EA040399B1 - COMPOUNDS FOR THE TREATMENT OF EPILEPSY, NEURODEGENERATIVE DISORDERS AND OTHER CNS DISORDERS - Google Patents

Description

Field of invention

This invention relates to novel compounds and novel compounds for use as a medicament for the prevention or treatment of epilepsy, neurodegenerative disorders and other CNS disorders. The present invention also relates to the use of said compounds for the manufacture of medicaments useful in the treatment of epilepsy. This invention also relates to methods for preparing these new compounds.

State of the art

Epilepsy is a neurological disorder in which affected patients are predisposed to experiencing epileptic seizures that can range from uncontrollable twitching to mild, transient loss of consciousness. Epilepsy has a prevalence of about 1% and is independent of socioeconomic status, age, or gender. The underlying causes of epilepsy (epileptogenesis) are varied and include both genetic and non-genetic risk factors (such as stroke or infection).

One mechanism underlying this disorder involves increased activity of voltage-gated calcium channels (VGCC), leading to increased neurotransmitter release, ^Ca2+ dysomeostasis, and neuronal hyperactivity, which may underlie, at least in part, epileptic seizures, TAU- phosphorylation and subsequent degeneration of neurons and death of neurons.

TAU is an intracellular protein capable of binding and therefore stabilizing and determining the structure and function of microtubules. In addition to this physiological function, TAU also plays a direct role in disorders characterized by insoluble tau aggregates or polymers, which are formed by the self-polymerization of tau monomers. The exact molecular mechanisms involved in TAU aggregation are unknown, but appear to involve (partial) denaturation or disruption of the formation of the tertiary structure of the TAU protein in conformations with a high propensity to self-organize into higher order structures.

An important aspect of TAU aggregation is its inherent cytotoxicity, which reduces cell integrity or even initiates cell death. One important aspect of toxic TAU aggregation in disease is the hyperphosphorylation of certain amino acid residues of the TAU protein. Hyperphosphorylation of TAU appears to facilitate the process of cytotoxic aggregation. Some of the kinases directly or indirectly involved in TAU hyperphosphorylation are mitogen-activated kinases ERK1 and/or ERK2. Intracellular Ca ²⁺ is an important trigger for the activation of ERK1 and/or ERK2 activity, and thus Ca ²⁺ can enhance toxic TAU aggregation.

Despite the fact that there are currently many treatment options for epilepsy, about 30% of patients are not treatable, indicating that there is a great medical need. In addition, existing AEDs (anti-epileptic drugs) have side effects such as cognitive impairment, which has a large negative impact on the quality of life of patients undergoing treatment. Finally, current treatments are purely symptomatic and do not delay or stop epileptogenesis or neuronal degeneration after a primary stroke.

Thus, there is a need in the art to develop improved, more potent therapeutic drugs that target the underlying molecular mechanisms of epilepsy, in specific treatments that reduce neuronal hyperactivity with neuroprotective properties and/or without compromising cognitive function.

The essence of the invention

In a first aspect of the present invention, there is provided a compound of formula (I), or a tautomer thereof, o

Where

R ¹ is selected from the group consisting of hydrogen and F; R ² is selected from the group consisting of hydrogen and F; R ³ is selected from the group consisting of hydrogen and F; R ⁴ is selected from the group consisting of hydrogen and F; R ⁵ is selected from the group consisting of hydrogen and F; provided that at least one of R ¹ , R ² , R ³ , R ⁴ or R ⁵ is not hydrogen; provided that said compound of formula (I) is not N-[2-(5fluoro-1H-indol-3-yl)ethyl]-5-[(3-fluorophenyl)methyl]isoxazole-3-carboxamide, or pharmaceutically acceptable salt.

- 1 040399

In another aspect, the compound has any of formulas (II), (III), (IV), (V) or (VI):

where R ¹ , R ² , R ³ and R ⁴ have the same meanings as defined in paragraph 1.

In another aspect, R ² , R ³ , R ⁴ and R ⁵ are hydrogen, and R ¹ is F.

In another aspect, R ¹ , R ² , R ⁴ and R ⁵ are hydrogen and R ³ is F.

In another aspect, R ² , R ⁴ and R ⁵ are hydrogen and R ¹ and R ³ are independently selected from the group consisting of hydrogen and F.

In another aspect, R ¹ , R ⁴ and R ⁵ are hydrogen and R ² and R ³ are independently selected from the group consisting of hydrogen and F.

In another aspect, R ¹ , R ³ and R ⁵ are hydrogen and R ² and R ⁴ are independently selected from the group consisting of hydrogen and F.

In another aspect, R ³ , R ⁴ and R ⁵ are hydrogen and R ¹ and R ² are independently selected from the group consisting of hydrogen and F.

In another aspect, R ² , R ³ , and R ⁵ are hydrogen and R ¹ and R ⁴ are independently selected from the group consisting of hydrogen and F.

In another aspect, the compound is selected from the group consisting of

-[(2,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazol-3-carboxamide;

N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(4-fluorophenyl)methyl]isoxazole-3-carboxamide;

-[(2,3-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazol-3-carboxamide;

5-[(2,5-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide;

N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(2-fluorophenyl)methyl]isoxazole-3-carboxamide;

5-[(3,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide;

5-[(3,5-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide.

In another aspect, a pharmaceutical composition is provided comprising one or more pharmaceutical auxiliary compounds and a therapeutically effective amount of a compound according to any one of claims 1-10 and a pharmaceutically acceptable salt thereof. In another aspect, the use of a compound or pharmaceutical composition as a drug is provided.

In another aspect, the use of a compound or pharmaceutical composition is provided as a medicament for the prevention and/or treatment of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis, drug withdrawal, tobacco, memory loss, dementia, schizophrenia. and panic.

In another aspect, the epilepsy is selected from the group consisting of refractory epilepsy, syndrome

- 2 040399

West, Dawes syndrome, benign rolandic epilepsy, Rasmussen syndrome, Lennox-Gastaut syndrome, Sturge-Weber syndrome, juvenile myoclonic epilepsy, absence epilepsy in children, epilepsy associated with idiopathic localization, temporal lobe epilepsy, partial seizures, simple partial seizures, tonic seizures, tonic-clonic seizures, clonic seizures, myoclonic seizures, absence seizures and atonic seizures, frontal lobe epilepsy, epilepsy with grand mal seizures, generalized epilepsy, idiopathic epilepsy, symptomatic epilepsy and cryptogenic epilepsy.

In another aspect, the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, diffuse Lewy body disease, amyotrophic lateral sclerosis, Niemann-Pick disease, Hallervorden-Spatz syndrome, Down's syndrome, neuroaxonal dystrophy, multiple system atrophy, Huntington's disease, frontal -temporal lobar degeneration (FTLD), cystic fibrosis and Creutzfeldt-Jakob disease.

Hereinafter, the present invention will be further described. In the following parts, various aspects of the present invention are defined in more detail. Each aspect thus defined may be combined with any other aspect or aspects, unless expressly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The independent and dependent claims set forth specific and preferred features of the present invention. Features of dependent claims may be combined with features of independent or other dependent claims, as appropriate.

Brief description of graphic materials

The following description of the drawings of specific embodiments of the present invention is merely exemplary in nature and is not intended to limit these statements, their application or use.

In FIG. 1, Part A is a graph showing the percentage of LDH infiltrated into the medium of each of M17-TAU P301L (P301L TAU) cells and M17-3.1 cells (vector) in the presence and absence of all-trans retinoic acid (RA). Part B is a graph showing the relative levels of cytosolic calcium in M17-TAU P301L cells in the presence and absence of retinoic acid (RA). **** indicate p<0.0001.

In FIG. 2 is a graph showing the percentage of media-infiltrated LDH activity of M17-TAU P301L cells in the presence of RA as a function of the concentration of isradipine added to the cells.

In FIG. 3 is a graph showing relative levels of cytosolic calcium in M17-TAU P301L cells after exposure to retinoic acid in the presence of vehicle or exemplary compound 6 at 625 nM. *** indicate p<0.001.

In FIG. 4 is a graph showing the relative number of viable primary neurons after interaction with diffusible amyloid-derived ligands (ADDL) in the presence of vehicle or exemplary compound 6. * indicates p<0.05 and ** indicates p<0.01.

In FIG. 5 is a graph showing cytosolic Ca ²⁺ levels in primary neurons after depolarization with 45 mM KCl in the presence of vehicle or exemplary compound 6. **** indicate p<0.0001.

In FIG. 6 is a graph showing the quantification of pathological phosphorylated TAU species determined by Western blotting of APP transgenic mouse brain extracts treated with vehicle or exemplary compound 6. The graphs show mean normalized TAU±SEM signals. ** indicate p<0.01 relative to animals treated with vehicle. TAU signals were generated using antibodies to: (A) AT8 p-TAU, indicating an antibody recognizing p-TAU phosphorylated at serine 202 and threonine 205; (B) AD2 p-TAU indicates an antibody recognizing p-TAU phosphorylated at serines 396 and 404; (C) TAU phosphorylated at T231; (D) TAU phosphorylated at S262.

In FIG. 7 shows the result of the Morris water maze test on APP transgenic mice treated with exemplary compound 6. (A) shows the length of the search path during the learning phase. The P value refers to APP transgenic mice treated with exemplary Compound 6 versus vehicle. Part B shows the ring crossing index (relative frequency of crossing the area of the imaginary platform) from the test run. ** indicates p<0.01.

In FIG. 8 is a graph showing wild-type and tgAPP mouse neuron firing rate as a function of electrical stimulation (step intensity (pA)) for exemplary compound 6. * and **** indicate p<0.05 and p<0.0001, respectively, wild-type compared to transgenic APP mice. # indicates p<0.05 of vehicle-treated APP transgenic mice compared to Compound 6.

