1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile

Abstract

The present invention relates to pharmacologically active 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile derivatives: [Case 1] TIFF2023533932000053.tif52134 relates to methods for the preparation of such compounds, pharmaceutical compositions containing them, and their use in the therapy and / or prevention of cognitive disorders.

Description

[Technical field]

[0001] The present invention relates to pharmacologically active compounds, more particularly 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile, its stereoisomers and salts thereof, methods for the preparation of such compounds, pharmaceutical compositions containing them and their use in the therapy and / or prevention of neurodegenerative disorders and / or cognitive dysfunction. [Background technology]

[0002] Prolyl oligopeptidase (EC 3.4.21.26) (POP), also known as prolyl endopeptidase (PREP), is a serine protease that catalyzes the hydrolysis of peptides at the C-terminal side of L-proline residues. It is widely distributed in mammals and can be purified from various organs, including the brain.

[0003] This enzyme plays an important role in the degradation of proline-containing neuropeptides that are associated with learning and memory functions (Wilk S et al., Life Sci. 1983; 33: 2149-57; O'Leary RM, O'Connor B, J. Neurochem. 1995; 65: 953-63).

[0004] The effect of prolyl oligopeptidase inhibition has been tested in the treatment of cognitive deficits associated with neurodegenerative processes. Parkinson's disease was produced in monkeys by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes depletion of substance P. Treatment with S-17092, a potent POP inhibitor, then increased performance in cognitive tasks (Schneider JS et al., Neuropsychopharmacology 2002;26(2):176-82). It has also been found that POP inhibition prevents the oligomerization of α-synuclein ex vivo (Myohanen TT et al., Br. J. Pharmacol. 2012;166(3):1097-113). In the case of Alzheimer's disease (AD), several in vivo experiments in animal models showed that POP inhibition resulted in neuroprotective and cognitive enhancing effects (Kato A et al., J. Pharmacol. Exp. Ther. 1997;283(1):328-35 and Toide K et al., Rev. Neurosci. 1998;9(1):17-29). Neuroprotective effects were originally observed by Katsube's group when cortical and cerebellar granule cells were prevented from age-induced apoptosis by treatment with the POP inhibitor ONO-1603 (Katsube N et al., J. Pharmacol. Exp. Ther. 1999;288(1):6-13).

[0005] Only a few clinical trials have been conducted with POP inhibitors in the treatment of cognitive deficits. In a Phase I clinical study, Morain's group (Morain P et al., Br. J. Clin. Pharmacol. 2000;50(4):350-9) found that the above-mentioned prolyl endopeptidase inhibitor S-17092 was cognitive enhancing and clearly dose-dependent in healthy elderly subjects. No adverse effects were detected. Subsequent studies suggested slight additional mood stabilization for this compound (Morain P et al., Neuropsychobiology 2007;55(3-4):176-83).

[0006] Prolyl oligopeptidase activity has been reported to be altered (postmortem) in several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Huntington's disease and multiple sclerosis (MS) (Mantle D et al., Clin. Chim. Acta 1996;249(1-2):129-39).

[0007] There is also a considerable amount of evidence indicating a role for neuroinflammation in the pathogenesis of neurodegenerative diseases such as AD, MS and Parkinson's disease (Hirsch EC et al., Lancet Neurol. 2009;8(4):382-97; Philips T et al., Lancet Neurol. 2011;10(3):253-63). POP is thought to be the main enzyme involved in the release of the anti-inflammatory tetrapeptide Ac-SDKP from Tβ4 in the brain (Yang F et al., Hypertension 2004;43(2):229-36; Nolte WM et al., Biochemistry 2009;48(50):11971-81). This suggests that inhibition of POP may be useful in reducing neuroinflammation and, as a result, POP inhibitors may be useful in the treatment of neurodegenerative diseases with an inflammatory component such as Alzheimer's disease and Parkinson's disease, and may in particular be useful in improving the cognitive impairment associated with these diseases.

[0008] Senile plaques spreading to brain cortical regions are a typical neuropathological feature of AD. The main protein constituent of these plaques is the amyloid β-peptide (Aβ). Aβ deposition causes neuronal dysfunction and brain death. This peptide is derived from the β-amyloid precursor protein (APP). Under normal conditions, APP is cleaved by α-secretase to generate soluble APPα, which prevents Aβ production.

[0009] Interestingly, POP inhibition increases intracellular IP3 levels, which may contribute to stimulating APPα production, which in turn would reduce Aβ generation.

[0010] In addition, Rossner (Rossner S et al., Neurochem. Res. 2005;30(6-7):695-702) found fewer POP-immunoreactive neurons in brain structures of AD patients affected by Aβ plaques.

[0011] In addition, substance P appears to be able to suppress the neurotoxic effects of β-amyloid protein (Kowall NW et al., Proc. Natl. Acad. Sci. USA 1991;88(16):7247-51). Prolyl oligopeptidase inhibitors inhibit the metabolism of substance P and help maintain levels of substance P that may suppress the neurotoxic effects of β-amyloid protein.

[0012] Considering the effects listed above, prolyl oligopeptidase inhibitors may be useful drugs for the treatment of Alzheimer's disease, helping to prevent brain damage and ameliorate the cognitive impairment associated with this disease.

[0013] Prolyl oligopeptidases are also associated with several factors that may be related to multiple sclerosis (MS). For example, POPs are involved in the regulation of microglial toxicity (Klegeris A et al., Glia 2008;56(6):675-85). Indeed, a direct relationship between POPs and MS has been established, with plasma POP activity being significantly reduced in patients with relapsing-remitting MS (RR-MS) (Tenorio-Laranga J et al., J Neuroinflammation 2010;7:23). Interestingly, this reduction correlated with the severity of disease symptoms, but not with the age of the patients. Instead, an inverse correlation between POP activity and age was observed in healthy controls, and in elderly controls, the levels were comparable to those found in MS patients.

[0014] The neuropathological hallmark of Parkinson's disease is the progressive degeneration of melanized dopaminergic neurons in the substantia nigra pars compacta, together with intracellular inclusions known as Lewy bodies. The main component of Lewy bodies is the 140 amino acid protein α-synuclein. Under certain conditions, α-synuclein monomers interact to form prefibrillar aggregates or protofibrils that can produce cytotoxic insoluble fibrils. These fibrils cannot be degraded by the proteasome, which impairs the function of this intracellular proteolytic system. This leads to the accumulation of α-synuclein protofibrils (and other proteins degraded by the proteasome) in the cytosol (Bennett MC, Pharmacol. Ther. 2005;105(3):311-31), and as a result, α-synuclein protofibrils are increased in the brains of Parkinson's disease patients. These fibrils have been associated with neurotoxicity in cell and mouse models overexpressing α-synuclein (Masliah E et al., Science 2000;287(5456):1265-9; Gosavi N et al., J. Biol. Chem. 2002;277(50):48984-92). Abnormal accumulation of misfolded α-synuclein can promote oxidative stress and lead to mitochondrial changes that can cause cell death (Hsu LJ et al., Am. J. Pathol. 2000;157(2):401-10). Furthermore, three point mutations in the α-synuclein gene (A53T, A30P, or E46K) are known to be involved in the pathogenesis of familial Parkinson's disease (Polymeropoulos MH et al., Science 1997;276(5321):2045-7; Zarranz JJ et al., Ann. Neurol. 2004;55(2):164-73).

[0015] The aggregation rate of α-synuclein was shown in vitro to be enhanced when the protein was incubated with a wild-type porcine POP clone, and this enhancement was dependent on the POP concentration (Brandt I et al., Peptides 2008;29(9):1472-8). Also, a mutant variant (S544A) lacking POP activity did not accelerate the aggregation rate.

[0016] The enhanced aggregation could also be prevented by the addition of POP inhibitors, suggesting that the effect was dependent on POP enzyme activity. Experimental data suggest that POP inhibitors can block the increase in α-synuclein aggregation induced by oxidative stress in neuroblastoma SH-SY5Y cells overexpressing human α-synuclein (Myohanen TT et al., Br. J. Pharmacol. 2012; 166(3): 1097-113). POPs colocalize with α-synuclein in SH-SY5Y cells, and this colocalization disappears after incubation with POP inhibitors, indicating an interaction between POPs and α-synuclein. Five days of treatment with POP inhibitors reduced the amount of soluble α-synuclein in the brains of A30P α-synuclein transgenic mice. In this regard, POP inhibitors have been reported to enhance α-synuclein clearance by modulating autophagy (Myohanen TT et al., Pharmacological Research 2020;151:104558).

[0017] Thus, inhibition of brain POP activity can prevent α-synuclein aggregation and, in this way, the formation of cytotoxic protofibrils present in Lewy bodies. POP inhibitors may therefore potentially have therapeutic value in the treatment of neurodegenerative disorders in which accelerated α-synuclein aggregation has been described.

[0018] Compounds capable of inhibiting POPs are effective in preventing experimental cognitive dysfunction induced by scopolamine in rats, suggesting that POP inhibitors have a function in alleviating memory dysfunction (Yoshimoto T et al., J. Pharmacobiodyn. 1987;10:730-5).

[0019] The effect of subchronic administration of rosmarinic acid, a noncompetitive POP inhibitor (with a relatively high IC50 value of 63.7 μM), was tested in the Morris water maze in rats and reported to enhance spatial memory (Park DH et al., Fitoterapia 2010;81(6):644-8).

