The subjects of the invention are new 4,6-disubstituted 2-(4-methylpiρerazin-l- yl)pyridine derivatives, a process for their preparation, pharmaceutical composition containing these compounds, their use, a method for modulating brain monoaminergic receptor activity and a monoaminergic receptor modulating agent. The invention relates to new chemical compounds, 2-(4-methylpiperazin-l-yl)pyridine derivatives, enantiomers thereof or a mixture of its enantiomers or their pharmaceutically permissible prodrugs and salts, pharmaceutical compositions containing these compounds, and methods of using any of these derivatives and compositions for the prevention or treatment of neurological or psychiatric diseases, especially related with the modulation or regulation of brain monoaminergic receptors such as serotonin receptors, preferably receptors selected from the group of: 5-HT ₆ , 5-HT _2A , 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α. These new compounds can be used for treating central nervous system diseases.

The neurotransmitter dopamine (DA) has a long association with normal functions such as motor control, cognition, and reward, as well as a number of syndromes including drug abuse, schizophrenia, and Parkinson's disease. The most widely considered neurochemical hypothesis of schizophrenia is the dopamine hypothesis, which postulates that symptoms of schizophrenia may result from excess dopaminergic neurotransmission particularly in mesolimbic and striatal brain regions, leading to positive symptoms and dopaminergic deficits in prefrontal brain regions, which are responsible for the negative symptoms. In the brain, this phenethylamine functions as a neurotransmitter, activating the five types of dopamine receptors - Di, D ₂ , D ₃ , D ₄ and D ₅ , and their variants. Dopamine is produced in several areas of the brain, including the substantia nigra and the ventral tegmental area. The dopamine hypothesis of psychosis is a model attributing symptoms of schizophrenia (like psychoses) to a disturbed and hyperactive dopaminergic signal transduction. The model draws evidence from the observation that a large number of antipsychotics have DA-antagonistic effects. Some of the most obvious evidence for this theory is from the effect of drugs such as amphetamine and cocaine. These drugs (and others like them) increase levels of dopamine in the brain and can cause psychosis, particularly after large doses or prolonged use. This is often referred to as 'amphetamine psychosis' or 'cocaine psychosis', but may produce experiences virtually indistinguishable from the positive symptoms associated with schizophrenia.

Some researchers have suggested that dopamine systems in the mesolimbic pathway may contribute to the 'positive symptoms' of schizophrenia whereas problems with dopamine function in the mesocortical pathway may be responsible for the 'negative symptoms'. Another important development was an accidental discovery that a group of drugs called the phenothiazines, including antipsychotics such as chlorpromazine, antagonized dopamine binding (particularly at receptors known as D ₂ dopamine receptors) and reduced positive psychotic symptoms. D ₂ receptor occupancy by antipsychotic drugs has been confirmed by a large number of imaging studies.

The caudate dopamine D ₂ receptor up-regulation has been related to the genetic risk for schizophrenia; i.e. higher dopamine D ₂ receptor density in caudate was associated with poorer performance on cognitive tasks involving corticostriatal pathways. Diminished dopamine activity within the prefrontal cortex has been also associated with many of the cognitive deficits that are observed in schizophrenia. It has been hypothesized that antipsychotic drugs used to treat schizophrenia restore normal activity by antagonizing the dopamine D ₂ receptor, which is also known to modulate key ionic currents in the prefrontal cortex.

However, the hypothesis that an under-active cortical dopamine system is responsible for schizophrenic symptoms has been challenged by evidence that newer atypical antipsychotic drugs are weak antagonists at the D ₂ receptor but potent antagonists at the serotonin 5-HT ₂ A receptor. Serotonin, which is believed to be the primary neurotransmitter associated with mood disorders, appears to have a causal role in schizophrenia. Serotonin hyperactivity may be related to dopamine hyperactivity; hence compounds that block 5 -HT receptors may impact the release of dopamine in certain dopamine-deficient areas of the brain, including the frontal and nigrostriatal tracts. As such, 5-HT receptor antagonism in these areas may help improve the negative symptoms of schizophrenia, while decreasing the incidence of extrapyramidal symptoms [Di Matteo et al. (2001) Trends Pharmacol Sci. May;22(5):229-32]. Serotonin receptor-based mechanisms have been postulated to play a critical role in the action of the new generation of antipsychotic drugs that are usually referred to as atypical because of their ability to achieve an antipsychotic effect with lower rates of extrapyramidal side effects compared to first-generation antipsychotic drugs such as haloperidol. Specifically, it has been proposed that potent 5-HT _2A receptor antagonism together with weak dopamine D ₂ receptor antagonism are the principal pharmacologic features that differentiate clozapine and other apparent atypical antipsychotic drugs from first-generation typical antipsychotic drugs. This hypothesis is consistent with the atypical features of quetiapine, olanzapine, risperidone, and ziprasidone, which are the most common treatments for schizophrenia in the United States and many other countries, as well as a large number of compounds in various stages of development.

5-HT _2A receptors were the first population of the entire serotoninergic family to be identified, due to an early discovery of ketanserin as their selective antagonist. The role of 5-HT _2A receptors in the regulation of a number of processes of the central nervous system (mood, appetite, sexual behavior, learning, and memory, etc.) and their dysfunctions (e.g. psychosis, depression, anxiety, and sleep disorders) has been well documented [Glennon, Dukat, ID Research Alert 1997, 2, 107-113; G. A. Kennett, Current Research in Serotonin 1998, 3, 1-18]. The mammalian 5-HT _2A receptor is a subtype of the 5-HT ₂ receptor which belongs to the serotonin receptor family and is a G protein coupled receptor (GPCR). This is the main excitatory receptor subtype among the GPCRs for serotonin, although 5-HT _2A may also have an inhibitory effect on certain areas such as the visual cortex and the orbitofrontal cortex.

Agonists acting at 5-HT _2A receptors located on the apical dendrites of pyramidal cells within regions of the prefrontal cortex are believed to mediate hallucinogenic activity. Basic research is supportive of the relevance of 5-HT _2A receptor blockade to antipsychotic drug action. Selective 5-HT _2A receptor antagonists, either alone or in combination with selective antagonists of other receptors, have been found to be effective in various animal models of psychosis. Other studies suggest that 5-HT _2A receptor blockade may play a key role in the treatment of negative symptoms, but only when D ₂ receptor blockade is absent or moderate. Efficacy to treat negative symptoms may be related to the ability of these agents to selectively increase dopaminergic activity in the prefrontal cortex since all these agents have been found to produce greater increases in dopamine release in the prefrontal cortex than the nucleus accumbens. The majority of atypical antipsychotics act via the 5-HT ₂ receptors [L. E. Schechter, et al. Curr. Opin. CPNS Invest. Drugs 1999, 1, 432-447; S. Miyamoto et al. Curr. Opin. CPNS Invest. Drugs 2000, 2, 25-39.; D.M.Weiner, et al., J. Pharmacol. Exp. Ther. 2001, 299, 268-276; B.L.Roth, et al., Pharmacol. Ther. 1998, 79, 231-257]. Antagonism of the 5-HT ₂ A receptor appears to improve negative symptoms and decrease extrapyramidal symptoms, while 5-HT ₂ c antagonism appears to result in weight gain.

Characterized in 1993, the 5-HT ₇ receptors belong to the G protein-coupled receptor family. Since their discovery, they have been the subject of intense research due to their widespread distribution in the brain, suggestive of multiple central roles. Studies utilizing selective inhibitors suggest that the 5-HT ₇ receptor modulates neuronal function in a number of brain areas including the hippocampus and thalamus. In turn, these findings suggest that 5-HT ₇ receptor-selective ligands might prove therapeutically useful for the treatment of psychiatric disorders.

The fact that such antipsychotics as risperidone and clozapine show a high affinity for 5-HT ₇ receptors and have features of antagonists has led to an assumption that these receptors may be important for mediating the unique actions of certain antipsychotic drugs. The role of 5-HT ₇ receptors in schizophrenia is corroborated by the observation

One of the newest members of the serotonin receptors family is the 5-HT ₆ receptor, a subtype localized almost exclusively in the CNS, predominating in brain regions associated with cognition and behavior. With the subsequent development of selective 5- HT ₆ receptor antagonists, preclinical studies in rodents and primates have elucidated the function of this receptor subtype in more detail. It is increasingly clear that blockade of 5- HT ₆ receptors leads to an improvement of cognitive performance in a wide variety of learning and memory paradigms and also results in anxiolytic and antidepressant-like activity. Manipulation of 5-HT ₆ receptor activity alters the transmission of several neurotransmitters important in memory: acetylcholine and glutamate, as well as dopamine, γ-aminobutyric acid (GABA), epinephrine (E), and norepinephrine (NE). Several 5-HT ₆ antagonists have been developed, advancing the understanding of the relationship between 5-HT ₆ blockade and memory consolidation in diverse learning paradigms. There is also evidence that 5-HT ₆ receptor activity affects anxiety behaviors and may be involved in the pathophysiology of schizophrenia.

