Syntheses of new ruthenium (II and III)-nitroimidazole complexes

Inorganica Chimica Acta 489 (2019) 100–107

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Research paper

Syntheses of new ruthenium (II and III)-nitroimidazole complexes a

b

Ian R. Baird , Kirsten A. Skov , Brian R. James a b

a,⁎

T

Department of Chemistry, The University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada Advanced Therapeutics, British Columbia Cancer Research Centre, Vancouver, BC V5Z 1L3, Canada

ARTICLE INFO

ABSTRACT

Keywords: Nitroimidazoles Metronidazole Etanidazole EF5 Ru complexes

Reactions of [Ru(DMF)6]Tf3 with 2- and 5-nitroimidzoles, including the so-called metronidazole (metro), etanidazole (SR2508), and EF5, of known biological value, have been used to synthesize: (a) hydrates of the three new RuII complexes [RuL6]Tf2,(L = 2NO2Im, 5NO2Im), and [Ru(2Me5NO2Im)5]Tf2; and (b) the two new RuIII complexes [Ru(DMF)4(SR2508)2]Tf3 and [Ru(DMF)2(EF5)2(EtOH)2]Tf3. Use of RuCl3٠3H2O as precursor generates hydrates of the complexes: fac- and mer-RuCl3(2NO2Im)3, mer-RuCl3(metro)3, RuCl3(SR2508)2(EtOH), RuCl3(EF5)2(EtOH), and trans-RuCl2(metro)4; a less well characterized complex is formulated RuCl3(2Me5NO2Im)3٠3CO2 containing what appears to be adsorbed CO2.

1. Introduction The UBC group here, mainly in collaboration with a group at the BC Cancer Agency, has been reporting papers on a wide range of imidazole (Im) complexes of RuII and RuIII for over 30 years, the interest being in their potential as radiosensitizers and hypoxic markers; the first publication appeared in 1986 [1], and the latest one in 2017 [2]. Complexes with nitroimidazoles (NO2Im), methylimidazoles (MeIm), and imidazolecarboxylates ligands, as well as Im itself, have been synthesised [1–7], sometimes with the co-ligands DMSO [3], DMF [4], diketonate [2], or chloride [1,3,5], depending on the Ru precursor used; both cationic and neutral complexes are known. This current paper reports on Ru-NO2Im complexes derived from [Ru(DMF)6]Tf3 (where Tf is the triflate anion CF3SO3), this extending our earlier study using this precursor with Im and MeIm [4]; also included here are related studies using RuCl3٠3H2O as precursor. In vitro evaluation of selected nitroimidazoles and some of the Ru-nitroimidazole complexes noted above has been studied [6]; the aim is to report elsewhere these findings with current on-going similar work that includes some of the new complexes reported in this current paper. Other groups have recently reported on RuII-2NO2Im systems, with interest in hypoxic markers (i.e. tracking oxygen status in tumour tissues) and cancer treatment agents [8,9]. 2. Experimental section 2.1. General 2-Nitroimidazole (2NO2Im), 4/5-nitroimidazole (4/5NO2Im), and

⁎

2-methyl-5-nitroimidazole (2Me5NO2Im) were Aldrich products; 2-(2methyl-5-nitro-1H-imidazol-1-yl)ethanol (metronidazole), and N-(2hydroxyethyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide (etanidazole, also called SR2508) were kindly donated by Drs. C. Koch (Univ. of Pennsylvania) and M. Taylor (MRC, Chilton), respectively, and were used as received. Chart 1 shows the metronidazole and etanidazole structures, as well as EF5, a pentafluorinated derivative of etanidazole synthesized by our procedure [7]; the 18F-EF5 radioactive derivative is of important value in PET imaging [10]. Other commercial chemicals were generally used as purchased, unless stated otherwise. Some solvents were dried using the appropriate drying agent and distilled prior to use: MeOH (Mg), EtOH (Mg), THF (Na-K alloy), Et2O (Na), acetone (K2CO3), and CH2Cl2 (CaH2); CDCl3, d6-acetone and d6-DMSO (from MSD Isotopes) were used immediately upon opening of an ampule to ensure dryness. Various silica materials from Merck were used for chromatography. Syntheses of complexes 1–12 were carried out in a Schlenk tube under air or N2, using the known [Ru(DMF)6]Tf3 [11] as precursor for 1–5, and RuCl3٠3H2O for 6–12; the trichloride (with 39% Ru) was donated by Colonial Metals Inc. Preparative TLCs used silica gel 60, 0.5 mm plates (20 cm × 20 cm) and a 20:1 CH2Cl2:MeOH eluent, unless stated otherwise. Column chromatography was performed using silica gel (230–400 mesh) that was packed down using a pressurized air-flow adapter; Rf values of the pure compounds were measured on Merck silica TLC Al sheets (silica gel 60F254). Dialysis bags (MWCO = 1000) were Sigma-Aldrich products. Elemental analyses (EA) were performed in this department on a Carlo Erba 1106 instrument, with data having an accuracy of ± 0.3%. IR spectra (listed as cm−1) were recorded

Corresponding author. E-mail address: [email protected] (B.R. James).

https://doi.org/10.1016/j.ica.2019.01.033 Received 13 December 2018; Received in revised form 26 January 2019; Accepted 27 January 2019 Available online 29 January 2019 0020-1693/ © 2019 Elsevier B.V. All rights reserved.

Inorganica Chimica Acta 489 (2019) 100–107

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5 NO2

4

N

N

3

2

1

Me metronidazole

OH

N

N

H N

75 °C, the nitroimidazole slowly dissolving during the reaction; after 5 min, the solution was dark green and then became dark blue after 45 min. The mixture was then stirred for 15 h to give a blue-black mixture; solvent was then removed, and EtOH (∼15 mL) added to the residue, the mixture then being sonicated to yield a blue-black suspension. The solid was collected, and washed with hot EtOH (5 × 10 mL). TLC analysis (toluene:MeOH, 15:1) of the solid revealed a single blue spot (Rf = 0) on the baseline and a faint band corresponding to free 4/5NO2Im (Rf = 0.65). The solid was then washed with boiling MeOH until no free 4/5NO2Im was detected in the filtrate by TLC, and then dried in vacuo for 3 d to yield 34.5 mg of 2 (58%). Anal. Calc. (found) for C20H18N18F6S2O18Ru٠3H2O: C, 21.22 (21.06); H, 2.14 (2.30); N, 22.27 (22.02). IR: 3458 (H2O); 3133, 2924, 2852 (C-H); 1654; 1509 (N-Oasym.); 1458; 1377 (N-Osym.); 1245, 1189, 1091, 994, 645. UV–Vis: 344, 672; for free 4/5NO2Im, 286. 1H NMR (CD3OD): δ 8.06 (s, Im-H2), 7.76 (s, Im-H4), 2.69; (D2O) δ 8.12 (s, Im-H2), 7.76 (s, Im-H4). 19F{1H} NMR (CD3OD: −1.34 (s, CF3SO3−).

R

O

NO2 R = CH 2CH2OH ; etanidazole (SR2508)

R = CH 2CF2CF3 ; EF5

Chart 1. Structures of some nitroimidazoles.

