Palladium-benzodiazepine derivatives as promising

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Dec 2, 2015 - Ábner Magalhães Nunes a, José Rui Machado Reys a, Heitor ...... [27] A. Mosset, J.P. Tuchagues, J.J. Bonnet, R. Haran, P. Sharrock, Inorg.
Journal of Inorganic Biochemistry 155 (2016) 129–135

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Palladium–benzodiazepine derivatives as promising metallodrugs for the development of antiepileptic therapies Walleska Bismaida Zacarias Galvão Barros a, Allysson Haide Queiroz da Silva a, Ana Soraya Lima Barbosa a,c, Ábner Magalhães Nunes a, José Rui Machado Reys a, Heitor Gomes de Araújo-Filho b, Jullyana de Souza Siqueira Quintans b, Lucindo José Quintans-Júnior b, Michel Pfeffer c, Valéria Rodrigues dos Santos Malta d, Mario Roberto Meneghetti a,⁎ a

Grupo de Catálise e Reatividade Química (GCaR), Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Av. Lourival de Melo Mota, s/n, Maceió, Alagoas CEP: 57.072-970, Brazil Laboratório de Neurociências e Ensaios Farmacológicos (LANEF), Departamento de Fisiologia, Universidade Federal de Sergipe (UFS), Av. Marechal Rondom, s/n, São Cristóvão, Sergipe CEP 49.000-100, Brazil c Laboratoire de Chimie et Systémique Organo-Métalliques, Institut de Chimie, UMR7177, 4 rue Blaise Pascal, Strasbourg 67000, France d Laboratório de Cristalografia e Modelagem Molecular (LaboCrMM), Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Av. Lourival de Melo Mota, s/n, Maceió, Alagoas CEP: 57.072-970, Brazil b

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Article history: Received 31 July 2015 Received in revised form 29 October 2015 Accepted 30 November 2015 Available online 2 December 2015 Keywords: Palladacycles Diazepam Benzodiazepine Convulsion Epilepsy

a b s t r a c t We synthesized two organometallic diazepam–palladium(II) derivatives by C–H activation of diazepam (DZP) with palladium salts, i.e., PdCl2 and Pd(OAc)2 (OAc = acetate). Both compounds obtained are air stable and were isolated in good yields. The anticonvulsant potential of the complexes, labeled [(DZP)PdCl]2 and [(DZP)PdOAc]2, was evaluated through two animal models: pentylenetetrazole (PTZ)- and picrotoxin (PTX)-induced convulsions. The organometallic DZP–palladium(II) acetate complex, [(DZP)PdOAc]2, significantly increased (p b 0.01 or p b 0.001) latencies and protected the animals against convulsions induced by PTZ and PTX, while the analogous chloro derivative, [(DZP)PdCl]2, was effective (p b 0.01) only in the PTZ model. These effects appear to be mediated through the GABAergic system. The possible mechanism of action of the DZP–palladium(II) complexes was also confirmed with the use of flumazenil (FLU), a GABAA-benzodiazepine receptor complex site antagonist. Herein, we present the first report of the anticonvulsant properties of organometallic DZP–palladium(II) complexes as well as evidence that these compounds may play an important role in the study of new drugs to treat patients with epilepsy. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is one of the most common serious neurological conditions worldwide, with an age-adjusted incidence of approximately 50 per

Abbreviations: AEDs, antiepileptic drugs; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; DZP, diazepam; [(DZP)PdCl]2, bis[2-{(7-chloro-1-methyl-2-oxo-2,3-dihydro1H-benzodiazepin-5-il)phenyl-κ 2 -C,N}-μ 2 -chloropalladium(II)]; [(DZP)PdOAc] 2 , bis[2-{(7-chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzodiazepin-5-il)phenyl-κ 2 C,N}-μ2-acetatopalladium(II)]; FLU, flumazenil; GABA, γ-aminobutyric acid; i.p., intraperitoneal; J, coupling constant; MALDI-TOF-MS, Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectroscopy; OAc, acetate; PBS, phosphate-buffered saline; PTX, picrotoxin; PTZ, pentylenetetrazole; Py, pyridine; δ, chemical shift. ⁎ Corresponding author. E-mail addresses: [email protected] (W.B.Z.G. Barros), [email protected] (A.H.Q. da Silva), [email protected] (A.S.L. Barbosa), [email protected] (Á.M. Nunes), [email protected] (J.R.M. Reys), [email protected] (H.G. de Araújo-Filho), [email protected] (J. de Souza Siqueira Quintans), [email protected] (L.J. Quintans-Júnior), [email protected] (M. Pfeffer), [email protected] (V.R. dos Santos Malta), [email protected] (M.R. Meneghetti).

http://dx.doi.org/10.1016/j.jinorgbio.2015.11.024 0162-0134/© 2015 Elsevier Inc. All rights reserved.