[0020] It has been found that patients with bipolar disorder have high levels of POP activity in serum. In recent years, POP has gained importance as a target for the treatment of this disease, especially due to its involvement in the metabolism of inositol-1,4,5-triphosphate (IP3). IP3 is a key molecule in the transmission of signals in the neuropeptide cascade. Through binding to specific receptors, neuropeptides induce an increase in IP3, which binds to its receptor on the membrane of the endoplasmic reticulum and induces the release of Ca2+, which is thought to play an important role in learning and memory. Recent findings show that POP modulates the concentration of IP3 (Komatsu Y J.Neurosci.1996;16:6342-52). Thus, it is known that gene disruption of POP in the eukaryotic Dictyostelium discoideum induces resistance to lithium through elevated IP3 (Schulz I et al., Eur. J. Biochem. 2002; 269: 5813-20) and also reduced the proteolytic activity of POP, which is responsible for high IP3 concentrations in human antisense glioma cells for POP, an effect also observed when these cells are treated with specific POP inhibitors (Williams RS et al., EMBO J. 1999; 18: 2734-45).

[0021] The IP3 signaling pathway is involved in the action of some pharmacotherapeutic mood stabilizers (lithium, carbamazepine and valproic acid), and defects in the mechanisms regulating IP3 signaling may cause bipolar disorder. Valproic acid, a mood stabilizer commonly used to treat bipolar disorder, also directly inhibits the activity of recombinant POP (Cheng L et al., Mol.CeII.Neurosci.2005;29:155-61). In summary, there is strong evidence that POP inhibitors are useful in preventing and / or treating bipolar affective disorder in mammals. Therefore, providing novel inhibitors of POP is of interest in the therapy of this disorder or disease.

[0022] In summary, the effects of several POP inhibitors in various cognitive tasks have been characterized, and there is a consensus that POP inhibitors have positive effects on learning and memory (Morain P et al., CNS Drug. Rev. 2002;8(1):31-52; Shinoda M et al., Eur. J. Pharmacol. 1996;305(1-3):31-8; Marighetto A et al., Learn. Mem. 2000;7(3):159-69; Toide K et al., Pharmacol. Biochem. Behav. 1997;56(3):427-34; Schneider JS et al., Neuropsychopharmacology 2002;26(2):176-82).

[0023] Although several patents and patent applications disclose POP inhibitors: WO2008 / 077978A1, WO2005 / 027934A1, JP2011-037874A2, WO2005 / 002624A1, WO2004 / 060862A2, WO03 / 04468A1; DE19603510A1, US2006 / 0100253A1 and US6,159,938A, there are only a few compounds under investigation in vivo (JTP-4819, S17092, Z-321, ONO-1603, Y-29794, ZTTA, Z-Pro-Prolinal, and KYP-2047). From this list, the first four inhibitors in the list have entered clinical trials and none of them have reached market launch.

[0024] WO2014 / 072498A1 discloses POP inhibitors that have high affinity for POPs and good ability to cross the blood-brain barrier to reach the brain where the inhibitor's action occurs when used to treat cognitive disorders, which are important properties for a compound that is a good candidate for use in the therapy of cognitive disorders.

[0025] In WO2014 / 072498A1, it was stated that the formulations of the compounds disclosed therein can be adapted for any route of administration. However, in the only in vivo examples disclosed in said application, the compounds were administered subcutaneously.

[0026] It would be advantageous to be able to administer POP inhibitors via the oral route, as this is most convenient from the patient's perspective.

[0027] Therefore, there is a need to identify new POP inhibitors, which are particularly well adapted for oral administration and efficiently reach the brain, the site of action, when said POP inhibitors are used in the therapy of cognitive disorders.Serotonin receptor antagonists (5-HT1A receptors) are commonly used as antidepressants.However, they induce significant sexual dysfunction (Uphouse L, Pharmacol.Biochem.Behav.2014;0:31-42), limiting their use for chronic therapy in adult population.For this reason, a POP inhibitor that has positive effects on cognition in mood disorders and does not have such adverse events will have a significant impact on the quality of life of patients. Summary of the Invention

[0028] The inventors have now successfully discovered that 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile, its stereoisomers, and their salts can not only inhibit POPs with high potency, but also exhibit high gastrointestinal penetration capabilities, achieve high brain exposure making them particularly effective when administered orally, and have slight binding to the 5-HT1A receptors avoiding negative side effects such as sexual dysfunction.

[0029] Thus, one aspect of the present invention is a compound of formula (I): [ka] The present invention relates to 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile, its stereoisomers, and pharma- ceutically acceptable salts thereof, having the formula:

[0030] Another aspect of the present invention relates to processes for the preparation of the compounds of formula (I) as defined above, their stereoisomers, and their pharma- ceutically acceptable salts.

[0031] Another aspect of the present invention relates to a medicament or pharmaceutical composition comprising at least one compound of formula (I) as defined above, its stereoisomers, and pharma- ceutically acceptable salts thereof, and a pharma- ceutically acceptable carrier, adjuvant, or vehicle.

[0032] Another aspect of the present invention relates to compounds of formula (I) as defined above, their stereoisomers and their pharma- ceutically acceptable salts for use as medicines, in particular for the prevention and / or treatment of cognitive and / or neurodegenerative disorders, such as synucleinopathies (diseases resulting from the accumulation of synuclein, such as Parkinson's disease, REM sleep behavior disorder, Lewy body disease or multiple system atrophy). In particular, the cognitive disorder may be associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar disorder, REM sleep behavior disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis.

[0033] Another aspect of the present invention relates to a method for the treatment or prevention of cognitive disorders in a mammal, characterized in that a therapeutic amount of a compound of formula (I) as defined above, its stereoisomers and their pharma- ceutically acceptable salts is administered to a patient in need of said treatment. In a particular embodiment, the disorder is a cognitive disorder associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar affective disorder, REM sleep behavior disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis.

[0034] Another aspect of the present invention relates to the use of compounds of formula (I) as defined above, their stereoisomers and their pharma- ceutically acceptable salts for the preparation of a medicament, in particular for the prevention and / or treatment of cognitive disorders, more particularly associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar disorder, REM sleep behavior disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis. These aspects and their preferred embodiments are further defined in the claims. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows the results of the POP inhibition assay for the compounds of Example 1, Comparative Example 3 and Comparative Example 4. [Diagram 2] FIG. 2 shows the results of the PAMPA assay for the compounds of Example 1, Comparative Example 3 and Comparative Example 4. [Diagram 3] FIG. 3 shows the results of the Caco-2 assay for the compounds of Example 1 and Comparative Example 3. [Figure 4] FIG. 4 shows the results of the in vivo brain exposure assay for the compounds of Example 1 and Comparative Example 3. [Diagram 5] 5a and 5b show the results of the NOR challenge assay for the compounds of Example 1 and Comparative Example 3. [Figure 6] FIG. 6 shows the results of binding to the 5-HT1A receptor for the compounds of Example 1 and Comparative Example 3. [Figure 7] FIG. 7 shows the IR spectrum of (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile. [Figure 8]FIG. 8 shows the H-NMR spectrum of (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile. [Figure 9] FIG. 9 shows the IR spectrum of (S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile. [Figure 10] FIG. 10 shows the H-NMR spectrum of (S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] In the context of the present invention, the following terms have the meanings detailed below.

[0037] The term "salt" should be understood as any form of active compound used according to the present invention, where said compound is in ionic form or charged, coupled with counter ions (cations or anions) or in solution. This definition also includes quaternary ammonium salts and complexes of active molecules with other molecules and ions, especially complexes formed through ionic interactions. This definition includes, in particular, physiologically acceptable salts, and this term should be understood as equivalent to "pharmacologically acceptable salts" or "pharmacologically acceptable salts".

[0038] The term "pharmaceutical acceptable salts" means, in the context of the present invention, any salt that is physiologically tolerable (meaning that it is not toxic, in particular as a result of the counterion), when used in a manner appropriate for the treatment applied or used, in particular in humans and / or mammals. These physiologically acceptable salts may be formed with cations or bases, and in the context of the present invention, in particular when used in humans and / or mammals, it is understood to be salts formed with at least one compound used according to the present invention - usually an acid (deprotonated) - for example an anion. These physiologically acceptable salts may also be formed with anions or acids, and in the context of the present invention, in particular when used in humans and / or mammals, it is understood to be salts formed with at least one compound used according to the present invention - usually protonated, for example at the nitrogen - for example a cation and at least one pharmacologically tolerable anion. This definition specifically includes, in the context of the present invention, salts formed with physiologically tolerable acids, i.e. salts of a particular active compound with a physiologically tolerable organic or inorganic acid, in particular when used in humans and / or mammals. Examples of salts of this type are those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.

[0039] The compounds of the present invention represented by formula (I) described above may include enantiomers resulting from the presence of three asymmetric centers in the molecule. Single enantiomers and any mixtures of two or more thereof are within the scope of the present invention.

[0040] Unless otherwise specified, the compounds of the present invention are also meant to include isotopically enriched forms, i.e., compounds which differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of at least one hydrogen atom by deuterium or tritium, or 13 C or 14 Replacement of at least one carbon by a C-enriched carbon, or15 Compounds having the present structure except for the replacement of at least one nitrogen with an N-enriched nitrogen are within the scope of this invention.

[0041] The compound of formula (I) or its salt is preferably in pharmaceutically acceptable form or substantially pure form. Pharmaceutically acceptable form means that it has a pharmaceutically acceptable level of purity, except for normal pharmaceutical additives such as diluents and carriers, and does not contain materials that are considered toxic at normal dosage levels. The purity level for drug substance is preferably greater than 50%, more preferably greater than 70%, and most preferably greater than 90%. In a preferred embodiment, it is greater than 95% of the compound of formula (I) or its salt.

