WO2010142642A1 - 4 -hydrazono- 1,4 -dihydropyridine derivatives for the treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention relates to pharmaceutical compositions comprising pyridinylidene hydrazone derivatives and their uses in the treatment or prevention of neurological and/or neurodegenerative disorders and/or diseases such as, e.g., Alzheimer's disease. Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis). Creutzfeldt-Jacob disease, Huntington's disease, HIV- related dementia, Lewis body dementia and motor neuropathy.

Description

4 -HYDRAZONO- 1 , 4 -DIHYDROPYRIDINE DERIVATIVES FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES

The present invention relates to pharmaceutical compositions comprising pyridinylidene hydrazone (also known as 4-hydrazono-l,4-dihydropyridine) derivatives and their uses in the treatment or prevention of neurological and/or neurodegenerative disorders and/or diseases such as, e.g., Alzheimer's disease, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson^'s disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt- Jacob disease, Huntington's disease, HIV -related dementia, Lewis body dementia and motor neuropathy.

Alzheimer's disease is a progressive neurodegenerative disease characterized by a loss of cognitive function and behavioral abnormalities. Immense efforts have been made to develop efficient strategies for ihe treatment of Alzheimer^'s disease because the prevalence has greatly increased. It is predicted that the number of Alzheimer's disease cases in the western world will double every twenty years and even triple in India and China with 29 million people in 2020, mostly due to increased human longevity (see Rajkumar, S. et al., WHO 2001).

Due to the multifactorial nature of the involved processes (Jakob-Roetne, R. et al.₅ Angew. Chem. 2009, 121, 3074), e.g. protein misfolding and aggregation, intracellular oxidative stress and free radical formation, elevated levels of trace metals, mitochondrial dysfunction and phosphorylation impairment, several approaches have been attempted to treat the progressive and eventually fatal neurodegenerative disease. (Gaggelli, E. et al., Chem. Rev. 2006, vol. 106: 1995-2044; Reddy, P.H, et al., Trends MoI Med. 2008, vol. 14: 45-53; Lin, M.T. et al., Nature 2006, vol. 443: 787-95). Among others, the most important current treatment strategies are the approved cholinesterase inhibitors and NMDA (N-methyl-D-aspartate) receptor antagonists, i.e. memantine, and substances in clinical trials, e.g. amyloid-β (Aβ) aggregation inhibitors, antioxidants, γ-secretase modulators, NGF mimics, peroxisome proliferator-activated receptors (PPAR) γ agonists, H3 antagonists, and HMG-CoA reductase inhibitors. (Roberson, E. D. et al., Science 2006, vol. 314: 781-784; http://www.alzforum.org/dis/tre/dre).

The current understanding, known as the cholinergic hypothesis, postulates that many of the cognitive, functional and behavioral symptoms experienced by patients with Alzheimer's disease are a result of deficiency in cholinergic neurotransmission. (Bartus, R. T. et al.. Science 1982, vol. 217:408-414). Hence, for the most part, inhibitors of the acetylcholinesterase (AChE). which enhance the acetylcholine (ACh) concentration in the brain, have been introduced to the market for treatment of mild-to-moderate Alzheimer^'s disease, e.g. tacrine, galanthamine, physostigmine. rivastigmine, and donepezil. (Holzgrabe, U. et al.,, Expert Opin. Ther. Targets 2007, vol. 11 : 161-179). Another approach is the development of dual inhibitors for AChE and butyrylcholinesterase (BuChE), as BuChE activity seems to correlate with AChE activity in AD and a cognitive improvement could be reached (Decker, M. et al., Bioorg. Med. Chem. 2008, 16, 4252; Kamal, M. A. et al., Neurochem. Res. 2008, 33, 745). Recently, dimebon, originally approved as a non-selective antihistamine, was discovered to weakly inhibit the butyrylcholinesterase and acetylcholinesterase, weakly block the NMDA receptor signaling pathway and inhibit mitochondrial permeability transition pore opening. A clinical trial phase III revealed significant improvement of the cognition of AD patients. (Doody, R. S. et al., Lancet 2008, vol. 372: 207-215).