In FIG. 9 is a graph showing post-hyperpolarization (AHP) single action potential amplitudes from fixed somatic current recordings using mouse brain sections incubated with vehicle or Compound 6. * indicates p<0.05.

Detailed Description of the Invention

Before describing the present invention, it should be understood that the present invention is not limited to the particular processes, methods, and compounds described, as such processes, methods, and compounds may, of course, vary. It should also be understood that the terminology used herein is not intended to be limiting, as the scope of this invention will only be limited by the appended claims.

When describing the compounds and methods of this invention, the terms used should be construed in accordance with the following definitions, unless the context requires otherwise.

As used in the description and the appended claims, the singular forms include both singular and plural references unless the context clearly dictates otherwise. For example, connection means one connection or more than one connection.

The terms comprising, contains, and consisting of, in the context of this document, are synonymous with including, includes, or containing, contains, and are inclusive or open-ended, and do not exclude additional, not listed, members, elements, or steps of the method. The terms containing, contains, and consisting of also include the term consisting of.

The term about in the context of this document, when referring to a measurable value such as a parameter, magnitude, time duration, and the like, is intended to cover variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less. and even more preferably ±0.1% or less of the specified value, if such changes are suitable for the disclosed invention. It should be understood that the meaning to which the modifier about refers is also specifically and preferably disclosed.

The term and/or, in the context of this document, when used in a list of two or more features means that any of the listed features can be used by itself or any combination of two or more of the listed features can be used. For example, if a list is described as containing groups A, B, and/or C, then the list may only contain A; only in; only with; combined A and B; in combination A and C, in combination B and C; or a combination of A, B and C.

The listing of numerical ranges by endpoints includes all integers and, if necessary, fractions referred to this range (for example, from 1 to 5, may include 1, 2, 3, 4, when referring to, for example, a number of elements, and may also include 1.5, 2, 2.75 and 3.80 when referring to measurements, for example). The endpoint enumeration also includes the endpoint values themselves (for example, 1.0 to 5.0 includes both 1.0 and 5.0). Any numerical range given in this document is intended to include all subranges included therein.

Reference throughout this specification to one embodiment or embodiment means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the occurrence of phrases in one embodiment or in an embodiment at different places in this specification does not necessarily refer to the same embodiment, but such an occurrence is possible. In addition, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure, in one or more embodiments. In addition, while some of the embodiments described herein include some but not other features included in other embodiments, combinations of features of different implementations should be within the scope of the invention and form different implementations, as will be appreciated by those skilled in the art. the field of technology. For example, in the following claims, any of the claimed embodiments may be used in any combination.

Unless otherwise indicated, all terms used in the disclosure of this invention, including technical and scientific terms, have the meaning generally accepted by a person skilled in the art to which this invention pertains. By way of further guidance, definitions of terms used in this specification are included for a better understanding of the gist of this invention.

When describing this invention, the terms used are to be construed in accordance with the following definitions, unless the context requires otherwise.

The terms described above and other terms used in the description are well understood by those skilled in the art.

Whenever used in this invention, the term compounds of this invention or a similar term is intended to include compounds of general formula (I) and any subgroup thereof. This term also refers to the compounds shown in table. 1, and their derivatives, Noxides, salts, solvates, hydrates, tautomeric forms, analogs, prodrugs, esters and metabolites, as well as their quaternized nitrogen-containing analogs. The Noxide forms of these compounds are intended to include compounds in which one or more nitrogen atoms

- 4 040399 oxidized to the so-called N-oxide.

The following are preferred provisions (features) and embodiments of the compounds and methods of this invention. Each statement and embodiments of the present invention thus defined may be combined with any other statement and/or implementation options, unless otherwise indicated. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. However, this invention is specifically covered by any or any combination of one or more of the following statements 1-15 and implementation options with any other aspects and/or implementation option.

1. The compound of formula (I) or its tautomer,

Where,

R ¹ is selected from the group consisting of hydrogen and F;

R ² is selected from the group consisting of hydrogen and F;

R ³ is selected from the group consisting of hydrogen and F;

R ⁴ is selected from the group consisting of hydrogen and F;

R ⁵ is selected from the group consisting of hydrogen and F;

provided that at least one of R ¹ , R ² , R ³ , R ⁴ or R ⁵ is not hydrogen; provided that said compound of formula (I) is not N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(3fluorophenyl)methyl]isoxazole-3-carboxamide, or a pharmaceutically acceptable salt.

2. The compound according to claim 1, having any of the formulas (II), (III), (IV), (V) or (VI):

- 5 040399

where R ¹ , R ² , R ³ and R ⁴ have the same meanings as defined in paragraph 1.

3. The compound according to claim 1 or 2, where R ² , R ³ , R ⁴ and R ⁵ are hydrogen, and R ¹ is F.

4. A compound according to claim 1 or 2, where R ¹ , R ² , R ⁴ and R ⁵ are hydrogen and R ³ is F.

5. A compound according to claim 1 or 2, wherein R ² , R ⁴ and R ⁵ are hydrogen and R ¹ and R ³ are independently selected from the group consisting of hydrogen and F.

6. A compound according to claim 1 or 2, wherein R ¹ , R ⁴ and R ⁵ are hydrogen and R ² and R ³ are independently selected from the group consisting of hydrogen and F.

7. A compound according to claim 1 or 2, wherein R ¹ , R ³ and R ⁵ are hydrogen and R ² and R ⁴ are independently selected from the group consisting of hydrogen and F.

8. A compound according to claim 1 or 2, wherein R ³ , R ⁴ and R ⁵ are hydrogen and R ¹ and R ² are independently selected from the group consisting of hydrogen and F.

9. A compound according to claim 1 or 2, wherein R ² , R ³ , and R ⁵ are hydrogen and R ¹ and R ⁴ are independently selected from the group consisting of hydrogen and F.

10. Compound according to claim 1, selected from the group consisting of

-[(2,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazol-3-carboxamide;

N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(4-fluorophenyl)methyl]isoxazole-3-carboxamide;

5-[(2,3-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide;

5-[(2,5-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide;

N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(2-fluorophenyl)methyl]isoxazole-3-carboxamide;

5-[(3,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide;

5-[(3,5-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide.

11. A pharmaceutical composition containing one or more pharmaceutical auxiliary compounds and a therapeutically effective amount of a compound according to any one of claims 1-10 and a pharmaceutically acceptable salt thereof.

12. The use of a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 as a drug.

13. The use of a compound according to any of claims 1-10 or a pharmaceutical composition according to claim 11 as a drug for the prevention and/or treatment of epilepsy, neuro-

- 6 040399 degenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis, drug withdrawal, tobacco, memory loss, dementia, schizophrenia and panic.

14. The use of claim 13 wherein the epilepsy is selected from the group consisting of refractory epilepsy, West syndrome, Dawes syndrome, benign Rolandic epilepsy, Rasmussen syndrome, Lennox-Gastaut syndrome, Sturge-Weber syndrome, juvenile myoclonic epilepsy, childhood absence epilepsy, epilepsy associated with idiopathic localization, temporal lobe epilepsy, partial seizures, simple partial seizures, tonic seizures, tonic-clonic seizures, clonic seizures, myoclonic seizures, absence seizures and atonic seizures, frontal lobe epilepsy, epilepsy with grand mal seizures, generalized epilepsy, idiopathic epilepsy, symptomatic epilepsy and cryptogenic epilepsy.

15. The use of claim 13 wherein the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, diffuse Lewy body disease, amyotrophic lateral sclerosis, Niemann-Pick disease, Hallervorden-Spatz syndrome, Down's syndrome, neuroaxonal dystrophy, multiple system atrophy , Huntington's disease, frontotemporal lobar degeneration (FTLD), cystic fibrosis, and Creutzfeldt-Jakob disease.

The compounds of this invention may be in the form of salts, preferably pharmaceutically acceptable salts, as generally described below. Some preferred, but non-limiting examples of suitable pharmaceutically acceptable organic and/or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference to the prior art mentioned below).

In case the compounds of the present invention contain an acid group as well as a basic group, the compounds of the present invention may also form internal salts, and such compounds are within the scope of the present invention. In case the compounds of this invention contain a hydrogen-donor heteroatom (eg NH), the present invention also provides salts and/or isomers formed by transferring said hydrogen atom to the main group or atom of the molecule.

Pharmaceutically acceptable salts of the compounds of formula (I) and any subgroups thereof include acid and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipine, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzat, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrophosphate/dihydrogen phosphate, pyroglutamate, sucrose, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate. Suitable base salts are formed from bases which form non-toxic salts. Examples include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc. Hemi-salts of acids and bases, such as polysulphate and hemicalcium salts, can also be formed. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference.

The compounds of this invention may exist in complex solid states ranging from fully amorphous to fully crystalline. The term amorphous refers to a state in which a substance has no long-range order at the molecular level and, depending on temperature, may exhibit the physical properties of a solid or liquid. Usually, such substances do not give characteristic X-ray patterns and, although they exhibit the properties of a solid, are more formally described as a liquid. When heated, a change in the properties of a solid to a liquid occurs, which is characterized by a change in state, usually of the second order (transition to a glassy state). The term crystalline refers to a solid phase in which the material has an ordered internal structure at the molecular level and produces a characteristic X-ray pattern with defined peaks. Such materials, when sufficiently heated, will also exhibit the properties of a liquid, but the transition from solid to liquid is characterized by a phase change, usually of the first order (melting point).

Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of the following methods: (i) by contacting a compound of formula (I) with the desired acid; (ii) by bringing into contact the compounds of formula (I) with the desired base; (iii) by removing a labile acid or alkaline protecting group from a suitable precursor of a compound of formula (I) or by ring-opening a suitable cyclic precursor, eg lactone or lactam, using the desired acid; or (iv) by converting one salt of the compound of formula (I) into another by contacting with a suitable acid or using a suitable ion exchange column.