[0042] As stated above, the term "pharmaceutically acceptable salt" refers to any salt that can provide (directly or indirectly) the compounds described herein when administered to a recipient.However, it will be recognized that non-pharmaceutically acceptable salts are also within the scope of the present invention, since they may be useful in the preparation of pharmaceutically acceptable salts, solvates and prodrugs.The preparation of salts can be carried out by methods known in the art.

[0043] As used herein, the terms "treat," "treating," and "treatment" include the eradication, elimination, amelioration, alteration, or management of a disease or condition, such as cognitive impairment.

[0044] As used herein, the terms "prevention," "preventing," "preventive," "prevent" and "prophylaxis" refer to the ability of the compounds of formula (I) to avert, minimize or impede the onset or occurrence of a disease or condition, such as cognitive impairment, before its onset.

[0045] Thus, "treating" or "treatment" and / or "preventing" or "prevention" generally refers to at least suppressing or ameliorating symptoms associated with a condition afflicting a subject, where suppression and amelioration are used broadly to refer to at least a reduction in a parameter, e.g., the magnitude of a symptom associated with the condition being treated, such as cognitive impairment. Thus, the methods of the present invention also include situations in which a condition is completely inhibited, e.g., prevented from occurring or stopped, e.g., terminated, such that the subject no longer experiences the condition. Thus, the methods include both preventing and managing cognitive impairment.

[0046] The terms "cognitive disorder" and "mild cognitive impairment" are used interchangeably herein to designate any condition characterized by deficits in mental activities associated with thinking, learning, or memory. Examples of such disorders include agnosia, amnesia, aphasia, apraxia, delirium, dementia, and learning disabilities.

[0047] Cognitive impairment may be associated with (and is often associated with) other conditions characterized by damage or loss of neurons or other structures involved in the transmission of signals between neurons (i.e., may be caused by or occur in the presence of other conditions). Thus, cognitive impairment may be associated with neurodegenerative diseases such as Alzheimer's disease, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal lobar degeneration, Huntington's disease, multiple sclerosis, normal pressure hydrocephalus, organic chronic brain syndrome, Parkinson's disease, Pick's disease, vascular dementia, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, etc.

[0048] Cognitive disorders may also be associated with other conditions that impair the normal functioning of the central nervous system, including psychiatric disorders, e.g., anxiety disorders, dissociative disorders, mood disorders, e.g., bipolar affective disorder, schizophrenia, and somatoform disorders and factitious disorder.

[0049] The compounds described herein may be used to treat agnosia, amnesia, aphasia, apraxia, delirium, dementia, learning disabilities and other cognitive disorders.

[0050] Examples of dementias that can be treated by the methods of the invention include AIDS dementia complex, Binswanger's disease, dementia with Lewy bodies, frontotemporal dementia, multi-infarct dementia, Pick's disease, semantic dementia and vascular dementia.

[0051] Examples of learning disorders that can be treated by the methods of the present invention include Asperger's syndrome, attention deficit disorder, attention deficit hyperactivity disorder, autism, childhood disintegrative disorder, Down's syndrome, and Rett's syndrome.

[0052] Examples of aphasia that can be treated by the methods of the present invention include progressive non-speech aphasia.

[0053] The compounds described herein may also be used to treat patients with mental activity deficits that are mild or do not otherwise significantly interfere with daily life.Mild cognitive impairment is an example of such a condition, and patients with mild cognitive impairment exhibit dementia symptoms (e.g., difficulty with language or memory), but the severity of these symptoms is such that a diagnosis of dementia may not be appropriate.The compounds described herein may also be used to treat mild cognitive impairment and other, similarly less severe forms of cognitive impairment.

[0054] Therefore, another aspect of the present invention is a method for the treatment or prevention of cognitive impairment in a mammal, comprising administering a therapeutic amount of a compound of the present invention to a patient in need of said treatment.

[0055] In a particular embodiment, the method of treatment or prevention is carried out by administering, by oral route, a therapeutic amount of a compound of the invention to a patient in need of said treatment.

[0056] In certain embodiments, the compounds of the invention are administered orally at a daily dose including 0.01 to 1.90 mg / kg, for example, 0.01, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85 or 1.90 mg / kg. The compounds of the invention may be administered once daily, twice daily, three times daily, four times daily, five times daily, or six times daily at any of the indicated daily oral doses listed above.

[0057] In certain embodiments of the invention, the compounds described herein may be used to treat patients with cognitive impairment associated with schizophrenia, bipolar affective disorder, Alzheimer's disease, or Parkinson's disease.

[0058] In certain embodiments of the invention, the compound has the following formula (Ia): [ka] has.

[0059] Specific individual compounds of the present invention under formula (I) include the compounds listed below: (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2R,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2R,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2R,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2R,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile, or a pharma- ceutically acceptable salt thereof.

[0060] The compounds of formula (I) defined above can be obtained by available synthetic procedures as illustrated by the following general schemes. [ka]

[0061] In the first step, the ester of formula (II) is dissolved or suspended in a polar organic solvent, such as ethanol (EtOH) or methanol (preferably a protic polar organic solvent), or in a mixture of polar organic solvents. An aqueous base solution is added and the hydrolysis reaction is carried out, typically under reflux, by maintaining the mixture at a temperature comprised between room temperature and the reflux temperature of the solvent mixture until completion of the hydrolysis, typically for a period of 0.5 to 4 hours, preferably 1 to 2 hours. The base solution is preferably of an inorganic nature, such as a dilute alkali, e.g. NaOH. The reaction mixture is then allowed to reach room temperature and is preferably concentrated to approximately one-fifth of the reaction volume. The reaction mixture is then slowly added to an acid solution, such as a 1M HCl solution, while cooling in an ice bath, to neutralize. If acidification results in a precipitation, the solid is filtered and washed with water to obtain the product of formula (VII). If no precipitate is obtained, the resulting solution is extracted several times with a suitable organic solvent, such as ethyl acetate, and the organic phase is dried and evaporated. The crude product of formula (VII) is purified by flash chromatography.

[0062] Deprotection of the amine of formula (III) is accomplished under mildly acidic conditions such as addition of a solution of hydrogen chloride in an organic solvent such as dioxane or using a TFA / DCM mixture at low temperatures ranging from 0° C. to room temperature. The reaction is stirred at room temperature for 1-3 hours. The solvent is then evaporated to dryness to give the hydrochloride or trifluoroacetate salt of the amine of formula (VI), depending on the acid used.

[0063] Compounds of formula (IX) are prepared under Schotten-Baumann conditions from carboxylic acids of formula (VII) and amines of formula (VI). Thus, a chlorinating agent such as oxalyl chloride is added to a solution of carboxylic acid of formula (VII) in an organic solvent such as toluene. The reaction is stirred for 1-2 hours at a temperature between 50° C. and 80° C. to form carboxylic acid chloride of formula (VIII). After evaporation of the solvent, the resulting crude is solubilized in an organic solvent such as THF and added to a basic aqueous solution of amine of formula (VI), typically aqueous NaOH solution of amine of formula (VI), at a low temperature such as 0° C. The reaction mixture is stirred for 1-2 hours at low temperature and 2-4 hours at room temperature. The solvent is then evaporated and the remaining aqueous fraction is adjusted to an acidic pH (3-4) by addition of HCl solution and extracted with ethyl acetate. The organic phase is washed with brine, dried, filtered and evaporated. The crude product is purified by flash chromatography, if necessary.

[0064] The product of formula (IX) is then coupled with pyrrolidine-2-carbonitrile of formula (IV) in the presence of a base such as N,N-diisopropylethylamine (DIEA) and assisted by a coupling reagent such as a carbodiimide. In particular, the compound of formula (IX) is dissolved in an aprotic organic solvent such as dichloromethane and added together with DIEA to a solid supported carbodiimide such as N-cyclohexylcarbodiimide, N'-methylpolystyrene. After 5 minutes, pyrrolidine-2-carbonitrile of formula (IV) and additional DIEA are added. The reaction is stirred at room temperature for 8-16 hours. The reaction mixture is then filtered and the remaining solid is washed with an aprotic organic solvent. The filtrate is evaporated to dryness. The crude product is then purified by preparative RP-HPLC.

[0065] Alternatively, compounds of formula (I) may be prepared as shown in the following scheme and described below. [ka]

[0066] An amine-functionalized resin, such as the Sieber amide resin of formula (X), is placed in a syringe equipped with a polyethylene porous disk. The resin is swollen by washing with a suitable organic solvent, such as dichloromethane (DCM) and dimethylformamide (DMF). When the amine group of the resin is protected (i.e., in the case of the Sieber amide resin), removal of the protecting group (such as the fluorenylmethoxycarbonyl (Fmoc) protecting group) is accomplished by treatment with an amine base solution, such as a piperidine solution in DMF. [ka]

[0067] After removal of the protecting group from the resin, the Fmoc-protected L-proline of formula (V) is attached to the resin using an activating agent such as triazole (i.e., TBTU) and an amine base such as DIEA in a suitable organic solvent such as DMF. The mixture is stirred for 1-2 hours. After filtration and washing, the extent of coupling may be followed using the Kaiser test and recoupling is performed if necessary. The Fmoc is removed by treatment with an amine base solution such as a piperidine solution in DMF and / or a mixture of piperidine / DBU / toluene / DMF to give the product of formula (XI). The removal of Fmoc may be assessed using the p-nitrophenyl ester NF31 test. [ka]

[0068] The product of formula (XI) is coupled with the product of formula (IX) using an activating agent such as PyBOP in a suitable organic solvent such as DMF in the presence or absence of an additive such as HOAt and an amine base such as DIEA to give the product of formula (XII). The mixture is manually stirred for a total reaction time of 1-2 hours. Systematic recoupling is carried out using the same amounts and times. The extent of coupling may be followed using the p-nitrophenyl ester NF31 test. [ka]

[0069] Alternatively, the product of formula (XII) may be obtained by stepwise coupling of product (XI) first with a compound of formula (XIII), followed by removal of the Fmoc protecting group and then coupling with a compound of formula (VII).

