VASORELAXATION EFFECT PROMOTED BY THE CALCIUM CHANNEL BLOCKER VERAPAMIL COORDINATED TO RUTHENIUM (II): CHEMICAL AND BIOLOGICAL PROPERTIES

VASORELAXATION EFFECT PROMOTED BY THE CALCIUM CHANNEL BLOCKER VERAPAMIL COORDINATED TO RUTHENIUM (II): CHEMICAL AND BIOLOGICAL PROPERTIES

Olivia Azevedo Nascimento ¹, Elisangela Fabiana Boffo ¹, Bruno Rodrigues Silva ², Juliana Cristina Biazzotto ², Lusiane Maria Bendhack ², Roberto Santana da Silva² and Renata Galvão de Lima ^{*1, 3}

Departamento de Química ¹, Universidade Federal da Bahia, Avenida Barão de Geremoabo number 147 , Ondina, Salvador Post Code 40170-115, BA, Brazil

Departamento de Física e Química ², Faculdade de Ciências Farmacêuticas de Ribeirão Preto-Universidade de São Paulo, Avenida do Café s/n, Monte Alegre, Ribeirão Preto Post Code 14040-903, SP, Brazil

Faculdade de Ciências Integradas do Pontal-Universidade Federal de Uberlândia ³, Rua 20 number 1600, Tupã, Ituiutaba Post Code 38304-402, MG, Brazil

ABSTRACT: This paper presents the synthesis and physical-chemical characterization of a new terpyridine (tpy) ruthenium complex coordinated to the L-type calcium blocker channels verapamil (vera) obtained from commercial pill. Elemental analysis (%C N H), infrared (IR) and ultraviolet-visible (UV-vis.) spectroscopy, mass spectrometry (MS), and ¹³CNMR (nuclear magnetic resonance), and HPLC (high performance liquid chromatography) confirmed the pure and structure of the ligand verapamil and of the ruthenium complex structurally designed as [Ru^IICl₂(tpy)(vera)] Cyclic voltammetry aided investigation of the electrochemical behavior of [RuIICl2(tpy)(vera)] based in the metal ion redox potential. Verapamil ligand and [RuIICl2(tpy)(vera)] induced vascular relaxation in endothelium-intact and endothelium-denuded aortic rings. Verapamil drug and [RuIICl2(tpy)(vera)] complex coupled to verapamil elicited similar pharmacological results. The calcium ion chelating effects (Kd) promoted for verapamil ligand and [RuIICl2(tpy)(vera)] complex were found based in the fluorescence technic. The smooth muscle cell images studied to [RuIICl2(tpy)(vera)] complex acting as a luminescent agent important as biological probe.

Ruthenium Complex, Verapamil,

L-Type Calcium Channel Blocker, Vascular Relaxation

INTRODUCTION: Voltage-sensitive calcium channel (L-type channels) blockers such as verapamil have found application in vasodilating therapy, being a great asset to treat hypertension and other cardiovascular diseases whose symptoms include abnormal arterial tone. Calcium entry through L-type channels in vascular smooth muscle cells (VSMC) contributes to vasoconstriction ¹. This contraction depends on increasing cytosolic calcium concentrations and subsequent activation of myosin-light chain kinase (MLCK) ².

Clinicians have prescribed calcium-channel blockers to treat hypertension and angina and to manage cardiac arrhythmias ³. Among these blockers is Verapamil (Fig.1), a drug belonging to the class of phenylalkylamines, which has been used in the therapy of some chronic conditions. This drug operates by selectively inhibiting Ca²⁺ influx through L-type calcium channels ⁴.

FIG. 1: CHEMICAL STRUCTURE OF VERAPAMIL.

Previous studies, based in molecular models, have suggested that, for verapamil to act, its relatively compact structure has to accommodate in the presence of calcium ion. Non-bonded interactions between the two dimethoxy aryl rings located on opposite ends of verapamil stabilize this drug in what is believed to be the most stable conformation of its isolated form ^4-7. Such conformational change may induce a different biological behavior and could be useful to describe the mechanism of action of this drug.