100,000 persons per year in developed countries [1]. Antiepileptic therapy can result in long-term remission in 60–70% of patients, but many patients require combination treatment to achieve optimal convulsion control, as monotherapy is ineffective at controlling seizures in 30–53% of patients. Despite the increase in available treatment options, patient outcomes have not improved significantly; thus, more effective therapies must be developed [2]. There is a clear need to continue to identify novel antiepileptic drugs (AEDs) that effectively control pharmacoresistant convulsions with minimal or no adverse events [3,4]. Shortly after their discovery in the late 1950s, benzodiazepines (BZDs) were implemented for use as anticonvulsants [5]. BZDs offered substantial advantages over previous medications [6], as noted by early clinicians, including high efficacy, rapid onset of action, and low toxicity [7]. Therefore, numerous advances in epilepsy treatment have been achieved with the use of BZDs. However, medicinal chemists have pursued the synthesis or semi-synthesis of new BZDs to improve their therapeutic activity and effectiveness and to reduce adverse events; for example, BZD consumption by the elderly can increase the

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risk of side effects or the use of potentially inappropriate medications, and BZDs are ineffective for certain types of epilepsy [4]. An important strategy in the development of new drugs involves the design and synthesis of bioactive species that contain metal elements in the molecular structure, i.e., metallodrugs [8,9]. A classic example of this type of compound is cisplatin and its derivatives, which are widely employed as anticancer agents [10–12]. One approach to the design and development of new efficient and potent metallodrugs is based on the association of well-known active organic drugs as ligands with metal elements, forming metal or organometallic complexes [9,12–16]. Utilizing this approach, in the present study, we pursued the synthesis of two BZD–palladium(II) organometallic complexes, bis[2-{(7-chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzodiazepin-5il)phenyl-κ 2 -C,N}-μ2 -acetatopalladium(II)], [(DZP)PdOAc]2, and bis[2-{(7-chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzodiazepin-5il)phenyl-κ2-C,N}-μ2-chloropalladium(II)], [(DZP)PdCl]2, and evaluated them as potential AEDs. Previous studies have reported the formation of metallacycle derivatives from BZDs with Pd(II) [16–19], Pt(II) [20], and Ru(II) [21]. All of these metallacycles were obtained via C–H activation reaction [22] and intramolecular coordination of the imine nitrogen. Moreover, Cinellu and coworkers [17] have already reported the synthesis of the [(DZP)PdCl]2 complex described in this work. However, to the best of our knowledge, there are no pharmacological studies related to the use of those BZD-metallacycle derivatives as potential AEDs. 2. Results and discussion 2.1. Chemistry The synthesis of the two palladacycles was quite simple and easy to accomplish. Two air stable solids were isolated in good yields (ca. 80%). Both DZP–palladium derivatives were obtained after cyclopalladation of DZP, via coordination of the imine nitrogen group and C–H activation of the phenyl substituent of the benzodiazepine moiety at the ortho position, using either PdCl2 or Pd(OAc)2 salts as palladium sources (Fig. 1). Structurally, both compounds were isolated as dimers with planar and

open-book shapes for [(DZP)PdCl]2 and [(DZP)PdOAc]2, respectively [23,24]. In solution, the two dimer complexes were present as a mixture of two possible isomers, the cisoid and transoid forms (Fig. 2), clearly detected by NMR studies [23,25]. 1 H NMR spectra of [(DZP)PdCl]2 (in DMSO-d6) and [(DZP)PdOAc]2 (in CDCl3), both at room temperature, indicated the anticipated presence of two isomers (Fig. 2), the transoid and cisoid, in 4:1 and 1:1 ratios, respectively. Notably, for the [(DZP)PdCl]2 complex, the transoid and cisoid isomers each clearly display one pair of diastereotopic signals attributable to the methylene hydrogens of the benzodiazepine ring, as well as one singlet related to the hydrogens of the NCH3 moiety. For the [(DZP)PdOAc]2 complex, similar patterns were observed for the analogous group of hydrogens in the transoid isomer. However, for the cisoid counterpart of [(DZP)PdOAc]2, two pairs of diastereotopic signals for the methylene hydrogens and two singlets for the methyl-N hydrogens were observed. Moreover, we verified that the hydrogens of the CH3COO-bridges for the transoid isomer gave rise to one singlet, whereas two singlets for these hydrogens were observed for the cisoid counterpart. This relative enhancement of the complexity of the cisoid-[(DZP)PdOAc]2 isomer is related to the geometry of the acetate-bridged dimer, commonly referred to as an open-book arrangement. In this case, the steric hindrance between the two planes formed by the square planar geometry of the palladium moieties provokes a twist of the two mirror images of the dimeric complex. This lowers the symmetry of the molecule; thus, most of the equivalent hydrogens of the two moieties become magnetically different. However, the complexity of the NMR data of the dimeric DZP– palladium(II) derivatives was drastically reduced with the formation of the monomeric counterparts in solution via simple addition, in situ, of Py-d5 to a solution of the respective dimers in the NMR tube (see Fig. 3 and experimental section). Only one isomer was observed for both the acetate and the chloro palladium derivatives. In fact, we observed and labeled all hydrogens of the DZP fragment (except the hydrogen replaced after ortho-metalation reaction), and in the case of the palladium acetate derivative, only one signal at 2.28 ppm was observed for the CH3COO ligand.

Fig. 1. Cyclopalladation reactions of diazepam (DZP) in the presence of different sources of Pd(II), forming [(DZP)PdCl]2 and [(DZP)PdOAc]2.

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Fig. 2. Schematic representation of the two possible isomers (transoid and cisoid) of [(DZP)PdCl]2 and [(DZP)PdOAc]2.