- 7 040399

All of these reactions are usually carried out in solution. The salt may precipitate out of solution and may be collected by filtration or may be recovered by evaporating the solvent. The degree of ionization in a salt can vary from fully ionized to almost non-ionized.

The compounds of this invention may also exist in unsolvated and solvated forms. The term solvate, in the context of this document, is used to describe a molecular complex comprising a compound of this invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol. The term hydrate is used when said solvent is water.

The currently accepted classification system for organic hydrates is that which defines an isolated site, channel or hydrates coordinated by metal ions - see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Britain, Marcel Dekker, 1995), incorporated herein in as a link. Isolated site hydrates are hydrates in which water molecules are isolated from direct contact with each other by bringing organic molecules into contact. In channel hydrates, water molecules lie in lattice channels where they sit next to other water molecules. In hydrates coordinated by metal ions, water molecules are bound to the metal ion.

If the solvent or water is closely related, the complex will have a well-defined stoichiometry, regardless of humidity. However, when the solvent or water is loosely bound, as in channel solvates and hygroscopic compounds, the water/solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

Also included within the scope of this invention are multicomponent complexes (other than salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and cocrystals. The latter are usually defined as crystalline complexes of neutral molecular components that are linked to each other through non-covalent interactions, but can also be a complex of a neutral molecule with a salt. Cocrystals can be obtained by melt crystallization, recrystallization from solvents, or physical grinding of the components together - see Chem Commun, 17, 1889-1896, O. Almarsson and M.J. Zaworotko (2004), incorporated herein by reference. For a general overview of multicomponent complexes, see J Pharm Sci, 64 (8), 12691288, by Haleblian (August 1975), incorporated herein by reference.

The compounds of this invention may also exist in a mesomorphic state (mesophase or liquid crystal) when exposed to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (melt or solution). Mesomorphism resulting from a change in temperature is described as thermotropic, while mesomorphism resulting from the addition of a second component such as water or another solvent is described as lyotropic. Compounds that can form lyotropic mesophases are described as amphiphilic and consist of molecules that are ionic (such as -COO ^- Na ⁺ , -COO-K + , or -SO3 ^- Na ⁺ ) or non-ionic (such as -N ^- N ⁺ ( CH3) ₃ ) polar head group. For more information, see Crystals and the Polarizing Microscope by NH Hartshorne and A. Stuart, ^4th Edition (Edward Arnold, 1970), incorporated herein by reference.

All references to compounds of formula (I) or any subgroups thereof include references to salts, solvates, multicomponent complexes and liquid crystals thereof, as well as solvates, multicomponent complexes and liquid crystals of their salts.

The compounds of this invention include compounds of formula (I) or any subgroups thereof as defined above, including all polymorphs and their crystal structures, their prodrugs and isomers (including optical, geometric and tautomeric isomers) as defined below, and isotopically labeled compounds of formula ( I).

Furthermore, while pharmaceutically acceptable salts are generally preferred with respect to salts of the compounds of this invention, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts which may, for example, be used in the isolation and/or purification of the compounds of this invention. invention.

This invention also generally covers all pharmaceutically acceptable prodrugs or predrugs of the compounds of formula (I) or any of their subgroups, for which a general reference is made to the prior art, cited below.

The term prodrug as used herein means pharmacologically acceptable derivatives such as esters, amides and phosphates such that the resulting in vivo biotransformation product of the derivative is the active drug. Reference to Goodman and Gilman (The Pharmacological Basis of Therapeutics, 8th Ed, McGraw-Hill, Int. Ed. 1992, Biotransformation of Drugs, p 13-15) describing prodrugs is generally included. Prodrugs of the compounds of this invention can be obtained by modifying the functional groups present

- 8 040399 in said component, such that the modifications are cleaved, either during conventional manipulations or in vivo, to the parent component. Typical examples of prodrugs are described, for example, in WO 99/33795, WO 99/33815, WO 99/33793 and WO 99/33792, all of which are incorporated herein by reference. Prodrugs are characterized by increased bioavailability and are readily metabolized to active inhibitors in vivo. The term prodrug, as used herein, means any compound that will be modified to form a drug species, the modification being either inside or outside the body, or before or after the predrug reaches the area of the body where the drug is to be used. facilities.

The scope of this invention includes all geometric isomers and tautomeric forms of the compounds of formula (I) or any of their subgroups, including compounds exhibiting more than one type of isomerism, and mixtures of one or more of them. Also included are acid addition or base salts in which the counterion is optically active, such as d-lactate or l-lysine, or a racemate, such as dl-tartrate or dl-arginine.

The cis/trans isomers can be separated using conventional methods well known to those skilled in the art, such as chromatography and fractional crystallization.

Conventional methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC).

Compounds of formula (I), or any subgroups thereof, may be prepared as described in the experimental section below using methods and reactions well known to those skilled in the art.

This invention also provides a pharmaceutical composition containing at least one compound according to this invention. The invention also provides a pharmaceutical composition comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier, adjuvant or diluent.

In some embodiments, this invention relates to the use of at least one compound of formula (I) or any subgroups thereof in (preparing a composition for) the prevention and/or treatment of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis , drug withdrawal, tobacco withdrawal, memory loss, dementia, schizophrenia and panic attacks.

In some embodiments, this invention relates to a method for the prevention and/or treatment of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis, drug withdrawal, tobacco, memory loss, dementia, schizophrenia, panic, comprising administering to a subject in need thereof, an effective amount of at least one compound of formula (I) or any of its subgroups, or a pharmaceutical composition containing said at least one compound of formula (I) or any of its subgroups.

In some embodiments, this invention relates to the use of at least one compound of formula (I) or any subgroups thereof in (preparing a composition for) the prevention and/or treatment of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis , drug withdrawal, tobacco withdrawal, memory loss, dementia, schizophrenia, panic attacks; more preferably epilepsy and neurodegenerative disorders, Parkinson's disease, Alzheimer's disease, Lewy body dementia, amyotrophic lateral sclerosis, Niemann-Pick disease, Hallervorden-Spatz disease, Down's syndrome, neuroaxonal dystrophy, multiple system atrophy, Huntington's disease, frontotemporal lobar degeneration (FTLD) ), multiple system atrophy, cystic fibrosis, Creutzfeldt-Jakob disease; even more preferably for the prevention and/or treatment of epilepsy and/or Alzheimer's disease.

In some embodiments, this invention relates to the use of at least one compound of formula (I) or any subgroups thereof in (preparing a composition for) the prevention and/or treatment of epilepsy, preferably epilepsy selected from the group consisting of refractory epilepsy, West syndrome , Dawes syndrome, benign rolandic epilepsy, Rasmussen syndrome, Lennox-Gastaut syndrome, West syndrome, Sterg-Weber syndrome, juvenile myoclonic epilepsy, childhood absence epilepsy, idiopathic localization-related epilepsy, temporal lobe epilepsy, partial seizures, simple partial seizure, tonic convulsions, tonic -clonic seizures, clonic seizures, myoclonic seizures, petit mal seizures and atonic seizures, frontal lobe epilepsy, grand mal seizure epilepsy, generalized epilepsy, idiopathic epilepsy, symptomatic epilepsy and cryptogenic epilepsy.

- 9 040399

In some embodiments, this invention relates to the use of at least one compound of formula (I) or any of its subgroups in (preparing a composition for) the prevention and/or treatment of pain disorders, and preferably pain disorders selected from the group consisting of acute pain, persistent pain, chronic pain, inflammatory pain and neuropathic pain.

In some embodiments, this invention relates to the use of at least one compound of formula (I) or any of its subgroups in (preparing a composition for) the prevention and/or treatment of anxiety disorders, and preferably anxiety disorders selected from the group consisting of panic attack, agoraphobia or specific phobia, obsessive-compulsive disorder, post-traumatic stress disorder, acute stress disorder, general anxiety disorder, eating disorder, substance-induced anxiety disorder, and unspecified anxiety disorder.

The term subject in the context of this document refers to a mammal. The subject is preferably a human, but can also be livestock, laboratory or domestic animals.

In some embodiments, at least one compound of formula (I) is used (for the preparation of a medicament) for the prevention and/or treatment of a disease selected from the group consisting of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis, drug withdrawal, tobacco, memory loss, dementia, schizophrenia and panic attacks, and/or to prevent, treat and/or alleviate complications and/or symptoms associated therewith.

The term effective amount, as used herein, means the amount of a drug or pharmaceutical that elicits a biological or medical response in a tissue, system, animal, or human, as requested by, for example, a researcher or clinician.

The term "therapeutically effective amount" means any amount that, when compared with a corresponding subject who has not received such an amount, results in an improvement in the treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a reduction in the rate of progression of the disease or disorder. The term also includes amounts effective to improve normal physiological function.

For use in therapy, therapeutically effective amounts of a compound of formula (I), as well as its tautomers, salts, hydrates, or solvates, may be administered as the crude chemical. In addition, the active ingredient may be presented as a pharmaceutical composition.

Accordingly, the invention also provides pharmaceutical compositions which comprise effective amounts of compounds of formula (I) or tautomers, salts, hydrates, solvates or prodrugs thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. The compounds of formula (I) or their tautomers, salts, hydrates or solvates are as described herein.

The compounds of this invention may be administered as the sole active ingredient or together, ie in fixed or free combination, with other therapeutic agents used in clinical practice for the treatment of the diseases listed above.

The compounds of this invention and other pharmaceutically active agents may be administered together or separately, and when administered separately, the administration may occur simultaneously or sequentially, in any order. The amounts of the compounds of this invention and other pharmaceutically active agent(s) and the relative timing of administration will be chosen to achieve the desired combined therapeutic effect. Administration in combination of a compound of formula (I) or its tautomer, salt, hydrate or solvate with other therapeutic agents may be in combination by administering simultaneously in (1) one pharmaceutical composition containing both compounds; or (2) a separate pharmaceutical composition, each containing one of the compounds. Alternatively, the combination may be administered separately sequentially, with one therapeutic agent administered first and the other second, or vice versa. Such sequential administration may be close in time or remote in time.