(using a KBr disc) on an ATI Mattson Genesis FTIR instrument; some major bands, especially below ∼1300 cm−1, are listed but are not assigned. UV–Vis spectra were recorded for MeOH solutions of the complexes (unless noted otherwise) on an HP 8452A diode array spectrophotometer, and are given as λmax (nm), sh = shoulder. The mass spectra were measured using +LSIMS or DCl on KRATOS Concept IIHQ or MS80 instruments, respectively; NH4+ solutions were used for DCI. Unfortunately only few of the Ru-nitroimidazole complexes displayed acceptable MS spectra using these techniques; MALDI, Electrospray, and EI mass spectrometers were also used, but none yielded useful MS data. The 1H and 19F{1H} NMR spectra, unless stated otherwise, were measured at room temperature (r.t., ∼20 ◦C) on a Varian XL300 spectrometer; the 1H shifts (s = singlet; t = triplet, q = quartet, br = broad) are referenced using residual protonated solvent δH signals: 7.24 (CDCl3), 3.30 (CD3OD), 2.49 (d6-DMSO), and 2.20 (d6-acetone); the 19F{1H} singlet of Tf is referenced to the fluorine shift of trifluoroacetic acid in D2O (external reference). Conductivity measurements, carried out on a RCM151B Serfass conductivity bridge (A.H. Thomas Co.) with a 3403 cell (Yellow Springs Instrument Co.), were calibrated using 0.0100 M aq. KCl solution (ΛM = 141.3 Ω−1 cm2 mol−1 at 25 °C, cell constant = 1.016 cm−1), and data are given in ΛM units ( ± 0.5). Determination of the μeff and number of unpaired electrons for selected paramagnetic RuIII complexes was performed at r.t. using the Evans method [12]; some details are given in the Supplementary Material S1.

2.2.3. [Ru(2Me5NO2Im)5]Tf2٠6H2O (3) As above, [Ru(DMF)6]Tf3 (149 mg, 0.151 mmol) and 2Me5NO2Im (118 mg, 0.928 mmol) were dissolved in MeOH (20 mL) under N2 to give a yellow solution that on being stirred at 75 °C for 5 h changed via green to finally blue; the mixture was then stirred for a further 12 h at 50 °C. Removal of the solvent in vacuo yielded a blue-black residue that (by TLC) contained three new products and unreacted 2Me5NO2Im. The residue was purified using column chromatography (THF:MeOH, 100:1 → 2:1): the nitroimidazole eluted first, followed by a turquoise band after the eluent polarity was increased. This band was dried to give a blue residue to which THF (15 mL) was added; after sonication (5 min) the blue solid was filtered off and washed with THF until the filtrate was free of the nitroimidazole (by TLC); the solid was dried in vacuo for 3 d to yield 20.4 mg of 3 (13%). Anal. Calc. (found) for C22H25N15F6S2O16Ru٠6H2O: C, 23.12 (23.41); H, 3.26 (3.34); N, 17.38 (17.43). IR: 3478 (H2O); 3158, 3046, 2894 (CH); 1569; 1504 (NOasym.); 1464; 1380 (N-Osym.); 1280, 1099, 1029, 815. UV–Vis: 356, 606, 710; for free 2Me5NO2Im, 300. 1H NMR (CD3OD): δ 7.97 (s, ImH4), 2.69, 2.40 (s, Im-2Me); (d6-DMSO) δ 12.86 (br s, Im-H1), 8.24 (s, Im-H4), 2.34 (s, Im-2Me). 19F{1H} NMR (d6-acetone): −1.28 (s, CF3SO3−).

2.2. Complexes synthesized from [Ru(DMF)6]Tf3 2.2.1. [Ru(2NO2Im)6]Tf2٠2H2O (1) In a Schlenk tube, [Ru(DMF)6]Tf3 (71.5 mg, 0.0724 mmol) was dissolved in MeOH (10 mL) under N2 to give a bright yellow solution; 2NO2Im (51.3 mg, 0.454 mmol), dissolved in hot MeOH (5 mL), was then added. The solution became green after being stirred at 70 °C for 1 h, and then slowly darkened, yielding a ‘blue mixture’ after 18 h with a blue precipitate on the walls of the tube; however, upon sonication of the mixture the solid dissolved. Addition of Et2O (25 mL) generated a dark blue precipitate that was collected and washed with Et2O (3 × 5 mL) and then hot MeOH, until no unreacted 2NO2Im was evident in the filtrate (using TLC). The isolated solid was dried in vacuo for 3 d at 80 °C to yield 20.9 mg of 1 (27%); this blue compound was insoluble in EtOH, THF, H2O, DMF, acetone, CH2Cl2, and toluene, slightly soluble in MeOH, and soluble in DMSO, although the solution slowly became yellow, probably via decomposition. Anal. Calc. (found) for C20H18N18F6S2O18Ru٠2H2O: C, 21.57 (21.62); H, 1.99 (1.75); N, 22.64 (22.57). IR: 3405 (H2O); 3253, 2912, 2854 (C-H); 1477 (N-Oasym.); 1360 (N-Osym.), 1326, 1256, 1153, 1029, 975, 803, 642. UV–Vis: 340, 596, 746; for free 2NO2Im, 314. 1H NMR (d6-DMSO): δ 6.22 (br s, overlapping signals of 2NO2Im ligands). 19F{1H} NMR (d6-DMSO): −1.38 (s, CF3SO3−). The NMR data were obtained from the blue solution, made by removing MeOH from the synthesis mixture and then adding d6-DMSO.

2.2.4. [Ru(DMF)4(SR2508)2]Tf3 (4) [Ru(DMF)6]Tf3 (149 mg, 0.151 mmol) and SR2508 (65.5 mg, 0.306 mmol) were dissolved in 5 mL EtOH under air in a Schlenk tube to give a yellow solution that was stirred for 24 h at 55 °C when the colour changed to green, blue, and finally purple. The solution volume was then reduced under vacuum to ∼2 mL when Et2O (30 mL) was added; over a period of 2 h, a purple film deposited on the inside of the tube, and a 5 min sonication released this film that was collected and washed with CH2Cl2 (4 × 10 mL). TLC analysis revealed the presence of two minor species that were removed via a second precipitation of the purple solid with acetone:Et2O (1:20). The purple solid was dried in vacuo at 50 °C for 3 d to give 84.2 mg of 4 (48%). Anal. Calc. (found) for C29H48N12O21S3F9Ru: C, 27.45 (27.25); H, 3.81(3.50); N, 13.25 (13.13). IR: 3337 (NeH); 3142, 2982, 2945 (CH); 1647 (C=ODMF; C=OSR2508 buried within this band); 1561; 1503 (N-Oasym.), 1432; 1360 (N-Osym.); 1281, 1251, 1167, 1064, 1029, 638. UV–Vis: 352, 546; for free SR2508, 316. 1H NMR (d6-DMSO): 22.46 (br s, 12H, DMF-Me1), 19.75 (br s, 12H, DMF-Me2), 8.35 (br t, 2H, -NH-), 7.62 (s, 2H, Im-H5), 7.17 (s, 2H, Im-H4), 5.20 (s, 4H, Im-CH2-), 3.42 (t, 4H, CH2-OH), 3.16 (q, 4H, -NH-CH2-). 19F{1H} NMR (d6-acetone): −1.53 (s, CF3SO3). 2.2.5. [Ru(DMF)2(EF5)2(EtOH)2]Tf3 (5) In a Schlenk tube, EF5 (35.2 mg, 0.117 mmol) was dissolved in 10 mL EtOH to give a colourless solution to which [Ru(DMF)6]Tf3 (50.6 mg, 0.0513 mmol) was added. The tube was sealed under air and the yellow solution was stirred at 50 °C for 24 h to give a pink-purple