[0070] The product of formula (XII), thoroughly washed with a suitable organic solvent such as DCM and dried, is transferred to a flask to which trifluoroacetic anhydride and pyridine are added in a small amount of organic solvent. The mixture is kept at a temperature of 20-40°C for 8-16 hours. The reaction mixture is then filtered and the resin is washed with the same organic solvent. The filtrate is collected and the solvent is evaporated to dryness. The crude obtained is dissolved in a suitable solvent such as ethyl acetate and washed with saturated NaHCO3 solution and 5% aqueous KHSO4 solution. The organic phase is dried, filtered and evaporated. The crude is taken up in H2O:CH3CN and lyophilized to obtain the peptide nitrile of formula (I).

[0071] Alternatively, the peptidyl resin of formula (XII) may be treated with a mixture of TFA / H2O / TIS for 1-2 hours. The resin is then filtered, washed with TFA, the filtrate is collected and the solvent is evaporated to dryness. The crude is resuspended in a mixture of H2O:CH3CN and lyophilized. The crude peptide amide obtained is taken up in a suitable organic solvent such as DCM and converted to a nitrile, for example in the presence of phosphorus pentoxide, titanium tetrachloride, thionyl chloride, trifluoroacetic anhydride / pyridine or triphenylphosphine / carbon tetrachloride. The mixture is kept at room temperature for 8-16 hours, the solvent is evaporated and the residue is taken up in ethyl acetate. The organic solution is then washed with aqueous KHSO4 and aqueous NaHCO3 solutions. Drying and evaporation of the organic phase gives the peptide nitrile of formula (I).

[0072] The crude product is purified by RP-HPLC.

[0073] Where the processes for preparing the compounds of the invention described above give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques, such as preparative chromatography. Where asymmetric centers exist, the compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution.

[0074] The compounds of formula (II), (III), (IV) and (V), as well as some of the compounds of formula (VII) used as starting materials, are either commercially available or may be prepared using methods well known to those skilled in the art.

[0075] Thus, in one aspect, the present invention provides a process for preparing a compound of formula (I), or a pharma- ceutically acceptable salt, isomer, prodrug or solvate thereof.

[0076] In one embodiment, the method comprises: a) Formula (IX): [ka] with a compound of formula (XI): [ka] (wherein polymer represents a polymer that is inert under the reaction conditions of the synthetic methods disclosed herein and that is insoluble but swellable in the solvents used herein, such as lightly crosslinked polystyrene and polyethylene glycol grafted polystyrene polymers), to produce a compound of formula (XII): [ka] obtaining a compound of formula (I), b) hydrolyzing the compound of formula (XII) to obtain a compound of formula (XIV): [ka] obtaining a compound of formula (I), c) subjecting the compound of formula (XIV) to conditions capable of converting the carboxamide group to a nitrile group to obtain a compound of formula (I). Including, Steps b) and c) can be carried out separately or in a one-pot reaction.

[0077] In another embodiment of the present invention, a process for preparing a compound of formula (I) or a pharma- ceutically acceptable salt, isomer, prodrug or solvate thereof comprises: a) Formula (IX): [ka] with a compound of formula (IV): [ka] with a compound of formula (I).

[0078] In yet another embodiment, the method comprises: a) Formula (XI): [ka] (wherein polymer represents a polymer that is inert under the reaction conditions of the synthetic methods disclosed herein and that is insoluble but swellable in the solvents used herein, such as lightly crosslinked polystyrene and polyethylene glycol grafted polystyrene polymers), by reacting a compound of formula (XIII): [ka] with a compound of b) removing the Fmoc protecting group; c) Formula (VII): [ka] with a compound of d) hydrolyzing the product obtained from the supported polymer to obtain a product of formula (I): [ka] A process for obtaining a compound of Includes.

[0079] It has been found that compounds of general formula (I) are useful in the treatment of cognitive disorders, particularly cognitive disorders associated with other diseases or conditions of the central nervous system.

[0080] In certain embodiments of the invention, the cognitive disorder is a cognitive disorder associated with a disease selected from the group consisting of schizophrenia, bipolar affective disorder, Alzheimer's disease and Parkinson's disease.

[0081] The present invention further provides a medicament or pharmaceutical composition comprising a compound of the invention or a pharmaceutical salt, derivative, prodrug or stereoisomer thereof, together with a pharma- ceutically acceptable carrier, adjuvant or vehicle for administration to a patient.

[0082] The auxiliary materials or additives of the pharmaceutical composition according to the invention can be selected from among carriers, excipients, carrying materials, lubricants, fillers, solvents, diluents, colorants, flavor modifiers such as sugars, antioxidants, binders, adhesives, disintegrants, antiadhesives, glidants and / or coagulants. In the case of suppositories, this may mean waxes or fatty acid esters, or preservatives, emulsifiers and / or carriers for parenteral application. The choice of these auxiliary materials and / or additives, as well as the amounts used, will depend on the form of application of the pharmaceutical composition.

[0083] The medicament or pharmaceutical composition according to the present invention may be in any form suitable for application to humans and / or animals, preferably humans, including infants, children and adults, and may be produced by standard procedures known to those skilled in the art.The formulation according to the present invention may thus be adapted for local or systemic application, in particular for dermal, transdermal, subcutaneous, intramuscular, intraarticular, intraperitoneal, intravenous, intraarterial, intravesical, intraosseous, intracavernous, intranasal, pulmonary, buccal, sublingual, ocular, intravitreal, transdermal, rectal, vaginal, oral, epidural, intrathecal, intraventricular, intracerebral, intralateral ventricular, intracisternal, intraspinal, perispinal, intracranial, delivery via needle or catheter with or without a pump device, or other application routes.

[0084] In the preferred embodiment of the present invention, pharmaceutical composition is in oral form, either solid or liquid.The dosage form suitable for oral administration can be tablet, pill, caplet, gel cap, chewing gum, capsule, granule, drop, syrup or liquid, and can contain conventional excipients known in the art, such as binder, such as syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; filler, such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tableting lubricant, such as magnesium stearate; disintegrant, such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agent, such as sodium lauryl sulfate.

[0085] In another embodiment, pharmaceutical composition is in the form of product for non-parenteral intranasal administration, preferably in the form of product for intranasal administration.Typically, intranasal administration is carried out by using nasal spray, squeeze bottle and liquid dropper as delivery device.For use in these devices, pharmaceutical composition is advantageously the solution or suspension of the compound of the present invention.

[0086] The compositions may be prepared by conventional blending, filling or tableting methods.The active agent may be distributed throughout these compositions by repeated blending operations using a large amount of filler.Such operations are conventional in the art.Tablets may be prepared, for example, by wet granulation or dry granulation, and may be optionally coated, particularly with enteric coating, according to the methods well known in normal pharmaceutical practice.

[0087] The pharmaceutical composition may also be adapted for parenteral administration in a suitable unit dosage form, e.g., as a sterile solution, suspension or reconstitutable dry preparation, aerosol or spray. Appropriate excipients, e.g., bulking agents, buffers or surfactants, may be used.

[0088] The compositions of the present invention may be formulated in dissolved form or as a deposit in a patch for transdermal application.

[0089] Dermal applications include ointments, gels, creams, lotions, suspensions or emulsions.

[0090] The preferred form of rectal application is by suppository.

[0091] The listed formulations are prepared using standard methods, such as those described or referenced in the Spanish and United States Pharmacopoeias and similar reference texts.

[0092] In one embodiment of the present invention, it is preferred that the compound of formula (I) is used in a therapeutically effective amount. The physician will determine the dosage of the therapeutic agent that is most suitable, which will vary depending on the form of administration and the particular compound selected, as well as the patient being treated, the patient's age, the type of disease, or the condition being treated. When the composition is administered orally, a larger amount of the active agent may be required to produce the same effect as if a smaller amount were given parenterally. The compounds are useful in the same manner as comparable therapeutic agents, and the dosage levels are of the same order of magnitude as these other therapeutic agents are commonly used. The active compounds are typically administered one or more times daily, for example, once, twice, three or four times daily, with typical total daily doses ranging from 0.011 to 1.90 mg / kg / day.

[0093] The compounds and compositions of the invention may be used with other drugs to provide a combination therapy, which may form part of the same composition or may be provided as separate compositions for administration at the same or different times.

[0094] In particular, the combination of at least one compound of formula (I) and at least one other drug may be formulated with at least a pharma- ceutically acceptable carrier, excipient, adjuvant or vehicle for their simultaneous, separate or sequential administration, meaning that the combination of the compound of formula (I) and the other drug may be administered as follows: a) As a combination where both are then always administered at the same time and are part of the same pharmaceutical formulation. b) As a combination of two units, each of which allows the possibility of simultaneous, sequential or separate administration. In a particular embodiment, the compound of formula (I) is administered simultaneously but independently of the other drug (i.e., in two units). In another particular embodiment, the compound of formula (I) is administered first, and then the other drug is administered separately or sequentially. In yet another particular embodiment, the other drug is administered first, and then the compound of formula (I) is administered separately or sequentially, as defined.

