa, I.; Starha, P.; Travnicek, Z. Eur. J. Inorg. Chem. 2010, 3441– 3448Google ScholarThere is no corresponding record for this reference.13Travnicek, Z.; Popa, I.; Cajan, M.; Zboril, R.; Krystof, V.; Mikulik, J. J. Inorg. Biochem. 2010, 104, 405– 417Google ScholarThere is no corresponding record for this reference.14Primik, M. F.; Muehlgassner, G.; Jakupec, M. A.; Zava, O.; Dyson, P. J.; Arion, V. B.; Keppler, B. K. Inorg. Chem. 2010, 49, 302– 311[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.15Ginzinger, W.; Arion, V. B.; Giester, G.; Galanski, M.; Keppler, B. K. Centr. Eur. J. Chem. 2008, 6, 340– 346[Crossref], [CAS], Google Scholar15Synthesis and structural peculiarities of gallium complexes with novel paullone derivativesGinzinger, Werner; Arion, Vladimir B.; Giester, Gerald; Galanski, Markus; Keppler, Bernhard K.Central European Journal of Chemistry (2008), (3), 340-346CODEN: CEJCAZ; ISSN:1895-1066. (Springer GmbH) 9-Bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-ylhydrazine was reacted with 2-acetylpyridine to give a Schiff base as a potential tridentate ligand. The reaction of this ligand with gallium chloride afforded complexes of 1:1 and 2:1 stoichiometry. The results of x-ray diffraction studies of the ligand and both gallium complexes are reported and compared with the data for a related gallium complex with a Schiff base obtained from 9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-ylhydrazine and 2-hydroxybenzaldehyde. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhtVCqsrjK md5=ac62530de701b40e4c8505b1a6fc97e516Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K. Organometallics 2007, 26, 6643– 6652[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.17Schmid, W. F.; John, R. O.; Muehlgassner, G.; Heffeter, P.; Jakupec, M. A.; Galanski, M.; Berger, W.; Arion, V. B.; Keppler, B. K. J. Med. Chem. 2007, 50, 6343– 6355[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.18Schmid, W. F.; Zorbas-Seifried, S.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Roller, A.; Galanski, M.; Chiorescu, I.; Zorbas, H.; Keppler, B. K. Inorg. Chem. 2007, 46, 3645– 3656[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.19Dobrov, A.; Arion, V. B.; Kandler, N.; Ginzinger, W.; Jakupec, M. A.; Rufinska, A.; Graf von Keyserlingk, N.; Galanski, M.; Kowol, C.; Keppler, B. K. Inorg. Chem. 2006, 45, 1945– 1950[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.20Filak, L. K.; Muehlgassner, G.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Arion, V. B.; Keppler, B. K. J. Biol. Inorg. Chem. 2010, 15, 903– 918[Crossref], [PubMed], [CAS], Google Scholar20Organometallic indolo[3,2-c]quinolines versus indolo[3,2-d]benzazepines: synthesis, structural and spectroscopic characterization, and biological efficacyFilak, Lukas K.; Muehlgassner, Gerhard; Jakupec, Michael A.; Heffeter, Petra; Berger, Walter; Arion, Vladimir B.; Keppler, Bernhard K.JBIC, Journal of Biological Inorganic Chemistry (2010), (6), 903-918CODEN: JJBCFA; ISSN:0949-8257. (Springer) The synthesis of Ru(II) and Os(II) arene complexes with the closely related indolo[3,2-c]quinolines N-(11H-indolo[3,2-c]quinolin-6-yl)-ethane-1,2-diamine (L1) and N'-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (L2) and indolo[3,2-d]benzazepines N-(7,12-dihydroindolo-[3,2-d][1]benzazepin-6-yl)-ethane-1,2-diamine (L3) and N'-(7,12-dihydroindolo-[3,2-d][1]benzazepin-6-yl)-N,N-dimethylethane-1,2-diamine (L4) [(η6-p-cymene)MII(L1)Cl]Cl, where M is Ru (4) and Os (6), [(η6-p-cymene)MII(L2)Cl]Cl, where M is Ru (5) and Os (7), [(η6-p-cymene)MII(L3)Cl]Cl, where M is Ru (8) and Os (10), and [(η6-p-cymene)MII(L4)Cl]Cl, where M is Ru (9) and Os (11), is reported. The compds. were comprehensively characterized by elemental anal., electrospray ionization mass spectrometry, spectroscopy (IR, UV-visible, and NMR), and x-ray crystallog. (L1·HCl, 4·H2O, 5, and 9·2.5H2O). Structure-activity relations with regard to cytotoxicity and cell cycle effects in human cancer cells as well as cyclin-dependent kinase (cdk) inhibition and DNA intercalation in cell-free settings were established. The metal-free indolo[3,2-c]quinolines inhibit cancer cell growth in vitro, with IC50 values in the high nanomolar range, whereas those of the related indolo[3,2-d]benzazepines are in the low micromolar range. In cell-free expts., these classes of compds. inhibit the activity of cdk2/cyclin E, but the much higher cytotoxicity and stronger cell cycle effects of indoloquinolines L1 and 7 are not paralleled by a substantially higher kinase inhibition compared with indolobenzazepines L4 and 11, arguing for addnl. targets and mol. effects, such as intercalation into DNA. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXkt1aqs7c%253D md5=c86f175ed0278bd443bcad6be29d171f21Lin, R.; Connolly, P. J.; Lu, Y.; Chiu, G.; Li, S.; Yu, Y.; Huang, S.; Li, X.; Emanuel, S. L.; Middleton, S. A.; Gruninger, R. H.; Adams, M.; Fuentes-Pesquera, A. R.; Greenberger, L. M. Bioorg. Med. Chem. Lett. 2007, 17, 4297– 4302Google ScholarThere is no corresponding record for this reference.22Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Connolly, P. J.; Chiu, G.; Li, S.; Emanuel, S. L.; Middleton, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 1243– 1245Google ScholarThere is no corresponding record for this reference.23Chiu, G.; Li, S.; Connolly, P. J.; Middleton, S. A.; Emanuel, S. L.; Huang, S.; Lin, R.; Lu, Y.PCT Int. Appl., WO 2006130673, 2006, 162 pp.Google ScholarThere is no corresponding record for this reference.24Lin, R.; Chiu, G.; Yu, Y.; Connolly, P. J.; Li, S.; Lu, Y.; Adams, M.; Fuentes-Pesquera, A. R.; Emanuel, S. L.; Greenberger, L. M. Bioorg. Med. Chem. Lett. 2007, 17, 4557– 4561Google ScholarThere is no corresponding record for this reference.25Chiu, G.; Yu, Y.; Lin, R.; Li, S.; Connolly, P. J.PCT Int. Appl., WO 2008048502, 2008, 43 pp.Google ScholarThere is no corresponding record for this reference.26Yu, Y.; Lin, R.; Connolly, P. J.PCT Int. Appl., WO 2008048503, 2008, 42 pp.Google ScholarThere is no corresponding record for this reference.27Peacock, A. F. A.; Sadler, P. J. Chem.