For pharmaceutical use, the compounds of this invention can be used in the form of the free acid or base, and/or in the form of a pharmaceutically acceptable acid addition salt and/or base (for example, prepared with a non-toxic organic or inorganic acid or base), in the form of a hydrate, solvate and /or complex, and/or in the form of a prodrug or predrug, such as an ester. As used herein, unless otherwise indicated, the term solvate includes any combination that may be formed by a compound of this invention with a suitable inorganic solvent (e.g., hydrates) or organic solvent such as, but not limited to, alcohols, ketones, esters. etc. Such salts, hydrates, solvates, etc. and their receipt will be clear to a person skilled in the art;

- 10 040399 refers, for example, to salts, hydrates, solvates, etc., described in US patent No. 6372778,

6369086,6369087 and 6372733.

Pharmaceutically acceptable salts of the compounds of this invention, ie in the form of water-soluble or oil-soluble or dispersible products, include the usual non-toxic or quaternary ammonium salts, which are formed, for example, from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hydrochloride, hexanoate. , hydrobromide, hydroiodide, 2hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Basic salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as salts of dicyclohexylamine, N-methyl-D-glucamine, and salts with amino acids. such as arginine, lysine and so on. In addition, the basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides, and others. Other pharmaceutically acceptable salts include the sulfuric acid salt of the ethanolate and sulfates.

Generally, for pharmaceutical use, the compounds of this invention may be formulated as a pharmaceutical preparation containing at least one compound of this invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more others. pharmaceutically active compounds.

By non-limiting examples, such a composition may be in a form suitable for oral administration, for parenteral administration (such as intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including in the eye), for administration by inhalation, skin patch, implant , suppository, etc. Such suitable administration forms, which may be solid, semi-solid, or liquid, depending on the route of administration, as well as methods and carriers, diluents, and excipients for use in their preparation, will be understood by those skilled in the art; once again, reference is made to, for example, US 6372778, 6369086, 6369087 and 6372733, as well as standard references, such as the latest edition of Remington's Pharmaceutical Sciences.

Some preferred but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, drops, sterile solutions. for injection and sterile packaged powders (which are usually reconstituted before use) for bolus administration and/or for continuous administration, which can be formulated with carriers, excipients and diluents which are themselves suitable for such compositions, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl propyl hydroxybenzoates, talc, magnesium stearate, food grade oils, vegetable oils and mineral oils, or suitable mixtures thereof. The compositions may optionally contain other pharmaceutically active substances (which may or may not result in a synergistic effect with the compounds of this invention) and other substances that are commonly used in pharmaceutical compositions, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents , disintegrants, bulking agents, fillers, preservatives, sweeteners, flavors, flow regulators, release agents, etc. The compositions can also be formulated to provide rapid, sustained or modified release of the active compound(s) contained therein, for example using liposomes or hydrophilic polymer matrices based on natural gels or synthetic polymers. In order to increase the solubility and/or stability of the compounds of the pharmaceutical composition of this invention, it may be advantageous to use α-, β- or γ-cyclodextrins or derivatives thereof. In addition, co-solvents such as alcohols can improve the solubility and/or stability of the compounds. In the preparation of aqueous compositions, the addition of salts of the compounds of this invention may be more suitable due to their increased solubility in water.

Suitable cyclodextrins are α-, β- or γ-cyclodextrins (CD) or their ethers and mixed ethers, wherein one or more hydroxyl groups of the anhydroglucose units of the cyclodextrin are substituted with alkyl, in particular methyl, ethyl or isopropyl, for example methylated random β-CD manner; hydroxyalkyl, in particular hydroxyethyl, hydroxypropyl or hydroxybutyl; carboxyalkyl, in particular carboxymethyl or carboxyethyl; alkylcarbonyl, in particular acetyl; alkoxycarbonylalkyl or carboxyalkoxyalkyl, especially carboxymethoxypropyl or carboxyethoxypropyl; alkylcarbonyloxyalkyl, especially 2acetyloxypropyl. Particularly noteworthy as complexing agents and/or solubilizers are β-CD, randomly methylated β-CD, 2,6-dimethyl-β-CD, 2-hydroxyethyl-in-CO, 2hydroxyethyl-γ-CD, 2-hydroxypropyl-γ- CD and (2-carboxymethoxy)propyl-β-CD and in particular 2hydroxypropyl-v-CD (2-HP-e-CD). The term mixed ether denotes cyclodextrin derivatives in which at least two hydroxyl groups of the cyclodextrin are esterified with different groups, such as, for example, hydroxypropyl and hydroxyethyl. An interesting way of formulating compounds in combination with a cyclodextrin or derivative thereof has been described in EP-A-721331. Although the compositions described herein contain antifungal active ingredients, they are equally interesting for formulating compounds. These compositions can also be made more palatable by adding pharmaceutically acceptable sweeteners and/or flavors. In particular, this invention provides a pharmaceutical composition containing an effective amount of a compound of this invention with a pharmaceutically acceptable cyclodextrin. The invention also provides cyclodextrin complexes consisting of a compound of the invention and a cyclodextrin.

Preference is given to compositions, formulations (and carriers, excipients, diluents, etc. for their use), routes of administration, etc., which are known per se, such as those described in US-4997834 and EP-A -0370498.

More specifically, the compositions may be formulated as a pharmaceutical composition containing a therapeutically effective amount of particles consisting of a solid dispersion of the compounds of the invention and one or more pharmaceutically acceptable water-soluble polymers.

The term solid dispersion defines a system in a solid state (as opposed to liquid or gaseous state) containing at least two components, with one component more or less evenly distributed throughout the other component or components. When said dispersion of the components is such that the system is chemically and physically homogeneous or completely homogeneous or consists of a single phase, as defined in thermodynamics, such a solid dispersion is called a solid solution. Solid solutions are preferred physical systems because the components in them are usually readily bioavailable to the organisms to which they are administered. The term solid dispersion also includes dispersions that are less homogeneous than solid solutions. Such dispersions are not chemically and physically homogeneous or contain more than one phase.

The water-soluble polymer is typically a polymer that has an apparent viscosity of 1 to 100 mPa s when dissolved in a 2% aqueous solution at 20°C. Preferred water-soluble polymers are hydroxypropyl methylcelluloses or HPMC. HPMC having a methoxy degree of substitution from about 0.8 to about 2.5 and a hydroxypropyl molar substitution from about 0.05 to about 3.0 are generally water soluble. The degree of methoxy substitution refers to the average number of methyl ether groups present per anhydroglucose unit of the cellulose molecule. Hydroxypropyl molar substitution refers to the average number of moles of propylene oxide that have reacted with each anhydroglucose unit of a cellulose molecule.

In addition, it may be convenient to formulate compounds in the form of nanoparticles that have a surface modifier adsorbed on their surface in an amount sufficient to maintain an effective average particle size of less than 1000 nm. Suitable surface modifiers may preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products, and surfactants. Preferred surface modifiers include nonionic and anionic surfactants.

Another interesting method of formulating the compounds of the invention involves a pharmaceutical composition in which the compounds are incorporated into hydrophilic polymers and coating this mixture as a film on a plurality of small beads, thus obtaining a composition with good bioavailability that can be conveniently formulated and suitable for preparation of pharmaceutical dosage forms for oral administration. These beads contain (a) a central, round or spherical core, (b) a film coating of a hydrophilic polymer and an antiretroviral agent, and (c) a seal-coated polymer layer. Materials suitable for use as cores in granules are varied, provided that these materials are pharmaceutically acceptable and have appropriate dimensions and hardness. Examples of such materials are polymers, inorganic substances, organic substances and saccharides and their derivatives.

The compositions can be obtained in a known manner, which usually includes mixing along

- 12 040399 at least one compound according to the invention with one or more pharmaceutically acceptable carriers and, if desired, in combination with other pharmaceutically active compounds, when necessary, under aseptic conditions. See references to US-A-6372778, A-6369086, A-6369087 and A6372733 and other prior art mentioned above, as well as to standard manuals such as the latest edition of Remington's Pharmaceutical Sciences.

The pharmaceutical compositions of the invention are preferably in unit dosage form and may be appropriately packaged in, for example, a box, blister, vial, vial, sachet, ampoule, or any other suitable single or multi-dose container or container (which may be appropriately labeled ); optionally with one or more package inserts containing product information and/or instructions for use. Typically, such dosage units will contain 1 to 1000 mg, and typically 5 to 500 mg, of at least one compound of the invention, such as about 10, 25, 50, 100, 200, 300, or 400 mg per dosage unit.

The compounds may be administered by various routes, including oral, ophthalmic, rectal, transdermal, subcutaneous, intravenous, intramuscular, or intranasal, depending primarily on the particular composition used and the condition being treated or prevented, and oral and intravenous administration are generally preferred. At least one compound of the invention is usually administered in an effective amount, by which is meant any amount of a compound of formula (I) above which, when appropriately administered, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Typically, depending on the condition to be prevented or treated and on the route of administration, such an effective amount is typically 0.01 to 1000 mg/kg, more typically 0.1 to 500 mg, such as 1 to 250 mg, such as about 5, 10, 20, 50, 100, 150, 200, or 250 mg/kg of patient body weight per day, such an effective amount can be administered as a single daily dose divided into one or more daily doses, or substantially continuously, for example with drip infusion. The amount to be administered, route of administration, and further treatment regimen may be determined by the attending physician depending on such factors as the age, sex, general condition of the patient, and the nature and severity of the disease/symptoms to be treated. See references to US A-6372778, A-6369086, A-6369087 and A-6372733 and other prior art mentioned above, as well as standard manuals such as the latest edition of Remington's Pharmaceutical Sciences.