Although literature works have described that calcium and verapamil interact in aqueous medium ⁸, it is possible to observe calcium release in the presence of other metal ions. Therefore, describing the biological mechanism through which verapamil operates is complicated: several different reactions may occur between this drug and other metal centers. The use of a transition metal ion could help to overcome this difficulty and allow for better interpretation of verapamil conformational change during the ion calcium interaction, contributing with knowledge in the area of bioinorganic chemistry ^9-12.

Therefore, the present study aimed to evaluate how Ca²⁺ interacts with the [Ru^IICl₂(tpy)(vera)] complex (where vera is verapamil, and tpy is 2,2′:6′,2′′-terpyridine) (Fig.2). The highly stable interaction between ruthenium (II) and unsaturated ligands (terpyridine ligand) motivated this investigation^13-16 —this interaction restricts the spatial structure of verapamil when its coordinated to the metal ion center. This could be an important step toward a better description of the biological mechanism through which phenylalkylamine drugs function.

To evaluate the potential effects of [Ru^IICl₂(tpy)(vera)], we studied the rat aorta relaxation induced by the new complex and measured the cytosolic calcium concentration in VSMC as imaged by fluorescence microscopy.

FIG. 2: CHEMICAL STRUCTURE FOR [Ru^IICl₂(tpy)(vera)] COMPLEX PROPOSED IN THIS WORK.

MATERIALS AND METHODS:

Apparatus: UV-vis. spectra were recorded on a Hitachi U-3501 spectrophotometer. Fluorescence spectra were acquired on Fluorolog-3 (JobinYvon-SPEX, Edison, NJ USA) spectrofluorometer equipped with an R928P Hamamatsu photomultiplier and a 450-W ozone-free Xe lamp, respectively. Infrared (IR) spectra were registered on a protegé 460 series FT-IR spectrometer, using solid samples pressed in KBr pellets. Cyclic voltammetry was conducted on an AUTOLAB^Ò model Drop Sense potentiostat/ galvanostat, employing a conventional three-electrode cell with a platinum disk working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode. The chromatograms were obtained on a Shimadzu system using UV–vis. detection, and SPD-M20A detector, an LC-20AT pump with CBM-10A interface, a DGU-20A3 degasser, and Chemstation software; a CLC-ODS column (5 mm, 250 mm, 6 mm i.d.; Shimadzu) was employed. The samples were dissolved in the appropriate mobile phase; the injection volume was 20 mL. Mass spectrometry was accomplished on an ultrOTOFQ-ESI-TOF Mass Spectrometer (Bruker Daltonics, Billerica, USA) operating in the positive mode.

The sample was infused at a flow rate of 100 mL h^-1, using methanol/water (20:80, v/v) as solvent. The MS calibration was carried out with the aid of a Na–TFA (10 mg mL^-1) solution. All the NMR experiments were recorded at 27 °C, on a Varian Inova 500 spectrometer 11.7 Tesla (¹³C at 125 MHz) equipped with a 5-mm direct detection probe from Cambridge Isotope Laboratories Inc. (USA), CDCl₃ (99.9%) was used as solvent. In vitro cell images were acquired under an Eclipse Ti fluorescent microscopy (Nikon). X-ray powder diffraction (XRD) experiment, a Shimadzu, model XRD-6000 diffractometer with cobalt tube (CoKα), operating voltage at 40.0 kV and 30.0 mA current was employed.

Dissociation constants (K_d) FLUO 3AM-Ca²⁺ (27 nmol L^-1) in the presence of verapamil (80 mmol L^-1) and of the [Ru^IICl₂(tpy)(vera)] complex (80 mmol L^-1) were obtained by a linear regression fit of the fluorescence intensity (I₀-I)/I₀ vs the concentration of the added calcium ion ([Ca²⁺)], according to the following equation (1):

(1)

FLUO 3AM-Ca²⁺ ® FLUO 3AM + Ca²⁺

where I₀ and I₀-I are the emission intensity of FLUO 3AM at a selected wavelength, in the absence and in the presence of the compounds, respectively.

Chemicals and Reagents:

RuCl₃.3H₂O; 2,2´:6´,2”-terpyridine; and FLUO 3AM were purchased as high purity reagents from Sigma Aldrich Chemicals (Brazil) and were used as supplied. All the preparations and measurements were carried out under argon atmosphere, protected from light.

Methanol, ethanol, chloroform and dichloromethane were stored over molecular sieves. Other chemicals were of reagent grade and were used without further purification. Column chromatography was performed with basic aluminum oxide (Sigma Aldrich-Brazil).