-Asian J. 2008, 3, 1890– 1899[Crossref], [PubMed], [CAS], Google Scholar27Medicinal organometallic chemistry: designing metal arene complexes as anticancer agentsPeacock, Anna F. A.; Sadler, Peter J.Chemistry - An Asian Journal (2008), (11), 1890-1899CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH Co. KGaA) A review. The field of medicinal inorg. chem. is rapidly advancing. In particular organometallic complexes have much potential as therapeutic and diagnostic agents. The C-bound and other ligands allow the thermodn. and kinetic reactivity of the metal ion to be controlled and also provide a scaffold for functionalization. The establishment of structure-activity relations and elucidation of the speciation of complexes under conditions relevant to drug testing and formulation are crucial for the further development of promising medicinal applications of organometallic complexes. Specific examples involving the design of Ru and Os arene complexes as anticancer agents are discussed. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhsVCjt73L md5=fa4f181bb367f2eea1e33c55399d122a28Bruijnincx, P. C. A.; Sadler, P. J. Adv. Inorg. Chem. 2009, 61, 1– 62Google ScholarThere is no corresponding record for this reference.29Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391– 401Google ScholarThere is no corresponding record for this reference.30Dougan, S. J.; Sadler, P. J. Chimia 2007, 61, 704– 715Google ScholarThere is no corresponding record for this reference.31Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003– 4018Google ScholarThere is no corresponding record for this reference.32Dyson, P. J. Chimia 2007, 61, 698– 703Google ScholarThere is no corresponding record for this reference.33Kuo, D. L. Tetrahedron 1992, 48, 9233– 9236Google ScholarThere is no corresponding record for this reference.34Lynch, B. M.; Khan, M. A.; Teo, H. C.; Pedrotti, F. Can. J. Chem. 1988, 66, 420– 428Google ScholarThere is no corresponding record for this reference.35Georg, G. I.; Tash, J. S.; Chakrasali, R.; Jakkaraj, S. R.PCT Int. Appl., WO 2006023704, 2006, 182 pp.Google ScholarThere is no corresponding record for this reference.36Berdini, V.; O’Brien, M. A.; Carr, M. G.; Early, T. R.; Navarro, E. F.; Gill, A. L.; Howard, S.; Trewartha, G.; Woolford, A. J.-A.; Woodhead, A. J.; Wyatt, P.PCT Int. Appl., WO 2005002552, 2005, 287 pp.Google ScholarThere is no corresponding record for this reference.37Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974, 233– 241Google ScholarThere is no corresponding record for this reference.38Kiel, W. A.; Ball, R. G.; Graham, W. A. G. J. Organomet. Chem. 1990, 383, 481– 496[Crossref], [CAS], Google Scholar38Carbonyl-η-hexamethylbenzene complexes of osmium. Carbon-hydrogen activation by (η-C6Me6)Os(CO)(H)2Kiel, William A.; Ball, Richard G.; Graham, William A. G.Journal of Organometallic Chemistry (1990), (1-3), 481-96CODEN: JORCAI; ISSN:0022-328X. Redn. of (η-C6-Me6)OS(CO)Cl2 with Zn-AcOH-MeOH gives (η-C6Me6)Os(CO)(Cl)H, which can be further reduced with Na[H2Al(OCH2CH2OCH3)2] to (η-C6Me6)Os(CO)H2 (I). Photolysis of I in hydrocarbons (benzene, cyclohexane, neopentane) results in formation of the C-H bond activation products η-C6Me6Os(CO)(R)(H) (II, R = C6H5, C6H11, CH2C(CH3)3, resp.) and free hexamethylbenzene. Independent syntheses of the hydrides II are described as well as syntheses of the complexes (η-C6Me6)Os(CO)(R)2 and (η-C6Me6)Os(CO)(R)Cl. The structure of (η-C6Me6)Os(CO)(cyclohexyl)2 detd. by single crystal X-ray diffraction is reported. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK3cXksF2ntb4%253D md5=c0fdf8c0541bb0776a41ac3db117b6ca39SAINT-Plus, version 7.06a and APEX2; Bruker–Nonius AXS, Inc.: Madison, WI, 2004.Google ScholarThere is no corresponding record for this reference.40Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112– 122[Crossref], [CAS], Google Scholar40A short history of SHELXSheldrick, George M.Acta Crystallographica, Section A: Foundations of Crystallography (2008), (1), 112-122CODEN: ACACEQ; ISSN:0108-7673. (International Union of Crystallography) An account is given of the development of the SHELX system of computer programs from SHELX-76 to the present day. In addn. to identifying useful innovations that have come into general use through their implementation in SHELX, a crit. anal. is presented of the less-successful features, missed opportunities and desirable improvements for future releases of the software. An attempt is made to understand how a program originally designed for photog. intensity data, punched cards and computers over 10000 times slower than an av. modern personal computer has managed to survive for so long. SHELXL is the most widely used program for small-mol. refinement and SHELXS and SHELXD are often employed for structure soln. despite the availability of objectively superior programs. SHELXL also finds a niche for the refinement of macromols. against high-resoln. or twinned data; SHELXPRO acts as an interface for macromol. applications. SHELXC, SHELXD and SHELXE are proving useful for the exptl. phasing of macromols., esp. because they are fast and robust and so are often employed in pipelines for high-throughput phasing. This paper could serve as a general literature citation when one or more of the open-source SHELX programs (and the Bruker AXS version SHELXTL) are employed in the course of a crystal-structure detn. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXhsVGhurzO md5=8f91f10be4a9df74b2a9dad522c71e6e41Burnett, M. N.; Johnson, G. K.ORTEPIII, Report ORNL-6895; Oak Ridge National Laboratory; Oak Ridge, TN, 1996.Google ScholarThere is no corresponding record for this reference.42Marko, D.; Schätzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.; Eisenbrand, G. Br. J. Cancer 2001, 84, 283– 289[Crossref], [PubMed], [CAS], Google Scholar42Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cellsMarko, D.; Schatzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.; Eisenbrand, G.British Journal of Cancer (2001), (2), 283-289CODEN: BJCAAI; ISSN:0007-0920. (Harcourt Publishers Ltd.) The bisindole indirubin has been described, more than 30 yr ago, as being clin. active in the treatment of human chronic myelocytic leukemia. However, the underlying mechanism of action has remained unclear. The authors have reported previously that indirubin and its analogs are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, the authors investigated the influence of indirubin and derivs. on CDK1/cyclin B kinase in human tumor cells at concns. known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G1/G0 phase of the cell cycle in the presence of 2 μM indirubin-3'-monoxime. At higher drug concns. (≥ 5 μM) an increase of the cell population in the G2/M phase is addnl. obsd. Cells synchronized in G2/M phase by nocodazole remain arrested in the G2/M phase after release, in the presence of indirubin-3'-monoxime (≥5 μM). After 24 h treatment with 10 μM indirubin-3'-monoxime a sub-G2 peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concns. of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a redn. of cyclin B in complex with CDK1 is obsd. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumor cells is a major mechanism by which indirubin derivs. exert their potent antitumor efficacy. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3MXht1Wgsrk%253D md5=117f30fb03c00c6169f207d24f348a0a43Zeng, Q.; Yao, G.; Wohlhieter, G. E.; Viswanadhan, V. N.; Tasker, A.; Rider, J. T.; Monenschein, H.; Dominguez, C.; Bourbeau, M. P.PCT Int. Appl., WO 2006044860, 2006, 61 pp.Google ScholarThere is no corresponding record for this reference.44Cui, J. J.; Deal, J. G.; Gu, D.; Guo, C.; Johnson, M. C.; Kania, R. S.; Kephart, S. E.; Linton, M. A.; McApline, I. J.; Pairish, M. A.; Palmer, C. L.PCT Int. Appl., WO 2009016460, 2009, 168 pp.Google ScholarThere is no corresponding record for this reference.45Kenda, B.; Quesnel, Y.; Ates, A.; Michel, P.; Turet, L.; Mercier, J.PCT Int. Appl., WO 2006128693, 2006, 258 pp.Google ScholarThere is no corresponding record for this reference.Cited ByThis article is cited by 23 publications.Mauricio García, Iván Romero, Jaime Portilla. Synthesis of Fluorescent 1,7-Dipyridyl-bis-pyrazolo[3,4-b:4′,3′-e]pyridines: Design of Reversible Chemosensors for Nanomolar Detection of Cu2+. ACS Omega 2019, 4 , 6757-6768. https://doi.org/10.1021/acsomega.9b00226Reece G. Kenny, Celine J. Marmion. Toward Multi-Targeted Platinum and Ruthenium Drugs—A New Paradigm in Cancer Drug Treatment Regimens?. Chemical Reviews 2019, 119 , 1058-1137. https://doi.org/10.1021/acs.chemrev.8b00271Marta Martínez-Alonso, Natalia Busto, Félix A. Jalón, Blanca R. Manzano, José M. Leal, Ana M. Rodríguez, Begoña García, and Gustavo Espino . Derivation of Structure–Activity Relationships from the Anticancer Properties of Ruthenium(II) Arene Complexes with 2-Aryldiazole Ligands. Inorganic Chemistry 2014, 53 (20) , 11274-11288. https://doi.org/10.1021/ic501865hGabriel E. Büchel, Anatolie Gavriluta, Maria Novak, Samuel M. Meier, Michael A. Jakupec, Olesea Cuzan, Constantin Turta, Jean-Bernard Tommasino, Erwann Jeanneau, Ghenadie Novitchi, Dominique Luneau, and Vladimir B. Arion . Striking Difference in Antiproliferative Activity of Ruthenium- and Osmium-Nitrosyl Complexes with Azole Heterocycles. Inorganic Chemistry 2013, 52 (11) , 6273-6285. https://doi.org/10.1021/ic400555kIryna N. Stepanenko, Angela Casini, Fabio Edafe, Maria S. Novak, Vladimir B. Arion, Paul J. Dyson, Michael A. Jakupec, and Bernhard K. Keppler . Conjugation of Organoruthenium(II) 3-(1H-Benzimidazol-2-yl)pyrazolo[3,4-b]pyridines and Indolo[3,2-d]benzazepines to Recombinant Human Serum Albumin: a Strategy To Enhance Cytotoxicity in Cancer Cells. Inorganic Chemistry 2011, 50 (24) , 12669-12679. https://doi.org/10.1021/ic201801eWilliam D.J. Tremlett, David M. Goodman, Tasha R. Steel, Saawan Kumar, Anna Wieczorek-Błauż, Fearghal P. Walsh, Matthew P. Sullivan, Muhammad Hanif, Christian G. Hartinger. Design concepts of half-sandwich organoruthenium anticancer agents based on bidentate bioactive ligands. Coordination Chemistry Reviews 2021, 445 , 213950. https://doi.org/10.1016/j.ccr.2021.213950Claudio Pettinari, Riccardo Pettinari, Nertil Xhaferai, Giuliano Giambastiani, Andrea Rossin, Laura Bonfili, Anna Maria Eleuteri, Massimiliano Cuccioloni. Binuclear 3,3′,5,5′-tetramethyl-1H,H-4,4′-bipyrazole Ruthenium(II) complexes: Synthesis, characterization and biological studies. Inorganica Chimica Acta 2020, 513 , 119902. https://doi.org/10.1016/j.ica.2020.119902Janos Sapi, Stéphane Gérard. Bicyclic 5-6 Systems: Three Heteroatoms 2:1. 2020,,https://doi.org/10.1016/B978-0-12-818655-8.00021-4Yi Ning, Xinwei He, Youpeng Zuo, Panyuan Cai, Mengqing Xie, Jian Wang, Yongjia Shang. Rhodium(II) Acetate‐Catalysed Cyclization of Pyrazol‐5‐amine and 1,3‐Diketone‐2‐diazo Compounds Using ‐Dimethylformamide as a Carbon‐Hydrogen Source: Access to Pyrazolo[3,4‐ ]pyridines. Advanced Synthesis Catalysis 2019, 361 (15) , 3518-3524. https://doi.org/10.1002/adsc.201900093Darko N. Pantić, Ljiljana E. Mihajlović-Lalić, Sandra Aranđelović, Siniša Radulović, Sanja Grgurić-Šipka. Synthesis, characterization and cytotoxic activity of organoruthenium(II)-halido complexes with 5-chloro-1 -benzimidazole-2-carboxylic acid. Journal of Coordination Chemistry 2019, 72 (5-7) , 908-919. https://doi.org/10.1080/00958972.2019.1583332Nadieh Dorostkar-Ahmadi, Abolghasem Davoodnia, Niloofar Tavakoli-Hoseini, Hossein Behmadi. Facile Synthesis of New 6-Alkylamino-1 -pyrazolo[3,4- ]pyridine-5-carbonitrile Derivatives. Journal of Heterocyclic Chemistry 2018, 55 (11) , 2635-2639. https://doi.org/10.1002/jhet.3285Shikha Singh, Parul Chauhan, Makthala Ravi, Prem P. Yadav. Eosin Y–Yb(OTf) catalyzed visible light mediated electrocyclization/indole ring opening towards the synthesis of heterobiaryl-pyrazolo[3,4- ]pyridines. New Journal of Chemistry 2018, 42 , 6617-6620. https://doi.org/10.1039/C8NJ00862KM.J. Chow, W.H. Ang. Organoruthenium(II)-Arene Complexes. 2017,,, 119-146. https://doi.org/10.1016/B978-0-12-803814-7.00004-6Sudipta Bhattacharyya, Kallol Purkait, Arindam Mukherjee. Ruthenium( ) p-cymene complexes of a benzimidazole-based ligand capable of VEGFR2 inhibition: hydrolysis, reactivity and cytotoxicity studies. Dalton Transactions 2017, 46 (26) , 8539-8554. https://doi.org/10.1039/C7DT00938KJayaraman Pitchaimani, Mamilla R. Charan Raja, Srinivasan Sujatha, Santanu Kar Mahapatra, Dohyun Moon, Savarimuthu Philip Anthony, Vedichi Madhu. Arene ruthenium( ) complexes with chalcone, aminoantipyrine and aminopyrimidine based ligands: synthesis, structure and preliminary evaluation of anti-leukemia activity. RSC Advances 2016, 6 (93) , 90982-90992. https://doi.org/10.1039/C6RA18504EKanidtha Hansongnern, Supojjanee Sansook, Thassani Romin, Arunpatcha Nimthong Roldan, Chaveng Pakawatchai. Crystal structure of [3-amino-2-(phenyldiazenyl)pyridine]chlorido(η -cymene)ruthenium(II) chloride. Acta Crystallographica Section E Crystallographic Communications 2015, 71 (10) , m185-m186. https://doi.org/10.1107/S2056989015017466Fang Wang, Xue Wang, Min-Xia Zhang, Yong-Hua Yang, Hai-Liang Zhu. Synthesis, biological evaluation and molecular modeling of 1H-benzo[d]imidazole derivatives as novel anti-tubulin polymerization agents. RSC Advances 2015, 5 (91) , 74425-74437. https://doi.org/10.1039/C5RA13746BPhilipp Anstaett, Gilles Gasser. Organometallic Complexes as Enzyme Inhibitors: A Conceptual Overview. 