In accordance with the method of the present invention, the specified pharmaceutical composition can be administered separately at different times during the course of therapy or simultaneously in separate or single combinations. Therefore, the present invention is to be understood as covering all such concurrent or alternate treatment regimens, and the term administration is to be interpreted accordingly.

For oral administration, the compositions of the present invention may be mixed with suitable additives such as excipients, stabilizers or inert diluents and may be administered by conventional means in suitable administration forms such as tablets, coated tablets, hard capsules, aqueous, alcohol or oil solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose or starch, in particular corn starch. In this case, the composition can be carried out both in the form of dry and wet granules. Suitable oil excipients or solvents are vegetable or animal oils such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as additional excipients for other forms of administration. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, fillers, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well known in the pharmaceutical composition art and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical compositions for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the invention or their physiologically acceptable salts in a pharmaceutically acceptable solvent such as ethanol or water, or a mixture of such solvents. If desired, the composition may also additionally contain other pharmaceutical excipients such as surfactants, emulsifiers and stabilizers, as well as a propellant.

For subcutaneous or intravenous administration, a compound of the invention, if desired, with

- 13 040399 commonly used substances such as solubilizers, emulsifiers or additional excipients are added to the solution, suspension or emulsion. The compounds of the invention can also be lyophilized and the resulting lyophilizates used, for example, for the preparation of injection or infusion compositions. Suitable solvents are, for example, water, physiological saline or alcohols, for example ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. Injectable solutions or suspensions may be formulated according to the known art using suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting agents. and suspending agents such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When administered rectally in the form of suppositories, these compositions may be prepared by mixing the compounds of the invention with a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols which are solid at ordinary temperatures but liquefy and/or dissolve in the rectum. to release the drug.

The compositions are of value in the veterinary field, which for the purposes of the present invention includes not only the prevention and/or treatment of diseases in animals, but also the enhancement of growth and/or weight gain of the animal and/or the quantity and/or quality of meat or other products, derived from economically important animals such as cattle, pigs, sheep, chicken, fish. Thus, in a further aspect, the invention relates to a (pharmaceutical) composition for veterinary use, which comprises at least one compound of the invention and at least one suitable carrier (ie, a carrier suitable for veterinary use). The invention also relates to the use of a compound of the invention in the preparation of such a composition.

Examples

The following examples are given for the purpose of illustrating the present invention and should in no way be interpreted as limiting the scope of the present invention.

Part A is the preparation of the compounds (intermediates and final compounds), while part B is the pharmacological examples.

Part A.

Compounds were prepared according to two different synthetic routes shown in Scheme 1 and Scheme 2 below.

Scheme 1

₊ III NaHCO ₃ * ^he L _0H EtOAc, H ₂ Ogo Η ₊ |l| NahCO ₃ ^N ' ^0H L _Br EtOAc, H ₂ O * H ^Na0H _r noA^ph EtOH/H ₂ O 5-E-tryptamine, HATU DIREAJMWA H CBr ₄ , P^RDHF u. ArB(OR);, Pd(Ph _a ) ₄ . _{Na 2} CO _{3 (} MeOCH _{2 ) 2 , H 2} O _{' TDGy} ° ^ΒΓ _H NaQH, EtOH/H ₂ O .0 '' ^N ' cTA 5-E-tryptamine, HATU, DIPEA * ^X DMF ^N ' cf

- 14 040399

Intermediate I - preparation of ethyl 5-(bromomethyl)isoxazole-3-carboxylate.

ABOUT

A solution of ethyl 2-chloro-2-(hydroxyimino)acetate (37.6 g, 240.66 mmol) in 200 ml of ethyl acetate was added dropwise at room temperature to a mixture of 3-bromoprop-1-yne (91 ml; 845 mmol) of bicarbonate sodium (71.48 g; 842 mmol), ethyl acetate (1200 ml) and water (12 ml) and the mixture was stirred at room temperature for 108 hours. The solid was filtered off and the filtrate was washed twice with water, dried over magnesium sulfate and concentrated at reduced pressure. The residue was purified by silica gel flash chromatography using a gradient of ethyl acetate (5-40%) in heptane to give 46.1 g (82%) of ethyl 5-(bromomethyl)isoxazole-3-carboxylate as a white solid. 1 H NMR (chloroform-d) δ: 6.74 (s, 1H), 4.50 (s, 2H), 4.45 (q, 2H), 1.42 (t, 3H).

Intermediate II - preparation of ethyl 5-(2,5-difluorobenzyl)isoxazole-3-carboxylate.

Ethyl 5(bromomethyl)isoxazole-3-carboxylate (1.2 g, 5.13 mmol), 2,5-difluorophenylboronic acid (0.928 g, 5.64 mmol), tetrakis( triphenylphosphine) palladium (0) (0.297 g; 0.256 mmol), sodium carbonate (1.09 g; 10.25 mmol) and a mixture of water (2 ml) and 1,2-dimethoxyethane (8 ml). The ampoules were hermetically sealed and heated at 130°C in a microwave oven for 20 min. The contents of twenty-five ampoules were combined, diluted with ethyl acetate and washed with water. The organic layer was concentrated under reduced pressure and the residue was purified by silica gel flash chromatography using a gradient of ethyl acetate (540%) in heptane to give 18.47 g (54%) of ethyl 5-(2,5-difluorobenzyl)isoxazole-3-carboxylate as a yellow oil.

¹ H NMR (chloroform-d) δ: 6.90-7.12 (m, 3H), 6.42 (s, 1H), 4.42 (q, 2H), 4.15 (s, 2H), 1.40 (t, 3H). ESI/APCI (+): 268 (M+H), 290 (M+Na).

Intermediate III - preparation of 5-(2,5-difluorobenzyl)isoxazole-3-carboxylic acid.

.ABOUT

HO-V nO/YjI

1M sodium hydroxide solution (206 ml; 206 mmol) was added to a solution of ethyl 5-(2,5difluorobenzyl)isoxazole-3-carboxylate (18.30 g; 68.48 mmol) in ethanol (20 ml). The mixture was stirred at room temperature for 2 hours. The solution was acidified to pH 1 by adding 12N hydrochloric acid. hydrochloric acid solution. The precipitate was collected by filtration and dried under reduced pressure to give 14.60 g (89%) of 5-(2,5-difluorobenzyl)isoxazole-3-carboxylic acid as a white solid.

1H NMR (DMSO-dg) δ: 7.17-7.34 (m, 3H), 6.59 (s, 1H), 4.27 (s, 2H).

Intermediate IV - preparation of ethyl 5-(hydroxymethyl)isoxazole-3-carboxylate.

—\ /° ° X hl/ .OH

ABOUT

Propargyl alcohol (11.42 ml; 191.36 mmol) was added to a mixture of ethyl 2-chloro-2(hydroxyimino)acetate (14.50 g; 95.68 mmol) and sodium bicarbonate (16.08 g; 191.36 mmol ) in ethyl acetate (400 ml) and water (40 ml) and stirred at room temperature for 24 h. The two phases were separated and the organic layer was concentrated under reduced pressure. The crude mixture was purified by flash chromatography on silica gel (eluting with 1 to 10% ethyl acetate in dichloromethane) to give 8.67 g (53%) of ethyl 5-(hydroxymethyl)isoxazole-3-carboxylate as an oil.

Intermediate V - preparation of 5-(hydroxymethyl)isoxazole-3-carboxylic acid.

A solution of sodium hydroxide in water (2M; 50 ml) was added to a mixture of ethyl 5-(hydroxymethyl)isoxazole-15 040399

3-carboxylate (8.67 g; 50.66 mmol) in ethanol (30 ml) and stirred vigorously for 2 hours. The solution was concentrated under reduced pressure, diluted with water and extracted with dichloromethane. The aqueous layer was acidified to pH 1 with 6N. hydrochloric acid and extracted several more times with ethyl acetate. The organic layer was dried and concentrated under reduced pressure to give 5.43 g (75%) of 5-(hydroxymethyl)isoxazole-3-carboxylic acid as a white solid.

Intermediate VI - preparation of tyl)isoxazole-3-carboxamide.

K-(2-(5-fluoro-III-indol-3-yl)ethyl)-5-(hydroxyme-

N,N-diisopropylethylamine (10.75 ml; 58.23 mmol) was added to a mixture of 2-(5-fluoro-Shindol-3-yl)ethanamine hydrochloride (5.00 g; 23.29 mmol), O-( 7-azabenzotriazol-1-yl)-K,K,K',K'tetramethyluronium (8.86 g; 23.29 mmol) and 5-(hydroxymethyl)isoxazole-3-carboxylic acid (3.67 g; 25. 62 mmol) in anhydrous DMF (40 ml) and stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and diluted with ethyl acetate, then washed with aqueous potassium hydrogen sulfate (1M) and aqueous sodium carbonate (1M). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluting with 0 to 10% methanol in dichloromethane) to give 5.26 g (74%) of N-(2-(5-fluoro-1H-indol-3-yl)ethyl)- 5-(hydroxymethyl)isoxazole-3carboxamide as a sticky yellowish solid.

Intermediate VII - Preparation of 5-(bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazole-3-carboxamide.

A solution of N-(2-(5-fluoro-III-indol-3-yl)ethyl)-5-(hydroxymethyl)isoxazole-3-carboxamide (5.30 g; 17.48 mmol) in THF (10 ml) was added to a solution of perbromomethane (8.69 g; 26.21 mmol) and triphenylphosphine (6.88 g; 26.21 mmol) in THF (60 ml). The resulting solution was stirred at room temperature for 2.5 hours. The solid was filtered off and the solution was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluting with 15 to 100% ethyl acetate in heptane) to give 2.64 g (41%) of 5-(bromomethyl)-K-(2-(5-fluoro-1H-indole-3 -yl)ethyl)isoxazole-3carboxamide as a white solid. ESI/APCI (+): 366, 368 (M+H). ESI/APCI (-): 366, 364 (M-H).