The 5-HT ₆ receptor has also recently been in the limelight as a drug target for cognition enhancement in Alzheimer's disease and other diseases where memory loss and learning complications are symptoms (e.g. addiction, depression, anxiety and schizophrenia). It is therefore construed to be a viable drug target in disorders where loss of these functions is a major burden to the patient and to caregivers. Especially in the case of Alzheimer's disease, the nootropic association of this receptor has attracted a significant amount of attention owing to the severe impairment of memory and cognition observed as a hallmark of this disease.

Cognitive impairments involving verbal learning, verbal delayed recall, working memory, vigilance, and executive functioning have a significant negative impact on social and occupational functioning of these patients.

The DSM-IV states that anxiety and psychosis, particularly paranoid delusions, are common in Alzheimer's disease. Benzodiazepines can be disinhibiting in such individuals and may exacerbate confusion and should be avoided if possible. Antipsychotic medications with high anticholinergic potential (e.g. thioridazine, chlorpromazine) may also affect memory adversely. While these agents have been favored in the past because of their tendency to produce sedation, newer agents such as olanzapine, risperidone, quetiapine and ziprasidone, have been reported to have lower incidences of neuroleptic- related side effects.

However, these products do not address the cognitive issue of Alzheimer's disease dementia and there is therefore a place in the therapeutic arsenal for a compound that would address both the memory impairments and the psychotic episodes of Alzheimer's disease patients.

A growing body of preclinical evidence is therefore supporting the use of serotonin 5- HT ₆ receptor antagonism as a promising mechanism for treating cognitive dysfunction, not only connected to neurodegenerative diseases, such as dementia of the Alzheimer type but also schizophrenia (Johnson, C. N.; Ahmed, M.; Miller, N. D. Curr. Opin. Drug Discov. Devel. 2008, 11, 642-654.; Mitchell, E. S.; Neumaier, J. F., Pharmacol. Ther. 2005, 108, 320-333).

It has become evident through analyses of the binding profiles of atypical antipsychotics on the 5-HT ₆ receptor, that antagonists at this receptor may have a role in enhancing cognitive function in schizophrenia. Recently, Li and colleagues [Li Z., Huang M., Prus A.J., Dai J. & Meltzer H.Y. Brain Research 2007, 1134(1), 70-78] studied the selective 5-HT ₆ receptor antagonist SB-399885 for its ability to potentiate efflux of dopamine induced by the typical neuroleptic haloperidol and the atypical neuroleptic risperidone, from the hippocampus and medial prefrontal cortex. Atypical neuroleptics, which include clozapine, olanzapine and risperidone, increase dopamine and acetylcholine levels due to stimulated release of these neurotransmitters. Microdialysis studies with S8- 399885 demonstrated that this compound was able to increase dopamine levels in hippocampal, but not in cortical, dialysates.

Activities mediated by both 5-HT ₆ and 5-HT ₇ receptor may address two major unmet needs in pharmacotherapy of schizophrenia: the efficacious treatment of cognitive symptoms and lack of weight gain in patients.

Paluchowska et al. provided structure-activity relationship studies of a series of novel 4,6-disubstituted 2-(l-piperazinyl) pyridines and pyrimidines were conducted to revise their model of serotonin 5-HT _2A receptor antagonist. The studies showed that they may constitute a new group of selective 5-HT _2A /5-HT _IA receptor antagonists, less potent than their pyrimidine analogs, though. For the discussed group of compounds it seems that introduction of the pyridine ring into the structure in place of the pyrimidine system is not a beneficial structural modification for 5-HT _2A receptor binding. Paluchowska et al. suggested that the central ring plays an important role in the ligand-receptor interaction. [Paluchowska M., Bojarski A., Bugno R., Charakchieva-Minol S., Wesolowska A. Arch. Pharm. Pharm. Med. Chem. 2003, 2, 104-110].

In the patent application WO 2006065706 (publ. 2006-06-22) N-biaryl and N- arylheteroaryl 2-substituted piperazine derivatives as modulators of the 5HT2C receptor useful for the treatment of disorders related thereto are described. The invention relates to certain N-biaryl and N-arylheteroaryl 2-substituted piperazine derivatives of Formula (Ia) that are modulators of the 5-HT _2C receptor. Accordingly, compounds of the invention are useful for the treatment of 5HT ₂ c receptor associated diseases or disorders, such as, obesity, Alzheimer's disease, erectile dysfunction and related disorders.

In the patent description US 4929726 (publ. 1990-05-29) novel diazines and their method of preparation are described. The invention relates to a method of preparation of substituted halogenodiazines which are useful as intermediates in the synthesis of novel unfused heterobicyclic compounds, and the products thereof. The reaction consists of the addition of an organolithium reagent with subsequent dehydrogenation of the addition product. It takes place in one reaction vessel, without isolation of the substituted halogenodihydro-diazine intermediate. The reactions proceed at moderate temperature and in a short amount of time, which decreases the probability of side reactions and increases yield. Furthermore, the workup step is conducted under two-phase conditions to prevent hydrolysis of the substituted halogenodiazine to a substituted hydroxydiazine. The substituted halogeno-diazines are used as intermediates in the synthesis of novel unfused heterobicyclic compounds containing an aromatic moiety, diazine, and another aromatic moiety, such as thiophene, benzene, or naphthalene, which have biological activity.

In the patent application WO 2009035671 (publ. 2009-03-19) substituted nitrogen- containing heteroaryl derivatives useful as modulators of the histamine H ₄ receptor are described. The invention relates to substituted nitrogen-containing heteroaryl derivatives, pharmaceutical compositions containing them and methods of using any of these derivatives and compositions for H ₄ receptor activity modulation and the treatment of states mediated by histamine H ₄ receptor activity.

In the patent description EP 0385237 Bl (publ. 1990-09-05) 2-(l-piperazinyl)-4- phenylcycloalkanopyridine derivatives, processes for the production thereof, and pharmaceutical composition containing the same are described. The invention concerns novel 2-(l-piperazinyl)-4-phenyl-cycloalkanopyridine derivatives of the formula wherein n is 3, 4, 5, 6 or 7; R<1> is hydrogen atom, Cl-ClO alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C6 cycloalkyl-(Cl-C4) alkyl, hydroxy-(C2-C6) alkyl, C1-C3 alkoxy-(C2-C6) alkyl, acyloxy-(C2-C6) alkyl, unsubstituted or substituted aroyl-(Cl-C6) alkyl, unsubstituted or substituted aryl, heteroaryl, or acyl; R<2> and R<3> are the same or different and are each hydrogen atom, halogen atom, C1-C6 alkyl, C1-C6 alkoxy, trifluoromethyl, or hydroxy; R<4>, R<5> and R<6> are the same or different and are each hydrogen atom, C1-C6 alkyl, or phenyl, or two of R<4>, R<5> and R<6> combine to form a single bond or C1-C3 alkylene; R<7> and R<8> are the same or different and are each hydrogen atom or C1-C3 alkyl; m is 2 or 3, or an acid addition salt thereof. These compounds are useful as a psychotropic drug.

In the patent application WO 2004041793 (publ. 2004-05-21) phenylalkyl and pyridylalkyl piperazine derivatives are described. The invention relates to compounds of the formula (1), pharmaceutical compositions containing them and their use in the treatment of central nervous system and other disorders.

Despite the described solutions and knowledge concerning the prevention or treatment of neurological or psychiatric diseases, especially related with the modulation or regulation of brain monoaminergic receptors there is still a need to create a new solution where modulation of monoaminergic receptors activity will be used. There is still a need to create a composition which might be use in the area of neurological and psychiatric diseases which cannot be cure with existing methods.

Neuroleptics are nowadays the major medicines used to treat schizophrenia. So far used, so-called first generation or typical neuroleptic drugs were first developed in the 1950s and used to treat psychosis. They were efficient inhibitors of dopaminergic receptors D ₂ and gave patients great relief, reducing psychotic (positive) symptoms (hallucinations, delusions). However, they had no effects on negative symptoms (flat affect, apathy, cognition and poverty of speech) and caused important side effects. Common side effects include: dry mouth, muscle stiffness, muscle cramps, tremors, extrapyramidal symptoms and weight-gain. Currently second generation neuroleptics or atypical neuroleptics are used, which additional essential part of the pharmacological profile is to block 5-HT receptors. Atypicals are a heterogeneous group of otherwise unrelated drugs united by the fact that they work differently from typical antipsychotics. Most share a common attribute of working on serotonin receptors as well as dopamine receptors. These include amongst others clozapine, quetiapine, olanzapine, risperidone, sertindole and ziprasidone. The atypical antipsychotics have found favor among clinicians and are now considered to be first line treatments for schizophrenia and are gradually replacing the typical antipsychotics. Most researchers agree that the defining characteristic of an atypical antipsychotic is the decreased propensity of these agents to cause extrapyramidal symptoms and an absence of sustained prolactin elevation.

