RU2841064C1 - 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to pharmaceutical chemistry and involves a compound of formula (I) and stereoisomers thereof, a method for production thereof, a pharmaceutical composition and use thereof.

EFFECT: compound of formula (I) and stereoisomers thereof, having prolyl oligopeptidase (POP) inhibitory activity and used to treat or prevent cognitive disorder.

10 cl, 11 dwg, 9 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to compounds having pharmacological activity, and more particularly to 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile, its stereoisomers and their salts, to methods for producing such compounds, pharmaceutical compositions containing them, and their use in the therapy and/or prevention of neurodegenerative disorders and/or cognitive impairment.

LEVEL OF TECHNOLOGY

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 end of L-proline residues. It is widely distributed in mammals and can be isolated from various organs, including the brain.

The enzyme plays an important role in the breakdown of proline-containing neuropeptides 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).

The effects of prolyl oligopeptidase inhibition have been tested in the treatment of cognitive deficits associated with neurodegenerative processes. Parkinson's disease was induced in monkeys by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a substance P-depleting neurotoxin. Subsequent treatment with S-17092, a potent POP inhibitor, improved performance on cognitive tasks (Schneider JS et al., Neuropsychopharmacology 2002 ; 26(2):176–82). Inhibition of POP was also found to prevent α-synuclein oligomerization ex vivo (Myöhänen 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 have shown 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 initially observed by Katsube's group when cortical and cerebellar cells were prevented from age-related apoptosis by treatment with the POP inhibitor ONO-1603 (Katsube N et al., J. Pharmacol. Exp. Ther. 1999 ; 288(1):6-13).

Clinical trials of POP inhibitors in the treatment of cognitive deficits have been conducted only in a few cases. In a phase I clinical trial, Morain's group (Morain P et al., Br. J. Clin. Pharmacol. 2000 ; 50(4):350-9) found that the aforementioned prolyl endopeptidase inhibitor S-17092 had cognitive-enhancing properties in healthy elderly subjects and had a clear dose-dependent effect; in addition, no side effects were observed. Later studies showed additional mild mood-stabilizing properties of this compound (Morain P et al., Neuropsychobiology 2007 ; 55(3-4):176-83).

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).

There is also a significant amount of evidence implicating 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 considered to be the major 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 POP inhibition may help reduce neuroinflammation, and therefore POP inhibitors may be used in the treatment of neurodegenerative diseases with an inflammatory component, such as Alzheimer's disease and Parkinson's disease, and in particular help improve the cognitive impairment associated with these diseases.

Senile plaques that extend to areas of the cerebral cortex are typical neuropathological features of AD. The main protein component of these plaques is β-amyloid peptide (Aβ). Aβ deposition causes neuronal dysfunction and death in the brain. This peptide is derived from the β-amyloid precursor protein (APP). Under normal conditions, APP is cleaved by α-secretase to form soluble APPα, which prevents Aβ formation.

Interestingly, POP inhibition increases intracellular IP3 levels, which may contribute to the stimulation of APPα production, which in turn reduces Aβ formation.

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

In addition, it appears that substance P may 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 substance P metabolism, helping to maintain substance P levels that may suppress the neurotoxic effects of β-amyloid protein.

Due to the above effects, it is believed that prolyl oligopeptidase inhibitors may be useful drugs for the treatment of Alzheimer's disease, helping to prevent brain damage and improve cognitive impairment associated with the disease.

Prolyl oligopeptidase has also been linked to several factors that may be relevant to multiple sclerosis (MS). For example, POP is involved in the regulation of microglial toxicity (Klegeris A et al., Glia 2008 ;56(6):675–85). Indeed, a direct link has been established between POP and MS; plasma POP activity was significantly reduced in patients with relapsing-remitting MS (RR-MS) (Tenorio-Laranga J et al., J Neuroinflammation 2010 ;7:23). Interestingly, the reduction correlated with the severity of disease symptoms rather than with the patient’s age. Instead, healthy controls showed an inverse correlation between POP activity and age, and elderly controls had levels comparable to those found in MS patients.

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 major component of Lewy bodies is a 140-amino acid protein, α-synuclein. Under certain conditions, α-synuclein monomers interact to form prefibrillar aggregates or protofibrils, which can create cytotoxic insoluble fibrils. These fibrils cannot be broken down by the proteasome, and they impair the function of this intracellular proteolytic system. This results in 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 consequence, an increase in the number of α-synuclein protofibrils in the brains of patients with Parkinson's disease. These fibrils have been associated with neurotoxicity in α-synuclein-overexpressing cells and mouse models (Masliah E et al., Science 2000 ; 287(5456):1265–9; Gosavi N et al., J. Biol. Chem. 2002 ; 277(50):489–84–92). Abnormal accumulation of misfolded α-synuclein can lead to mitochondrial changes that can promote oxidative stress and induce cell death (Hsu LJ et al., Am. J. Pathol. 2000 ; 157(2):401-10). In addition, three point mutations (A53T, A30P, or E46K) in the α-synuclein gene 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).

It has been shown in vitro that the rate of α-synuclein aggregation was increased when the protein was incubated with a wild-type POP clone, and this increase was dependent on the POP concentration (Brandt I et al., Peptides 2008 ; 29(9):1472-8). Furthermore, a mutant variant lacking POP activity (S544A) did not increase the rate of aggregation.

The increased aggregation could also be prevented by the addition of POP inhibitors, suggesting that the effect depends on the enzymatic activity of POP. Experimental data suggest that POP inhibitors can block the increased α-synuclein aggregation induced by oxidative stress in SH-SY5Y neuroblastoma cells overexpressing human α-synuclein (Myöhänen TT et al., Br. J. Pharmacol. 2012 ; 166(3):1097–113). POP colocalizes with α-synuclein in SH-SY5Y cells, and this colocalization disappears after incubation with POP inhibitors, indicating an interaction between POP and α-synuclein. A 5-day treatment with a POP inhibitor 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 (Myöhänen TT et al., Pharmacological Research 2020 ; 151:104558).

Thus, inhibition of POP activity in the brain may prevent α-synuclein aggregation and thereby prevent the formation of cytotoxic protofibrils present in Lewy bodies. POP inhibitors may therefore have potential therapeutic value in the treatment of neurodegenerative diseases in which accelerated α-synuclein aggregation has been described.

Compounds capable of inhibiting POP are effective in preventing experimental cognitive impairment induced by scopolamine in rats, suggesting that POP inhibitors function to alleviate mnemonic dysfunctions (Yoshimoto T et al., J. Pharmacobiodyn. 1987 ; 10:730-5).

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

Patients with bipolar disorder have been found to have high levels of POP activity in the serum. In recent years, POP has gained importance as a target for the treatment of this disorder, particularly due to its involvement in the metabolism of inositol 1,4,5-triphosphate (IP3). IP3 is a key molecule in the signal transduction pathway of neuropeptides. By binding to specific receptors, neuropeptides induce an increase in IP3, which binds to its receptor on the endoplasmic reticulum membrane and induces the release of Ca2+, which is thought to play a critical role in learning and memory. Recent findings have shown that POP modulates IP3 concentrations (Komatsu Y J Neurosci. 1996 ; 16:6342–52). Thus, disruption of the POP gene in the eukaryote Dictyostelium discoideum is known to induce lithium resistance by increasing IP3 levels (Schulz I et al., Eur. J. Biochem. 2002 ; 269:5813-20), and also reduces the proteolytic activity of POP, which results in high levels of IP3 in glioma cells, antisense to human POP. This effect is also observed when these cells are treated with specific POP inhibitors (Williams RS et al., EMBO J. 1999 ; 18:2734-45).

The IP3 signaling pathway is involved in the action of several therapeutic mood stabilizer drugs (lithium, carbamazepine, and valproic acid), and defects in the mechanisms that regulate IP3 signaling may cause bipolar disorder. Moreover, valproic acid, a mood stabilizer commonly used to treat bipolar disorder, directly inhibits recombinant POP activity (Cheng L et al., Mol. CeII. Neurosci. 2005 ; 29: 155–61). Thus, there is compelling evidence that POP inhibitors are useful for the prevention and/or treatment of bipolar disorder in mammals. Thus, the development of new POP inhibitors is of interest for the therapy of this disorder or disease.