Verapamil extraction methods:

Verapamil was obtained from a local market formulation containing 240 mg of verapamil hydrochloride as coated tablets from Sandoz^Ò (Santana Pharmacy, Salvador-BA). First, the pill was ground in a mortar, and the resulting powder was sonicated in 10 mL of CHCl3, for 15 minute.After that, the suspension was centrifuged at 3000 rpm for 20 minute, to separate the liquid from the solid. The yellow chloroform extract was dried under vacuum, to give a solid.

Final mass = 230 mg. Mass analysis: Calcd. for [C₂₇H₃₈N₂O₄.1.3H₂O] (MM. 478.02 g/mol): C, 67.78; H, 8.49; N, 5.85. Found: C, 67.28; H, 7.44; N, 6.05. ¹³C NMR (CDCl₃, 50.3 MHz) δ: 20.7 (CH₂), 34.9 (CH₂), 38.1 (CH₂), 40.2 (N-CH₃), 53.3 (N-CH₂), 55.4 (N-CH₂), 55.91 (O-CH₃), 55.96 (O-CH₃), 56.0 (O-CH₃), 56.3 (O-CH₃), 109.4 (CH), 111.2 (CH), 111.6 (CH), 112.0 (CH), 118.8 (CH), 120.2 (CH), 121.2 (CN), 129.7 (C), 148.3 (C-O), 148.7 (C-O), 149.4 (C-O), 149.4 (C-O). Its show X-ray powder diffractograms analyses characteristic of verapamil. HCl single enantiomer ^17-18 demonstrated by high-intensity diffraction peaks detected at 2q = 14.55^◦; 18.10^◦; 19.00^◦ (Fig. 3).

The extracted sample was characterized by ¹³C NMR spectroscopy (Fig. 4), and data were compared with published results¹⁹. FTIR: n(C-O-C) 2949 cm^-1 (methoxy groups), n(CN) 2234 cm^-1 (saturated alkyl nitrile), n(C=N) 1608 1591 1518 cm^-1 (benzene ring) ¹⁸.

FIG. 3: XRD RESULTS OBTAINED VERAPAMIL HYDROCHLORIDE EXTRACTED SAMPLE.

FIG. 4: ¹³C NMR SPECTRA FOR VERAPAMIL LIGAND IN CDCl₃. IN SIDE: VERAPAMIL MOLECULAR STRUCTURE WITH CARBON NUMBERING SCHEME.

Synthesis of the ruthenium complexes:

[Ru^III(tpy)Cl₃]: The [Ru^III(tpy)Cl₃] precursor complex was synthesized following a previously reported method¹³.

[Ru^IICl₂(tpy)(vera)]: The [Ru^IICl₂(tpy)(vera)] complex was synthesized using a method similar to that described by Suen and others^14,16 with some modifications. 247 mg (0.53 mmol) of the [Ru^III(tpy)Cl₃] complex was added to 30 mL of ethanol/water (25:75 % v/v), followed by addition of 500 μL of triethylamine and 20 mg of LiCl. The mixture was heated at reflux for 15 minute, when the color changed from brown to purple. Next, 240 mg (0.53 mmol) of the extracted verapamil dissolved in ethanol (10 mL) was added to the purple solution. After 4 h under reflux at 80 ⁰C, the purple mixture was cooled, and the volume was reduced to a minimum by rotary evaporation. The crude products were separated by classic chromatography on alumina oxide. The fraction eluted with methanol/dichloromethane (10:90% v/v) contained the target [Ru^IICl₂(tpy)(vera)] compound, isolated as the third fraction. Yield: 77 mg (32%). Mass analysis =. Calcd. for [RuCl₂(C₄₂H₄₉N₅O₄)] (MM. 859.84 g/mol): C, 58.66; H, 5.74; N, 8.14. Found: C, 59.00; H, 5.64; N, 8.00.

Vascular Reactivity Studies Vessel preparation:

Male Wistar rats (400–450 g) were killed by decapitation in accordance with the Ethics Committee for Animal Research, Ribeirão Preto Campus, University of São Paulo, Brazil.