The deposition of Aβ in the brain is the pathological hallmark of Alzheimer^'s disease and one of the potential reasons of the neuronal damage. (Hamley, I. W., Angew. Chem. Int. Ed. Engl. 2007, vol. 46: 8128-8147). Aβ is a fragment of about 37 to 42 amino acids excised from the transmembrane region of the amyloid precursor protein (APP) by the sequential action of β-site APP cleaving enzyme (BACE = β-secretase) and γ-secretase, eventually resulting in neurotoxicity, tau hyperphosphorylation and Aβ oligomerization. aggregation and finally plaque formation. Consequently, inhibitors of BACE and γ-secretase are prime targets for Alzheimer's disease drug development. (Olsen, R. E. et al., Aπnu. Rep. Med. Chem. 2007, vol. 42: 27-47). Tarenflurbil, a γ-secretase inhibitor, is currently in clinical trial phase III as well as the BACE-I inhibitor Way-258131. (Wilcock. G.K. et al., Lancet Neurol. 2008, vol. 7: 483-493). Anti-amyloid disease-modifying treatments directly target the Aβ protein, e.g. tramiposate is a small-molecule glycosarninoglycan mimetic which binds to soluble Aβ, and thereby stops the formation of the amyloid plaques. (SantaMaria, T. et al., MoI. Neurodegeneration 2007, vol. 2: 1-12). Additionally, vaccination and anti-Aβ monoclonal antibodies, e.g. bapineuzumab, are variations of the amyloid concept.

Certain substances of peptidic and non-peptidic origin with anti-amyloid potency have been reported. (Austen, B.M. et al., Biochemistry 2008, vol. 47: 1984-1992). Among the latter are endogenous compounds such as melatonin, in addition to rifampicin, benzofuran, benzothiazole, hydroxyindole, curcumin-derived pyrazoles and isoxazoles, and naphthylazo derivatives, phenols, classic antibiotics, e.g. tetracycline, doxycycline and minocycline as well as cyclodextrins. (Talaga, P., Mini Rev. Med Chiem. 2001, vol. 1 :175-186; Henriksen, G. et al., J Med. Chem. 2007, vol. 50: 1087-1089; Cohen, T. et al., Biochemistry 2006, vol. 45: 4727-4735; Narlawar, R. et al., ChemMedChem 2008, vol. 3: 165-172; Yona, R.L. et al., ChemMedChem 2008, vol. 3, 66; Shoval, E. L. et al., Amyloid 2007, vol. 14: 73-87; Forloni, G. et al., FEBS Letters 2001, vol. 487: 404-407; Choi, Y. et al., Neuropsychopharmacol. 2007, vol. 32: 2393-2404; Danielsson, J. et ai. Biochemistry 2004, vol. 43: 6261-6269). Other researchers have reported several isoindoline and piperidine derivatives inhibiting amyloid protein aggregation and deposition. (WO 00/76969; WO 03/033489). In addition, the role of AChE is not only the hydrolysis of the neurotransmitter ACh, but also the acceleration of the aggregation of AB into amyloid fibrils. The peripheral anionic site (PAS) of AChE gorge seems to be responsible for this activity. (Johnson, G. et al., Biochem. Biophys. P_^es. Commun. 1999, vol. 258: 758 762; De Ferrari, G. V. et al., Biochemistry 2001, vol. 40: 10447-10457). Within this context, it is expected that even a peripheral site blocker prevents the AB peptide to interact with AChE, and, thus, inhibit the fibril formation process. Studies regarding development of such AChE inhibitors have been reported for coumarine derivatives. (Bartolini, M. et al., Biochem. Pharmacol. 2003, vol. 65: 407-416; Piazzi, L. et al., J. Med. Chem. 2003, vol. 46: 2279-2282).