2014,,, 1-42. https://doi.org/10.1002/9783527673438.ch01Meng Li, Bao-Xiang Zhao. Progress of the synthesis of condensed pyrazole derivatives (from 2010 to mid-2013). European Journal of Medicinal Chemistry 2014, 85 , 311-340. https://doi.org/10.1016/j.ejmech.2014.07.102Brendan Gleeson, Patrick J. Carroll, Larry G. Sneddon. Functionalized ferratricarbadecaboranyl complexes for potential anticancer applications. Journal of Organometallic Chemistry 2013, 747 , 51-61. https://doi.org/10.1016/j.jorganchem.2012.12.001Claudio Pettinari, Riccardo Pettinari, Corrado Di Nicola, Fabio Marchetti. Half-Sandwich Rhodium(III), Iridium(III), and Ruthenium(II) Complexes with Ancillary Pyrazole-Based Ligands. 2013,,, 269-284. https://doi.org/10.1002/9781118742952.ch21Rui-Rong Ye, Zhuo-Feng Ke, Cai-Ping Tan, Liang He, Liang-Nian Ji, Zong-Wan Mao. Histone-Deacetylase-Targeted Fluorescent Ruthenium(II) Polypyridyl Complexes as Potent Anticancer Agents. Chemistry - A European Journal 2013, 19 (31) , 10160-10169. https://doi.org/10.1002/chem.201300814Kelly J. Kilpin, Paul J. Dyson. Enzyme inhibition by metal complexes: concepts, strategies and applications. Chemical Science 2013, 4 , 1410. https://doi.org/10.1039/c3sc22349cFiguresReferencesSupport InfoAbstractHigh Resolution ImageDownload MS PowerPoint SlideChart 1Chart 1. Compounds Reported in This Work with Atom Numbering Schemes for NMR Signal AssignmentaHigh Resolution ImageDownload MS PowerPoint SlideChart aUnderlined compounds have been characterized by X-ray diffraction (XRD).Scheme 1Scheme 1. Synthesis of 5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (3)aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (i) KMnO4, NaOH, 3 h, 100 °C, H2SO4;(35) (ii) methanol, H2SO4, reflux, 6–8 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, 2.5–3 h; and (vi) KMnO4, NaOH, 3 h, 100 °C, HCl, 24–35% (v + vi).Scheme 2Scheme 2. Synthesis of 1-Methoxymethyl-2,3-diaminobenzene (5)aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents 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, chromatographic purification, 65–75% yield; (iii) 10% Pd/C, ethanol, H2, room temperature, 18–24 h, 100% yield.Scheme 3Scheme 3. Synthesis of 3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines L1–L3aHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (i) CDI, dry DMF, room temperature, 20–24 h; (ii) 1,2-diaminobenzene or 5, dry DMF, 80–85 °C; 5–20 h; and (iii) glacial AcOH, 120–125 °C, 2.5–4.5 h, chromatographic purification.Figure 1Figure 1. Part of the 1H,1H ROESY plot of L3.High Resolution ImageDownload MS PowerPoint SlideScheme 4Scheme 4. Coordination of L3 (7b-L3 (left) and 4b′-L3 (right) Tautomers)High Resolution ImageDownload MS PowerPoint SlideFigure 2Figure 2. Fragment of the crystal structure of 11b·4H2O showing nitrogen atoms N3 and N4, which act as proton donors in intermolecular hydrogen bonding interactions N3–H···Cl2 [N3···Cl2 3.067(7) Å, N3–H···Cl2 177.3°] and N4–H···O3 [N4···O3 2.671(9) Å, N4–H···O3 170.9°]; thermal ellipsoids have been drawn at 50% probability level. Selected bond lengths and angles: Os–Cl1, 2.421(2) Å; Os–N1, 2.072(7) Å; Os–N5, 2.096(7) Å; Os–C(arene)av, 2.198(32) Å; C1–N2, 1.349(11) Å; N2–N1, 1.366(10) Å; N1–C6, 1.357(11) Å; C6–C7, 1.428(12) Å; C7–N5, 1.345(11) Å; N5–C13, 1.391(11) Å; N1–Os–N5, 75.6(3)°; N1–Os–Cl1, 85.5(2)°; N5–Os–Cl1, 83.4(2)°.High Resolution ImageDownload MS PowerPoint SlideFigure 3Figure 3. ORTEP plot of the structure of the cation [OsIICl(η6-p-cymene)(L2)]+ in 12b·2CH3OH·2H2O. Thermal ellipsoids have been drawn at the 50% probability level. Selected bond lengths and angles: Os–Cl1, 2.4043(1) Å; Os–N1, 2.083(3) Å; Os–N5, 2.097(3) Å; Os–C(arene)av, 2.197(26) Å; C1–N2, 1.361(5) Å; N2–N1, 1.344(4) Å; N1–C6, 1.336(5) Å; C6–C7, 1.447(6) Å; C7–N5, 1.335(5) Å; N5–C13, 1.380(5) Å; N1–Os–N5, 74.73(13)°, N1–Os–Cl1, 84.29(10)°; and N5–Os–Cl1, 83.25(10)°.High Resolution ImageDownload MS PowerPoint SlideFigure 4Figure 4. ORTEP plot of the complex [RuIICl(η6-p-cymene)(L3–H)] (13c). Thermal ellipsoids have been drawn at 30% probability level. Selected bond lengths and angles: Ru–Cl, 2.399(2) Å; Ru–N1, 2.067(6) Å; Ru–N5, 2.089(5) Å; Ru–C(arene)av, 2.186(31) Å; C1–N2, 1.366(8) Å; N2–N1, 1.353(7) Å; N1–C6, 1.362(7) Å; C6–C7, 1.426(9) Å; C7–N5, 1.335(8) Å; N5–C13, 1.398(8) Å; N1–Ru–N5, 75.9(2)°; N1–Ru–Cl, 87.35(17)°; and N5–Ru–Cl, 85.73(17)°.High Resolution ImageDownload MS PowerPoint SlideFigure 5Figure 5. Fragment of the crystal structure of [RuIICl(η6-p-cymene)(L3)]Cl·[RuIICl(η6-p-cymene)(L3–H)]·CH3OH (13d·CH3OH) showing the intermolecular hydrogen bonding N2a–H···N2b [N2a···N2bi (−x + 1, –y + 1, –z + 1), 2.814(7) Å; N2a–H···N2bi, 163.3°]. Selected bond lengths and angles: Ru1a–Cl1a, 2.3952(17) Å; Ru1a–N1a, 2.078(5) Å; Ru1a–N5a, 2.099(5) Å; Ru1a–C(arene)av, 2.194(33) Å; C1a–N2a, 1.358(8) Å; N2a–N1a, 1.354(7) Å; N1a–C6a, 1.350(8) Å; C6a–C7a, 1.441(9) Å; C7a–N5a, 1.335(8) Å; N5a–C13a, 1.381(8) Å; N1a–Ru1a–N5a, 75.6(2)°; N1a–Ru1a–Cl1a, 84.45(15)°; and N5a–Ru1a–Cl1a, 85.05(15)°.High Resolution ImageDownload MS PowerPoint SlideFigure 6Figure 6. ORTEP plot of [OsIICl(η6-p-cymene)(L3–H)] in [OsIICl(η6-p-cymene)(L3)]Cl·[OsIICl(η6-p-cymene)(L3–H)]·0.75CH3OH·0.25H2O (13e·0.75CH3OH·0.25H2O). Thermal ellipsoids have been drawn at the 40% probability level. Selected bond lengths and angles: Os1a–Cl1a, 2.402(2) Å; Os1a–N1a, 2.068(7) Å; Os1a–N5a, 2.086(7) Å; Os1a–C(arene)av, 2.192(25) Å; C1a–N2a, 1.349(10) Å; N2a–N1a 1.369(9) Å; N1a–C6a, 1.361(10) Å; C6a–C7a, 1.431(11) Å; C7a–N5a, 1.339(10) Å; N5a–C13a, 1.398(11) Å; N1a–Os1a–N5a, 75.1(3)°; N1a–Os1a–Cl1a, 83.56(18)°; and N5a–Os1a–Cl1a, 84.2(2)°.High Resolution ImageDownload MS PowerPoint SlideFigure 7Figure 7. Concentration–effect 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.High Resolution ImageDownload MS PowerPoint SlideFigure 8Figure 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 (( ▲) G0/G1, (○) S, and (■) G2/M phase). Note the different concentration scales.High Resolution ImageDownload MS PowerPoint SlideFigure 9Figure 9. Concentration-dependent effects of metal-free ligands (L1–L3) as well as corresponding ruthenium (11a–13a) and osmium complexes (11b–13b), on kinase activities of recombinant Cdk1/cyclin B (top) and Cdk2/cyclin E (bottom).High Resolution ImageDownload MS PowerPoint SlideReferencesARTICLE SECTIONSJump To This article references 45 other publications. 1Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2009, 9, 153– 166[Crossref], [PubMed], [CAS], Google Scholar1Cell cycle, CDKs and cancer: a changing paradigmMalumbres, Marcos; Barbacid, MarianoNature Reviews Cancer (2009), (3), 153-166CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group) A review. Tumor-assocd. cell cycle defects are often mediated by alterations in cyclin-dependent kinase (CDK) activity. Misregulated CDKs induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, mammalian CDKs are essential for driving each cell cycle phase, so therapeutic strategies that block CDK activity are unlikely to selectively target tumor cells. However, recent genetic evidence has revealed that, whereas CDK1 is required for the cell cycle, interphase CDKs are only essential for proliferation of specialized cells. Emerging evidence suggests that tumor cells may also require specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide therapeutic benefit against certain human neoplasias. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXit1ans78%253D md5=2a7eb34b2381e288b3f43ec1bffc0aec2Senderowicz, A. M. Oncogene 2003, 22, 6609– 6620Google ScholarThere is no corresponding record for this reference.3Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770– 1783[Crossref], [PubMed], [CAS], Google Scholar3Cyclin-dependent kinase pathways as targets for cancer treatmentShapiro, Geoffrey I.Journal of Clinical Oncology (2006), (11), 1770-1783CODEN: JCONDN; ISSN:0732-183X. (American Society of Clinical Oncology) A review. Cyclin-dependent kinases (cdks) are crit. regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor-dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B-responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, esp. in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compds. potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD28XksVKgtbg%253D md5=420a31b47f6600625f7d499a4b8a5fc94Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101, 2511– 2526[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.5Malumbres, M.; Pevarello, P.; Barbacid, M.; Bischoff, J. R. Trends Pharmacol. Sci. 2008, 29, 16– 21Google ScholarThere is no corresponding record for this reference.6Bible, K. C.; Kaufmann, S. H. Cancer Res. 1997, 57, 3375– 3380Google ScholarThere is no corresponding record for this reference.7Bible, K. C.; Boerner, S. A.; Kirkland, K.; Anderl, K. L.; Bartelt, D., Jr.; Svingen, P. A.; Kottke, T. J.; Lee, Y. K.; Eckdahl, S.; Stalboerger, P. G.; Jenkins, R. B.; Kaufmann, S. H. Clin. Cancer Res. 2000, 6, 661– 670[PubMed], [CAS], Google Scholar7Characterization of an ovarian carcinoma cell line resistant to cisplatin and flavopiridolBible, Keith C.; Boerner, Scott A.; Kirkland, Kathryn; Anderl, Kari L.; Bartelt, Duane, Jr.; Svingen, Phyllis A.; Kottke, Timothy J.; Lee, Yean K.; Eckdahl, Steven; Stalboerger, Paul G.; Jenkins, Robert B.; Kaufmann, Scott H.Clinical Cancer Research (2000), (2), 661-670CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research) Flavopiridol, the first inhibitor of cyclin-dependent kinases to enter clin. trials, has shown promising antineoplastic activity and is currently undergoing Phase II testing. Little is known about mechanisms of resistance to this agent. In the present study, we have characterized an ovarian carcinoma cell line [OV202 high passage (hp)] that spontaneously developed drug resistance upon prolonged passage in tissue culture. Std. cytogenetic anal. and spectral karyotyping revealed that OV202 hp and the parental low passage line OV202 shared several marker chromosomes, confirming the relatedness of these cell lines. Immunoblotting demonstrated that OV202 and OV202 hp contained similar levels of a variety of polypeptides involved in cell cycle regulation, including cyclin-dependent kinases 2 and 4; cyclins A, D1, and E; and proliferating cell nuclear antigen. Despite these similarities, OV202 hp was resistant to flavopiridol and cisplatin, with increases of 5- and 3-fold, resp., in the mean drug concns. required to inhibit colony formation by 90%. In contrast, OV202 hp and OV202 displayed indistinguishable sensitivities to oxaliplatin, paclitaxel, topotecan, 1,3-bis(2-chloroethyl)-1-nitrosourea, etoposide, doxorubicin, vincristine, and 5-fluorouracil, suggesting that the spontaneously acquired resistance was not attributable to altered P-glycoprotein levels or a general failure to engage the cell death machinery. After incubation with cisplatin, whole cell platinum and platinum-DNA adducts measured using mass spectrometry were lower in OV202 hp cells than OV202 cells. Similarly, after flavopiridol exposure, whole cell flavopiridol concns. measured by a newly developed high performance liq. chromatog. assay were lower in OV202 hp cells. These data are consistent with the hypothesis that acquisition of spontaneous resistance to flavopiridol and cisplatin in OV202 hp cells is due, at least in part, to reduced accumulation of the resp. drugs. These observations not only provide the first characterization of a flavopiridol-resistant cell line but also raise the possibility that alterations in drug accumulation might be important in detg. sensitivity to this agent. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3cXhs1akt7s%253D md5=eab27239203f5f7c8b0229ec4d7d93c78Bible, K. C.; Lensing, J. L.; Nelson, S. A.; Lee, Y. K.; Reid, J. M.; Ames, M. M.; Isham, C. R.; Piens, J.; Rubin, S. L.; Rubin, J.; Kaufmann, S. H.; Atherton, P. J.; Sloan, J. A.; Daiss, M. K.; Adjei, A. A.; Erlichman, C. Clin. Cancer Res. 2005, 11, 5935– 5941[Crossref], [PubMed], [CAS], Google Scholar8Phase 1 Trial of Flavopiridol Combined with Cisplatin or Carboplatin in Patients with Advanced Malignancies with the Assessment of Pharmacokinetic and Pharmacodynamic End PointsBible, Keith C.; Lensing, Janet L.; Nelson, Sacha A.; Lee, Yean K.; Reid, Joel M.; Ames, Matthew M.; Isham, Crescent R.; Piens, Jill; Rubin, Stacie L.; Rubin, Joseph; Kaufmann, Scott H.; Atherton, Pamela J.; Sloan, Jeffrey A.; Daiss, Michelle K.; Adjei, Alex A.; Erlichman, CharlesClinical Cancer Research (2005), (16), 5935-5941CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research) Purpose: Flavopiridol, a cyclin-dependent kinase inhibitor, transcription inhibitor, and DNA-interacting agent, was combined with cisplatin or carboplatin to establish toxicities, evaluate pharmacokinetics, and examine its effects on patient cancers and levels of selected polypeptides in patient peripheral blood mononuclear cells (PBMC). Exptl. Design: Therapy was given every 3 wk. Stage I: cisplatin was fixed at 30 mg/m2 with escalating flavopiridol. Stage II: flavopiridol was fixed at the stage I max. tolerated dose (MTD) with escalation of cisplatin. Stage III: flavopiridol was fixed at the stage I MTD with escalation of carboplatin. Results: Thirty-nine patients were treated with 136 cycles of chemotherapy. Neutropenia was seen in only 11% of patients. Grade 3 flavopiridol/CDDP toxicities were nausea (30%), vomiting (19%), diarrhea (15%), dehydration (15%), and neutropenia (10%). Flavopiridol combined with carboplatin resulted in unexpectedly high toxicities and one treatment-related death. Stable disease ( 3 mo) was seen in 34% of treated patients, but there were no objective responses. The stage II MTD was 60 mg/m2 cisplatin and 100 mg/m2/24 h flavopiridol. As given, CDDP did not alter flavopiridol pharmacokinetics. Flavopiridol induced increased p53 and pSTAT3 levels in patient PBMCs but had no effects on cyclin D1, phosphoRNA polymerase II, or Mcl-1. Conclusions: Flavopiridol and cisplatin can be safely combined in the treatment of cancer patients. Unexpected toxicity in flavopiridol/carboplatin-treated patients attenuates enthusiasm for this alternative combination. Anal. of polypeptide levels in patient PBMCs suggests that flavopiridol may be affecting some, but not all, of its known in vitro mol. targets in vivo. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2MXos1Cisr4%253D md5=2a0cd103f2eead0b4720d4ab3ea600e89Coley, H. M.; Shotton, C. F.; Kokkinos, M. I.; Thomas, H. Gynecol. Oncol. 2007, 105, 462– 469[Crossref], [PubMed], [CAS], Google Scholar9The effects of the CDK inhibitor seliciclib alone or in combination with cisplatin in human uterine sarcoma cell linesColey, Helen M.; Shotton, Christine F.; Kokkinos, Maria I.; Thomas, HilaryGynecologic Oncology (2007), (2), 462-469CODEN: GYNOA3; ISSN:0090-8258. (Elsevier) Objectives: Inhibition of cyclin-dependent-kinases (CDKs) represents an interesting approach in cancer therapy. We have explored this in cell lines of human uterine sarcoma-tumors assocd. with poor survival, chemo-unresponsiveness and deregulation of cell cycle components. We studied the effects of the CDK inhibitor seliciclib (CYC202, R-roscovitine) when used alone or in combination with cisplatin. Methods: Cell lines used: SK-UT-1, SK-UT-1b and SK-LMS-1, the cytotoxicity of seliciclib and cisplatin was measured by the MTT assay. In combination with cisplatin the effects of seliciclib were examd. by isobologram anal. CDK2 levels were examd. at mRNA and protein level by immunoblotting and PCR. We also looked at the effects of seliciclib on p53-dependent response of cells to seliciclib using immunoblotting. The effects of combination treatment were analyzed using annexin V and PI staining by flow cytometric anal. Results: IC50 values for seliciclib were 10.5, 7.1 and 25.7 μM, for SK-UT-1, SK-UT-1b and SK-LMS-1 resp., P53 in the SK-UT-1b (wild-type) and SK-LMS-1 lines (mutant) showed a wild-type response with induction seen with seliciclib treatment for 24 and 48 h. Seliciclib caused a decrease in CDK2 mRNA and protein over 72 h. A combination of cisplatin and seliciclib was synergistic in all three cell lines. Effects of combination treatment were an enhancement in apoptosis as judged by the emergence of a sub-G1 population in cell cycle anal. and a sub-G1 population with PI staining. Conclusions: Our data demonstrate the effectiveness of seliciclib as a single agent and when used in combination with cisplatin where the effects are synergistic. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXksVOnu7k%253D md5=85796769ec1921ca2d149e82c26b77d010Travnicek, Z.; Popa, I.; Cajan, M.; Herchel, R.; Marek, J. Polyhedron 2007, 26, 5271– 5282Google ScholarThere is no corresponding record for this reference.11Malon, M.; Travnicek, Z.; Marysko, M.; Zboril, R.; Maslan, M.; Marek, J.; Dolezal, K.; Rolcik, J.; Krystof, V.; Strnad, M. Inorg. Chim. Acta 2001, 323, 119– 129Google ScholarThere is no corresponding record for this reference.12Dvorak, L.; Popa, I.; Starha, P.; Travnicek, Z. Eur. J. Inorg. Chem. 2010, 3441– 3448Google ScholarThere is no corresponding record for this reference.13Travnicek, Z.; Popa, I.; Cajan, M.; Zboril, R.; Krystof, V.; Mikulik, J. J. Inorg. Biochem. 2010, 104, 405– 417Google ScholarThere is no corresponding record for this reference.14Primik, M. F.; Muehlgassner, G.; Jakupec, M. A.; Zava, O.; Dyson, P. J.; Arion, V. B.; Keppler, B. K. Inorg. Chem. 2010, 49, 302– 311[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.15Ginzinger, W.; Arion, V. B.; Giester, G.; Galanski, M.; Keppler, B. K. Centr. Eur. J. Chem. 2008, 6, 340– 346[Crossref], [CAS], Google Scholar15Synthesis and structural peculiarities of gallium complexes with novel paullone derivativesGinzinger, Werner; Arion, Vladimir B.; Giester, Gerald; Galanski, Markus; Keppler, Bernhard K.Central European Journal of Chemistry (2008), (3), 340-346CODEN: CEJCAZ; ISSN:1895-1066. (Springer GmbH) 9-Bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-ylhydrazine was reacted with 2-acetylpyridine to give a Schiff base as a potential tridentate ligand. The reaction of this ligand with gallium chloride afforded complexes of 1:1 and 2:1 stoichiometry. The results of x-ray diffraction studies of the ligand and both gallium complexes are reported and compared with the data for a related gallium complex with a Schiff base obtained from 9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-ylhydrazine and 2-hydroxybenzaldehyde. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhtVCqsrjK md5=ac62530de701b40e4c8505b1a6fc97e516Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K. Organometallics 2007, 26, 6643– 6652[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.17Schmid, W. F.; John, R. O.; Muehlgassner, G.; Heffeter, P.; Jakupec, M. A.; Galanski, M.; Berger, W.; Arion, V. B.; Keppler, B. K. J. Med. Chem. 2007, 50, 6343– 6355[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.18Schmid, W. F.; Zorbas-Seifried, S.