The thoracic aorta was quickly removed, dissected free, and cut into 4-mm-long rings. Next, the relaxation induced by these compounds in endothelium-intact (E⁺) and endothelium-denuded (E^-) arteries was investigated. In the latter case, the endothelium was mechanically removed. The aortic rings were placed between two stainless-steel stirrups and connected to an isometric force transducer (Letica Scientific Instruments), to measure tension in the vessels. The rings were placed in a 10 mL organ chamber containing Krebs solution with the following composition (mol L^-1 x 10³): NaCl 130, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 14.9, glucose 5.5, and CaCl₂ 1.6. The solution was maintained at pH 7.4 and 37 ⁰C, gassed with 95% O₂ and 5% CO₂.

The rings were initially stretched to a basal tension of 1.5 g (previously determined by length–tension relationship experiments), before allowing them to equilibrate for 60 minute in the bath, which was changed every 15–20 minute. Endothelial integrity was qualitatively assessed on the basis of the degree of relaxation elicited by acetylcholine (ACh, 1 x 10^-6 mol L^-1) in the presence of contractile tone induced by phenylephrine (1 x 10^-7 mol L^-1)²⁰. The efficacy (E_max) and potency (pD₂) of [Ru^IICl₂(tpy)(vera)] and verapamil in inducing vascular relaxation was examined.

Culture Cell:

VSMC (A7R5) was obtained from the Rio de Janeiro Cell Bank (BCRJ/UFRJ). Cells were seeded in a Petri dish with a 1 cm diameter laminated hole containing DMEM, 10% fetal bovine serum, and 5% penicillin. The cells were then cultured in a humidified incubator at 37 ^oC for 24 h, under 5% CO_2.

Fluorescence Microscopy:

RESULTS AND DISCUSSION: Verapamil hydrochloride is a chiral drug that is commercially available as a racemic mixture, usually termed racemate ¹⁸. HPLC analysis confirmed the purity of extracted verapamil where the retention time was 2.94 minutes (Fig. 5, see experimental section for HPLC conditions), and the spectrum recorded for this peak presented the typical ultraviolet bands (at 225 and 277 nm) of this drug^{17, 21}.

FIG.5: CHROMATOGRAM OF VERAPAMIL EXTRACTED. ISOCRATIC ELUTION WITH (%) 80:20 ACETONITRILE: WATER, WAS USED AT A CONSTANT FLOW-RATE OF 1.0 ml min^-1, UV DETECTION 270 nm AND 25 ± 0, 1 ⁰C.

Next, we synthesized the [Ru^IICl₂(tpy)(vera)] complex from [Ru^III(tpy)Cl₃], by substituting one chloride in the ruthenium coordination sphere with verapamil, followed by ruthenium(III)  reduction with triethylamine. Substitution of a chloride of [Ru^III(tpy)Cl₃] by an incoming ligand (along with ruthenium(III) reduction) resulted in formation of trans-[Ru^II(tpy)LCl₂] complexes, like previously synthesized when L= CH₃CN, CH₃CH₂CN and pyridine ¹⁴. In this proposed, we infer that the chloride ligands in the [Ru^IICl₂(tpy)(vera)] complex are in trans position.

The IR spectrum of [Ru^IICl₂(tpy)(vera)] in KBr pellet displayed the band corresponding to nitrile stretching at 2241 cm^-1 (Fig. 6), resembling the band relative to CºN vibration in the IR spectrum of free verapamil (2235 cm^-1) ¹⁸.

FIG. 6: INFRARED SPECTRUM FOR [Ru^IICl₂(tpy)(vera)] COMPLEX (A) AND VERAPAMIL EXTRACT (B) IN KBr PELLET.

FIG.7: MASS SPECTRA FOR [Ru^IICl₂(tpy)(vera)] COMPLEX IN METHANOL.

The spectroscopic behavior of ruthenium (II) terpyridine complexes containing a nitrile ligand is usually discussed in texts about spectroscopic transitions, including the ligand centered and metal-ligand charge transfer bands. The UV-vis. spectrum of [Ru^IICl₂(tpy)(vera)] in dichloromethane (Fig. 8) displayed bands similar to those detected for trans-[RuCl₂(tpy)(CH₃CN)] and trans-[RuCl₂(tpy)(CH₃CH₂CN)]¹⁴.