2.2.2. [Ru(5NO2Im)6]Tf2٠3H2O (2) As above, [Ru(DMF)6]Tf3 (55.3 mg, 0.0560 mmol) was dissolved in MeOH (15 mL) under N2 to give a yellow solution. 4/5NO2Im (44.4 mg, 0.392 mmol) was then added and the yellow-white slurry was stirred at 101

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solution; the volume was then reduced to ∼1 mL and Et2O (10 mL) was added. After 1 h at r.t. a purple precipitate was seen on the inside of the flask; the solvent was decanted off, and the volume reduced to ∼1 mL followed by addition of Et2O. This procedure was repeated several times until purple solid ceased precipitating from the solution. The combined purple solids were dissolved in MeOH (10 mL), and the solution transferred to a flask where the MeOH was removed to yield a microcrystalline, dark purple solid; its TLC analysis revealed two minor pink products (both with Rf > 0). From the purple band with Rf = 0, the purple solid was loaded onto a fine frit and washed with warm CH2Cl2 until the pink colour was absent in the filtrate. The pure, purple solid was then dried in vacuo for 3 d at r.t. (26.9 mg of 5, 38%). Anal. Calc. (found) for C29H40N10F19S3O19Ru: C, 25.04 (24.99); H, 2.90 (2.99); N, 10.07 (9.92). IR: 3411 (N-H); 3061, 2929 (CH); 1686 (C=OEF5); 1645 (C=ODMF); 1562; 1491 (N-Oasym.); 1429; 1352 (NOsym.); 1251, 1199, 1160, 1030, 639. UV–Vis: 344, 550; for free EF5, 316. 1H NMR (d6-acetone): δ 22.16 (s, DMF-Me1), 19.88 (s, DMF-Me2), 8.20 (br s, Im-H4&5), 5.61 (s, CO-CH2), 4.16 (br s, CH2-CF2), 3.40 (q, CH3CH2OH), 1.14 (t, CH3CH2OH). 19F{1H} NMR (d6-acetone): −1.74 (s, CF3SO3−), −8.13 (s, -CF3), −45.23 (s, -CF2-).

2.3.3. “RuCl3(2Me5NO2Im)3٠3CO2” (8) As above, 2Me5NO2Im (1.43 g, 11.2 mmol) was added to an EtOH solution (25 mL) of RuCl3٠3H2O (319 mg, 1.23 mmol), and the tube was evacuated and then purged with N2. The mixture was stirred at 70 °C for 5 h, when the orange-brown solution was taken to dryness. TLC analysis revealed a series of products and so the mixture was purified using column chromatography (THF). The bands eluted in the following order: band 1 (colourless), band 2 (yellow), band 3 (orange), band 4 (colourless, unreacted 2Me5NO2Im). Analysis of band 3 revealed the presence of 2Me5NO2Im, and so this band was purified further using preparative TLC (THF); the use of preparative TLC for compound purification from the initial product mixture was also successful. The dark orange solid (132 mg of 8, 18%) became brown-black after exposure to air for about 3 h. band 1: LR-MS [DCI(+)]: 270 (M+ (for C8H8N6O4 + NH4+), 253 (M+ + H), 238 (M+ − Me), 220, 206 (M+ − NO2). UV–Vis (MeOH): 218, 238, 278; for free 2Me5NO2Im, 300. 1H NMR (200 MHz, CDCl3): δ 7.02 (s, 14.4, Im-H4), 5.05 (s, 6.2, unassigned), 2.31 (s, 19.5, Im-2Me), 1.47 (s, 172.1, unassigned). See Results and discussion band 3: Anal. Calc. for C12H15N9O6Cl3Ru (8): C, 24.48; H, 2.57; N, 21.41 (with ٠3CO2: C, 25.00; H, 2.10; N, 17.49); found C, 24.93; H, 2.02; N, 17.76. IR: 3457 (N-H); 3153, 2919, 2858, 1137 (C-H); 2370, 2337 (CO2); 1623; 1508 (N-Oasym.), 1476; 1380 (N-Osym.); 1220, 1137. UV–Vis: 212, 234, 358. 1H NMR (CD3OD): [initial spectrum] δ 17.25 (br s, Im-H4, 1H), −3.95 (br s, Im-2Me, 6H), −5.05 (br s, Im-2Me, 3H), −7.90 (br s, Im-H4, 2H); [spectrum after 6 d] δ −3.85 (br s, Im-H4, 3H), −8.65 (br s, Im-2Me, 9H). μeff = 1.7B.M. See Results and discussion.

2.3. Complexes synthesized from RuCl3٠3H2O 2.3.1. mer-RuCl3(2NO2Im)3٠4H2O (6) In a Schlenk tube, 2NO2Im (202 mg, 1.78 mmol) was dissolved in 7 mL EtOH, and the mixture was heated to 75 °C to give a colourless solution. RuCl3٠3H2O (154 mg, 0.595 mmol) was then added with a further 5 mL EtOH. The tube was sealed under air and the mixture was stirred at reflux for 5 h, during which the original orange mixture became green-blue and then a dark blue-black solution. TLC analysis revealed side-products other than the desired dark blue band (Rf = 0). The EtOH was removed to give a blue-black solid that was treated with 15 mL EtOAc and the mixture then sonicated and stirred for 1 h. The resulting, filtered off solid contained (by TLC) 2NO2Im that was removed by washing with hot EtOAc. The solid was washed through the frit with MeOH, this then being removed in vacuo to yield a blue-black solid (87.0 mg of 6, 27%). Anal. Calc. (found) for C9H9N9O6Cl3Ru٠4H2O: C, 17.47 (17.75); H, 2.77 (2.97); N, 17.19 (17.24). IR: 3415 (H2O); 3135, 2923, 2856 (C-H); 1707; 1660; 1508 (NOasym.); 1370 (N-Osym.); 1326, 1224, 1149, 1066, 965, 697. UV–Vis: 210, 268, 368. 1H NMR (d6-DMSO): δ 14.40 (br s, 3H, NH), 7.88 (s, 1H, Im-H5, trans to Cl), 7.82 (s, 2H, Im-H5, trans to 2NO2Im), 7.64 (s, 1H, Im-H4, trans to Cl), 7.37 (s, 2H, Im-H4, trans to 2NO2Im).

