ES2659828A2 - Use of 4H-pyrano [2,3-c] pyrazole derivatives in the treatment of neurodegenerative diseases (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

The present invention relates to the use of 3-alkyl-6-amino-4-aryl-1,4-dihydropyrano [2,3-c] pyrazole-5-carbonitrile derivatives with inhibitory activity of the glycogen synthase kinase kinase 3 β, inducer effect of the transcription factor Nrf2, and neuroprotective capacity. Another aspect of the invention relates to the use of the derivatives object of this invention for the treatment of diseases whose pathogenesis intervenes in the abnormal activity of this enzyme and/or oxidative stress and/or diseases that occur with deregulation of gene activity of phase II activated by the factor Nrf2, such as diseases that have an inflammatory component or neurodegenerative diseases. (Machine-translation by Google Translate, not legally binding)

Description

USE OF DERLV ADOS 4H-PIRANOI2,3-clPlRAZOL IN THE TREATMENT OF NEURODEGENERA TIV AS DISEASES.

FIELD OF THE INVENTION

The present invention is mainly framed in the pharmaceutical sector with applications aimed at the prevention and / or treatment of diseases and
any type of condition or damage that involves abnormal enzyme activity

3 ~ glycogen synthase kinase (GSK3 ~) or that is subject to high levels of oxidative stress and, in particular, in the identification of chemical compounds useful in the preventive and / or therapeutic treatment of diseases that occur with a
inflammatory component and / or neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disease that
it constitutes the main cause of dementia among the elderly population (Ballard et al. 2011, Lancet, 377: 10 19-3 1). This pathology affects more than 36 million people to
global level and, due to the continuous aging of the population, the 11.5 million patients are expected to be reached by 2050 (AD Report, 2009). The increasing number of people affected, together with the enormous socio-health costs involved, highlight the imperative need to find new treatments that prevent and even stop the progression of the disease.

The clinical outcome of AD patients is characterized by the gradual loss of memory and, consequently, progressive deterioration of cognitive abilities. This symptomatology is due to neuronal death, which occurs mostly at the level of the cholinergic system. Histopathologically, AD is characterized by the presence of two protein aggregates: intracellular neurofibrillary tangles (NFTs), formed by accumulations of hyperphosphorylated tau protein (Lee et al., 2001, Annu Rev Neurosci, 24: 1121-59) and senile extracellular plaques, accumulations of the ~ -amiloid (A ~) peptide (Vinters, 1987, Stroke, 18: 311-24). In addition, other factors are involved in the pathophysiology of AD, such as mitochondrial dysfunction (Volgyi et al., 2015, Curr Alzheimer Res, 12: 655-72), increased levels of oxidative stress (Pratico, 2008, Trends Pharmacol Sci , 29: 609- (5) neuroinflammation (Latta et al. 2015, Neuroscience, 302: 103- (1) And excitotoxicity (Zadori et al., 2014, J Alzheimers Dis, 42 Suppl 3: S 177-87) among others Due to the close interrelationship between all these pathophysiological mechanisms, AD is considered a multifactorial disease (Iqbal et al., 2000, Neurobiol Aging, 21: 901-2; djscussion 03-4).

NFTs constitute one of the main histopathological marks of AD patients. Tau is a microtubule-associated protein involved in the stabilization of the cytoskeleton. In AD, tau is hyperphosphorylated by different kinases, inducing their dissociation of microtubules and, as a consequence, they cause destabilization of the cytoskeleton, synaptic dysfunction and neuronal death (Martin et al. 2011, Neurochem In !, 58: 458-71) . Once dissociated, tau is added to form crHF helical paired filaments) that give rise to the NFTs. In AD patients, the degree of phosphorylation of Tau is higher than in normal conditions and there seems to be a clear correlation between the hyperphosphorylation of this protein and the loss of cognitive abilities of these patients (Grundke-Iqbal et al., 1986, Proc Na! / Acad. Sci. USA, 83: 4913-7, Greenberg et al., 1992, J Biol. Chem., 267: 564-9, Arriagada et al., 1992, Neurology, 42: 631-9 ). Pathological phosphorylation of tau is mediated by different kinases (Grundke-Iqbal et al., 1986, Proc Nall Acad Sci USA, 83: 4913-7), of which, the glycogen synthase kinase 3 ~ enzyme (GSK3 ~) is the main responsible (Martin et al., 2013, Ageing Res Rev,

12: 289-309). This enzyme is over-activated and over-expressed in AD (Leroy et al., 2007, Neuropa! Hol Appl Neurobiol, 33: 43-55), correlating the progression of its disease with cognitive impairment. In addition, the proapoptotic nature of tau contributes to the neuronal death that occurs in AD (Mines et al., 2011, In! J Alzheimers Dis, 2011: 861072). Therefore, GSK3 ~ constitutes an important target for the treatment of AD, since the inhibitors of this enzyme are able to reduce the neurodegeneration caused by tau biperphosphorylation and the formation of FTs. The EA is part of a set of pathologies known as taupatias, in which the biperphosphorylation of tau occurs and the subsequent formation of NFTs. Taupatias include, in addition to AD, amyotrophic lateral sclerosis, post-encephalic parkinsonism, progressive supranuclear paralysis, Gerstmann-Straussler-Scbeinker disease,

Hallervorden-Spatz, Creutzfeldt-Jakob disease Down syndrome,

Niemann-Pick disease and Pick's disease.

One of the main risk factors for the development of AD and other neurodegenerative diseases is oxidative stress, given the special vulnerability of the CNS against it. The main cellular defense against damage caused by reactive oxygen and nitrogen species (ERO and ERN) is the pathway

rt2-EpRE (Zhang et al., 2013, Prog Neurobiol 100: 30-47). Nrt2 (factor 2 related to the nuclear factor erythroid) is a transcription factor that induces the expression of antioxidant and anti-inflammatory enzymes through their binding to AD regulatory sequences known as electrophilic response elements (EpRE). Under normal conditions, rt2 is sequestered in the cytosol by the Keap 1 repressor protein, which promotes its ubiquitination and its degradation by the proteasome (ltoh et al. 1999, Gene Dev, 13: 76-86). In the presence of oxidizing or electrophilic species, rt2 dissociates from Keap 1 and translocates to the nucleus. Once there, it heterodimerizes with Maf proteins and e binds to EpRE sequences, inducing the expression of cytoprotective genes that allow the elimination of toxic stimulation (Nguyen et al., 2004, Free Radie Biol Med, 37: 433-41).

This important cell defense pathway is deregulated in several neurodegenerative diseases, such as AD, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis and stroke (Ramsey et al., 2007, J Neuropathol Exp Neurol, 66: 75 -85). In particular, in AD the Nrt2 factor is preferentially located in the cytosol, despite high levels of oxidative stress. The induction of this transcription factor favors neuronal survival and the improvement of cognitive abilities in different models of AD (Kanninen et al., 2008, Mol Ce! L Neurosei, 39: 302-1 3, Kanninen et al., 2009 , Proe Natl Aead Sei USA, 106: 16505-10). On the other hand, Nrt2 facilitates the autophagy removal of phosphorylated tau protein (Jo et al., 2014, Na! Cornrnun, 5: 3496). Therefore, the induction of rt2 has positioned itself as a promising neuroprotective target for the treatment of AD and other neurodegenerative diseases.

The GSK3p enzyme is one of the factors that induce the cytosolic location of the rt2 factor observed in AD, despite the high oxidative stress that occurs in this disease. The inhibition of this enzyme with lithium favors nuclear accumulation and increased transcriptional activity of Nrt2 (Red et al., 2008, Mol Cel! Neurosci, 39: 125-32). This kinase is capable of negatively regulating Nrt2 through two mechanisms; a) on the one hand, GSK3p phosphorylates directly to Nrt2 (Red et al., 2008, J Neurochem, 105: 192-202), so that it is recognized by ubiquitin ligase p-TrCP (Cbowdhry et al., 2013, Oncogene , 32: 3765-81), promoting its ubiquitination and its consequent degradation by the proteasome. b) On the other hand, GSK3p is capable of activating the Fyn kinase, which translocates to the nucleus, inducing the export of rt2 to the 01 cite (Jain et al., 2007, J Biol Chem,

282: 16502-10), again to be degraded by the proteasome. In this way, the overactivation and overexpression of GSK3p in AD contributes to further increase the levels of oxidative stress and with it, neurodegeneration.

On the other hand, both in AD and in other neurodegenerative diseases, oxidative stress and the formation of aberrant protein aggregates produce a glial overactivation generating a chronic neuroinflammation situation (Glass et al., 2010, Cel !, 140: 918-34 ). As a consequence of this neuroinflammation, more protein aggregates and oxidizing species are generated, exacerbating neuronal death. The over-activation of GSK3p increases microglial activation in several neurodegenerative diseases, including AD (Morrison et al., 2002, Adv Exp Med Biol, 513: 41-86). This kinase increases the expression of proinflammatory cytokines such as IL-lp, IL-6 YTNFa (Martin et al., 2005, Natlmmunol, 6: 77784), Yde oxidative stress generating enzymes such as iNOS (Yuskaitis et al., 2009, Cel! Signal, 21: 264-73), while lowering levels of anti-inflammatory cytokines such as IL-I O (Martin et al., 2005, Natlmmunol, 6: 777-84). In this sense, the inhibition of GSK3p increases the tolerance of astrocytes to inflammatory stimuli (Beurel et al., 2010, Neuroscience, 169: 1063-70), reduces the production of proinflammatory cytokines (IL-6, IL-12 Y lNFy) And increases the levels of the anti-inflammatory cytokine IL-IO in mice treated with lipopolysaccharide (LPS) (Martin et al., 2005, Nat Immunol, 6: 777-84). Therefore, GSK3p also contributes to the neuroinflammation that occurs in AD and other neurodegenerative diseases.

Finally, there is a balance between the rt2-EpRE pathway and that of the transcription factor NF-KB (main effector of inflammation) (Bellezza et al., 2010, Cancers (Base /), 2: 483-97), which induces the expression of proinflammatory cytokines and iNOS. Therefore, the dysfunction of the Nrt2-EpRE in AD and the negative regulation of Nrt2 mediated by GSK3p contribute significantly to the neuroinflammatory component of these pathologies.

In the patent documents W02014059383, W02012050517, EP2321295, W02009156859, W02011156889, W02012116362, W02012145420 W02012149478, W02013067036, W02013132124, P201300667, P20140084 and various products or treatments based on various chemical or natural compounds in various diseases which owe their action to the inhibition of the GSK3f3 enzyme or the induction and / or modulation of JUta Nrt2-EPRE as a neuroprotective strategy.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the use of compounds with structure 3-alkyl 1-6-amino4-aryl-l, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile with inhibitory capacity of the GSK3 enzyme (3, and / or with the inductive capacity of JUta rt2-EPRE, with the antioxidant, anti-inflammatory and neuroprotective effects that this entails.In the present invention, the inclusion of the inhibitory capacity of the GSK3f3 enzyme and the inducing capacity are described for the first time. of the ril transcription factor, in a single molecule, thanks to the structural modifications made, to give rise to a new compound that includes these activities.The compounds object of the present invention have an inductive capacity of rt2, so they can potentially be useful in the preventive and / or therapeutic treatment of neurodegenerative diseases More specifically, the object of the present invention is to provide new compounds useful as active ingredients of a medicament, that allow the prevention and / or treatment of diseases such as AD.

Therefore, in one aspect, the invention relates to a compound of formula

(1) (defined below), its salts, prodrugs or olvates. Said compound of formmu (I) can be used in the treatment of neurodegenerative diseases and / or diseases that are related to a dysfunction of the tau protein causing its aggregation such as the so-called taupatias such as AD, lateral sclerosis amyotrophic, post-encephalic parkinsonism, progressive supranuclear paralysis, Gerstmann-Straussler-Scheinker disease,

5 Hallervorden-Spatz disease, Creutzfeldt-Jacob disease, Down syndrome, iemann-Pick disease and Pick disease.

In one aspect, the invention includes a pharmaceutical composition that

comprises a compound of formula (I), or a salt, a prodrug or a solvate

pharmaceutically acceptable thereof.

In another aspect, the invention relates to a pharmaceutical composition comprising a compound of formula (1), or a salt, a prodrug or a solvate thereof, and a pharmaceutically acceptable carrier.

In another aspect, the invention protects the use of said compound of formula (1), or its pharmaceutically acceptable salts, prodrugs or solvates, in the preparation of a pharmaceutical composition for the prevention or treatment of

neurodegenerative diseases, or in ischemic-cerebral diseases. In the context of the present invention, the following terms have the meaning detailed below: When the term "independently selected" is used, the substituents referred to (eg R groups, such as R groups "R2, R3 , R., ¡Rs, ~

or X or Y or Z or variables such as "n") may be identical or different, or where appropriate when specified. The term "alkyl" refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms that do not contain

25 unsaturations, having one to eight carbon atoms and which is linked to the rest of the molecule by a single bond. Preferably, it refers to a linear or branched aliphatic chain radical having between 1 and 6, preferably between 1 and 3 ("C'_3 alkyl") carbon atoms, and which is attached to the rest of the molecule by a simple bond. This term includes, for example and in a non-limiting sense,

Methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. The alkyl radicals may be optionally substituted by one or more substituents independently selected from the group consisting of halogens, hydroxyl, alkoxides, carboxy, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "alkoxy" refers to a group -O-alkyl, where alkyl is as previously defined. Preferably alkoxy is methoxy.

The term "halogen" refers to bromine, chlorine, iodine or fluorine. Preferably, halogen and fluorine or chlorine or bromine.

The term "haloalkyl" refers to an alkyl radical, as previously defined, which is substituted by one or more halogens, as they have also been previously defined, including for example, and in a non-limiting sense, trifluoromethyl, trichloromethyl, 2 , 2,2, -trifluoroethyl, l-fluoromethyl-2-fluoroethyl, etc.

The term "alkoxycarbonyl" refers to a radical of formula -C (O) OR where R is an alkyl radical as previously described. Alkoxycarbonyl radicals may include, for example, and in a non-limiting sense, methoxy, ethoxy, propoxy, etc.