FIG. 8: ELECTRONIC SPECTRA IN UV-VIS. REGION OF [Ru^IICl₂(tpy)(vera)] COMPLEX [3,70 x 10^-5 mol L^-1] IN CH₂Cl₂.

The bands in the UV region were attributed to the intraligand (IL) p®p* transition centered mainly on the terpyridine and verapamil ligands. In the visible region, bands at 380 and 520 nm emerged, characterized as singlet metal-to-ligand charge transfer (¹MLCT) transitions from the ruthenium t₂g state to the p* orbital on the terpyridine ligand (Table 1). Both verapamil and [Ru^IICl₂(tpy)(vera)] exhibited fluorescence emission under UV excitation only (Table 1).

TABLE 1: ELECTRONIC DATE AND REDOX POTENTIAL FOR VERAPAMIL AND [Ru^IICl₂(tpy)(vera)] COMPLEX IN CH₂Cl₂

a: ref.²¹ , b: excitation wavelenght at 290 nm in DMSO

The Ru^II/Ru^III reversible process occurring at E_1/2= + 0.52 V vs Ag/AgCl (Fig. 9) was consistent with results obtained for similar ruthenium terpyridine complexes¹⁴.

Wavenumber (cm^-1)

FIG. 9: CYCLIC VOLTAMOGRAM FOR [RuCl₂(tpy)(vera)] COMPLEX [1.0 x 10^-3mol L^-1] in CH₂Cl₂. SCAN RATE 100 mV s^-1.

In addition to the physicochemical analyses, pharmacological experiments helped to determine how much the ruthenium metal ion influenced the pharmacological effect of verapamil.

To this end, we dissolved [Ru^IICl₂(tpy)(vera)] in a 1% dimethylsulfoxide (DMSO)/Krebs buffer solution, due to the hydrophobic characteristics of the ruthenium complex. At 37 ⁰C, no spectroscopic alterations in the spectrum of the [Ru^IICl₂(tpy)(vera)] solution occurred along time (Fig. 10), which indicated the chemical stability of the complex in this medium.

FIG.10: UV-VISIBLE SPECTRAL BEHAVIOR Of [Ru^IICl₂(tpy)(vera)] COMPLEX IN KREBS BUFFER SOLUTION: 1% DMSO AND 37 ⁰C (SOLID LINE) AFTER 30 MIN. (DASH LINE), 45 MIN. (DOT LINE) AND 1 H (DASH DOT LINE).

Fig.11 depicts the concentration-effect curves for the rat aorta relaxation induced by increased concentrations of verapamil and the [Ru^IICl₂(tpy)(vera)] complex on endothelium-intact (E⁺) and endothelium-denuded (E^-) aortic rings pre-contracted with phenylephrine. For E⁺ aortic rings, both compounds induced relaxation in a concentration-dependent way. In both cases, the maximum effect was reached at the same concentration, with no differences in the potency calculated from the pD₂ values (Table 2).

FIG. 11: VASCULAR RELAXATION INDUCED BY VERAPAMIL AND [Ru^IICl₂(tpy)(vera)] ON RAT INTACT (E+) AORTIC RINGS. RESPONSES ARE REPRESENTED AS THE PERCENTAGE OF THE RELAXATION INDUCED BY COMPOUNDS ON THE PRE CONTRACTION WITH PHENYLEPHRINE. DATA ARE SHOWN AS MEAN ± SEM.

TABLE 2: E_max AND pD₂ VALUES TO PERCENTAGE (%) OF VASCULAR RELAXATION INDUCED BY VERAPAMIL AND [RuCl₂(tpy)(vera)] COMPLEX IN INTACT (E⁺) OR DENUDED (E^-) AORTA FROM RAT.

Data are shown as mean ± SEM of “n” independent experiments

Phenylalkylamines like verapamil are important in hypertension therapy. They act by blocking Ca²⁺ inflow into the cells, across the plasma membrane⁵. Previous studies have suggested that verapamil stabilizes as a relatively compact structure where non-bonded interactions between the two dimethoxy aryl rings hold Ca^{2+ 5-6}.

We decided to investigate whether the similar pharmacological effects observed for verapamil and [Ru^IICl₂(tpy)(vera)] originated from the capacity of the methoxy and alkyl nitrile functional groups on verapamil to hold Ca²⁺ via electrostatic interactions ^5-6. FLUO 3-AM, a chelating fluorescence calcium probe with maximum emission at 550 nm under visible excitation, aided us in this investigation.