The solid-state structure of [(DZP)PdClPy] was unambiguously determined via single-crystal X-ray diffraction (see Fig. 4). Crystals of this monomer were obtained from the slow crystallization of the complex in a CHCl3/hexane solution. The crystallographic data and details of the structure determination of [(DZP)PdClPy] are presented in Table S1 (see in the Supplementary data), and selected bond lengths and angles of [(DZP)PdClPy] are given in Table 1. In the palladacycle formation, cis stereochemistry was evident between C(13)–Pd– N(4) with an angle of 81°, and the coordination sphere of Pd, including C(13), N′(1), Cl(2), and N(4), is essentially planar (the sum of the angles about the Pd atom is 360°). As expected, the trans stereochemistry between N(4)–Pd–N(1′) was observed, with an angle of ca. 175°. The trans N–Pd–N isomer is always obtained in the bridge-splitting reaction of dimeric palladacycles upon reaction with L ligands such as pyridines, phosphines and isonitriles [26]. Moreover, the typical boat conformation of the benzodiazepinic ring is also maintained after cyclopalladation [27]. In order to estimate an important physicochemical parameter related to the biodisponibility of a drug, we evaluated the solubility of both DZP–palladium(II) complexes in different aqueous media. For that, we

essay to prepare three different solutions: in water:DMSO (9:1), in phosphate-buffered saline (PBS;) solution, and in bovine serum albumin (BSA) in PBS solution. This last solution is often used as model protein concentration in blood plasma in lab experiments [28]. Like DZP, both complexes are just very slightly soluble in PBS solution, and just slightly soluble in water:DMSO (9:1). Nevertheless, also like DZP, both complexes increased their solubility in BSA aqueous solution, particularly [(DZP)PdOAc]2, (see Table S2 and Fig. S1 in Supplementary data). Indeed, those complexes, like other BZDs, seem also to highly bond to plasma proteins [29], increasing the permanence of the drug in the organism. It is important to remark that no significant modifications of these solutions/suspensions are observed in terms of solubility or color at least for 6 h (see Figs. S2 and S3 in Supplementary data), suggesting a good stability of the complexes. Despite some slightly modifications of their UV–vis absorption spectra starts to be observed after 24–48 h in solution, suggesting some degree of decomposition after long time in solution (see Fig. S4 in Supplementary data), no traces of colloidal palladium are observed (even after one week in solution), as well, no color modification of the solutions/suspensions. Briefly, the structure of the

Fig. 3. The monomeric forms of [(DZP)PdOAc]2 and [(DZP)PdCl]2 attained following the coordination of Py, [(DZP)PdOAcPy] and [(DZP)PdClPy], respectively.

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Fig. 4. ORTEP view and atomic numbering of [(DZP)PdClPy] showing the atom labeling and the 50% probability ellipsoids.

palladacycles seems to be preserved at the conditions tested. Additional studies are in course in order to verify possible biotransformations of these metallodrug prototypes by an organism [30,31].

2.2. Pharmacological studies The pharmacology of BZDs has garnered intense interest, particularly in the search for promising new 1,4-BZD candidates for the treatment of various diseases such as cancer [32], depression [33], allergy [34], and fungal infections [35]. Specifically, the present study investigated the anticonvulsant effect of two organometallic compounds synthesized from DZP, a member of the 1,4-BZD family. The antiepileptic potential of the [(DZP)PdCl]2 and [(DZP)PdOAc]2 complexes was assessed via the two most widely used animal models employed in the screening of new agents: pentylenetetrazole (PTZ)and picrotoxin (PTX)-induced convulsions in mice [36–38]. The discovery of novel AEDs relies upon preclinical studies employing animal models prior to the introduction of AEDs into human volunteers [38–40]. For these studies, DZP (6 mg/kg; 21 μmol/kg) was adopted as the standard drug, as both complexes are derived from it, which allowed for important correlations between the complexes and the free ligand. Additionally, the specific amount of [(DZP)PdCl]2, [(DZP)PdOAc]2 or DZP were dissolved in an aqueous solution of Tween 80 (4%). The Tween 80 is inert at this concentration (data not shown). Agents were administrated by intraperitoneally (i.p.) at a dose volume of 0.1 mL/10 g.

Table 1 Selected bond lengths [Å] and angles [°] for [(DZP)PdClPy]. Atoms

Distances [Å] and angles [°]

C(13)–Pd Cl(2)–Pd N(1′)–Pd N(4)–Pd C(2)–O(2) C(2)–N(1) C(5)–N(4) C(5)–C(12) C(12)–C(13) C(13)–Pd–N(4) C(13)–Pd–N(1′) N(4)–Pd–Cl(2) N(1′)–Pd–Cl(2) N(4)–Pd–N(1′) Cl(2)–Pd–N(1′)-C(5′) C(11)–C(5)–N(4)–C(3)

1.984(5) 2.4074(14) 2.043(4) 2.026(4) 1.216(6) 1.380(7) 1.302(6) 1.464(7) 1.413(6) 80.86(18) 93.74(19) 96.20(12) 89.21(13) 174.58(17) 74.78 1.91