Thus, the effects of several POP inhibitors in a variety of cognitive tasks have been characterized, and there is a consensus that POP inhibitors have beneficial 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).

A number of patents and patent applications have described POP inhibitors: WO 2008/077978 A1, WO 2005/027934 A1, JP 2011-037874 A2, WO 2005/002624 A1, WO 2004/060862 A2, WO 03/04468 A1; DE 196 03 510 A1, US 2006/0100253 A1 and US 6159938 A, but only a few compounds have been studied in vivo (JTP-4819, S-17092, Z-321, ONO-1603, Y-29794, ZTTA, Z-Pro-Prolinal and KYP-2047). Of this list, the first four inhibitors on the list have undergone clinical trials and none of them have reached the market.

WO 2014/072498 A1 describes POP inhibitors with high affinity for POP and good ability to cross the blood-brain barrier to reach the brain, where the inhibitor's effect in the treatment of cognitive disorders is manifested. This is an important feature of the compounds, allowing them to be used for the treatment of cognitive disorders.

WO 2014/072498 A1 discloses that the composition of the compounds disclosed therein can be adapted for any route of administration. However, only in the in vivo examples disclosed in that application were the compounds administered subcutaneously.

It would be an advantage to be able to administer POP inhibitors orally, as this is most convenient from the patient's point of view.

Therefore, there is a need to find new POP inhibitors that are particularly well adapted for oral administration and that effectively reach the brain, which is the site of action when these POP inhibitors are used in the treatment of cognitive disorders. Serotonin receptor antagonists (5-HT1A receptor) are commonly used as antidepressants. However, they cause significant sexual dysfunction (Uphouse L, Pharmacol. Biochem. Behav. 2014 ; 0:31-42), which limits their use for chronic therapy in the adult population. For this reason, a POP inhibitor with a positive effect on cognitive function in affective disorders and without such adverse effects would have a significant impact on the patient's quality of life.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have successfully discovered that 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile, its stereoisomers and its salts are not only able to inhibit POP with high efficiency, but also exhibit high penetrating ability in the gastrointestinal tract, provide high effect on the brain, which allows them to be particularly effective when administered orally, and have little binding to the 5-HT1A receptor, which avoids negative side effects such as sexual dysfunction.

Thus, one aspect of the invention relates to 1-[1-(4-benzyloxy-3,5-difluoro-benzoyl)-4-fluoro-pyrrolidine-2-carbonyl]-pyrrolidine-2-carbonitrile having the formula (I):

(where the asterisk in the formula indicates the presence of chiral centers), its stereoisomers and their pharmaceutically acceptable salts.

Another aspect of the present invention relates to processes for preparing a compound of formula (I) as defined above, its stereoisomers and its pharmaceutically acceptable salts.

Another aspect of the present invention relates to a medicinal product or pharmaceutical composition comprising at least one compound of formula (I) as defined above, its stereoisomers and their pharmaceutically acceptable salts and a pharmaceutically acceptable carrier, adjuvant or vehicle.

Another aspect of the invention relates to a compound of formula (I) as defined above, its stereoisomers and its pharmaceutically acceptable salts for use as a medicinal product, in particular for the prevention and/or treatment of cognitive disorders and/or neurodegenerative disorders such as synucleinopathies (diseases associated with the accumulation of synuclein such as Parkinson's disease, REM sleep behavior disorder, dementia with Lewy bodies or multiple system atrophy). In particular, the cognitive disorders may be 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, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis.

Another aspect of the invention relates to a method for treating or preventing cognitive disorders in a mammal, wherein a patient in need of said treatment is administered a therapeutic amount of a compound of formula (I) as defined above, its stereoisomers and its pharmaceutically acceptable salts. In a particular embodiment of the invention, the disorder is a cognitive disorder associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar affective disorder, rapid eye movement disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis.

Another aspect of the invention relates to the use of a compound of formula (I) as defined above, its stereoisomers and pharmaceutically acceptable salts thereof for the manufacture of a medicament, in particular for the prevention and/or treatment of cognitive disorders and, more particularly, a cognitive disorder associated with a disease selected from the group consisting of schizophrenia, major depressive disorder, bipolar affective disorder, rapid eye movement disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis. These aspects and their preferred embodiments of the invention are further defined in the claims.

DESCRIPTION OF FIGURES

Figure 1 shows the results of the POP inhibition assay for the compounds of Example 1, Comparative Example 3, and Comparative Example 4.

Figure 2 shows the results of PAMPA analysis for the compounds of Example 1, Comparative Example 3, and Comparative Example 4.

Figure 3 shows the results of Caco-2 analysis for the compounds of Example 1 and Comparative Example 3.

Figure 4 shows the results of the in vivo brain effect analysis for the compounds of Example 1 and Comparative Example 3.

Figures 5a and 5b show the results of the NOR problem analysis for the connections of Example 1 and Comparative Example 3.

Figure 6 shows the 5-HT1A receptor binding results for the compounds of Example 1 and Comparative Example 3.

Figure 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 shows ^{the 1} H-NMR spectrum of (S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile.

Figure 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 shows ^{the 1} 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 INVENTION

In the context of the present invention, the following terms have the meanings described in detail below.

The term "salt" is to be understood as any form of the active compound used according to the invention, in which said compound is in ionic form or charged and bound to a counterion (cation or anion) or is in solution. This definition also includes quaternary ammonium salts and complexes of the active molecule with other molecules and ions, in particular complexes formed by ionic interactions. The definition includes, in particular, physiologically acceptable salts; this term is to be understood as equivalent to "pharmacologically acceptable salts" or "pharmaceutically acceptable salts".

The term "pharmaceutically acceptable salts" in the context of the present invention means any salt which is physiologically tolerable (usually this means that it is not toxic, in particular due to the counterion) when used in an appropriate manner for the treatment applied or used, in particular on humans and/or mammals. These physiologically acceptable salts can be formed with cations or with bases, and in the context of the present invention are understood as salts formed by at least one compound used according to the invention, usually an acid (deprotonated), such as an anion, especially when used on humans and/or mammals. These physiologically acceptable salts can also be formed with anions or with acids, and in the context of the present invention are understood as salts formed by at least one compound used according to the invention, usually protonated, for example at nitrogen, such as a cation, and at least one physiologically tolerable anion, especially when used on humans and/or mammals. This definition specifically includes, in the context of the present invention, a salt formed with a physiologically tolerated acid, i.e. salts of a specific active compound with a physiologically tolerated organic or inorganic acid, especially when used on humans and/or mammals. Examples of salts of this type are 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.

The compounds of the present invention represented by the above-described formula (I) may include enantiomers resulting from the presence of three chiral centers in the molecule. Individual enantiomers and any mixture of two or more of them are within the scope of the present invention.

Unless otherwise indicated, the compounds of the present invention also include isotopically labeled forms, i.e., compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the present invention includes compounds having the present structure except for the replacement of at least one hydrogen atom with deuterium or tritium, or the replacement of at least one carbon atom with ^{a 13} C- or ¹⁴ C-enriched carbon, or the replacement of at least one nitrogen atom with a ¹⁵ N-enriched nitrogen.

The compounds of formula (I) or their salts are preferably in a pharmaceutically acceptable or substantially pure form. By pharmaceutically acceptable form is meant, inter alia, a form having a pharmaceutically acceptable level of purity, excluding conventional pharmaceutical additives such as diluents and carriers, and not including substances considered toxic at normal dosages. Purity levels of the drug substance are preferably above 50%, more preferably above 70%, most preferably above 90%. In a preferred embodiment of the invention, they constitute more than 95% of the compound of formula (I) or its salts.