It has to be stressed that modified APP processing and/or the generation of extracellular plaques containing proteinaceous depositions are not only known from Alzheimer' s pathology but also from subjects suffering from other neurological and/or neurodegenerative diseases and/or disorders. These diseases and/or disorders comprise, inter alia, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt- Jacob disease, Huntington's disease, HIV-related dementia, Lewis body dementia and motor neuropathy.

In order to prevent, treat and/or ameliorate neurodegenerative diseases and/or disorders related to the deposition of amyloid plaques, compounds have been developed which inhibit both AChE and amyloid fibrils formation and, in particular, AChE, BuChE and amyloid fibrils formation.

Considering the severe defects associated with neurodegenerative diseases and/or disorders related to the deposition of amyloid plaques, compounds for treating and/or preventing these diseases and/or disorders are highly desirable.

In the context of this invention, it was surprisingly found that the pyridinylidene hydrazone derivatives described herein, in particular the compounds of formula (I), inhibit acetylcholinesterase as well as Aβ fibril formation significantly and are further able to cross the blood-brain barrier which is thought to be due to their pK_a values and lipophilicity. In particular, these compounds inhibit both acetylcholinesterase and butyrylcholinesterase, inhibit Aβ fibril formation, and destruct already formed Aβ fibrils. These compounds are particularly useful in the medical intervention of, e.g., neurodegenerative diseases/disorders like Alzheimer's disease, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt- Jacob disease, Huntington's disease, HIV-related dementia, Lewis body dementia, motor neuropathy and the like.

Accordingly, the present invention relates to pharmaceutical compositions comprising a compound of formula (I)

(I) or a pharmaceutically acceptable salt, solvate or prodrug thereof. L¹ is a linking group selected from a covalent bond, optionally substituted Ci_-I2 alkylene, optionally substituted C_2-I2 alkenylene or optionally substituted C_2-12 alkynylene. The above optionally substituted groups may be substituted with one or more (such as, e.g., one, two, three or four) groups independently selected from hydroxyl, Ci_-4 alkoxy, Ci_-4 alkylamino, (C i-₄ alkyl)(Ci_₄ alkyl)amino or halogen. The above optionally substituted groups may be interrupted by one or more (such as, e.g., one, two, three or four) groups independently selected from -O-, -S-, -CO-, -NH-, -NH-CO- or -CO-NH-. Preferably, they are not interrupted or they are interrupted by one or two groups independently selected from -0-, - S- or -NH-. More preferably, they are not interrupted or they are interrupted by one or two groups independently selected from -O- or -S-. Even more preferably, they are not interrupted. Preferably, L¹ is optionally substituted Cj .(, alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene or hexylene) or a covalent bond. More preferably, L¹ is optionally substituted Ci_-3 alkylene or a covalent bond. Even more preferably, L¹ is a covalent bond.

L² is a linking group selected from a covalent bond, optionally substituted Ci_-J2 alkylene, optionally substituted C_2-12 alkenylene or optionally substituted C_2-12 alkynylene. The above optionally substituted groups may be substituted with one or more (such as, e.g., one, two, three or four) groups independently selected from hydroxyl, Ci -₄ alkoxy, C i_-4 alkylamino, (Ci_-4 alkyl)(Ci_₄ alkyl)amino or halogen. The above optionally substituted groups may be interrupted by one or more (such as, e.g., one, two, three or four) groups independently selected from -O- -S-, -CO-, -NH-, -NH-CO- or

-CO-NH-. Preferably, they are not interrupted or they are interrupted by one or two groups independently selected from -0-, -S- or -NH-. More preferably, they are not interrupted or they are interrupted by one or two groups independently selected from -O- or -S-. Even more preferably, they are not interrupted. Preferably, L¹ is optionally substituted Ci_-8 alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene or octylene). More preferably, L¹ is an optionally substituted Cj_-4 alkylene. Even more preferably, L is ethylene or propylene.