2.3.4. mer-RuCl3(metro)3٠2H2O (9) In a Schlenk tube, RuCl3٠3H2O (87.2 mg, 0.364 mmol) and metronidazole (278 mg, 1.62 mmol) were added to 15 mL EtOH; the partially soluble metronidazole dissolved on heating the mixture to 65 °C. After 10 min, the initial brown solution turned orange and an orange precipitate became evident, and after 3 h, a bright orange slurry was present. The tube was then submersed in an ice-bath for 30 min; the orange solid was then collected and washed with warm EtOH (5 × 10 mL). TLC analysis revealed that the orange solid (Rf = 0.70) contained two minor side-products with lower Rf values. The orange solid was dissolved in minimal MeOH and the solution then loaded onto a silica gel column (THF:MeOH, 20:1); the orange band (the 2nd to elute) was collected in a number of fractions that were combined and taken to dryness to yield an orange solid (99.3 mg of 9, 38%). Anal. Calc. (found) for C18H27N9O9Cl3Ru٠2H2O: C, 28.56 (28.73); H, 4.13 (3.99); N, 16.65 (15.96). UV–Vis: 298, 476; for free metronidazole, 312. 1 H NMR (400 MHz, d6-acetone): δ 12.05 (br s, 1H, Im-H4), 10.20 (br s, 2H, CH2), 8.25 (br s, 2H, CH2), 4.85 (s, 3H, Im-2Me), 4.55 (br s, 2H, −OH), 4.35 (s, 6H, Im-2Me), 3.55 (br s, 1H, −OH), −1.20 (br s, 4H, CH2), −3.10 (br s, 4H, CH2), −9.21 (br s, 2H, Im-H4); peak assignments are tentative and are based on the integration ratios and trends observed for complexes 7 and 8, of similar composition and geometry. μeff = 1.8B.M.

2.3.2. fac- and mer-RuCl3(5NO2Im)3٠0.5 EtOH ٠H2O (7) Similar to above, 4/5NO2Im (finely ground, 212 mg, 1.88 mmol) was added to an EtOH solution (10 mL) of RuCl3٠3H2O (97.3 mg, 0.406 mmol); the tube was evacuated, and then purged with N2. The slurry (due to low solubility of 4/5NO2Im) was stirred at 70 °C for 16 h; cooling to r.t. precipitated unreacted 4/5NO2Im that was filtered off using Schlenk techniques. Subsequent, dropwise addition of Et2O to the filtrate gave an orange precipitate that was collected, washed with Et2O (3 × 5 mL) and then dried in vacuo at r.t. (76.5 mg of 7, 34%). After about a month, the sealed filtrate revealed formation of small orange crystals that were filtered off; attempts at removing solvent under vacuum resulted in the crystals becoming opaque, consistent with solvent within the crystal lattice. Anal. Calc. (found) for C9H9N9O6Cl3Ru٠0.5EtOH٠H2O: C, 20.44 (20.42); H, 2.40 (2.31); N, 21.45 (21.28). LR-MS: 547 (M+ − H), 441 (M+ − 3Cl), 435 (M+ − 4/ 5NO2Im). IR: 3148, 2972, 2875 (C-H); 1559; 1525 (N-Oasym.); 1480; 1379 (N-Osym.); 1228, 1090, 814, 643. UV–Vis: 288, 350(sh). 1H NMR (d6-DMSO): [precipitate, fac] δ 13.24 (br s, Im-H2 and 4), 3.39 (q, CH3CH2OH), 1.05 (t, CH3CH2OH); [crystals, mer] δ 4.71 (br s, 2H, 5NO2Im trans to Cl), 3.35 (q, CH3CH2OH), 1.03 (t, CH3CH2OH), −14.15 (br s, 4H, 5NO2Im mutually trans). μeff = 1.5B.M.

2.3.5. RuCl3(SR2508)2(EtOH)٠3H2O (10) In a Schlenk tube, RuCl3٠3H2O (82.9 mg, 0.346 mmol) and SR2508 (260.1 mg, 1.21 mmol) were dissolved in 10 mL EtOH to yield an orange-brown solution. The mixture was stirred under 1 atm N2 at 75 °C for 2 h with a colour change to green and then dark blue, including formation of a blue-black precipitate; this was collected, washed with EtOH (3 × 10 mL) and dried in vacuo at r.t. for 3 d to yield 136 mg of 10, 62%). Anal. Calc. (found) for C16H26N8O9Cl3Ru٠3H2O: C, 26.11 (26.07); H, 4.38 (3.91) ; N, 15.23 (14.84). IR: 3327 (br, H2O + N-H); 3124, 2944, 2878 (CH); 1761; 1677 (C=O); 1627; 1506 (N-Oasym.), 1421, 1351 (N-Osym), 1064, 766, 509. UV–Vis (H2O): 326, 522, 760. 1H 102

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NMR (d6-DMSO): no peaks observed other than those corresponding to free EtOH. The UV–Vis spectrum in water changed with time, showing shifts of λmax to 322 and 518 nm, with the 760 nm band remaining unchanged; after 8 h at r.t., the solution was taken to dryness to yield a purple solid that was dried in vacuo at r.t. Anal. Calc. (found) for C14H22N8O9Cl3Ru٠2H2O [RuCl3(SR2508)2(H2O) ٠2H2O]: C, 24.38 (24.45); H, 3.80 (3.62); N, 16.24 (16.06). IR: 3319 (br, H2O + N-H); 3108, 2938 (C-H); 1672 (C=O); 1631; 1494 (N-Oasym.); 1358 (N-Osym.); 1189, 1110, 1059. μeff = 1.9B.M. (D2O, see Supplementary Material, S1). Conductivity of 10 (H2O): ΛM increased over ∼48 h from ∼35 to 372 ohm1mol−1cm2 (blue → purple → burgundy), implying a final 3:1 electrolyte.

complexes were fully characterized including X-ray structures. The reduction of RuIII to RuII was attributed to either the imidazole and/or the MeOH solvent used in the syntheses, both these reagents being known to reduce transition metals, including RuIII species [13–16]. The first part of the experimental work reported in this paper describes similar methodology extended to a range of nitroimidazoles to form the L = 2NO2Im and 5NO2Im complexes; however, with L = 2Me5NO2Im, the product is [RuIIL5]Tf2; all these three RuII complexes (1–3) contain solvated H2O, and the reductant is almost certainly MeOH (see below). In contrast, from the reaction of the RuIII precursor with SR2508 in EtOH, [RuIII(DMF)4(SR2508)2]Tf3 is isolated and, similarly with EF5, the product is [RuIII(DMF)2(EtOH)2 (EF5)2]Tf3, neither species being solvated. Section 2.3 of the experimental section describes use of the commercially available RuCl3٠3H2O as the precursor, although this is known to be a mixture of RuIII and RuIV species [17]; worth noting is that mmol amounts of RuCl3٠3H2O used in the syntheses are based on the molecular weight of a trichloride hydrate. Use of EtOH as solvent in these syntheses leads to isolation of mer-RuCl3(2NO2Im)3, fac- and merRuCl3(5NO2Im)3, RuCl3(metro)3, RuCl3(SR2508)2(EtOH), and RuCl3(EF5)2(EtOH); reaction with 2Me5NO2Im gives a less well characterized product, tentatively and speculatively written as “RuCl3(2Me5NO2Im)3٠3CO2”. The data are consistent with MeOH being the reductant in the [Ru(DMF)6]Tf3 reactions, whereas EtOH is not a sufficiently strong reductant. The RuIII products from the more readily available RuCl3٠3H2O as precursor for the reactions in EtOH support this suggestion. The reaction with metronidazole does generate RuCl2(metro)4 and, although carried out in MeOH, does require an H2 pre-treatment for the reduction. The reduction of RuCl3 in refluxing MeOH is known to form an ill-defined “blue solution” [18]. The syntheses using either of the Ru precursors are not simple, often requiring TLC to remove unreacted nitroimidazole; the yields are moderate at best, varying from 13 to 62%.