As noted previously, the term "pharmaceutically acceptable salts" refers to any salt that, when administered to a recipient, is capable (directly or indirectly) of providing a compound as described herein. However, it should be understood that pharmaceutically unacceptable salts are also within the scope of the invention, as they may be useful in the preparation of pharmaceutically acceptable salts, solvates, and prodrugs. The preparation of salts can be accomplished by methods known to those skilled in the art.

As used herein, the terms "treat," "treatment," and "cure" include eliminating, removing, reversing, alleviating, modifying, or controlling a disease or condition, such as a cognitive disorder.

As used herein, the terms “prevention,” “preventing,” “preventive,” “prevent” and “prophylaxis” refer to the ability of a compound of formula (I) to prevent, minimize or delay the onset or progression of a disease or condition, such as a cognitive disorder, before it occurs.

Therefore, by "therapy" or "treatment" and/or "prevention" or "prophylaxis" is generally meant at least suppression or improvement of symptoms associated with a condition affecting a subject, where suppression and improvement are used in a broad sense to mean at least a decrease in the value of a parameter, for example a symptom, associated with a condition that is treated, for example a cognitive disorder. Thus, the method of the present invention also includes situations where the condition is completely suppressed, for example prevented or stopped, for example terminated, such that the subject no longer experiences the condition. Thus, the present method includes both the prevention and treatment of a cognitive disorder.

The terms "cognitive disorder" and "mild cognitive impairment" are used interchangeably herein to refer to any condition characterized by a deficit in mental processing related to thinking, learning, or memory. Examples of such disorders include agnosia, amnesia, aphasia, apraxia, delirium, dementia, and learning disorders.

Cognitive impairment may be (and often is) associated with (that is, may be caused by or occur in the presence of) other conditions characterized by damage to or loss of neurons or other structures involved in the transmission of signals between neurons. 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, dementia with Lewy bodies, multiple system atrophy, and progressive supranuclear palsy.

Cognitive impairment may also be associated with other conditions that disrupt the normal functioning of the central nervous system, including mental disorders such as anxiety disorders, dissociative disorders, mood disorders such as bipolar disorder, schizophrenia, and somatoform and factitious disorders.

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

Examples of dementias that can be treated with the methods of the invention include AIDS dementia complex, Binswanger's disease, dementia with Lewy bodies, frontotemporal dementia, dementia with multiple infarcts, Pick's disease, semantic dementia, and vascular dementia.

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

Examples of aphasia that can be treated with the methods of the invention include progressive non-fluent aphasia.

The compounds described herein may also be used to treat patients with cognitive impairment that is mild or that does not otherwise significantly interfere with daily life. Mild cognitive impairment is an example of such a condition: a patient with mild cognitive impairment exhibits symptoms of dementia (e.g., difficulty with speech or memory), but the severity of these symptoms is such that a diagnosis of dementia may be inappropriate. The compounds described herein may be used to treat mild cognitive impairment and other, less severe forms of cognitive impairment.

Thus, another aspect of the present invention is a method for treating or preventing cognitive disorders in a mammal, wherein a therapeutic amount of a compound of the invention is administered to a patient in need of said treatment.

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

In a particular embodiment, the compounds of the invention are administered orally at a daily dose of from 0.01 to 1.90 mg/kg, such as 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, 1.50, 1.60, 1.65, 1.70, 1.75, 1.80, 1.55, 1.60, 1.65, 1.70, 1.75, 1, 1.85 or 1.90 mg/kg. The compounds of the invention can be administered in any of the above daily oral doses once a day, twice a day, three times a day, several times a day, five times a day or six times a day.

In a specific embodiment of the present invention, the compounds described herein may be used to treat patients with cognitive impairment associated with schizophrenia, bipolar disorder, Alzheimer's disease, or Parkinson's disease.

In a specific embodiment of the present invention, the compounds have the following formula (Ia):

Specific individual compounds of the invention falling within 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 their pharmaceutically acceptable salts.

The compounds of formula (I) defined above can be prepared by available synthetic methods as shown in the following general schemes:

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

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

A compound of formula (IX) is prepared from a carboxylic acid of formula (VII) and an amine of formula (VI) under Schotten-Baumann reaction conditions. Accordingly, a chlorinating agent such as oxalyl chloride is added to a solution of a carboxylic acid of formula (VII) in an organic solvent such as toluene. The reaction mixture is stirred at a temperature of from 50 °C to 80 °C for 1-2 hours to ensure the formation of a carboxylic acid chloride of formula (VIII). After evaporation of the solvent, the resulting crude product is solubilized in an organic solvent such as THF and added to an aqueous basic solution of an amine of formula (VI), typically an aqueous solution of an amine of formula (VI) in NaOH, at a reduced temperature such as 0 °C. The reaction mixture is stirred at a reduced temperature for 1 to 2 hours and at room temperature for 2 to 4 hours. The solvent is then evaporated, the pH of the remaining aqueous fraction is adjusted to acidic (3-4) by adding HCl solution and extracted with ethyl acetate. The organic phase is washed with saturated saline solution, dried, filtered and evaporated. If necessary, the crude product is purified by flash chromatography.

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 with a coupling reagent such as carbodiimide. Specifically, the compound of formula (IX) is dissolved in an aprotic organic solvent such as dichloromethane and added to a carbodiimide, for example, a carbodiimide on a solid support such as N-cyclohexylcarbodiimide, N'-methylpolystyrene, together with DIEA. After 5 min, pyrrolidine-2-carbonitrile of formula (IV) and additional DIEA are added. The reaction mixture is stirred at room temperature for 8 to 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.

Alternatively, compounds of formula (I) can be prepared as shown in the following scheme and described below:

An amine-functionalized resin, such as the Sieber amide resin of formula (X), is placed in a syringe fitted with a porous polyethylene disk. The resin swells upon washing with appropriate organic solvents such as dichloromethane (DCM) and dimethylformamide (DMF). When the amine group of the resin is protected (e.g., in the case of the Sieber amide resin), removal of the protecting group (such as the fluorenylmethoxycarbonyl (Fmoc) protecting group) is achieved by treatment with a solution of an amine base such as a solution of piperidine in DMF.

After deprotection of the resin, the Fmoc-protected proline of formula (V) is coupled to the resin using an activating agent such as a 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 degree of coupling can be monitored using the Kaiser test, and recoupling is performed if necessary. Fmoc is removed by treatment with an amine base solution such as a solution of piperidine in DMF and/or a mixture of piperidine/DBU/toluene/DMF to give the product of formula (XI). Fmoc removal can be assessed using the NF31 p-nitrophenyl ether test.

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

Alternatively, the product of formula (XII) can also be prepared 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).

The product of formula (XII), washed thoroughly 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 maintained at a temperature of 20 to 40 °C for 8 to 16 hours. The reaction mixture is then filtered and the resin is washed with the same organic solvent. The filtrates are collected and the solvent is evaporated to dryness. The resulting crude product is dissolved in a suitable solvent such as ethyl acetate and washed with a saturated solution of NaHCO ₃ and 5% aq. KHSO ₄ . The organic phase is dried, filtered and evaporated. The crude product is dissolved in a mixture of H ₂ O:CH ₃ CN and lyophilized to give the peptide nitrile of formula (I).

Alternatively, the peptidyl resin of formula (XII) can be treated with TFA/ _H2O /TIS for 1-2 hours. The resin is then filtered and washed with TFA, the filtrates are collected and the solvent is evaporated to dryness. The crude product is resuspended in _H2O : _CH3CN and lyophilized. The resulting crude peptide amide is taken up in an appropriate organic solvent such as DCM and converted to the 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 to 16 hours, the solvent is evaporated and the residue is dissolved in ethyl acetate. The organic solution is then washed with aq. _KHSO4 and aq. NaHCO ₃ solution. Drying and evaporation of the organic phase yields the peptide nitrile of formula (I).

The crude product was purified by RP-HPLC.

When the above-described processes for preparing the compounds of the invention yield mixtures of stereoisomers, these isomers can be separated by conventional techniques such as preparative chromatography. When chiral centers are present, the compounds can be obtained in racemic form or individual enantiomers can be obtained either by enantiospecific synthesis or by resolution.