The side-effect profile of atypical antipsychotics, though improved compared to their "typical" predecessors, remains clearly a highly problematic feature of their drug profile. All of the leading atypicals are generally perceived to have similar efficacy. Recent data from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study revealed that 74% of patients of a 1,432 patient cohort who received at least one dose of antipsychotic medication discontinued medication before 18 months, mostly due to insufficient efficacy or intolerable side-effects. Moreover, according to Datamonitor primary physician research, psychiatrists across the seven major markets believe that positive symptoms (32%) are the most clinically important domain of schizophrenia to treat. Treatment of negative symptoms (22%) is regarded as the second most important domain to treat followed by cognitive impairment (16%), impairment of daily function (16%) and disorganized thinking (14%). Treatment of positive symptoms of schizophrenia is regarded as the most important by psychiatrists since these symptoms, if left untreated, result in psychosis, which has the greatest propensity to lead to hospitalization, incarceration or homelessness, over a relatively short period of time. However, positive symptoms of schizophrenia are well-controlled by both typical and atypical antipsychotics. If negative symptoms are left untreated, patients may show extremes of emotion, which are often inappropriate. Sudden interruptions in their train of thought or speech may occur, making it very difficult for other people to understand them. Consequently patients tend to become isolated from society, and have difficulty in holding down an employment. If negative symptoms are left untreated the quality of life and health of the patient deteriorates over the long term.

Similarly, cognitive impairment, a long-term symptom of schizophrenia, which leads to a more gradual decline in quality of life as compared to positive symptoms, is currently not correctly addressed by the existing therapeutic arsenal.

Marketed atypical antipsychotics have all shown comparable efficacy in the treatment of positive symptoms of schizophrenia, although at present, data indicates that their efficacy in the treatment of negative symptoms is poorly defined.

The goal of the present invention is to address two major unmet needs in pharmacotherapy of schizophrenia: the efficacious treatment of cognitive symptoms and to reduce the weight gain propensity associated with existing therapeutic arsenal. This invention proposes to address both positive symptoms through the dopamine hypothesis and negative symptoms through its affinity to serotonin receptors, with the special focus on cognitive impairment mediated by the 5-HT ₆ receptor. Cognitive impairment - the overall weakness of intelligence, as well as the selective impairment of certain aspects of attention, memory, visual-spatial functions, or language ability - is now generally regarded as a core deficit of schizophrenia.

The embodiment of such a stated goal and the solution of problems described in the state of the art dealing with treatment of neurological and psychiatric diseases, especially related with the modulation or regulation of brain monoaminergic receptors have been achieved in the present invention by providing a family of compounds, formulated or unformulated to pharmaceutical standard, to be used for the treatment of positive and negative symptoms of schizophrenia, schizoaffective or schizoid disorders, schizophreniform disorders, delusional disorders and other psychotic disorders, whether due to general medical condition or related to substance abuse. The present invention can also be used in the treatment of mood disorders such as bipolar disorders and depressive disorders, anxiety disorders, attacks of unconsciousness, and diseases that cause cognitive disorders (such as Alzheimer's disease or senile dementia or dementia due to other general medical condition and amnestic disorders) and concentration problems (for example attention deficit hyperactivity disorder), or autism. Due to its central acting mode of action the present invention also be used in the treatment of thermoregulatory disorders, such as hypothermia, sleep disorders, including rapid eye movement sleep, symptoms connected with excessive stress or nociception disorders, as well as for treating opioid-induced respiratory depression, migraine, haematomas, for preventing cerebral ischaemia, and for treating peristaltic movement disorders, such as Irritable Bowel Syndrome, and miction disorders, including cystitis and excessive bladder contractility.

The subject of the present invention is a compound of Formula I-P:

formula I-P or an enantiomer thereof or a mixture of its enantiomers or its pharmaceutically acceptable salt, or pharmaceutically acceptable prodrugs, where prodrugs are functional derivatives of said compounds and which are readily converted to the active moiety in vivo or pharmaceutically active metabolites, wherein:

R ¹ represents an unsubstituted or substituted aromatic or heteroaromatic group containing C and/or S atoms,

R ² represents a branched or unbranched C _1-I2 aliphatic group or C _1-12 alicyclic (mono or polycyclic) group.

Preferably, R ¹ represents an unsubstituted or substituted aromatic or heteroaromatic group containing C and/or S atoms wherein aromatic or heteroaromatic group is selected from but not limited to phenyl, 2-thienyl, 3-thienyl, 1-naphthyl, 2-naphthyl, 3-benzo[b]thienyl or 2- benzo[c]thienyl and is unsubstituted or is substituted with one or more substituents independently selected from the group consisting of but not limited to halo (Cl, Br, F, I), hydroxy or alkoxy (-0-Ci _-4 alkyl) and wherein R ¹ is selected preferably from the group consisting of 2-thienyl, 3-thienyl, phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3 -fluorophenyl, 4-fluorophenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4- methoxyphenyl, 1-naphthyl, 2-naphthyl, 3-benzo[b]thienyl or 2-benzo[c]thienyl, except 1- methyl-4-(6-methyl-4-thiophen-2-yl-pyridin-2-yl)-piperazine and 1 -methyl-4-(6-methyl-4- phenyl-pyridin-2-yl)-piperazine.

Preferably, R ² represents a branched or unbranched C _M2 aliphatic group or C _M2 alicyclic (mono or polycyclic) group, wherein R ² is Ci _-12 alkyl (branched or unbranched) or C _1- I ₂ cycloalkyl mono or poly-, where R ² is selected preferably from the group consisting of methyl, ethyl, propyl, isopropyl, 1-buthyl, 2-buthyl, isobuthyl, tert-buthyl, 1-pentyl, 2- pentyl, 3-pentyl, isopentyl, 1-hexyl, 2-hexyl, 3-hexyl, isohexyl or cyclohexyl, 1- decahydronaphthyl, 2-decahydronaphthyl, 1-adamantyl or 2-adamantyl groups. Preferably, a compound is selected from the group consisting of: 6-methyl-4-(3-thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 4-(2-methoxyphenyl)-6-methyl-2-(4-methylpiperazin-l-yl)pyrid ine, 4-(3 -methoxyphenyl)-6-methyl-2-(4-methylpiperazin- 1 -yl)pyridine, 4-(4-methoxyphenyl)-6-methyl-2-(4-methylpiperazin-l-yl)pyrid ine, 4-(3 -benzo [b]thienyl)-6-methyl-2-(4-methylpiperazin- 1 -yl)pyridine, 6-(tert-buthyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-ethyl-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-isopropyl-4-(3-thienyl)- 2-(4-methylpiperazin- 1 -yl)pyridine, 6-( 1 -buthyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-( 1 -pentyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-( 1 -adamantyl)-4-(3 -thienyl)- 2-(4-methylpiperazin- 1 -yl)pyridine, 6-(isopentyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-( 1 -hexyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 6-(cyclohexyl)-4-(3 -thienyl)-2-(4-methylpiperazin- 1 -yl)pyridine, 4-(4-fluorophenyl)-6-methyl-2-(4-methylpiperazin- 1 -yl)pyridine, and their pharmaceutically acceptable salts, and pharmaceutically acceptable prodrugs where prodrugs are functional derivatives of said compounds and which are readily converted to the active moiety in vivo and pharmaceutically active metabolites.

Preferably, pharmaceutically acceptable salts are selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p- toluenesulfonate and pamoate and the like.

The invention also relates to pharmaceutically acceptable prodrugs of the compounds described herein, and methods employing such pharmaceutically acceptable prodrugs. The term "prodrug" means a precursor of a designated compound that, following administration to a subject, yields the compound in vivo via a chemical or physiological process such as solvolysis or enzymatic cleavage, or under physiological conditions (e.g. a prodrug on being brought to physiological pH is converted to the compound of the present invention). A "pharmaceutically acceptable prodrug" is a prodrug that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to the subject. Illustrative procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985. The present invention also relates to pharmaceutically active metabolites of the compounds of the invention, and uses of such metabolites in the methods of the invention. A "pharmaceutically active metabolite" means a pharmacologically active product of metabolism in the body of a compound of the present invention or salt thereof. Prodrugs and active metabolites of a compound may be determined using routine techniques known or available in the art.

The next subject of invention is a process for the preparation compound of formula (I-P) or an enantiomer thereof or a mixture of its enantiomers or its pharmaceutically acceptable salts of compounds of Formula (I-P), pharmaceutically acceptable prodrugs where prodrugs are functional derivatives of said compounds and which are readily converted to the active moiety in vivo of compounds of Formula (I-P), or pharmaceutically active metabolites of compounds of Formula (I-P), as claimed in any one of claims 1 to 5, characterized in that

formula I-P, wherein:

R ¹ represents an unsubstituted or substituted aromatic or heteroaromatic group containing C and/or S atoms,

R ² represents a branched or unbranched aliphatic group with 1-12 carbon atoms or C _1-I2 alicyclic (mono or polycyclic) group, comprising:

reacting 2-(l-benzotriazolyl)acetonitrile (compound of formula II) with a compound of formula (III) - α,β-unsaturated ketone and a compound of formula (IV), wherein compound of formula IV is selected from the group consisting of diethylamine, piperazine, pyrrolidine or morpholine in an anhydrous organic solvent where said anhydrous organic solvent is an anhydrous polar aprotic solvent.

Preferably, a compound of formula (I-P) is synthesized in the presence of inorganic or organic acids, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate and the like, and an anhydrous organic solvent where said anhydrous organic solvent is an anhydrous polar aprotic solvent.