The term "cycloalkyl" refers to a saturated or partially saturated mono or polycyclic aliphatic group, having between 3 and 10, preferably between 3 and 6 carbon atoms that is attached to the rest of the molecule by means of a single bond, including for example, and in a non-limiting sense, cyclopropyl, cyclobutyl, cyclobexyl, cyclopentyl, etc.

The term "amino" refers to a radical of the formula -NH2 •

The term "aryl" refers to an aromatic group having between 6 and 18, preferably between 6 and 10 carbon atoms, comprising 1, 2 or 3 aromatic nuclei, linked by means of a carbon-carbon bond or condensates, including for example and in a non-limiting sense phenyl, naphthyl, diphenyl, indenyl, phenanthryl, etc.

The term "heterocycle" refers to a 3 to 10 membered ring radical, preferably a 5 or 6 membered ring, consisting of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and which may be partially or fully saturated, or it may be aromatic ("beteroaryl"). For the purposes of this invention, the heterocycle may be a monocyclic, bicyclic or tricyclic ring system, which may include condensed ring systems. Examples of such beterocycles include, but are not limited to, pyrrolidine, piperidine, piperazine, morpholine, tetrabidrofuran, benzimidazole,

benzothiazole, furan, pyrrole, pyridine, pyrimidine, isothiazole imidazole, indole, puTina,

quinoline, thiadizol. As understood in this technical area, there may be a certain degree of substitution in the radicals defined above. The references of the present

With respect to the substituted groups, they indicate that the specified radical may be substituted in one or more positions available with one or more substituents. Such substituents include, for example, and in a non-limiting sense, C6-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl, aryl, heterocycle, halogen, -CN, -N02, -CF3, -N ( Ra) (Rb), -OR :, -SR !, -C (O) Re, -C (O) OR [,

10 C (O) (Rg) (Rh), -OC (O) R¡; in which Ra. Rb Re, R !, Re, Rr, Rg, Rh and R¡ are independently selected from hydrogen, alkyl, C¡-C6, aryl, heterocycle and trifluoromethyl.

One aspect of the present invention relates to a compound of formula (1):

where

(one)

I

I

R and R¡ are selected from the group consisting of: A hydrogen atom;

Alkyl optionally substituted by one, two, or three halogen atoms selected from fluorine, chlorine and bromine; cycloalkyl (C3-C6); alkoxy (C¡

C6); cycloalkoxy (C3-C6); cyano and nitro; and / or phenyl optionally substituted by one, two or three groups selected independently from fluorine; chlorine; bromine; optionally alkyl

I

substituted by one, two, or three halogen atoms selected from fluorine,

I

chlorine and bromine; cycloalkyl (C) -C6); (C¡-C6) alkoxy; cycloalkoxy (C) -C6); cyano and nÜro; or a heteroaryl group selected from 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinjlo, 2-pyrazinyl, 3-pyrazinyl, 2-pyrrolyl, 3-pyrrolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 3isothiazolyl, 4-isothiazoyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 3pyrazolyl, 4-pyrazolyl, 1,2,3-oxadiazol-4-yl, 1,2,3-oxadiazol-5-yl, 1,2,4oxadiazol-3-yl, l, 2,4-oxadiazol-5-yl, l, 2,5-oxadiazol-3-yl, 1,3,4oxadiazol-2-yl, 1,2,3- thiadiazol-4-yl, 1,2,3-thiadiazol-5-yl, 1,2,4-thiadiazol3-i lo, 1,2,4-thiadiazol-5-yl, 1,2,5-thiadiazol-3 -yl, 1,3,4-thiadiazol-2-yl, 1,2,3-triazol-4-yl, 2-4-triazol-3-yl and 5-tetrazolyl, the heteroaryl group being optionally substituted by one, two or three groups independently selected from fluorine, chlorine, bromine, alkyl, cycloalkyl (C) -C6), alkoxy (C ¡-C6), cycloalkoxy10 (C3-C6), cyano and nitro; R2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl or heteroaryl, optionally substituted by one, two or three groups independently selected from fluorine, chlorine, bromine, alkyl,

I

alkoxy, cycloalkyl (C3-C6) cycloalkoxy (C3-C6), cyano, nitro and carboxylate or;

R) is selected from the group consisting of hydrogen, (C-C6) alkyl, cycloalkyl (C) -C6) and phenyl, optionally substituted by one, two or three I groups independently selected from fluorine, chlorine, bromine,

(C ¡-C6) alkyl, (C¡-C6) alkoxy, cycloalkyl (C3-C6), (C) -C6 cycloalkoxy), cyano, nitro and carboxylate;

I

~ and Rs are selected from the group consisting of hydrogen, alkyl, (C3-C6) cycloalkyl, acetyl, phenyl or heteroaryl, optionally substituted

by one, two or three groups independently selected from fluorine, chlorine, bromine, (C¡-C6) alkyl, a1coxyl (C¡-C6), cycloalkyl (C3-C6),

cycloalkoxy (C) -C6), or two groups can together form a

I

cycloalkyl group (C} -C6) or - (CH2k (where n = O, 1, 2, 3, 4, 5, 6) 0 -

CH = CH-CH = CH-;

X is selected from an oxygen atom, a nitrogen atom or a

sulfur atom, -SO- or S02;

5

and is selected from an oxygen atom, a nitrogen atom or a

sulfur atom, -SO- or S02;

and its salts, preferably pharmaceutically acceptable salts, prodrugs or

Solvates

The term "pharmaceutically acceptable" preferably refers to

I O

molecular compositions and entities that are physiologically tolerable and not

normally produce an allergic reaction or a similar unfavorable reaction, such

such as gastric disorders, dizziness and the like, when administered to a human being or

animal. The term "pharmaceutically acceptable" means that it is approved by

a regulatory agency such as the European medicine agency or agency

fifteen

US regulator, or that is included in the US Pharmacopoeia or other

Pharmacopoeia generally recognized for use in animals and, more

Particularly in human beings.

The term "salts" as used herein refers to any salt of the

compound of formula (1) which, when administered to a subject, is capable of

twenty

providing (directly or indirectly) said compound of formula (1). The term

"subject" includes any animal, for example, a mammal, including beings

humans. The preparation of said salts can be carried out by methods

Conventionals known to those skilled in the art. By way of illustration, a salt

of a compound of formula (1) can be synthesized by conventional methods to

25

starting from a compound of formula (1) containing a basic moiety. Usually,

such salts are prepared, for example, by reacting the free base forms of

the compounds of formula (1) with a stoichiometric amount of the appropriate acid

in water or in an organic solvent or in a mixture of water and a solvent

organic. In general, non-aqueous medium such as ether, ethyl acetate, are preferred.

30

ethanol, isopropanol or acetonitrile.

In a particular embodiment, said salts of the compound of formula (1) are

pharmaceutically acceptable salts, that is, salts that can be administered to a

subject and provide a compound of formula (1) in a biological compartment of said individual. Such pharmaceutically acceptable salts include acid addition salts, for example, salts formed from organic and inorganic acids, such as hydrobromic, hydrochloric, nitric phosphoric, sulfuric, acetic, adipic, aspartic benzenesulfonic, benzoic, citric, ethanesulfonic, formic, fumaric, glutamic, lactic, maleic, malic, malonic, mandelic, methanesulfonic, 1,5-naphthalenedisulfonic, oxalic, pivotal, propionic, p-toluenesulfonic, succinic, tartaric and the like, as well as metal salts, being selected the metal between sodium, pota io, lithium, calcium, magnesium, zinc, aluminum and the like, or ammonium salts,

or a salt formed from organic bases, such as 2-amino-l-butanol, 2-arnyl-2-ethyl-l, 3-propanediol, 2-amino-2-methyl-1, 3-propanediol, benzathine, benzyldimethylamine, c1oroprocaine , choline, dibenzylmethylamine, diethanolamine, diisopropanolamine, ethylenediamine dimethylsteramine meglumine, 2-methyl-2-amino-l-propanol, a monoamine-glycol, monoethanolamine, monoisopropanolamine, morpholine, N, dibenzylethyl-2-amino-amino-amino-amino -l-propanol, N, -dimethylaniline, procaine, pyridine, quinoline, t-butyl dimethylamine, triethanolamine, triethylamine, trihydroxymethylaminomethane, triisopropanolamine, trimethylamine and the like, and salts with amino acids such as glycine, lysine, arginine, taurine, histurine, taurine Alanine, valine, cysteine and the like.

In another particular embodiment, some substituent groups may be added to the compounds object of the invention, to make them susceptible to salt formation. For example, acid functional groups that can form stable salts with cations and basic functional groups that form stable salts with acids. Generally, there must be a difference of at least three units in the pKa values of the compound used as a drug and the counterion. For derivative compounds that are very weak bases, the choice to form salts is preferably a strong acid, such as hydrochloric acid (pKa = -6.1), sulfuric acid (pKal = -3.0, pKa2 = 1.96), or methanesulfonic acid (pK. = -1,2) to ensure the protonation of the compound. Compounds that are more basic can form salts with weak acids, such as phosphoric acid (pKa l = 2 15, pKa2 = 7.2, pKa3 = 12.38), tartaric (pK. = 2.93), acetic (pK . = 476), and benzoic (pKa = 4.2). For compounds that are very weak acids, strongly basic cations, such as sodium (PK. = 14.8), potassium (pK. = 16.0) or calcium (pKa = 12.9), are preferred to ensure deprotonation of the compound. . Compounds that are more acidic can form salts with weaker cations, such as zinc (PKa = 8.96), choline (pKa = 18.9) and diethanolamine (pK. = 9.65). Representative functional groups for the formation of stable salts listed based on the relative value of their acid-base strength include, but are not limited to, suLphonic acid (PKat = -1.2, pKa2 = -0.7), carboxylic acid (pKa = 4, 7) and imide (pKa = 8.2).

In another particular embodiment, said salts of the compound of formula (I) are pharmaceutically acceptable salts, which may be useful in the preparation of pharmaceutically acceptable salts of the compound of formula (1), or of its prodrugs or solvates.

The term "prodrug" is used, in this description, in the broadest sense, and includes any compound derived from a compound of formula (1) which, when administered to a subject, is capable of providing, directly or indirectly, a compound of formula (I), or a pharmaceutically acceptable salt thereof, in said subject. Advantageously, said derivative is a compound that increases the bioavailability of the compound of formula (I) when administered to a subject (for example, by making a compound of formula (l) administered orally more easily absorbed by blood), or that enhances the release of a compound of formula (1) in a biological compartment (for example, the brain or the

lymphatic system) in relation to the original compound (without derivatizing). The nature of said derivative is not critical, as long as it can be administered to a subject and

I

provide a compound of formula (1) in a biological compartment of said subject. Such derivatives will be apparent to those skilled in the art, and include, depending on the functional groups present in the molecule and without limitation,

following derivatives of the present compounds: esters, amino acid esters, phosphate esters, sulphonate esters of metal salts, carbamates and amides.

The preparation of said prodrugs can be carried out by conventional methods known to those skilled in the art. Said methods will be chosen according to the derivatization to be introduced in the compound of formula (I). Illustrative examples of some methods for producing prodrugs of active compounds I can be found, for example, in Krogsgaard-Larsen al. "Textbook of Drug

design and Discovery "Taylor & Francis (April 2002). In a particular embodiment,

I

said pro-drug is an amide and its obtaining is carried out by conventional methods of amide formation, for example, by reacting the compound of formula (1) as a free base with an organic acid or with an appropriate organic acid derivative, by For example, with a pharmaceutically acceptable organic acid such as acetic, adipic, aspartic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric formic, glutamic, lactic, maleic, malic, malonic, mandelic, methanesulfonic, 1,5-naphthalenedisulfonic, oxalic, pivotal , propionic, p-toluene ultrasonic, succinic, tartaric and the like.

The compounds of formula (1), or their salts, may be in crystalline form either as free compounds or as solvates, both forms being included within the scope of the present invention. The term "solvate" as used herein includes any compound formed by combining molecules of a solvent with molecules or ions of a compound of formula (1) or a salt thereof; said solvent may be an organic solvent, for example, an alcohol, or an aqueous solvent, for example, water, in which case the solvate is called "hydrate."

In a particular embodiment, said solvate is a pharmaceutically acceptable solvate, that is, it can be administered to a subject and provide (directly or indirectly) a compound of formula (1) or a salt thereof. In another embodiment

I

In particular, said solvate is not fanatically acceptable but can be used in the preparation of fannaceutically acceptable solvates of the compound of formula (1) or

I

of its salts. The preparation of said solvates can be carried out by conventional methods I known to those skilled in the art, by contacting the

compound of formula (1) or a salt thereof with the appropriate solvent. In a particular embodiment, the compounds of formula (1) or their salts,

I

prodrugs or solvates will preferably be in a pure or pharmaceutically acceptable form. A pharmaceutically acceptable form excluding normal pharmaceutical additives as diluents and carriers, and not including materials considered toxic at normal dosage levels. The level of purity of the compounds will preferably be greater than 50%, more preferably

equal to greater than 70%, even more preferably equal to greater than 90%. In a

I

I

preferred embodiment the purity of the compound of formula (1), or its salts, prodrugs

or solvates, will be greater than 95%.

The compounds of the present invention represented by the formula (I) described above may include enantiomers depending on the presence of chiral centers or geometric isomers, depending on the presence of multiple bonds (for example Z, E). The enantiomeric or diastereoisomeric geometric isomers of the compounds of formula (1) and mixtures thereof are within the scope of the present invention.

In another particular embodiment, the compounds subject to this invention give pharmaceutically acceptable compositions comprising the compounds of formula 1 with a pharmaceutically acceptable carrier, for example, pharmaceutical composition that includes one or more compounds of formula 1, alone or in combination with one or more additional therapeutic agents as a mixture with pharmaceutically acceptable excipients.

The term "pharmaceutically acceptable excipient" means one or more solids, or compatible liquids, diluents or encapsulation substances that are likely to be administered to an object.