Addition of the calcium ion solution to FLUO 3-AM in DMSO solution prompted a progressive, [Ca²⁺]-dependent increase in the fluorescence intensity of the emission spectrum (Fig. 12A) (Calcium Signaling Protocols 1999). Plotting of (I₀ - I)/I₀ versus [Ca²⁺] afforded a satisfactory straight line (Fig. 12B). The dissociation constant (K_d) of  FLUO 3-AM in the presence of rising ion Ca²⁺ concentration was 1.15 mmol L^-1.This value was close to that reported for this same fluorescence probe in buffer solution (pH 7.2), at 22 ⁰C is equal 3.90 mmol L^{-1 25}.

In the presence of verapamil and [Ru^IICl₂(tpy)(vera)], the K_d of FLUO 3AM increased to 2.62 and 4.10 mmol L^-1, respectively. On the basis of the dissociation equation (1), the higher K_d values indicated a competitive chelating effect between the tested compounds and FLUO 3-AM for the calcium ion.

FIG. 12: FLUORESCENCE SPECTRA OF FLUO 3AM (27 nmol L^-1) WITH ADDED [CA²⁺] (0 – 0.6 mmol L^-1) IN DMSO AT ROOM TEMPERATURE (» 25 ⁰C) (A) AND PLOTTING OF (I₀ - I)/I₀ VERSUS [Ca²⁺] GAVE A SATISFACTORY STRAIGHT LINE (B).

Next, we assessed the calcium chelating activity of free verapamil and [Ru^IICl₂(tpy)(vera)] in vitro, using VSMC A7R5 and FLUO 3-AM. Under visible light excitation, it was possible to observe the fluorescence emission of FLUO 3-AM chelated with Ca²⁺ in the VSMC (Fig. 13B). Incubation with verapamil suppressed this fluorescence (Fig. 13D). These results evidenced competitive Ca²⁺ chelation between FLUO 3-AM and verapamil, corroborating the results in. Incubation with [Ru^IICl₂(tpy)(vera)] Fig. 12 provided a similar outcome (Fig. 13F).

Moreover, VSMC incubated with [Ru^IICl₂(tpy)(vera)] displayed fluorescence emission under ultraviolet light excitation (Fig. 13G), which emerged from the ruthenium complex located in the area of the cell nucleus. Verapamil did not prompt this effect in the cell (results not shown). Taken together, these results reflected efficient internalization of the ruthenium complex by the cell as compared with verapamil ²⁶.

FIG.13: FLUORESCENCE MICROSCOPY ANALYSES FOR SMOOTH MUSCLE CELL A7R5 IN PRESENCE OF FLUO 3AM (10 mmol L^-1) (A), UNDER VISIBLE LIGHT EXCITATION (B), AFTER FREE VERAPAMIL (0.20 mmol L^-1) (C), UNDER VISIBLE LIGHT EXCITATION (D). MUSCLE CELL IN PRESENCE OF FLUO 3AM (10 mmol/L) AND [Ru^IICl₂(tpy)(vera)] COMPLEX (0.20 mmol L^-1) (E), UNDER VISIBLE (F) LIGHT EXCITATION.

CONCLUSION: The mechanisms through which the [Ru^IICl₂(tpy)(vera)] complex induces muscular relaxation remain unknown. However, the present study has shown that ruthenium verapamil compounds interact with calcium ions via the same electrostatic interaction involving the methoxy and alkyl nitrile substituents present in verapamil. The preliminary pharmacological results evidenced that [Ru^IICl₂(tpy)(vera)] induced vascular smooth muscle relaxation, indicating the potential therapeutic action of ruthenium complexes as calcium blockers. Compared with verapamil, [Ru^IICl₂(tpy)(vera)] offers the advantage of acting as a luminescent biological probe.

ACKNOWLEDGMENTS: This work was supported by grants from FAPESB PPP0055/2010. C, H, and N analyses were conducted at the Chemistry Department of the Federal University of Minas Gerais. Dr. Norberto Peporine Lopes is gratefully acknowledged for assistance with mass spectrum analysis.

AUTHOR CONTRIBUTIONS: All the authors participated in the design, preparation, interpretation of the cited studies, and review of the manuscript.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest.