2.3.6. RuCl3(EF5)2(EtOH)٠3H2O (11a) and RuCl3(EF5)2 (11b) In a 100 mL round-bottom flask, RuCl3٠3H2O (29.6 mg, 0.114 mmol) and EF5 (101 mg, 0.334 mmol) were dissolved in 15 mL EtOH, and the brown-orange mixture was stirred at 60 °C under 1 atm N2 for 2 h. A small aliquot was then removed and diluted with EtOH in a 1 cm UV–Vis cuvette, the spectrum of the pale purple solution revealing a λmax at 530 nm. The remaining mixture was then diluted by addition of 35 mL EtOH and stirred in air at reflux for 6 h; the final dark purple solution contained (via TLC analysis) unreacted EF5 and a single product (Rf = 0). The volume was reduced to ∼5 mL under vacuum and the solution then transferred to a dialysis bag (MWCO = 1000) that was sealed and submersed in EtOH (1 L); stirring for 16 h resulted in a pink external EtOH solution and a purple-black internal EtOH solution which, according to TLC, contained no free EF5. The EtOH was removed from the internal solution to yield the purple 11a (45.0 mg, 49%). Anal. Calc. (found) for C18H20N8O7F10Cl3Ru٠3H2O: C, 23.71(24.09); H, 2.87 (2.65); N, 12.29 (11.68). LR-MS [-LSIMS]: 912 (M+ + 3H2O), 894 (M+ + 2H2O), 876 (M+ + H2O), 301 (EF5). IR: 3318 (H2O + N-H); 3117, 2984 (C-H); 1762; 1686 (C]O); 1555; 1492 (N-Oasym.); 1346 (N-Osym.); 1195, 1155, 1104, 1023. UV–Vis: 308, 530. 1 H NMR (CD3OD): trace δ signals due to free EF5. μeff = 1.9B.M. (d6acetone). Drying the purple solid at 80 °C in vacuo resulted in a green solid: Anal. Calc. (found) for C16H14N8O6F10Cl3Ru [RuCl3(EF5)2] (11b): C, 23.67 (23.86); H, 1.74 (1.99); N, 13.80 (13.48). Dissolution of the green solid in EtOH revealed no 530 nm peak, but after ∼1 h the solution became purple with a 530 nm λmax. Dissolution of the green solid in H2O over 5 days gave a pink solution with an increase in conductivity to that of a 3:1 electrolyte (Table S2), that is likely cis-[Ru (EF5)2(H2O)4]3+ (see Results and discussion).

3.2. Complexes formed from [Ru(DMF)6]Tf3 3.2.1. Hexakis(nitroimidazole)-RuII complexes, and RuIII DMF-SR2508 and -EF5 complexes Analogous to the results obtained for the imidazole and methylimidazole complexes [4], nitroimidazole species were also isolated in the RuII oxidation state, when the syntheses were carried out in MeOH, the in situ reductant of the RuIII, since nitroimidazoles themselves are oxidants. Indeed, their ready acceptance of electrons is the key property for their use as radiosensitizers (see Introduction) [1,2,19]. Reaction of excess nitroimidazole with [Ru(DMF)6]Tf3 in refluxing MeOH (under an inert atmosphere) produces aqua-solvated species of [RuL6]Tf2 when L = 2NO2Im and 5NO2Im (complexes 1 and 2, respectively), and [RuL5]Tf2 when L = 2Me5NO2Im (3). The reaction of 2NO2Im with [Ru(DMF)6]Tf3 in CD3OD was monitored by 1H NMR spectroscopy, and the data support successive displacement of DMF ligands by 2NO2Im (see Figs. 1 and 2): as the reaction proceeds, the new 1H peaks likely correspond to [Ru(DMF)6x(2NO2Im)x]Tf3 complexes, where x = 2, 4, 6. A coordinated 2NO2Im ligand exhibits a strong trans effect due to the electron-withdrawing NO2-group [20], and the 1H NMR peak pattern (Fig. 2) suggests that the DMF ligands are displaced two at a time. The upfield shift of the Me signals of the remaining DMF ligands that occurs upon substitution is not understood; however, all RuIII-imidazole [16,21,22] and -nitroimidazole [20] complexes display upfield signals (δ −5 to −20). Hence, successive addition of nitroimidazoles to give 1, and/or the change in the oxidation state, might lead to this upfield shift (a → b → c, see Figs. 1 and 2); however, the Ru is probably reduced to the 2+ state after substitution of all the DMF ligands, as the NMR data show paramagnetic isotropic shifts for each species. The observation of broad peaks in the upfield region of the spectrum (δ −14 to −23) also supports the formation of a RuIII-2NO2Im complex prior to reduction to

2.3.7. trans-RuCl2(metro)4٠H2O (12) In a Schlenk tube, RuCl3٠3H2O (106 mg, 0.441 mmol) was dissolved in 10 mL MeOH and the resulting brown solution was stirred under a stream of H2 at 65 °C, the colour changing over 3 h to navy blue (via orange and green). Metronidazole (308 mg, 1.80 mmol) was then added, and the mixture became yellow-green after 20 min, and then brown after 1 h. Stirring for 14 h under 1 atm H2 generated a purple precipitate and a red-brown filtrate. The solid was isolated, washed with MeOH (3 × 10 mL) and dried in vacuo (46.9 mg of 12, 26%). Anal. Calc. (found) for C24H32N12O12Cl2Ru٠H2O: C, 33.11 (33.09); H, 3.94 (4.21); N, 19.31 (18.97). IR: 3385 (H2O); 3151, 3112, 3028, 2930, 2882 (C-H); 1738; 1647 (C-OH); 1544; 1472 (N-Oasym.); 1425; 1345 (NOsym.); 1259, 1179, 1146, 1056, 866, 828, 740. UV–Vis (MeOH): 304, 494. 1H NMR (200 MHz, d6-acetone): δ 7.91 (s, Im-H4), 4.23 (br s, CH2), 3.91 (br s, OH), 3.60 (br s, CH2), 2.05 (s, Im-2Me). 3. Results and discussion 3.1. General comments About 20 years ago, our group reported the syntheses of the complexes [RuIIL6]Tf2 from the RuIII precursor [Ru(DMF)6]Tf3,where L = imidazole, N-methylimidazole, and 5-methylimidazole [4]. The 103

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D D

D Ru D

3+ D Da

D

+ 2 Im -2D

D

2/3+

Im

D

Ru

Db

Im

Im

+ 2 Im -2D

D

Im Ru Im

2/3+ Dc Im

+ 2 Im, e-2D

Im Im

Im Ru Im

2+ Im Im

1

34 D = DM F Im = 2NO2Im

Fig. 1. Proposed stepwise DMF displacement by 2NO2Im for the [Ru(2NO2Im)6]2 (1) synthesis.