2.2.1. Pentylenetetrazole (PTZ)-induced convulsions PTZ is a convulsant agent frequently used in animal models for the induction of convulsions [40]. PTZ is a GABAergic non-competitive antagonist that does not interact directly with GABA receptors, but instead blocks GABA-mediated Cl− influx [41,42]. Several studies have proposed that the pharmacological effect of PTZ is at least partly mediated via interactions with the GABAA receptor [36,40]. Thus, PTZ-induced convulsions are a very important animal model to use to search for new AEDs [39]. Both compounds were effective as anticonvulsants in PTZ-induced convulsions. For all tested doses, acute treatment with [(DZP)PdOAc]2 significantly (p b 0.001) increased the latency of convulsions when compared to the respective vehicle (control group); however, [(DZP)PdCl]2-treated mice only demonstrated this same outcome at the highest dose. At 3.0 and 6.0 mg/kg, [(DZP)PdOAc]2 significantly reduced the incidence of convulsion episodes by 100% (p b 0.001), whereas at 1.5 mg/kg, it had a 90% effect (p b 0.001). Compared to the vehicle-treated group, the incidence of convulsion episodes was reduced in [(DZP)PdCl]2-treated mice by 60% (p b 0.01) at 6.0 mg/kg, whereas at 1.5 and 3.0 mg/kg, it had no significant effects. Treatment with both BZD–palladium(II) organometallic complexes significantly (p b 0.01 or p b 0.001) reduced the number of deaths (Table 2). Moreover, DZP was considered the standard BZD agonist, and DZP-treated mice were significantly protected in all observed parameters. According to the results herein, the [(DZP)PdOAc]2 palladacycle, used in the pretreatment of convulsions chemically induced with PTZ, demonstrated anticonvulsant effects that were significantly superior to [(DZP)PdCl]2 in in vivo tests. Our initial suggestion for the potential anticonvulsant mechanism of [(DZP)PdOAc]2 and [(DZP)PdCl]2 was based in their association with GABAA-BZD receptors because both are benzodiazepine derivatives. Thus, we considered their possible antagonism when faced with flumazenil (FLU), a selective and competitive GABAA-BZD receptor antagonist [43]. When the potential involvement of GABAA-BZD receptors was assessed, we demonstrated that the administration of FLU (10 mg/kg, i.p.) significantly antagonized the anticonvulsant effect of both DZP-palladacycles (Table 2). Here, as with DZP, [(DZP)PdOAc]2 and [(DZP)PdCl]2 were completely antagonized by FLU, both in convulsion latency and the percentage of animals that exhibited convulsions. This effect strongly corroborates the hypothesis that these complexes (or their metabolites) act on the same DZP binding site on the GABAA receptors [44,45]. However, studies using binding techniques may assist in further confirming our hypothesis. The remarkable anticonvulsant effect of [(DZP)PdOAc]2 is not entirely understood, but it may be associated with its pharmacokinetics features, such as its relatively higher solubility in comparison to [(DZP)PdCl]2, or even its stability in biological media (see in the Supplementary data). Thus, further studies are ongoing to elucidate the mechanism of action of DZP–palladium(II) derivatives, as well as their potential use as anticonvulsant or antiepileptic agents or for the treatment of chronic pain, as anticonvulsants have been widely used for this purpose. 2.2.2. Picrotoxin (PTX)-induced convulsions To better understand the anticonvulsant effects demonstrated by [(DZP)PdOAc]2 and [(DZP)PdCl]2, we investigated the alterations in GABA content in mice exposed to the chemoconvulsive substance picrotoxin (PTX). PTX is a noncompetitive antagonist for the GABAA receptor chloride channels that acts at a different site than DZP [46]. According to Loscher, chemoconvulsive agents, such as PTX, may be useful for the discovery of new anticonvulsant agents that have not demonstrated pharmacoresistance to several types of epilepsy [39]. We found that [(DZP)PdOAc]2-treated mice demonstrated significant (p b 0.01) protection against convulsions induced by PTX (Table 3) at a dose of 6 mg/kg, suggesting that this compound may act

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Table 2 Effect of acute treatment with [(DZP)PdOAc]2, [(DZP)PdCl]2 or DZP in the presence and absence of flumazenil (FLU) during pentylenetetrazole (PTZ)-induced convulsions in mice. Treatment

Dose (mg/kg) [μmol/kg]

Latency (s)a

% Convulsions

% Death

Vehicle [(DZP)PdOAc]2 [(DZP)PdOAc]2 [(DZP)PdOAc]2 [(DZP)PdCl]2 [(DZP)PdCl]2 [(DZP)PdCl]2 DZP [(DZP)PdOAc]2 + FLU [(DZP)PdCl]2 + FLU DZP + FLU

– 1.5 [1.7] 3.0 [3.3] 6.0 [6.7] 1.5 [1.8] 3.0 [3.5] 6.0 [7.0] 6.0 [21] 6.0 + 10 [6.7 + 32,970] 6.0 + 10 [7.0 + 32,970] 6.0 + 10 [21 + 32,970]

328.2 ± 14.9 875.1 ± 22.3c 900.0 ± 0.0c,# 900.0 ± 0.0c,# 376.9 ± 22.7 321.5 ± 35.3 644.2 ± 31.0b 900.0 ± 0.0c 410.1 ± 53.8 393.8 ± 48.4 311.5 ± 33.0