REFERENCES:

1. a) Zhorov BS, Tikhonov DB, Ligand action on sodium, potassium, and calcium channels: role of permeant ions, Trends in Pharmacological Sciences, 2013; 34: 154-161
2. b) McDonald TF, Pelzer S, Trautwein W, Pelzer DJ, Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiological Review, 1994; 74: 365-507.
3. Catterall W, Signaling complexes of voltaje-gated sodium and calcium channels. Neuroscience Letters, 2010; 486: 107-116.
4. Bombig MTN, Póvoa R, Interações e associações de medicamentos no tratamento anti-hipertensivo – Antagonistas dos canais de cálcio. Revista Brasileira Hipertensão, 2009; 16: 226-230.
5. a) Bergson P, Lupkind G, Lee SP, Duban ME, Hanck DA. Verapamil block of T-type calcium channels, Molecular Pharmacology, 2011; 79: 411-419. b) Sandmann S, Unger T, L- and T-type calcium channel blockade – the efficacy of the calcium channel antagonist mibefradil. Journal of Clinical and Basic Cardiology, 1999; 2: 187-201.
6. a) Lacinová L, T-type calclium channels blockers-new and notable. General Physiology and Biophysics, 2011; 30: 403-409. b) Awasthi A, Yadav A, Phenylalkylamines as calcium channel blockers. Journal of Chemical Science, 2007; 119: 565-570.
7. [a) Singh K, Kumar M, Pavadai E, Naran K, Warner DF, Ruminsk PG, Chibale K, Synthesis of new verapamil analogues and their evaluation in combination with rifampicin against Mycobacterium tuberculosis and molecular docking studies in the binding site of efflux protein Rv1258c. Biiorganic & Medicinal Chemistry Letters, 2014; 24: 2985-2990. b) Zhorov BS, Ananthanarayanan VS, Docking of verapamil in a synthetic Ca²⁺ channel: formation of a ternary complex involving Ca²⁺ Archives of Biochemistry and Biophysics, 1997; 341: 238-244.
8. a) St-Onge M, Dube PA, Goselin S, Guimont C, Godwin J, Archambault PM, Chauny JM, Frenette AJ, Darveau M, Le Sage, Treatment for calcium channel blocker poisoning: A systematic review. Clinical Toxicology, 2014; 52: 926-944. b)Tetreault S, Ananthanarayanan VS, Interaction of Calcium Channel Antagonists with Calcium: Structural Studies on Verapamil and Its Ca²⁺ Journal of Medical Chemistry, 1993; 36: 1017-1023.
9. Cheng RCK, Tikhonov DB, Zhorov BS Structural model of phenylalkylamine binding to L-type calcium channels. The Journal of Biological Chemistry, 2009; 284: 28332-28342.
10. Sarkar K, Sen K, Some drugs in action: metal ions do influence in activity. International Journal of Pharmaceutical Science and Research, 2015; 6: 1-13.
11. Motswainyana WM, Ajibade PA, Anticancer Activities of Mononuclear Ruthenium(II) Coordination Complexes, Advances in Chemistry, 2015; 2015: 1-21.
12. Haas KL, Franz KJ, Application of Metal Coordination Chemistry To Explore and Manipulate Cell Biology, Chemical Review, 2009; 109: 4921-4960.
13. Navarro M, Gabbiani C, Messori L, Gambino, D. Metal-based drugs for malaria, trypanosomiasis and leishmaniasis: recent achievements and perspectives, Drug Discovery Today, 2010; 15: 1070-1086.
14. a) El Batal H, Guo K, Li X, Wesdemiotes C, Morefield CN, Newkome, GR, Perylene-Based Bis-, Tetrakis-, and Hexakis(terpyridine) Ligands and Their Ruthenium(II)–Bis(terpyridine) Complexes: Synthesis and Photophysical Properties. European Journal of Organic Chemistry, 2013; 18: 3640-3644. b)Sullivan BP, Calvert JM, Meyer TJ, Cis-trans isomerism in (trpy)(PPh₃)RuC1₂. Comparisons between the chemical and physical properties of a cis-trans isomeric pair, Inorganic Chemistry, 1980; 19: 1404-1407.
15. Suen HF, Wilson SW, Pomerantz M, Walsh JL Photosubstitution reactions of terpyridine complexes of ruthenium (II). Inorganic Chemistry, 1989; 28: 786-791.