RuII. Spin density calculation could be informative about the upfield shift of the Me signals. The isolated 1 was insoluble in all common solvents; the 1H NMR spectrum, obtained by removing MeOH from the reaction mixture and then dissolving the residue in d6-DMSO, shows one broad singlet (δ 6.22) for all 6 equiv. 2NO2Im ligands; the H4- and H5-Im proton signals overlap, and free 2NO2Im was observed in the in situ spectrum at δ 7.35. The upfield proton shift signals upon coordination of the 2NO2Im to RuII follow a different trend to those seen for the Im and MeIm complexes, where H5 shifts downfield and H4 shifts upfield [4]; the coordination removes the degeneracy of H4 and H5 (that are equivalent in the free ligand due to tautomerization), and only one broad singlet is seen. The broadening is likely an electronic effect incurred by the 2nitro group, as this phenomenon is not seen, for example, in the RuII2MeIm complexes [4]. Of note, no C2 peak is seen in the 13C NMR spectra of free 2-nitroimidazoles [10], possibly because of the electronwithdrawing ability of the NO2 group. [Ru(5NO2Im)6]Tf2 (2) was soluble in D2O, sparingly soluble in MeOH, and insoluble in other solvents; the penta-coordinated 2Me5NO2Im analogue (3) was sparingly soluble in MeOH and in DMSO. Coordination of 5NO2Im and 2Me5NO2Im to RuII leads to an upfield shift in the 1H signals due to the π-donor ability of RuII. For 2, the H2 and H4 peaks are observed at δ 8.06 and 7.76 in CD3OD, respectively (cf. δ 8.35 and 7.85 for the free ligand). For 3, the coordination upfield shifts of H4 in d6-DMSO (δ 8.28 to 8.24), and that for the 2Me group (δ 2.36 to 2.34) are small. For 2 and 3 in CD3OD, an unidentified 1H signal appears at δ 2.69. The elemental analysis for 1 is consistent with a hexa-coordinated 2NO2Im formulation with 2 solvate H2O molecules. The presence of H2O is supported by solid state IR data (νO-H at 3405 cm−1) and is commonly observed for several of the isolated nitroimidazole complexes (e.g. complexes 2 and 3 contain 3 and 6 mol of H2O, respectively); such solvation was also established crystallographically within the structures [Ru(Im)6]CO3٠5H2O and [Ru(NMeIm)6]Cl2٠2H2O [16,23]. The (2NO2Im)6 species 1 was not expected because of the steric

position of the NO2-group. Others have reported that reactions of Ru with NMeIm and 5MeIm do not occur with 2MeIm or 1,2-Me2Im using the same conditions [20,23,24], and we have reported that reaction of 2MeIm with [Ru(DMF)6]Tf3 gives [Ru(DMF)2(2MeIm)4]Tf2 [4]. A ChemDraw SymApps model reveals that the Me and NO2 groups are of similar size, and presumably the NO2 planarity allows for a (2NO2Im)6 configuration in 1. In 3, the steric demand of the 2Me position could dominate, as only five 2Me5NO2Im ligands coordinate. Of note, the complex RuIIH(CO)(PPh3)2L, where L is a deprotonated, N-O bonded ‘2Me5NO2Im’, has been characterized crystallography [25]; such ƞ2bonding within one 2Me5NO2Im ligand in 3 would imply a 6-coordinate RuIII species, but the elemental analysis and NMR data are consistent with the given formulation. In the hexa-coordinated 2, the nitroimidazole is assumed to involve the less sterically hindered 5NO2 tautomer, comparable to data found for the structurally characterized [Ru(5MeIm)6]Tf2 [4]; complexes with 4MeIm can exist, but are isolated in much smaller quantities than the 5MeIm isomer [26]. The qualitative UV–Vis spectra for complexes 1–3 reveal similarities and differences. Each spectrum shows a high intensity band in the 340–360 nm range that likely corresponds to a ligand π → π* transition or the t2(Ru) → π*(L) transition [27]. Complexes 1 and 3 contain two other bands (596 and 746 nm for 1, and 606 and 710 nm for 3): the more intense higher energy band is thought to correspond to the spinallowed singlet-singlet transition, and the weaker band is likely the corresponding singlet-triplet transition allowed by the strong spin-orbit coupling in ruthenium [28,29]. Complex 2 shows just one extra band (low intensity at 672 nm) that is presumably a d-d transition. The IR spectra for 1–3 reveal that coordination of the nitroimidazole to RuII shifts both the asymmetric and symmetric νNO bands to slightly lower energies, as observed before [30]; e.g., in 1, these respective shifts of the 2-NO2 group are 8 and 10 cm−1 lower from those of the free ligand (1485 and 1370 cm−1). That these bands for 2 (1509 and 1377 cm−1) are similar to those of the 3 (1504 and 1380 cm−1) implies a 5NO2 tautomer in 2: a 4NO2 tautomer is likely to have higher energy νNO values, as seen in the difference between those of free MF5 (1473 and 1368 cm−1, a 2Me5NO2Im) and those of free 2M4NF5 (1506 and

free 2NO2Im free DMF

a

a

b

b

c

c

Fig. 2. 1H NMR spectrum in CD3OD during synthesis of 1; see Fig. 1 for labelling of a, b and c. At zero time, the spectrum shows only broad Me signals at δ 22.5 and 19.8, in agreement with the literature data for [Ru(DMF)6]Tf3 [11]. At the end of the reaction, only the broad singlet of 1 at δ 6.2 is seen. 104

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HN O2N Cl HN

b N NO2

a

a N

Ru

Cl

b

Cl

N NO2 a NH

mer-RuCl3(2NO2Im)3

ppm Fig. 3. 1H NMR spectrum for 6 in d6-DMSO.

1400 cm−1, a 2Me4NO2Im) [10]. Complexes 1–3 were insoluble in most common solvents, except for 2 being soluble in aqueous media. In attempts to synthesize water-soluble complexes (of use for in vitro testing), just 2 equiv. of SR2508 and EF5 were reacted with [Ru(DMF)6]Tf3. The products were the RuIII species, [Ru(DMF)4(SR2508)2]Tf3 (4) and [Ru (DMF)2(EF5)2(EtOH)2]Tf3 (5), respectively; use of MeOH instead of EtOH was unsuccessful due to an apparent instability of these imidazoles in the MeOH. The in situ reduction in the syntheses of the [RuL6]Tf2 complexes (L = Im, 2NO2Im, 5NO2Im) could depend on the number of coordinated (nitro)imidazole ligands and/or the MeOH solvent; this uncertainty was not pursued. The 1H NMR data for 4 at δ 22.46 and 19.75 correspond to the Me groups of coordinated DMF [11]; the 1H peaks of coordinated SR2508 display no significant isotropic shifts like those seen for nitroimidazoles (and imidazoles) within anionic chloro-Ru(III) species [16,26,31]. The IR spectrum of 4 shows νC=O for coordinated DMF at 1647 cm−1 (cf. νC=O of 1673 cm−1 for free DMF); νC=O for the SR2508 ligand (1667 cm−1 for free SR2508) is probably buried within the DMF band. IR bands at 3337 and 3142 cm−1 (attributed to νNH and νCH, respectively) and at 1503 and 1360 cm−1 (νNOasym and νNOsym; cf. 1487 and 1365 cm−1 for free ligand) are from the SR2508 ligand. The 1H NMR spectrum of 5 reveals the presence of EF5, EtOH and DMF, but surprisingly only the shifts for the coordinated DMF Me-groups are paramagnetically shifted (δ 22.16, 19.88); the EF5 and EtOH signals are broadened and shifted downfield from those of the free ligands, as expected upon coordination to the ‘electron deficient’ RuIII. The 19F{1H} signals of δ −8.13 and −45.23 in d6-acetone are essentially the same as those of free EF5 (δ −8.10 and −45.19). The IR spectrum shows νC=O bands for coordinated DMF (1645 cm−1) and EF5 (1686 cm−1). Complexes 4 and 5 have similar electronic spectra, with bands around 350 and 550 nm. Attempts to obtain a mass spectrum for 5 were unsuccessful.