A and B are each independently selected from optionally substituted aromatic 5- to 10- membered monocyclic or bicyclic ring systems, which optionally contain one or more (such as, e.g., one. two, three or four) ring heteroatoms independently selected from N, O or S. The above mentioned ring systems may be substituted with one ore more (such as, e.g., one, two, three, four, five, six or seven) groups independently selected from halogen (such as, e.g., chloro), C_1-4 alkyl, C_2-4 alkenyl, €₂=₄ alkynyl, C_1-4 halogenalkyl, €2=4 halogenalkenyl, C₂-₄ halogenalkynyl, Cj_-4 alkoxy, C₂-4 alkenyloxy, C2-4 alkynyloxy, Ci_-4 halogenalkoxy, C₂-₄ halogenalkenyloxy, C_2-4 halogenalkynyloxy, cyano, nitro, C ₁.₄ alkylthio, C_2-4 alkenylthio or C₂-4 alkynylthio. Preferably, the above mentioned ring systems are unsubstituted or substituted with one, two, three, four or five groups independently selected from halogen, Ci_-4 alkyl, Cj_-4 alkoxy, C_1-4 halogenalkyl, C_1-4 halogenalkoxy or cyano. More preferably, the above mentioned ring systems are unsubstituted or substituted with one, two, three, four or five groups independently selected from halogen, Ci_-4 halogenalkyl, Ci_-4 halogenalkoxy or cyano. Even more preferably, the above mentioned ring systems are unsubstituted or substituted with one, two or three halogen.

Preferably, A and B are each independently selected from optionally substituted aromatic 5- or 6-membered monocyclic ring systems, which optionally contain one, two or three heteroatoms, independently selected from N, O or S. Accordingly, non-limiting examples of A and/or B are phenyl, furanyi, thiofuranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl (such as, e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl or furazanyl.

More preferably, A and B are each independently selected from optionally substituted aromatic 6-membered monocyclic ring systems, which optionally contain one or two heteroatoms independently selected from N, O or S. Even more preferably, A and B are each independently an optionally substituted phenyl.

The following compounds are preferred examples of the compounds of formula (I):

compound (i) compound (ii)

compound (iii) compound (iv)

Again in the context of medical intervention these compounds are particularly useful in the amelioration and/or treatment of neurodegenerative diseases/disorders, in particular neurodegenerative diseases/disorders involving abnormal Aβ biology and/or Aβ plaque formation and/or ACnE physiology.

For the skilled person in the field of synthetic chemistry, various ways for the preparation of the compounds of formula (I) will be readily apparent. For example, the 4-hydrazono-l,4- dihydropyridine (formerly called pyridinylidene hydrazone) derivatives of the present invention can be prepared according to the following approach, as illustrated in Scheme (I):

Scheme (I)

Accordingly, 4-hydrazinopyridine is condensed with an aldehyde derivative (OHC-IΛA; such as, e.g., dichlorobenzaldehyde) to give the respective hydrazone. The hydrazone is then coupled with an aryl-halogenate derivative or a heteroaryl-halogenate derivative (B-L -Hal) to give the compound of formula (I). In the above scheme, L¹, L², A and B have the meanings described herein above and Hal is halogen (such as, e.g., bromine).

As also documented in the appended examples, compounds of the formula (I), in particular the compounds (i) to (iv), have been found to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and the formation of amyloid fibrils. Without being bound by theory, it is believed that the compounds of formula (I) inhibit AChE by interacting with two separate sites of the enzyme, the active site and the peripheral binding site. Accordingly, the compounds described herein are particularly useful in the amelioration of diseases/disorders involving abnormal AChE physiology and/or amyloid biology. The compounds described herein are useful in altering pathological conditions associated with AChE and/or Aβ physiology.

In the context of this invention and as known in the art, acetylcholinesterase (AChE) is an enzyme that hydrolytically degrades acetylcholine, producing choline and acetate. The compounds described herein are useful for inhibiting acetylcholinesterase (AChE), in particular human acetylcholinesterase (e.g., Gene Accession No. NM_000665.3) However, acetylcholinesterases from other species are also inhibited.

In the context of this invention and as known in the art, butyrylcholinesterase (BuChE) is an enzyme that hydrolytically degrades different choline esters, such as, e.g., butyrylcholine. The compounds described herein are useful for inhibiting butyrylcholinesterase (BuChE), in particular human butyrylcholinesterase (e.g., Gene Accession No. NM_000055) However, butyrylcholinesterases from other species are also inhibited.