16. Coe BJ, Thompson DW, Culbertson CT, Schoonover JR, Meyer TJ, Synthesis and photophysical properties of mono(2,2’-2”-terpyridine) complexes of ruthenium (II), Inorganic Chemistry, 1995; 34: 3385-3395.
17. de Lima RG, Sauaia MG, Bonaventura D, Tedesco AC, Bendhack LM, da Silva RS, Influence of ancillary ligand L in the nitric oxide photorelease by the [Ru(L)(tpy)NO]³⁺ complex and its vasodilator activity based on visible light irradiation, Inorganica Chimica Acta, 2006; 359: 2543-2549.
18. Yoshida MI, Gomes ECL, Soares CDV, Cunha AF, Oliveira MA, Thermal Analysis Applied to Verapamil Hydrochloride Characterization in Pharmaceutical Formulations, Molecules, 2010; 15: 2439-2452
19. [a) Baviskar D, Sharma R, Jain D, Modulation of drug release by utilizing pH-independent matrix system comprising water soluble drug verapamil hydrochloride. Pakistan Journal of Pharmaceutical Sciences, 2013; 26: 137-144.
20. b) Rustichelli C, Gamberini MC, Ferioli V, Gamberini G, Properties of the racemic species of verapamil hydrochloride and gallopamil hydrochloride. International Journal of Pharmaceutics, 1999; 178: 111-120.
21. Srinivasan V, Sivaramakrishnan H, Karthikeyan B, Detection, Isolation and Characterization of Principal Synthetic Route Indicative Impurities in Verapamil Hydrochloride, Scientia Pharmaceutica, 2011; 79: 555-568.
22. a) Jarret C, Lekic M, Smithe CL, Pusec CM, Sweazea KL. Mechanism mediated vasodilatation in system arteries from mouring doves (Zenaida macrouca). Journal of Comparative Physiology B, 2013; 183: 959-967. b)Furchgott RF, Zawadzki JV, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 1980; 288: 373-376.
23. a) Chen A, Tsao MJ, Chuang JF, Lin CH, Electrochemical determination of Verapamil with a microchip embedded with gold nanoelectrode ensemble electrodes. Electrochimica Acta, 2013; 89: 700-707. b) Rahman N, Ahmadkhan N, Azmi SNH, Optimized and Validated Spectrophotometric Methods for the Determination of Verapamil Hydrochloride in Drug Formulations. Science Asia, 2005; 31: 341-348.
24. Clarke RE, Ford PC, Benzonitrile and acetonitrile complexes of ruthenium ammines. Inorganic Chemistry, 1970; 9: 227-235.
25. Perekalen DS, Trifonova EH, Nelyubena YV, Kudinov AR, Synthesis of cyclohexadienyl ruthenium arene complexes by replacement of acetonitrile ligands in [(η⁵-C₆H₇)Ru(MeCN)₃]⁺. Journal of Organometallic Chemistry, 2014; 754: 1-4.
26. a) Gu M, Wang YD, Zhao XG, Gu ZM, Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system. Rapid Communications in Mass Spectrometry, 2006; 20: 764-770. b) Sun L, Zhang S, Zhong D, In vitro identification of metabolites of verapamil in rat liver microssomes. Acta Pharmacologica Sinica, 2004; 25: 121-128.
27. Calcium Signaling Protocols (Methods in Molecular Biology, Volume 114) (1999), D. Lambert, Ed., pp 125–133, Humana Press.
28. Lo KKW, Lee TKM, Lau JSY, Poon WL, Cheng S.H. Luminescent biological probes derived from ruthenium (II) estradiol polypiridine complexes. Inorganic Chemistry, 2008; 47: 200-2008.
    

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    How to cite this article:

    Nascimento OA, Boffo EF, Silva BR, Biazzotto JC, Bendhack LM, Santana da Silva R and Galvão de Lima R: Vasorelaxation Effect Promoted By the Calcium Channel Blocker Verapamil Coordinated To Ruthenium (Ii): Chemical and Biological Properties. Int J Pharm Sci Res 2015; 6(9): 3733-43. doi: 10.13040/IJPSR.0975-8232.6(9).3733-43.

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