In a particular preferred embodiment, the invention relates to compounds of

formula (1) in which: R is hydrogen; RI is selected from the group comprising an alkyl optionally substituted by one or two groups independently selected from fluorine, chlorine, and bromine; (C1-C6) alkoxy, nitro and amino. Preferably R is methyl; R2 is selected from the group comprising a phenyl aromatic ring optionally substituted by one, two or three groups independently selected from fluorine, chlorine, (C1-C6) alkyl, optionally substituted by one, two, or three halogen atoms selected from fluorine, chlorine and bromine; (C1-C6) alkoxy, cyano and nitro; or a heterocycle optionally substituted by one, two or three groups independently selected from fluorine, chlorine, (C 1 -C 6) alkyl, alkoxy and nitro; R3 is the cyano group; ~ and R5 are hydrogen;

X is nitrogen; and is oxygen;

and its salts, prodrugs or solvates, preferably, its pharmaceutically acceptable salts.

Particularly preferred compounds of formula (1) of the present invention are the following:

•

6-amino-4-phenyl-3-meti 1-1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-3-meti 1-4- (2-methoxy feni 1) -L, 4-dibydropyran [2,3-c] pyrazol-5carbonitrile.

•

6-amino-3-methyl-4- (3-methoxyphene1) -1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

•

6-amino-3-methyl-4- (4-methoxyphenyl) -1,4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

•

6-amino-3-methyl-4- (2-tol i 1) -1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-3-meti 1-4- (3-tol il) -1, 4-di bidropi rano [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-3-methyl-4- (4-tolyl) -1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-4- (4-fluorophenyl) -3-methyl-l, 4-dibydropyran [2,3-c] pyrazol-5carbonitrile.

I

•

6-amino-4- (2-cJorophenyl) -3-meti 1-1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amioo-4- (3-cJorophenyl) -3-methyl-1, 4-dibydropyran [2,3-c] pyrazol-5-carbonitrile. I

•

6-amino-4- (4-chloropheni 1) -3-meti 1-1, 4-dibydropyran [2,3-c] pyrazol-5-carbonitrile.

I

• 6-amino-4- (2-bromopheni 1) -3-meti1-1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

I

•

6-amino-4- (4-bromopheni 1) -3-meti1-1, 4-dibydropyran [2,3-c] pyrazol-5carbonitrile. I

•

6-amino-3-methyl-4- (2-o itrofeo i 1) -1, 4-di h idropi rano [2,3-c] pi razol-5-carbonitri lo.

•

6-amino-3-meti 1-4- (3-nitrofeoi 1) -1, 4-dibydropyran [2,3-c] pyrazol-5-carbonitri lo. I

•

6-amino-3-methyl-4- (4-0 itrofeoj 1) -1, 4-di bidropyran [2,3-c] pyrazol-5-carbonitrile.

I

•

6-amino-4- (2-furyl) -3-meti 1-1, 4-d and bidropi raoo [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-3-meti 1-4- (2-tieni 1) -1, 4-dibydropyran [2,3-c] pyrazol-5-carbonitri lo. I

I

I

•

6-am ino-3-meti 1-4- (3-pyridi 1) -1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-3-methyl-4- (4-pyridyl) -1,4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

The compounds of formula (1) of the present invention exhibit activity

5 GSK3f3 enzyme inhibitor and rt2 factor inducing activity, so they can be used in the prevention or therapy of neurodegenerative diseases, e.g., AD, amyotrophic lateral sclerosis, post-encephalic parkinsonism, progressive supranuclear paralysis, disease of Gerstmann-Straussler-Scheinker, the Hallervorden-Spatz ED, the Creutzfeldt-Jacob disease, the syndrome of

10 Down, iemann-Pick disease and Pick's disease and / or in the treatment of other neurodegenerative diseases in which neuron loss occurs, as is the case of stroke (stroke).

For administration to a subject in need of treatment, the compounds of formula (1) of the present invention are conveniently formulated.

15 with the excipients suitable for administration by any appropriate route, for example, orally, parenterally, subcutaneously, intramuscularly, intravascularly or rectally, preferably orally.

Therefore, in other aspects, the invention includes a pharmaceutical composition, hereinafter the pharmaceutical composition of the invention, which comprises a compound of formula (1), or a pharmaceutically acceptable compound thereof, together

with one or more pharmaceutically acceptable excipients.

In a particular embodiment, the pharmaceutical composition of the invention is presented in a pharmaceutical form of oral administration, either in solid or liquid form. Illustrative example of pharmaceutical forms of administration by

Oral routes include tablets, granulated capsules, solutions, uspensions, etc., for which they will include appropriate pharmaceutically acceptable excipients, such as binders, diluents, disintegrants, lubricants, bumectants, etc., and can be prepared by conventional methods. The pharmaceutical composition of the invention can also be adapted for parenteral administration (e.g., via

30 intramuscularly, intravenously etc.), in the form of, for example, sterile freeze-dried solutions, suspensions or products, in the appropriate dosage form; in this case,

said pharmaceutical compositions will include suitable excipients, such as

buffers, surfactants, etc. The pharmaceutical composition of the invention also

it can be adapted for subcutaneous administration in the form of, for example,

sterile solutions or suspensions, in the appropriate dosage form; in this

5

case, said pharmaceutical compositions will include suitable excipients, such

such as buffers, surfactants, etc. Also, the pharmaceutical composition of the

invention can be adapted for rectal administration for which

will include suitable excipients compatible with the compounds of formula (I) of

the invention. The formulations can be prepared according to conventional methods

THE

such as those described in the Spanish, European or State pharmacopoeias

United States of America, or similar reference texts, for example "Treaty of

Farmacia Galenica "by C. Faulí i Trillo, 10 Edition, 1993, Luzán 5, S.A. de

Editions

The compound of formula (1) of the invention will be administered in an amount.

fifteen

therapeutically effective that will generally depend on the efficacy of the compound of

formula (1) chosen, of the severity of the pathology to be treated, etc. However,

It will typically be administered at daily doses between 0.1 and 100 mg of

compound of formula (1) per kg of body weight, more preferably the doses

Daily will be between 2 and 5 mg / kg body weight.

twenty

In another aspect, the invention protects the use of a compound of formula (1),

its pharmaceutically acceptable salts, prodrugs or solvates, in the preparation of

a pharmaceutical composition for the prevention or treatment of a disease

neurodegenerative, such as AD, and / or an ischemic-cerebral disease (stroke).

Neurodegenerative diseases are those, often of cause

25

known, in which progressive degeneration of the nervous system occurs in

any of its parts or in its entirety. For a detailed description of them

see, for example, the monograph "Eurodegenerative diseases"

Coordinators José M "Segovia de Arana and Francisco Mora Teruel, edited by Serie

Farmaindu tria scientist, Madrid, July 2002.

30

Ischemic-cerebral diseases constitute an acute pathology, which

takes place as a result of the interruption of the blood supply to a party

of the brain, or when the rupture of a blood vessel occurs with the consequent

cerebral haemorrhage. Although these diseases are not included in the group of neurodegenerative diseases, it should be borne in mind that, secondary to an ischemic-cerebral accident, neurodegeneration also occurs in those affected areas. Hence the usefulness of treating these diseases with the compounds of the invention.

The administration of the compounds of formula (I) of the invention, their pharmaceutically acceptable salts, prodrugs or solvates, can be carried out alone or in combination with additional drugs such as drugs useful for the treatment of neurodegenerative disease or an ischemiccerebral disease, to provide a combination therapy; said additional drugs may be part of the same pharmaceutical composition of the invention which comprises the compound of formula (1) and / or its salts, pro-pharmaceutically acceptable drugs or solvates, or not, in which case, they will be administered simultaneously or sequentially. to the administration of the pharmaceutical composition of the invention. Illustrative, non-limiting examples of such additional drugs that may be employed to provide a combination therapy include agents such as memantine (an NMDA type glutamate receptor blocker approved for use in the advanced stages of Alzheimer's disease), vitamins , anti inflammatory or antidepressants.

EMBODIMENT OF THE INVENTION

The present invention is further illustrated by the following examples, which are not intended to be limiting in scope.

The compounds whose biological activity is the subject of the present invention were synthesized following the following organic synthesis procedures. General procedure: a solution of 5-methyl-lH-pyrazol-3 (2H) -one (1 equiv, mmo!), Malononitrile (1 equiv, lmo!), The corresponding aromatic aldehyde (1 equiv, l mmol) and Ammonium acetate (1 equiv, 1 mmo!) in ethanol (2 mL) was heated at reflux for 5 hours. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude was recrystallized from ethanol or purified by

column chromatography with Hice gel using dichloromethane / methanol mixtures

as eluent to obtain pure products.

Example 1: 6-amino-4-phenyl-3-methyl-1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

Following the procedure described, 5-methyl-lH-pyrazol-3 (2H) -one (98 mg, 1

mmol), malononitrile (66 mg, 1 mmol), benzaldehyde (106 mg, I mmol) and acetate

ammonium (77 mg, 1 mmo!) in EtOH (2 rnL). Reaction time: 5 h. Crystallized in

10 EtOH, to obtain compound 1 as a white solid (217 mg, 86%). Rf: 0.47 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) 8 1.78 (s, 3H, C-3CH)), 4.59 (s, IH, CH-4), 6.90 (s, 2H, C-6 H2 ), 7.11-7.27 (m, 3H, ArH), 7.27-7.38 (m, 2H, ArH), 12, II (s, lH, NH-1) ppm; 13C NMR (63 MHz, DMSO-do) 8 160.91 (C-6), 154.80 (C7a), 144.50 (C-4CAr), 135.61 (C-3), 128.49 (2xCHAr ), 127.52 (2xCHAr), 126.79

15 (CHAr), 120.88 (CN), 97.68 (C-3a), 57.15 (C-5), 36.25 (CH-4), 9.79 (C-3CH)) ppm; IR (KBr) v 3368, 3164 2191, 1647 cm · l; MS (API-ES +) miz: Tracing for CI4HI2 40: 252 1011; found: [(M + Ht] 253.1086; found: [(M + at J 275.0909. Elemental analysis calc. (%) for C14H12N40: C 66.65, H 4.79, 22.21; found: C 66.43, H 4.82, N 22.08;

Example 2: 6-amino-3-methyl-4- (2-methoxyphenyl) -1,4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

Following the crito procedure, 5-methyl-1 H-pyrazol-3 (2H) -one (98 mg, 1

mmo!), malononitrile (66 mg, 1 mmol) 2-methoxybenzaldehyde (136 mg, 1 mmol) and ammonium acetate (77 mg, l mmol) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 2 as a gray solid (245 mg, 87 5%). Rf: 0.40 (DCM-MeOH 8%); 'H NMR (250 MHz, DMSO-do) 8 1.78 (s, 3H, C3CH3), 3.78 (s, 3H, OCH3), 4.96 (s, IH, CH-4), 6.80 (s, 2H, C-6NH2), 6.89 (t, 1 = 7.3 Hz, IH, ArH), 6.94-7.04 (m, 2H, ArH), 7.19 (t, 1 = 7.6 Hz, IH, ArH), 12.00 (s, IH, NH-I) ppm; 3C NMR (63 MHz, DMSO-do) 8 16151 (C-6), 156.36 (C-7a *), 155.09 (ArCOCH3 *), 135.08 (C-3), 132.13 ( C-4CAr), 128.65 (CHAr), 127.95 (CHAr),

10 120.96 (CN), 120.84 (CHAr), 111.30 (CHAr), 97.85 (C-3a) 56.33 (ArCOCH3), 55.59 (C-5), 29.15 ( CH-4), 9.55 (C-3CH3) ppm; IR (KBr) v 3371, 3151, 2192, 1654, 1256 cm- '; MS (API-ES +) miz: Calc. For C '5H'4 40 2: 282,1117; found: [(M + Ht J 283,1195; found: [(M + at J 305,1039. Elemental analysis calc. (%) found C'5H'4N40 2: C 63.82, H 5.00, N 19 , 85; found: C 63.52, H 5.08, N 19.59.

Example 3: 6-amino-3-methyl-4- (3-methoxyphenyl) -1 4-dihydropyran [2,3-c Jpirazol-5carbonitrile.

I

I

Following the procedure described, 5-methyl-1 H-pyrazol-3 (2H) -one (98 mg, 1 I 20 mmol), malononitrile (66 mg, 1 mmol), 3-methoxybenzaldehyde (136 mg, 1 mmol) and

ammonium acetate (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 b. Flash chromatography DCMlMeOH (0-5%), to obtain compound 3 as a

I

pale yellow solid (235 mg, 83%). Rf: 0.40 (DCM-MeOH 8%); 'H NMR (250 MHz, DMSO-do) 8 1.81 (s, 3H, C-3CH)), 3.72 (s, 3H, OCH3), 4.57 (s, lH, CH-4)

I

25 6.69-6.75 (m, 2H, ArH), 6.80 (dd, 1 = 8.0, 1.8 Hz, lH, ArH), 6.88 (s, 2H, C-6NH2) , 7.24 (t, 1 = 8.0 Hz, LH, ArH), 12.10 (s, lH, H-1) ppm; \ 3C NMR (63 MHz, DMSO-

I

do) 8 160.93 (C-6), 159.24 (ArCOCH) *), 154.76 (C-7a *), 146.16 (C-4CAr), 135.62

(C-3), 129.62 (CHAr), 120.85 (CN), 119.70 (CHAr), 113.51 (CHAr), 111.67 (CHAr),

I

I

9750 (C-3a), 56.97 (C-5), 54.99 (ArCOCH3), 36, 18 (CH-4), 9.84 (C-3CH3) ppm; GO

(KBr) v 3383, 3161, 2184, 1641, 1263 cm-I; MS (API-ES +) miz: Calc_ for C1sHI4 402: 282 1117; found: [(M + Ht] 283,1223; found: [(M + a) J 305,1038. Elemental analysis calc_ (%) for ClsH14N402: C 63.82, H 5.00, N 19.85;

5 found: C 63.60, H 5.07, 19.51.