The next subject of invention is a pharmaceutical composition, comprising a therapeutically effective amount of at least one compound selected from the group consisting of compounds as described above.

The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. The active agents of the invention are used, alone or in combination with one or more additional active ingredients, to formulate pharmaceutical compositions of the invention. A pharmaceutical composition of the invention comprises an effective amount of at least one active agent in accordance with the invention. As known in pharmaceutical technology, at least one pharmaceutically acceptable carrier may be comprised in embodiments of pharmaceutical compositions according to this invention.

Preferably, the therapeutically effective amount provided in the treatment is administered in an amount of about 0.01 to 1,000 mg/kg at least once a day for the duration of the treatment. Preferably, the said composition is parenterally, vaginally, rectally, sublingually, transdermally, locally or orally administered administered.

Preferably, the said composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a binder, diluent, lubricant, disintegrating agent, effervescing agent, dyestuff, sweetener, wetting agent, and mixtures thereof wherein carrier is chosen from solid or liquid carriers, whether sterile or not. Pharmaceutically acceptable carrier is chosen from solid or liquid carriers, whether sterile or not. Solid carriers suitable for use in the composition of the invention include one or more substances which potentially act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aides, binders, tablet-disintegrating agents or encapsulating materials. In powders, the carrier and compound is a finely divided solid. In tablets, said compound is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Said powders and tablets contain from 5 to 99% by weight of the compound. Solid carriers suitable for use in the composition of the invention include calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Any pharmaceutically acceptable liquid carrier suitable for preparing solutions, suspensions, emulsions, syrups and elixirs can be employed in the composition of the invention. Is that case, the compound is dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or pharmaceutically acceptable oil or fat, or a mixture thereof. Said liquid composition contain additionally or not other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers, osmo-regulators, or the like.

Examples of liquid carriers suitable for oral and parenteral administration include water, particularly containing additives as above, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution, alcohols, including monohydric alcohols and polyhydric alcohols, such as glycols or their derivatives, or oils such as fractionated coconut oil, cottonseed oil and arachid oil. For parenteral administration the carrier may also be an oily ester such as ethyl oleate or isopropyl myristate.

Preferably, a pharmaceutical composition, is a sterile solution or suspension suitable for intramuscular, intraperitoneal, intravenous or subcutaneous injection. Preferably, a composition, is suitable for oral administration either liquid or solid composition form.

Preferably, the medicament is an immediate, extended or slow- release formulation. The preparation may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories. In an embodiment, the compositions are formulated for intravenous infusion, topical administration, or oral administration.

Preferably, therapeutically effective amount is provided in the treatment of a disease, disorders or medical condition that is selected from the group consisting of schizophrenia, schizoaffective or schizoid disorders, schizophreniform disorders, delusional disorders and other psychotic disorders, whether due to general medical condition or related to substance abuse, in the treatment of mood disorders such as bipolar disorders and depressive disorders, anxiety disorders, attacks of unconsciousness, and diseases that cause cognitive disorders, such as Alzheimer's disease or senile dementia or dementia due to other general medical condition and amnestic disorders, and concentration problems, such as attention deficit hyperactivity disorder, or autism or thermoregulatory disorders, such as hypothermia, sleep disorders, including rapid eye movement sleep, symptoms connected with excessive stress or nociception disorders, as well as for treating opioid-induced respiratory depression, migraine, haematomas, for preventing cerebral ischaemia, and for treating peristaltic movement disorders, such as Irritable Bowel Syndrome, and miction disorders, including cystitis and excessive bladder contractility,disorders. Preferably, a composition is for the prevention or treatment of neurological or psychiatric diseases, especially related with the modulation or regulation of brain monoaminergic receptors such as serotonin receptors, preferably receptors selected from the group of: 5- HT ₆ , 5-HT ₂ A, 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α.

The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of a disease or disorder. When referring to modulating the target receptor, a "therapeutically effective amount" means an amount sufficient to at least affect the activity of such receptor. Measuring the activity of the target receptor may be performed by routine analytical methods. Target receptor modulation is useful in a variety of settings, including assays. In addition, effective amounts or doses of the active agents of the present invention may be ascertained by routine methods such as modeling, dose escalation studies or clinical trials, and by taking into consideration routine factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician.

The amount varies according to the size, age and response pattern of the patient, the severity of the disorders, the judgment of the attending physician and the like.

The next subject of invention is a use of the compound of formula I for the preparation of a pharmaceutical composition as described above.

Preferably, for the prevention or treatment of neurological or psychiatric diseases, as described above.

The next subject of invention is a method for modulating monoaminergic receptors activity, characterized in that monoaminergic receptors are such as serotonin receptors, preferably receptors selected from the group of: 5-HT ₆ , 5-HT _2A , 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α wherein monoaminergic receptors are exposed to an effective amount of at least one compound of Formula (I), an enantiomer thereof or a mixture of its enantiomers or pharmaceutically acceptable salts of compounds of Formula (I) or pharmaceutically acceptable prodrugs of compounds of Formula (I), or pharmaceutically active metabolites of compounds of Formula (I) as as described above.

Preferably, monoaminergic receptor are such as serotonin receptors, preferably receptors selected from the group of: 5-HT ₆ , 5-HT _2A , 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α is in a subject with a disease, disorders or medical condition that is selected from the group consisting of schizophrenia, schizoaffective or schizoid disorders, schizophreniform disorders, delusional disorders and other psychotic disorders, whether due to general medical condition or related to substance abuse. The present invention can also be used in the treatment of mood disorders such as bipolar disorders and depressive disorders, anxiety disorders, attacks of unconsciousness, and diseases that cause cognitive disorders, such as Alzheimer's disease or senile dementia or dementia due to other general medical condition and amnestic disorders, and concentration problems, such as attention deficit hyperactivity disorder, or autism or thermoregulatory disorders, such as hypothermia, sleep disorders, including rapid eye movement sleep, symptoms connected with excessive stress or nociception disorders, as well as for treating opioid-induced respiratory depression, migraine, haematomas, for preventing cerebral ischaemia, and for treating peristaltic movement disorders, such as Irritable Bowel Syndrome, and miction disorders, including cystitis and excessive bladder contractility.

The next subject of invention is a monoaminergic receptor modulating agent as described above, characterized in that monoaminergic receptors are such as serotonin receptors, preferably receptors selected from the group of: 5-HT ₆ , 5-HT ₂ A, 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α wherein monoaminergic receptors are exposed to an effective amount of at least one compound of Formula (I), an enantiomer thereof or a mixture of its enantiomers or pharmaceutically acceptable salts of compounds of Formula (I) or pharmaceutically acceptable prodrugs of compounds of Formula (I), or pharmaceutically active metabolites of compounds of Formula (I) as described above.

Preferably, the monoaminergic receptors are such as serotonin receptors, preferably receptors selected from the group of: 5-HT ₆ , 5-HT ₂ A, 5-HT ₇ , dopamine receptors, preferably D ₂ and adrenergic receptors, preferably α is in a subject with a disease, disorders or medical condition that is selected from the group consisting of schizophrenia, schizoaffective or schizoid disorders, schizophreniform disorders, delusional disorders and other psychotic disorders, whether due to general medical condition or related to substance abuse. The present invention can also be used in the treatment of mood disorders such as bipolar disorders and depressive disorders, anxiety disorders, attacks of unconsciousness, and diseases that cause cognitive disorders, such as Alzheimer's disease or senile dementia or dementia due to other general medical condition and amnestic disorders, and concentration problems, such as attention deficit hyperactivity disorder, or autism or thermoregulatory disorders, such as hypothermia, sleep disorders, including rapid eye movement sleep, symptoms connected with excessive stress or nociception disorders, as well as for treating opioid-induced respiratory depression, migraine, haematomas, for preventing cerebral ischaemia, and for treating peristaltic movement disorders, such as Irritable Bowel Syndrome, and miction disorders, including cystitis and excessive bladder contractility.

The attached figures facilitate a better understanding of the nature of the present invention.

Figure 1 presents effects of compound #9 on Incidental Learning in C57/B1 Mice - difference of Transfer Latencies before and after treatment, (mean +/- interpercentile range).

Figure 2 presents compound #9 administered in a range of doses (0.125-0.5 mg/kg) significantly protected rats from ketamine-induced deficits in extradimensional shift performance as compared with vehicle-ketamine conditions. The action of compound #9was comparable to that of sertindole at a dose of 2.5 mg/kg (*** : pO.OOl) Figure 3 presents effects of compound #9 on scopolamine-induced learning deficit in passive avoidance test in rats. All rats except vehicle controls were treated with scopolamine (1 mg/kg, SC) 30 min, and compound #9 (0.125, 0.25, 0.5 mg/kg, IP; 60 min) or sertindole (2.5 mg/kg, PO, 120 min), respectively, before training trial. The memory retention trial was carried out 24 hours later. Results represent the median ± interquartille range of the test dark chamber entrance latencies in seconds. Data were analyzed with a non-parametric Kruskal-Wallis ANOVA with drug treatment as a between-subject factor. ANOVA revealed significant effect of drug treatment: Kruskal-Wallis statistic: 17.14, P<0.01. Symbols: *p<0.05 vs. the scopolamine-treated group's latency; Dunn's post-hoc test. N= 14 for vehicle and scopolamine-treated rats and 10 for all other groups. The following specific Examples are set forth to illustrate the invention and to aid in the understanding of the invention, and are not intended and should not be construed to limit in any way the invention set forth in the claims which follow thereafter.

