Ruthenafuran Complexes Supported by the Bipyridine-Bis(diphenylphosphino)methane Ligand Set: Synthesis and Cytotoxicity Studies

Mononuclear and dinuclear Ru(II) complexes cis-[Ru(κ2-dppm)(bpy)Cl2] (1), cis-[Ru(κ2-dppe)(bpy)Cl2] (2) and [Ru2(bpy)2(μ-dpam)2(μ-Cl)2](Cl)2 ([3](Cl)2) were prepared from the reactions between cis(Cl), cis(S)-[Ru(bpy)(dmso-S)2Cl2] and diphosphine/diarsine ligands (bpy = 2,2′-bipyridine; dppm = 1,1-bis(diphenylphosphino)methane; dppe = 1,2-bis(diphenylphosphino)ethane; dpam = 1,1-bis(diphenylarsino)methane). While methoxy-substituted ruthenafuran [Ru(bpy)(κ2-dppe)(C^O)]+ ([7]+; C^O = anionic bidentate [C(OMe)CHC(Ph)O]− chelate) was obtained as the only product in the reaction between 2 and phenyl ynone HC≡C(C=O)Ph in MeOH, replacing 2 with 1 led to the formation of both methoxy-substituted ruthenafuran [Ru(bpy)(κ2-dppm)(C^O)]+ ([4]+) and phosphonium-ring-fused bicyclic ruthenafuran [Ru(bpy)(P^C^O)Cl]+ ([5]+; P^C^O = neutral tridentate [(Ph)2PCH2P(Ph)2CCHC(Ph)O] chelate). All of these aforementioned metallafuran complexes were derived from Ru(II)–vinylidene intermediates. The potential applications of these metallafuran complexes as anticancer agents were evaluated by in vitro cytotoxicity studies against cervical carcinoma (HeLa) cancer cell line. All the ruthenafuran complexes were found to be one order of magnitude more cytotoxic than cisplatin, which is one of the metal-based anticancer agents being widely used currently.


Reactions between the Metal Precursors and Phenyl Ynone
Reactions between phenyl ynone HC≡C(C=O)Ph and metal precursors 1, 2, and [3](Cl)2 were investigated. While the ynone did not react with [3](Cl)2, the reactions between HC≡C(C=O)Ph and metal precursors 1 and 2 led to different products, depending on the reaction conditions (Scheme 3a

Reactions between the Metal Precursors and Phenyl Ynone
Reactions between phenyl ynone HC≡C(C=O)Ph and metal precursors 1, 2, and [3](Cl)2 were investigated. While the ynone did not react with [3](Cl)2, the reactions between HC≡C(C=O)Ph and metal precursors 1 and 2 led to different products, depending on the reaction conditions (Scheme 3a). A mixture of methoxy-substituted ruthenafuran