Example 4: 6-amino-3-methyl-4- (4-methoxyphenyl) -1,4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

OMe

Following the procedure described, 5-methyl-lH-pyrazol-3 (2H) -one (98 mg, I mmol), malononitrile (66 mg, 1 mmol), 4-methoxybenzaldehyde (136 mg, 1 mrnol) and ammonium acetate (77 mg, I mmol) in EtOH (2 mL). Reaction time: 5 h. Flash chromatography DCMlMeOH (0-5%), to obtain compound 4 as a yellow solid (200 mg, 71%). Rf: 0.40 (DCM-MeOH 8%); IH NMR (250 MHz, 15 DMSO-d6) b 1.78 (s, 3H, C-3CH3), 3.73 (s, 3H, OCH3), 4.53 (s, lH, CH-4), 6 , 83 (s, 2H, C-6NH2), 6.87 (d, J = 8.7 Hz, 2H, ArH), 7.07 (d, J = 8.7 Hz, 2H, ArH), 12, 07 (s, lB, Hl) ppm; I3C NMR (63 MHz, DMSO-d6) b 161.04 (C-6), 158.31 (ArCOCH3 *), 155.10 (C-7a *), 136.85 (C-4CAr), 135.90 (C-3), 128.86 (2xCHAr), 121.22 (CN), 114.11 (2xCHAr), 98.24 (C-3a), 57.90 (C-5), 55.35 (ArCOCH3 ), 35.78 (CH-4), 10.12

20 (C-3CH3) ppm; IR (KBr) v 3231 3122, 2190, 1639, 1258 cm-I; MS (API-ES +) miz: Calc. For C1sHI4 40 2: 282,1117; found: [(M + Ht J 283,1200; found: [(M + at J 305,1024. Elemental analysis calc. (%) for ClsH14N40 2: C 63.82, H 5.00, N 19.85; found : C 63.73, H 4.98, 19.62.

Example S: 6-amino-3-methyl-4- (2-tolyl) -1,4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

Following the procedure described, 5-methyl-1 H-pyrazol-3 (2H) -one (98 mg, 1 mmo!), Malononitrile (66 mg, 1 mmol), 2-methylbenzaldebide (120 mg, I mmol) and acetate ammonium (77 mg, I mmol) in EtOH (2 mL). Reaction time: 5 h.

5 Crystallized from EtOH, to obtain compound 5 as a gray solid (235 mg, 88%). Rf: 0.44 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) 15 1.68 (s, 3H, C3CH3), 2.28 (s, 3H, ArCCH3), 4.84 (s, IH, CH-4), 6.86 ( s 2H, C-6NH2), 6.96-7.01 (m, IH, ArH), 7.09-7.18 (m, 3H, ArH), 12.09 (s, IH, HI) ppm; 13C NMR (63 MHz, DMSO-do) 15 160.80 (C-6), 155.11 (C-7a), 141.95 (C-4CAr), 135.39 (ArCCH3 *),

10 135.01 (C-3 *), 130.54 (CHAr), 128.95 (CHAr), 126.64 (CHAr), 126.39 (CHAr), 120.81 (CN), 97.58 ( C-3a), 56.69 (C-5), 33.02 (CH-4), 18.99 (ArCCH3), 9.60 (C3CH3) ppm; IR (KBr) v 3362, 3152, 2191.1650 cm · l; MS (API-ES +) miz: Calc. For C1sHI4 40: 266.1168; found: [(M + Htl 267, 1229; found: [(M + atl 289,1055. Elemental analysis calc. (%) for C 1sH I4 40: C 67.65, H 5.30, N 21.04;

15 found: C 67.51, H 5.31, 20.99.

Example 6: 6-amino-3-meti 1-4- (3-tol i 1) -1,4-dihydropyran [2,3-c lpi razol-5-carbonitri lo.

I

I

Following the procedure described, 5-methyl-1H-pyrazol-3 (2H) -one (98 mg, 1

20 mmol), malononitrile (66 rng, I mmol), 3-rnetylbenzaldehyde (120 mg, I mmol) and ammonium acetate (77 mg, I mmol) in EtOH (2 mL). Reaction time: 5 h. I Crystallized from EtOH, to obtain compound 6 as a yellow solid (223 mg,

I

84%) Rf: 0.44 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) 15 1.78 (s, 3H, C3CH3), 2.27 (s, 3H, ArCCH3), 4.54 (s, 1H, CH-4), 6.87 ( s, 2H, C-6 H2), 6.92-7.00

I

(m, 2H, ArH), 7.03 (d, J = 7.6 Hz, lH, ArH), 7.20 (t, J = 7.8 Hz, IH, ArR), 12.09 (s, lH, NH-1) ppm; 13C NMR (63 MHz, DMSO-d6) 8 160.88 (C-6), 154.76 (C-7a), 144.47 (C-4CAr), 137.53 (ArCCH3 *), 135.58 ( C-3 *), 128.32 (CHAr), 127.92 (CHAr), 127.49 (CHAr), 124.72 (CHAr), 120.89 (eN), 97.70 (C-3a), 57.18 (C-5), 36.20 (CH-4), 21.12

5 (ArCCH3), 9.81 (C-3CH3) ppm; IR (KBr) v 3366.3175, 2191.1646 cm-I; MS (API-ES +) miz: Calco for CIsHI4 40: 266,1168; found: [(M + Htl 267,1276; found: [(M + atJ 289,1103. Calcium elementary analysis (%) for ClsH14N40: C 67.65, H 5.30, 21.04; found: C 67.66 , H 5.41, 20.82.

Example 7: 6-amino-3-methyl-4- (4-to1-yl) -1,4-dihydropyran [2,3-c lpyrazol-5-carbonitrile.

I

15 20 25

Following the procedure described, 5-methyl-1H-pyrazol-3 (2R) -one (98 mg, I m01ol), malononitri10 (66 mg, I O1mol), 4-methylbenzaldebide (120 mg, 1 mmol) and ammonium acetate ( 77 mg, 1 mL01) in EtOH (2 mL). Reaction time: 5 b. Crystallized from EtOH, to obtain compound 7 as a white solid (215 mg, 81%). Rf: 0.42 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-d6) 81.78 (s, 3H, C3CH3), 2.27 (s, 3H, ArCCH3), 4.54 (s, lH, CH-4), 6.84 (s , 2H, C-6NH2), 7.04 (d, J = 7.8 Hz, 2H, ArR), 7.12 (d, J = 7.8 Hz, 2H, ArR), 12.08 (s, lH, Hl) ppm; 13c NMR (63 MHz, DMSO-d6) 8 160.78 (C-6), 154.77 (C-7a) 141.52 (C-4CAr), 135.73 (ArCCH3 *), 135.54 (C -3 *), 129.02 (2xCHAr), 127.38 (2xCHAr), 120.85 (eN), 97.74 (C-3a), 57.34 (C-5), 35.85 (CH- 4) 20.66 (ArCCH3) 9.78 (C-3CH3) ppm; IR (KBr) v 3372, 3184, 2191.1645 cm- '; MS (APT-ES +) miz: Calco for ClsH 14N40: 266.1168; found: [(M + Ht l 267,1241; found: [(M + Natl 289,1163. Elemental analysis cale. (%) for CIsH I4 40: C 67,65, H 5,30, 21.04; found : C 67.55, H 5.26, 21.03. I I I

I

I

I

I

I

I

Example 8: 6-amino-4- (4-fluorophenyl) -3-methyl-1, 4-dihydropyran [2,3-c] pyrazol-5

carbonitrile

F

Following the procedure described, 5-methyl-1H-pyrazol-3 (2H) -one (98 mg, l

Rmol), malononitrile (66 mg, 1 mmol), 4-fluorobenzaldehyde (124 mg, 1 mmol) and ammonium acetate (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 h. Flash chromatography DCMlMeOH (0-4%), to obtain compound 8 as a pale yellow solid (197 mg, 73%). Rf: 0.46 (DCM-8% MeOH); 'H NMR (250 MHz, DMSO-do) 8 1.78 (s, 3H, C-3CH) 4.63 (s, 1H, CHA), 6.91 (s, 2H, C-6NH2),

10 7.09-7.25 (m, 4H, ArH), 12.13 (s, IH, NH-1) ppm; 13C NMR (63 MHz, DMSO-d6) 8 160.97 (d, J = 242.4 Hz, ArCF), 160.86 (C-6), 154.73 (C-7a), 140.71 (d , J = 3.0 Hz, C4CAr), 135.66 (C-3), 129.38 (d, J = 8.2 Hz, 2xCHAr), 120.77 (eN), 115.21 (d, J = 21.4 Hz, 2xCHAr), 97.52 (C-3a) 57.04 (C-5), 35.44 (CH-4), 9.77 (C-3CH) ppm; '9F NMR (235 MHz, DMSO-do) 8-116.64 (m) ppm; IR (KBr) v 3220, 3122, 2193, 1642

15 cm "; MS (API-ES +) miz: Cale. For C'4HIIFN40: 270.0917; found: [(M + Ht] 271.099; found: [(M + Nan 293.0814. Anal is elemental cale. ( %) for C'4H "FN40: C 62.22, H 4.10, N 20.73; found: C 62.26, H 4.15, N 20.62.

Example 9: 6-aminoA- (2-c1orophenyl) -3-methyl-1 4-dihydropyran [2,3-c] pyrazol-5

20 carbonitrile. 4'

Following the procedure described, 5-meti1-1H-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitrile (66 mg, 1 mmo1), 2-c1-benzobenzaldehyde (141 mg, 1 rmol) and ammonium acetate ( 77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 9 as a white solid (243 mg, 85%). Rf: 0.47 (DCM-MeOH 8%); IH NMR (250 MHz, DMSO-do) 8 1.76 (s, 3H, C3CH3), 5.06 (s, IH, CH-4), 6, 96 (s, 2H, C-6NHÜ, 7.11-7.37 (m, 3H, ArH), 7.41 (d,

J = 7.3 Hz, LH, ArH), 12.13 (s, 1 H, H-1) ppm, 13 C NMR (63 MHz, DMSO-d6) 8 161, 32 (C-6), 154.97 (C-7a), 140.96 (C-4CAr), 135.39 (C-3), 131, 97 (ArCCl), 130.76 (CHAr), 129.52 (CHAr), 128.64 (CHAr ), 127.81 (CHAr), 120.47 (CN), 96.87 (C-3a), 55.68 (C-5), 33.48 (CH-4), 9.57 (C-3CH3 ) ppm; 1R (KBr) v 3385, 3156 2187, 1650,

l

1045 cm MS (API-ES +) miz: Tracing for C14HI ICIN40: 286.0621; found:

10 [(M + Ht J 287.0679; found: [(M + Na) j 309.0527; found: [(2M + Ht J 573.1356; found: [(2M + at J 595.1180. Calcium elemental analysis. (%) for CI4H IICI 40: C 58.65, H 3.87, 19.54; found: C 58.26, H 3.91, N 19.44.

Example 10: 6-amino-4- (3-chlorophenyl) -3-methyl-1, 4-dihydropyran [2,3-c] pyrazol-515 carbonitrile.

Following the procedure described, 5-methyl-IH-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitrile (66 mg, 1 mmol), 3-chlorobenzaldebide (141 mg, 1 rmol) and ammonium acetate ( 77 mg, 1 mmo \) in EtOH (2 rnL). Reaction time: 5 b.

Crystallized from EtOH, to obtain compound 10 as a pale yellow solid (220 mg, 77%). Rf: 0.47 (DCM-8% MeOH); IH R.MN (250 MHz, DMSO-do) 8 1.80 (, 3H, C-3CH3), 4.66 (s, IH, CH-4), 6.99 (s, 2H, C-6NH2) 7.14 (d, J = 7, l Hz, lH, ArH), 7.20 (s, IH, Arl!), 7.25-7.45 (m, 2H, Arl!), 12.18 ( s, IH, NH-I) ppm; 13C NMR (63 MHz, DMSO-do) 8 161.06 (C-6), 154.73 (C-7a), 147.14 (C-4CAr), 135.78 (C-3),

25 133.09 (ArCCI), 130.55 (CHAr), 127.29 (CHAr), 126.90 (CHAr), 126.37 (CHAr), 120.75 (CN), 97.07 (C-3a ), 56.47 (C-5), 35.82 (CH-4), 9.83 (C-3CH3) ppm; 1R (KBr) v 3366, 3175, 2191, 1646, 1070 cm · l; MS (API-ES +) miz: Calc. For C1 4H IICIN40: 286.0621; found: [(M + H) j 287.0708; Found: [(M + a) j 309.0538.

Calcium elementary analysis (%) for C'4HIICIN40: C 58.65, H 3.87, 19.54;

Found: C 58.42, H 3.86, N 19.45.

