WO2012127030A1 - Arylpiperazines as neuroprotective agents - Google Patents

Abstract

The present invention generally relates to novel arylpiperazines. In particular, these arylpiperazines can be used as neuroprotective agents. The invention also relates to a process for the manufacture of the novel compounds. Further, the invention relates to the use of the novel arylpiperazines in the treatment of diseases associated with, accompanied by or caused by mitochondrial stress.

Description

Arylpiperazines as neuroprotective agents

The present invention generally relates to novel arylpiperazines. In particular, these arylpiperazines can be used as neuroprotective agents. The invention also relates to a process for the manufacture of the novel compounds. Further, the invention relates to the use of the novel arylpiperazines in the treatment of diseases associated with, accompanied by or caused by mitochondrial stress. Nitrogen monoxide (NO), also known as nitric oxide, is a free-radical gas, freely diffusible through biological membranes. It is synthesized through sequential oxidation of L-arginine to L-citrulline catalyzed by nitric oxide synthase (NOS). Three different forms of NO synthase - endothelial, neuronal and inducible, have been identified. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed and generate physiologically vital amounts of NO involved in maintenance of vascular tone and regulation of neurotransmission (Moncada 1993). Inducible NOS (iNOS), on the other hand, produces much larger (nanomolar) amounts NO in response to various proinflammatory stimuli, which in the brain represents a defensive mechanism against certain infectious pathogens (Pannu 2006). The overproduction of NO by iNOS has been tightly linked to neuroinflammation and neurodegeneration associated with traumatic/ischemic injuries, Alzheimer's, Parkinson's and Krabbe's disease, as well as with demyelinating conditions such as those observed in multiple sclerosis (MS), periventricular leukomalacia, spinal cord injury (SCI) and X-linked adrenoleukodystrophy (X-ALD) (Pannu 2006). NO- mediated cell damage is a consequence of its highly reactive nature. It reacts with superoxide radicals to generate reactive nitrogen species (RNS) such as dinitrogen trioxide (N₂O₃) and peroxynitrite (ONOO-), causing toxicity through complexation with iron in iron-containing enzyme systems (Drapier 1988), oxidation of protein sulfhydryl groups (Radi 1991 ), nitration of proteins, nitrosylation of nucleic acids and DNA strand breaks (Wink 1991 ). The main outcome of excessive NO production is apoptotic death of various cell types, including neurons (Heales 1999). N-arylpiperazine subunit is part of a variety of pharmacologically interesting compounds, which act as dopamine and serotonine ligands, calcium blockers, antipsychotics, antihypertensive drugs or acetylcholinesterase inhibitors (Romero 2006). Dopamine receptor ligands used for symptomatic therapy of Parkinson's disease show neuroprotective effects under a variety of neurodegenerative conditions (Chen 2008, Uberti 2002, Carvey 1997, Kitamura 1998; Yang 2008, Park 2009, Kato 2008) and the neuroprotective action of dopamine D1 antagonists has also been demonstrated (Cools 2002, Sonsalla 1986). Although some of the reported effects have been directly linked to the binding to dopamine receptors (Chen 2008), it appears that non-receptor dependent pathways could also be involved (Ramirez 2003, Gu 2004; Matsuo 2010). Non-receptor-mediated neuroprotection by dopaminergic ligands may include free radical scavenging activity against hydroxyl radicals and nitric oxide (Gomez-Vargas 1998, Pardo 1995) and subsequent prevention of neuronal mitochondrial damage and apoptosis (Gille 2002, Uberti 2004). According to the present invention, novel arylpiperazine-based dopamine receptor ligands were synthesized. It was found that these arylpiperazines show neuroprotective action in cell culture, with a significant selectivity for

NO.

Thus, a first aspect of the invention relates to a compound of the general formula I, II or III

II

111

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹ is selected from the group consisting of:

C-i-Ci₂-alkyl, C₃-C₁₂-cycloalkyl, C₆-Ci₀-aryl, and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxy I, OCOR⁴, amino, Ci-Ce-a!kylamino, Ci-Ce-dialkylamino, CrC₆-(halo)alkyl, Ci-C₆-(halo)alkoxy and/or COOR⁴;

R² is selected from the group consisting of:

Ce-C-io-aryl and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxyl, OCOR⁴, amino, Ci-C₆-alkylamino, Ci-Ce-dialkylamino,

Ci-C₆-(ha!o)alkyl, C C₆-(halo)alkoxy and/or COOR⁴;

R³ is independently at each occurrence selected from the group consisting of: hydrogen, halogen, hydroxyl, d-C₂-(halo)alkyl, and Ci-C₂-(halo)alkoxy; R⁴ is independently at each occurrence selected from the group consisting of: hydrogen and C C₂-alkyl; and

n is 1-10.

Preferably, the inventive compounds have neuroprotective activity In particular, the compounds according to the invention are capable of protecting human neuroblastoma cells from oxidative stress induced by nitric oxide (NO) as described in the Examples.

For example, cells incubated with a compound of formula I, II, or III at a concentration of 2.5 μΜ show a reduction in NO-induced cell damage of at least 15%, which may be determined using an acid phosphatase assay. Alternatively, incubation of cells with a compound of formula I, II, or III at a concentration of 10 μΜ leads to a reduction in NO-induced cell damage of at least 30%. In a further alternative, treatment of cells with a compound of formula I, II, or III at a concentration of 10 μΜ reduces the amount of superoxide in e.g. SNP-treated cells by at least 2.5%. In yet a further alternative, treatment of cells with a compound of formula I, II, or III at a concentration of 10 μΜ prevents changes of more than 30% in activation/inhibition of signalling molecules such as Akt, JNK, ERK, and AMPK in e.g. SNP-treated cells. Activation/inhibition may be determined by analyzing phosphorylation of the respective signalling molecule by e.g. immunoblotting. Most preferably, a compound according to the invention shows all biological effects described above.

A "pharmaceutically acceptable salt" refers to salts or complexes of a compound of formula I, II, or III. Examples of such salts include, but are not limited to, base addition salts formed by reaction of a compound of formula I, II, or III, with an organic or inorganic base, e.g. ammonia or a hydroxide, carbonate or bicarbonate of a metal cation, which is preferably selected from alkali metals (e.g. sodium, potassium or lithium), and alkaline earth metals (e.g. calcium or magnesium), or with an organic primary, secondary or tertiary alkyl amine. Also encompassed are acid addition salts formed with inorganic acids (e.g. hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid), as well as salts formed with organic acids, e.g. aliphatic monocarboxylic and dicarboxylic acids, aromatic acids, and sulfonic acids. Non-limiting examples of such acids are acetic acid, benzoic acid, (+)-camphor-10- sulfonic acid, citric acid, gluconic acid, lactic acid, methanesulfonic acid, propionic acid, oxalic acid, succinic acid, tartric acid, trifluoroacetic acid, and triphenylacetic acid. A .pharmaceutically acceptable solvate" may e.g. be a hydrate.

The term„(halo)alkyl" according to the present invention relates to an alkyl group which optionally contains at least one halo, e.g. F, CI, Br or I substituent up to perhalogenation. The term „alkyl" means a monovalent linear or branched, saturated or unsaturated hydrocarbon moiety, consisting of carbon and hydrogen atoms, wherein the number of carbon atoms is defined by a subscript number, e.g. „Ci-Ci₂". Non-limiting examples include methyl, ethyl, ethenyl, ethinyl, propyl, isopropyl, allyl, n-butyl, isobutyl, tert-butyl, butenyl, hexyl, octyl, and dodecyl.

„Cycloalkyl" means a monovalent saturated or partially unsaturated carbocyclic moiety, preferably consisting of one or two rings. Non-limiting examples include cyclopropyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexadienyl, decalinyl, norbornyl etc.

The term„aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 10 carbon atoms having a single ring or two condensed rings. Preferred aryl groups include phenyl and naphthyl.

The term„heteroaryl" as used herein refers to a monocyclic radical of 5 to 7 ring atoms containing one, two, or three ring heteroatoms selected from nitrogen, oxygen, and sulfur, the remaining ring atoms being carbon. Preferred examples include, but are not limited to furanyl, imidazolyl, isoxazolyl, oxazolyl, pyridyl, pyridazinyl, pyrazinyl, thiazolyl, and thiophenyl. "Alkoxy" means a moiety of the formula -OR, wherein R is an a Iky I moiety as defined herein. Non-limiting examples of alkoxy moieties include methoxy, ethoxy, and isopropoxy.

..Substituted" means that one or more functional groups (..substituents") are attached to one or more carbon atoms of an alkyl, cycloalkyi, aryl, or heteroaryl moiety as defined herein. Preferably, a given moiety is substituted with one, two, three, four, or five independently selected substituents. In a preferred embodiment, the compound according to the invention has the general formula I or II. In this embodiment, R¹, R², R³, R⁴, and n may have a preferred meaning as defined herein. In a further preferred embodiment, the compound according to the invention has the general formula I.

In yet a further preferred embodiment of the invention, the compound has the general formula I, II or III,

wherein R¹ is selected from the group consisting of:

d-Ce-alkyl, phenyl, and nitrogen-containing C₆-heteroaryl, each unsubstituted or substituted with halogen, hydroxy, Ci-C₆-(halo)alkyl, and/or Ci-C₆-(halo)alkoxy, and

R², R³, R⁴, and n may have preferred meaning as defined herein.