Reactions between the Metal Precursors and Phenyl Ynone
Reactions between phenyl ynone HC≡C(C=O)Ph and metal precursors 1, 2, and [3](Cl) 2 were investigated. While the ynone did not react with [3](Cl) 2 , the reactions between HC≡C(C=O)Ph and metal precursors 1 and 2 led to different products, depending on the reaction conditions (Scheme 3a). A mixture of methoxy-substituted ruthenafuran The molecular structures for [4](ClO4) and [5](OTf)⋅CH2Cl2 were determined by Xray crystallography ( Figure 2). The α-metallafuran structure (metal fragment at the α-carbon atom of the original furan) in [4] + is apparently a result of a rearranged [HC≡C(C=O)Ph + − OMe] structure on Ru center, therefore the formation of [4] + was due to a combination of an alkyne−vinylidene rearrangement of HC≡C(C=O)Ph on the Ru center, followed by a nucleophilic attack by − OMe (originated from the solvent MeOH) and Oynone coordination to Ru (Scheme 3b). The five-membered metallacycle is essentially planar with the sum of interior angle close to 540° (539.5°). The Ru−C and C−C distances in the metallacycle (1.964 (6)   The molecular structures for [4](ClO 4 ) and [5](OTf)·CH 2 Cl 2 were determined by X-ray crystallography ( Figure 2). The α-metallafuran structure (metal fragment at the α-carbon atom of the original furan) in [4] + is apparently a result of a rearranged [HC≡C(C=O)Ph + − OMe] structure on Ru center, therefore the formation of [4] + was due to a combination of an alkyne-vinylidene rearrangement of HC≡C(C=O)Ph on the Ru center, followed by a nucleophilic attack by − OMe (originated from the solvent MeOH) and O ynone coordination to Ru (Scheme 3b). The five-membered metallacycle is essentially planar with the sum of interior angle close to 540 • (539.5 • ). The Ru-C and C-C distances in the metallacycle (1.964(6) and 1.413(8)-1.414(8) Å, respectively) revealed the partial double bond character in the Ru-C and C-C bonds and supported the resonance representation (see Scheme 1c). [5] + features a neutral tridentate PˆCˆO pincer ligand [(Ph) 2 PCH 2 P(Ph) 2 CCHC(Ph)O], which could be formed as a result of an alkyne-vinylidene rearrangement of HC≡C(C=O)Ph on the Ru center, followed by a nucleophilic attack by a P dppm atom and O ynone coordination to Ru (Scheme 3b). The [Ru(PˆCˆO)] moiety is a bicyclic system comprising a planar αmetallafuran (sum of interior angle = 539.9 • ) fused with a five-membered CˆP-chelate ring adopting an envelope conformation with the CH 2 unit on the dppm as the flap. Again, the Ru-C and C-C distances in the metallacycle (1.967(4) and 1.375(5)-1.441(5) Å, respectively) revealed the partial double bond character in the Ru-C and C-C bonds.  (7), Ru1−O1 2.127(4), C1−O2 1.339 (7), The formation of [4] + and [5] + in the reaction between HC≡C(C=O)Ph and 1 revealed that there are two competing reactions for the Ru−vinylidene intermediate, namely intermolecular nucleophilic attack by − OMe originated from the solvent MeOH, and intramolecular nucleophilic attack by the auxiliary ligand dppm. However, such competition was not observed in the reaction between HC≡C(C=O)Ph and 2 in MeOH, where methoxysubstituted ruthenafuran [Ru(bpy)(κ 2 -dppe)(C^O)] + ([7] + ) was found to be the only product (Scheme 3a). The difference in reactivity between 1 and 2 may be attributed to their difference in natural bite angle (72° for dppm; 85° for dppe) [48], as bidentate ligands are known to be more labile due to the ring strain. The molecular structure for [7](OTf)⋅CH2Cl2 was also determined by X-ray crystallography (  The formation of [4] + and [5] + in the reaction between HC≡C(C=O)Ph and 1 revealed that there are two competing reactions for the Ru-vinylidene intermediate, namely intermolecular nucleophilic attack by − OMe originated from the solvent MeOH, and intramolecular nucleophilic attack by the auxiliary ligand dppm. However, such competition was not observed in the reaction between HC≡C(C=O)Ph and 2 in MeOH, where methoxysubstituted ruthenafuran [Ru(bpy)(κ 2 -dppe)(CˆO)] + ([7] + ) was found to be the only product (Scheme 3a). The difference in reactivity between 1 and 2 may be attributed to their difference in natural bite angle (72 • for dppm; 85 • for dppe) [48], as bidentate ligands are known to be more labile due to the ring strain. The molecular structure for [7](OTf)·CH 2 Cl 2 was also determined by X-ray crystallography ( Figure 3); the structural parameters on the metallafuran moiety (Ru-C, C-C, and C-O distances are 1.982(3), 1.393(4)-1.399(4), and 1.276(3) Å, respectively; sum of interior angle = 539.8 • ) were found to be very similar to those in [4] Attempts were made to improve the yield of the phosphonium-ring-fused bicyclic ruthenafuran [5] + by changing the reaction solvent from MeOH to THF. While no reaction was observed between HC≡C(C=O)Ph and 1 in dry THF, performing the reaction in a mixture of THF and H 2 O led to the formation of a mixture of [5] + and carbonyl complex [Ru(bpy)(κ 2 -dppm)(CO)(Cl)] + ([6] + , Scheme 3a). The role of H 2 O in this reaction is unknown, and the carbonyl complex is likely a result of an oxidative cleavage of a vinylidene species [49,50] uct (Scheme 3a). The difference in reactivity between 1 and 2 may be attributed to their difference in natural bite angle (72° for dppm; 85° for dppe) [48], as bidentate ligands are known to be more labile due to the ring strain. The molecular structure for [7](OTf)⋅CH2Cl2 was also determined by X-ray crystallography ( Figure 3); the structural parameters on the metallafuran moiety (Ru−C, C−C, and C−O distances are 1.982(3), 1.393(4)−1.399(4), and 1.276(3) Å , respectively; sum of interior angle = 539.8°) were found to be very similar to those in [4] + .