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

Thus, in one aspect, the present invention relates to methods for preparing a compound of formula (I) or a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof.

In one embodiment of the invention, the method comprises the steps of:

a) interaction of the compound of formula (IX):

with a compound of formula (XI):

wherein polymer means a polymer that is inert under the reaction conditions of the synthesis process described herein and insoluble but swellable in the solvents used in this case, such as low crosslinked polystyrene and polyethylene glycol grafted polystyrene polymers.

to obtain a compound of formula (XII):

b) hydrolysis of the compound of formula (XII) to obtain a compound of formula (XIV):

And

c) subjecting the compound of formula (XIV) to conditions capable of converting the carboxamide group into a nitrile group to yield a compound of formula (I):

where steps b) and c) can be carried out separately or in the same reactor.

In another embodiment of the present invention, a process for preparing a compound of formula (I) or a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof comprises the following steps:

a) interaction of the compound of formula (IX):

with a compound of formula (IV):

In another embodiment, the method comprises the following steps:

a) interaction of the compound of formula (XI):

wherein the polymer means a polymer that is inert under the reaction conditions of the synthetic method described herein and insoluble but swellable in the solvents used herein, such as low cross-linked polystyrene and polyethylene glycol-grafted polystyrene polymers,

with a compound of formula (XIII):

b) removal of the Fmoc protecting group,

c) reaction with a compound of formula (VII):

d) hydrolyzing the obtained product from the carrier polymer to obtain a compound of formula (I):

It has been found that compounds of general formula (I) can be used in the treatment of cognitive disorders, in particular cognitive disorders associated with other diseases or conditions of the central nervous system.

In a specific embodiment 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.

The present invention further relates to medicaments or pharmaceutical compositions for administration to a patient, comprising a compound of the present invention or a pharmaceutical salt, derivative, prodrug or stereoisomer thereof together with a pharmaceutically acceptable carrier, adjuvant or vehicle.

Auxiliary materials or additives for the pharmaceutical composition of the present invention can be selected from carriers, excipients, auxiliary materials, lubricants, fillers, solvents, diluents, colorants, flavors such as sugars, antioxidants, binders, adhesives, disintegrants, anti-adhesives, glidants and/or agglutinating agents. In the case of suppositories, this may imply waxes or fatty acid esters, or preservatives, emulsifiers and/or carriers for parenteral use. The choice of these auxiliary materials and/or additives and the amounts used will depend on the type of use of the pharmaceutical composition.

The medicinal product or pharmaceutical composition of the present invention may be in any form suitable for use in humans and/or animals, preferably humans, including infants, children and adults, and may be prepared by standard methods known to those skilled in the art. Thus, the composition according to the invention can be adapted for local or systemic use, in particular for cutaneous, transdermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, intravenous, intra-arterial, intravesical, intraosseous, intracavernous, intranasal, pulmonary, buccal, sublingual, ocular, intravitreal, transdermal, rectal, vaginal, oral, epidural, intrathecal, intraventricular, intracerebral, intracerebroventricular, intracisternal, intraspinal, perispinal, intracranial, by means of needles or catheters with or without pump devices, or other routes of administration.

In a preferred embodiment of the present invention, the pharmaceutical compositions are in oral form, solid or liquid. Suitable dosage forms for oral administration may be tablets, pills, caplets, gelatin capsules, chewing gums, capsules, granules, drops, syrups or solutions and may contain conventional excipients known in the art, such as binding agents, for example syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents, such as sodium lauryl sulfate.

In another embodiment of the invention, the pharmaceutical compositions are in the form of products for non-parenteral intranasal administration, preferably in the form of products for intranasal administration. Typically, intranasal administration is carried out using nasal sprays, bottles and droppers as delivery devices. For use with these devices, the pharmaceutical compositions are preferably liquid solutions or suspensions of the compounds of the invention.

The compositions can be prepared by conventional mixing, filling or tabletting processes. Repeated mixing operations can be used to distribute the active agent throughout the compositions in which large amounts of excipients are used. Such operations are conventional in the art. Tablets can be prepared, for example, by wet or dry granulation and, optionally, coated according to methods well known in normal pharmaceutical practice, in particular enteric coating.

The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or reconstitutable dry preparations, aerosols or sprays in suitable dosage form. Adequate excipients such as fillers, buffering agents or surface-active agents may be used.

The composition according to the invention can be prepared in the form of deposits in dissolved form or in the form of patches for transdermal use.

Skin applications include ointments, gels, creams, lotions, suspensions or emulsions.

A suitable form for rectal use is suppositories.

The compositions mentioned may be prepared using standard methods such as those described or mentioned in the Spanish and US Pharmacopoeias and similar reference literature.

In one embodiment of the invention, it is preferred that the compound of formula (I) is used in therapeutically effective amounts. The physician will determine the most appropriate dosage of the present therapeutic agents and will vary depending on the form of administration and the particular compound selected, and will further vary depending on the patient being treated, the age of the patient, the type of disease or condition being treated. When the composition is administered orally, larger amounts of the active agent may be required to obtain the same effect as with a smaller amount administered parenterally. The compounds can be used in the same manner as comparable therapeutic agents, and the dosage level is of the same order of magnitude as that normally used with such therapeutic agents. The active compounds are typically administered one or more times per day, for example, 1, 2, 3 or 4 times per day, with typical total daily doses in the range of 0.011 to 1.90 mg/kg/day.

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

In particular, the combination of at least one compound of formula (I) and at least one other medicinal product can be formulated for its simultaneous, separate or sequential administration with at least a pharmaceutically acceptable carrier, additive, adjuvant or vehicle. This implies that the combination of a compound of formula (I) and another medicinal product can be administered as follows:

a) In the form of a combination, which is part of the same medicinal product, both drugs are always administered simultaneously.

b) In the form of a combination of two units, each of which allows for simultaneous, sequential or separate administration. In an embodiment of the invention, the compound of formula (I) is administered independently of the other drug (i.e. in two doses), but at the same time. 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 specified.

In the context of the present invention, the following acronyms and abbreviations have been used, the meanings of which are described in detail below:

AcOEt
AD
AUC
BBB
Boc
BSA
DBU
DCM
DMEM
DI
DIEA
DMF
DMSO
EtOH
Fmoc
FPLC
FTIR
HBSS
HOAT
hPOP
IP
IP3
IPTG
L.B.
MALDI-TOF
MK-801
M.S.
NMR
NOR
OD
PAMPA
PBS
PC
P.E.
pETM10
PI
POP
P.S.
PyBOP
RP-HPLC
SD
SDS-PAGE
TBTU
TEER
TFU
THF
TIS
Tris
Tβ4
UHPLC
ZGP-AMC Ethyl acetate
Alzheimer's disease
Area under the curve
Blood-brain barrier
tert -Butoxycarbonyl
Bovine serum albumin
1,8-Diazabicyclo[5.4.0]undec-7-ene
Dichloromethane
Dulbecco's Modified Eagle Medium
Discrimination Index
N, N' -Diisopropylethylamine
Dimethylformamide
Dimethyl sulfoxide
Ethanol
9-Fluorenylmethoxycarbonyl
Fast protein liquid chromatography
Fourier transform infrared spectrometer
Hanks Balanced Salt Solution
1-Hydroxy-7-azabenzotriazole
Human prolyl oligopeptidase
Intraperitoneally
Inositol triphosphate
Isopropyl β-D-1-thiogalactopyranoside
Lysogenic broth
Time-of-flight matrix-assisted laser desorption/ionization
Dizocilpine
Multiple sclerosis
Nuclear magnetic resonance
Recognizing new objects
Optical density
Parallel analysis of permeability of artificial membrane
Phosphate buffered saline
Phosphatidylcholine
Phosphatidylethanolamine
Plasmid pETM10
Phosphatidylinositol
Prolyl oligopeptidase
Phosphatidylserine
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
Reversed phase high performance liquid chromatography
Standard deviation
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
O -(Benzotriazol-1-yl)- N, N,N',N' -tetramethyluronium tetrafluoroborate
Transendothelial electrical resistance
Trifluoroacetic acid
Tetrahydrofuran
Triisopropylsilane
Tris(hydroxymethyl)aminomethane
Protein thymosin beta-4
Ultra Performance Liquid Chromatography
( N -Benzyloxycarbonyl-Gly-Pro-methylcoumarinyl-7-amide)

The following examples are merely illustrative of some embodiments of the invention and are in no way to be construed as limiting it.