Example 1 6-(l-buthyl)-4-(3-thienyl)-2-(4-methylpiperazin-l-yI)pyridin e,

Preparation of 6-(l-buthyl)-4-(3-thienyl)-2-(4-methylpiperazin-l-yl)pyridin e: equimolar amounts (3 mmol) of α,β-unsaturated ketone (l-Thiophen-3-yl-but-2-en-l-one), 2-(l- benzotriazolyl)acetonitrile and N-methylpiperazine were refluxed in ethanol (10 mL) for 48 h. the solvent was evaporated, and the residue was dissolved in CHC13 (20 mL), washed with 10% NaOH then with brine, and dried over anhydrous MgSO4. After evaporation of the solvent the residue was purified by column chromatography (SiO ₂ , CHCl ₃ /Me0H = 49/1).

The absorbent was SiO ₂ or/and neutral Al ₂ O ₃ , and the eluents were the systems: CHCl ₃ ZCH ₃ OH in a volume ratio of 19:1 or/and ethyl acetate/hexane in a volume ratio of 1 :2, respectively. The purity of the products was checked by the TLC method on aluminium plates covered with neutral Al ₂ O ₃ , using, as an eluent, ethyl acetate/hexane system in a volume ratio of 1 :2.

For the obtained compounds (described below) the ¹ H NMR spectrum was recorded.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,39; 2.35 (s, 3H); 2.44 (s, 3H); 2.54 (dd, J = 5.1 i 4.9 Hz, 4H); 3.60 (t, J = 5.1 Hz, 4H); 6.62 (s, IH); 6.72 (s, IH); 7.38 (dd, J = 2.3 i 1.8 Hz, 2H); 7.55 (dd, J = 2.6 i 1.8 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [°C] for salts: 505,545; 163-165 (acetone / hexane 1:1) Example 2 4-(2-methoxyphenyl)-6-methyl-2-(4-methylpiperazin-l-yl)pyrid ine

The title compound was prepared according to the methods as described in Example 1 with l-(2-Methoxy-phenyl)-but-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,38; 2.34 (s, 3H); 2.44 (s, 3H); 2.53 (t, J = 5.1 Hz, 4H); 3.58 (dd, J = 5.1 i 4.9 Hz, 4H); 3.81 (s, 3H); 6.59 (s, IH); 6.67 (s, IH); 6.96-7.04 (m, 2H); 7.28-7.37 (m, 2H).

Molecular weight; t _t (beginning of crystallization) [°C] for salts: 333,861; 239-241 (acetone)

Example 3 4-(3-methoxyphenyl)-6-methyl-2-(4-methylpiperazin-l-yl)pyrid ine

The title compound was prepared according to the methods as described in Example 1 with l-(3-Methoxy-phenyl)-but-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 2.35 (s, 3H); 2.46 (s, 3H); 2.54 (t, J = 5.1 Hz, 4H); 3.62 (dd, J = 5.1 i 4.9 Hz, 4H); 3.86 (s, 3H); 6.61 (s, IH); 6.71 (s, IH); 6.93 (ddd, J = 8.2, 2.6 i 1.0 Hz, IH); 7.10 (dd, J = 2.5 i 1.6 Hz, IH); 7.16 (ddd, J = 7.6, 1.6 i 1.0 Hz, IH); 7.35 (t, J = 7.9 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 370,321; 254-256 (acetone) Example 4

4-(4-methoxyphenyl)-6-methyl-2-(4-methylpiperazin-l-yl)py ridine dihydrochloride

The title compound was prepared according to the methods as described in Example 1 with l-(4-Methoxy-phenyl)-but-2-en-l-on used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,40; 2.35 (s, 3H); 2.45 (s, 3H); 2.54 (dd, J = 5.1 i 4.9 Hz, 4H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 3.84 (s, 3H); 6.59 (s, IH); 6.70 (s, IH); 6.96 (ddd, J = 9.0, 2.8 i 2.0 Hz, 2H); 7.53 (ddd, J = 9.0, 2.8 i 2.0 Hz, 2H).

Molecular weight; t _t (beginning of crystallization) [°C] for salts: 370,321 ; 245-247 (acetone)

Example 5 4-(3-benzo[b]thienyl)-6-methyl-2-(4-methyIpiperazin-l-yl)pyr idine fumarate

The title compound was prepared according to the methods as described in Example 1 with l-Benzo[b]thiophen-3-yl-but-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 2.36 (s, 3H); 2.48 (s, 3H); 2.55 (dd, J = 5.1 i 4.9 Hz, 4H); 3.63 (dd, J = 5.1 i 4.9 Hz, 4H); 6.64 (s, IH); 6.72 (s, IH); 7.36- 7.44 (m, 2H); 7.47 (s, IH); 7.86-7.95 (m, 2H).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 439,534; 164-166 (acetone) Example 6 6-(tert-buthyl)-4-(3-thienyl)-2-(4-methylpiperazin-l-yl)pyri dine fumarate

The title compound was prepared according to the methods as described in Example 1 with 4,4-dimethyl-l-thiophen-3-yl-pent-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDCB, 60MHz, TMS) [δ ppm] for base: 0,42; 1,3 (s, 9H); 2,35 (s, 3H); 2,4- 2,65 (m, 4H); 3,5-3,8 (m, 4H); 6,7 (s, IH); 6,95 (s, IH); 7,35-7,45 (m, 2H); 7,55-7,65 (m, IH).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 431,554; 159-160 (ethanol)

Example 7 6-ethyI-4-(3-thienyl)-2-(4-methylpiperazin-l-yl)pyridine fumarate

The title compound was prepared according to the methods as described in Example 1 with l-thiophen-3-yl-pent-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 60MHz, TMS) [δ ppm] for base: 0,59; 1,3 (t, J=8Hz, 3H); 2,35 (s, 3H); 2,4-2,9 (m, 6H); 3,5-3,8 (m, 4H); 6,7 (s, IH); 6,8 (s, IH); 7,3-7,5 (m, 2H); 7,5-7,7 (m, IH).

Molecular weight; t, (beginning of crystallization) [ ⁰ C] for salts: 403,500; 146-148 (ethanol) Example 8 6-isopropyl-4-(3-thienyl)- 2-(4-methyIpiperazin-l-yl)pyridine fumarate

The title compound was prepared according to the methods as described in Example 1 with 4-methyl-l-thiophen-3-yl-pent-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,46; 1.29 (d, J = 6.9 Hz, 6H); 2.36 (s, 3H); 2.54 (dd, J = 5.1 i 4.9 Hz, 4H); 2.88-2.98 (m, IH); 3.63 (dd, J = 5.1 i 4.9 Hz, 4H); 6.63 (d, J = 1.0 Hz, IH); 6.72 (d, J - 1.3 Hz, IH); 7.38 (d, J = 2.3 Hz, 2H); 7.55 (dd, J = 2.3 i 2.0 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 417,528; 130-132 (acetone)

Example 9 6-(l-buthyl)-4-(3-thienyl)-2-(4-methylpiperazin-l-yl)pyridin e fumarate

The title compound was prepared according to the methods as described in Example 1 with l-thiophen-3-yl-hept-2-en-l-one used as α,β-unsaturated ketone.

R _f ; ¹ H NMR (CDCl ₃ , 300MHz, TMS) [δ ppm] for base: 0,54; 0.94 (dd, J = 7.4 i 7.2 Hz, 3H); 1.34- 1.46 (m, 2H); 1.67-1.78 (m, 2H); 2.35 (s, 3H); 2.54 (dd, J = 5.1 i 4.9 Hz, 4H); 2.67 (dd, J = 8.0 i 7.7 Hz, 2H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 6.63 (d, J = 1.0 Hz, IH); 6.71 (d, J = 1.0 Hz, IH); 7.38 (d, J = 2.1 Hz, 2H); 7.55 (dd, J = 2.3 i 2.0 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 431,555; 149-151 (acetone) Example 10 6-(l-pentyI)-4-(3-thienyl)-2-(4-methylpiperazin-l-yl)pyridin e fumarate

The title compound was prepared according to the methods as described in Example 1 with l-thiophen-3-yl-oct-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDCB, 300MHz, TMS) [δ ppm] for base: 0,58; 0.90 (dd, J = 7.2 i 6.9 Hz, 3H); 1.30-1.39 (m, 4H); 1.69-1.79 (m, 2H); 2.35 (s, 3H); 2.54 (t, J = 5.1 Hz, 4H); 2.66 (t, J = 7.7 Hz, 2H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 6.63 (d, J = 1.0 Hz, IH); 6.76 (d, J = 1.0 Hz, IH); 7.38 (d, J = 2.3 Hz, 2H); 7.55 (dd, J = 2.3 i 2.1 Hz, IH)..