100 10e 0e 0e 100 70 60d 0 100 100 100

60 30d 10e 0e 50 60 20e 0e 50 60 30d

n = 8, per group. #p b 0.001 (one-way ANOVA and Turkey's test), significantly different from [(DZP)PdCl]2-treated group (1.5 mg/kg). a Values represent mean ± S.E.M. b p b 0.01. c p b 0.001 (one-way ANOVA and Turkey's test), significantly different from control group. d p b 0.01. e p b 0.001 (Fisher's test), significantly different from control group.

as a GABAA receptor agonist by increasing chloride influx via brain chloride channels. Thus, we showed that [(DZP)PdOAc]2 produces a protective activity against PTX-induced convulsions. This lack of an anticonvulsant profile in the PTX model suggests that [(DZP)PdOAc]2 may act through the GABAergic system but most likely does not bind to the same site as PTX. In contrast, acute treatment with [(DZP)PdCl]2 at all doses was unable to produce an anticonvulsant profile in this model (Table 3). Another interesting finding was that the DZP was most effective when compared to higher dose of [(DZP)PdOAc]2 against PTX-induced convulsions. This high efficacy of DZP on PTX test has been previously described by other study [47]. Both anticonvulsant tests assessed in present study are based on the drug action on the GABAA receptors [38,39]. PTZ and PTX have different sites on the GABAA receptor, so some drugs can possess anticonvulsant activity in PTZ test, but have no effect on PTX test or vice versa [48]. According to Richter and coworkers [49], a total of 19 GABAA receptor subunits (α 1–6, β 1–3, γ 1–3, δ, ε, π, θ, ρ 1–3) have been identified in mammalian brain. Classical benzodiazepines, such as DZP, predominantly exert their action via GABAA Rs composed of α 1βγ 2, α 2βγ 2, α 3βγ 2 and α 5βγ 2 subunits and are known to bind at the extracellular α-γ interface. However, new allosteric modulators of GABAA receptors has been described, so the benzodiazepine site are suitability for study of new drugs or structure-based drug design [49], which may be reasonable to understand the effects of DZP-palladium(II) complexes. Thus, DZP–palladium(II) complexes seems to act at the same receptor of DZP, but it is possibly that acting on different sites of the same GABAA receptor. This possibility could bring the hypothesis that

Table 3 Effect of acute treatment with [(DZP)PdOAc]2, [(DZP)PdCl]2 or DZP in picrotoxin (PTX)induced convulsions in mice. Treatment

Dose (mg/kg) [μmol/kg]

Latency (s)a

% Convulsions

% Death

Vehicle [(DZP)PdOAc]2 [(DZP)PdOAc]2 [(DZP)PdCl]2 [(DZP)PdCl]2 DZP

– 3.0 [3.3] 6.0 [6.7] 3.0 [3.5] 6.0 [7.0] 6.0 [21]

454.6 ± 28.0 588.3 ± 21.5 734.2 ± 65.9b 487.4 ± 23.8 524.1 ± 41.5 980.0 ± 77.2c

40 10d 20d 50 40 10d

20 0 10 0 0 0

n = 8, per group. #p b 0.05 (one-way ANOVA and Turkey's test), DZP-treated group significantly different from [(DZP)PdOAc]2-treated group (higher dose). a Values represent mean ± S.E.M. b p b 0.01. c p b 0.001 (one-way ANOVA and Turkey's test), significantly different from control group. d p b 0.01 (Fisher's test), significantly different from control group.

DZP–palladium(II) complexes do not produce some side-effects or pharmacoresistant, for example, which are common in the DZP treatment [50,51]. It would be interesting to double-check this possibility in future studies. 3. Conclusions With the work herein, we demonstrated that DZP–palladium(II) derivatives [(DZP)PdOAc]2 and [(DZP)PdCl]2 display significant anticonvulsant action. Their effects, much like DZP, appear to be mediated by GABAA-BZD receptors. In comparison, the [(DZP)PdOAc]2 palladacycle, used in the pretreatment of convulsions chemically induced by PTZ, demonstrated anticonvulsant effects that were significantly superior to [(DZP)PdCl]2. Additionally, we discovered that only [(DZP)PdOAc]2 showed significant protection against convulsions induced by PTX, suggesting that it may act as GABAA receptor agonist by increasing chloride influx via brain chloride channels. Thus, we showed that at the higher doses, [(DZP)PdOAc]2 but not [(DZP)PdCl]2 produced protective activity against convulsions induced by PTX. Thus, this lack of an anticonvulsant profile in the PTX model suggests that [(DZP)PdOAc]2 may act through the GABAergic system but most likely does not bind to the same site as PTX. The remarkable anticonvulsant effect of [(DZP)PdOAc]2 is not entirely understood, but it may be associated with its pharmacokinetics, such as its relatively higher solubility in blood plasma in comparison to [(DZP)PdCl]2, or even its stability in biological media. However, our findings offer exciting evidence to further investigate whether these compounds could be useful in treating different types of epilepsy, offering great relief for patients with a pharmacoresistant profile. Additional studies will continue to elucidate the mechanism of action of the DZP– palladium(II) derivatives and their level of toxicity and will also define the potential clinical uses of these BDZ-palladacycle complexes as anticonvulsant and antiepileptic agents. 4. Experimental section 4.1. Chemistry 4.1.1. General procedure All reagents and solvents were purchased from Sigma-Aldrich Chemical Co., except diazepam, which was a product of Laboratório Instituto Vital Brazil Filho. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer using TMS as an internal standard. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. MALDI-TOF-MS spectra were obtained without matrix using an AutoflexIII Maldi-TOF-TOF 200 mass