In yet a further preferred embodiment of the invention, R¹ is phenyl, which is unsubstituted or substituted with halogen, hydroxyl, and/or CrC₆-alkoxy, and

R², R³, R\ and n may have a preferred meaning as defined herein.

In yet a further preferred embodiment of the invention, R² is selected from the group consisting of:

phenyl and Cs-Cr-heteroaryl, each unsubstituted or substituted with halogen, hydroxyl, amino, CrC₆-(halo)alkyl, and/or Ci-C₆-(halo)alkoxy, and R¹, R³, R⁴, and n may have a preferred meaning as defined herein.

In yet a further preferred embodiment of the invention, R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with halogen, hydroxyl, amino and/or Ci-C₆-alkoxy, and

R\ R³, R⁴, and n may have a preferred meaning as defined herein.

In yet a further preferred embodiment of the invention, the compound has the general formula I, wherein R² is selected from the group consisting of: phenyl, 2-pyridyl, 3-pyridyl, and pyridazinyl, each unsubstituted or substituted with halogen, hydroxy I, amino and/or Ci-Ce-alkoxy. In yet a further preferred embodiment of the invention, the compound has the general formula II, wherein R² is selected from the group consisting of: phenyl, 2-pyridyl, and 3-pyridyl. The phenyl group may be unsubstituted or substituted with halogen, amino, and/or Ci-C₆-alkoxy. The 2-pyridyl or 3- pyridyl group may be unsubstituted or substituted, preferably monosubstituted in the ortho-position, with halogen, hydroxy, amino, and/or Ci-Ce-alkoxy.

In certain further preferred embodiments of the compound of formula I, II, or III, R³ is hydrogen, and R , R², R⁴, and n may have a preferred meaning as defined herein.

In still further preferred embodiments of the compound of formula I, II, or III, R⁴ is hydrogen, and R¹, R², R³, and n may have a preferred meaning as defined herein.

In yet a further preferred embodiment of the invention, n is 1 -6. In a more preferred embodiment, n is 2.

More preferably, the inventive compound has the general formula I, II, or III, wherein

R¹ is phenyl, which is unsubstituted or substituted with halogen, hydroxyl, and/or Ci-Ce-alkoxy;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with halogen, hydroxyl, amino and/or Ci-Ce-alkoxy; and

n is 2.

Still more preferably, R¹ is unsubstituted phenyl;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with hydroxyl;

R³ and R⁴ each are hydrogen; and n is 2.

Still more preferably, the compound has the general formula II, wherein R¹ is unsubstituted phenyl;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, and 3-pyridyl, each unsubstituted or substituted with hydroxyl, with phenyl preferably being unsubstituted and 2-pyridyl and 3- pyridyl preferably being unsubstituted or monosubstituted in the ortho- position,

R³ and R⁴ each are hydrogen; and n is 2.

Still more preferably, the compound has the general formula I, wherein R¹ is unsubstituted phenyl;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, pyridazinyl, each unsubstituted or substituted with hydroxyl;

R³ and R⁴ each are hydrogen; and n is 2.

Most preferably, the compound according to the invention is selected from the group consisting of:

N-{4-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide,

N-{3-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide,

2-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide, 2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-isonicotinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyi}-isonicotinamide, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide,

2-Hydroxy-N-{4-[2-(4-pheny!-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide, 2-Hydroxy-N-{3-[2-(4-phenyi-piperazin-1 -y!)-ethy!]-phenyl}-nicotinamide, 4-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide, and

4-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide, and pharmaceutically acceptable salts or solvates thereof (i.e. of any one of the above compounds).

In a particularly preferred embodiment, the compound according to the invention is selected from the group consisting of:

N-{4-[2-(4-Phenyl-piperazin-1 -y!)-ethyl]-phenyl}-benzamide,

N-{3-[2-(4-Phenyl-piperazin-1 -yl)-ethyl]-phenyi}-benzamide,

2-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide,

2-Hydroxy-N-{4-[2-(4-pheny!-piperazin-1-y!)-ethyl]-phenyl}-nicotinamide, 2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide, 4-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide, and pharmaceutically acceptable salts or solvates thereof.

In another particularly preferred embodiment, the compound according to the invention is N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolin- amide or a pharmaceutically acceptable salt or solvate thereof.

A further aspect of the invention relates to a process for the manufacture of a compound of the general formula I, II or 111, which comprises the steps of:

(i) reacting an arylcarboxylic acid of general formula IV a), IV b), or IV c)

VI c)

(ii) reducing said compound of formula VI a), VI b), or VI c) with a suitable reducing agent,

(iii) hydrogenating the product of step (ii),

(iv) reacting the product of step (iii) with a carboxylic acid of the general formula R²COOH, and

(v) optionally isolating the resulting compound of formula I, II, or III, wherein R¹ is selected from the group consisting of:

Ci-Ci2-alkyl, C₃-C ₂-cycloalkyl, C₆-Ci₀-aryl, and C₃-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxy!, OCOR⁴, amino, Ci-C₆-alkylamino, Ci-C₆-dialkylamino, C₁-C₆-(halo)alkyl, Ci-C₆-(halo)alkoxy and/or COOR⁴;

R² is selected from the group consisting of:

C₆-Ci₀-aryl and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxyl, OCOR⁴, amino, Ci-C₆-alkylamino, Ci-C₆-dialkylamino, d-Ce-ihaloJalkyl, C₁-C₆-(halo)alkoxy and/or COOR⁴;

R³ is independently at each occurrence selected from the group consisting of: hydrogen, halogen, hydroxyl, C₁-C₂-(halo)alkyl, and C C₂-(halo)alkoxy; R⁴ is independently at each occurrence selected from the group consisting of: hydrogen and CrC₂-alkyl; and wherein n is 0-9.

In a preferred embodiment of said process, the aryicarboxyiic acid of step (i) has the general formula VII

0₂N wherein n is 0-9.

In a further preferred embodiment of said process, the amine of step (i) has the general formula VIII

wherein R¹ has the meaning as defined supra.

In yet a further preferred embodiment of the process, the reaction product of step (i) has the general formula IX

IX

wherein R¹ and n have the meaning as defined supra.

In a more preferred embodiment of the process, R¹ is phenyl, R² is selected from phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, and pyrazinyl, each unsubstituted or substituted with hydroxy I; R³ and R⁴ are hydrogen at each occurrence; and n is 1.

Suitable reducing agents for use in the reduction step (ii) are e.g. diborane (B₂H₆), lithium aluminium hydride (LAH; LiAIH₄), and diisobutyialuminium hydride (DIBAL).

Suitable catalysts for hydrogenating the niiro group (N0₂) in step (iii) are transition metals of groups 8, 9, 10 and 11 of the periodic table, particularly nickel (e.g. Raney nickel), platinum, palladium, rhodium, and ruthenium.

According to yet a further aspect of the present invention, the compound of formula I, II, or III is for use in medicine. Said use is preferably a use in human medicine, but the compounds may also be used for veterinary purposes. In specific embodiments, it may be preferred to use compounds of formula I. The compound of formula I, II, or III is preferably administered to a subject in need thereof, e.g. a human subject, as a pharmaceutical composition. Thus, a further aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula I, II, or III, or a pharmaceutically acceptable salt or solvate thereof, and further comprising pharmaceutically acceptable excipients, adjuvants, diluents and/or carriers.

As exemplary excipients, disintegrators, binders, fillers, and lubricants may be mentioned. Examples of disintegrators include agar-agar, algins, calcium carbonate, cellulose, colloid silicon dioxide, gums, magnesium aluminium silicate, methylcellulose, and starch. Examples of binders include hydroxymethyl cellulose, hydroxypropylcellulose, microcrystalline cellulose, and polyvinylpyrrolidone. Examples of fillers include calcium carbonate, calcium phosphate, tribasic calcium sulfate, calcium carboxymethy!ce!lulose, cellulose, dextrin, dextrose,¹ fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl laureate, ethyl oleate, glycerin, glyceryl palmitostearate, glycols, hydrogenated vegetable oil, magnesium oxide, mannitol, poloxamer, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, stearates, and talc.

Commonly used buffer substances, colorants, consistency-improving agents, diluents, emollients, flavour-improving agents, preservatives, salts for varying the osmotic pressure, solubilizers, stabilizers, wetting and emulsifying agents, masking agents and antioxidants come into consideration as pharmaceutical adjuvants.

Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting-point wax, cocoa butter, water, alcohols, polyols, glycerol, vegetable oils and the like.

The pharmaceutical composition may also comprise at least one further active agent, e.g. one or more further organic or inorganic molecule. The composition may be used alone, without further medication. Alternatively, the composition may be used in combination with other medicaments, e.g. medicaments for treating neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, ALS, and stroke.

In particular, the inventive compound of formula I, II, or III, or a pharmaceutical composition comprising a compound of formula I, II, or III, is for use in the treatment of a disease, disorder or condition associated with, accompanied by or caused by mitochondrial stress.

The present invention also relates to a method for the treatment of a disease, disorder or condition associated with, accompanied by or caused by mitochondrial stress, comprising administering a pharmaceutically effective amount of a compound of formula I, II or III to a subject in need thereof.

Mitochondria are cellular organelles composed of two membranes. An inner mitochondrial membrane (IMM), which is organized into so-called cristae, is surrounded by an outer membrane, which encloses the entire organelle. The space surrounded by the IMM is called matrix and harbours the majority of the mitochondrial proteins, as well as the mitochondrial genome. Between the two membranes, there is an intermembrane space.