In the context of this invention and as known in the art, Aβ (amyloid β, A-beta, Aβ4, or β-A4) is a predominantly fibrillar peptide found in extracellular neuritic plaques; (see Koo PNAS 1999 vol. 96: 9989-9990, Glenner BBRC 1984 vol. 12: 1131). It is of note that Aβ has several naturally occurring forms comprising 39 amino acids (Aβ39), 40 amino acids (Aβ40), 41 amino acids (Aβ41), 42 amino acids (Aβ42) or 43 amino acids (Aβ43); (see Sinha PNAS 1999 vol. 96: 11094-1053; Price Science 1998 vol. 282: 1078-1083; WO 00/72880 or Hardy TINS 1997 vol. 20: 154). The human forms are referred to as the Aβ39, Aβ40, Aβ 41, Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acid sequence (starting from the N-terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. In Aβ41, AB40, AB39, the C-terminal amino acids A, IA, VIA are missing, respectively. In the Aβ43- form an additional threonine reside is at the C-terminus of the above depicted sequence. Shorter peptides derived from the Aβ and/or the above amino acid sequences, for example HHQKLVFFAED (see also appended examples), can also form plaques.

Accordingly, the compounds of formula (I), in particular the compounds (i) to (iv), can be used to treat or prevent neurological and/or neurodegenerative disease and/or disorders, such as Alzheimer's Disease. Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson^'s disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt- Jacob disease, Huntington's disease, HIV-related dementia, Lewis body dementia and motor neuropathy. In particular, in the context of this invention, the compounds are useful in the prevention, amelioration and/or treatment of Alzheimer's disease and Down's syndrome.

As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon including straight chain and/or branched chain groups.

As used herein, the term "alkenyl" refers to an unsaturated hydrocarbon including straight chain and/or branched chain groups, comprising at least one carbon-to-carbon double bond.

As used herein, the term "alkynyl" refers to an unsaturated hydrocarbon including straight chain and/or branched chain groups, comprising at least one carbon-to-carbon triple bond.

As used herein, the term "alkyl ene" refers to an alkanediyl group including straight chain and/or branched chain groups.

As used herein, the term '"alkenylene" refers to an alkenediyl group including straight chain and/or branched chain groups, and comprising at least one carbon-to-carbon double bond.

As used herein, the term "alkynylene" refers to an alkynediyl group including straight chain and/or branched chain groups, and comprising at least one carbon-to-carbon triple bond. As used herein, the term "halogenalkyl" refers to an alkyl group substituted with halogen. Likewise, as used herein, the term "halogenalkenyl" refers to an alkenyl group substituted with halogen and the term "halogenalkynyl" refers to an alkynyl group substituted with halogen.

As used herein, the term "alkoxy" refers to an alkyl group covalently bonded to an O, i.e. an -O-alkyl-group. Likewise, as used herein, the term "alkenyloxy" refers to an alkenyl group covalently bonded to an O and the term "alkynyloxy" refers to an alkynyl group covalently bonded to an O.

As used herein, the term "halogenalkoxy" refers to an alkoxy group substituted with halogen. Likewise, as used herein, "halogenalkenyloxy" refers to an alkenyloxy group substituted with halogen and "halogenalkynyloxy" refers to an alkynyloxy group substituted with halogen.

As used herein, the term "alkylamino" refers to an alkyl group covalently bonded to a N, i.e. an -NH-alkyl group. Likewise, as used herein, (alkyl)(alkyl)amino refers to two independent alkyl groups each covalently bonded to the same N, i.e. an -N(alkyl)-alkyl group.

As used herein, the term "alkylthio" refers to an alkyl group covalently bonded to a S, i.e. an -S-alkyl group. Likewise, as used herein, the term "alken)'lthio" refers to an alkenyl group covalently bonded to a S and the term "alkynylthio" refers to an alkynyl group covalently bonded to a S.