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 445,582; 121-123 (acetone)

Example 11 6-(l-adamantyl)-4-(3-thienyl)- 2-(4-methyIpiperazin-l-yl)pyridine fumarate

The title compound was prepared according to the methods as described in Example 1 with 3-adamantan-l-yl-l-tiophen-3-yl-propenone used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,63; 1.78 (s, 6H); 2.00 (s, 6H); 2.09 (s, 3H); 2.36 (s, 3H); 2.55 (dd, J = 5.1 i 4.9 Hz, 4H); 3.64 (t, J = 4.9 Hz, 4H); 6.63 (d, J = 0.8 Hz, IH); 6.80 (d, J = 1.0 Hz, IH); 7.38 (dd, J = 2.4 i 1.8 Hz, 2H); 7.54 (dd, J = 2.3 i 2.0 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [°C] for salts: 509,668; 220-222 (acetone) Example 12 6-(isopentyl)-4-(3-thienyI)-2-(4-methylpiperazin-l-yl)pyridi ne fumarate

The title compound was prepared according to the methods as described in Example 1 with 6-methyl-l-thiophen-3-yl-hept-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,58; 0.95 (d, J = 6.4 Hz, 6H); 1.58-1.65 (m, 3H); 2.36 (s, 3H); 2.55 (t, J = 5.1 Hz, 4H); 2.68 (t, J = 7.7 Hz, 2H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 6.62 (d, J = 1.0 Hz, IH); 6.71 (d, J = 1.0 Hz, IH); 7.38 (d, J = 2.0 Hz, 2H); 7.55 (dd, J = 2.3 i 2.0 Hz, IH).

Molecular weight; t, (beginning of crystallization) [°C] for salts: 445,582; 137-138 (acetone)

Example 13 6-(l-hexyl)-4-(3-thienyl)-2-(4-methyIpiperazin-l-yl)pyridine fumarate

The title compound was prepared according to the methods as described in Example 1 with l-thiophen-3-yl-non-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,60; 0.89 (dd, J = 7.2 i 6.7 Hz, 3H); 1.25-1.40 (m, 6H); 1.68-1.78 (m, 2H); 2.36 (s, 3H); 2.55 (dd, J = 5.1 i 4.9 Hz, 4H); 2.66 (t, J = 7.7 Hz, 2H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 6.63 (s, IH); 6.70 (s, IH); 7.38 (d, J = 2.1 Hz, 2H); 7.55 (dd, J = 2.3 i 2.1 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 459,608; 129-131 (acetone) Example 14 6-(cyclohexyl)-4-(3-thienyI)-2-(4-methylpiperazin-l-yl)pyrid ine fumarate

The title compound was prepared according to the methods as described in Example 1 with 3-cyclohexyl-l-thiophen-3-yl-propenone used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,59; 1.20-1.61 (m, 5H); 1.72- 1.76 (m, IH); 1.82-1.86 (m, 2H); 1.94-1.98 (m, 2H); 2.36 (s, 3H); 2.53-2.62 (m, 5H); 3.62 (dd, J = 5.1 1 4.9 Hz, 4H); 6.63 (d, J = 1.0 Hz, IH); 6.71 (s, IH); 7.38 (d, J = 2.0 Hz, 2H); 7.54 (t, J = 2.0 Hz, IH).

Molecular weight; t _t (beginning of crystallization) [°C] for salts: 457,593; 170-172 (acetone)

Example 15 4-(4-fluorophenyl)-6-methyl-2-(4-methylpiperazin-l-yl)pyridi ne fumarate

The title compound was prepared according to the methods as described in Example 1 with l-(4-fluoro-phenyl)-but-2-en-l-one used as α,β-unsaturated ketone.

Rf; IH NMR (CDC13, 300MHz, TMS) [δ ppm] for base: 0,45; 2.35 (s, 3H); 2.45 (s, 3H); 2.54 (t, J = 5.1 Hz, 4H); 3.61 (dd, J = 5.1 i 4.9 Hz, 4H); 6.56 (s, IH); 6.67 (s, IH); 7.11 (t, J = 8.7 Hz, 2H); 7.51-77.57 (m, 2H).

Molecular weight; t _t (beginning of crystallization) [ ⁰ C] for salts: 401,436; 178-180 (acetone) Examples 16-24 Biological Testing

For biological tests the obtained compounds (described below) were converted into appropriate salts (mono- or dihydrochloride or fumarate, respectively) in the solution of acetone or ethanol or acetone/hexane mixture in a volume ratio of 1 : 1

Example 16 Biological Testing: Binding Assay on Cloned Human 5-HT ₆ receptor

The activity of the compounds of the invention against 5-HT ₆ receptor was tested using the following binding assay.

Membranes from HEK 293 cells stably expressing human 5-HT ₆ receptor were used according to similar procedure used for 5-HT ₇ binding (Holenz J., et al; J. Med Chem. 2005, 48, 1781-95.). Briefly, the membranes (20 μg protein per tube) were incubated in 50 mM TrisHCl buffer (pH 7.4) containing 10 mM MgCl ₂ and 0.5 mM EDTA, in the presence of 7 - 9 concentrations of test drug and 2 nM [ ³ H]- LSD (79.2 Ci/mmol). Nonspecific binding was defined in the presence of 10 μM of methiothepin. After a 1-h incubation at 37 ⁰ C, the assay samples were rapidly filtered through Whatman GF/B filters and subsequently washed with ice-cold 50 mM Tris buffer (pH 7.4) using a Brandel harvester.

The activity of selected compounds of the invention against 5-HT ₆ receptor are represented by Kj values:

Table 1. 5-HT ₆ receptor binding affinities.

Example 17 Biological Testing: Binding Assay on Rat Cortex 5-HT _2A receptor

Radioligand binding experiments for 5-HT _2A receptors were conducted in the cortex of the rat brain according to the published procedures. The radioligand used was [ ³ H]-ketanserin (60 Ci/mmol, NEN Chemicals). The Kj values were determined from at least three competition binding experiments in which 10 drug concentrations, run in triplicate, were used. The Cheng and Prusoff equation was used for Kj calculations.

Results for the compounds tested in this assay are presented as an average of results obtained:

Table 2. 5-HT _2A receptor binding affinities.

Example 18 Biological Testing: Binding Assay on Rat Hippocampus 5-HT ₇ receptor

The serotonin 5-HT ₇ receptor binding assay was performed using rat hypothalamic membranes, according to the method described by Aguirre et al. (Aguirre, N.; Ballaz, S.; Lasheras, B.; Del Rio, J. Eur. J. Pharmacol. 1998, 346, 181) with minor modifications. In brief, hypothalami dissected from male Wistar rats (200 - 250 g) were frozen at -8O ⁰ C prior to the preparation of radioligand binding homogenate. On the day of experiment hypothalami were allowed to defrost, then immediately homogenized in 20 volumes of 50 mM Tris-HCl buffer (pH 7.4 at 23°C) and centrifuged at 48,00Og for 10 min at 4°C. The supernatant was removed, resulting pellet rehomogenized and incubated at 37°C for 15 min, to remove endogenous serotonin. After incubation, the homogenate was centrifuged twice under the same conditions as before. The final pellet was resuspended in assay buffer (50 mM) Tris- HCl containing 0.01 mM pargyline, 4 mM CaC12, and 0.1% ascorbate. Aliquots of membranes (10 mg original wet tissue weight) were incubated in the presence of 3 IM (±)- pindolol (to eliminate binding to 5-HT receptors) with 0.5 nM [3HJ-5-CT (specific activity, 34.5 Ci/mmol; NEN) and eight concentrations of the displacing drug. Nonspecific binding was determined using 10 IM of serotonin. After incubation at 23 °C for 120 min, the reaction was terminated by rapid filtration through a Whatman GF/B filter.

Table 3. 5-HT ₇ receptor binding affinities.

Example 19 Biological Testing: Binding Assay on Rat Hippocampus D ₂ receptor

Dopamine D ₂ binding assays. The preparation of rat striatal membranes was conducted as described previously (Ossowska, G.; Nowak, G.; Kata, R.; Klenk-Majewska, B.; Danilczuk, Z.; Zebrowska-Lupina, I. J. Neural Transm. 2001, 108, 311.) The final tissue concentration for D2 receptor binding was 3 mg original wet weight mL ^~ . All the assays were carried out in 50 mM potassium phosphate buffer (pH 7.4). The radioligand used was [3H]-spiperone (15.70 Ci/mmol, NEN Chemicals) in the presence of 50 nM ketanserin to prevent radioligand binding to 5-HT _2A receptors. Displacement experiments were performed in a total volume of 1.2 mL. Assay tubes (in triplicate) containing: 0.1 mL of 1 nM [3H]-spiperone, 0.1 mL competing drug, or 0.1 mL of vehicle (total binding) and 1 mL of tissue were incubated at 37°C for 30 min. Nonspecific binding was determined using 0.1 mL of 5 μM butaclamol.

Table 4. D2 receptor binding affinities.

Example 20 Biological Testing: Binding Assay on Rat Hippocampal 5-HTi _A receptor

Radioligand binding experiments for 5 -HTi _A receptors were conducted using rat hippocampal membranes. The radioligand used was [ ³ HJ-8-OH-DPAT [8-hydroxy-2-(di-n- propylamino)tetralin, 190 Ci/mmol, Amersham]. The K, values were determined from at least three competition binding experiments in which 10 drug concentrations, run in triplicate, were used. The Cheng and Prusoff equation was used for K, calculations.