Example 11: 6-amino-4- (4-chlorophenyl) -3-methyl-l, 4-dibydropyran [2,3-c] pyrazol-55 carbonitrile.

he

Following the procedure described, 5-methyl-IH-pyrazol-3 (2H) -one (98 mg, 1

mmol), malononitrile (66 mg, 1 mmo \), 4-chlorobenzaldebide (141 mg, 1 mmol) and

acetate

ammonium (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 b.

the

Crystallized from EtOH, to obtain compound 11 as a white solid (252 mg,

88%) Rf: 0.49 (DCM-8% MeOH); 'H NMR (250 MHz, DMSO-do) 8 1.79 (s, 3H, C

3CH3), 4.63 (s, I H, CH-4), 6.94 (s, 2H, C-6NH2), 7, 19 (d, J = 8.4 Hz, 2H, ArH), 7.38

(d, J = 8.4 Hz, 2H, ArH), 12.14 (s, IH, NH-1) ppm; \ 3C NMR (63 MHz, DMSO-do) 8

160.69 (C-6), 154.48 (C-7a), 143.29 (C-4CAr), 135.46 (C-3), 131, 01 (ArCCI), 129.16

fifteen

(2xCHAr), 128.25 (2xCHAr), 120.46 (eN), 96.98 (C-3a), 56.50 (C-5), 35.33 (CH-4),

9.53 (C-3CH3) ppm; IR (KBr) v 3404, 3166, 2185, 1649, 1075 cm- '; MS (API-ES +)

miz: Tracing for C'4H "CI

40: 286.0621; Found: [(M + H) J 287.0666; found:

[(M + Nat] 309.0493. Calcium elementary analysis (%) for C '~ IICIN40: C 58.65, H

3.87, N 19.54; Found: C 58.55, H 3.88, N 19.55.

twenty

Example

12: 6-amino-4- (2-bromophenyl) -3-methyl-1, 4-dibydropyran [2,3-c] pyrazol-5

carbonitrile

Following the procedure described, 5-methyl-IH-pyrazol-3 (2R) -one (98 mg, 1

mmol), malononitrile (66 mg, 1 mmol), 2-bromobenzaldehyde (185 mg, 1 mmol) and ammonium acetate (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 12 as a white solid (271 mg, 5 82%). Rf: 0.49 (DCM-8% MeOH); 'H NMR (250 MHz, DMSO-d6) or 1.76 (s, 3H, C3CH3), 5.07 (s, IH, CH-4), 6.97 (s, 2H, C-6NH2), 7, 07-7.27 (m, 2H, ArR), 7.35 (t, J = 7.4 Hz, IR, ArR), 7.58 (d, J = 7.7 Hz, IH, ArR), 12 , 15 (s, lH, NH-2) ppm; 13c NMR (63 MHz, DMSO-do) o161, 24 (C-6), 154.91 (C-7a), 142.60 (C-4CAr), 135.42 (C-3), 132.66 ( CHAr), 130.99 (CHAr), 128.95 (CHAr), 128.45 (CHAr), 122.44

10 (ArCBr) 120.38 (CN), 97.06 (C-3a), 55.92 (C-5), 35.86 (CH-4), 9.71 (C-3CH3) ppm · IR ( KBr) v 3386, 3142, 2187, 1651, 1047 cm- '; MS (API-ES +) miz: Calco for C'4H I, BrN40: 330.0116; found: [(M + Hn 331.0198; found: [(M + Nat] 353,0007. Calcium elementary analysis (%) for C'4HI, BrN40: C 50.77, H 3.35, 16.92; Found: C 50.61, H 3.34, N 16.81

Example 13: 6-amino-4- (4-bromophenyl) -3-methyl-1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

20 Following the procedure described, 5-methyl-lH-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitril0 (66 mg, 1 romol), 4-bromobenzaldehyde (185 mg, 1 rumo \) and acetate ammonium (77 mg, 1 mmo \) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 13 as a pale yellow solid (305 mg, 92%). Rf: 0.50 (DCM-MeOH 8%); IH NMR (250 MHz, DMSO-d6) or 1.79 25 (s, 3H, C-3CH3), 4.62 (s, IH, CH-4) 6.95 (s, 2H, C-6NH2), 7 , 14 (d, J = 8.4 Hz, 2H, ArCR), 7.51 (d, J = 8.4 Hz, 2H, ArCR), 12.15 (s, lH, NH-1) ppm, 13c NMR (63 MHz, DMSO-d6) or 160.94 (C-6), 154.72 (C-7a), 143.95 (C-4CAr), 135.70 (C-3), 131, 41

(2xCHAr), 12978 (2xCHAr), 120.70 (ArCBr *), 119.78 (CN *), 97, 15 (C-3a), 56.64

(C-5), 35.63 (CH-4), 9.78 (C-3CH3) ppm, IR (KBr) v 3392, 3177, 2188, 1638, 1073 cm-1; MS (API-ES +) miz: Calc. For C14R 11BrN40: 3300116; found: [(M + Ht J 331.0 196; found: [(M + a) l 352.9992. Elemental analysis calc. (%) for

C14H 11BrN40: C 50.77, H 3.35, N 16.92; Found: C 50.78, H 3.46, N 17.03.

Example 14: 6-amino-3-methyl-4- (2-nitrophenyl) -1,4-dibydropyran [2,3-c] pyrazol-5carbonitrile. 4'

Following the procedure described, 5-methyl-IH-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitrile (66 mg, 1 mmol) 2-nitrobenzaldehyde (124 mg, 1 mmol) and ammonium acetate ( 77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 14 as a pale yellow solid (280 mg, 94%). Rf: 0.42 (O M-MeOH 8%); 1 H NMR (250 MHz, OMSO-d6) () 1.76 15 (s, 3H, C-3CH3), 5.09 (s, IH, CH-4), 7.04 (s, 2H, C-6NH2 ), 7.32 (dd, J = 7.8, 1.0 Hz, IH, ArH), 7.49 (dt, J = 8.0, 1.0 Hz, IH, ArH) 7.66 (dt , J = 7.6, 1.0 Hz, lH ArH), 7.85 (dd, J = 8, 1, 1.0 Hz, IR, ArH), 12.21 (s, IH, HI) ppm; 13C NMR (63 MHz, DMSOd6) () 161.18 (C-6), 154.96 (C-7a), 149.15 (ArCN02), 137.62 (C-4CAr *), 135.72 (C3 *), 133.40 (CHAr), 131, 29 (CHAr), 128.35 (CHAr), 123.60 (CHAr), 120.26 (CN),

20 96.39 (C-3a), 55.99 (C-5), 31, 39 (CH-4), 9.51 (C-3CH3) ppm; IR (KBr) v 3409, 3159, 2184, 1650, 1593, 1345 cm -1; MS (API-ES +) miz: Calc. For C14H 11 50: 297.0862; found: [(M + Ht] 298.0968; found: [(M + Nat] 320.0784; found: [(2M + Ht] 595, 1886. Elemental analysis caIc. (%) for CI4H11N50): C 56, 56, H 3.73, 23.56; found: C 56.37, H 3.79, 23.39.

Example 15: 6-amino-3-methyl-4- (3-nitrophenyl) -1,4-dihydropyran [2,3-c] pyrazol-5-carbonyl lo.

Following the procedure described, 5-methyl-IH-pyrazol-3 (2H) -one (98 mg, 1

mmol), malononitrile (66 mg, 1 mmol), 3-nitrobenzaldehyde (124 mg, 1 rumol) and

ammonium acetate (77 mg, 1 mmol) in EtOH (2 roL). Reaction time: 5 h.

Crystallized from EtOH, to obtain compound 15 as a yellow solid (270 mg, 91%). Rf: 0.44 (DCM-8% MeOH). IH NMR (250 MHz, DMSO-d6) 8 1.80 (s, 3H, C-3CH3), 4.88 (s, IH, CH-4), 7.06 (s, 2H, C-6NH2), 7.59-7.72 (m, 2H, ArH), 8.03 (s, IH, ArH), 8.12 (dt, J = 6.5, 2.4 Hz, IH, ArH), 12, 21 (s, lH, NH-l) ppm; 13C NMR (63 MHz, DMSO-d6) 8 161, 51 (C-6), 155.05 (C-7a), 148.23 (ArCN02 *), 147.20 (C

10 4CAr *), 136.27 (C-3), 134.78 (CHAr), 130.64 (CHAr), 122.37 (CHAr), 122.22 (CHAr), 120.91 (eN), 97 , 03 (C-3a), 56.44 (C-5), 35.98 (CH-4), 10.13 (C-3CH3) ppm; I

IR (KBr) v 3412, 3162, 2193, 1644, 1593, 1345 cm-; MS (API-ES +) miz: Calc. For CI4HIINsO: 297.0862; found: [(M + H) "J 298.0896; found: [(M + Nan 320.07 11; found: [(2M + Ht J 595.1766; found: [(2M + Na)" J 617, 1568. 15 Elemental analysis cale (%) for CI4HIINs0 3: C 56.56, H 3.73, N 23.56; found:

C 56.15, H 3.80, N 23.27. Example J6: 6-amino-3-methyl-4- (4-nitrophenyl) -1,4-dihydropyl1o [2,3-c] pyrazol-5carbonitrile.

Following the procedure described, 5-methyl-1H-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitri10 (66 mg, 1 mmo!), 4-nitrobenza! Dehyde (124 mg,! Mmo!) and ammonium acetate (77 mg, 1 mmo!) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 16 as a yellow solid (275 20

mg, 93%). Rf: 0.42 (DCM-8% MeOH); 'H NMR (250 MHz, DMSO-do) 8 1.80 (s,

3H, C-3CH3), 4.83 (s, 1 H, CH-4), 7.06 (s, 2H, C-6NH2), 7.46 (d, J = 8, 7 Hz, 2H, ArH ) 8.21 (d, J = 8.7 Hz, 2H, KrH), 12.2 1 (s, lH, HI) ppm; '3C NMR (63 MHz, DMSOdó) 8 161, 18 (C-6), 154.70 (C-7a), 152.15 (ArCN02), 146.41 (C-4CAr), 135.92 (C- 3),

5 128.88 (2xCHAr), 123.95 (2xCHAr), 120.55 (eN), 96.59 (C-3a), 55.90 (C-5), 35.90 (CH-4), 9 , 78 (C-3CH3) ppm; IR (KBr) v 3410, 3219, 2194, 1640, 1511, 1347 cm "; MS (API-ES +) miz: Calc. For C'4HIIN50: 297.0862; found: [(M + Ht] 298.0951; found: [(M + Nat J 320.0757. Elemental analysis calc. (%) for C '~ IIN503: C 56.56, H 3.73, N 23.56; found: C 56.40, H 3, 79, N 23.21,

Example 17: 6-amino-4- (2-furyl) -3-methyl-1, 4-dihydropyran [2,3-c Jpirazol-5carbonitri lo.

Following the procedure described, 5-methyl-1H-pyrazol-3 (2H) -one (98 mg, 1

15 romol), malononitrile (66 mg, lmol), 2-furancarboxaldehyde (96 mg, 1 blunt!) And ammonium acetate (77 mg, 1 mmol) in EtOH (2 roL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 17 as a gray solid (200 mg, 83%). Rf: 0.42 (DCM-8% MeOH); 'H NMR (250 MHz, DMSO-d6) 8 1.97 (s, 3H, C3CH3), 4.77 (s, IH, CH-4), 6, 17 (d, J = 3.0 Hz, lB , ArH), 6.37 (dd, J = 3.0, 1.9 Hz, lH,

20 ArH), 6.96 (s, 2H, C-6NH2), 7.53 (s, IH, ArH), 12.16 (s, 1B, NH-I) ppm; 3C NMR (63 MHz, DMSO-do) 8 161.51 (C-6), 155.74 (C-7a *), 154.84 (C-4CAr *), 142.32 (CHAr), 135, 87 (C-3), 120.65 (CN), 110.28 (CHAr), 105.68 (CHAr), 95.14 (C-3a), 53.97 (C-5), 29.83 ( CH-4), 9.62 (C-3CH3) ppm; IR (KBr) v 3345, 3162, 2185, 1647 cm "; MS MS (API-ES +) miz: Calc. For C'2HION402: 242,0804; found:

25 [(M + Ht J 243.0861; found: [(M + Nat] 265.0697; found: [(2M + Nat J 507.1506. Elemental tracing analysis (%) for C'2H, oN402: C 59 , 50, H 4.16, N 23.13; found: C 59.31, H 4.20, N 22.67.

Example 18: 6-am ino-3-meti 1-4- (2-tieni1) -1, 4-dibydropyran [2,3-c] pyrazol-5

carbonitrile

3. 4·

Following the procedure described, 5-methyl-lH-pyrazol-3 (2H) -one (98 mg, 1

5 mmol), malononitrile (66 mg, 1 mmol), 2-thiophenecarboxaldehyde (112 mg, 1 mmol) and ammonium acetate (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 h. Crystallized from EtOH, to obtain compound 18 as a pale yellow solid (201 mg, 78%). Rf: 0.43 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) or 1.91 (s, 3H, C-3CH3), 4.99 (s, 1H, CH-4), 6.73-7, 11 (m, 4H, ArH Y C-6NHÚ, 7.38 (d,

10 1 = 4.9 Hz, 1H, ArH), 12.18 (s, lH, NH-1) ppm; 13C NMR (63 MHz, DMSO-d6) or 161.01 (C-6), 154.65 (C-7a), 150, 15 (C-4CAr), 136.39 (C-3), 126.88 (CHAr), 125.37 (CHAr), 124.74 (CHAr), 121.00 (CN), 97.92 (C-3a), 57.89 (C-5), 31.75 (CH-4 ), 10, II (C-3CH)) ppm; IR (KBr) v 3339, 3160, 2189, 1646 cm-I; MS (API-ES +) miz: Calco for CI2N40S: 258.0575; Found: [(M + Ht] 259.0665; Found: [(M + Nat]

HIO15 281, 0496; Found: [(2M + Nat] 539,1085. Elemental analysis caLco (%) for 0S: C 55.80, H 3.90, N 21.69, S 12.41; Found: C 55.45, H 3 , 82,

CI2HION4 21, 46, S 12.27.

Example 19: 6-amino-3-methyl-1-4- (3-pyridyl) -1,4-dibydropyran [2,3-c] pyrazol-520 carbonitrile.

Following the procedure described, 5-methyl-1H-pyrazol-3 (2H) -one (98 rng, 1 rnmol), malononitrile (66 mg, 1 mmol), 3-pyridinecarboxaldehyde (07 mg, 1 mmol) and ammonium acetate (77 mg, 1 mmol) in EtOH (2 mL). Reaction time: 5 b.

Crystallized from EtOH, to obtain compound 19 as a gray solid (215 mg, 85

%). Rf: 0.20 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) or 1.78 (s, 3H, C

3CH3), 4.69 (s, lH, CH-4), 7.01 (s, 2H, C-6NH2), 7.35 (dd, J = 7.9, 48Hz, lH, ArH),

7.52 (dd, J = 7.9, 1.7 Hz, IH, ArH), 8.45 (s, 2H, ArH), 12.19 (s, 1B, NH-I) ppm; J3C

5 NMR (63 MHz, DMSO-do) or 161, 11 (C-6), 154.78 (C-7a), 148.82 (CHAr), 148.32

(CHAr), 139.78 (C-4CAr) 135.74 (C-3), 135.23 (CHAr), 123.95 (CHAr), 120.71

(CN), 96.82 (C-3a), 56.23 (C-5), 33.68 (CH-4), 9.77 (C-3CH3) ppm; IR (KBr) v 3388,

3165, 2190, 1644 cm · l; MS (API-ES +) miz: Calco for C 13 HIINsO: 253.0964;

found: [(M + Ht J 254,1037; found: [(M + Nat J 276.0859. Elemental analysis 10 calc. (%) for CJ3HII 50: C 61.65, H 4.38, 27.65; Found: C 61.63 H 4.28

27.48.