The scope of the present invention embraces all pharmaceutically acceptable salt forms of the compounds of formula (I) as defined herein and as described herein, which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of a carboxylic acid group with a physiologically acceptable cation as they are well known in the art. Exemplary base addition salts comprise, for example, alkali metal salts such as sodium or potassium salts; alkaline-earth metal salts such as calcium or magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, diethanol amine salts or ethylenediamine salts; aralkyl amine salts such as N,N- dibenzylethylenediamine salts, benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts. benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts or lysine salts. Exemplary acid addition salts comprise, for example, mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts, nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts or perchlorate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, nicotinate, benzoate, salicylate or ascorbate salts; sulfonate salts such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p- toluenesulfonate (tosylate), 2-naphthalenesulfonate, 3-phenylsulfonate or camphorsulfonate salts; and acidic amino acid salts such as aspartate or glutamate salts.

The scope of the present invention also embraces solid forms of the compounds of formula (I) as defined herein and as described herein, in any solvated form, including, for example, solvates with water, e.g. hydrates, or solvates with organic solvents such as, e.g., methanol, ethanol or acetonitrile, i.e. as a methanolate, ethanolate or acetonitrilate, respectively.

The scope of the present invention further embraces pharmaceutically acceptable prodrugs of the compounds of formula (1) as defined herein and as described herein. Such pharmaceutically acceptable prodrugs are derivatives of the respective compounds which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds of formula (I), which are pharmaceutically active in vivo. Prodrugs of the compounds of formula (I) may be formed in a conventional manner with a functional group of said compounds such as with an amino, hydroxy or carboxy group. The prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier. Amsterdam 1985). Prodrugs include acid derivatives well known to the person skilled in the art, such as, e.g., esters prepared by reaction of the parent acidic compound with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a suitable amine. When a compound employed in the present invention has a carboxyl group, an ester derivative prepared by reacting the carboxyl group with a suitable alcohol or an amide derivative prepared by reacting the carboxyl group with a suitable amine is exemplified as a prodrug. An especially preferred ester derivative as a prodrug is methylester, ethylester, n- propylester, isopropylester, n-butylester, isobutylester, tert-butylester, morpholinoethylester or N,N-diethylglycolamidoester. When a compound employed in the present invention has a hydroxy group, an acyloxy derivative prepared by reacting the hydroxyl group with a suitable acylhalide or a suitable acid anhydride is exemplified as a prodrug. An especially preferred acvloxy derivative as a prodrug is -O(=O)CH₃, -OCC=O)-C₂H₅, -OC(O)-Ctert-Bu). -OC(O)-Ci₅H₃I, -0C(O)-(m-C00Na-Ph), -OCC=O)-CH₂CH₂COONa, -0(C-O)-CH(NH₂)CH₃ or -OC(=O)-CH₂-N(CH₃)₂. When a compound employed in the present invention has an amino group, an amide derivative prepared by reacting the amino group with a suitable acid halide or a suitable mixed anhydride is exemplified as a prodrug. An especially preferred amide derivative as a prodrug is -NHC(=O)-(CH₂)₂OCH₃ or -NHCC=O)-CH(NH₂)CH₃.

The compounds of the present invention, in particular the compounds of formula (I), can be used as medicaments. As such, the compounds of the present invention may be administered per se or may be formulated as pharmaceutical compositions. In particular, the present invention provides for compounds for use in the treatment or prevention of neurological and/or neurodegenerative disorders and/or diseases associated with amyloid plaque formation. According!)', the present invention also provides for compounds for the preparation of a medicament for the treatment or prevention of neurodegenerative diseases and/or disorders associated with amyloid plaque formation.