[0095] In the context of the present invention, the following acronyms and abbreviations are used, the meanings of which are detailed below: AcOEt Ethyl acetate AD Alzheimer's disease Area under the AUC curve BBB Blood-brain barrier Boc tert-butoxycarbonyl BSA Bovine Serum Albumin DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCM Dichloromethane DMEM Dulbecco's Modified Eagle's Medium DI identification index DIEA N,N'-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl sulfoxide EtOH Ethanol Fmoc 9-Fluorenylmethoxycarbonyl FPLC Fast Protein Liquid Chromatography FTIR Fourier Transform Infrared Spectrometer HBSS Hanks Balanced Salt Solution HOAt 1-Hydroxy-7-azabenzotriazole hPOP human prolyl oligopeptidase IP intraperitoneal IP3 Inositol Triphosphate IPTG Isopropyl β-D-1-thiogalactopyranoside LB Lysogeny Broth MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time of Flight MK-801 Dizocilpine MS multiple sclerosis NMR nuclear magnetic resonance NOR New Object Recognition OD optical density PAMPA Parallel Artificial Membrane Permeability Assay PBS Phosphate Buffered Saline PC Phosphatidylcholine PE Phosphatidylethanolamine Plasmid pETM10 PI Phosphatidylinositol POP Prolyl Oligopeptidase PS Phosphatidylserine PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RP-HPLC Reversed-phase high-performance liquid chromatography SD standard deviation SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TEER Transendothelial Electrical Resistance TFA Trifluoroacetic acid THF Tetrahydrofuran TIS Triisopropylsilane Tris Tris(hydroxymethyl)aminomethane Tβ4 Thymosin beta-4 protein UPLC Ultra High Performance Liquid Chromatography ZGP-AMC (N-benzyloxycarbonyl-Gly-Pro-methylcoumarinyl-7-amide) EXAMPLES

[0096] The following examples are merely illustrative of certain embodiments of the invention and are not to be construed as limiting in any way.

[0097] Specific synthetic conditions used for the preparations described in the examples Step A Hydrolysis of an ester of formula (II) to a carboxylic acid of formula (VII) The ester of formula (II) (1 mmol) is solubilized in 95% EtOH. NaOH (3.7 mmol) is added and the reaction is maintained at reflux for approximately 2 hours. It is then allowed to reach room temperature. The reaction mixture is concentrated to approximately 15-20 mL, and the solution is then slowly added to a 1M HCl solution while cooling in an ice bath. The white solid precipitate is collected by filtration, washed with water and thoroughly dried before the next synthetic step. In case no precipitation occurs, the resulting solution is extracted with AcOEt (3x) and the organic phase is dried and evaporated. The crude product is purified by flash chromatography, if necessary.

[0098] Step B Deprotection of the Boc-protected amine of formula (III) to obtain an amine of formula (VI) The Boc-protected amine of formula (III) (1 mmol) is slowly added to 4 M HCl in dioxane (20 ml) at 0° C. The reaction is stirred at room temperature for 2 hours. The solvent is then evaporated to dryness to give the hydrochloride salt of the amine of formula (VI).

[0099] Step C Coupling of an amine of formula (VI) to a carboxylic acid of formula (VII) by formation of a carboxylic acid chloride of formula (VIII) Oxalyl chloride (1.5 mmol) is added to a solution of carboxylic acid (VII) (1 mmol) in toluene (5 ml). The reaction is stirred at 50° C. for 1.5 hours to form the carboxylic acid chloride of formula (VIII). After evaporation of the solvent, the crude obtained is solubilized in THF and added to an amine of formula (VI) (1.1 mmol) in aqueous NaOH at 0° C. The reaction mixture is stirred at 0° C. for 1.5 hours and at room temperature for 3 hours. The THF is then evaporated and the remaining aqueous fraction is adjusted to an acidic pH (3-4) by addition of 1 M HCl solution and extracted with ethyl acetate. The organic phase is washed with brine, dried, filtered and evaporated. The crude product of formula (IX) is purified by flash chromatography, if necessary.

[0100] Step DCoupling of the product of formula (IX) to pyrrolidine-2-carbonitrile of formula (IV) in solution The product of formula (IX) (1.2 mmol) is dissolved in DCM and added to N-cyclohexylcarbodiimide, N'-methylpolystyrene (3 mmol) together with DIEA (1 mmol). After 5 min, (S)-pyrrolidine-2-carbonitrile of formula (IV) (1 mmol) and DIEA (1 mmol) are added. The reaction is stirred overnight at room temperature. The reaction mixture is then filtered and the remaining solid is washed with DCM. The filtrate is evaporated to dryness. The crude product is then purified by preparative RP-HPLC and lyophilized to give the product of formula (I).

[0101] Step E : General procedure for synthesis on solid phase Resin swelling / pretreatment: Sieber amide resin of formula (X) (1 equivalent) is placed in a syringe equipped with a polyethylene porous disk. The resin is swelled by washing with DCM and DMF. Removal of the fluorenylmethoxycarbonyl (Fmoc) protecting group is achieved by treatment with a 20% solution of piperidine in DMF. [ka]

[0102] Fmoc-protected L-proline (4 eq.) of formula (V) is then attached to the resin using TBTU (4 eq.) and DIEA (8 eq.) in DMF. The mixture is manually stirred intermittently for 90 min. After filtration and washing, the extent of coupling is monitored using the Kaiser test, and recoupling is performed if necessary. Fmoc is removed by treatment with a 20% piperidine solution in DMF, followed by a piperidine / DBU / toluene / DMF (20:5:5:70) solution to obtain the product of formula (XI). Fmoc removal is assessed using the p-nitrophenyl ester NF31 test (described in Madder, A. et al., Eur. J. Org. Chem. 1999; (11): 2787-91). [ka]

[0103] The product of formula (IX) (2 equivalents) is coupled to the product of formula (XI) using PyBOP (2 equivalents), HOAt (6 equivalents) and DIEA (6 equivalents) in DMF to obtain the product of formula (XII). The mixture is manually stirred intermittently for a total reaction time of 90 minutes. Systematic recoupling is performed using the same amounts and times. The extent of coupling is monitored using the p-nitrophenyl ester NF31 test.

[0104] Alternatively, the product of formula (XIII) (4 eq.) is coupled to the product of formula (XI) using PyBOP (4 eq.), HOAt (12 eq.) and DIEA (12 eq.) in DMF. The mixture is manually stirred intermittently for a total reaction time of 90 min. The extent of coupling is monitored using the p-nitrophenyl ester NF31 test and recoupling is performed if necessary. The Fmoc group is removed by treatment with a 20% piperidine solution in DMF and a piperidine / DBU / toluene / DMF (20:5:5:70) solution. The product of formula (VII) (4 eq.) is then incorporated using PyBOP (4 eq.), HOAt (12 eq.) and DIEA (12 eq.) in DMF to give the product of formula (XII). The mixture is manually stirred intermittently for a total reaction time of 90 min. The extent of coupling is monitored using the p-nitrophenyl ester NF31 test and recoupling is performed if necessary. [ka]

[0105] The product of formula (XII), thoroughly washed with DCM and dried, is transferred to a round-bottom flask and trifluoroacetic anhydride (5 eq.) and pyridine (10 eq.) in DCM (approximately 2 mL / 100 mg) are added. The mixture is kept at room temperature overnight. The reaction mixture is then filtered and the resin is washed with DCM. The filtrate is collected and the solvent is evaporated to dryness. The crude obtained is dissolved in AcOEt and washed with saturated NaHCO3 solution and 5% aqueous KHSO4 solution. The organic phase is dried, filtered and evaporated. The crude is taken up in H2O:CH3CN (1:1) and lyophilized to obtain the peptide nitrile of formula (I).

[0106] Alternatively, the peptidyl resin of formula (XII) may be treated with a mixture of TFA / H2O / TIS (95:2.5:2.5, approximately 2-5 mL / 100 mg) for 1-2 hours. The resin is then filtered, washed with TFA, the filtrate is collected and the solvent is evaporated to dryness. The crude is resuspended in a mixture of H2O:CH3CN (1:1) and lyophilized. The resulting crude peptide amide is taken up in DCM and trifluoroacetic anhydride (5 eq.) and pyridine (10 eq.) are added. The mixture is kept at room temperature overnight, the solvent is evaporated and the residue is taken up in AcOEt. The organic solution is then washed with 5% aqueous KHSO4 and 5% aqueous NaHCO3. Drying and evaporation of the organic phase gives the peptide nitrile of formula (I).

[0107] The crude product is purified by RP-HPLC.