The most prominent function of mitochondria is their role in the regulation of cellular metabolism and, most importantly, in the oxidative phosphorylation, a central step in the production of energy in the form of ATP. The content of mitochondria differs according to the cell type. Cells which have high energy consumption, such as muscle and nerve cells, are particularly rich in mitochondria.

A problem in the process of oxidative phosphorylation is that a small part of the electrons transported in the respiratory chain is responsible for the formation of reactive oxygen species (ROS) such as the dioxide anion (O₂ ^~-, also known as superoxide) and hydrogen peroxide (H₂0₂). ROS lead to propagation of free radicals and can oxidize cellular lipids, nucleotide bases, and proteins. For example, O₂ ^"- can react with nitric oxide (NO) to form toxic compounds such as peroxy nitrite (ONOO ). Inhibition of components of the mitochondrial respiratory chain may ultimately lead to apoptosis of cells.

A state where cells, e.g. human ceils, produce increased amounts of oxidants, e.g. ROS, leading to an increased release of free radicals and resulting in cellular degeneration is referred to as oxidative or mitochondrial stress. Mitochondrial stress seems to be involved in several disorders, including neurodegenerative and cardiovascular diseases. Thus, in a preferred embodiment of the invention, the compound of formula I, II or III as described supra is for use in the treatment of a neurodegenerative disorder. Another preferred embodiment of the invention is a method for the treatment of a neurodegenerative disorder. Preferably, said neurodegenerative disorder is selected from amyotrophic lateral sclerosis, Alzheimer's Disease, Parkinson's Disease and stroke.

In a most preferred embodiment, said neurodegenerative disorder is amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig' s disease).

In another most preferred embodiment, said neurodegenerative disorder is Alzheimer's Disease.

In another most preferred embodiment, said neurodegenerative disorder is Parkinson's Disease.

In another most preferred embodiment, said neurodegenerative disorder is stroke.

The present invention shall be further illustrated by the following Figures and Examples, which are not intended to limit the invention.

Figure Legends

Figure 1. Synthetic route and chemical structures of the arylpiperazine dopaminergic ligands. (A) Synthetic pathways for N-{4-[2-(4-phenyl- piperazin-1-yl)-ethyl]-phenyl}-arylamides and N-{3-[2-(4-phenyl-piperazin- 1 -yl)-ethyl]-phenyl}-arylamides. (B) Synthetic pathways for 5-[2-(4-phenyl- piperazin-1 -yl)-ethy!]-2-pyridin-4-yl-1 H-benzoimidazole.

Figure 2. The effect of arylpiperazines on SNP- and H₂0₂-induced toxicity in SH-SY5Y neuroblastoma cells. (A) SH-SY5Y cells were incubated with different concentrations of SNP or H₂0₂ and the cell viability was determined by acid phosphatase assay after 24 h. (B) Cells were pretreated with different arylpiperazines (10 μΜ) for 30 min and then exposed to SNP (2 mM) or H₂O_z (100 μΜ). The cell viability was determined by acid phosphatase assay after 24 h. (C, D) Cells were pre- incubated for 30 min with various concentrations of compound 6a (up to 10 μΜ, C) or 10 μΜ of compound 6a (D) before addition of 2 mM SNP. After 24 h, cell viability was measured by acid phosphatase assay (C), whereas cell morphology was analyzed by inverted microscopy (D). (E) PMA-differentiated SH-SY5Y cells were exposed to SNP (2 mM) and/or compound 6a (10 μΜ) and acid phosphatase activity as a measure of cell viability was assessed after 24 h. The presented data are mean ± SD values from a representative of at least three independent experiments (^*p < 0.05).

Figure 3. The inhibitory effect of compound 6a on SNP-induced depolarization of mitochondrial membrane and apoptosis in SH-SY5Y cells. (A, B, C, D) SH-SY5Y cells were pretreated with compound 6a (10 μΜ) for 30 minutes and then exposed to SNP (2 mM). After 24 h, cells were stained with annexin V-FITC/Pl (A), PI (B), ApoStat (C) or JC-1 (D), and phosphatidylserine externalization (A), DNA fragmentation (B), caspase activation (C) or mitochondrial membrane potential (D) was examined by flow cytometry. The representative dot plots and histograms (A, B, C) or mean + SD values (D) from three independent experiments are presented (^*p < 0.05 and ^#p < 0.05 refer to untreated and SNP-treated cells, respectively).

Figure 4. The effect of compound 6a on superoxide and NO levels in SH- SY5Y cells following exposure to chemically or cell-derived NO. (A, B) SH- SY5Y cells were pre-incubated with compound 6a (10 μΜ) for 30 minutes and then treated with SNP (2 mM). After 24 h, flow cytometry was used to determine intracellular levels of superoxide anion in DHE-stained cells (A) or NO in DAF-stained cells (B). (C, D) Rat peritoneal macrophages were incubated without or with LPS (5 pg/ml) and rat interferon-γ (10 ng/ml), in the absence or presence of different concentrations of compound 6a. Nitrite accumulation was measured after 24 h and 48 h using Griess reaction (C), while intracellular NO content in DAF-stained cells was determined after 24 h by flow cytometry (D). (E, F) SH-SY5Y cells were co-incubated with LPS + IFN-y-stimulated rat peritoneal macrophages in the absence or presence of different concentrations of compound 6a. After 24 h and 48 h, nitrite concentrations were measured using Griess method (E), while the cell viability was determined by acid phosphatase assay (F). The representative histogram (A) or mean + SD values (B-F) from three independent experiments are presented (^*p < 0.05 and *p < 0.05 refer to untreated and SNP-treated cells, respectively).

Figure 5. The effect of compound 6a on SNP-induced cell death- regulating signalling pathways in SH-SY5Y cells. SH-SY5Y cells were treated with compound 6a (10 μΜ) for 30 minutes before addition of SNP (2 mM). Activation (phosphorylation) of Akt, JNK, ERK and AMPK was analyzed by immunoblotting after 8 h (the data from one of two experiments with similar results are shown). Figure 6. The effect of compound 6a is not dopamine receptor-mediated. SH-SY5Y cells were first incubated with D1/D2 receptor blocker (+)butac!amol (10 μΜ), then with compound 6a (10 μΜ) after 30 minutes and then with SNP (2 mM) after additional 30 min. Cell viability was assessed by acid phosphatase assay after 24 h. The data are presented as mean + SD of triplicates from one of two experiments with similar results (^"p < 0.05 refers to cells treated with SNP alone).

Examples

Example 1 : Synthesis and characterization of N-arylpyperazine derivatives

Overview

Synthetic route and chemical structures of the compounds synthesized in the present study are shown in Figure 1. Acylation of N-phenylpiperazine using 4-nitrophenylacetic acid gave rise to 2-(nitrophenyl)-1 -(4-phenyl- piperazin-1 -yl)-ethanones (1 a,b). Amides 1a, b were converted to 1 -nitro- phenethyl-4-phenyl-piperazines 2a,b using diborane in tetrahydrofuran (THF). Reduction of nitro compounds 2a,b by Ra-Ni/hydrazine provided 1 -aminophenethyl-4-phenyl-piperazines 3a, b. Target compounds 4a,b- 12a,b were obtained by condensation of anilines 3a,b with corresponding aromatic carboxylic acid in presence of propylphosphonic acid anhydride (PPAA) in N-Dimethylformamide (DMF). All compounds were characterized by NMR spectroscopy and mass spectroscopy.

General

A Stuart SMP10 apparatus (Bibby Sterlin Ltd, Stone, UK) was used to determine melting points, presented here as uncorrected. ¹H-NMR (at 300 MHz) and ¹³C-NMR (at 50 MHz) spectra were recorded on a Bruker AC300 (Bruker, Karlsruhe, Germany) with CDCI₃ as a solvent, and, unless otherwise stated, are reported in ppm using tetramethylsilane as the internal standard. The mass spectra were determined by field desorption mass spectroscopy on a Finnigan Mat 8230 mass spectrometer (Finnigan, Bremen, Germany). For analytical thin-layer chromatography Merck (Darmstadt, Germany) F-256 plastic-backed thin-layer silica gel plates were used. Chromatographic purifications were performed on Merck-60 silica gel columns, 230-400 mesh ASTM, under medium pressure (dry column flash chromatography). All reagents and solvents used herein were obtained from Aldrich and were used without further purification. Solutions were routinely dried over anhydrous MgS0₄ prior to evaporation.

Synthesis of 4-f2~(4-Phenyl-piperazin-1-yi)-ethvi]-nitrophenylacetic acid (1a) and 3-[2-(4-Phenyl-Diperazin-1-vl)-ethvll-nitrophenylacetic acid (1b) A solution of nitrophenylacetic acid (9.5 g, 52.2 mmol), 9.0 g phenylpiperazine (9.0 g, 58.5 mmoi), DCC (10.7 g, 52.2 mmol) and 100 mg DMAP in 300 ml dry THF was stirred overnight at room temperature. Obtained precipitated dicyclohexyl urea was filtrated off, washed with 50 ml THF and combined filtrates were evaporated in vacuo. Oily residue was dissolved in 150 ml ethyl acetate and allowed to crystallise in a refridgerator. Obtained products were isolated by filtration and dried.