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spectrometer (Bruker Daltonics). IR spectra were recorded on a Fourier Transform Infrared spectrometer (Shimadzu, IR Prestige-21 model). UV–Vis absorption spectra were recorded on a Uv–Vis-NIR spectrophotometer (Shimadzu, UV-3600 model). Elemental analyses of C, H and N were carried out on a Perkin-Elmer CHN 2400 elemental analyzer. For the X-ray structure determination of [(DZP)PdClPy], a fragment with approximate dimensions of 0.092 × 0.101 × 0.247 mm was cut from a transparent light yellow crystal and used for intensity-data collection at 293(2) K. The crystals were mounted on a goniometer in an Enraf–Nonius Kappa geometry CCD diffractometer with graphitemonochromated Mo Kα (k = 0.71073 Å) radiation. The final unit cell parameters were based on all reflections. Data were collected with the COLLECT program, and the integration and scaling of the reflections were performed with the HKL Denzo–Scalepack software package [52, 53]. Absorption correction was carried out by the multiscan method [53]. The structures were solved by direct methods with SHELXS-97 [54], and the models were refined by full-matrix least-squares on F2 with SHELXL-97 [55]. All hydrogen atoms were stereochemically positioned and refined with the riding model [56]. The ORTEP views were prepared with ORTEP-3 for Windows [57]. The hydrogen atoms on the aromatic rings were refined isotropically, each with a thermal parameter 20% greater than the equivalent isotropic displacement parameter of the atom to which it was bound. 4.1.2. Synthesis and characterization of bis[2-{(7-chloro-1-methyl-2-oxo2,3-dihydro-1H-benzodiazepin-5-il)phenyl-κ2-C,N}-μ2acetatopalladium(II)]- [(DZP)PdOAc]2 DZP (1.00 mmol, 0.285 g) was added to a suspension of powdered palladium(II) acetate (0.89 mmol, 0.200 g) in acetic acid (15 mL). This mixture was heated at reflux temperature during 1 h. The brown-red solution thus obtained was filtered to remove traces of metallic palladium, and the solvent was removed under vacuum. The resulting solid was dissolved in chloroform (5 mL) and n-hexane was added the solution until complete precipitation of the desired compound has occurred. It was filtered and dried under vacuum, to obtain an orange solid. Yield: 377 mg, 84%. 1 H NMR (CDCl3, 400 MHz, ppm). Major isomer: δ 7.8–6.4 (aromatic hydrogens); 4.36 (d, J = 12.80 Hz, 2H, H3a); 3.32 (d, J = 12.68 Hz, 2H, H3b); 3.32 (s, 6H, H18); 2.25 (s, 6H, H20). Minor isomer: δ 7.8–6.4 (aromatic hydrogens); 4.23 (d, J = 12.93 Hz, 1H, H3a); 3.80 (d, J = 13.06 Hz, 1H, H3b); 3.68 (d, J = 12.93 Hz, 1H, H3c); 3.26 and 3.16 (s, 6H, H18); 3.04 (d, J = 12.93 Hz, 1H, H3d); 2.32 and 2.31 (s, 6H, H20). 1H NMR (CDCl3 + Py-d5, 400 MHz, ppm): δ 7.63 (s, 1H, H6); 7.61 (d, J = 2.27 Hz, 1H, H8); 7.35 (d, J = 8.69 Hz, 1H, H9); 7.11 (d, J = 7.56 Hz, 1H, H17); 7.01 (m, 2H, H15 and H16); 6.28 (d, J = 7,56 Hz, 1H, H14); 4.89 (d, J = 12.85 Hz, 1H, H3b); 3.81 (d, J = 12.85 Hz, 1H, H3a); 3.39 (s, 3H, H18); 2.28 (s, 3H, H20). 13C NMR (CDCl3, 100 MHz, ppm): δ 181.90 (C19), 177.96 (C5), 168.24 (C2), 156.59 (C13), 144.18 (C11), 141.34 (C10), 132.42 (C6), 132.32 (C15), 130.06 (C8), 129.57 (C12), 128.97 (C16), 128.81 (C17), 125.49 (C7), 124.05 (C14), 123.23 (C9), 55.70 (C3), 35.07 (C18), and 24.59 (C20). MALDI-TOF (without matrix): m/z [M-OAc] found for C36H30Cl2N4O6Pd2 was 839. IR (in KBr, νmáx/cm− 1): 3109–3012 (ν C–H aromatic); 1687 (ν C = O and ν C = N); 1562 and 1417 (ν as/s COO). Elemental analysis — theoretical: C, 48.13; H, 3.37; N, 6.24 and experimental: C, 48.51; H, 3.58; and N, 6.11. 4.1.3. Synthesis and characterization of bis[2-{(7-chloro-1-methyl2-oxo-2,3-dihydro-1H-benzodiazepin-5-il)phenyl-κ 2-C,N}-μ 2 chloropalladium(II)]- [(DZP)PdCl]2 Lithium chloride (5.00 mmol, 0.212 g) was added to a suspension of PdCl2 (1.00 mmol, 0.177 g) in methanol (10 mL). The solution was heated at 60 °C, until the PdCl2 was dissolved (ca. 10 min). DZP (1.00 mmol, 0.2847 g) was added to the filtered solution of Li2PdCl4 thus obtained, and it was heated at reflux temperature for 1.5 h. The mixture was cooled to RT, and the precipitate was filtered, washed with methanol