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Roller, A.; Galanski, M.; Chiorescu, I.; Zorbas, H.; Keppler, B. K. Inorg. Chem. 2007, 46, 3645– 3656[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.19Dobrov, A.; Arion, V. B.; Kandler, N.; Ginzinger, W.; Jakupec, M. A.; Rufinska, A.; Graf von Keyserlingk, N.; Galanski, M.; Kowol, C.; Keppler, B. K. Inorg. Chem. 2006, 45, 1945– 1950[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.20Filak, L. K.; Muehlgassner, G.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Arion, V. B.; Keppler, B. K. J. Biol. Inorg. Chem. 2010, 15, 903– 918[Crossref], [PubMed], [CAS], Google Scholar20Organometallic indolo[3,2-c]quinolines versus indolo[3,2-d]benzazepines: synthesis, structural and spectroscopic characterization, and biological efficacyFilak, Lukas K.; Muehlgassner, Gerhard; Jakupec, Michael A.; Heffeter, Petra; Berger, Walter; Arion, Vladimir B.; Keppler, Bernhard K.JBIC, Journal of Biological Inorganic Chemistry (2010), (6), 903-918CODEN: JJBCFA; ISSN:0949-8257. (Springer) The synthesis of Ru(II) and Os(II) arene complexes with the closely related indolo[3,2-c]quinolines N-(11H-indolo[3,2-c]quinolin-6-yl)-ethane-1,2-diamine (L1) and N'-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (L2) and indolo[3,2-d]benzazepines N-(7,12-dihydroindolo-[3,2-d][1]benzazepin-6-yl)-ethane-1,2-diamine (L3) and N'-(7,12-dihydroindolo-[3,2-d][1]benzazepin-6-yl)-N,N-dimethylethane-1,2-diamine (L4) [(η6-p-cymene)MII(L1)Cl]Cl, where M is Ru (4) and Os (6), [(η6-p-cymene)MII(L2)Cl]Cl, where M is Ru (5) and Os (7), [(η6-p-cymene)MII(L3)Cl]Cl, where M is Ru (8) and Os (10), and [(η6-p-cymene)MII(L4)Cl]Cl, where M is Ru (9) and Os (11), is reported. The compds. were comprehensively characterized by elemental anal., electrospray ionization mass spectrometry, spectroscopy (IR, UV-visible, and NMR), and x-ray crystallog. (L1·HCl, 4·H2O, 5, and 9·2.5H2O). Structure-activity relations with regard to cytotoxicity and cell cycle effects in human cancer cells as well as cyclin-dependent kinase (cdk) inhibition and DNA intercalation in cell-free settings were established. The metal-free indolo[3,2-c]quinolines inhibit cancer cell growth in vitro, with IC50 values in the high nanomolar range, whereas those of the related indolo[3,2-d]benzazepines are in the low micromolar range. In cell-free expts., these classes of compds. inhibit the activity of cdk2/cyclin E, but the much higher cytotoxicity and stronger cell cycle effects of indoloquinolines L1 and 7 are not paralleled by a substantially higher kinase inhibition compared with indolobenzazepines L4 and 11, arguing for addnl. targets and mol. effects, such as intercalation into DNA. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXkt1aqs7c%253D md5=c86f175ed0278bd443bcad6be29d171f21Lin, R.; Connolly, P. J.; Lu, Y.; Chiu, G.; Li, S.; Yu, Y.; Huang, S.; Li, X.; Emanuel, S. L.; Middleton, S. A.; Gruninger, R. H.; Adams, M.; Fuentes-Pesquera, A. R.; Greenberger, L. M. Bioorg. Med. Chem. Lett. 2007, 17, 4297– 4302Google ScholarThere is no corresponding record for this reference.22Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Connolly, P. J.; Chiu, G.; Li, S.; Emanuel, S. L.; Middleton, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 1243– 1245Google ScholarThere is no corresponding record for this reference.23Chiu, G.; Li, S.; Connolly, P. J.; Middleton, S. A.; Emanuel, S. L.; Huang, S.; Lin, R.; Lu, Y.PCT Int. Appl., WO 2006130673, 2006, 162 pp.Google ScholarThere is no corresponding record for this reference.24Lin, R.; Chiu, G.; Yu, Y.; Connolly, P. J.; Li, S.; Lu, Y.; Adams, M.; Fuentes-Pesquera, A. R.; Emanuel, S. L.; Greenberger, L. M. Bioorg. Med. Chem. Lett. 2007, 17, 4557– 4561Google ScholarThere is no corresponding record for this reference.25Chiu, G.; Yu, Y.; Lin, R.; Li, S.; Connolly, P. J.PCT Int. Appl., WO 2008048502, 2008, 43 pp.Google ScholarThere is no corresponding record for this reference.26Yu, Y.; Lin, R.; Connolly, P. J.PCT Int. Appl., WO 2008048503, 2008, 42 pp.Google ScholarThere is no corresponding record for this reference.27Peacock, A. F. A.; Sadler, P. J. Chem.-Asian J. 2008, 3, 1890– 1899[Crossref], [PubMed], [CAS], Google Scholar27Medicinal organometallic chemistry: designing metal arene complexes as anticancer agentsPeacock, Anna F. A.; Sadler, Peter J.Chemistry - An Asian Journal (2008), (11), 1890-1899CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH Co. KGaA) A review. The field of medicinal inorg. chem. is rapidly advancing. In particular organometallic complexes have much potential as therapeutic and diagnostic agents. The C-bound and other ligands allow the thermodn. and kinetic reactivity of the metal ion to be controlled and also provide a scaffold for functionalization. The establishment of structure-activity relations and elucidation of the speciation of complexes under conditions relevant to drug testing and formulation are crucial for the further development of promising medicinal applications of organometallic complexes. Specific examples involving the design of Ru and Os arene complexes as anticancer agents are discussed. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhsVCjt73L md5=fa4f181bb367f2eea1e33c55399d122a28Bruijnincx, P. C. A.; Sadler, P. J. Adv. Inorg. Chem. 2009, 61, 1– 62Google ScholarThere is no corresponding record for this reference.29Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391– 401Google ScholarThere is no corresponding record for this reference.30Dougan, S. J.; Sadler, P. J. Chimia 2007, 61, 704– 715Google ScholarThere is no corresponding record for this reference.31Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003– 4018Google ScholarThere is no corresponding record for this reference.32Dyson, P. J. Chimia 2007, 61, 698– 703Google ScholarThere is no corresponding record for this reference.33Kuo, D. L. Tetrahedron 1992, 48, 9233– 9236Google ScholarThere is no corresponding record for this reference.34Lynch, B. M.; Khan, M. A.; Teo, H. C.; Pedrotti, F. Can. J. Chem. 1988, 66, 420– 428Google ScholarThere is no corresponding record for this reference.35Georg, G. I.; Tash, J. S.; Chakrasali, R.; Jakkaraj, S. R.PCT Int. Appl., WO 2006023704, 2006, 182 pp.Google ScholarThere is no corresponding record for this reference.36Berdini, V.; O’Brien, M. A.; Carr, M. G.; Early, T. R.; Navarro, E. F.; Gill, A. L.; Howard, S.; Trewartha, G.; Woolford, A. J.