Syntheses of both neutral [42] and anionic [16,21–23,31,43] RuIIIimidazole complexes are well investigated, but the only reported RuIIInitroimidazole complex is [4NO2ImH][RuCl4(5NO2Im)2], this study also noting that the analogous 2Me5NO2Im complex could not be obtained pure [20]. The first report on the use of the readily available RuCl3٠3H2O in syntheses (in EtOH) of imidazole complexes appeared in 1986 [42], examples including formation of the chloro-bridged, Ru2III dimers [Ru2L4Cl6]٠2H2O, [Ru2L’4Cl6] ٠4H2O and [Ru2L’’3(H2O)Cl6], where L = N(vinyl)Im or 4MeIm; L’ = Im or 2Et(4/5MeIm); L’’ = NMeIm or 2MeIm; the more sterically demanding 2Me(1-vinyl)Im gave a monomer mer-RuCl32Me(1-vinyl)3]٠H2O. Later reported syntheses are those of the monomeric mer-complexes RuCl3(NMeIm)2(DMSO) [44], RuCl3(Im)3 [45] and RuCl3(Im)2(DMAD), where DMAD = dimethyladenine [16]. Our EA and spectroscopic data for products from reactions of RuCl3٠3H2O with nitroimidazoles are consistent with monomeric RuIII species. Complexes 6 and 7 provide further examples of the many Ru-Im complexes that contain solvated H2O molecules (see above); 6 shows a strong IR νOH band at 3415 cm−1, and weak bands at 1707 and 1660 cm−1 could also be due to H2O. The 1H NMR spectrum in d6DMSO showed significantly more H2O than present in the solvent, but the amount could not be quantified. Of note, the 2NO2Im signals were little shifted from those of the free ligands, a phenomenon common to all RuIII-2NO2Im complexes (4–6, 10, 11) synthesized during this work. Complex 6 has two sets of two peaks in about a 2:1 ratio (Fig. 3); the 2NO2Im coordination prevents ligand tautomerization, resulting in separate signals for H4 and H5, the 2:1 ratio thus implying a mer-geometry. 1H assignments are based on proximity of the H-atom to the RuIII, and electronegativity arguments where the H-atoms trans to Cl are downfield to those of the mutually trans 2NO2Im ligands; the lower intensity peaks thus correspond to the 2NO2Im protons trans to the Cl. Isolation of the pale orange 7 required Schlenk techniques, because the complex decomposes in air to a black tar over ∼2 h. Drying the solid at ∼80 °C also resulted in decomposition, and so the complex was dried at r.t.; this likely relates to the presence of the EtOH solvate. The 1 H NMR spectrum has one broad peak at δ 13.24 for the H2 and H4 protons that is assigned to three equivalent 5NO2Im ligands in a fac geometry. Over a month, the spectrum changed to two broad peaks at δ 4.71 and −14.15 in a 1:2 ratio, consistent with a more thermally stable mer isomer (Fig. 4); the δ −14.15 signal is similar to that of δ −16.00 for trans-[4NO2ImH][RuCl4(5NO2Im)2] [20]. These data show that the 1 H signals for 5NO2Im ligands trans to Cl are downfield relative to those of mutually trans 5NO2Im ligands. The IR spectra for the isolated solid and crystals of 7 (unfortunately not of X-ray quality) were the same: of four strong, broad bands observed between 1350 and 1650 cm−1, two

3.3. Complexes formed from RuCl3٠3H2O Advantages of chloro-containing nitroimidazole complexes are: (i) they undergo solvolysis in aqueous media [21,31]; (ii) the solvolysis product can bind to the nitrogen bases of DNA [32,33] and histidines in serum proteins [34,35]; and (iii) coordinated nitroimidazoles (to Pt, Ru and Rh) are useful radiosensitizers [1,2,36,37]. In RuIII complexes, the Ru-N bonds are substitution inert [16,38] and thus the species generally remain intact prior to reduction at neutral pH [39], although exceptions are known [40]. Antitumour-active RuIII complexes are likely pro-drugs that are transformed by in vivo reduction into more active DNA-binding RuII complexes [39,41]. 105

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NO2

NO2

HN

HN N

Cl O2N

Ru

N

HN

N Cl

Cl O2N

NO2

N

HN

NH

Cl

N

Ru

Cl Cl

N NH O2N

mer-RuCl mer-RuCl 3(5NO 2Im) 3 3 3(5NO 2Im)

fac-RuCl3(5NO2Im)3 Fig. 4. Geometrical isomerization of 7 in d6-DMSO.

originate from N-O stretching (νNOasym 1525 and νNOsym 1379 cm−1 based on values of 1522 and 1381 cm−1 for trans-[PPh4] [RuCl4(5NO2Im)2]) [20]; specific bands to mer- and fac-isomers could not be identified. The other two bands (1480 and 1559 cm−1) likely correspond to stretching modes of the Im ring. The reaction of RuCl3٠3H2O with 2Me5NO2Im containing the sterically demanding 2Me-group, in contrast to the reaction of this imidazole with [Ru(DMF)6]Tf3 that simply formed complex 3, resulted in some remarkable and incompletely understood findings. As well as an orange RuIII product 8 [formulated RuCl3(2Me5NO2Im)3٠3CO2, see below], two organic products were isolated using preparative TLC. The first band (highest Rf) gave a colourless oil that by DCI(+) MS was a ‘dimer’ formed by loss of two Hatoms from 2Me5NO2Im (Fig. 5 shows plausible structures), but IR and NMR data were not helpful; the UV–Vis spectrum revealed a π → π* band at 278 nm, slightly higher in energy than that of 2Me5NO2Im (300 nm). This dimer was unstable in solution and slowly (or rapidly at higher temperatures) became yellow at r.t. in CDCl3 or MeOH; this refers to the band 2 species mentioned in the experimental data for 8 (Section 2.3); the MS readout for this yellow species showed a parent mass of 438 suggesting even further conglomeration of the ring system. Complex 8, like 7, was air-sensitive, and the EA did not support a RuCl3(2Me5NO2Im)3 formulation. The IR spectrum (Fig. S2) showed bands at 2370 and 2337 cm−1 consistent with the presence of ‘free/ adsorbed’ CO2 (cf. 2360 and 2339 cm−1) in the complex’s lattice, and a formulation with an added 3 mol of CO2 leads to an acceptable EA. When a KBr pellet of 8 was heated to > 200 °C for 5–10 s, the resulting IR spectrum revealed the loss of the CO2 bands; the NO2Im band intensities also decreased suggesting decomposition of the complex. The UV–Vis spectrum for 8 had a 360 nm band similar to those observed for 6 (368 nm) and 7 (350 nm), consistent with coordination of an NO2Im ligand to RuIII [23,31,46]. The 1H NMR spectrum (Fig. S3) initially revealed the presence of two inequivalent 2Me5NO2Im ligands in a 2:1 ratio, supporting a mer geometry. Analogous to data for mer-7, a downfield signal (δ 17.25) is observed for the H4-atom of the ligand trans to Cl, with the corresponding 2Me peak at δ −5.05, while the corresponding peaks for the mutually trans nitroimidazole ligands are observed at δ −7.90 and −3.95, respectively. After 6 days the spectrum shows only two signals (δ −3.85 and −8.65), comparable to those