[0108] Example 1 (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile [ka] Commercially available Fmoc-protected L-proline (Fmoc-L-Pro-OH) of formula (V) (249.7 mg, 0.7 mmol), (2S,4R)-4-fluoro-1-Fmoc-pyrrolodine-2-carboxylic acid of formula (III) (533 mg, 1.5 mmol) and 4-benzyloxy-3,5-difluorobenzoic acid of formula (VII) (271 mg, 1 mmol) are coupled sequentially on commercially available Sieber amide resin (333.3 mg, 0.2 mmol, 1 equiv.) by stepwise coupling as described in step E above. Purification by RP-HPLC gives 156 mg (0.341 mmol) of the final product. Melting point: 137.6-139.1°C. FTIR: 1642, 1582, 1517, 1414, 1381, 1317, 1230, 1203, 1172, 1064, 1036cm -1 . 1 H-NMR (CDCl3, 400MHz) δ(ppm): 7.42(2H,m), 7.35(3H,m), 7.12(2H,m), 5.30(1H,m), 5.23(1H,m), 4.9 3(1H,dd), 4.82(1H,m), 3.87(2H,m), 4.08 / 3.69(2H,m), 2.58 / 2.36(2H,m), 2.26(2H,m), 2.24(2H,m).

[0109] Example 2 (S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile [ka] This compound was prepared according to the method described in Example 1, but replacing (2S,4R)-4-fluoro-1-Fmoc-pyrrolodine-2-carboxylic acid with (2S,4S)-4-fluoro-1-Fmoc-pyrrolodine-2-carboxylic acid. Purification by RP-HPLC gives 138 mg (0.302 mmol) of the final product. Melting point: 135-137°C. FTIR: 3483, 2954, 1653, 1581, 1518, 1437, 1417, 1383, 1356, 1321, 1234, 1203, 1172, 1065, 1039, 1005, 960, 901, 856, 762, 737, 696cm -1 .

[0110] Comparative Example 3: (S)-1-((2S)-1-(4-(benzyloxy)-3,5-dimethoxybenzoyl)-4,4-difluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile [ka] Starting from commercially available Sieber amide resin (250 mg, 0.19 mmol, 1 eq.), commercially available Fmoc-L-proline (Fmoc-L-Pro-OH) (258 mg, 0.77 mmol) and ((S)-1-(4-(benzyloxy)-3,5-dimethoxybenzoyl)-4,4-difluoropyrrolidine-2-carboxylic acid) (161 mg, 0.38 mmol), the product is prepared according to procedure E described above. Purification by RP-HPLC gives 18 mg (0.036 mmol) of the final product.

[0111] Comparative Example 4: (S)-1-((S)-1-(4-(benzyloxy)-3-fluorobenzoyl)-4,4-difluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile [ka] Commercially available Fmoc-protected L-proline (Fmoc-L-Pro-OH) (150 mg, 0.45 mmol), Fmoc-4,4-difluoro-L-proline (166 mg, 0.45 mmol) and 4-benzyloxy-3-fluorobenzoic acid (109 mg, 0.45 mmol) are sequentially coupled onto commercially available Sieber amide resin (200 mg, 0.15 mmol, 1 equiv.) by the stepwise coupling described above in step E. Purification by RP-HPLC gives 14 mg (0.030 mmol) of the final product.

[0112] Pharmacology Data Determination of the inhibitory effect of novel compounds on (human) prolyl oligopeptidase activity. Expression and purification of prolyl oligopeptidase (POP) POP was obtained by expression in E. coli and affinity purification using His-tail fusions according to literature procedures (Tarrago T et al., ChemBioChem 2006;7:827-33) summarized below.

[0113] hPOP Expression: E. coli BL21 competent cells were transformed with pETM10 hPOP. To induce expression, a preculture of LB medium (50 mL) containing kanamycin (50 μg / mL) was inoculated with one colony and grown overnight at 37°C. The following day, two cultures (500 mL) of LB medium were inoculated with the overnight culture (10 mL). The inoculated culture was incubated at OD 595The cells were grown at 37 °C and 220 rpm until the pH reached 1.2 (2.5-3 h). IPTG was then added (1 mM final concentration) and induction was performed overnight at 25 °C. The cells were harvested (3500 g, 15 min, 4 °C) and the pellet was suspended in suspension buffer (50 mL) [Tris-HCl, pH 8 (50 mM), NaCl (300 mM), imidazole (1 mM)] and sonicated at 50% and 0.5 pulse intensities using 4 cycles (each consisting of 15 s of sonication and 15 s of rest) and the samples were kept on ice. After sonication, the samples were centrifuged (40000 g, 30 min, 4 °C) and the supernatant was used immediately for POP purification. An AKTA Explorer FPLC system was used for purification. The supernatant was applied to a HiTrapQuelating column (5 mL) pre-equilibrated with 5 column volumes of suspension buffer at a flow rate of 1 mL / min. The column was washed with suspension buffer until the absorbance at 280 nm returned to basal level. The column was then rinsed with 5 volumes of washing buffer (50 mM Tris-HCl, pH 8, 300 mM NaCl, 30 mM imidazole). Elution was performed with 4 volumes of elution buffer (50 mM Tris-HCl, pH 8, 300 mM NaCl, 500 mM imidazole). Fractions (4 mL) were collected during the entire elution. POP activity was checked in all fractions and the positive ones were analyzed by SDS-PAGE and stained with Biosafe Comassie Stain G-250. The positive fractions were collected and desalted by using a HiPrep26 / 10 desalting column with Tris-HCl (50 mM, pH 8) as buffer. Recombinant hPOP was quantified by Bio-Rad protein assay with BSA as standard. Aliquots of the recombinant enzyme were prepared, immediately frozen in liquid nitrogen and stored at -80°C.

[0114] POP inhibition assay POP activity was determined according to the method described by Toide et al. (Toide K et al., J. Pharmacol. Exp. Ther. 1995;274:1370-8) using ZGP-AMC (N-benzyloxycarbonyl-Gly-Pro-methylcoumarinyl-7-amide) as the POP substrate. Reactions were performed in 96-well microtiter plates, which allows multiple reactions to be followed simultaneously. For each reaction, activity buffer (134 μl, 100 mM Na / K phosphate buffer, pH 8.0) was preincubated for 15 min at 37 °C with hPOP (ranging from 20 to 60 nM depending on the activity of the hPOP batch) and the corresponding new compound solution (3 μl). Stock solutions of new compounds were prepared in DMSO (100 mM) and dilutions were prepared from this stock solution with DMSO.

[0115] After preincubation, ZGP-AMC (10 μl, 3 mM in 40% 1,4-dioxane) was added (3 μl, 1.5 mM in 40% 1,4-dioxane, condition B) and the reaction was incubated for 1 h at 37° C. The reaction was stopped by adding sodium acetate (150 μl, 1 M, pH 4) and the formation of AMC was measured fluorescently. The excitation and emission wavelengths were 360 / 40 nm and 485 / 20 nm, respectively.

[0116] Several concentration points (ranging from 25 pM to 400 μM) were measured for each compound. The inhibitory activity against prolyl oligopeptidase was calculated according to Equation 1. For each new compound, the fluorescence was measured in the presence (a) and absence (b) of hPOP. The maximum fluorescence (0% inhibitory activity) was obtained from a sample of hPOP in the absence of inhibitory compound. To estimate the inhibitory potency of the new compounds, the activity was plotted against the log concentration of the compound and fitted to a sigmoidal curve using GraphPad Prism software, and the IC, defined as the concentration of compound required to inhibit 50% of POP activity, was calculated. 50 Values ​​were determined from the resulting curve.

number

[0117] The new compounds show high inhibitory potency against human prolyl oligopeptidase. The results are summarized in Table 1. [Table 1]

[0118] The data in Table 1 are presented in Figure 1, in which the bars represent the mean values ​​± standard deviation (SD).

[0119] Determination of compound permeability Parallel artificial membrane permeability assay (PAMPA) The parallel artificial membrane permeability assay (PAMPA) described in Kansy M et al., J. Med. Chem. 1998;41(7):1007-10 was used to determine the ability of compounds to cross the blood-brain barrier (BBB) ​​by passive diffusion (Di L et al., Eur. J. Med. Chem. 2003;38(3):223-32). The effective permeability (P e) were measured at an initial concentration of 200 μM. Buffer solutions were prepared from commercial concentrates according to the manufacturer's instructions. The pH was adjusted to 7.4 using 0.5 M NaOH solution. Stock solutions of new compounds were prepared in DMSO and diluted with buffer solution to a final concentration of 200 μM (0.5% DMSO content). The PAMPA sandwich was separated and each donor well was filled with 200 μL of compound solution. The acceptor plate was placed on the donor plate, ensuring that the underside of the membrane was in contact with the buffer. A mixture of 4 μL of phospholipids (20 mg / mL) in dodecane was added to the filter of each well and 200 μL of buffer solution was added to each acceptor well. The plate was covered and incubated for 4 h at room temperature in a saturated humidity atmosphere under orbital agitation at 100 rpm. After 4 hours, the contents of the acceptor and donor compartments were analyzed by HPLC: 150 μL of each well from the donor plate and 150 μL of each well from the acceptor plate were transferred to HPLC vials and each sample was eluted with reverse phase C 18 The column (150 mm x 4.6 mm x 5 μm, 100 Å) was injected (100 μL per injection from the acceptor well and 10 μL per injection from the donor well for the t0 reference). Transport was also confirmed by MALDI-TOF spectroscopy.

[0120] The phospholipid mixture used was a porcine polar brain lipid extract provided by Avanti polar lipids with the following composition: 12.6% phosphatidylcholine (PC), 33.1% phosphatidylethanolamine (PE), 18.5% phosphatidylserine (PS), 4.1% phosphatidylinositol (PI), 0.8% phosphatidic acid and 30.9% other compounds.