General Procedures
All reactions were performed under an argon atmosphere using standard Schlenk techniques unless otherwise stated. All reagents were used as received, and solvents for reactions were purified by a PureSolv MD5 solvent purification system. cis(Cl), cis(S)-[Ru(bpy)(dmso-S) 2 Cl 2 ] (bpy = 2,2 -bipyridine; dmso = dimethyl sulfoxide) were prepared in accordance with the literature methods [64]. 1 13 C HMBC NMR spectra were recorded on Bruker 600 AVANCE III FT-NMR spectrometer. Peak positions were calibrated with solvent residue peaks as internal standard. The 31 P{ 1 H} NMR spectra were referenced to external P(C 6 H 5 ) 3 (−4.7 ppm) [65]. Labeling scheme for H, C and P atoms in the NMR assignments is shown in Figure 4. Electrospray mass spectrometry was performed on a PE-SCIEX API 3200 triple quadrupole mass spectrometer, and the reported or simulated mass values correspond to the most abundant isotopic peaks in the experimental or simulated spectra, respectively. Elemental analyses were performed on an Elementar Vario Micro Cube carbon-hydrogen-nitrogen elemental microanalyzer. Fourier transform infrared (FTIR) spectra were recorded at room temperature using Pekin Elmer "Spectrum 100" FTIR Spectrometer. HeLa (human cervical carcinoma) cell line was preserved by our laboratory.   [65]. Labeling scheme for H, C and P atoms in the NMR assignments is shown in Figure 4. Electrospray mass spectrometry was performed on a PE-SCIEX API 3200 triple quadrupole mass spectrometer, and the reported or simulated mass values correspond to the most abundant isotopic peaks in the experimental or simulated spectra, respectively. Elemental analyses were performed on an Elementar Vario Micro Cube carbon-hydrogen-nitrogen elemental microanalyzer. Fourier transform infrared (FTIR) spectra were recorded at room temperature using Pekin Elmer "Spectrum
Method 2: A mixture of cis(Cl), cis(S)-[Ru(bpy)(dmso-S) 2 Cl 2 ] (0.825 mmol) and dppm/ dppe/dpam (0.825 mmol) was refluxed in MeOH (50 mL) under argon for 16 h. During refluxing, the color of the solution changed from orange to deep red. Upon cooling to room temperature and the removal of all solvent by reduced pressure, the reaction mixture was dissolved in a minimum amount of CH 2 Cl 2 and added to Et 2 O (300 mL) to yield red precipitates. These precipitates were pure enough for further reactions and could further be recrystallized as mentioned in Method 1.