-A.; Woodhead, A. J.; Wyatt, P.PCT Int. Appl., WO 2005002552, 2005, 287 pp.Google ScholarThere is no corresponding record for this reference.37Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974, 233– 241Google ScholarThere is no corresponding record for this reference.38Kiel, W. A.; Ball, R. G.; Graham, W. A. G. J. Organomet. Chem. 1990, 383, 481– 496[Crossref], [CAS], Google Scholar38Carbonyl-η-hexamethylbenzene complexes of osmium. Carbon-hydrogen activation by (η-C6Me6)Os(CO)(H)2Kiel, William A.; Ball, Richard G.; Graham, William A. G.Journal of Organometallic Chemistry (1990), (1-3), 481-96CODEN: JORCAI; ISSN:0022-328X. Redn. of (η-C6-Me6)OS(CO)Cl2 with Zn-AcOH-MeOH gives (η-C6Me6)Os(CO)(Cl)H, which can be further reduced with Na[H2Al(OCH2CH2OCH3)2] to (η-C6Me6)Os(CO)H2 (I). Photolysis of I in hydrocarbons (benzene, cyclohexane, neopentane) results in formation of the C-H bond activation products η-C6Me6Os(CO)(R)(H) (II, R = C6H5, C6H11, CH2C(CH3)3, resp.) and free hexamethylbenzene. Independent syntheses of the hydrides II are described as well as syntheses of the complexes (η-C6Me6)Os(CO)(R)2 and (η-C6Me6)Os(CO)(R)Cl. The structure of (η-C6Me6)Os(CO)(cyclohexyl)2 detd. by single crystal X-ray diffraction is reported. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK3cXksF2ntb4%253D md5=c0fdf8c0541bb0776a41ac3db117b6ca39SAINT-Plus, version 7.06a and APEX2; Bruker–Nonius AXS, Inc.: Madison, WI, 2004.Google ScholarThere is no corresponding record for this reference.40Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112– 122[Crossref], [CAS], Google Scholar40A short history of SHELXSheldrick, George M.Acta Crystallographica, Section A: Foundations of Crystallography (2008), (1), 112-122CODEN: ACACEQ; ISSN:0108-7673. (International Union of Crystallography) An account is given of the development of the SHELX system of computer programs from SHELX-76 to the present day. In addn. to identifying useful innovations that have come into general use through their implementation in SHELX, a crit. anal. is presented of the less-successful features, missed opportunities and desirable improvements for future releases of the software. An attempt is made to understand how a program originally designed for photog. intensity data, punched cards and computers over 10000 times slower than an av. modern personal computer has managed to survive for so long. SHELXL is the most widely used program for small-mol. refinement and SHELXS and SHELXD are often employed for structure soln. despite the availability of objectively superior programs. SHELXL also finds a niche for the refinement of macromols. against high-resoln. or twinned data; SHELXPRO acts as an interface for macromol. applications. SHELXC, SHELXD and SHELXE are proving useful for the exptl. phasing of macromols., esp. because they are fast and robust and so are often employed in pipelines for high-throughput phasing. This paper could serve as a general literature citation when one or more of the open-source SHELX programs (and the Bruker AXS version SHELXTL) are employed in the course of a crystal-structure detn. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXhsVGhurzO md5=8f91f10be4a9df74b2a9dad522c71e6e41Burnett, M. N.; Johnson, G. K.ORTEPIII, Report ORNL-6895; Oak Ridge National Laboratory; Oak Ridge, TN, 1996.Google ScholarThere is no corresponding record for this reference.42Marko, D.; Schätzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.; Eisenbrand, G. Br. J. Cancer 2001, 84, 283– 289[Crossref], [PubMed], [CAS], Google Scholar42Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cellsMarko, D.; Schatzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.; Eisenbrand, G.British Journal of Cancer (2001), (2), 283-289CODEN: BJCAAI; ISSN:0007-0920. (Harcourt Publishers Ltd.) The bisindole indirubin has been described, more than 30 yr ago, as being clin. active in the treatment of human chronic myelocytic leukemia. However, the underlying mechanism of action has remained unclear. The authors have reported previously that indirubin and its analogs are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, the authors investigated the influence of indirubin and derivs. on CDK1/cyclin B kinase in human tumor cells at concns. known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G1/G0 phase of the cell cycle in the presence of 2 μM indirubin-3'-monoxime. At higher drug concns. (≥ 5 μM) an increase of the cell population in the G2/M phase is addnl. obsd. Cells synchronized in G2/M phase by nocodazole remain arrested in the G2/M phase after release, in the presence of indirubin-3'-monoxime (≥5 μM). After 24 h treatment with 10 μM indirubin-3'-monoxime a sub-G2 peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concns. of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a redn. of cyclin B in complex with CDK1 is obsd. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumor cells is a major mechanism by which indirubin derivs. exert their potent antitumor efficacy. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3MXht1Wgsrk%253D md5=117f30fb03c00c6169f207d24f348a0a43Zeng, Q.; Yao, G.; Wohlhieter, G. E.; Viswanadhan, V. N.; Tasker, A.; Rider, J. T.; Monenschein, H.; Dominguez, C.; Bourbeau, M. P.PCT Int. Appl., WO 2006044860, 2006, 61 pp.Google ScholarThere is no corresponding record for this reference.44Cui, J. J.; Deal, J. G.; Gu, D.; Guo, C.; Johnson, M. C.; Kania, R. S.; Kephart, S. E.; Linton, M. A.; McApline, I. J.; Pairish, M. A.; Palmer, C. L.PCT Int. Appl., WO 2009016460, 2009, 168 pp.Google ScholarThere is no corresponding record for this reference.45Kenda, B.; Quesnel, Y.; Ates, A.; Michel, P.; Turet, L.; Mercier, J.PCT Int. Appl., WO 2006128693, 2006, 258 pp.Google ScholarThere is no corresponding record for this reference.Supporting InformationSupporting InformationARTICLE SECTIONSJump ToAssigned NMR (1H, 13C, 15N) signals for L1–L3, 11a–13a, 11b–13b (Tables S1–S3); ORTEP 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)(L2)]+ in 12b (Figure S2); stability of complexes in solution; time-dependent UV–vis spectra of L1, 11a, and 11b in MeOH (Figure S3); time-dependent UV–vis spectra of L3, 13a, and 13b in MeOH (Figure S4); time-dependent UV–vis spectra of 11a in H2O for 48 h (Figure S5); crystallographic data for 2·0.5H2O, 11b·4H2O, 12b, 12b·2CH3OH·2H2O, 13c, 13d·CH3OH, and 13e·0.75CH3OH·0.25H2O (in CIF format). 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