[0121] After 4 hours, the effective permeability (P e ) was calculated using Equation 2, the percentage of transport (T%) was calculated using Equation 3, and compound retention in the phospholipid membrane (R%) was calculated using Equation 4.

number

number

number

[0122] Based on the index Pe values ​​shown in Table 2, the novel compounds show good permeability through the BBB (Table 3). [Table 2] [Table 3]

[0123] The data in Table 3 are presented in Figure 2, in which the bars represent the mean values ​​± standard deviation (SD).

[0124] Caco-2 assay Caco-2 cells are widely used as an in vitro model to predict human drug absorption. The Caco-2 cell line is derived from a human colorectal carcinoma, and when cultured, the cells spontaneously differentiate into a monolayer of polarized enterocytes.

[0125] Drug permeability across Caco-2 cell monolayers has been shown to correlate well with in vivo absorption in men (Zhao YH et al., 2001, J. Pharmaceut. Sci. 90:749-784) and has become a well-established in vitro method for prediction of intestinal absorption (Kansy M et al., 2001, Pharmacacokinetic optimization in drug research, Eds. Testa B et al., 447-646).

[0126] Experiments were performed using the CacoReay permeability kit (ReadyCell, Barcelona, ​​Spain).

[0127] The same week of receipt of the kit, the transport medium for the cells was liquefied according to the user manual provided with the kit, and the transport medium was then replaced with fresh Dulbecco's Modified Eagle Medium (DMEM, low glucose).

[0128] After 3 days, equivalent to day 20 of cell culture, the quality of the barrier system and the presence of tight junctions between the cells were checked by measuring the transendothelial electrical resistance (TEER) of the cell barrier, which was >200 Ω cm 2 A TEER value of 0.001 (or higher) indicates that the barrier system is acceptable for absorption assays. The electrodes were sterilized by immersion in ethanol (70%) for 30 min under UV light. The electrodes were then equilibrated for 30 min in pre-warmed DMEM medium at room temperature. TEER measurements were performed at room temperature and had a value of 1357 ± 168 Ω cm. 2 The mean value of 0.01 was obtained to confirm the integrity of the cell barrier.

[0129] Prior to the permeability assay, prepare transport buffer (HBSS (1x)-Ca 2+ / Mg 2+ ) and warmed to 37°C to avoid temperature stress of the cells during the experiment. In parallel, the compounds to be tested were added to the transport buffer (HBSS(1x)-Ca 2+ / Mg 2+) at a final concentration of 50 μM containing 0.5% DMSO, an amount that is well tolerated by cells and does not affect barrier integrity. Compounds were tested at a concentration of 50 μM.

[0130] Prior to the permeability assay, the DMEM in the apical and basal compartments was removed by aspiration. A volume of 10 μL of medium was left in each compartment to prevent the cells from drying out and damaging their barrier properties. Both compartments were rinsed with transport buffer, and the basal (bottom) compartment was filled with 300 μL of transport buffer and the apical (top) compartment with 75 μL of transport buffer.

[0131] The contents of the basolateral compartment were completely removed by aspiration and refilled with 250 μL of transport buffer. The contents of the apical compartment were also removed by aspiration, leaving 10 μL of transport buffer to prevent the cells from drying out. 65 μL of the compound solution to be tested was added to each well.

[0132] For transport experiments (apical to basal), cells were incubated with solutions containing compounds for 2 h at 37° C. The contents of the compartments were then collected and analyzed by HPLC (column C18 Sunfire 100 mm×4.6 mm, 3.5 mm, 100 Å, Waters; flow rate 1 mL / min; gradient 0-100% BA=0.045% trifluoroacetic acid in H2O and B=0.036% trifluoroacetic acid in acetonitrile in 8 min, detection at 220 nm). All experiments were performed in triplicate.

[0133] After removing the samples from the compartments, the basal compartment was refilled with 250 μL of transport buffer and the apical compartment with 65 μL of Lucifer Yellow (LY) solution at a concentration of 20 μM in transport buffer to check the integrity of the cell barrier during the assay. The cell barrier was incubated with the LY solution for 1 h at 37° C. LY is used as a barrier integrity marker for the permeability assay. After incubation, the contents of the compartments were collected and analyzed using a fluorometer.

[0134] For a fully functional Caco-2 barrier, the permeability of LY should be less than 0.7% after 1 hour. After the assay, LY permeability was below this transport average indicating the barrier integrity of the barrier during compound evaluation.

[0135] In the Caco-2 permeability assay, the percentage of transport (T%) for a compound was calculated according to the following formula:

number

[0136] As shown above, the gastric permeability of the compound of Example 1 is much higher than that of Comparative Example 3. In the figure, the bars represent the mean value ± standard deviation (SD).

[0137] The data in Table 4 are presented in FIG.

[0138] In vivo brain exposure For each compound, 24 adult male mice, 7-8 weeks old on the day of the experiment, were used for the study and randomly assigned among the groups. The experiment was performed in three groups of animals as described in the table below. [Table 5]

[0139] Following compound administration, peripheral blood was obtained at several time points after either intraperitoneal (Comparative Example 3) or oral (Example 1) dosing.

[0140] Brains were also collected after transcardial perfusion with phosphate-buffered saline. All samples were stored at -80°C until bioanalysis.

[0141] Plasma samples were thawed at room temperature (RT) and samples were protein precipitated with 2 volumes of acetonitrile containing 20 ng / mL phenacetin as an internal standard. Samples were mixed thoroughly followed by centrifugation at 2,200×g for 20 min. Supernatants were diluted 1:1 with 150 mM phosphate buffered saline (pH 7.4) and pipetted into a UPLC 96-well plate and awaited analysis.

[0142] Brain samples were prepared for analysis by weighing, thawing at RT, and homogenizing the tissue in a bead homogenizer with four volumes of phosphate-buffered saline. The homogenate was treated similarly to the plasma samples.

[0143] Reference (calibration) samples were prepared similarly to the actual study samples after spiking blank plasma and brain homogenate to concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10000ng / mL for Example 1 and Comparative Example 3. Quality control samples were prepared similarly to the actual study samples after spiking blank plasma and brain homogenate to concentrations of 20, 200 and 2000ng / mL for Example 1 and Comparative Example 3.

[0144] Levels of the compounds of Comparative Example 3 and Example 1 were quantified from mouse plasma and brain. Samples were analyzed using a tailored bioanalytical method using UPLC / QE-Orbitrap-Mass Spectrometry.

[0145] The concentration of compound (ng / mL) over time was plotted and the area under the curve (AUC) in plasma and brain samples was calculated using Graphpad Prism™ 5.0. The plasma to brain ratio was calculated as follows:

number

[0146] The data in Table 5 is presented in FIG.

[0147] As shown above, when using the compound of Example 1 via oral administration, it is possible to achieve higher brain exposures than those achieved with other compounds, even when the compound is administered intraperitoneally.

[0148] Microsomal stability (rat) Microsomal stability is useful for predicting whether a compound will survive first-pass metabolism and achieve systemic exposure after oral administration, and is also useful for predicting the half-life of a compound in the bloodstream.

[0149] The stability of the compounds in pooled rat (Sprague-Dawley) liver microsomes (cat. no. 452501) was investigated according to the instructions provided by the supplier (BD Bioscience). Stock solutions (500 μM) of the compounds to be tested were prepared in DMSO. 2 μL of the compounds were incubated at a final concentration of 1 μM in the following mixture: - 713 μL H2O - 200 μL of 0.5 M potassium phosphate (pH 7.4) - 50 μL of NADPH regenerating solution A (BD Bioscience, Catalog No. 451220) - 10 μL of NADPH regenerating solution B (BD Bioscience, Catalog No. 451220)

[0150] The resulting mixture was warmed to 37° C. for 5 min. Then, 25 μL of liver microsomes were added at a final concentration of 0.5 mg / mL. The mixture was incubated at 37° C. with orbital stirring (100 rpm). At selected time points, 100 μL aliquots were extracted and mixed with 100 μL of acetonitrile. The resulting precipitate was shaken on a vortex for 1 min. The samples were kept at 4° C. for 30 min, after which the samples were centrifuged at 20,000×g at 4° C. for 30 min. The supernatant was filtered and then analyzed by UPLC-MS. All stability studies were performed in duplicate. [Table 7]

[0151] These data suggest that Comparative Example 3 and Comparative Example 4 cannot overcome metabolic first-pass after oral administration. Therefore, they will not reach the systemic circulation after oral administration. In fact, Comparative Example 3 shows no efficacy after oral administration, and very low drug plasma levels are detected.

[0152] PK in mice Pharmacokinetics of the compound after intravenous bolus administration was carried out in male Swiss Albino mice. The study was conducted as per the guidelines of the Institutional Animal Ethics Committee (IAEC) and in compliance with the requirements of the Committee for the Control and Supervision of Experiments on Animals (CPCSEA), India.

[0153] The study was conducted with 12 mice per compound using a fractional sampling design (n=3 / time point) to generate a composite profile. Compounds were dissolved in 2% Tween 80® in saline (0.9 NaCl) at a concentration of 0.5 mg / mL. Mice were administered a single dose of 1 mg / kg compound solution by intravenous bolus through the lateral tail vein using a 1 mL BD syringe guided by a 26G needle at a dose volume of 2 mL / kg.

[0154] Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24 and 48 hours after dosing (n=3 per time point). At each time point, 120 μL of blood was collected by retro-orbital plexus puncture and transferred to a labeled microtube containing 200 mM K2EDTA solution (20 μL per mL of blood). Blood samples were centrifuged at 5000×g for 5 minutes at 4±2° C. Plasma samples were stored at or below −60° C. until bioanalysis.