Synthesis of [4](OTf) and [5](OTf)
A mixture of 1-phenylprop-2-yn-1-one (HC≡C(C=O)Ph, 0.14 mmol) and cis-[Ru(bpy) (dppm)Cl 2 ] (0.07 mmol) were refluxed in MeOH (50 mL) under argon for 16 h, during which the metal precursor gradually dissolved and the color of the reaction mixture changed from red to deep red. Upon cooling to room temperature, a saturated aqueous NaOTf solution (5 mL) was added into the reaction mixture, and the mixture was concentrated to about 5 mL by reduced pressure to give a suspension of brown red solids. The solids were then collected by suction filtration, washed with deionized water (10 mL × 3) and finally Et 2 O (10 mL × 3). The separation of the crude products [4](OTf) and [5](OTf) was performed by column chromatography. Conditions: basic alumina, CH 2 Cl 2 /(CH 3 ) 2 CO 9:1 (v/v) as eluent gave [4](OTf) as the first band (orange); the second band (purple) containing [5](OTf) was then eluted with a CH 2 Cl 2 /(CH 3 ) 2 CO 7:3 (v/v) mixture. Analytically pure orange crystals of [4](OTf) and deep purple crystals of [5](OTf) were obtained by the recrystallization of the collected bands via layering of n-hexane onto a CH 2 Cl 2 solution of the complexes.

Synthesis of
The synthesis of [5](OTf) and [6](OTf) was similar to that of [4](OTf), except that a mixture of THF and H 2 O (40 and 10 mL, respectively) was used as the reaction solvent. During refluxing, the metal precursor gradually dissolved and the color of the reaction mixture changed from yellowish red to purple red. Upon cooling to room temperature, the mixture was concentrated to about 10 mL by reduced pressure and the resultant aqueous solution was washed with Et 2 O (10 mL × 2). A saturated aqueous NaOTf solution (5 mL) was added to the aqueous phase, and the resultant mixture was then extracted with CH 2 Cl 2 (30 mL × 3). The organic phases were dried over anhydrous MgSO 4 . After the removal of MgSO 4 by a simple filtration, the filtrate was concentrated to give a dark purple red oil. The separation of the crude products [5](OTf) and [6](OTf) was performed by column chromatography. Conditions: basic alumina, CH 2 Cl 2 /(CH 3 ) 2 CO 7:3 (v/v) as eluent gave [5](OTf) as the first band (purple); the second band (yellow) containing [6](OTf) was then eluted with a CH 2 Cl 2 /(CH 3 ) 2 CO 5:5 (v/v) mixture. The yield of [5](OTf) prepared by this method was found to be 63%.
[6](OTf The procedure for the synthesis of [7](OTf) was the same as that for the synthesis of [4](OTf), except that cis-[Ru(bpy)(dppe)Cl 2 ] was used instead of cis-[Ru(bpy)(dppm)Cl 2 ]. The color of the reaction mixture was found to be orange during refluxing. The crude product, [7](OTf), was eluted as an orange band using a CH 2 Cl 2 /(CH 3 ) 2 CO 8:2 (v/v) mixture as eluent on a basic alumina column. Analytically pure orange crystals of [7](OTf) were obtained by the recrystallization of the collected band via layering of n-hexane onto a CH 2 Cl 2 solution of the complex. [7](OTf

Synthesis of [8](OTf)
The procedure for the synthesis of [8](OTf) was the same as that for [6](OTf), except that cis-[Ru(bpy)(dppe)Cl 2 ] was used instead of cis-[Ru(bpy)(dppm)Cl 2 ]. During refluxing, the metal precursor gradually dissolved and the color of the reaction mixture changed from deep yellow to pale yellow. The crude product, [8](OTf), was eluted as a yellow band using a CH 2 Cl 2 /(CH 3 ) 2 CO 5:5 (v/v) mixture as eluent on a basic alumina column. Analytically pure yellow crystals of [8](OTf) were obtained by the recrystallization of the collected band via layering of n-hexane onto a CH 2 Cl 2 solution of the complex. [8](OTf