EXAMPLES

SPECIFIC SYNTHESIS CONDITIONS USED IN THE PREPARATIONS DESCRIBED IN THE EXAMPLES

Method A : Hydrolysis of an ester of formula (II) to a carboxylic acid of formula (VII)

The ester of formula (II) (1 mmol) was dissolved in 95% EtOH. NaOH (3.7 mmol) was added and the reaction mixture was heated under reflux for about 2 h. It was then allowed to reach room temperature. The reaction mixture was concentrated to about 15-20 ml, then this solution was slowly added to 1 M HCl while cooling in an ice bath. The white solid precipitate was collected by filtration, washed with water and dried well before the next step of the synthesis. In case of no precipitate, the resulting solution was extracted with AcOEt (3x), the organic phase was dried and evaporated. The crude product was purified by flash chromatography if necessary.

Method B : Deprotection of a Boc-protected amine of formula (III) to give 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 h. The solvent is then evaporated to dryness to give the hydrochloride salt of the amine of formula (VI).

Method C : Coupling an amine of formula (VI) with a carboxylic acid of formula (VII) by forming a carboxylic acid chloride of formula (VIII).

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

Method D : Coupling of the product of formula (IX) with pyrrolidine-2-carbonitrile of formula (IV) in solution

The product of formula (IX) (1.2 mmol) was 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) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then filtered and the remaining solid was washed with DCM. The filtrate was evaporated to dryness. The crude product was then purified by preparative RP-HPLC and lyophilized to yield the product of formula (I).

Method E : General solid phase synthesis method

Resin swelling/conditioning: Sieber amide resin of formula (X) (1 equiv.) is placed in a syringe fitted with a porous polyethylene disc. The resin is swollen by DCM and DMF washes. Removal of the fluorenylmethoxycarbonyl (Fmoc) protecting group is achieved by treatment with 20% piperidine in DMF.

The Fmoc-protected proline of formula (V) (4 equiv) is then coupled to the resin using TBTU (4 equiv) and DIEA (8 equiv) in DMF. The mixture is stirred periodically by hand for 90 min. After filtration and washing, the degree of coupling is monitored by a Kaiser test and recoupling is performed if necessary. Fmoc is removed to give the product of formula (XI) by treatment with 20% piperidine in DMF and then with piperidine/DBU/toluene/DMF (20:5:5:70). Fmoc removal is assessed by the NF31 p-nitrophenyl ether test (described by Madder, A. et al., Eur. J. Org. Chem. 1999 ; (11):2787-91).

The product of formula (IX) (2 equiv) is coupled with the product of formula (XI) to give the product of formula (XII) using PyBOP (2 equiv), HOAt (6 equiv) and DIEA (6 equiv) in DMF. The mixture is stirred periodically by hand throughout the reaction time of 90 min. Systematic re-coupling is carried out using the same amounts and times. The degree of coupling is monitored using the NF31 p-nitrophenyl ether test.

Alternatively, the product of formula (XIII) (4 equiv) is coupled with the product of formula (XI) using PyBOP (4 equiv), HOAt (12 equiv) and DIEA (12 equiv) in DMF. The mixture is stirred periodically by hand throughout the 90 min reaction time. The degree of coupling is monitored using the NF31 p-nitrophenyl ester test and re-coupling is performed if necessary. The Fmoc group is removed by treatment with 20% piperidine in DMF and by treatment with piperidine/DBU/toluene/DMF (20:5:5:70). The product of formula (VII) (4 equiv) is then introduced using PyBOP (4 equiv), HOAt (12 equiv) and DIEA (12 equiv) in DMF to afford the product of formula (XII). The mixture is stirred periodically by hand throughout the entire reaction time of 90 min. The degree of binding is monitored using the NF31 p-nitrophenyl ether test and re-binding is carried out if necessary.

The product of formula (XII), washed thoroughly with DCM and dried, is transferred to a round-bottomed flask and trifluoroacetic anhydride (5 equiv.) and pyridine (10 equiv.) in DCM (approximately 2 ml/100 mg) are added. The mixture is kept at room temperature overnight. Then the reaction mixture is filtered and the resin is washed with DCM. The filtrates are collected and the solvent is evaporated to dryness. The resulting crude product is dissolved in AcOEt and washed with saturated NaHCO ₃ solution and 5% aq. KHSO ₄ solution. The organic phase is dried, filtered and evaporated. The crude product is dissolved in H ₂ O:CH ₃ CN (1:1) and lyophilized to give the peptide nitrile of formula (I).

Alternatively, the peptidyl resin of formula (XII) can be treated with TFA/ _H2O /TIS (95:2.5:2.5, ca. 2-5 mL/100 mg) for 1-2 h. The resin is then filtered and washed with TFA, the filtrates are collected, and the solvent is evaporated to dryness. The crude product is resuspended in _H2O : _CH3CN (1:1) and lyophilized. The resulting crude peptide amide is dissolved in DCM and trifluoroacetic anhydride (5 equiv.) and pyridine (10 equiv.) are added. The mixture is kept at room temperature overnight, the solvent is evaporated, and the residue is dissolved in AcOEt. The organic solution is then washed with 5% aq. _KHSO4 and 10% aq. _NaHCO3 . Drying and evaporation of the organic phase yields the peptide nitrile of formula (I).

The crude product was purified by RP-HPLC.

Example 1

(S)-1-((2S,4R)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile

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 (533 mg, 1.5 mmol) of formula (III) and 4-benzyloxy-3,5-difluorobenzoic acid (271 mg, 1 mmol) of formula (VII) were sequentially coupled with commercially available Sieber amide resin (333.3 mg, 0.2 mmol, 1 equiv) via a stepwise coupling as described above in method E. Purification by RP-HPLC afforded 156 mg (0.341 mmol) of the desired product.

mp 137.6-139.1°C. FTIR: 1642, 1582, 1517, 1414, 1381, 1317, 1230, 1203, 1172, 1064, 1036 cm ^-1 . ^1H -NMR ( _CDCl3 , 400 MHz) δ (ppm): 7.42 (2H, m), 7.35 (3H, m), 7.12 (2H, m), 5.30 (1H, m), 5.23 (1H, m), 4.93 (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).

Example 2

(S)-1-((2S,4S)-1-(4-(benzyloxy)-3,5-difluorobenzoyl)-4-fluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile

This compound was prepared according to the method described in Example 1, but with (2S,4S)-4-fluoro-1-Fmoc-pyrrolodine-2-carboxylic acid replaced by (2S,4S)-4-fluoro-1-Fmoc-pyrrolodine-2-carboxylic acid. Purification by RP-HPLC gave 138 mg (0.302 mmol) of the desired product.

mp 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, 696 cm ^-1 .

Comparative example 3:

(S)-1-((2S)-1-(4-(benzyloxy)-3,5-dimethoxybenzoyl)-4,4-difluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile

The product was prepared according to procedure E above starting from commercially available Sieber amide resin (250 mg, 0.19 mmol, 1 equiv), 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). Purification by RP-HPLC afforded 18 mg (0.036 mmol) of the desired product.

Comparative example 4:

(S)-1-((S)-1-(4-(benzyloxy)-3-fluorobenzoyl)-4,4-difluoropyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile

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) were sequentially coupled with commercially available Sieber amide resin (200 mg, 0.15 mmol, 1 equiv) via a stepwise coupling as described in Method E above. Purification by RP-HPLC afforded 14 mg (0.030 mmol) of the desired product.