until the filtrate was colorless. The yellow precipitate was dried in high vacuum. Yield: 320 mg, 75%. 1 H NMR (DMSO-d6, 400 MHz, ppm). Major isomer: δ 7.9–7.6 (m, 6H, H6, H8 and H9); 7.3–6.9 (br sig, 6H, H15, H16 and H17); 5.52 (br sig, 2H, H14); 4.63 (d, J = 12.72 Hz, 2H, H3a); 3.94 (d, J = 12.72 Hz, 2H, H3b); 3.30 (s, 6H, H18). Minor isomer: δ 7.9–7.6 (m, 6H, H6, H8 and H9); 7.3– 6.9 (br sig, 6H, H15, H16 and H17); 4.41 (d, J = 12.72 Hz, 1H, H3a); 3.88 (d, J = 12.72 Hz, 1H, H3b); 3.16 (s, 3H, H18). 1H NMR (CDCl3 + Py-d5, 400 MHz, ppm): δ 7.62 (s, 1H, H6); 7.59 (d, J = 2.55 Hz, 1H, H8); 7.33 (d, J = 8.56 Hz, 1H, H9); 7.06 (m, 3H, H15, H16 and H17); 6.28 (br sig, 1H, H14); 5.84 (br sig, 1H, H3b); 3.79 (d, J = 12.50 Hz, 1H, H3a); 3.38 (s, 3H, H18). 13 C NMR (DMSO-d6, 100 MHz, ppm): 178.04 (C5), 167.07 (C2), 153.99 (C13), 145.43 (C11), 142.20 (C10), 136.61 (C6), 132.91 (C15), 130.55 (C8), 130.08 (C12), 129.37 (C16), 129.19 (C17), 128.82 (C7), 125.03 (C14), 124.45 (C9), 54.47 (C3), 35.02 (C18). MALDI-TOF (without matrix): m/z [M-Cl] found for C32H24Cl4N4O2Pd2 was 816. IR (in KBr, νmáx/cm−1): 3103–3021 (ν C–H aromatic); 1683 (ν C = O and ν C = N). Elemental analysis — theoretical: C, 45.15; H, 2.84; N, 6.58 and experimental: C, 44.81; H, 2.89; and N, 6.48. 4.2. Pharmacology 4.2.1. Animals and treatment Male Swiss mice (30–34 g), 2–3 months old, were used throughout this study. The animals were randomly housed in appropriate cages at 22 ± 1 °C on a 12 h light/dark cycle (lights on 06:00–18:00) with free access to pellet diet (Purina®, Brazil) and tap water. All experiments involving behavioral analysis were carried out by the same visual observer. Experimental protocols were approved by the Federal University of Sergipe Animal Care and Use Committee (CEPA/UFS # 91/11). 4.2.2. PTZ- and PTX-induced convulsions The method previously described by Swinyard and coworkers [5] was employed to induce convulsions in mice using PTZ or PTX, with minor modifications [40]. The mice were divided into groups (n = 8 animals per group). One group received Tween 80 (4%), while a second one was treated with DZP (6.0 mg/kg, i.p.). The remaining groups received different douses of [(DZP)PdOAc]2 (acute dose: 6.0 mg/kg [6.7 μmol/kg], i.p.) or [(DZP)PdCl]2 (acute dose: 6.0 mg/kg [7.0 μmol/kg], i.p.). After 60 min of drug administration, the mice were treated with PTZ (60 mg/kg, i.p.) or PTX (8.0 mg/kg, i.p.). The latency and percent of clonic or generalized convulsions were recorded. To measure these parameters, the animals were observed individually for 15 min (900 s) or 20 min (1200 s) for the PTZ- or PTX-induced convulsion tests, respectively. The incidence of deaths was monitored until 48 h after the injection of convulsant agents. 4.2.3. Effects of flumazenil (FLU) on PTZ-induced convulsions The effect of selective GABAA-BZD receptor antagonist flumazenil (FLU) [58] on the anticonvulsant activity of [(DZP)PdOAc]2 and [(DZP)PdCl]2 was assessed. In the experimental groups, mice were pretreated with FLU (10 mg/kg, i.p.) 15 min before the administration of [(DZP)PdOAc]2 (6.0 mg/kg [6.7 μmol/kg], i.p.), [(DZP)PdCl]2 (6.0 mg/kg [7.0 μmol/kg], i.p.) or DZP (6.0 mg/kg [21 μmol/kg], i.p). After 60 min of drug administration, the mice were treated with PTZ (60 mg/kg, i.p.). 4.3. Statistical analysis The data obtained were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test. Differences were considered statistically significant when p b 0.05. All statistical analyses were performed using Graph Pad Prism 5.0 (Graph Pad Prism Software Inc., San Diego, CA, USA).

