**1. Introduction**

256 Bioenergetics

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> Mitochondria are important regulators of cellular functions and energy metabolism, therefore mitochondrial dysfunction leads to a compromised energy-generating system, deteriorated cellular homeostasis and neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease (Shapira, 1999; 2009). Hence, the protection of mitochondria, even their repair mechanisms at the level of complex I, may be a key strategy in limiting mitochondrial damage and ensuring cellular integrity (Dawson Dawson, 2003). Thus, in addition to traditionally used antiparkinsonian drugs, which are focused on the activation of the dopaminergic system, different mitochondria-protecting agents are being used in clinics for the treatment of Parkinson's disease. For instance, agents with antioxidant properties, such as melatonin (Esposito & Cuzzocrea, 2010), coenzyme Q10 and creatine (Kones, 2010), lipoic acid (De Araújo et al., 2011), and the extract of Hyoscyamus niger seeds (Sengupta et al., 2011), are currently used to treat Parkinson's disease.

> Recently, antihypertensive drugs of the calcium antagonistic series, which belong to 1,4 dihydropyridine (DHP) class and are capable of penetrating the blood-brain barrier (e.g., nifedipine, nimodipine), were shown to significantly reduce the risk of developing Parkinson's disease (Becker et al., 2008; Ritz et al., 2010). This was explained by blocking Ltype calcium channels in the dopaminergic neurons of the substantia nigra, where elevated calcium ion concentrations initiate cell death (Sulzeret & Schmitz, 2007). However, the mechanism of the antiparkinsonian action of DHPs is not yet understood.

> Our investigation of DHP compounds showed that many of them are capable of protecting mitochondrial processes (Fernandes et al., 2003, 2005, 2008, 2009). For instance, the most

Targeting the Mitochondria

concentrations of 0.1% (v/v) and less.

Fig. 1. The structures of AV-6-93 (A) and diflurone (B).

one-fifth of the culture medium was changed on DIV 3 (day 3 *in vitro*).

**2.3 Primary culture of rat cortical neurons** 

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 259

bis(1-adamantyloxycarbonyl)-4-(2-difluoromethoxyphenyl)-1,4-dihydropyridine] (Fig. 1B) were synthesised at the Latvian Institute of Organic Synthesis, 21 Aizkraukles Street, Riga, LV-1006. AV-6-93 and diflurone were dissolved in 100% DMSO and further diluted to

Chemicals for the mitochondrial studies were obtained from Sigma Chemical Company (St

Primary cultures were prepared from 1-day-old Wistar rat pups, according to the method of Alho et al., 1988, with minor modifications. Briefly, cortices were dissected in ice-cold Krebs-Ringer solution (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM KH2PO4, 15 mM glucose, 20 mM HEPES, pH 7.4, containing 0.3% bovine serum albumin) and trypsinised in 0.8% trypsin-EDTA (Invitrogen, U.K.) for 10 min at 37 °C, followed by trituration in 0.008% DNAse I solution containing 0.05% soybean trypsin inhibitor (both obtained from Surgitech AS, Estonia). Cells were resuspended in Eagle's basal medium with Earle's salts (BME, Invitrogen, U.K.), containing 10% heat-inactivated foetal bovine serum (FBS, Invitrogen, U.K.), 25 mM KCl, 2 mM GlutaMAXTM-I (Invitrogen, U.K.) and 100 g/mL gentamycin. Cells were plated onto poly-L-lysine- (Sigma Chemical Co., MO, USA) coated 48-well plates at a density of 1.8 x 105 cells/cm2. The medium was changed to NeurobasalTM-A medium containing 2 mM GlutaMAXTM-I with B-27 supplement and 100 μg/mL gentamycin 2.5 hr later. Cultures were incubated for 6 days in a 5% CO2/95% air atmosphere at 37 C, and

Louis, MO, USA); chemicals for cytotoxicity studies mentioned in 2.3. and 2.4.

active compound cerebrocrast {4-[2-(difluoromethoxy)phenyl-2,6-dimethyl-1,4 dihydropiridine-3,5-dicarboxylic acid di(2-propoxyethyl)diester}, which has shown neuroprotective effects in different neurodeficiency models (Klusa, 1995), decreased mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in rat cerebellar granule cells (Klimaviciusa et al., 2007). In isolated mitochondria of rat liver, cerebrocrast inhibited the inner mitochondrial anion channel, Ca2+-induced opening of the mitochondrial membrane permeability transition pore and permeabilization of the mitochondrial inner membrane (Vicente et al., 2006). In addition, it normalized oxidative phosphorylation and increased adenosine triphosphate (ATP)-induced contraction in swollen mitochondria of isolated rat skeletal muscle (Velena et al., 1997). Cerebrocrast and its congeners also protected against histopathological changes caused by azidothymidine, known to be a mitochondrial toxin (Pupure et al., 2008).

The present study investigates two novel DHP compounds, cerebrocrast analogues containing structure elements that may enhance the delivery of molecules through the blood-brain barrier and improve their access to mitochondria. The compounds are composed of either one adamantane moiety in position 3 (AV-6-93) or two adamantane moieties in positions 3 and 5 (diflurone) of the DHP ring. We suggest that these DHP structures may possess mitochondria-protecting and antiparkinsonian activity due to both the adamantane moiety, which can be considered to be an important functional unit, and the DHP structure, which may serve as the carrier molecule. Adamantane molecules were previously used in the design of neuroprotective drugs. For example, amantadine (1-aminoadamantane) is used in antiparkinsonian drugs with mechanisms focused on NMDAreceptor gated ion channels (Kornhuber et al., 1991). Adamantane derivatives, particularly memantine, are reported as neuroprotective agents against mitochondrial toxicity *in vivo* (Rojas et al., 2008) and *in vitro* (McAllister et al., 2008). Memantine may act directly on dopamine D2High receptors (Seeman et al., 2008), whereas amantadine may stimulate the synthesis and release of dopamine in the rat striatum (Spilker & Dhasmana, 1973), which is beneficial in the treatment of Parkinson's disease. Aminoadamantane derivatives 4-(1 adamantylamino)-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-(1-adamantylammonio)-1 hydroxy-2,2,6,6-tetramethylpiperidinium dihydrochloride were also synthesised as antiparkinsonian drugs (Skolimowski et al., 2003). However, compounds with adamantane moieties attached to the DHP structure have not yet been synthesised.