Results for the compounds tested in this assay are presented in Table 5 as an average of results obtained. Table 5. 5-HT ₁ A receptor binding affinities.

Example 21 Biological Testing: Binding Assay on cloned adrenergic a _{ receptor

(Xi Receptor binding experiments were conducted according to following protocol. [ ³ H]Prazosin (26 Ci/mmol, NEN Chemicals) was used for labelling αi receptors. The membrane preparation and assay procedure were carried out according to the published procedures (Maj I , Klimek V, Nowak G (1985) Eur Pharmacol 119, 1 13-116; Mogilnicka E, Nielsen M (1986) Eur Pharmacol 122, 369-372) with slight modifications. The cortex tissue of the rat brain was homogenized in 20 vol (w/v) of ice-cold Tris- HCI buffer (50 mM, pH = 7.4) with an Ultra Turrax homogenizer. The homogenate was centrifuged at 25 000 x g for 10 min, and the resulting pellet was suspended in the same volume of Tris- HC] buffer, and was recentrifuged. The final pellet was resuspended in 170 vol (w/v) of Tris-HCl buffer (50 nM, pH = 7.4). [ ³ H]Prazosin in a volume of 100 μl was added to aliquots ( 1.7 mL) of the membrane suspension, and the samples were incubated at 25 °C for 30 min. The total incubation volume of 2 mL was filtered through Whatman GF/B glass filters, and was then washed with a cold buffer (3 x 5 mL) using a Brandel cell harvester. Non-specific binding of [3H]prazosin was obtained in the presence of phentolamine (200 μl, final concentration 10 ^*6 M). The final [ ³ H]prazosin concentration was 3 x 10 ^" M and the concentration of the analyzed compounds ranged from 10 ^*10 to 10 ^"3 M. Ki values were determined from at least three independent experiments, run in triplicate:

Table 6. a _\ receptor binding affinities.

Example 22

Biological Testing: Investigation of the influence of compound #9 on incidental learning in mice.

Subjects

Male C57BL/ mice (Charles River, Germany), 22-24 g of body weight were group-housed in the standard laboratory cages and kept in a temperature controlled colony room (21 ± 2oC) with a 12-hr light/dark cycle (light on: 07:00, off: 19:00). Commercial food and tap water were available ad libitum. Each experimental group consisted of 9-11 mice per treatment. All mice were used only once. During the adaptation period, mice were carried into the testing room where they were weighed and handled by moving them from one standard home cage to another in the proximity of the apparatus. This adaptation phase was intended to reduce the stress associated with handling, injections, and exposure to the apparatus.

Apparatus

The elevated plus maze allows investigation of cognitive as well as anxiolytic effects of treatment. In this particular test, unlike other tests of memory (e.g., food-reinforced T-maze or footshock-reinforced passive avoidance), mice use a repertoire of native sensory cues and a natural behavioral output. The elevated plus maze reliably assesses the memory of exploring a specific spatial location. The shorter latency to enter the "safe" space on the second exposure serves as the measure of intact cognitive functioning (the shorter, the better memory is) [Itoh J, et al. (1991) Eur J Pharmacol 194: 71-76]. Conversely, treatment with amnestic compounds like NMDA receptor antagonist MK-801 [Popik P. et al. Neuropsychopharmacology 28 (3):457-467, 2003; Popik P. et al. Neuropsychopharmacology 31 ( 1 ) : 160- 170, 2006] resulted in much less pronounced shortening of the latency to enter the "safe" compartment on second test. The maze was made of black Plexiglas and consisted of a central platform (5 x 5 cm) from which two open (5 x 30 cm) and two enclosed (5 x 30 x 15 cm) arms extended (Lister RG (1987) Psychopharmacology 92: 180-185). The apparatus was elevated to a height of 50 cm above the floor. During testing, location of the mouse was monitored through a closed circuit TV camera positioned directly above the apparatus. There was an ambient indirect lighting (~ 55 Lux), comprising of fluorescent bulbs. The floors were repeatedly washed and dried to keep the apparatus free of urine and feces.

Procedure

In the first test, mice were individually placed at the end of one open arm facing away from the central platform. The latency of each mouse to find and enter (with four paws) one of the enclosed arms was measured up to 90 s (test #1 latency) and mice were allowed to freely explore the apparatus for the following 150 sec. The second test was carried out 6 days later. As in the first test, mice were individually placed at the end of one open arm facing away from the central platform, and the latency to enter one of enclosed arms was measured again (test #2 latency). The apparatus was cleaned and dried after each animal. Before the first test, the mice were pretreated with distilled water (vehicle) or 0.1, 0.3 or 1 mg/kg of the tested compound or 2.5 mg/kg of sertindole (Sigma) used as a "positive" control. The tested compound was diluted in distilled water, while sertindole was diluted in a small amount of 0.1 N HCl and distilled water. All solutions were made fresh the day of experiment. The doses of the tested compound were based on earlier reports, the dose of sertindole was chosen based on our unpublished observations indicating its positive effect in another procedure measuring cognitive capacity of animals. Vehicle and the tested compound were administered IP 60 min before the test #1, and sertindole was administered PO, 2 h before the test #1. Both compounds were given in the volume of 10 ml/kg.

Effects of compound #9 on Incidental Learning in C57/B1 Mice - difference of Transfer Latencies before and after treatment are presented on Fig. 1.

Example 23

Biological Testing: Investigation of the protection from ketamine-induced cognitive deficit in the attentional set shifting task in rats by compound #9

Rationale

Antagonists of N-methyl-D-aspartate (NMDA) receptor (e.g. ketamine, phencyclidyne, dizocilpine) are widely used to model schizophrenia-like symptoms in laboratory animals, including those of cognitive functions. Inhibition of NMDA receptor may induce impairments specifically related to cognitive inflexibility as assessed in attentional set- shifting task (ASST) in rodents. For example, subchronic administration of phencyclidine (PCP) produced selective deficits in extradimensional (ED) set-shifting in rats (Rodefer et al, 2008, Neuropsychopharmacology 33: 2657-2666). Similarly, acute administration of ketamine specifically impaired rats' performance at the ED stage of ASST [Nikiforuk et al., 2009, Eur Neuropsychopharmacol. 2009 Sep 1. [Epub ahead of print]).

Although atypical antipsychotic medications appear to demonstrate a promising effect on cognitive functioning [Harvey and Keefe 2001, Am J Psychiatry 158: 176-184], several specific dysfunctions including cognitive inflexibility are not normalized by these treatments [Goldberg et al, 2007, Arch Gen Psychiatry 64: 1115-1122]. Nevertheless, animal studies have demonstrated that subchronic PCP-induced ED deficits were ameliorated by the atypical antipsychotic sertindole [Rodefer et al, 2008, Neuropsychopharmacology 33: 2657-2666; Goetghebeur and Dias 2009, Psychopharmacology (Berl) 202: 287-293]. Similarly, the 5-HT6 receptor antagonist SB 271046 attenuated PCP-induced set-shifting deficits. In contrast, either classical antipsychotic (haloperidol) or second-generation antipsychotics (clozapine, olanzapine and risperidone) were ineffective in treating set-shifting deficits.

Methods Subjects

Male Sprague-Dawley rats (Charles River, Germany) weighting 250-280 g on the arrival were used in this study. They were initially group-housed (five rats/cage) in a temperature (21±1°C) and humidity (40-50%) controlled colony room under 12/12-h light/dark cycle (lights on at 06:00 hours). Rats were allowed to acclimatize for at least 7 days before the start of the experimental procedure. For one week prior to the testing the rats were individually housed and mildly food deprived (15g of food pellets per day), with ad libitum access to water. Behavioral testing was carried out during the light phase of the light/dark cycle. The experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Apparatus

Testing was conducted in a modified wired rat housing cage (length x width x height: 42 x

32 x 22 cm) with a white plywood wall dividing half of the length of the cage into two sections. During the testing, one digging ceramic pot (internal diameter of 10.5 cm and the depth of 4 cm) was placed in each section. Each pot was defined by a pair of cues along with two stimulus dimensions. To mark each pot with a distinct odor, 5 μl of a flavoring essence (Dr. Oetker®, Poland) was applied on a piece of blotting paper fixed to the external rim prior to use. A different pot was used for each combination of the digging medium and odor, and only one odor was ever applied to a given pot. The bait (a one third of Honey Nut Cheerio, Nestle®) was placed on the bottom of the "positive" pot and was buried in the digging medium.

Procedure

The procedure was adopted from [Birrell and Brown 2000] and entailed three days for each rat:

Day 1, habituation: rats were habituated to the testing area and trained to dig in the pots filled with sawdust to retrieve the food reward. Once the rats had eaten the Cheerio, they were placed in the apparatus and were given three trials to retrieve reward from both of sawdust-filled baited pots. With each exposure, the bait was covered with an increasing amount of sawdust.