Example 20: 6-amino-3-meti 1-4- (4-pyridi 1) -1, 4-dibydropyran [2 3-cJpyrazol-5carbonitrile.

Following the procedure described, 5-methyl-lH-pyrazol-3 (2H) -one (98 mg, 1 mmol), malononitrile (66 mg, 1 mmol), 4-pyridinecarboxaldebide (107 mg, I mmol) and ammonium acetate ( 77 mg, 1 mmol) in EtOH (2 rnL). Reaction time: 5 b. Crystallized from EtOH, to obtain compound 20 as a white solid (212 mg,

20 84%). Rf: 0.20 (DCM-8% MeOH); IH NMR (250 MHz, DMSO-do) or 1.81 (s, 3H, C3CH3), 4.66 (s, lH, CH-4), 7.04 (s, 2H, C-6NH2), 7, 19 (d, J = 5.8 Hz, 2H, ArH), 8.51 (d, J = 5.8 Hz, 2H, ArH), 12.20 (s, lH, NH-I) ppm; 13C NMR (63 MHz, DMSO-do) or 16126 (C-6), 154.76 (C-7a), 152.86 (C-4CAr), 149.95 (2xCHAr), 135.82 (C-3 ), 122.79 (2xCHAr), 120.55 (eN), 96.26 (C-3a), 55.56 (C-5), 35.54 (CH-4), 9.76 (C

l

3CH3) ppm; IR (KBr) v 3315, 3025, 2169, 1657 cm; MS (API-ES +) miz: Calc. For C13H IIN50: 253.0964; found: [(M + Ht J 254.1068. Elemental analysis calc. (%) for CI3HII 50: C 61, 65, H 4.38, 27.65; found: C 61, 29, H 4.40, N 27.34.

1. BIOLOGICAL ACTIVITIES STUDIED IN THE COMPOUNDS OF THE INVENTION.

Example 21 Measurement of the inhibitory activity of the GSK313 enzyme

The inhibitory capacity of GSK3 ~ of the compounds was determined with the Kinase-Glo luminescence method. The compounds were studied at four concentrations (O, 1, 1, 10, and 11M) to obtain inhibition curves and calculate their Clso values. The injector of GSK3 ~ SB216763 was used as the reference compound. The products were dissolved in Tris buffer (40 mM, pH 7.5) supplemented with MgCl2 (20 mM), BSA (O, I mg / mL) and dithiothreitol (DTT, 50 11M). A blank and a control were included in each plate, corresponding to the activity of the enzyme in the absence of inhibitor.

In each well, 10 IlL of GSK3 ~ (1.25 ng / IlL) and 10 IlL of compound were added at the desired concentration. After 30 min, 10 IlL I of the GSK3 ~ peptide substrate (111M), and 10 IlL of ATP (25 11M) were added. After 1 hour, it

I

measured the amount of ATP produced following the instructions in the measurement kit. The

I

Enzymatic activity was calculated by refracting the measured luminescence to that detected in the absence of inhibitor. The concentration of compound that produces 50% of I inhibition of GSK3 ~ activity (CIso) was calculated by extrapolation of the curve of

I

activity. The results of the inhibition of GSK3 ~ (CIso,! -1M) are shown in Table 1. I Table 1

I

Inhibition of the GSK3p enzyme by the compounds of the invention, and the reference compound S8216763. Data are shown as the mean ± E.E. I of three experiments in duplicate at four different concentrations.

I

Compound

R2 GSK3tl CIso (jtM)

SB 216763

- 0.034 ± 0.0 1

one

Ph > 30

2

o-OCI-l) -Ph 16.4 ± 1.8

3

m-OCI-l) -Ph 7.20 ± 0.7

I I I

I

I

4

p-OCH) -Ph 6.60 + 1.7

5

o-CH) -Pb > 30

6

m-CH) -Ph 21.4 + 4.6

7

p-CH) -Ph 17.5 + 1, 9

8

p-F-Ph 17.9 + 1.5

9

o-CI-Ph > 30

10

m-CI-Ph 20.5 + 8.0

eleven

p-CI-Ph 6.29 ± 2.6

12

o-Br-Ph > 30

13

p-Br-Ph 3.77 ± 2.05

14

o-N02-Ph 3.89 + 1, 9

fifteen

m-N02-Ph 7.20 ± 1, 6

16

p-N02-Pb 16.3 ± 2.8

17

2-furil 14.8 + 4.8

18

2-thienyl > 30

19

3-pyridyl > 30

twenty

4-Pyridyl 8.28 ± 0.7

Example 22

5 Measurement of the induction of the rf2-EDRE pathway by measuring Luciferase activity in MCF7 cells stably transfected with the ARE-LUC Plasmid (AREc32).

10 For the induction study of the Nrf2 transcription factor, the AREc32 cell line was used which is stably transfected with the luciferase reporter gene linked to the EPRE sequence. In the presence of electrophiles or oxidative stress, the Nrf2 factor translocates to the nucleus and binds to EPRE sequences, activating the expression of luciferase, forming a proportional amount thereof. AREc32 cells are

15 cultured in DMEM medium with glutamax, supplemented with 10% fetal bovine serum (SBF), 1% penicillin-streptomycin and 1.6% geneticin (G418). The cells were grown in 75 cm2 bottles with 11 mL of the specific medium, incubated at 37 ° C and 5% CO2 and 1: 4 passes were made every 4-6 days when the cells reached 80% confluence. For the experiments, the cells are

20 cultured in white 96-well plates, at a density of 2 × 10 04 cells / well in 100 ml. / Well of culture medium. After 24 b in culture, the cells were treated with

the compounds object of the invention at the desired concentrations (0.3, 3, 10 and 30 flM) for 24 h at 3rC and 5% CO2. In each plate a baseline variable, a variable with tert-butyl hydroquinone (1 flM sulforaphane, positive control) and the problem variables were included, in a final volume of 100 flL.

Table 2 Induction of the transcription factor Nrf2 by the compounds of the invention, and the sulforaphane reference compound. Data are shown as the mean ±

E.E. of at least three experiments in duplicate at four different concentrations. *** p <0.001; ** p <0.01; * p <0.05 compared to baseline

Compound

R2 Relative Luciferase activity (response to baseline)

Concentrations

0, / pM 0.3pM / pM 8f1M

Sulforaphane

1.35 ± 0.3 1.52 ± 0.1, ** 3.59 ± 0.3 *** 11, 18 ± 0.5 ***

Concentrations

0.3 flM 3 JlM JOpM 30flM

I

Ph 1.05 ± 0.09 1.28 ± 0.10 1.47 ± 0.13 ** 2, 13 ± 0.11 ***

2

o-OMe-Ph 1.09 ± 0.06 1.26 ± 0.06 ** 1.35 ± 0.04 *** 1.77 ± 0.09 ***

3

117-0Me-Ph 0.96 ± 0.08 1.23 ± 0.05 1.93 ± 0.09 *** 2.99 ± 0.21 ***

4

p-OMe-Ph 1.10 ± 0.03 1.22 ± 0.04 1.57 ± 0.12 *** 1.72 ± 0.11 ***

5

o-Me-Ph 1.13 ± 0.05 1.2 1 ± 0.10 * 1.39 ± 0.02 *** 1.51 ± 0.04 ***

6

m-Me-Ph 1.39 ± 0.16 1.58 ± 0.27 1.86 ± 0.2 1 * 2.35 ± 0.26 ***

7

p-Me-Ph 1.08 ± 0.10 1.18 ± 0.06 1.56 ± 0.06 *** 2.30 ± 0.15 ***

8

p-F-Ph 1.15 ± 0.08 1.27 ± 0.09 1.75 ± 0.07 *** 2.72 ± 0.22 ***

9

o-CI-Ph 1.32 ± 0.10 * 1.47 ± 0.11 ** 1.63 ± 0.10 ** 2.07 ± 0.06 ***

10

m-CI-Ph 0.98 ± 0.03 1.11 ± 0.05 2.54 ± 0.24 *** 4.09 ± 0.20 ***

II

p-CI-Ph 1.02 ± 0.02 1.53 ± 0.09 * 1.99 ± 0.17 *** 3.92 ± 0.24 * "

12

o-Br-Ph 1.07 ± 0.04 1.07 ± 0.06 1.25 ± 0.12 * 1.60 ± 0.07 ***

13

p-Br-Ph 1, 11 ± 0.08 1.42 ± 0.06 2, 15 ± 0, 18 *** 6.09 ± 0.41 * ..

14

0 ° 2-Ph 1.12 ± 0.05 1, 18 ± 0.02 2.51 ± 0.11 *** 2.56 ± 0.15 ***

fifteen

II1-N02-Ph 1.22 ± 0.08 1.30 ± 0.10 1.48 ± 0, IO ** 2.39 ± 0.15 ***

16

p-N02-Ph 1.17 ± 0.09 1.19 ± 0.04 1.95 ± 0.15 * 4.31 ± 0.51 ***

17

2-furil 1.10 ± 0.05 1.41 ± 0.06 1.98 ± 0.24 * 3.12 ± 0.41 ***

18

2-thienyl 1.29 ± 0.09 * 1.39 ± 0.11 * 1.46 ± 0.09 ** 2.32 ± 0.15 ***

19

3-pyridyl 1.09 ± 0.07 1.30 ± 0.07 2.38 ± 0.25 *** 5.09 ± 0.29 ***

twenty

4-Pyridyl 1.20 ± 0.06 1.69 ± 0.07 3.20 ± 0.14 *** 10.3 ± 0.76 ***

After 24 b incubation with the compounds under study, activity was measured of luciferase by a bioluminescence test, for which the kit was used

"Luciferase assay system" (Promega E 1500). After the incubation period, the treatments were removed and the cells were washed with 100 µL of PBS O, 1M. After the wash was removed, 20 µL of the "Iysis buffer" reagent was added to each well. After 10 min, the plate was introduced in a multi-well luminescence reader, FluoStar optima (BMG Labtech).

The measure of luminescence in arbitrary units is directly proportional to the amount of luciferase in each of the wells and this, directly proportional to the induction of the rf2 factor for each of the products under study at the established concentration. The measurements were made in duplicate and the values were normalized with respect to the luminescence of the baseline taking its luciferase expression value as 1.

The results of induction of the rf2 factor obtained for the compounds object of the invention described as examples 1 to 20 are shown in Table 2, expressed as induction, taking as values the values of the untreated cells.

Example 23 Study of the neuroprotective capacity of the compounds object of the invention against models of toxicity induced by oxidative stress

SH-SY5Y neuroblastoma cell culture

SH-SY5Y [ECACC 94030304] cells, from passes between 5 and 16 after thawing, were maintained in a Dulbecco-modified Eagle medium (DMEM) containing 15 non-essential amino acids and supplemented with 10% fetal bovine serum, 1 mM glutamine, 50 units / mL of penicillin and 50 J.lglmL of streptomycin (GffiCO reagents, Madrid Spain). The cells were seeded in containers containing supplemented medium and kept in an incubator at 37 ° C in a humid atmosphere with 5% CO2 by passing 1: 4 twice a week. For the experiments, the cells were cultured in 48-well plates at a density of 1 x 10 05 cells / well. For cytotoxicity experiments the cells were treated with the drugs before reaching confluence, in DMEM with 1% fetal bovine serum.

Mixed culture of rat glial cells:

Glial cell cultures were prepared from cerebral cortex of 3-4 days old Sprague-Dawley rats. After removing the blood vessels and meninges, the forebrain was disintegrated in DMEMIF 12 medium. Once disintegrated, the cells were seeded in 96-well plates, at a density of 3x 105 cells / mL, in DMEMIF 12 medium with 20% of fetal bovine serum (SBF) and 1% penicillin / streptomycin (100 mg / mL; 100 f.lg / mL), and were incubated at 37 ° C and 5% CO2. After 5 days, the medium was replaced by DMEMlFI2 with 10% SBF. The cells were kept in culture between 7-10 days before performing the treatment.

Measurement of lipopolysaccharide-induced nitrite production (LPS) in mixed cultures of rat glial cells

The cells were pre-incubated with each of the treatments at the indicated concentrations (3, 10, 30 and 60 f.lM) for 24 h. After this period, the treatment was withdrawn and the cells were incubated with LPS (1 f.lg / mL) coincided with the treatments at the desired concentrations. A baseline variable and a positive control (sulforaphane) were included in each experiment. The production of nih-itos was measured after 18 h after treatment with LPS by the modified Griess method. An equal volume of Griess reagents was added to 75 ~ of supernatant from each sample. After 5 minutes of incubation at room temperature, the abbance at 550 nrn was measured in a multi-well reader (Fluostar Galaxy; BMG Labtech, Offenburg Germany). Data were referenced with respect to baseline, taking this value as 100% nitrite release.

Measurement of cell viability: 3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide MIT

The parameter that was used to measure cell viability was the metabolic reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MIT). The MIT technique consists of an indirect measure of cell viability. This process is

performed by the mitochondrial enzyme succinate dehydrogenase cells

metabolically active, which transfomulates the MTT of a hydrophilic compound of

yellow color (tetrazolium salt) to a violet, bidrophobic compound (salt of

formazan). The amount of living cells is proportional to the amount of formazan

5

formed (Mosmann, 1983, J lmmunol Melhods, 65: 55-63). Apoptotic cells

They have their mitochondria damaged and will not perform the process. Therefore, this method

allows to measure survival and cell proliferation, as well as determine the

cytotoxicity of potential therapeutic agents. To determine cell viability

in SH-SY5Y cells, 30 ~ 1 MTT well (5 mg / mL) is added and, after 2 h, the

the

medium is removed without losing the formazan crystals, which dissolve in 300 µl of

DMSO Subsequently, the samples are transferred to a 96-well plate to

measure the absorbance of the samples at 570 no. All MTT trials are

They performed in triplicate. It is quantified spectrophotometrically with the reader

Optimum absorbance / fluorescence FluoStar. The absorbance values obtained

l5

with the toxic alone and with each compound in the presence of the toxic they were subtracted from the value

of absorbance obtained in baseline conditions, without treatment. The value obtained from

the subtraction of the baseline absorbance values less toxic only, was considered 100%

of death and the values obtained with the compounds in the presence of toxic

normalized as percentages of value dicbo. To calculate the percentage of

twenty

survival, these values were subtracted from 100.