4-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-nitrophenylacetic acid (1a): Yield: 15.0 g; mp 147-150 °C; NMR: δ 8.17 (d, J=8.5 Hz, 2H, ArH); 7.42 (d, J=8.5 Hz, 2H, ArH); 7.42 (t, J=7 Hz, 2H, ArH); 7.89 (m, 3H, ArH); 3.86 (s, 2H, CH₂); 3.80 (s, 2H, CH₂); 3.63 (s, 2H, CH₂); 3.15 (s, 2H, CH₂CO); 3.08 (s, 2H, CH₂). MS: m/e (100) 325.4 (M+). 3-[2-(4-Phenyl-piperazin-1 -yl)-ethyl]-nitrophenylacetic acid (1b): Yield: 8.5 g; mp 124-126 °C; NMR.-δ 8.11 (m, 2H, ArH); 7.61 (d, J=8.5 Hz, 1 H, ArH); 7.49 (t, J=7 Hz, 1 H, ArH); 7.27 (t, J=7 Hz, 2H, ArH); 6.89 (m, 3H, ArH); 3.84 (s, 2H, CH₂); 3.80 (s, 2H, CH₂); 3.65 (s, 2H, CH₂CO); 3.13(s, 4H, CH₂). MS: m/e (100) 325.4 (M+)

Synthesis of 4-[2-(4-Phenyl-piperazin- 1-yl)-eth yll-nitrobenzene (2a) and 3- [2-(4-Phenyl-piperazin-1-yl)-ethyl]-nitrobenzene (2b)

Under nitrogen atmosphere, diborane (130 ml, 1 M solution in THF) was added dropwise to a solution of nitrophenylacetamides 1a,b (14.0 g, 44.0 mmol) in 100 ml THF at ice bath temperature. Upon completion of addition, the mixture was first stirred for 60 min at room temperature and then refluxed for 120 min. Excess of diborane was removed by careful addition of 80 ml 5 N HCI and refluxing for additional 60 min. Finally, the reaction mixture was left to reach room temperature, alkalinized with 20% NaOH and extracted with ethyl acetate. Upon drying over MgS0₄ organic extracts were concentrated in vacuo and the crude products recrystallized from ethyl acetate. 4-[2-(4-Phenyl-piperazin-1 -yl)-ethyl]-nitrobenzene (2a): Yield: 10.0 g; mp 137-140 °C; NMR: δ 8.13 (d, J=8.5 Hz, 2H, ArH); 7.36 (d, J=8.5 Hz, 2H, ArH); 7.26 (t, J=7 Hz, 2H, ArH); 6.92 (d, J=8 Hz, 2H, ArH); 6.65 (t, J=8 Hz, 1 H, ArH); 3.27 (m, 4H, CH₂); 2.94 (t, J=7 Hz, 2H, CH₂); 2.67 (m, 4H, CH₂). MS: m/e (100) 311.4 (M+)

3- [2-(4-Phenyl-piperazin-1 -yl)-ethyl]-nitrobenzene (2b): Yield: 9.5 g; mp 88-90 °C; NMR:6 8.09 (s, 1 H, ArH); 8.06 (d, J=7.5Hz, 1 H, ArH); 7.55 (d, J=7.5 Hz, 1 H, ArH); 7.44 (t, J=7.5 Hz, 1 H, ArH); 7.24 (m, 2H, ArH); 6.89 (d, J=8 Hz, 2H, ArH); 6.65 (t, J=8 Hz, H, ArH); 3.22 (m, 4H, CH₂); 2.94 (t, J=7 Hz, 2H, CH₂); 2.67 (m, 4H, CH₂). MS: m/e (100) 311.4 (M+)

Synthesis of 4-[2-(4-Phenyl-piperazin-1-vl)-ethvl]-phenvlamine (3a) and 3-

[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenylamine (3b)

Ra-Ni (0.4-0.5 g) was added in small portions to a stirred solution of the nitro compounds 2a,b (6.0 g, 20 mmol), 7.2 ml (90 mmol) hydrazine hydrate, 20 ml ethanol and 20 ml 1 ,2-dichloroethane at 30 °C. After the addition of Ra-Ni was completed, the reaction temperature was increased to 40-45 °C by external heating. After 60 min, the reaction mixture was filtered through celite. Amines 3a,b were recovered from filtrate after solvent was removed in vacuo and recrystallization from ethyl acetate.

4- [2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenylamine (3a): Yield: 3.8 g; mp 202-205 °C; NMR:5 7.25 (m, 4H, ArH); 7.00 (t, J=7 Hz, 2H, ArH); 6.92 (d, J=8 Hz, 4H, ArH); 6.64 (t, J=7 Hz, 1 H, ArH); 6.62 (d, J=8 Hz, 2H, ArH); 3.27 (m, 4H, CH₂); 3.32 (m, 4H, CH₂); 2.69 (m, 8H, CH₂). MS: m/e (100) 281.4 (M+)

3-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenylamine (3b): Yield: 3.6 g; mp 105-108 °C; NMR:6 7.23 (t, J=8 Hz, 2H, ArH); 6.93 (d, J=8 Hz, 3H, ArH); 6.64 (t, J=8 Hz, 1 H, ArH); 6.42 (s, 1 H, CH₂); 6.36 (m,2H, CH₂); 3.27 (m, 4H, CH₂); 3.32 (m, 4H, CH₂); 2.69 (m, 8H, CH₂). MS: m/e (100) 281 .5 (M+)

Synthesis of 5-[2-( 4-Phen yl-piperazin- 1 -yl)-eth yl]-2-p yridin-4-yl- 1 H-benzo- imidazole (14)

isonicotinic acid (393 mg, 3.3 mmol), diamine 13 (890 mg, 3.0 mmol) and 4.0 ml 4 N HCI were heated in an autoclave to 180 °C for 6 h. After cooling to ambient temperature, 15 ml of 10% NaHC0₃ was added and the obtained product was extracted with chloroform. The solvent was removed in vacuo and crude benzimidazole 14 was purified by silica gel column chromatography using a gradient of methanol (0-2%) in dichloromethane and recrystallized from EtOH.

Yield: 410 mg; mp 220-223 °C; ¹H NMR: δ 13.15 (s, H, NH); 8.74 (d, J=5 Hz, 2H, ArH); 8.07 (d, J=5 Hz, 2H, ArH); 7.64 (d, J=4 Hz, 1 H, ArH); 7.46 (s, 1 H, ArH); ); 7.20 (m, 3H, ArH); 6.92 (d, J =7 Hz, 1 H, ArH); 6.75 (d, J =7 Hz, 2H, ArH); 3.12 (m, 4H); 2.79 (m, 2H); 2.58 (m,6H). MS: m/e (100) 383.3 (M+) General procedure for the synthesis of N-{4-[2-(4-Phenyl-piperazin-1-vl)- ethyl]-phenyl}-arylamides and N-{3-f2-(4-Phenyl-piperazin-1-yl)-ethyl]- phenvD-arylamides (4a,b-12a,b)

Aryl carboxylic acids (2.2 mmol), amines 3a,b (560 mg, 2.0 mmol), 1 .0 ml triethyl amine, and 1 .8 ml 50% PPAA, were stirred in 7 ml DMF at room temperature for 16 h, subsequently diluted with 200 ml ethyl acetate and extracted 2 times with 50 ml 8% NaHCO₃ and 50 ml H₂O, each. Organic phase was dried over MgSO₄, filtered and concentrated in vacuo. Obtained products were purified by silica gel column chromatography using a gradient of methanol (0-5 %) in dichloromethane. Most amides crystallized from ethyl acetate as free bases. Amides 6b-8b were converted to oxalates using solution of anhydrous oxalic acid in diethyl ether and recrystallized from ethanol. N-{4-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide (4a): Yield: 590 mg; mp 203-208°C; ¹H NMR : 510.19 (s, 1 H, NH); 7.94 (d, J=8 Hz, 2Η, ArH); 7.68 (d, J=8 Hz, 2Η, ArH); 7.56 (m, 3H, ArH); 7.20 (m, 4H, ArH); 6.92 (d, J=8.5 Hz, 2H, ArH); 6.76 (t, J=7 Hz, H), 3.12 (m, 6H); 2.745 (m, 2H); 2.57 (m,4H). MS: m/e (100) 385.4 (M+)

2-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-benzamide

(5a): Yield: 220 mg; mp 170 °C (dec); ¹H NMR : δ 1 1 .93 (s, 1 H, NH); 7.76 (m, 2H, ArH); 7.32 (d, J=8 Hz, 2H, ArH); 7.32 (m, 2H, ArH); 7.21 (m, 4H, ArH); 7.13 (t, J=7 Hz, 1 H); 6.93 (m, 4H, ArH); 6.780 (t, J=7 Hz, 1 H); 3.18 (m, 6H); 2.81 (m, 2H); 2.71 (m, 2H). MS: m/e (100) 401 .4 (M+)

N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-picolinamide (6a): Yield: 470 mg; mp 158-160 °C; ¹H NMR : 510.57 (s, 1 H, NH); 8.73 (d, J=4 Hz, 2H, ArH); 8.17 (d, J=6 Hz, 2H, ArH); 8.06 (t, J=7 Hz, 1 H); 7.81 (d, J=8 Hz, 2H, ArH); 7.66 (m, 1 H, ArH); 7.24 (m, 4H, ArH); 6.91 (d, J=8.5 Hz, 2H, ArH); 6.76 (t, J=7 Hz, 1 H), 3.1 1 (m, 4H); 2.74 (m, 2H); 2.55 (m, 6H). MS: m/e (100) 386.3 (M+) N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-nicotinamide (7a): Yield: 570 mg; mp 203-205 °C; ¹H NMR: 5 10.38 (s, 1 H, NH); 9.09 (s, 1 H, ArH); 8.74 (d, J=5 Hz, 1 H, ArH); 8.27 (d, J=8 Hz, 1 H, ArH); 7.67 (d, J=8 Hz, 2H, ArH); 7.55 (m, 1 H, ArH); 7.22 (m, 4H, ArH); 6.92 (d, J=8.5 Hz, 2H, ArH); 6.76 (t, J=7 Hz, 1 H), 3.12 (m, 4H); 2.77 (m, 2H); 2.58 (m,6H). MS: m/e (100) 386.4 (M+)