Day 2, training: the rats were trained on a series of simple discriminations (SD), to a criterion of six consecutive correct trials. For these trials, the rats had to learn to associate the food reward with an odor cue (e.g., arrack vs. orange, both pots filled with sawdust) and/or digging medium (e.g., plastic balls vs. pebbles, no odor). All rats were trained using the same pairs of stimuli. The positive and negative cues for each rat were presented randomly and equally. These training stimuli were not used again in later testing trials. Day 3, testing: in a single test session the rats performed a series of increasingly difficult discriminations. The first four trials at the beginning of each discrimination stage were a discovery period (not included in six trials to criteria), in which the rat was allowed to dig in both pots regardless of where it first began to dig. In the subsequent trials, an incorrect choice terminated the trial. Digging was defined as any distinct displacement of the digging media with either the paw or the nose; rat could investigate the digging pot by sniffing or touching without displacing material. Testing was continued at each stage until the rat reached the criterion of six consecutive correct trials, after which testing proceeded to the next stage. In the simple discrimination (SD), involving only one stimulus dimension, the pots differed along one of two dimensions (e.g., an odor or a digging medium). For the compound discrimination (CD), the second (irrelevant) dimension was introduced, but the correct and incorrect exemplars of the relevant dimension remained constant. For the reversal of this discrimination (Revl), the exemplars and relevant dimension were unchanged, but the previously correct exemplar was now incorrect and vice versa. The intra-dimensional (ID) shift was then presented, comprising new exemplars of both the relevant and irrelevant dimensions, with the relevant dimension remaining the same as previously. The ID discrimination was then reversed (Rev 2), so that formerly positive exemplar became the negative one. For extra-dimensional (ED) shift, a new pair of exemplars was again introduced, but this time a relevant dimension was also changed. Finally, the last stage was the reversal (Rev 3) of ED discrimination problem. The order of discriminations was always the same (i.e. SD, CD, Rev 1, ID, Rev 2, ED, and Rev3). For one-half of the animals, the discriminations began with an odor as a relevant dimension. For another half, they began with a digging medium as the salient cue. The exemplars were always presented in pairs and varied so that only one animal within each treatment group received the same combination. The following pairs of exemplars were used: Pair 1 : odor: lemon vs. almond, medium: cotton wool vs. crumpled tissue; Pair 2: odor: spicy vs. vanilla, medium: metallic filler vs. shredded paper; Pair 3: odor: rum vs. cream, medium: clay pellets vs. silk. The assignment of each exemplar in a pair as being positive or negative at a given stage and the left-right positioning of the pots in the test apparatus on each trial were randomized.

Drugs

Ketamine (10% aqueous solution (115.34 mg/ml) Biowet, Pulawy, Poland) was dissolved in sterile physiological saline. Sertindole (Sigma, Poland) was dissolved in a minimum amount of 0.1 M hydrochloric acid and diluted with saline. Ketamine (10 mg/kg, SC) was administered 75 min, whereas compound #9 (0.125, 0.25, 0.5 mg/kg, IP) and sertindole (2.5 mg/kg, PO) were administered 90 and 120 min, respectively, before testing. The drugs or physiological saline were administered in a volume of 1 ml/kg of body weight. Ketamine, at the doses used in the present study has been shown to affect behavioral assays sensitive to disruptive effects of PCP-like agents [Kos et al, 2006]. The dose of sertindole and administration schedule was based on the previous report showing its efficacy in alleviating PCP-induced impairments in ASST. Data analysis

The number of trials required to achieve the criterion of six consecutive correct responses as well as the number of errors made were recorded for each rat and for each discrimination problem. However, because the analyses of these measures yielded the same results, only the number of trials to criterion parameter is reported. Data were calculated using three- way mixed-design ANOVAs followed by the Newman-KeuFs post-hoc test. The alpha value was set at p<0.05 level. The data fulfilled criteria of normal distribution. Statistical analyses were performed with the use of Statistica 7.0 for Windows.

RESULTSAND CONCLUSION

In the present study compound #9 administered in a range of doses (0.125-0.5 mg/kg) significantly protected rats from ketamine-induced deficits in ED shift performance as compared with vehicle-ketamine conditions. The action of compound #9 was comparable to that of sertindole at a dose of 2.5 mg/kg (fig. 2). In sum, compound #9 ameliorated ketamine-induced deficits specifically related to cognitive inflexibility observed in schizophrenia.

Example 24

Biological Testing: Antiamnestic efficacy of compound #9 in passive avoidance paradigm in rats

Rationale

The aim of this experiment was to assess whether compound #9 could affect memory- disturbing effects on scopolamine, an unspecific antagonist of muscarinic cholinergic receptors. The amnestic effects of scopolamine are well known and their reversal has been used to ascertain potential memory facilitating effects of compounds. To address this issue the effects of three doses of compound #9 (0.125, 0.25 and 0.5 mg/kg, IP) on scopolamine- induced amnesia were determined in the passive avoidance test. The effects of sertindole (2.5 mg/kg, PO), an atypical antipsychotic on scopolamine-induced amnesia were also assessed, as requested by study sponsor.

Subjects

Experiments were performed on male Wistar Han rats (Charles River Laboratories, Germany), weighting 290-320 g. They were group-housed (five rats/cage) in a temperature (21±1°C) and humidity (40-50%) controlled colony room under 12/12-h light/dark cycle (lights on at 06:00 hours). Rats were allowed to acclimatize for at least 2 days before the start of the experimental procedure. Behavioral testing was carried out during the light phase of the light/dark cycle. The experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Apparatus and procedure

Passive avoidance test relies on the natural tendency of animals to escape dangerous places. Normal rats and mice placed on the well-lit and open area, having such possibility almost immediately escape into the dark and enclosed area. However, if they experience an unpleasant/stressful stimulus in the dark (initially "safe") compartment, on subsequent testing they show a passive avoidance: passive indicates an inhibition of a natural tendency, i.e., the lack of activity. This is the simplest test used in behavioral laboratories to assess learning and memory processes. The apparatus (30 x 30 x 30 cm) consisted of the dark chamber with the electrified grid floor (Coulbourn Instruments, USA). One of the walls of the dark compartment was equipped with an entrance (the guillotine door). An open platform (30 x 5 cm) protruded from the entrance, and served as the starting point for each subject. The platform was painted white and the apparatus was placed so that the platform was positioned 65 cm above the floor of experimental room. The open platform was illuminated by the electric bulb (25 W) placed 20 cm above the platform. Thus, the platform was well-lit and opened to the experimental room. Prior to each experimental session, each rat was placed separately in a holding cage and allowed to adapt to the cage for 15 min. The experiment consisted of the habituation, training and testing sessions. During the habituation session rats were placed on an open platform facing away from the open guillotine door. The latency to enter the darkened compartment was recorded by a stopwatch. Once the rat entered the dark compartment, the door was closed. After 10 s the rat was placed back into the holding cage. During the training session — carried out on the next day -- rats were once again placed individually on an open and well-lit platform and the latency to enter dark compartment was recorded. This procedure was repeated for 3 times. After the third trial, as soon as the rat had entered the dark compartment, the door was closed and the animal received the footshock delivered through the grid floor (1 mA for 3 s, Coulbourn Instruments, USA animal shocker). The rat was removed from the dark compartment ~ 10 s after the shock administration. The testing session consisted of a single trial without the footshock and was performed 24 h after the training. The latency to enter the dark compartment was measured. An upper cutoff time of 600 s was set. Drugs

Scopolamine (Sigma-Aldrich, 1 mg/kg SC) or its vehicle (0.9% physiological saline) was administered 30 min prior the shock trial. Sertindole (Sigma-Aldrich, 2.5 mg/kg PO) was administered 120 min prior the shock trial, compound #9 (0.125, 0.25 or 0.5 mg/kg IP) was administered 60 min prior the shock trial.

Data analysis

Since the data did not display normal distribution, the results (the latencies to enter the dark compartment during the testing session) are presented as median +/- interquartille range. Statistics involved Kruskal-Wallis non-parametric one-way ANOVA followed by Dunn's post-hoc comparison tests.

RESULTSAND CONCLUSION

During the initial training, all the rats needed less than 90 s to enter the dark compartment. Twenty-four hours after the electric shock training trial, most vehiclepretreated rats did not re-enter the darkened compartment. The median latency was increased to 600 s, indicating that the rats acquired the memory of the aversive stimulus associated with the darkened compartment. Pretreatment with scopolamine (1 mg/kg) significantly reduced the re-entry latency (to 256.5 s) as compared with vehicle control (p<0.05). compound #9 (at 0.5 mg/kg, but not other doses) protected rats from scopolamine-induced amnesia, as evidenced by a significant (p<0.05) difference between vehicle-scopolamine and compound #9-scopolamine treated groups test entrance latencies. Sertindole administered at the dose of 2.5 mg/kg did not affect the scopolamine-induced memory deficit.

Effects of compound #9 on scopolamine-induced learning deficit in passive avoidance test in rats are presented on Fig. 3.

CONLUSION

It was found that compound #9 at the only dose of 0.5 mg/kg reversed the amnestic effects of scopolamine on passive avoidance acquisition. This result demonstrates that compound #9 may exhibit the memory-enhancing effects in certain behavioral paradigms, probably mediated by cholinergic receptors.