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Transparency declarations The authors declare no conflicts of interest. Acknowledgments The authors thank FINEP, CAPES, CNPq, FAPEAL, FAPITEC, and CapesCofecub-1421/2010 for financial support. WBZGB, AHQS, and ASLB express their appreciation for fellowships granted by CAPES. LJQJ and MRM thank CNPq for research fellowships. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2015.11.024. References [1] P. Sardo, G. Ferraro, BMC Neurosci. 8 (2007) 47, http://dx.doi.org/10.1186/14712202-8-47. [2] J.L. Becerra, J. Ojeda, E. Corredera, J.R. Gimenez, CNS Drugs 25 (2011) 3–16, http://dx. doi.org/10.2165/1159572-S0-000000000-00000. [3] D.A.M. Araújo, R.A. Mafra, A.L.P. Rodrigues, V. Miguel-Silva, P.S.L. Beirão, R.N. de Almeida, L. Quintans Jr., M.F.V. de Souza, J.S. Cruz, Br. J. Pharmacol. 140 (2003) 1331–1339, http://dx.doi.org/10.1038/sj.bjp.0705471. [4] A.T.R. Couto, D.T. Silva, C.C. Silvestre, D.P. Lyra Jr., L.J. Quintans Jr., Eur. J. Clin. Pharmacol. 69 (2013) 1343–1350, http://dx.doi.org/10.1007/s00228-012-1439-7. [5] E.A. Swinyard, A.W. Castellion, J. Pharmacol. Exp. Ther. 151 (1966) 369–375 (DOI:?). [6] A. McDowall, S. Owen, A.A. Robin, Br. J. Psychiatry 112 (1966) 629–631 (DOI:?). [7] E. Costa, A. Guidotti, C.C. Mao, A. Suria, Life Sci. 17 (1975) 167–185 (DOI:?). [8] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391–401, http://dx.doi.org/10. 1039/b707077m. [9] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197–206, http://dx. doi.org/10.1016/j.cbpa.2007.11.013. [10] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467–2498, http://dx.doi.org/10. 1021/cr980421n. [11] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307–320, http://dx.doi.org/10. 1038/nrd1691. [12] X. Meng, M.L. Leyva, M. Jenny, I. Gross, S. Benosman, B. Fricker, S. Harlepp, P. Hebraud, A. Boos, P. Wlosik, P. Bischoff, C. Sirlin, M. Pfeffer, J.-P. Loeffler, C. Gaiddon, Cancer Res. 69 (2009) 5458–5466, http://dx.doi.org/10.1158/0008-5472. CAN-08-4408. [13] C. Supan, G. Mombo-Ngoma, M.P. Dal-Bianco, C.L.O. Salazar, S. Issifou, F. Mazuir, A. Filali-Ansary, C. Biot, D. Ter-Minassian, M. Ramharter, P.G. Kremsner, B. Lell, Antimicrob. Agents Chemother. 56 (2012) 3165–3173, http://dx.doi.org/10.1128/ AAC.05359-11. [14] D. Dive, C. Biot, ChemMedChem 3 (2008) 383–391, http://dx.doi.org/10.1002/cmdc. 200700127. [15] A. Samanta, G.K. Ghosh, I. Mitra, S. Mukherjee, J.C.K. Bose, S. Mukhopadhyay, W. Linert, S.C. Moi, RSC Adv. 4 (2014) 43516–43524, http://dx.doi.org/10.1039/ C4RA06137C. [16] J. Spencer, R.P. Rathnam, B.Z. Chowdhry, Future Med. Chem. 2 (2010) 1441–1449, http://dx.doi.org/10.4155/fmc.10.226. [17] M.A. Cinellu, S. Gladiali, G. Minghetti, S. Stoccoro, F. Demartin, J. Organomet. Chem. 401 (1991) 371–384, http://dx.doi.org/10.1016/0022-328X(91)86234-H. [18] J. Spencer, R.P. Rathnam, M. Motukuri, A.K. Kotha, S.C.W. Richardson, A. Hazrati, J.A. Hartley, L. Malec, M.B. Hursthouse, Dalton Trans (2009) 4299–4303, http://dx.doi. org/10.1039/B819061E. [19] J. Spencer, B.Z. Chowdhry, A.I. Mallet, R.P. Rathnam, T. Adatia, A. Bashall, F. Rominger, Tetrahedron 64 (2008) 6082–6089, http://dx.doi.org/10.1016/j.tet. 2008.01.059. [20] S. Stoccoro, M.A. Cinellu, A. Zucca, G. Minghetti, F. Demartin, Inorg. Chim. Acta 215 (1994) 17–26, http://dx.doi.org/10.1016/0020-1693(93)03699-B. [21] J. Perez, V. Riera, A. Rodriguez, D. Miguel, Organometallics 21 (2002) 5437–5438, http://dx.doi.org/10.1021/om0205288. [22] M. Albrecht, C − H Bond Activation, in: J. Dupont, M. Pfeffer (Eds.), Palladacycles: Synthesis, Characterization and Applications, Wiley-VCH, Weinheim 2008, pp. 13–31. [23] M.L. Zanini, M.R. Meneghetti, G. Ebeling, P.R. Livotto, F. Rominger, J. Dupont, Inorg. Chim. Acta 350 (2003) 527–536, http://dx.doi.org/10.1016/S0020-1693(03)00011-2.

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