N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-isonicotinamide (8a): Yield: 470 mg; mp 198-200 °C; Ή NMR: 5 10.44 (s, 1 H, NH); 8.77 (d, J=5 Hz, 2H, ArH); 7.84 (d, J=5 Hz, 2H, ArH); 7.66 (d, J=8 Hz, 2H, ArH); 6.92 (d, J=8.5 Hz, 2H, ArH); 6.75 (t, J=7 Hz, 1 H), 3.12 (m, 4H); 2.75 (m, 2H); 2.58 (m,6H). MS: m/e (100) 386.2 (M+)

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide (9a): Yield: 560 mg; mp 190-191 °C; H NMR: 5 10.66 (s, 1 H, NH); 9.63 (s, 1 H, ArH); 9.47 (d, J=5 Hz, 1 H, ArH); 8.00 (d, J=5 Hz, 2H, ArH); 7.66 (d, J=8 Hz, 2H, ArH); 7.25 (d, J=8 Hz, 2H, ArH); 7.19 (t, J=7 Hz, 2H, ArH), 6.91 (d, J=7 Hz, 2H, ArH); 6.74 (t, J=7 Hz, 1 H); 3.1 1 (m, 4H); 2.76 (m, 2H); 2.56 (m,6H). MS: m/e (100) 387.3 (M+)

N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide (10a): Yield: 510 mg; mp 200-202 °C; ¹H NMR: δ 10.68 (s, 1 H, NH); 9.29 (s, 1 H, ArH); 8.92 (s, 1 H, ArH); 8.81 (s, 1 H, ArH); 7.80 (d, J=8 Hz, 1 H, ArH); 7.27 (m, 4H, ArH); 6.91 (d, J=8 Hz, 2H, ArH); 6.76 (t, J=7 Hz, H); 3.14 (m, 4H); 2.75 (m, 2H); 2.58 (m,6 H). MS: m/e (100) 387.2 (M+)

2-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyi}-nicotinamide (11a): Yield: 210 mg; 238-240 °C; ¹H NMR :δ 12.12 (s, 1 H, NH); 8.45 (d, J=8 Hz, 1 H, ArH); 7.80 (d, J=5 Hz, 1 H, ArH); 7.59 (d, J=9 Hz, 1 H, ArH); 7.20 (m, 4H, ArH); 6.91 (d, J=9 Hz, 2H, ArH); 6.76 (t, J=7 Hz, 1 H); 3.14 (m, 4H); 2.74 (m, 2H); 2.58 (m,6H). MS: m/e (100) 402.2 (M+)

4-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-nicotinamide (12a): Yield: 290 mg; 261-262 °C; ¹H NMR: δ 9.89 (s, 1 H, NH); 8.20 (d, J = 2 Hz, 1 H, ArH); 7.92 (dd, J, = 2 Hz, J₂ = 10 Hz, H, ArH); 7.58 (d, J = 8.5 Hz, 1 H, ArH); 7.19 (m, 5H, ArH); 6.91 (d, J =8.5 Hz, 2H, ArH); 6.76 (t, J=7 Hz, 1 H, ArH); 6.35 (d, J = 10 Hz, 1 H, ArH); 3.1 1 (m, 4H); 2.72 (m, 2H); 2.57 (m,6H). MS: m/e ( 00) 402.2 (M+) N-{3-[2-(4-Phenyl-piperazin-1 -yl)-ethyl]-phenyl}-benzamide (4b): Yield: 370 mg; 186-189 °C; Ή NMR: δ 10.18 (s, 1 H, NH); 7.84 (d, J=7 Hz, 2H, ArH); 7.67 (s, 1 H, ArH); 7.92 (t, J=7 Hz, 2H, ArH); 7.52 (t, J = 7.0 Hz, 2H, ArH); 7.25 (d, J =7.0 Hz, 1 H, ArH); 7.19 (t, J=7.0, 2H, ArH); 6.99 (d, J =7.0 Hz, 1 H, ArH); 6.92 (d, J =7.0 Hz, 2H, ArH); 6.79 (t, J=7.0, 1 H, ArH); 3.1 1 (m, 4H); 2.72 (m, 2H); 2.57 (m,6H). MS: m/e (100) 385.4 (M+)

2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide

(5b): Yield: 360 mg; 1 12-115 °C; Ή NMR: δ 7.79 (d, J = 2 Hz, 2H, ArH); 7.64 (d, J=2 Hz, 1 H, ArH); 7.37 (s, 2H, ArH); 7.18 (m, , 6H, ArH); 6.92 (d, J=7 Hz, 2H, ArH); 6.79 (t, J=7 Hz, 1 H, ArH); 3.12 (m, 4H); 2.77 (m, 2H); 2.57 (m,6H). MS: m/e ( 00) 401.4 (M+)

N-{3-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-picolinamide (6b): Yield: 260 mg; mp 214-215 °C; H NMR: δ 10.54 (s, 1 H, NH); 8.73 (d, J=7 Hz, 1 H, ArH); 8.08 (d, J=7 Hz, 1H, ArH); 7.94 (s, 5H, ArH); 7.81 (s, 1 H, ArH); 7.71 (m, 3H, ArH); 7.27 (t, J=7 Hz, 1 H, ArH); 7.19 (t, J=7 Hz,2H, ArH); 6.99 (d, J=7 Hz, 1 H, ArH); 6.92 (d, J=7 Hz, 2H, ArH); 6.73 (t, J=7 Hz, 1 H, ArH); 3. 1 (m, 4H); 2.72 (m, 2H); 2.59 (m,6H). MS: m/e (100) 386.4 (M+)

N-{3-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-nicotinamide (7b): Yield: 400 mg; mp 195-197 °C; H NMR (d₆DMSO): δ 10.48 (s, 1 H, NH); 8.76 (d, J=4.5 Hz, 1 H, ArH); 8.29 (d, J=7 Hz, 1 H, ArH); 7.78 (s, 5H, ArH); 7.78 (m, 4H, ArH); 7.34 (t, J=7 Hz, H, ArH); 7.25 (t, J=7 Hz, 2H, ArH); 7.06 (d, J=7 Hz, 1 H, ArH); 7.01 (d, J=7 Hz, 1 H, ArH); 6.85 (d, J=7 Hz, 2H, ArH); 3.39 (m, 4H); 3.39 (m, 2H); 3.01 (m,6H). MS: m/e (100) 386.3 (M+)

N-{3-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-isonicotinamide (8b): Yield: 260 mg; mp 163-165 °C; H NMR (c/₆DMSO): δ 10.52 (s, 1 H, NH); 8.79 (d, J=4.5 Hz, 2H, ArH); 7.85 (d, J=4.5 Hz, 2H, ArH); 7.78 (s, 1 H, ArH); 7.59 (d, J=7 Hz, 1 H, ArH); 7.34 (t, J=7 Hz, 1 H, ArH); 7.25 (t, J=7 Hz, 2H, ArH); 7.07 (d, J=7 Hz, 1 H, ArH); 7.00 (d, J=7 Hz, 1 H, ArH); 6.65 (d, J=7 Hz, 2H, ArH); 3.39 (m, 4H); 3.39 (m, 2H); 3.01 (m,6H). MS: m/e ( 00) 386.3 (M+) N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide (9b): Yield: 160 mg ; mp 168-170 °C; H NMR (d₆DMSO): δ 10.65 (s, 1 H, NH); 9.64 (s, 1 H, ArH); 9.47 (d, J=4.5 Hz, 1 H, ArH); 8.09 (d, J=4.5 Hz, 1 H, ArH); 7.765 (s, 5H, ArH); 7.30 (t, J=7 Hz, 1 H, ArH); ); 7.19 (t, J=7 Hz, 2H, ArH); 7.05 (d, J=7 Hz, 1 H, ArH); 6.92 (d, J=7 Hz, 1 H, ArH); 6.75 (d, J=7 Hz, 2H, ArH); 3.12 (m, 4H); 2.79 (m, 2H); 2.58 (m,6H). MS: m/e (100) 387.3 (M+)

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide (10b): Yield: 80 mg ; mp 151 -153 °C; ¹H NMR: δ 10.64 (s, 1 H, NH); 9.29 (s, 1 H, ArH); 8.92 (s, 1 H, ArH); 8.81 (s, 1 H, ArH); 7.81 (s, 1 H, ArH); 7.71 (d, J=8 Hz, 1 H, ArH); 7.28 (t, J=7 Hz, 1 H, ArH); 7.19 (t, J=7 Hz, 2H, ArH); 7.03 (d, J=8 Hz, 2H, ArH); 6.91 (d, J=8 Hz, 2H, ArH); 6.75 (t, J=7 Hz, 1 H, ArH); 6.57 (t, J=6 Hz, 1 H, ArH); 3.13 (m, 4H); 2.78 (m, 2H); 2.59 (m, 6H). MS: m/e (100) 387.3 (M+)