PHARMACOLOGICAL DATA

DETERMINATION OF THE INHIBITORY EFFECT OF NEW COMPOUNDS ON THE ACTIVITY OF (HUMAN) PROLYL OLIGOPEPTIDASE

Expression and purification of prolyl oligopeptidase (POP)

POP was obtained by expression in E. coli and affinity purification using a His-tail fusion according to the method described in the literature (Tarragó T et al., ChemBioChem 2006 ; 7 :827-33), summarized below:

hPOP expression: Competent E. coli BL21 cells were transformed with pETM10 hPOP. To induce expression, one colony was inoculated with a preculture of LB medium (50 ml) containing kanamycin (50 μg/ml) and grown overnight at 37°C. The next day, two cultures of LB medium (500 ml) were inoculated with the overnight culture (10 ml). The inoculated cultures were grown at 37°C and 220 rpm until the OD595 was 1.2 (2.5-3 h). IPTG (final concentration 1 mM) was then added and induced 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 using four cycles (each consisting of 15 sec sonication and 15 sec rest) at 50% intensity and 0.5 pulses, the sample was stored on ice. After sonication, the sample was centrifuged (40,000 g , 30 min, 4 °C) and the supernatant was immediately used for POP purification. An ÅKTA explorer FPLC system was used for purification. The supernatant was loaded at 1 ml/min onto a HiTrapQuelating column (5 ml) pre-equilibrated with 5 column volumes of suspension buffer. The column was washed with suspension buffer until the absorbance at 280 nm returned to the baseline. The column was then washed with 5 volumes of wash 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 throughout the elution. POP activity was checked in all fractions and positive fractions were analyzed by SDS-PAGE and stained with Biosafe Comassie Stain G-250. Positive fractions were collected and desalted using a HiPrep 26/10 Desalting column with Tris-HCl (50 mM, pH 8) as buffer. Recombinant hPOP was quantified using the Bio-Rad protein assay with BSA as standard. Aliquots of the recombinant enzyme were prepared, immediately frozen with liquid nitrogen, and stored at -80°C.

POP inhibition assays

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 carried out in 96-well microtiter plates, which allowed several reactions to be monitored 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 lot) and a solution of the corresponding new compound (3 μl). A stock solution of the new compound was prepared in DMSO (100 mM), and dilutions with DMSO were prepared from this stock solution.

After pre-incubation, Z-G-P-AMC (10 μl, 3 mM in 40% 1,4-dioxane) (3 μl, 1.5 mM in 40% 1,4-dioxane, under condition B) was added 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 AMC formation was measured fluorimetrically. Excitation and emission wavelengths were 360/40 and 485/20 nm, respectively.

For each compound, several concentration points (ranging from 25 pM to 400 μM) were measured. The inhibitory activity against prolyl oligopeptidase was calculated according to equation 1. For each new compound, fluorescence was measured in the presence ( a ) and absence ( b ) of hPOP. Maximum fluorescence (0% inhibitory activity) was obtained from the hPOP sample in the absence of inhibitory compounds. To evaluate the inhibitory activity of a new compound, the activity was plotted against the logarithmic concentration of the compound, fitted to a sigmoid curve using GraphPad Prism software, and the IC ₅₀ value, defined as the concentration of the compound required to inhibit 50% of the POP activity, was established from the resulting curve.

Where:

a corresponds to the fluorescence intensity in the presence of substrate+test compound+hPOP

b corresponds to the fluorescence intensity in the presence of substrate+test compound

c corresponds to the fluorescence intensity in the presence of substrate+hPOP

d corresponds to the fluorescence intensity in the presence of the substrate.

The new compounds exhibit high inhibitory activity against human prolyl oligopeptidase. The results are summarized in Table 1.

Table 1: Inhibition of human prolyl oligopeptidase. Connection (example no.) IC ₅₀ (nM) SD Note 1 45.0 7.01 Connect by note 3 48.7 20.3 Connect by note 4 365.3 103.6

The data in Table 1 are presented in Fig. 1. In this figure, the bars represent the mean±standard deviation (SD).

DETERMINATION OF PERMEABILITY OF COMPOUNDS

Parallel Artificial Membrane Permeability Analysis (PAMPA)

The parallel artificial membrane permeability assay (PAMPA) described by 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 ) of compounds was measured at an initial concentration of 200 μM. The buffer solution was prepared from a commercial concentrate according to the manufacturer's instructions. The pH was adjusted to 7.4 with 0.5 M NaOH. A stock solution of a new compound was prepared in DMSO and diluted with the buffer solution to a final concentration of 200 μM (DMSO content 0.5%). The PAMPA sandwich was separated and each donor well was filled with 200 μl of the compound solution. The acceptor plate was placed in the donor well, ensuring that the underside of the membrane was in contact with the buffer. 4 μl of the phospholipid mixture (20 mg/ml) in dodecane were added to the filter of each well, and 200 μl of the buffer solution were added to each acceptor well. The acceptor plate was placed in the donor well, ensuring that the underside of the membrane was in contact with the buffer. 4 μl of the phospholipid mixture (20 mg/ml) in dodecane were added to the filter of each well, and 200 μl of the buffer solution were added to each acceptor well. The plate was covered and incubated at room temperature in a saturated humidified atmosphere for 4 h with orbital shaking at 100 rpm. After 4 h, 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, injecting each sample onto a _C18 reversed-phase column (150 mm × 4.6 mm × 5 μm, 100 Å) (100 μl/injection from acceptor wells, 10 μl/injection from donor wells and _for t0 standards). Transport was also confirmed by MALDI-TOF spectrometry.

The phospholipid mixture used was a porcine brain polar 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.

The effective permeability ( P _e ) after 4 hours was calculated using equation 2, the percent transfer (T%) was calculated using equation 3 and the compound retention (R%) of the phospholipid membrane using equation 4:

Where:

t denotes time (h)

C _A (t) denotes the concentration of the compound in the acceptor well at time t , C _D (t) denotes the concentration of the compound in the donor well at time t

and C _D (t ₀ ) denotes the concentration of the compound in the donor well at time t ₀ .

Based on the indicative _Pe values shown in Table 2, the new compounds exhibit good permeability across the BBB (Table 3).

Table 2: Indicative P _e values Approximate P values _e (cm/sec) Transport within the CNS P _e ≥4⋅10 ^-6 Good 2∙10 ^-6 ≤P _e <4∙10 ^-6 Doubtful P _e <2⋅10 ^-6 Bad

Table 3: Effective permeability (P _e ) and percentage of transport of new compounds Connection (example no.) P _e ( × 10 ^-6 cm/sec) SD %T SD % R SD 1 4.7 0,1 18.1 0.4 24 3.5 Connect by note 3 22.14 5.86 29.92 4.80 10.93 1.60 Connect by note 4 - - 10.43 2.06 - -

The data in Table 3 are presented in Fig. 2. In this figure, the bars represent the mean±standard deviation (SD).

Caco-2 Analysis

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

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

The experiments were carried out using the CacoReay permeability measurement kit (ReadyCell, Barcelona, Spain).

In the same week after receiving the kit, the cell transport medium was diluted according to the user manual included with the kit, after which the transport medium was replaced with fresh Dulbecco's modified Eagle's medium (DMEM, low glucose).

After three days, corresponding to the 20th day of cell culture, the quality of the barrier system and the presence of tight junctions between cells were checked by measuring the transendothelial electrical resistance (TEER) of the cell barrier. A TEER value of 200 ^Ω⋅cm2 (or higher) indicates that the barrier system is suitable for the uptake assay. The electrodes were sterilized by immersing them in ethanol (70%) for 30 min under UV light. After that, the electrode was equilibrated for 30 min in DMEM medium pre-warmed to room temperature. TEER measurements were performed at room temperature, obtaining an average value of 1357±168 ^Ω⋅cm2 , confirming the integrity of the cell barrier.