Culture of human hetocarcinoma cells He G2

The HepG2 human hepatocarcinoma line was cultured in Eagle's

minimum essential medium (EMEM) supplemented with 15 non-essential amino acids,

25

I mM sodium pyruvate, 10% inactivated bovine fetal serum, 100 units / ml of

penicillin and 100 µg / ml streptomycin. The cells were kept in flasks

said medium containing at 37 ° C in a humid atmosphere with 5% CO2 and 95% of

air. For toxicity tests, HepG2 cells were plated

96-well transparent, with a density of lxl05 cells per well. When

30

the cells reached 80% confluence, the medium was removed and replaced by

fresh medium containing the compounds at the desired concentrations (10, 30, 10

and 300 flM). After 24 b, cell viability was determined by the MIT reduction method.

Europrotection exerted by the compounds object of this invention 1-5:

The inhibition of the GSK3 ~ enzyme combined with the activation of the Nrt2-EPRE pathway by the compounds exerts a neuroprotective effect against oxidative stress models, due to the expression of various cytoprotective genes. Therefore, we study the neuroprotective capacity of the compounds object of this patent in in vitro models of cytotoxicity induced by oxidative stress. The neuroprotective effect of the compounds in bumano neuroblastoma cells was evaluated against oxidative stress produced by rotenone and oligomycin A, blockers of the mitochondrial respiratory chain as described in Hal1iwel et al. 1992 (Halliwell, 1992, J Neurochem, 59: 1609-23, Newbouse et al., 2004, Toxicol Sci, 79: 137-46).

Europrotection against oxidative stress induced by the combination of rotenone

(30 fl ==. L ~: "= ::> '=== - = -' =" ">" ": - = -

In this study, a 24 b pre-incubation was performed with the synthesized compounds at a concentration of 1 flM and a 24 h co-incubation of these in the presence of rotenone / oligomycin A (30 flMlIO flM, respectively). After 24b, cell viability was evaluated by the MIT reduction method.

With this protocol, information is obtained on all the potential biological activities present in the structure under study. In this model, being present during the 24 hours prior to the incubation of the toxic, the compound is able to show its inductive capacity of the Nrt2 factor favoring cell survival. In addition, it is also present while the cells are exposed to the toxic stimulus, the mixture of rotenone / oligomycin A that causes the appearance of a large amount of free radicals inside the cell. the cell and induce cell apoptosis. The percentages of and protection are summarized in Table 3.

Table 3 Percentage of survival and cellular protection produced by the compounds of the invention and melatonin, at the concentration of 1 J.1M against the toxicity induced by the combination of rotenone and oligomycin A. Data are expressed.

5 as the mean ± E.E. of four triplicate experiments, performed with four different batches of cells. ### p <0.001 compared to baseline. ••• p <0.001; "p <0.01; 'p <0.05 compared with the rote / olig combination.

Compound

R2 Rot / Olig (30/10) Pre-incubation

% Survival

% Protection

Basal

100

Rote / Olig

50.79 ± 1.8311 ##

Melatonin

67.72 ± 3.12 *** 34.89 ± 4.44

one

Ph 72, 15 ± 3.11 *** 40.06 ± 5.37

2

o-OMe-Ph 71, 60 ± 3.80 *** 39.07 ± 6.84

3

m-OMe-Ph 77.28 ± 2.54 *** 55.33 ± 3.83

4

p-OMe-Ph 71, 99 ± 2.10 *** 38.80 ± 4.51

5

o-Me-Ph 61, 99 ± 2.00 *** 26.15 ± 3.45

6

m-Me-Ph 74.40 ± 4.40 *** 48.91 ± 8.22

7

p-Me-Ph 69.06 ± 2.88 *** 40.49 ± 4.45

8

p-F-Ph 73, 10 ± 3.84 *** 46.29 ± 7.54

9

o-CI-Ph 73.35 ± 2.79 *** 47.05 ± 5.03

10

m-CI-Ph 74.59 ± 2.29 *** 49.83 ± 3.12

eleven

p-CI-Ph 69.26 ± 2.47 *** 39, 18 ± 3.34

12

o-Br-Ph 60.96 ± 2.01 *** 24.03 ± 3.42

13

p-Br-Ph 66.76 ± 2.62 *** 35.61 ± 4.69

14

o-N02-Pb 67.54 ± 3.41 ** 30.30 ± 5.06

fifteen

m-N02-Ph 72.73 ± 2.50 *** 4 ~ 18 ± 3.54

16

p-N02-Ph 68.49 ± 3.20 ** 32.32 ± 4.83

17

2-furil 73.72 ± 1.74 *** 47.93 ± 1.91

18

2-tieni l 74.05 ± 2.14 *** 48.56 ± 3.11

19

3-pyridyl 78.64 ± 2.77 *** 57.91 ± 4.57

twenty

4-Pyridyl 67.12 ± 2.39 *** 36.42 ± 3.58

10 Europrotection against okadaic acid-induced toxicity (20 nM): In this study, a 24-b pre-incubation was performed with the compounds object of the invention at a concentration of IJ.M and a 18-b co-incubation of these in the presence of okadaic acid (20 nM). After 18 b, cell viability

15 was evaluated by the MIT reduction method. Through this essay you study

the neuroprotective potential derived from the inhibition of the GSK3 enzyme (3, since this

It is the main kinase responsible for phosphorylating the Tau protein. In addition, induction

of the rfl factor also induces the synthesis of autophagy-related proteins

which facilitate the removal of hyperphosphorylated tau protein.

Table 4 Percentage of cellular protection produced by the compounds of the invention, melatonin and SB216763, at the concentration of 1 IlM, against the toxicity induced by okadaic acid. Data are expressed as the mean ± E.E. of four triplicate experiments, performed with four different batches of

10 cells ### p <0.001 compared to baseline. *** p <0.001; "p <0.01; * p <

0.05 compared to okadaic acid.

Compound

R2 Ac. okadaic (20 nM)

% Survival

% Protection

Basal

100

Ac. okadic

47.14 ± 3.41 ###

Melatonin

65.53 ± 3.05 *** 34.56 ± 6.28

SB216763

67.41 ± 4.82 *** 38.03 ± 6.43

one

Ph 62.04 ± 4.54 ** 27.77 ± 5.49

2

o-OMe-Ph 67.20 ± 5.51 *** 39.84 ± 8.07

3

m-OMev 76.34 ± 2.57 *** 56.20 ± 7.07

4

p-OMe-Ph 68.36 ± 6.28 ** 41, 07 ± 12.5

5

o-Me-Ph 67.38 ± 2.78 *** 38.10 ± 4.96

6

m-Me-Ph 77.86 ± 3.20 *** 55.44 ± 4.96

7

p-Me-Ph 69.86 ± 3.14 *** 45.00 ± 2.93

8

p-F-Ph 72.58 ± 4.54 *** 48.03 ± 5.04

9

o-CI-Ph 68.46 ± 4.59 *** 40.22 ± 6.61

10

m-CI-Ph 70.55 ± 3.90 *** 42.63 ± 8.82

eleven

p-CI-Ph 69.97 ± 5.58 *** 37.46 ± 5.54

12

o-Br-Ph 70.07 ± 4.74 *** 46.41 ± 5.82

13

p-Br-Ph 76.29 ± 3.21 *** 53, 75 ± 7.75

14

o-N02-Ph 69.09 ± 1.26 *** 34.81 ± 4.09

fifteen

m-N02-Ph 72.44 ± 4.01 *** 48.64 ± 5.16

16

p-N02 -Ph 64.97 ± 4.62 *** 34.02 ± 5.77

17

2-furil 70.90 ± 3.72 *** 45, 12 ± 5.05

18

2-thienyl 67.73 ± 7.51 ** 42.26 ± 11.8

19

3-pyridyl 65.75 ± 2.01 *** 35.32 ± 4.64

twenty

4-Pidridil 73, 08 ± 1.99 *** 50, l6 ± 3.78

The induction of rt2 combined with the inhibition of the enzyme GSK3 ~ in a

Single molecule demonstrates an interesting neuroprotective profile against toxicity induced by okadaic acid. This toxic is an inhibitor of the enzymes protein phosphatases l and 2A (PP I and PP2A), so it induces biperphosphorylation and aggregation of tau, as well as oxidative stress, ultimately causing cell death. As the results in Table 4 indicate, the compounds object of this invention showed a good neuroprotective profile in this in vi / ro EA model, with a minimum protection percentage of 27.7% (1, R = H), Y a maximum of 56.6% (6, R = m-CH3). With the exception of compound 1 (R = H), all precursors showed better neuroprotective capacity against OA than melatonin. In addition, most were better neuroprotectors than SB216763.

Reduction of nitrite production induced by lipopolysaccharide (l! J.g / rnL) in mixed glial rat cultures: Anti-inflammatory effect:

As discussed in the introduction, the GSK3p enzyme also plays an important role in neuroinflammation. The activity of this enzyme has been related to an increase in the production of iNOS (Yuskaitis et al., 2009, Cell Signa !, 21: 264-73) and pro-inflammatory cytokines (Martin et al., 2005, Na! Immunol , 6: 777-84). On the other hand, Nrt2 induces the expression of both antioxidant and anti-inflammatory enzymes. Since the compounds object of the invention have demonstrated the ability to inhibit GSK3p and induce rt2, we study the potential anti-inflammatory effect of the compounds object of this invention. To do this, we evaluate its ability to reduce the production of nitrite induced by lipopolysaccharide (LPS) in primary cultures of rat glia, which is a model of glial overactivation. The study was carried out following a pre-and co-incubation protocol. After 7 days in culture, the rat glial cells were pre-incubated with the compounds at four concentrations (3, 10, 30, and 11M) for 18 b. Then, the medium was removed and the cells were co-incubated with the compounds at the mentioned concentrations and LPS (1 IlglmL) for 24 b. Finally, the presence of nitrites in the supernatant was measured by the Griess colorimetric method. Nitrite production is directly proportional to the activity of the OS enzyme, whose expression increases in glial cells when

are activated by LPS.

Table 5

5 Anti-inflammatory effect induced by the compounds of the invention, the sulforaphane reference compound. Data are shown as the mean ±

E.E. of four experiments in triplicate at four different concentrations. • ** p <0.001; •• P <0.01; • P <0.05 compared to the nitrite production induced by LPS.

Compound

Rz % reduction in nitrite release induced by LPS in mixed cultures of rat glia

Concentrations

O.3pM I pM 3pM IOPM

Sulforaphane

19.8 ± 4.80 ** 4 1.09 ± 8.62 *** 79.0 ± 6.81 *** 100 ± 2.55 * **

Concentrations

3pM IOPM 30pM 60pM

one

Ph 9.98 ± 5.54 28.5 ± 7, 16 27.6 ± 5.07 43.2 ± 9.99 **

2

o-OM e-Ph 19.5 ± 8.40 40.6 ± 6.02 46, 1 ± 4.85 59.7 ± 7.51 *

3

m-OM e-Ph 31.4 ± 3.7 1 * 60.9 ± 5.37 *** 84, 2 ± 4.23 * ** 99, I ± 1.88 ** *

4

p-OMe-Ph 39, 1 ± 2.29 47.2 ± 8.79 ** 63, 8 ± 4.46 *** 79.9 ± 5.92 ***

5

o-M e-Ph 44.3 ± 10.6 * 64.0 ± 5.44 *** 91, 7 ± 4.08 *** 100 ± 2.14 ***

6

m-Me-Ph 34.8 ± 5.37 ** 45, I ± 5.37 * ** 80.5 ± 3.28 *** 94.4 ± 0.95 ***

7

p-Me-Ph 11.7 ± 3.89 * 27.2 ± 5.35 *** 62.3 ± 6, 12 *** 9 1.7 ± 3.80 ***

8

p-F-Ph 19.2 ± 6.80 * 49.5 ± 4, 16 *** 90, 1 ± 2.25 ** * 100 ± 2.27 ***

9

o-CI-Ph 14.6 ± 5.40 14.9 ± 2.88 14.1 ± 2.7 19.3 ± 13.6

10

m-CI-Ph 45.9 ± 7.99 * 52.5 ± 7, 13 ** 64.8 ± 5, 11 ** 87.7 ± 3.53 ***

eleven

p-CI-Ph 23.4 ± 8, 19 36, I ± 3.78 ** 43, 4 ± 5, 15 * 49.6 ± 7, 15 *** 54.7 ± 4.47 ** 62.0 ± 8.94 *** 88.9 ± 5, 15 ***

12

o-Br-Ph 32.4 ± 5.57

13

p-Br-Ph 28.9 ± 6.54 * 43, I ± 5.52 ** 60.3 ± 3.36 *** 73.8 ± 2.86 ** *

14

o-NOz-Ph 47.3 ± 9.54 ** 7 1.8 ± 3.70 * ** 95.1 ± 1.64 *** 100 ± 2.76 * **

fifteen

m-NOz-Ph 12.8 ± 1.37 25.7 ± 5.18 32.5 ± 3.34 38.6 ± 4.97

16

p-NOr Ph 15, 1 ± 3.89 35.7 ± 5.72 57.7 ± 5.79 * 90.6 ± 5.71 ***

17

2-furil 9.95 ± 1.8 41, 2 ± 4.62 *** 65.2 ± 4.33 *** 80.0 ± 4.17 * **

18

2-thienyl 20.5 ± 9.67 27.1 ± 6.85 26.3 ± 1.3 27.5 ± 5.39

19

3-pyridyl 28.9 ± 3.49 *** 62.9 ± 5.52 *** 86, I ± 4.26 *** 100 ± 5.20 ***

twenty

4-Pyridyl 10.7 ± 2.69 25.6 ± 4.55 ** 48.6 ± 4.04 *** 70.6 ± 4.40 ** *

In this way, the lower the amount of nitrites detected, the less

the expression of i OS, and greater the anti-inflammatory effect observed. The values obtained are shown in table 5.