Preparation of Compounds of the Invention

Example 1 Preparation of 5-(2,5-difluorobenzyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide.

N-ethyldiisopropylamine (25.65 ml; 148.37 mmol) was added to a stirred mixture of 2-(5-fluoro-1H-indol-3-yl)ethane-1-amine hydrochloride (13.00 g; 59.35 mmol) , 5-(2,5-difluorobenzyl)isoxazole-3carboxylic acid (14.19 g; 59.35 mmol) and O-(7-azabenzotriazol-1-yl)-K,K,K',K'tetramethyluronium hexafluorophosphate ( 22.57 g; 59.35 mmol) in anhydrous DMF (90 ml). The mixture was stirred at room temperature for 60 hours and then concentrated under reduced pressure. The residue was taken up in ethyl acetate, and the solution was washed with water and saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography using a gradient of ethyl acetate (1-10%) in dichloromethane to give 20.02 g of a yellowish solid which was recrystallized from a mixture of dichloromethane and neptane to give 19.06 g (80%) of 5-(2. 5-difluorobenzyl)-K-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3

- 16 040399 carboxamide in the form of a white solid. Or 5-(bromomethyl)-N-(2-(5-fluoro-III-indol-3yl)ethyl)isoxazole-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml) together with (2,5-difluorophenyl)boronic acid (0.068 g; 0.430 mmol), sodium carbonate (0.086 g; 0.430 mmol), tetrakis(triphenylphosphine)palladium (0) (0.024 g; 0.020 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.038 g (23%) of 5-(2,5-difluorobenzyl)-N-(2-(5-fluoro-1H-indole -3yl)ethyl)isoxazole-3-carboxamide as a white solid.

¹ H NMR (DMSO-d6) δ: 10.92 (br. s, 1H), 8.82 (t, 1H), 7.1-7.38 (m, 6H), 6.90 (td, 1H ), 6.53 (s, 1H), 4.26 (s, 2H), 3.48 (q. 2H), 2.88 (t, 2H). ESI/APCI (+): 400 (M+H). ESI/APCI (-): 398 (M-H).

Example 2 Preparation of 5-(2,4-difluorobenzyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide.

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml ) together with (2,4-difluorophenyl)boronic acid (0.068 g; 0.430 mmol), sodium carbonate (0.086 g; 0.430 mmol), tetrakis(triphenylphosphine)palladium (0) (0.024 g; 0.020 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.0462 g (28%) of 5-(2,4-difluorobenzyl)-N-(2-(5fluoro-1H-indole -3-yl)ethyl)isoxazole-3-carboxamide as a white solid.

1 H NMR (300 MHz, DMSO) δ: 10.92 (br. s, 1H); 8.82 (t, 1H); 7.39-7.57 (m, 1H); 7.21-7.36 (m, 4H); 7.11 (td, 1H); 6.90 (td, 1H); 6.50 (s, 1H); 4.24 (s, 2H); 3.49 (q, 2H); 2.89 (t, 2H). ESI/APCI (+): 400 (M+H). ESI/APCI (-): 398 (M-H).

Example 3 Preparation of 5-(3,4-difluorobenzyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide.

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml ) together with (3,4-difluorophenyl)boronic acid (0.068 g; 0.430 mmol), sodium carbonate (0.086 g; 0.430 mmol), tetrakis(triphenylphosphine)palladium (0) (0.024 g; 0.020 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.065 g (40%) of 5-(3,4-difluorobenzyl)-N-(2-(5-fluoro-1Hindole-3 -yl)ethyl)isoxazole-3-carboxamide as a pale yellow solid.

1H NMR (300 MHz, DMSO-d ₆ ) δ: ppm 10.92 (br. s, 1H); 8.81 (t, 1H) 7.36-7.49 (m, 2H); 7.28-7.35 (m, 2H); 7.25 (d, 1H); 7.12-7.21 (m, 1H); 6.90 (td, 1H); 6.54 (s, 1H); 4.23 (s, 2H); 3.48 (q, 2H); 2.89 (t, 2H). ESI/APCI (+): 400 (M+H). ESI/APCI (-): 398 (M-H).

Example 4 Preparation of 5-(3,5-difluorobenzyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide.

- 17 040399

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml ) together with (3,5-difluorophenyl)boronic acid (0.068 g; 0.430 mmol), sodium carbonate (0.086 g; 0.430 mmol), tetrakis(triphenylphosphine)palladium (0) (0.024 g; 0.020 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.068 g (41%) of 5-(3,5-difluorobenzyl)-M(2-(5-fluoro-1H-indole- 3-yl)ethyl)isoxazole-3-carboxamide as a white solid.

1H NMR (300 MHz, DMSO-d6) δ ppm 10.93 (br. s, 1H); 8.83 (t, 1H); 7.03-7.42 (m, 6H); 6.91 (td, 6H); 6.59 (s, 1H); 4.27 (s, 2H); 3.50 (q, 2H); 2.90 (t, 2H). ESI/APCI (+): 400 (M+H). ESI/APCI (-): 398 (M-H).

Example 5 Preparation of N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-5-(4-fluorobenzyl)isoxazole-3-carboxamide.

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml ) along with (4-fluorophenyl)boronic acid (0.086 g; 0.430 mmol), N,Ndiisopropylethylamine (0.151 ml; 0.819 mmol), [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (11) (0.033 g; 0.040 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.088 g (57%) of N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-5-(4 -fluorobenzyl)isoxazole-3-carboxamide as a white solid.

1H NMR (300 MHz, DMSO-d6) δ ppm 10.92 (br. s, 1H); 8.80 (t, 1H); 7.11-7.42 (m; 7H); 6.81-6.99 (m, 1H); 6.51 (s, 1H); 4.21 (s, 2H); 3.48 (q, 2H); 2.88 (t, 2H). ESI/APCI (+): 382 (M+H). ESI/APCI (-): 380 (M-H).

Example 6 Preparation of 5-(2,3-difluorobenzyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide.

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.200 g; 0.546 mmol) was dissolved in DME (3 ml) and water (1 ml ) along with (2,3-difluorophenyl)boronic acid (0.129 g; 0.819 mmol), N,N-diisopropylethylamine (0.201 ml; 1.09 mmol), bis(diphenylphosphino)ferrocene]dichloropalladium (ll) (0.045 g; 0.056 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude mixture was purified by silica gel flash chromatography (eluting with 20 to 100% ethyl acetate in heptane) to give 0.015 g (7%) of 5-(2,3difluorobenzyl)-N-(2-(5-fluoro-1H-indole-3 -yl)ethyl)isoxazole-3-carboxamide as a white solid.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 10.85-11.04 (m, 1H); 8.82 (t, 1H); 7.45-7.30 (1H, m); 7.28-7.36 (m, 2H); 7.17-7.27 (m, 3H); 6.90 (td, 1H); 6.55 (s; 1H); 4.33 (s; 2H); 3.48 (d, 2H); 2.88 (t, 2H). ESI/APCI (+): 400 (M+H). ESI/APCI (-): 398 (M-H).

- 18 040399

Example 7 Preparation of X-(2-(5-fluoro-1H-indol-3-yl)ethyl)-5-(2-fluorobenzyl)isoxazole-3-carboxamide.

5-(Bromomethyl)-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)isoxazol-3-carboxamide (0.150 g; 0.409 mmol) was dissolved in DME (3 ml) and water (1 ml ) together with (2-fluorophenyl)boronic acid (0.086 g; 0.430 mmol), K,K-diisopropylethylamine (0.151 ml; 0.819 mmol), bis(diphenylphosphino)ferrocene]dichloropalladium (ll) (0.033 g; 0.040 mmol) and heated overnight at 90°C. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate, the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude mixture was purified by silica gel flash chromatography (eluting with 0 to 10% ethyl acetate in dichloromethane) to give 0.068 g (44%) of N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-5- (2-fluorobenzyl)isoxazole-3-carboxamide as a white solid.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 10.93 (br. s, 1H); 8.66-8.96 (m, 1H); 7.13-7.52 (m, 7H); 6.79-7.00 (m, 1H); 6.51 (s, 1H); 4.26 (s, 2H); 3.49 (q, 2H); 2.89 (t, 2H). ESI/APCI (+): 382 (M+H). ESI/APCI (-): 380 (M-H).

Part B.

Example 8 Construction of a cell line overexpressing the TAU gene.

The TAU expression plasmid was constructed by subcloning the human TAU-P301L cDNA (encoding TAU with proline 301 substituted with a leucine residue) into the mammalian expression vector pcDNA3.1, resulting in the plasmid pcDNA3.1-TAU P301L. Plasmids pcDNA3.1 and pcDNA3.1-TAU P301L were transfected into human neuroblastoma cells (BM17; ATCC No. CRL-2267) and independent clonal lines with stably integrated plasmids into the genome were selected. This resulted in cell lines named M17-3.1 and M17-TAU (P301L) (transfected with pcDNA3.1 and pcDNA3.1-TAU P301L, respectively). Expression of the TAU P301L genes in cell lines was confirmed by Western analysis.

Example 9 Use of cells expressing P301L TAU as a model for neuronal degeneration.

P301L TAU expression in M17-TAU (P301L) cells was found to have increased toxicity compared to control cells expressing wild-type TAU (M17-TAUwt).

In degenerated or dead cells, lactate dehydrogenase (LDH) leaks out of the cells into the extracellular environment due to loss of plasma membrane integrity. This principle was used to determine cytotoxicity by quantifying the level of leaked LDH into the culture medium in relation to the sum of the total LDH activity from live and dead cells.

The detailed procedure for determining cytotoxicity was as follows.

