N-valplroyl-aminoacids as new potential antiepileptic drugs: synthesis, characterization and in vitro studies on stability

Epilepsy, affecting at least 50 million persons worldwide, is one of the most common neurological disorders. Despite the significant advances in understanding epileptogenic mechanisms and in counteracting their pathological consequences, this clinical condition still has to be faced of treating more effectively the symptoms (epileptic seizures) and of preventing their unfavourable evolution. So far, research has been unsuccessful involved in developing effective antiepileptic drugs (AEDs) capable of preventing the development of the pathogenic process, set in motion by different etiological factors, that leads ultimately to chronic epilepsies .[1, 2] So, a substantial need remains to develop new AEDs with better safety, less toxicity, and higher efficacy [3, 4]. Valproic acid, VPA, is one of the four most widely prescribed AEDs. Besides its wide use in both generalized and partial epilepsies, VPA has also gained widespread use in recent years for the treatment of bipolar disorders, neuropathic pain and for prophylactic treatment of migraine. [5,6] However the use of VPA is limited by two rare but potentially life-threatening side effects, hepatotoxicity, induced from the formation of metabolite(s) with a terminal double bond, specifically 4-ene-VPA, [7] and teratogenicity, associated with the parent compound itself [8]. In a previous work we reported the synthesis of aminoacidic ester derivatives of VPA as resulted of chemical conjugation of VPA with esters of essential neutral aminoacids, with the aim of modifying the physicochemical properties relevant to bioavailability, such as solubility or lipophilicity, improving the efficacy and reducing unwanted side or toxic effects of VPA [9]. We had reported also the synthesis of N-valproyl-L-tryptophan, that has shown adequate physicochemical characteristics to permeate biological membranes and antiepileptic activity at lower concentration than VPA. [10,11] In this paper, we focused our research on synthesis and characterization of new aminoacidic compounds with potential antiepileptic activity: N-Valproyl-L-Leucine (ValLeu), N-Valproyl-L-methionine (ValMet) and N-Valproyl-L-Histidine (ValHist). The conjugation could consent to obtain VPA derivatives, lacking of structural characteristics usually implicated on VPA teratogenicity, and avoiding formation of possible hepatotoxic metabolites. The aminoacidic derivatives of VPA was successfully obtained covalent linking carboxyl group of drug with aminic group of L-aminoacids, by synthesis involving two main steps. The first step, described in our previous work [9] was modified by adding DMAP as further coupling agent together with DCC. The structures of obtained compounds were assigned on the basis of respective analytical data-sets, FT-IR, MS and 1H and 13C-NMR spectral data. Since the drug lipophilicity is an important factor conditioning brain uptake, the apparent partition coefficient (Papp) could be used as simple descriptor of ability to cross the BBB: values of log Papp within -0.2 to 1.3 have been described as optimal for cerebral transport; on the other hand higher values than these could reduce the rate of transport inside the membrane [12, 13]. Apparent partition coefficient (Papp) of ValLeu, ValMet and ValHist were determined in n-octanol /phosphate buffer pH 7.4 solution and expressed as Log Papp. The determined Log Papp resulted -0.11,- 1,02 and -1,61 respectively. The Log DpH7.4 values indicate that ValLeu, ValMet are adequate to cross biological membranes and in particular BBB barrier while ValHist value is too low, probably due to the fact that was obtained as hydrochloride. Compared to others drug administration routes, the oral one remains the most preferred as it implies ease of administration as well as high patient compliance. However, the transit through the gastrointestinal tract could constitute a limiting step to bioavailability as a consequence of degradation correlated to the environmental pH. In view of a possible administration of ValLeu, ValMet and ValHist by oral route, studies on their chemical stability were performed in simulated gastro-intestinal buffer (37°C, pH 1.2 to 8.0) and monitored by HPLC analysis. The experiments demonstrated that ValLeu, ValMet and ValHist remained unchanged up to 24 h, and didn’t produce degradation products or potential metabolites. This behaviour indicates high stability at pH conditions of gastro-intestinal tract. Since compounds containing amide functional group could be susceptible of hydrolysis by plasma and/or cerebral enzymes, our experiments were focused on the evaluation of stability of ValLeu, ValMet and ValHist in these biological environments. Otherwise, plasma stability of drug candidates plays an important role in drug discovery and development; it is essential for maintaining acceptable drug concentration and half-life in order to achieve desirable pharmacological effects [14]. Experimental data highlighted that ValLeu, ValMet and ValHist remained unmodified up to 24 h in plasma environment. In rat brain homogenate ValLeu, ValMet and ValHist didn’t undergo cleavage after 24 h, indicating that synthetized compounds have also good stability to cerebral enzymes. References [1] Arroyo S, Brodie MJ, Avanzini G, Baumgartner C, Chiron C, Dulac O, French JA, Serratosa JM Is refractory epilepsy preventable? Epilepsia, (2002) 43, 437–444. [2] Walker MC, White HS, Sander JWAS. 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