In this study, we tested novel compounds *in vitro* to assess their influence on mitochondrial processes in primary cultures of rat cortical neurons, using mitochondrial toxin MPP+, and on isolated rat liver mitochondria.

## **2. Materials and methods**

#### **2.1 Animals**

Male Wistar rats (250-350 g), housed at 22 ± 2 ºC under artificial light for a 12-h light/dark cycle and with access to water and food *ad libitum*, were used for these experiments. All of the experimental procedures were performed in accordance with the guidelines of Directive 86/609/EEC "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (1986) and were approved by the National Ethics Committee.

#### **2.2 Chemicals**

AV-6-93 [2,6- dimethyl-3-(1-adamantyloxycarbonyl)-4-(2-difluoromethoxyphenyl)-5-[(2 propoxy)ethoxycarbonyl]-1,4-dihydropyridine] (Fig. 1A) and diflurone [2,6- dimethyl-3,5-

active compound cerebrocrast {4-[2-(difluoromethoxy)phenyl-2,6-dimethyl-1,4 dihydropiridine-3,5-dicarboxylic acid di(2-propoxyethyl)diester}, which has shown neuroprotective effects in different neurodeficiency models (Klusa, 1995), decreased mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in rat cerebellar granule cells (Klimaviciusa et al., 2007). In isolated mitochondria of rat liver, cerebrocrast inhibited the inner mitochondrial anion channel, Ca2+-induced opening of the mitochondrial membrane permeability transition pore and permeabilization of the mitochondrial inner membrane (Vicente et al., 2006). In addition, it normalized oxidative phosphorylation and increased adenosine triphosphate (ATP)-induced contraction in swollen mitochondria of isolated rat skeletal muscle (Velena et al., 1997). Cerebrocrast and its congeners also protected against histopathological changes caused by azidothymidine,

The present study investigates two novel DHP compounds, cerebrocrast analogues containing structure elements that may enhance the delivery of molecules through the blood-brain barrier and improve their access to mitochondria. The compounds are composed of either one adamantane moiety in position 3 (AV-6-93) or two adamantane moieties in positions 3 and 5 (diflurone) of the DHP ring. We suggest that these DHP structures may possess mitochondria-protecting and antiparkinsonian activity due to both the adamantane moiety, which can be considered to be an important functional unit, and the DHP structure, which may serve as the carrier molecule. Adamantane molecules were previously used in the design of neuroprotective drugs. For example, amantadine (1-aminoadamantane) is used in antiparkinsonian drugs with mechanisms focused on NMDAreceptor gated ion channels (Kornhuber et al., 1991). Adamantane derivatives, particularly memantine, are reported as neuroprotective agents against mitochondrial toxicity *in vivo* (Rojas et al., 2008) and *in vitro* (McAllister et al., 2008). Memantine may act directly on dopamine D2High receptors (Seeman et al., 2008), whereas amantadine may stimulate the synthesis and release of dopamine in the rat striatum (Spilker & Dhasmana, 1973), which is beneficial in the treatment of Parkinson's disease. Aminoadamantane derivatives 4-(1 adamantylamino)-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-(1-adamantylammonio)-1 hydroxy-2,2,6,6-tetramethylpiperidinium dihydrochloride were also synthesised as antiparkinsonian drugs (Skolimowski et al., 2003). However, compounds with adamantane

In this study, we tested novel compounds *in vitro* to assess their influence on mitochondrial processes in primary cultures of rat cortical neurons, using mitochondrial toxin MPP+, and

Male Wistar rats (250-350 g), housed at 22 ± 2 ºC under artificial light for a 12-h light/dark cycle and with access to water and food *ad libitum*, were used for these experiments. All of the experimental procedures were performed in accordance with the guidelines of Directive 86/609/EEC "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (1986) and were approved by the National

AV-6-93 [2,6- dimethyl-3-(1-adamantyloxycarbonyl)-4-(2-difluoromethoxyphenyl)-5-[(2 propoxy)ethoxycarbonyl]-1,4-dihydropyridine] (Fig. 1A) and diflurone [2,6- dimethyl-3,5-

known to be a mitochondrial toxin (Pupure et al., 2008).

moieties attached to the DHP structure have not yet been synthesised.

on isolated rat liver mitochondria.

**2. Materials and methods** 

**2.1 Animals** 

Ethics Committee.

**2.2 Chemicals** 

bis(1-adamantyloxycarbonyl)-4-(2-difluoromethoxyphenyl)-1,4-dihydropyridine] (Fig. 1B) were synthesised at the Latvian Institute of Organic Synthesis, 21 Aizkraukles Street, Riga, LV-1006. AV-6-93 and diflurone were dissolved in 100% DMSO and further diluted to concentrations of 0.1% (v/v) and less.

Chemicals for the mitochondrial studies were obtained from Sigma Chemical Company (St Louis, MO, USA); chemicals for cytotoxicity studies mentioned in 2.3. and 2.4.

Fig. 1. The structures of AV-6-93 (A) and diflurone (B).