From the respective M17-3.1 and M17-TAU (P301L) precultures, cells were seeded at 2500 cells/cm ² in Optimem reduced serum without phenol red (Gibco, Cat. 31985-047) supplemented with 1% fetal calf serum, 1 mM pyruvate sodium, 1x essential amino acids, 500 mcg/ml G418 0.5x antibiotic/antifungal. After 3 h incubation at 37° C./5% CO ₂ 1 volume of Optimem reduced serum (same as above; except without calf serum) with 2.5 μM all-trans retinoic acid (ATRA) was added. Cells were further incubated for 7 days. Subsequently, LDH activity was determined using the Promega Cytotox 96 non-radioactive cytotoxicity assay (catalog number G1780) according to the supplier's instructions.

In FIG. 1A shows that M17-TAU P301L cells, but not M17-3.1 cells, have a relatively high level of LDH leaked into the medium, demonstrating toxicity specifically induced by TAU P301. Highlighting the sensitivity of the TAU(301) expressing neuroblastoma cell line to retinoic acid-induced differentiation.

Example 10 Use of cells expressing P301L TAU as a model for calcium dyshomeostasis.

Cystic calcium was measured by loading cells with media containing Fura-2 AM (SigmaAldrich), a cell-permeable Ca ²⁺ fluorescent probe. Fura-2-AM was dissolved in DMSO plus 20% pluronic acid (F-127) (Invitrogen) 1:1 and diluted in medium to a final concentration of 0.5 μM. Probenecid (Sigma-Aldrich) was added to this loading medium to a final concentration of 2.5 mM. Then the culture medium was replaced with the loading medium and after incubation for 1 h at 37°C, the cells were washed twice and the medium was replaced with HBSS (Gibco) supplemented with 0.2%

- 19 040399

FBS and 0.02 M HEPES. Changes in cytosolic calcium levels were then measured using a FlexStation 3 microplate reader (Molecular Devices) and quantified ratiometrically by calculating changes in the amount of cytosolic Ca ²⁺ bound to Fura-2 (fluorescence intensity at 340 nm) relative to the amount of Ca ^{2 +} unbound to Fura-2 (fluorescence intensity at 380 nm). The data were processed using SoftMax Pro 5.4.6 software (Molecular Devices).

In FIG. 1B shows that P301L TAU expressing cells in which toxicity was induced by ATRA according to the method of Example 9 had elevated levels of cytosolic calcium.

Example 11 Use of P301L TAU expressing cells as a model for neurodegeneration in the screening of compounds for the treatment of epilepsy.

A cell line model with TAU-induced cytotoxicity allows the identification of compounds that can be used in transgenic animals and humans for the treatment of epilepsy. Isradipine is a voltage-gated calcium channel (VGCC) inhibitor, a well-known target for the treatment of epilepsy, and isradipine is active in models of epilepsy. In FIG. 2 shows the reduction in toxicity of RA-incubated cells that were treated with isradipine, a voltage-gated calcium channel (VGCC) inhibitor.

Example 12 Use of TAU Expressing Cells in Screening Example Compounds of the Invention.

The M17-TAU P301L cell line made it possible to evaluate the ability of new compounds to counteract TAU cytotoxicity. Active inhibitors of TAU cytotoxicity were found to inhibit LDH leakage from M17-TAU P301L cells treated as described in Example 9. The efficacy (activity) of the compounds was determined by testing the compounds at various concentrations ranging from ineffective (at a relatively low concentration) to effective concentration by their ability to reduce LDH activity in retinoic acid-incubated M17-TAU P301L cells. These measurements were used to calculate the EC ₅₀ values.

Typical compounds according to this invention are shown in table. 1 with their chemical structure and their EC ₅₀ value (expressed in μg/ml) as determined in Example 12 in the TAU induced toxicity experiment.

- 20 040399

Table 1

In addition, the compounds were tested to reduce elevated levels of cytosolic calcium. In the presence of compounds, these levels of cytosolic calcium decreased. In FIG. 3 shows that the compounds alleviate calcium dyshomeostasis under conditions of toxicity in the M17-TAU P301L cell line model described in Example 9.

Example 13 Ex-vivo Inhibition of Oligomeric Amyloid Beta Toxicity.

An exemplary compound of this invention was tested for its ability to inhibit toxicity induced by oligomeric amyloid beta (Abo). Neurons from rat embryos were harvested and cultured using standard methods (such as those used in Schlager et al., 2014. Cell reports, 8(5), pp. 1248-56.). Differentiated neurons were infected with Avo and their viability was quantified. Avo treatment resulted in a significant decrease in neuronal viability, while viability was strongly restored in Avo-treated neurons in the presence of 100 ng/ml Compound 6 (FIG. 4). These results demonstrate that Compound 6 greatly reduces Avo-induced neuronal cell death.

Detailed methods.

On the day in vitro, 19 days after isolation, primary hippocampal neurons were transfected with Marcks-GFP to visualize the neurons by fluorescence microscopy.

- 21 040399

Preparing ADDL for generating Αβο.

Preparation of ADDLs (diffusing ligands derived from Abeta) (representing Αβο) was performed according to Klein (Klein, 2002). Neurochemistry international, 41(5), pp. 345-52.). Abeta 1-42 was purchased from AnaSpec Inc. and dissolved in HFIP to homogenize the peptide. The HFIP was then removed by lyophilization and the Abeta film was dried at -20° C. overnight over a drying agent. The Abeta film was then redissolved in 100% DMSO and further diluted (1/25) in Ham's F12 medium. Controls were prepared by adding equal amounts of DMSO to Ham's F12 medium. Solutions Abeta and controls were incubated overnight at 4°C. The solution was centrifuged at 14000xg for 10 min at 4°C, the supernatants were transferred to fresh tubes and the protein concentration was determined using NanoDrop®.

Cell processing.

On in vitro day 21, primary rat hippocampal neurons were treated with an equal volume of control/ADDL solution of 0 and 1000 nM ADDL. Cultures were kept at 37° C. in 5% CO ₂ for 24 h until PFA fixation. Viability was assessed using a live-dead assay from the Thermo Fisher catalog, catalog number: L3224.

Fixing.

Neurons were fixed in 4% PFA/sucrose for 10 min at room temperature.

Cells were permeialized using buffer containing 0.1% Triton-X/0.1% NaCit/PBS. After washing, coverslips containing neurons were inverted onto a drop of mounting medium (H1000, Vector Laboratories) (without DAPI), dried at room temperature, and sealed.

Example 14 Inhibition of VGCC activity ex-vivo.

Compound 6 was tested for its ability to inhibit VGCC activity in primary neurons. Neurons from mouse embryos were harvested and cultured using standard techniques (such as those used in Schlager et al., 2014. Cell reports, 8(5), pp. 1248-56). To stimulate VGCC activity, neurons were depolarized using 45 mM KCl and incubated with vehicle or compound at a concentration of 1.5 μM. Calcium influx was measured using the Fura2 fluorescent cytosolic Ca ²⁺ reagent. In FIG. 5 shows that in compound-treated neurons, Ca ²⁺ influx was significantly reduced after KCl depolarization, indicating that the compound inhibited VGCC activity.

Detailed methods.

Changes in cytosolic Ca ²⁺ concentration were measured after cells were loaded with Fura-2 AM (SigmaAldrich), a cell-permeable Ca ²⁺ fluorescent probe. Briefly, Fura-2-AM was dissolved in DMSO with 20% pluronic acid (F-127) (Invitrogen) 1:1 and diluted in medium to a final concentration of 0.5 μM. Probenecid (Sigma-Aldrich) was added to this loading medium to a final concentration of 2.5 mM. The culture medium was then replaced with loading medium and after incubation for 1 h at 37° C., the cells were washed twice and the medium replaced with HBSS (Gibco) supplemented with 0.2% FBS and 0.02 M HEPES. Changes in cytosolic calcium levels were then measured using a FlexStation 3 microplate reader (Molecular Devices) and quantified ratiometrically by calculating changes in the amount of cytosolic Ca ²⁺ bound to Fura-2 (fluorescence intensity at 340 nm) relative to the amount of Ca ^{2 +} unbound to Fura-2 (fluorescence intensity at 380 nm). The data were processed using SoftMax Pro 5.4.6 software (Molecular Devices).

Example 15 Inhibition of pathologies caused by TAU in vivo.

Transgenic mice APP * PS1 at the age of 5 months (The Journal of Neuroscience, September 1, 2000, 20(17):6452-6458) were injected daily subcutaneously with 20 mg/kg of compound 6 for 2 weeks.

At the end of the administration period, the mice were sacrificed and the respective brains were used for biochemical analysis. Western analysis of brain extracts using phospho-TAU specific antibodies showed that, compared to vehicle-treated mice, Compound 6 more effectively reduced TAU phosphorylation, demonstrating the in vivo effect of reducing the number of pathological TAU species in the brain with Compound 6 (Fig. 6 ).

Example 16 In Vivo Effects on Cognitive Function in a Mouse Model of Alzheimer's Disease.

Transgenic APP mice aged 4 months (THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 10, Issue of March 5, pp. 6483-6492, 1999) were injected subcutaneously with compound 6 at a dose of 20 mg/kg daily for 8 weeks . Cognitive function was assessed using the Morris water maze test. During the learning phase, which assesses the effectiveness of learning, mice have a reduced search path over time compared to vehicle-treated mice (FIG. 7, part A). After the learning phase, a test was conducted in which the platform was removed from the water bath to evaluate the spatial memory of the platform's location.

This test revealed (FIG. 7, Part B) a higher ring crossing index score (which is the number of swims across the platform platform in the target area adjusted for swims through corresponding areas in other quadrants) compared to vehicle-treated animals.

- 22 040399

Both scores indicate that compound-treated mice had improved cognitive function nearly identical to wild-type controls compared to vehicle-treated mice.

Example 17 Normalization of neuronal hyperactivation in an ex-vivo mouse model of Alzheimer's disease.

Brain sections of wild-type and tgAPP mice were incubated with DMSO or compound 6.

Neurons were stimulated with increasing currents and the frequency of action potentials (pulse emission frequency) was measured.