N

N Me

N

N

N

NO2

N Me

O2N

Me

NO2

NO2 Me

N

of the mutually trans nitroimidazole ligands in a mer complex, and the δ −5.05 and downfield peaks have disappeared; this spectrum may be that of a chloro-bridge dimer species (see above) formed by ligand dissociation of 2Me5NO2Im, which is observed in the δ 0–15 region. The synthesis, EA, and the spectroscopic data for 8 were completely reproducible. However, the source of the CO2 remains a mystery in this complicated 2Me5NO2Im system and has not been pursued – a possibility is that CO2 could be adsorbed on the weakly basic TLC plates used in the preparation of the complex. From the reaction of RuCl3٠3H2O with metronidazole, a 5-NO2Im derivative (Chart 1), the mer-RuCl3L3٠2H2O (9) was isolated, whereas SR2508 and EF5 (2NO2Im derivatives, Chart 1) gave the complexes RuCl3L2(EtOH) ٠3H2O(10 and 11a),presumably because of the bulkier 2NO2Im ligands. The EtOH is weakly bonded and readily displaced by H2O; e.g., an initially blue aqueous solution of 10 became purple over 8 h, from which RuCl3(SR2508)2(H2O)٠2H2O was isolated; this exchange reaction was monitored by UV–Vis spectroscopy, the 326 and 522 nm peaks of 10 changing to 322 and 518 nm. The EtOH of 11a was also removed by vacuum at 80 °C to give RuCl3(EF5)2 (11b) as a green solid; this process was reversible, and dissolution of the 5-coordinate species in EtOH gave an initially green solution that after 1 h became purple with a 530 nm peak, identical to that of an EtOH solution of 11a. The solvolysis of the antitumour complex [RuCl4Im2] − at r.t. involves three successive aquation steps [22], the first correlating well with the initial binding profile of the species to DNA [47]. For [RuCl4(5NO2Im)2] −, the aquation reaction (in D2O) is much slower, not being detected by 1H NMR until after 3 days [20]. The solvolysis of 10 and 11b was more readily followed by conductivity changes in H2O and, compared to [RuCl4Im2]−, the Cl− ligands are relatively labile; e.g., for 11b, the first and second aquation steps are complete in ∼8 and 18 h, respectively, compared to 22 h for the first aquation step of [RuCl4Im2] −. Complete displacement of the final Cl− ligand in 11b takes ∼120 h, whereas the 3rd aquation step of [RuCl4Im2] − is only 30% complete after 8 months [22]. These findings are consistent with the scheme shown in Fig. 6, in which an 2NO2Im ligand leads to labilization of a trans chloride; the third Cl− is more difficult to displace as it is trans to H2O. The paramagnetic susceptibilities in solution of some trichloro RuIII complexes (7–11) were measured by Evans’ NMR solution method (see Supplementary) and, in each case, μeff values correspond to the presence of one unpaired electron in a low spin, +3 oxidation state. Attempts to synthesize (dichloro)nitroimidazole-RuII complexes from “Ru blue” solutions (formed by reducing RuCl3٠3H2O with H2 [18]) were successful only in one case, namely trans-RuCl2(metro)4 (12). The coordination of the metronidazole changed its UV–Vis π → π* transition from 312 to 304 nm, and an MLCT band seen at 494 nm is typical of RuII heterocyclic complexes [48]. The 1H NMR spectrum contains broad signals, typical for nitroimidazole complexes even for a diamagnetic RuII species. The assignments were made according to coupling observed in the 2D COSY spectrum, and the relative

NH Me

HN O2N

N NO2 N

NH Me

Fig. 5. Possible configurations of a dimer formed from 2Me5NO2Im. 106

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Cl L

Cl

Cl

Ru

L

+ H2O

-EtOH

EtOH

H2O L

OH2 Ru OH2

OH 2 L

3+

Cl L

+ H2O H2O L - Cl

Cl Ru OH 2

Cl Ru OH2

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Cl L

OH2 L

+ H2O - Cl 2+

- Cl

Cl L

Cl Ru OH2

OH2

+

L

+ H2O

Fig. 6. Successive aquation of 10 (L = SR2508) and 11a (L = EF5).

integrations of the peaks. The presence of only a single set of ligand peaks suggests a trans geometry for 12. 4. Conclusions Five new Ru complexes of nitroimidazoles have been synthesized using [Ru(DMF)6]Tf3 as precursor; seven other complexes are made from the commercially available RuCl3٠3H2O as precursor. The nitroimdazoles used are 2- or 5-NO2 derivatives, including metronidazole, etanidazole and EF5, of known biological value. The aim is to extend our library of Ru complexes of many imidazole ligands gathered over 30 years, and report soon on a selection of such species regarding their in vitro properties such as accumulation (and aerobic and hypoxic toxicity) in cells, binding to DNA, and radiosensitizing properties; of the complexes reported in this paper, data for those containing SR2058 (4 and 10) or EF5 (5, 11a) are currently being collected. Acknowledgements We thank the Natural Sciences and Engineering Research Council of Canada (NSERC, Grant CRDP J 234014) and the Medical Research Council of Canada for financial support, C. Koch (University of Pennsylvania) and M. Taylor (MRC, Chilton) for etanidazole and metronidazole, respectively, and Colonial Metals Inc. for the RuCl3٠3H2O. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ica.2019.01.033. References [1] P.K.L. Chan, K.A. Skov, B.R. James, N.P. Farrell, J. Radiat. Oncol. Biol. Phys. 12 (1986) 1059–1062. [2] I.R. Baird, B.O. Patrick, K.A. Skov, B.R. James, Inorg. Chim. Acta 466 (2017) 565–577. [3] P.K.L. Chan, K.A. Skov, B.R. James, Int. J. Radiat, Biol. 5249 (1987). [4] I.R. Baird, S.J. Rettig, B.R. James, K.A. Skov, Can. J. Chem. 76 (1998) 1379–1388. [5] D.C. Kennedy, B.R. James, Inorg. Chem. Commun. 78 (2017) 32–36. [6] I.R. Baird, Ph.D. Thesis, Univ. of B.C., Vancouver, 1999. [7] I.R. Baird, K.A. Skov, B.R. James, S.J. Rettig, C.J. Koch, Synth. Commun. 28 (1998) 3701–3709.

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