2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-nicotinamide (11 b): Yield: 260 mg; mp 237-240 °C; H NMR: δ 12.14 (s, 1 H, NH); 8.42 (d, J=7 Hz, 1 H, ArH); 7.80 (d, J=6 Hz, 1 H, ArH); 7.54 (m, 2H, ArH); 7.21 (d, J=7 Hz, 1 H, ArH); 7.14 (t, J=8 Hz, 2H, ArH); 6.98 (d, J=7 Hz, 1 H, ArH); 6.91 (d, J=8, 2H, ArH); 6.75 (t, J=7 Hz, 1 H, ArH); 6.57 (t, J=6 Hz, 1 H, ArH); 3.12(m, 4H); 2.77 (m, 2H); 2.59 (m,6H). MS: m/e (100) 402.2 (M+)

4-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide (12b): Yield: 200 mg; mp 205-208 °C; Ή NMR: δ 12.09 (s, 1 H, NH); 9.90 (s, 1 H, ArH); 8.18 (d, J=2 Hz, 1 H, ArH); 7.95 (dd, J,=2 Hz, J₂=10 Hz,1 H, ArH); 7.57 (s, 1 H, ArH);7.53 (d, J=7 Hz, 1 H, ArH), 7.22 (m, 4H, ArH); 6.96 (d, J=7 Hz, 1 H, ArH); 6.91 (d, J=7 Hz, 2H, ArH); 6. 57 (t, J=7 Hz, 1 H, ArH); 6.39 (t, J=7 Hz, 1 H, ArH), 3.12 (m, 4H); 2.77 (m, 2H); 2.59 (m,6H). MS: m/e (100) 402.2 (M+)

Material and Methods for cell culture experiments (see examples 2-6)

Cell cultures

All chemicals were from Sigma (St. Louis, MO) unless stated otherwise. The human neuroblastoma cell line SH-SY5Y was obtained from American Type Culture Collection. Rat peritoneal macrophages were isolated from Albino Oxford rats as previously described (Harhaji 2006), in accordance with the Declaration of Helsinki. The neuroblastoma cell line was grown in Modified Eagle Medium and F12 cell culture medium (1 :1) supplemented with 10% fetal calf serum, 2 mM L-glutamine, nonessential amino acids and penicillin/streptomycin. Before co-cultivation with SH-SY5Y cells, rat peritoneai macrophages were initially grown in a HEPES (25 mM)-buffered RPMI 1640 cell culture medium supplemented with 5% fetal calf serum, 2 mM L-glutamine, 10 mM sodium pyruvate and penicillin/streptomycin. The cells were maintained at 37°C in a humidified atmosphere with 5% C0₂. The SH-SY5Y cells were prepared for experiments using the conventional trypsinization procedure with trypsin/EDTA. For the measurement of acid phosphatase activity (cell viability) or flow cytometric analysis cells were incubated in 96-well flat-bottom plates (1.5 x 10⁴ cells per well) or 24-well flat-bottom plates (1.2 x 10⁵ cells per well), respectively. For co-cultivation experiments, peritoneal macrophages (1 x 10⁵ cells per well, 24-well plate) were initially grown in a HEPES (25 mM)-buffered RPMl 1640 cell culture medium supplemented with 5% fetal calf serum, 2 mM L-glutamine, 10 mM sodium pyruvate and penicillin/streptomycin. After 4-hour incubation non-adherent cells were washed out and SH-SY5Y cells were added (1.5 x 10⁴ cells per well). SH-SY5Y cells were differentiated using phorbol 2-myristate 13-acetate (PMA). Cells were seeded in 96-well flat-bottom plates (2 x 10³ cells per well) and PMA (80 nM) was added 24 h after seeding. Medium was changed every 3 days and cultures were allowed to differentiate for 6 days. Undifferentiated cells were rested for 24 h and treated with NO-releasing chemical sodium nitroprusside (SNP) and/or other agents, as described below and in Figure legends.

Cellular acid phosphatase activity assay for cell viability

Cell viability was determined after 24-hour treatment by measuring the activity of a lysosomal enzyme acid phosphatase (Connolly 1986). At the end of incubation, cultivating medium was removed, and the acid phosphatase substrate p-nitrophenyl phosphate (10 mM) was added. The reaction was stopped after 1 h of incubation at 37°C by addition of 0.1 M NaOH. The color development, corresponding to the number of viable cells, was monitored by automated microplate reader at 405 nm. After subtracting the background value of NaOH alone, the results were presented as a percentage of the viability of untreated cells, which were regarded as 100% viable.

Apoptosis analysis

Apoptotic cell death was assessed by flow cytometry analysis of DNA fragmentation in cells stained with the DNA-binding dye propidium iodide (PI; BD Pharmingen, San Diego, CA) as previously described (Harhaji- Trajkovic 2009). Red PI fluorescence (FL2) was analyzed with a FACSCalibur flow cytometer (BD, Heidelberg, Germany), using a peak fluorescence gate to exclude cell aggregates. Cell distribution among cell cycle phases was determined with Cell Quest Pro software and hypodiploid cells in the sub-G₀/Gi compartment were considered apoptotic. Apoptosis was also analyzed by double staining with f!uoresceinisothiocyanate (FITC)-conjugated annexin V and PI, in which annexin V binds to the apoptotic cells with exposed phosphatidylserine, while PI labels the late apoptotic/necrotic cells with a damaged membrane. Staining was performed according to the manufacturer's instructions (BD Pharmingen), and flow cytometry was performed on a FACSCalibur flow cytometer. The percentage of early apoptotic (annexin PI^~~) and late apoptotic/necrotic (annexin7PI⁺) cells was determined using Cell Quest Pro software.

Caspase activation

Activation of caspases, apoptosis executioner enzymes, was measured by flow cytometry after labeling the cells with a cell-permeable, FITC- conjugated pan-caspase inhibitor (ApoStat; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Increase in green fluorescence (FL1) is a measure of caspase activity within individual cells of the treated population. The results are expressed as the percentage of cells containing active caspases.

Measurement of superoxide and reactive nitrogen species (RNS)

The production of superoxide and RNS was measured using redox- sensitive fluorochromes dihydroethidium (DHE) and diaminofluorescein (DAF) (both from Invitrogen, Paisley, UK), which are fairly selective for 0₂ ^" and NO/ONOO-, respectively (Zhao 2003, Roychowdhury 2002). DHE (20 μΜ) and DAF (20 μΜ) were incubated with cells for 30 min at the end of the treatment. Cells were then detached by trypsinization and washed with phosphate buffered saline. The mean intensity of red (FL2) fluorescence (DHE) or green (FL1 ) fluorescence (DAF), corresponding to ROS or RNS levels, respectively, was determined using a FACSCalibur flow cytometer (BD). Nitrite accumulation, which reflects NO production, was measured using Griess reaction as previously described (Harhaji 2006).

Mitochondrial depolarization

The mitochondrial depolarization was assessed using JC-1 (R&D Systems), a lipophilic cation susceptible to changes in mitochondrial membrane potential with property of aggregating upon membrane polarization, forming an orange-red fluorescent compound. If the potential is disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The cells were stained with JC-1 as described by the manufacturer, and the green monomer and the red aggregates were detected by flow cytometry. The results are presented as a green/red fluorescence ratio (geomean FL1/FL2), the decrease and increase of which reflect mitochondrial hyperpolarization and depolarization, respectively.

Immunoblot

Western blot followed by protein detection with specific antibodies was used for assessing the activation of Akt, AMP-activated protein kinase (AMPK) and mitogen-activated protein kinases ( APK) Jun-N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). The cells were lysed in a lysis buffer (30 mM Tris-HCI pH 8.0, 150 mM NaCI, 1 % NP-40, 1 mM phenylmethylsulfonylfluoride and protease inhibitor cocktail; all from Sigma-Aldrich, St. Louis, MO) on ice for 30 min, centrifuged at 14000 x g for 15 min at 4°C, and the cell lysates were collected. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred to a nitrocellulose membranes (Bio-Rad, Marnes-la-Coquette, France). Following . incubation with anti-phospho-JNK, anti-JN , anti- phospho-ERK, anti-ERK, anti-phospho-AMPK, anti-AMPK, anti-phospho- Akt, anti-Akt (Cell Signaling Technology, Beverly MA), or anti- -actin antibodies (Abeam, Cambridge, MA) as primary antibodies and peroxidase-conjugated goat anti-rabbit IgG (Southern Biotech, Birmingham, AL) as a secondary antibody, specific protein bands were visualized using enhanced chemiluminescence reagents for Western blot analysis (Amersham Pharmacia Biotech, Piscataway, NJ). The signal intensity was determined by densitometry and the results were presented as phospho/total protein signal ratio, which was arbitrarily set to 1 in control.

Statistics

The statistical significance of differences was analyzed by t-test or ANOVA followed by the Student-Newman-Keuls test. A value of p<0.05 was considered significant.