[0155] Plasma samples were analyzed using an LC-MS / MS method with an LLOQ of 1.06 ng / mL. Pharmacokinetic parameters in plasma were calculated using the non-compartmental analysis tool of validated Phoenix WinNonlin® software (version 6.3). Area under the concentration-time curve (AUC last ) was calculated by the linear trapezoidal rule. max ) and time to peak plasma concentration (Tmax) were observed values. Clearance (CL) and volume of distribution (Vss) were predicted values. [Table 8]

[0156] Comparative Example 4 showed very high clearance after intravenous (iv) administration in mice, indicating that the compound undergoes high metabolic first-pass (consistent with the microsomal stability data) and suggesting that the compound does not reach the systemic circulation after oral administration.

[0157] Effects of novel compounds on learning and memory in animal models of cognitive impairment

[0158] New compounds were evaluated for their effectiveness as cognitive enhancers in pharmacological models for cognitive dysfunction. The effects of new compounds were evaluated in untreated and MK-801-treated rodents (mice or rats). MK-801 is a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors that impairs animal performance in various learning and memory paradigms (Castellano C et al., Curr. Drug Targets 2001;2:273-83.; Riedel G et al., Behav. Brain Res. 2003;140:1-47). MK-801 also produces a variety of effects on rodent behavior, including deficits in sensory processing, hyperkinesis, stereotypy and ataxia. The behavioral phenotype induced by MK-801 treatment has been widely used as an animal model of cognitive deficits (Bardgett ME et al., Brain Res. Bull. 2003;60:131-42; Van der Staay FJ et al., Behav. Brain Res. 2011;220:215-29; Mutlu O et al., Pharmacol. Biochem. Behav. 2011;99:557-65).

[0159] To determine whether the tested compounds act as cognitive enhancers, their ability to restore normal cognitive behavior was tested by widely used tests such as the novel object recognition test (Dere E et al., Neurosci. Biobehav. Rev. 2007;31:673-704; Boess FG et al., J. Pharmacol. Exp. Ther. 2007;321:716-25); passive or inhibitory avoidance task (Sarter M et al., Psychopharmacology (Berl) 1992;107:144-59); Morris water maze (D'Hooge R et al., Brain Res. Rev. 2001;36:60-900); and the T-maze transfer task (Boess FG et al., Neuropharmacology 2004;47:1081-92; Spowart-Manning L et al., Behav. Brain 2004;321:716-25); Res.2004;151:37-46).

[0160] As representative examples for the evaluation of new POP inhibitors, the protocols followed for each of the behavioral tests and the results obtained in the object recognition and passive avoidance tests are described.

[0161] Novel object recognition task The novel object recognition (NOR) task is based on the natural preference of rodents to explore novel objects (Ennaceur A et al., Behav. Brain Res. 1988;31:47-59). It is a relevant non-reward test for studying visual learning and memory deficits. Briefly, the NOR task procedure consisted of three trials: habituation, training and retention. Each animal was habituated to a circular arena with a diameter of 40 cm for 10 minutes in the absence of objects (habituation session). The next day, animals were placed in the circular arena for 10 minutes for training trials, in which two identical objects were placed in symmetrical positions. This procedure was performed for two consecutive days. On the third day, one of the objects was replaced by a different object. The object that was not used in the training trials was used as a novel object in the retention trials. The animals were then allowed to freely explore for 10 minutes, and the time spent exploring each object was recorded. Animals were expected to spend more time exploring novel objects, which is a sign of intact recognition memory. A discrimination index was calculated as follows: time spent exploring the new object minus time spent exploring the old object, divided by the total time exploring both objects, and multiplied by 100. A higher discrimination index was considered to reflect greater memory retention.

[0162] Compounds administered by the oral route Experiments were performed using 8-9 week old male C57 / Bl6 mice (n=7-9 animals per group). Compounds were dissolved using a mixture of 5% Tween 80® in saline (0.9% NaCl) to obtain a solution with a strength of 0.5 mg / mL.

[0163] The test was divided into three parts: habituation, training and testing. On the first day, the animals were habituated to the arena (circular, 40 cm diameter) for 10 min. On the next two days, the animals were allowed to freely explore the two identical objects placed in the arena for 10 min. In the test session (day 4), one of the objects was replaced by a novel object. Prior to this session, 35 min before testing, the animals were administered the compound under study (5% Tween 80® in saline, 5 mg / kg) or vehicle (5% Tween 80® in saline, 1 mL per 100 g body weight) by os (po). To induce cognitive deficits, dizocilpine (MK-801) was administered subcutaneously at a dose of 0.2 mg / kg in saline to the negative control group or the drug test group 20 min before testing. The baseline control group was injected with saline.

[0164] After drug administration, each mouse was allowed to freely explore the objects for 10 min. Discrimination index (DI) was defined as the parameter for evaluation.

number

[0165] The experiment was recorded using a webcam placed 1.5 m above the arena. The distance traveled by the mice as a percentage of time explored in the circular arena area was analyzed using Panlab SMART 2.0 software.

[0166] Compounds administered by intraperitoneal route Experiments were performed using 8-9 week old male C57 / Bl6 mice (n=7-9 animals per group). Compounds were dissolved using a mixture of 5% Tween 80® in saline (0.9% NaCl) to obtain a solution with a strength of 0.5 mg / mL.

[0167] The test was divided into three parts: habituation, training and testing. On the first day, the animals were habituated to the arena (circular, 40 cm diameter) for 10 min. On the next two days, the animals were allowed to freely explore the two identical objects placed in the arena for 10 min. In the test session (day 4), one of the objects was replaced by a novel object. Prior to this session, 35 min before testing, the animals were intraperitoneally administered the compound under study (5% Tween 80® in saline, 5 mg / kg) or vehicle (5% Tween 80® in saline, 1 mL per 100 g body weight). To induce cognitive deficits, dizocilpine (MK-801) was administered subcutaneously in saline at a dose of 0.2 mg / kg 20 min before testing to the negative control group or drug test group. The baseline control group was injected with saline.

[0168] After drug administration, each mouse was allowed to freely explore the objects for 10 min. Discrimination index (DI) was defined as the parameter for evaluation.

number

[0169] The experiment was recorded using a webcam placed 1.5 m above the arena. The distance traveled by the mice as a percentage of time explored in the circular arena area was analyzed using Panlab SMART 2.0 software.

[0170] The results obtained when administering the compound of Example 1 and the compound of Comparative Example 3 are provided in the table below. [Table 9]

[0171] The data in Table 8 are presented in Figure 5a (ip administration) and Figure 5b (po administration), in which the bars represent the mean ± sem of the animals. * P < 0.05, ** P < 0.01, ***P<0.001 versus basal (basal MK 801) cognitive deficit condition (one-way ANOVA test followed by Dunnett post-hoc test).

[0172] As shown above, the compound of Example 1 is superior to Comparative Example 3 when it is administered by the oral route, making it particularly suitable for oral administration.

[0173] Selectivity profile (5-HT1A binding) The experiments were performed using a SafetyScreen44® panel from Cerep / Eurofins. Compounds were tested at a concentration of 100 μM (in 1% DMSO).

[0174] Compound binding was calculated as the percentage inhibition of binding of the radiolabeled ligand specific for each target.

[0175] Results are expressed as percent inhibition of control specific binding obtained in the presence of the test compound.

number

[0176] Results showing greater than 50% inhibition are considered to represent a significant effect of the test compound. [Table 10]

[0177] The data in Table 9 are presented in FIG.

Claims

1. Formula (I): 【Chemistry 1】 wherein the asterisk represents an asymmetric center, a stereoisomer thereof, or a salt of said compound or stereoisomer.

2. The compound of formula (I) has the formula (Ia): 【Chemistry 2】 2. The compound of claim 1 , which is a compound of the formula:

3. The compound is (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2R,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2R,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (S)-1-((2R,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (R)-1-((2R,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile 2. The compound of claim 1, a stereoisomer thereof, or a salt of said compound or stereoisomer, selected from:

4. A process for preparing a compound of formula (I) as defined in any one of claims 1 to 3, comprising a) Formula (IX): 【Chemistry 3】 with a compound of formula (XI): 【Chemistry 4】 to form a compound of formula (XII): 【Chemistry 5】 obtaining a compound of formula (I), b) hydrolyzing the compound of formula (XII) to obtain a compound of formula (XIV): 【Chemistry 6】 obtaining a compound of formula (I), c) subjecting said compound of formula (XIV) to conditions capable of converting the carboxamide group to a nitrile group to obtain said compound of formula (I). Including, Steps b) and c) can be carried out separately or in a one-pot reaction. method.

5. A process for preparing a compound of formula (I) according to any one of claims 1 to 3, a stereoisomer thereof, or a salt of said compound or stereoisomer, comprising the steps of: Formula (IX): 【Chemistry 7】 with a compound of formula (IV): 【Chemistry 8】 with a compound of formula (I).

6. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 3, a stereoisomer thereof, or a pharma- ceutically acceptable salt of said compound or stereoisomer, and a pharma- ceutically acceptable carrier, adjuvant or vehicle.

7. A pharmaceutical composition for the treatment and / or prevention of cognitive impairment, comprising a compound of formula (I) according to any one of claims 1 to 3, a stereoisomer thereof, or a salt of the compound or stereoisomer.

8. 8. The pharmaceutical composition of claim 7, wherein the cognitive disorder is a cognitive disorder associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar disorder, REM sleep behavior disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, or amyotrophic lateral sclerosis.

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