Before the permeability assay, transport buffer (HBSS (1×)-Ca ²⁺ /Mg ²⁺ ) was prepared and warmed to 37°C to avoid thermal stress of the cells during the experiment. In parallel, the tested compounds were dissolved to a final concentration of 50 μM in transport buffer (HBSS (1×)-Ca ²⁺ /Mg ²⁺ ). Containing 0.5% DMSO, an amount that is well tolerated by the cells and does not affect the integrity of the barrier. Compounds were tested at a concentration of 50 μM.

Before permeability analysis, DMEM was removed from the apical and basal compartments by aspiration. 10 μl of medium was left in each compartment to prevent drying of the cells and disruption of their barrier properties. Both compartments were washed with transport buffer, the basal (lower) compartment was filled with 300 μl, and the apical (upper) compartment with 75 μl of transport buffer.

The contents of the basal 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 cell desiccation. 65 μl of test compound solution were added to each well.

For apical-basal transport experiments, cells were incubated with compound-containing solutions for 2 h at 37°C. Compartments were then extracted 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% B over 8 min, A=0.045% trifluoroacetic acid in _H2O and B=0.036% trifluoroacetic acid in acetonitrile, detection at 220 nm). All experiments were performed in triplicate.

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

For a fully functional Caco-2 barrier, LY permeability should be less than 0.7% at 1 hour. After analysis, LY permeability was below this average transport value, indicating barrier integrity during compound evaluation.

In the Caco-2 permeability assay, the percent transport (T%) for compounds was calculated according to the following equation:

where t denotes time (hour), C _A (t) denotes the concentration of the compound in the basal compartment (acceptor) at time t, C _D (t ₀ ) denotes the concentration of the compound in the apical compartment (donor) at time 0.

Table 4: Permeability (Caco-2 analysis) Connection (example no.) %T SD 1 45 3 Connect by note 3 5.7 1

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

The data of Table 4 are presented in Fig. 3.

Effects on the brain in vivo

For each compound, twenty-four adult male mice aged 7-8 weeks on the day of the experiment were used, which were randomly assigned to the groups. The experiments were carried out on 3 groups of animals, as shown in the table below:

Group Treatment, route/dose, volume of administration Number of mice per group Plasma and brain sample time 1 No treatment (control group) N=3 Pre-dose 2 Compound no. 3, i/p, 5 mg/kg, 10 ml/kg N=21
(3 animals for each sampling time) T0+5 min, T0+30 min, T0+1h, T0+2h, T0+4h, T0+7h, T0+24h 3 Ave. 1, po, 5 mg/kg, 10 ml/kg N=21
(3 animals for each sampling time) T0+5 min, T0+30 min, T0+1 h, T0+2 h, T0+4 h, T0+7 h, T0+24 h mg, milligram; ml, milliliter; h, hour; kg, kilogram; min, minute; N, number of animals; i/p, intraperitoneally; p/o, orally; T, time; min, minute

After compound administration, terminal blood was collected at several time points following intraperitoneal (Example 3) or oral administration (Example 1).

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

Plasma samples were thawed at room temperature (RT) and proteins were precipitated with a twofold volume of acetonitrile containing 20 ng/ml phenacetin as an internal standard. Samples were mixed and then centrifuged for 20 min at 2200 g. Supernatants were diluted 1:1 with 150 mM phosphate-buffered saline (pH 7.4) and pipetted into UHPLC 96-well plates to await analysis.

Brain samples were weighed, thawed at room temperature, and prepared for analysis by homogenizing the tissue in a bead homogenizer with 4 times the volume of phosphate-buffered saline. The homogenate was processed similarly to plasma samples.

Reference (calibration) samples were prepared in the same way as the actual study samples, after adding pure plasma and brain homogenate to and concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 ng/ml of the compound according to Example 1 and the compound according to Example 3.

Levels of compounds of Example 3 and Example 1 were quantified from mouse plasma and brain. Samples were analyzed using a targeted bioanalytical method using UHPLC/QE-orbitrap mass spectroscopy.

The concentration (ng/mL) of compounds was plotted against time and the area under the curve (AUC) in plasma and brain tissue was calculated using Graphpad PrismTM 5.0. The plasma to brain ratio was calculated as follows:

Table 5: Effects on the brain Connection (example no.) %BBB 1 25.3 Connect by note 3 21

The data of Table 5 are presented in Fig. 4.

As shown above, when the compound of Example 1 is administered orally, it is possible to achieve a higher effect on the brain than that achieved by other compounds, even when said compounds are administered intraperitoneally.

Microsomal stability (rat)

Microsomal stability can be used to predict whether a compound will pass through first-pass metabolism and achieve systemic exposure after oral administration. It can also predict the half-life of a compound once it enters the bloodstream.

The stability of compounds in pooled rat liver microsomes (Sprague-Dawley) (catalog #452501) was studied according to the instructions provided by the supplier (BD Bioscience). A stock solution (500 μM) of the test compound was prepared in DMSO. Two μL of compounds were incubated at a final concentration of 1 μM in the following mixture:

- 713 µl _H2O

- 200 µl 0.5 M potassium phosphate (pH 7.4)

- 50 µl NADPH A regenerating system solution (BD Bioscience, cat. #451220)

- 10 µl NADPH B system regeneration solution (BD Bioscience, cat. no. 451200)

The resulting mixture was heated to 37°C for 5 min. Then, 25 μl of liver microsomes at a final concentration of 0.5 mg/ml were added. The mixture was incubated at 37°C with orbital shaking (100 rpm). At selected time points, 100 μl aliquots were extracted and mixed with 100 μl of acetonitrile. The resulting pellet was vortexed for 1 min. The samples were incubated at 4°C for 1 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 UHPLC-MS. All stability tests were performed in duplicate.

Table 6: Microsomal stability (rat) Connection (example no.) Half-life (min) Note 1 12 Connect by note 3 <5 Connect by note 4 <5

These data suggest that the compound of comparative example 3 and the compound of comparative example 4 will not undergo the first step metabolism after oral administration. Therefore, it will not reach the systemic circulation after oral administration. Indeed, the compound of comparative example 3 did not show any efficacy after oral administration, as the drug levels detected in plasma were very low.

PK study in mice

The pharmacokinetics of the compounds following intravenous bolus administration were studied in male Swiss Albino mice. The study was conducted in accordance with the guidelines of the Institutional Animal Ethics Committee (IAEC) and in accordance with the requirements of the Committee for the Control and Supervision of Experiments on Animals (CPCSEA), India.

The study was conducted in 12 mice per compound using a sparse sampling design (n=3/time point) to obtain a composite profile. Compounds were dissolved in 2% Twee80® in saline (0.9 NaCl) at a concentration of 0.5 mg/mL. Mice were administered a single 1 mg/kg dose of compound solution by intravenous bolus via the lateral tail vein using a 1 mL BD syringe with a 26G needle at a dose volume of 2 mL/kg.

Blood samples were collected (n=3 per time point) at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 h post-dose. At each time point, 120 μL of blood were collected by retro-orbital plexus puncture and transferred to a labeled microcentrifuge tube containing 200 mM 200 mM _K2EDTA (20 μL per mL of blood). Blood samples were centrifuged at 5000 g for 5 min at 4 ± 2°C. (20 μL per mL of blood). Blood samples were centrifuged at 5000 g for 5 min at -60°C prior to bioassay.

Plasma samples were analyzed by LC-MS/MS with a LLOQ of 1.06 ng/mL. Plasma pharmacokinetic parameters were calculated using the non-compartmental analysis tool of the validated Phoenix WinNonlin® software (version 6.3). The area under the concentration-time curve (AUC _last ) was calculated using the linear trapezoidal rule. Peak plasma concentration (C _max ) and time to peak plasma concentration (T max ) were observed values. Clearance (CL) and volume of distribution (Vss) were predicted values.

Table 7: Pharmacokinetics (mice) Connection (example no.) AUC _last (ng h/ml) CL (ml/min/kg) Vss (l/kg) 1 127 99.6 1.19 Connect by note 3 131 126 0.64 Connect by note 4 22.7 697 9.06

The compound of Comparative Example 4 shows very high clearance after intravenous (i.v.) administration to mice, indicating that the compound undergoes high first-pass metabolism (consistent with microsomal stability data), suggesting that the compound will not enter the systemic circulation after oral administration.