Example 2: Arylpyperazines protect neuroblastoma cells from NO- mediated toxicity

The potential neuroprotective effect of arylpiperazines was investigated in neuron-like SH-SY5Y cells treated for 24 h with NO donor SNP or hydrogen peroxide. Both toxins in a dose dependent manner decreased cell viability, as demonstrated by acid phosphatase activity assay (Fig 2A). The compounds were screened at the highest non-toxic concentration, which was 10 μΜ - at 20 μΜ, several ligands have demonstrated a slight reduction in cell numbers (data not shown). While none of the investigated ligands showed protective effects on H₂O₂ induced toxicity, the ligands reduced SNP-induced cell damage (Fig 2B). Compound 6a (N-{4-[2-(4- phenyl-piperazin-1 -yl)-ethyl]-phenyl}-picolinamide) was among the most effective ligands and it was therefore chosen for further examination. An additional investigation demonstrated that neuroprotective action of 6a was dose-dependent (Fig. 2C). In accordance with the results of the cytotoxicity assay, the addition of 6a significantly prevented morphological changes associated with SNP-induced cell death, as assessed by phase contrast microscopy analysis (Fig 2D). The ability of 6a to protect neurons from NO-induced neuronal damage was confirmed in PMA-differentiated SH-SY5Y cells (Fig 2E). Example 3: Arylpyperazines reduce mitochondrial depolarization, caspase activation and apoptosis

Apoptotic events in SH-SY5Y cells treated with SNP were determined using double staining with annexin V-FITC and propidium iodide, which detect phosphatidylserine externalization and cell membrane damage, respectively. Compound 6a significantly decreased the number of annexin⁺PI^~ (early apoptotic) and annexin⁺PI⁺ (late apoptotic/necrotic) cells in SNP-treated SH-SY5Y cultures (Fig 3A). Accordingly, 6a markedly reduced SNP-triggered increase in number of apoptotic, hypodiploid cells with fragmented DNA (sub-G₀/Gi compartment of the cell cycle prophile), as confirmed by DNA staining with PI (Fig 3B). SNP-induced activation of apoptosis-executing enzymes of caspase family, monitored by fluorescent caspase inhibitor ApoStat, was also partially prevented by co-treatment of SH-SY5Y cells with 6a (Fig 3C). NO-induced caspase activation and apoptosis were associated with mitochondrial depolarization, reflected in a decrease in red/green fluorescence ratio of mitochondrial-binding fluorochrome JC-1 (Fig. 3D). Compound 6a alone caused hyper- polarization of mitochondrial membrane and restored mitochondrial potential in SNP-exposed cells (Fig. 3D).

Example 4: Arylpyperazines reduce superoxide production in neuroblastoma cells exposed to NO We next investigated if compound 6a influenced SNP-generated intracellular production of reactive oxygen and nitrogen species in SH- SY5Y cells. The increase in superoxide anion and NO/peroxynitrite content in SNP-exposed cells was demonstrated using fluorescent dyes DHE and DAF, respectively (Fig. 4A, B). While treatment with 6a markedly reduced the amount of superoxide in SNP-treated cells, we observed only a slight non-significant decrease in NO/peroxynitrite concentration (Fig. 4A, B). To evaluate the ability of 6a to interfere with cell-derived NO, we used rat peritoneal macrophages stimulated for NO production with LPS and IFN-γ. DAF-measured intracellular NO concentration expectedly increased upon stimulation, but did not significantly change upon the co- treatment with compound 6a (Fig. 4C). The inability of 6a to affect LPS + IFN-induced NO production was also confirmed by Griess reaction-based measurement of the time-dependent nitrite accumulation as another indicator of NO production (Fig. 4D). Accordingly, 6a did not significantly affect NO production in co-cocultures of LPS+IFN-y-stimulated peritoneal macrophages and SH-SY5Y cells (Fig. 4E). On the other hand, it partially protected SH-SY5Y cells from the cytotoxic effect of LPS+IFN-y-activated macrophages (Fig. 4F). The observed cytotoxicity was NO-dependent, as confirmed by the ability of NO synthase inhibitors aminoguanidine and L- NAME to improve viability of SH-SY5Y cells (data not shown). It therefore appears that compound 6a somehow interferes with the cytotoxic action of NO without exerting significant NO-scavenging ability. Example 5: Arylpyperazines modulate apoptosis-associated signalling pathways in NO-treated neuroblastoma cells

Oxidative stress, including that induced by NO, triggers various intracellular signalling pathways involved in regulation of cell death. We therefore examined the influence of compound 6a on the activation of Akt survival pathway (Virdee 1999), as well as on the activation of JNK, ERK and AMPK pathways, which are involved in cell death induction (Lam 2008, Li 2004, Shibata 2006, Chalimoniuk 2009, Ishikawa 2003, Ishikawa 2000, McCullough 2005, Chen 2010). Immunoblot analysis of enzyme phosphorylation (activation) demonstrated that the treatment with SNP for 8 h inhibited Akt and activated JNK, ERK and AMPK in SH-SY5Y cells (Fig. 5). SNP-induced changes in activation of signalling molecules were prevented by treatment with 6a, which restored phosphorylation of Akt and downregulated activation of JNK, ERK and AMPK in SNP-treated cells (Fig. 5). Example 6: Arylpiperazine-mediated neuroprotection is independent of dopamine receptor binding

Finally, we investigated if the protective effect of compound 6a depends on binding to dopamine receptors. To that aim, SH-SY5Y ceils were incubated with (+)butaclamol, a non-selective, high affinity D1 and D2 receptors blocker and then exposed to 6a. However, (+)butaclamol pretreatment did not affect either SNP-induced decrease in cell viability or the protective action of compound 6a, thus indicating that the observed protection from NO cytotoxicity did not depend on dopamine receptor binding (Fig. 6).

Discussion

In the present study we investigated the ability of novel ary!piperazine- based dopaminergic ligands to protect SH-SY5Y human neuroblastoma cell line from nitric oxide (NO)-induced oxidative stress and subsequent apoptosis. Acid phosphatase activity assay for cell viability revealed that arylpiperazines were able to protect both undifferentiated and phorbol myristate acetate-differentiated SH-SY5Y neuron-like cells from the cytotoxic effect of NO donor sodium nitroprusside (SNP), but not hydrogen peroxide. The flow cytometry analysis with appropriate fluorochromes confirmed that the observed neuroprotective effect was associated with the reduction in SNP-induced mitochondrial membrane depolarization, caspase activation and subsequent phosphatidylserine externalization and DNA fragmentation as the hallmarks of apoptotic cell death. Arylpiperazine treatment reduced superoxide production, but not intracellular accumulation of NO in SNP-exposed SH-SY5Y cells, as determined by dihydroethidium staining and diaminofluorescein staining/nitrite measurement, respectively. Similar results were obtained by using rat macrophages as a source of NO. Immunoblot analysis revealed that arylpiperazine treatment diminished SNP-triggered activation of pro- apoptotic signalling pathways (Jun-N-terminal kinase, extracellular signal- regulated kinase, AMP-activated protein kinase) and prevented SNP- mediated down-regulation of anti-apoptotic Akt activity. Neuroprotective effect of arylpiperazines was apparently independent of binding to dopamine receptors, as it was not affected by pretreatment with butaclamol, a high-affinity D1/D2 receptor blocker. The ability to prevent NO-induced modulation of cell death-associated signalling pathways, mitochondrial damage, oxidative stress and subsequent neuronal apoptosis makes arylpiperazine dopaminergic ligands plausible candidates for development as potential therapeutics for neurodegenerative disorders. The above examples demonstrate the neuroprotective action of novel arylpiperazine-based dopamine receptor ligands in NO-exposed SH-SY5Y neuroblastoma cells. The most effective compound, 6a, prevented NO- induced depolarization of mitochondrial membrane, oxidative stress and alterations of intracellular signalling pathways involved in apoptotic cell death. Interestingly, the observed effects were apparently not due to a direct scavenging of NO and were independent of dopamine receptor binding. Also, the neuroprotective action of novel arylpiperazines was relatively selective for NO, as they were unable to protect SH-SY5Y cells from the toxic effect of hydrogen peroxide.

The NO donor SNP has previously been used for induction of apoptosis in a number of neuron-like cell lines (Pytlowany 2008; Lim 2009). NO and its toxic metabolite, peroxynitrite (ONOO^~), inhibit components of the mitochondrial respiratory chain, thus leading to depolarization of mitochondrial membrane and subsequent release of small molecules such as cytochrome c, which activate caspases, the main apoptosis-executing enzyme family (Heaies 1999). Accordingly, the compounds of the present invention, e.g. compound 6a, exerted neuroprotective effect through mitochondrial membrane stabilization, causing repolarization of mitochondrial membrane and subsequent decrease in caspase activation and DNA fragmentation. Other neuroprotective dopamine receptor ligands, such as pramipexole and talipexole, display similar mode of action, accumulating in the mitochondria and restoring their membrane potential following MPP⁺ induced apoptotic damage in SHSY5Y cells (Abramova 2002; Kakimura 2001 ). While the exact intracellular site of action of the inventive compounds such as compound 6a remains to be pinpointed, it is nevertheless evident that the protective effect occurs as a consequence of reducing superoxide, rather than NO content, following exposure of cells to SNP. It seems conceivable to assume that compound 6a somehow reduces the production of O₂ ^■ by NO-damaged mitochondria, thus attenuating the direct toxicity of superoxide and preventing its combination with NO and subsequent generation of toxic peroxynitrite. There is a growing body of evidence suggesting that ROS and RNS act together to mediate damage in neurodegenerative disease (Floyd 1999; Calabrese 2000; Contestabile 2003), but some data argue that prevention of ONOO^" formation is not sufficient to decrease cell damage because NO itself is equally or more toxic (Meij 2004). Since compound 6a was markedly less efficient in preventing neuronal damage caused by peroxynitrite donor SIN-1 (Tovilovic et al., unpublished observation), it seems plausible that NO, rather than ONOO^", was the critical nitrogen species initiating degeneration of SH-SY5Y cells in our experimental conditions. However, the effect of 6a was apparently not selective for nitrosonium ion (NO⁺) generated by SNP, as neuroprotection was also observed with LPS + IFN- stimulated macrophages which release iNOS-derived NO. Importantly, the latter points to a potential therapeutic value of the arylpiperazines in preventing CNS damage in neurodegenerative/neuroinflammatory diseases in which macrophage-like microglial cells are major source or neurotoxic inflammatory mediators, including NO (Brown 2010).