As can be seen in the table, the compounds object of the invention showed a good anti-inflammatory capacity, with percentages of reduction of nitrite production in the micromolar range. The greatest anti-inflammatory effect was shown by compound 14 (R = o-N02), with an ECso of only 3.9 flM.

Toxicological properties of the compounds object of this invention

In general, compounds with inducing capacity of transcription factor

rfl can present a bepatotoxic effect, due to the electrophilic character that generally characterizes them. The derivatives object of this invention were designed to avoid this toxicity and improve its safety profile. The liver plays an essential role in the metabolism of endogenous and exogenous compounds. Hepatocytes capture these compounds and, after metabolizing them, are eliminated by different routes. The metabolized compounds usually have less pharmacological activity and less toxicity than the corresponding active ingredients. However, in certain cases the active substances or their corresponding metabolites can be toxic to bepatocytes. Hepatic toxicity is one of the main causes for drug withdrawal from preclinical studies. The HepG2 cell line shows the majority of the genotypic and phenotypic characteristics of normal bepatic cells, and they possess the typical functions of the German bepatic cells. In addition, these cells have been defined by the EMA (European Medicines Agency) as a model of bepatotoxicity by measuring the induction of apoptosis and / or cell survival. Therefore, the safety profile of the compounds object of the present invention was evaluated in the HepG2 bepatocarcinoma cell line. HepG2 cells were cultured in flask T75 using as EMEM culture medium supplemented with non-essential amino acids, 10% fetal bovine serum, 50 units / rnL of penicillin and 50! -Lg / mL streptomycin (GmCO reagents, Madrid, Spain ). The cultures were kept in an incubator with a gas mixture of 0 2 / C02 (95/5%) at 37 ° C with a humid atmosphere, passing 1: 2 passes twice a week when they reached a confluence of 90%. All experiments were performed on cells that were between passes 5 and 15. For the experiments, the cells were cultured in 96-well plates at a density of 4 × 1 04 cells / well

in 200¡tL of culture medium. After 24 b in culture, the maintenance medium was removed and replaced with 100 µl of the corresponding solution of the compounds object of the invention in culture medium at the desired concentrations (10, 30 and 100 Y300 ~ LM). The compounds were studied in triplicate, including in each experiment 3 wells without treatment, which was considered 100% feasibility (baseline conditions). After 24 b incubation with the treatments, survival was assessed by the MTT reduction method. In order to study their safety profile further, their effect on the cells used for neuroprotection experiments, the human neuroblastoma cell line, was also evaluated

10 SH-SY5Y. The maintenance and culture methods of this cell line have been described above. For toxicity experiments, SH-SY5Y cells were cultured in 96-well plates at the density of 6 × 1 04 cells / well. The experimental conditions were the same as those used for the study of

toxicity in HepG2 cells. 15 The results obtained are shown in Table 6 expressed as the lethal dose 50 (DLso), or concentration necessary to reduce by 50% the viability of the basal conditions (without treatment) measured by the MTT reduction method. In general, all compounds showed low toxicity in the human neuroblastoma line SH-SY5Y. On the other hand, none of them showed toxicity in the HepG2 hepatocarcinoma line, so that the compounds object of this invention would show low or no hepatotoxicity.

Table 6 DLso of the compounds of the invention, measured as a reduction in cell viability in the HepG2 hepatocarcinoma line and in the SH-SY5Y neuronal line. Data are expressed as the mean ±

E.E. of four triplicate experiments, performed with four different batches of cells.

Compound

R SH-SY5Y LDso (~ M) HepG2 LDso (jlM)

Melatonin

> lOO > lOO

S8216763

> lOO > lOO

one

Ph > 100 > 100

2

o-OMe-Ph > 100 > 100

3

m-OMe-Ph > 100 > 100

4

p-OMe-Ph > 100 > 100

5

o-Me-Ph > 100 > 100

6

m-Me-Ph > 100 > 100

7

p-Me-Ph > 100 > 100

8

p-F-Ph > 100 > 100

9

o-C I-Ph > 100 > 100

10

m-CI-Ph > 100 > 100

eleven

p-CI-Ph > 100 > 100

12

o-Br-Ph > 100 > 100

13

p-Br-Ph > 100 > 100

14

o-NOrPh > 100 > 100

fifteen

m-N02-Ph > 100 > 100

16

p-N02-Ph > 100 > 100

17

2-furi l > 100 > 100

18

2-tieni l > 100 > 100

19

3-Piridi l > 100 > 100

twenty

4-Pyridyl > 100 > 100

Claims (17)

1. Use of a compound of formula 1 (1) with the substituents specified as GSK3 enzyme iobibidor (3 and Nrf2 5 factor inducer

(one)

where

10

R and Rt are selected from the group consisting of:

A hydrogen atom;

Alkyl optionally substituted by one, two, or three halogen atoms

selected from fluorine, chlorine and bromine; cycloalkyl (C) -C6); alkoxy (C t-

C6); cycloalkoxy (C) -C6); cyano and nitro; I

fifteen

phenyl optionally substituted by one two or three groups selected

independently between fluorine; chlorine bromine; optionally alkyl

substituted by one two, or three halogen atoms selected from fluorine,

chlorine and bromine; cycloalkyl (C) -C6); alkoxy (C t-C6); cycloalkoxy (C) -C6) '

cyano and nitro; or

twenty

a heteroaryl group selected from 2-pyridyl, 3-pyridyl, 4-pyridyl,

3-pyridazinyl, 4-pyridazinyl, 2-pyrimjdinyl, 4-pyrimidinyl, 5-pyrimidinyl,

2-pyrazinyl, 3-pyrazinyl, 2-pyrrolyl, 3-pyrrolyl, 2-furyl, 3-furyl, 2

thienyl, 3-thienyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-thiazolyl, 4

thiazolyl, 5-thiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 3

25

isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 3

pyrazolyl, 4-pyrazolyl, 1,2,3-oxadiazol-4-yl, 1,2,3-oxadiazol-5-yl, 1,2,4

oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,5-oxadiazol-3-yl, 1,3,4

oxadiazol-2-yl, 1,2,3-thiadiazol-4-yl, 1,2,3-thiadiazol-5-yl, 1,2,4-thiadiazole

3-yl, 1,2,4-thiadiazol-5-yl, l, 2,5-thiadiazol-3-yl, 1,3,4-thiadiazol-2-yl, 1,2,3 triazol-4-yl, I, 2,4-triazol-3-yl and 5-tetrazolyl, the heteroaryl group being optionally substituted by one, two or three selected groups independently between fluorine, chlorine, bromine, alkyl, cycloalkyl (C3-C6), (C¡-C6) alkoxy, (C3-C6) cycloalkoxy, cyano and nitro; R2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl or heteroaryl, optionally substituted by one, two or three groups independently selected from fluorine, chlorine, bromine, alkyl, alkoxy, cycloalkyl (C3-C6), cycloalkoxy (C) -C6), cyano, nitro and carboxylate or; R3 is selected from the group consisting of hydrogen, (C¡-C6) alkyl, cycloalkyl (C3-C6) and phenyl, optionally substituted by one, two or three groups independently selected from fluorine, chlorine, bromine, (C¡-C6) alkyl, (C¡-C6) alkoxy, (C3-C6) cycloalkyl, (C3-C6) cycloalkoxy, cyano, nitro and carboxylate; R, ¡and Rs are selected from the group consisting of hydrogen, alkyl, cycloalkyl (C) -C6), acetyl, phenyl or heteroaryl, optionally substituted by one, two or three groups independently selected from fluorine, chlorine, bromine, (C¡-C6) alkyl, (C¡-C6) alkoxy, (C3-C6) cycloalkyl, cycloalkoxy (C3-C6), or two groups can together form a cycloalkyl group (C3-C6) or - (CH2k (where n = O, 1, 2, 3, 4, 5, 6), or - CH = CH-CH = CH-; X is selected from an oxygen atom, a nitrogen atom or a sulfur atom, -SO- or S02; and is selected from an oxygen atom, a nitrogen atom or sulfur atom, -SO- or S02; and its salts, preferably pharmaceutically acceptable salts, prodrugs or solvates. 2. Use of a compound according to claim 1, wherein R is hydrogen; R, is selected from the group comprising an alkyl optionally substituted by one or two groups independently selected from fluorine, chlorine and bromine; alkoxy (C, -C6), nitro and amino. Preferably R is methyl; R2 is selected from the group comprising a phenyl aromatic ring optionally substituted by one, two or three groups independently selected from fluorine, chlorine, (C, -C6) alkyl, optionally substituted by one, two, or three halogen atoms selected from fluorine , chlorine and bromine; (C, -C6) alkoxy, cyano and nitro; or a heterocyclic optionally substituted by one, two or three groups independently selected from fluorine, chlorine, alkyl (CI-C6), alkoxy and nitro R3 is the cyano group; R, ¡and Rs are hydrogen; X is nitrogen; and is oxygen; and its salts, prodrugs or solvates, preferably, its pharmaceutically acceptable salts. 3. Use of a compound according to claim 2 as a GSK3 inhibitor (3 and inducer of the Nrf2 factor selected from:

or 6-amino-4-phenyl-3-meti1-1, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

or 6-amino-3-methyl-4- (2-methoxyphenyl) -1, 4-dihydropyran [2,3-c) pyrazol-5carbonitrile.

or 6-amino-3-methyl-4- (3-methoxyphenyl) -1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

or 6-amino-3-methyl-4- (4-methoxyphenyl) -1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

or 6-amino-3-meti 1-4- (2-to li1) -1, 4-dihydropyran [2,3-c] pyrazol-5-carboni tri lo.

or 6-am ino-3-meti 1-4- (3-toli 1) -1, 4-d i-propyran [2,3-c] pyrazol-5-carbonyl lo.

or 6-am ino-3-meti 1-4- (4-tol i 1) -1, 4-dih idropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-4- (4-tluorophenyl) -3-methyl-1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

•

6-amino-4- (2-c1oropheni1) -3-meti1-1, 4-di b idropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-4- (3-c1orophenyl) -3-methyl-l, 4-dihydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-4- (4-c1orophenyl) -3-methyl-l, 4-dibydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-4- (2-bromopheni1) -3-meti1-1, 4-dihydropyran [2,3-c] pyrazol-5carbonitrile.

•

6-amino-4- (4-bromophenyl) -3-methyl-l, 4-dibydropyran [2,3-c] pyrazol-5-carbonyl lo.

•

6-amino-3-methyl-4- (2-nitrophenyl) -1, 4-di b idropi rano [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-3-methyl-4- (3-nitrophenyl) -1,4-dibydropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-3-methyl-4- (4-nitrophenyl) -1,4-dibydropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-4- (2-furi 1) -3-methyl-l, 4-dibydropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-3-methyl-4- (2-thieni 1) -1,4-di bidropyran [2,3-c] pyrazol-5-carbonitrile.

•

6-amino-3-meti 1-4- (3-pi ridyl) -l, 4-dihydropyran [2,3-c] pyrazol-5-carbonitri lo.

•

6-amino-3-methyl-4- (4-pyridyl) -1,4-dibydropyran [2,3-c] pyrazol-5-carbonitrile.

Four.

A pharmaceutical composition comprising a compound of formula (1) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, prodrug or solvate thereof, and a pharmaceutically acceptable excipient.

5.

Use of a formula compound (1) according to any one of claims 1 to 3, or of a pharmaceutically acceptable salt, prodrug or solvate thereof, in the preparation of a famlaceutical composition with inhibitory effect of GSK3f3 and inductive effect of Nrf2 and / or neuroprotective and / or anti-inflammatory.

6.

Use of a compound of formula (1) according to any one of claims I to 3, or of a pharmaceutically acceptable salt, prodrug or solvate thereof, in the preparation of a pharmaceutical composition for the prevention or treatment of

a central and / or peripheral neurodegenerative disease or an ischemic-cerebral disease (stroke). 7. Use according to claim 6, wherein said neurodegenerative disease is ALzheimer's disease. 8. Use according to claim 6, wherein said neurodegenerative disease is amyotrophic lateral sclerosis. LO 9. Use according to claim 6, wherein said neurodegenerative disease is multiple sclerosis. 10. Use according to claim 6, wherein said neurodegenerative disease is post-encephalic parkinsonism. J]. Use according to claim 6, wherein said neurodegenerative disease is progressive supranuclear paralysis. 12. Use according to claim 6, wherein said neurodegenerative disease is Gerstrnann-Straussler-Scbeinker disease. 13. Use according to claim 6, wherein said neurodegenerative disease is Hallervorden-Spatz disease. 14. Use according to claim 6, wherein said neurodegenerative disease is Creutzfeldt-Jacob disease. 15. Use according to claim 6, wherein said neurodegenerative disease is the Down's Syndrome. 30

16.

Use according to claim 6 wherein said neurodegenerative disease is iemann-Pick disease.

17.

Use according to claim 6, wherein said neurodegenerative disease is Pick's disease.

5] 8. Use according to claim 6, wherein said neurodegenerative disease is cerebral stroke. 19. Use according to any of claims 6 to 18, wherein a compound of formula (I), or a pharmaceutically acceptable salt, prodrug or solvate thereof It is for the manufacture of a medicament for oral administration,
parenteral, subcutaneous, intramuscular, intravascular or rectal. 20. Use according to any of claims 6 to 18, wherein said compound of Formula (1), or a pharmaceutically acceptable salt, prodrug or solvate thereof, is administered in a daily dose comprised between 0.1 and 100 mglkg body weight. 21. Use according to claim 20, wherein said compound of formula (n, or a pharmaceutically acceptable salt thereof, is administered in a daily dose comprised between 2 and 5 mg / kg body weight.