EFFECTS OF NOVEL COMPOUNDS ON LEARNING AND MEMORY IN AN ANIMAL MODEL WITH COGNITION IMPAIRMENT

The new compounds were evaluated for their efficacy as cognitive enhancers in a pharmacological model of cognitive impairment. The effects of the new compounds were assessed in untreated rodents and in rodents treated with MK-801 (mice or rats). MK-801 is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist 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 has a variety of behavioral effects in rodents, including sensory processing deficits, hypermotility, stereotypy, and ataxia. The behavioral phenotype induced by MK-801 treatment has been widely used as an animal model of cognitive impairment (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).

To determine whether the compounds under study act as cognitive enhancers, their ability to restore normal cognitive behavior was tested using 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); the passive or inhibitory avoidance task (Sarter M et al., Psychopharmacology (Berl) 1992 ; 107:144–59); the Morris water maze (DʼHooge R et al., Brain Res. Rev. 2001 ; 36:60–90); and the T-maze alternation task (Boess FG et al., Neuropharmacology 2004 ; 47:1081-92; Spowart-Manning L et al., Behav. Brain Res. 2004 ; 151:37-46).

As an illustrative example for the evaluation of new POP inhibitors, the protocol followed for each of the behavioral tests is described, as well as the results obtained in the object recognition test and the passive avoidance test.

The task of recognizing new objects

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 suitable 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 40 cm diameter circular arena for 10 min in the absence of objects (habituation session). The next day, the animal was placed in the circular arena for 10 min for a training trial, and two identical objects were placed in a symmetrical position. This phase was carried out for two consecutive days. On the third day, one of the objects was replaced by a different object. The object not used in the training trial was used as a novel object in the retention trial. The animals were then allowed to freely explore for 10 min, and the time spent exploring each object was recorded. The animal was expected to spend more time exploring the new object, which was a sign of intact recognition memory. The discrimination index was calculated as follows: time spent exploring the new object minus time spent exploring the old object, divided by the total time spent exploring both objects, and multiplied by 100. A higher discrimination index was thought to reflect greater memory retention.

Compounds administered orally

The experiment was performed on male C57/Bl6 mice aged 8-9 weeks (n=7-9 animals per group). The compounds were dissolved in a mixture of 5% Tween80® in physiological solution (0.9% NaCl) to obtain a solution concentration of 0.5 mg/ml.

The test was divided into three parts: habituation, training and test. On the first day, the animals were accustomed to the arena (round, 40 cm in diameter) for 10 minutes. On the following two days, the animals were allowed to freely explore two identical objects placed in the arena for 10 minutes. In the test sessions (day four), one of the objects was replaced by a new one. Before this session, thirty-five minutes before the test, the animals were orally administered the test compounds (5% Tween80® in saline, 1 ml per 100 g body weight). To induce cognitive deficit, dizocilpine (MK-801) was administered subcutaneously at a dose of 0.2 mg/kg in saline 20 min before the test in the negative control group or in the drug testing groups. The initial control group was administered saline.

After drug administration, each mouse was allowed to freely explore the objects for 10 minutes. The discrimination index (DI) was determined as the evaluation parameter:

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 spent in the circular arena areas was analyzed using Panlab SMART 2.0 software.

Compounds administered intraperitoneally

The experiment was performed on male C57/Bl6 mice aged 8-9 weeks (n=7-9 animals per group). The compounds were dissolved in a mixture of 5% Tween80® in physiological solution (0.9% NaCl) to obtain a solution concentration of 0.5 mg/ml.

The test was divided into three parts: habituation, training and test. On the first day, the animals were accustomed to the arena (round, 40 cm in diameter) for 10 minutes. On the following two days, the animals were allowed to freely explore two identical objects placed in the arena for 10 minutes. In the test sessions (day four), one of the objects was replaced by a new one. Before this session, thirty-five minutes before the test, the animals were intraperitoneally injected with the test compounds (5% Tween80® in saline, 5 mg/kg) or (5% Tween80® in saline, 1 ml per 100 g body weight). To induce cognitive deficit, dizocilpine (MK-801) was administered subcutaneously at a dose of 0.2 mg/kg in saline 20 min before the test in the negative control group or in the drug testing groups. The initial control group received saline.

After administration of the drug, each mouse was allowed to freely explore the objects for 10 minutes. The discrimination index (DI) was determined as the evaluation parameter:

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 spent in the circular arena areas was analyzed using Panlab SMART 2.0 software.

The results obtained by introducing the compound of Example 1 and the compound of Comparative Example 3 are presented in the table below.

Table 8: NOR task test Compound % discrimination index Intraperitoneally Control 39.5±3.9** MK 801 (p/k) 21±4.7 MK 801 (s/c)+pr. 1 (w/b) 30.3±3.9 MK 801 (p/k)+connection by pr. 3 (v/b) 42.7±3.3*** Orally Control 53.3±2.7*** MK 801 (p/k) 16.6±5.9 MK 801 (s/c)+pr. 1 (p/o) 42.1±5.1** MK 801 (p/k)+connection by pr. 3 (p/o) 20.1±2.9

The data from Table 8 are presented in Fig. 5a (intraperitoneal administration) and Fig. 5b (oral administration). In these figures, bars represent the mean±SE for n animals. *P < 0.05, **P < 0.01, ***P < 0.001 compared to baseline (MK 801 baseline) cognitive deficit conditions (one-way ANOVA followed by Dunnett's post hoc test).

As shown above, the compound of Example 1 is superior to the compound of Example 3 when administered orally, making it particularly suitable for oral administration.

Selectivity profile (binding to 5-HT1A)

The experiment was performed using the SafetyScreen44® panel from Cerep/Eurofins. Compounds were tested at a concentration of 100 μM (in 1% DSMO).

Compound binding was calculated as the percentage inhibition of radiolabeled ligand binding specific to each target.

Results are expressed as percentage inhibition of control specific binding.

,

obtained in the presence of test compounds.

Results showing inhibition greater than 50% are considered to reflect significant effects of the test compounds.

Table 9: 5-HT1A receptor binding Compound % inhibition of control specific binding SD Ex. 1 -6.3 1,2 Connected by Avenue 3 64.6 5.6

The data of Table 9 are presented in Fig. 6.

Claims (36)

1. Compound of formula (I) (where asterisks denote chiral centers) or its stereoisomers. 2. A compound according to claim 1, having formula (Ia): or its stereoisomers. 3. The compound according to claim 1, wherein said compound is selected from the following: (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. 4. A process for producing a compound of formula (I) as defined in any one of claims 1 to 3, which comprises a) interaction of the compound of formula (IX): with a compound of formula (XI): to obtain a compound of formula (XII): b) hydrolysis of the compound of formula (XII) to obtain a compound of formula (XIV): And c) subjecting the compound of formula (XIV) to conditions capable of converting the carboxamide group into a nitrile group to yield a compound of formula (I): where steps b) and c) can be carried out separately or in the same reactor. 5. A process for producing a compound of formula (I) as defined in any one of claims 1 to 3, or its stereoisomers, which comprises reacting a compound of formula (IX): with a compound of formula (IV): 6. A pharmaceutical composition for the treatment or prevention of a cognitive disorder, comprising a therapeutically effective amount of a compound of formula (I) as defined in any one of claims 1 to 3, or a stereoisomer thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle. 7. Use of a compound of formula (I) as defined in any one of claims 1 to 3 for the treatment or prevention of a cognitive disorder. 8. Use of a compound as defined in 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 affective disorder, rapid eye movement disorder, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis. 9. Use of a compound of formula (I) as defined in any one of claims 1 to 3 for the manufacture of a medicinal product for the treatment or prevention of a cognitive disorder. 10. The use as defined in claim 9, wherein the cognitive 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, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration or amyotrophic lateral sclerosis.