The activation of MAPK superfamily members JNK and ERK has been reported to contribute to NO-induced apoptotic death of neuronal cell lines and primary neurons (Lam 2008, Li 2004, Shibata 2006, Chalimoniuk 2009, Ishikawa 2003, Ishikawa 2000). The pro-apoptotic role of JNK and ERK in NO-exposed neurons was mediated by phosphorylation of Bcl-2 family members Bcl-2 and Bcl-x(L) and subsequent translocation of Bax from cytosol to mitochondria, causing depolarization of the mitochondrial membrane (Lam 2008, Ishikawa 2003). In addition to ERK and JNK, activation of the energy balance-sensing enzyme AMPK has been shown to contribute to neuronal damage by NO- or hydrogen peroxide-mediated oxidative stress (McCullough 2005, Chen 2010). On the other hand, survival signals, such as protein kinase B/Akt, can inhibit sustained ERK1/2 activation and thereby promote neuronal survival (Virdee 1999), while the activation of Akt signaling pathway causes inhibition of AMPK activity in various cell types (Kovacic 2003; Tzatsos 2007; Hu 2008). These data are consistent with the ability of NO to activate JNK, ERK and AMPK, as well as to reduce Akt phosphorylation and cause mitochondrial depolarization in the present study. Moreover, the neuroprotective effect of compounds such as 6a was associated with significant reduction of JNK, ERK and AMPK activation, and recovery of Akt phosphorylation and mitochondrial membrane potential in NO-exposed SH-SY5Y cells. However, it still remains to be explored whether the observed neuroprotection was primarily mediated through modulation of cell death- related signaling pathways or direct scavenging of mitochondria-produced superoxide. These two mutually non-exclusive mechanisms, including the exact role of MAP kinase, AMPK and Akt pathway modulation in arylpiperazine-mediated neuroprotection is currently under investigation in our laboratory.

Considering the role of DA receptors in dopamine ligand-mediated neuroprotection, both DA receptor-dependent and -independent effects have been reported for different dopamine ligands (Mitchell 2002; Cools 2002; Moussa 2006; Kitamura 1998; Gu 2004). In our study, a non- selective high-affinity D1/D2 receptor antagonist (+) butaclamol did not influence the neuroprotective effect of compound 6a, thus indicating that neuroprotection was probably independent of binding to DA receptors.

Most compounds were selectively active in SNP tests (Examples 2-5) and/or in an 6-OHDA (6-hydroxydopamine, also known as oxidopamine or 2,4,5-trihydroxyphenethylamine) test, but were inactive in a H₂O₂ test.

In conclusion, our data suggest that arylpiperazine dopaminergic ligands prevent NO-induced modulation of cell death-associated signalling pathways, mitochondrial damage, oxidative stress and subsequent apoptosis of neuron-like SH-SY5Y cells. Having in mind the role of mitochondrial aberrations, metabolic imbalance and resulting oxidative stress in development of Alzheimer's and Parkinson's disease (Andersen 2004; Sayre 2001 ; Halliwell 2006), arylpiperazine dopaminergic ligands may be suitable candidates for development as potential therapeutics for these and other neurodegenerative conditions.

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Claims

Claims

1. A compound of the general formula I, II or III

III

or a pharmaceutically acceptable salt or solvate thereof,

wherein

R¹ is selected from the group consisting of:

Ci-Ci2-alkyl, C₃-Ci₂-cycloalkyl, C₆-Ci₀-aryl, and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxyl, OCOR⁴, amino, Ci-C₆-alkylamino, d-Ce-dialkylamino, Ci-C₆-(halo)alkyl, Ci-C₆-(halo)- alkoxy and/or COOR⁴;

R² is selected from the group consisting of:

C₆-Cio-aryl and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxyl, OCOR⁴, amino, Ci-C₆-alkylamino, Ci-C₆-dialkyl- amino, C C₆-(halo)alkyl, d-C₆-(halo)alkoxy and/or COOR⁴;

R³ is independently at each occurrence selected from the group consisting of:

hydrogen, halogen, hydroxyl, C₁-C₂-(halo)alkyl, and Ci-C₂-(halo)alkoxy; R⁴ is independently at each occurrence selected from the group consisting of:

hydrogen and CrC₂-alkyl; and n is 1-10.

2. The compound according to claim 1 , which has neuroprotective activity.

3. The compound according to claim 1 or claim 2, which has the general formula I or II.

4. The compound according to any one of the preceding claims,

wherein

R is selected from the group consisting of.

d-Ce-alkyl, phenyl, and nitrogen-containing C₆-heteroaryl, each unsubstituted or substituted with halogen, hydroxy, Ci-C₆-(halo)alkyl, and/or CrC₆-(halo)alkoxy.

5. The compound according to any one of the preceding claims,

wherein

R is phenyl, which is unsubstituted or substituted with halogen, hydroxy I, and/or Ci-C₆-alkoxy.

6. The compound according to any one of the preceding claims,

wherein

R² is selected from the group consisting of:

phenyl and C₅-C₇-heteroaryl, each unsubstituted or substituted with halogen, hydroxy I, amino, Ci-C₆-(halo)alkyl, and/or C₁-C₆-(halo)alkoxy.

7. The compound according to any one of the preceding claims,

wherein

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with halogen, hydroxyl, amino and/or d-Ce-alkoxy.

8. The compound according to any one of the preceding claims,

wherein R³ is hydrogen.

9. The compound according to any one of the preceding claims,

wherein

R⁴ is hydrogen.

10. The compound according to any one of the preceding claims,

wherein

n is 1-6.

11 . The compound according to any one of the preceding claims,

wherein

n is 2.

12. The compound according to any one of the preceding claims,

wherein

R is phenyl, which is unsubstituted or substituted with halogen, hydroxyl, and/or Ci-C₆-alkoxy;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with halogen, hydroxyl, amino and/or d-Ce-alkoxy; and

n is 2.

13. The compound according to any one of the preceding claims,

wherein

R¹ is unsubstituted phenyl;

R² is selected from the group consisting of:

phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridazinyl, pyrazinyl, each unsubstituted or substituted with hydroxyl;

R³ and R⁴ each are hydrogen; and

n is 2.

14. The compound according to any one of the preceding claims, wherein the compound is selected from the group consisting of:

N-{4-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide,

N-{3-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide,

2-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide, 2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-benzamide, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-nicotinamide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-isonicotinamide, N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-isonicotinamide, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyridazine-4-carboxylic acid amide,

N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide,

N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-pyrazine-2-carboxylic acid amide,

2-Hydroxy-N-{4-[2-(4-phenyi-piperazin-1-yl)-ethyl]-phenyl}- nicotinamide,

2-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}- nicotinamide,

4-Hydroxy-N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}- nicotinamide,

4-Hydroxy-N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}- nicotinamide, and

pharmaceutically acceptable salts or solvates thereof.

15. The compound N-{4-[2-(4-phenyl-piperazin-1 -yl)-ethyl]-phenyl}-picolin amide, or a pharmaceutically acceptable salt or solvate thereof.

16. The compound according to any one of the preceding claims for use in medicine, particularly for use in human medicine.

17. The compound for use according to claim 16 for use in the treatment of a disease, disorder or condition associated with, accompanied by or caused by mitochondrial stress.

18. The compound for use according to claim 16 or claim 17 for use in the treatment of a neurodegenerative disorder.

19. The compound for use according to claim 18, wherein said neurodegenerative disorder is selected from amyotrophic lateral sclerosis, Alzheimer's Disease, Parkinson's Disease and stroke.

20. A pharmaceutical composition comprising the compound according to any one of claims 1 to 15, and further comprising pharmaceutically acceptable excipients, adjuvants, diluents and/or carriers.

21 . Process for the manufacture of a compound of the general formula I, II or 111,

comprising the steps of:

(i) reacting an arylcarboxylic acid of general formula IV a), IV b), or IV c)

IV a) IV b) IV c) with an amine of general formula V

VI c)

(ii) reducing said compound of formula VI a), VI b) or VI c) with a suitable reducing agent,

(iii) hydrogenating the product of step (ii),

(iv) reacting the product of step (iii) with a carboxylic acid of the general formula R²COOH, and

(v) optionally isolating the resulting compound of formula I, II or III, wherein R¹, R² and R³, at each occurrence, have the meaning as defined in claim 1 , and wherein n is 0-9.

22. A method for the treatment of a disease, disorder or condition associated with, accompanied by or caused by mitochondrial stress, comprising administering a pharmaceutically effective amount of a compound according to any one of claims 1 to 15 to a subject in need thereof.

23. The method according to claim 22, wherein said disease, disorder or condition is a neurodegenerative disorder.

24. The method according to claim 23, wherein said neurodegenerative disorder is selected from amyotrophic lateral sclerosis, Alzheimer's Disease, Parkinson's Disease and stroke.