In tgAPP mice, the frequency of action potentials at higher stimuli was higher. Incubation of sections with exemplary Compound 6 significantly reduced the frequency compared to wild type (FIG. 8). These data indicate that in tgAPP mice neuronal activity is less restricted or unrestricted as seen in wild-type mice, and that the compound reduces this neuronal hyperactivity in tgAPP mice.

Sharp slices of the brain.

Acute sagittal brain sections of DT or hAPP mice were prepared by decapitation of mice after isoflurane anesthesia. The brain was quickly removed and immersed for 3-4 min in ice-cold freshly prepared cutting artificial cerebrospinal fluid (aCSF cutting fluid) containing (in mM) 214 sucrose, 2.5 KCl, 2 CaCl ₂ , 2 MgSO ₄ , 1.25 NaH ₂ PO ₂ , 26 NaHCO ₃ and 10 glucose and oxygenated with a ratio of 95% 02/5% CO ₂ . 350 μm sagittal sections were generated using a vibratome (VT 1000S; Leica Microsystems) and incubated in standard oxygenated aCSF (in mM: 125 NaCl, 2.5 KCl, 2 CaCl ₂ , 2 MgSO ₄ , 1.25 NaH ₂ PO ₂ , 26 NaHCO ₃ and 10 glucose, osmolarity 305 mOsm) at 34°C for 20 min. Incubation was continued at room temperature (RT) for another 1 hour, after which each section was transferred to a submerged recording chamber and continuously perfused with carbonized aCSF.

Recording of CA1 single action potential fixation (sAP) and frequency of pulse emission.

Potential clamped somatic or dendritic (>200 µm from the soma) recordings were performed at room temperature (24 to 28°C) and sections were continuously perfused with carboxylated standard aCSF supplemented with control or test REM0043039 at 2 µM. For registration of whole cells, pipettes for clamping the potential were filled with a solution containing (in mm) 140 K-gluconate, 5 NaCl, 2 MgCl ₂ , 10 HEPES, 0.5 EGTA, 2 MgATP, 0.4 NaGTP, osmolarity 305, pH was adjusted to 7.25 with KOH. The soma or dendrite of CA1 large pyramidal neurons were identified and potential fixed after visual approximation of a recording pipette using a combination of infrared light optics and differential interference contrast (DIC). The potential clamping electrodes had a resistance of about 5 and 14 μΩ when filled for somatic and dendritic recording, respectively. Recordings were terminated when the series resistance exceeded 40 µΩ. The signals were digitized and low-pass filtered at 10 kHz. The signal was amplified with an Axopatch 200B amplifier, digitized with a Digidata 155 interface, and sampled with a Clampex 10 (Molecular Devices, CA).

A single spike was induced by administering a 2 ms depolarizing current pulse (35 pA) and action potential (AP) parameters were at baseline and after 25 min of vehicle or compound perfusion.

CA1 dendritic cell firing rates were recorded in response to hyperpolarization and depolarization stages (-0.4 to +0.45 nA, 0.05 nA steps) after 1 hour perfusion with vehicle or compound at 2 μM. The average number of action potentials (pulse frequency) was plotted against the intensity of the current step.

Example 18 Compound Tests in a Cainite Epilepsy Model

The compounds were tested in a rat model of kainite epilepsy.

Pharmacological evaluation of the antiepileptic activity of the compounds was performed in a mouse model using kainic acid (KA) (Groticke et al., 2008, Experimental Neurology, 213, pp. 71-83; Dietrich et al., Aug 2016, Conf Proc IEEE Eng Med Biol Soc , pp. 4005-4008). This model shows recurrent seizures after an initial CA-induced status epilepticus (SE) and a latent epileptogenic phase of 2 to 3 weeks.

CA was injected into the CA1 region of the dorsal hippocampus, followed by surgical implantation of ipsilateral bipolar electrodes for EEG recording. Based on the appearance of non-convulsive SE caused by KA, mice were selected for inclusion in the study. EEG video monitoring once or twice a week for 1 h was used to record hippocampal discharges (HPD) over time. The severity of behavioral (convulsive) seizures was assessed according to Racine (Racine RJ, 1972, ElectroencephalogrClin Neurophysiol., 38(1):1-12).

Daily subcutaneous administration of compound (25 mg/kg) or vehicle administration was initiated before or after the initial kainate-induced SE. Compound-treated mice showed a greater than 20% reduction in kainate-induced HPD and/or spontaneous seizures compared to vehicle-treated mice.

Example 19 Compound increases subsequent hyperpolarization of action potentials.

Improving A-type potassium current has therapeutic potential for the treatment of epilepsy, as it reduces neuronal excitability.

Action potentials (AP) were analyzed after electrical stimulation of mouse brain slices (FIG. 9). The subsequent hypopolarization of AP sections incubated with compound 6 was increased, indicating that the compound facilitates the repolarization of neurons to a resting state.

The experiment was carried out essentially as described in Example 17.

Example 20 Use of the Calcium Dysomeostasis Model with Cells Overexpressing TAU.P301L in Screening for Compounds of the Invention.

Compounds were tested for the reduction of elevated levels of cytosolic Ca ²⁺ in a cellular model of calcium dyshomeostasis (FIG. 1B) as described in Example 10. The efficacy (activity) of the compounds was determined by testing the compounds at various concentrations, ranging from ineffective to effective concentrations, for their the ability to reduce the level of cytosolic Ca ²⁺ associated with Fura-2 (fluorescence intensity at 340 nm) relative to the amount of Ca ²⁺ not associated with Fura-2 (fluorescence intensity at 380 nm).

Typical compounds according to this invention are presented in table. 2 with their chemical structure and EC5o value reducing cytosolic Ca ²⁺ (expressed in μg/ml).

Example 21 Use of an ex-vivo model of KCl-induced calcium influx in the screening of compounds for the treatment of epilepsy and other neurodegenerative diseases involving calcium dyshomeostasis.

-

Claims (14)

Compounds were tested at 10 μM for their ability to inhibit Ca ²⁺ influx into primary neurons (FIG. 5) as described in Example 14. Primary neurons treated with vehicle in which the compound had not been dissolved were used as a negative control. The percentage (%) of Ca ²⁺ influx inhibition was calculated relative to vehicle-treated neurons. The results are shown in table 3. Table 3 Compound (%) inhibition of cytosolic Ca ²⁺ influx 1 43 2 48 3 42 4 39 5 42 6 43 7 56 CLAIM 1. The compound of formula (I) or its tautomer, ABOUT (I) where R ¹ is selected from the group consisting of hydrogen and F; R ² is selected from the group consisting of hydrogen and F; R ³ is selected from the group consisting of hydrogen and F; R ⁴ is selected from the group consisting of hydrogen and F; R ⁵ is selected from the group consisting of hydrogen and F; provided that at least one of R ¹ , R ² , R ³ , R ⁴ or R ⁵ is not hydrogen; provided that said compound of formula (I) is not N-[2-(5-fluoro-1H-indol-3-yl)ethyl]5-[(3-fluorophenyl)methyl]isoxazole-3-carboxamide, or pharmaceutically acceptable salt. 2. The compound according to claim 1, having any of the formulas (II), (III), (IV), (V) or (VI) ABOUT - 25 040399 3. The compound according to claim 1 or 2, where R ² , R ³ , R ⁴ and R ⁵ are hydrogen, and R ¹ is F. 4. The compound according to claim 1 or 2, where R ¹ , R ² , R ⁴ and R ⁵ are hydrogen and R ³ is F. 5. A compound according to claim 1 or 2, wherein R ² , R ⁴ and R ⁵ are hydrogen and R ¹ and R ³ are independently selected from the group consisting of hydrogen and F. 6. A compound according to claim 1 or 2, wherein R ¹ , R ⁴ and R ⁵ are hydrogen and R ² and R ³ are independently selected from the group consisting of hydrogen and F. 7. A compound according to claim 1 or 2, wherein R ¹ , R ³ and R ⁵ are hydrogen and R ² and R ⁴ are independently selected from the group consisting of hydrogen and F. 8. A compound according to claim 1 or 2, wherein R ³ , R ⁴ and R ⁵ are hydrogen and R ¹ and R ² are independently selected from the group consisting of hydrogen and F. 9. A compound according to claim 1 or 2, wherein R ² , R ³ , and R ⁵ are hydrogen and R ¹ and R ⁴ are independently selected from the group consisting of hydrogen and F. 10. The compound according to claim 1, selected from the group consisting of 5-[(2,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide; N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(4-fluorophenyl)methyl]isoxαzol-3-carboxamide; 5-[(2,3-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide; 5-[(2,5-difluorophenyl)methyl]-K-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide; N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-5-[(2-fluorophenyl)methyl]isoxαzol-3-carboxamide; 5-[(3,4-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide; 5-[(3,5-difluorophenyl)methyl]-N-[2-(5-fluoro-1H-indol-3-yl)ethyl]isoxazole-3-carboxamide. 11. A pharmaceutical composition containing one or more pharmaceutical auxiliary compounds and a therapeutically effective amount of a compound according to any one of claims 1 to 10 and a pharmaceutically acceptable salt thereof. 12. The use of a compound according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 as a medicine. 13. The use of a compound according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 as a drug for the prevention and/or treatment of epilepsy, neurodegenerative disorders, pain disorders, anxiety disorders, depression, bipolar disorder, psychosis, drug withdrawal drugs, tobacco, memory loss, dementia, schizophrenia and panic attacks. 14. Use according to claim 13, wherein the epilepsy is selected from the group consisting of refractory epilepsy, West syndrome, Dawes syndrome, benign Rolandic epilepsy, Rasmussen syndrome, Lennox-Gastaut syndrome, Sturge-Weber syndrome, juvenile myoclonic epilepsy, childhood absence epilepsy , epilepsy associated with idiopathic localization, temporal lobe epilepsy, partial seizures, simple partial seizures, tonic seizures, tonic-clonic seizures, -