Accordingly, within the scope of the present invention are pharmaceutical compositions comprising a compound of formula (I) as defined herein and as described herein or a pharmaceutically acceptable salt, solvate or prodrug thereof. Thus the invention also provides a pharmaceutical composition which comprises a therapeutically effective amount of a compound of formula (I) in admixture with one or more pharmaceutically acceptable excipients. such as. e.g., carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, and/or antioxidants. The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20th Edition. The pharmaceutical excipient(s) can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition according to the invention comprises a therapeutically effective amount of at least one compound of formula (I) as defined herein and as described herein or a pharmaceutically acceptable acceptable salt, solvate or prodrug thereof, and one or more pharmaceutically acceptable excipients as described herein.

The compounds or the pharmaceutical compositions of the invention may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g. as a tablet, capsule or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g. subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g. through mouth or nose). gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, and vaginal.

If said compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally. intrathecaily, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds or pharmaceutical composiiions are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com. potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC). hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate. stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similat type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Alternatively, said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel. hydro gel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.

Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For topical application to the skin, said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax. 2-octyldodecanol, benzyl alcohol and water.

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

A proposed, yet non-limiting dose of the compounds according to the present invention for administration to a human (of approximately 70 kg body weight) may be 0.1 mg to 1 g, preferably 1 mg to 500 mg, and more preferably 50 mg to 100 mg, of the active ingredient per unit dose. The unit dose may be administered, for example, 1 to 4 times per day. The dose will depend on the route of administration. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient as well as the severity of the condition to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian.

The term "treatment" of a disorder or disease as used herein is well known in the art. "Treatment'^" of a disorder or disease implies that a disorder or disease has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typical!}' shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e. diagnose a disorder or disease).

"Treatment" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g. no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). "Treatment" of a disorder or disease may also lead to a partial response (e.g. amelioration of symptoms) or complete response (e.g. disappearance of symptoms) of the subject/patient suffering from the disorder or disease. "Amelioration" of a disorder or disease may. for example, lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (e.g. the exemplary responses as described herein above).

Treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).

Also the term "prevention" of a disorder or disease as used herein is well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease as defined herein may, in particular, benefit from a prevention of the disorder or disease. Said subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard assays, using, for example, genetic markers or phenotypic indicators. It is io be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in said patient/subject (for example, said patient/subject does not show any clinical or pathological symptoms). Thus, the term "prevention" comprises the use of compounds of the present invention or pharmaceutical compositions comprising said compounds before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.

The subject or patient, such as the subject in need of treatment or prevention, may be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig. a hamster, a rat, a mouse), a murine (e.g. a mouse), a canine (e.g. a dog), a feline (e.g. a cat), an equine (e.g. a horse), a primate, a simian (e.g. a monkey or ape), a monkey (e.g. a marmoset, a baboon), an ape (e. g. gorilla, chimpanzee, orangutang, gibbon), or a human. The meaning of the terms "eukaryote". '"animal^*', "mammal", etc. is well known in the art and can, for example, be deduced from Wehner und Gehring "Zoologie^5* (1995; Thieme Verlag). In the context of this invention, it is particularly envisaged that animals are to be treated which are economically, agronomically or scientifically important. Scientifically important organisms include, but are not limited to, mice, rats, rabbits, fruit flies like Drosophila melagonaster and nematodes like Caenorhabditis elegans. Non-limiting examples of agronomically important animals are sheep, cattle and pig, while, for example, cats and dogs may be considered as economically important animals. In one embodiment of the present invention, the subject is a human.

In this specification, a number of documents including patent applications and journal publications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is also illustrated by the following illustrative figures. The appended figures show:

Figure 1: A) Inhibition of fibril formation by compound (iii) (i.e., the compound of Example 3; denoted as "Ex. 3") in comparison to DUO 3 and a negative control using amyloid β- protein (1-40); B) Inhibition of fibril formation by compound (iii) (i.e., the compound of Example 3; denoted as "Ex. 3") in comparison to DUO 3 and a negative control using Aβ peptide (HHQKLVFF AED); C) Inhibition of fibril formation by compounds (i), (ii), (iii) and (iv) (i.e., the compounds of Examples 1 , 2, 3 and 4, respectively; denoted as "Ex. 1", "Ex. T\ "Ex. 3" and "Ex. A^"'\ respectively) in comparison to DUO 3 and a negative control using Aβ peptide (HHQKLVFF AED); D) Inhibition of fibril formation by compound (ii) (i.e., the compound of Example 2: denoted as "Ex. 2") at concentrations of 0.5 mM, 0.05 mM and 0.005 mM and compound (iii) (i.e., the compound of Example 3; denoted as "Ex. 3") at concentrations of 0.5 mM, 0.05 mM and 0.005 mM in comparison to DUO 3 and a negative control using Aβ peptide (HHQKLVFF AED); and E) Effect of compound (iii) (i.e., the compound of Example 3; denoted as "5d") at concentrations of 0.5 mM, 0.05 mM and 0.005 mM, respectively, and a negative control using Aβ peptide (HHQKL VFF AED) on fibril destruction; the destruction occurs very quickly within the first minutes as can be seen from the low starting fluorescence emission in comparison to the control; after a further small decrease within the next 5 min the emission stays constant at least until 15 h (data not shown). Examples

The invention will now be described by reference to the following examples, which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

General procedure for the synthesis of the following 1 -substituted 4-((2,6- dichlorobenzylidene)hydrazono)-l,4-dihydropyridine derivatives:

The following exemplary compounds of the formula (I) were synthesized by the condensation of 4-hydrazinopyridine (synthesized according to Mann F. G et al., J. Chem. Soc. 1959. 3830- 3834) with 2,6-dichlorobenzaldehyde (Douglas, W. A. et al., J. Med. Chem., 1977, vol. 20: 939-943) followed by alkylation with the corresponding alkylhalogenates.

A mixture of 0.01 mol of 4-(2-(2,6-dichlorobenzylidene)hydrazinyl)pyridine hydrochloride and 0.02 mol of the corresponding bromide (such as, e.g., benzylbromide for Example 1, phenylethylbromide for Example 2, phenylpropylbromide for Example 3. and 2.6- dichlorobenzylbromide for Example 4) in EtOH (20 ml) was refluxed for 24 h. After cooling the mixture, the salt was obtained and converted to the free base by means of partitioning between CH₂CI₂ and 2 N NaOH. The organic layer was evaporated to dryness and the residue recrystallized from EtOH/H₂O (2/1).

Alternatively, p-dichlorobenzaldehyde can be used to make 4-(2-(4- chlorobenzylidene)hydrazinyl)pyridine hydrochloride. To a solution of 7.5 mmol of 4- Pyridylhydrazine hydrochlorid in EtOH (35ml) and H₂O (35ml) a solution of 9 mmol of p- chlorobenzaldehyde in EtOH (5ml) was added. After heating the reaction mixture on the steam bath for 3min, the mixture was concentrated to remove most of the EtOH, cooled, filtered and recrystallized from EtOH/H2O (2/1). Yield: 65% Example 1: 4-((2,6-dichIorobenzyIidene)hydrazono)-l-benzyl-l,4-dihydropyridine

Yield 62%; yellowish oil; IR (KBr); v_maks 1644, 1510, 1393, 1174, 825, 774 cm-¹; ¹H NMR (CH₃OH-J-/): δ ppm 5.01 (2H, s, CH₂-Ph), 6.39 (IH, dd, J=2.5/7.5 Hz,), 7.19-7.46 (HH, m), 8.50 (I H, s, N=CH); ¹³C NMR

δ ppm 60.33 (t), 108.54 (d), 112.30 (d), 128.73 (d), 129.60 (d), 130.20 (d), 130.24 (d), 130.56 (d), 132.96 (s), 135.86 (s), 137.61(s), 139.82 (d), 140.31 (d), 145.73 (d), 163.12 (s). EI-MS m/z (% relative intensity): 357 (M+2), 355 (M+), 154 (34), 152 (100), 125 (26). 91(40), 92 (40), 63 (28). (C₁₉H₁₅N₃Cl₂) C, H, N.

Example 2: 4-((2,6-dichIorobenzylidene)hydrazono)-l-(2-phenyIethyI)-l,4- dihydropyridine

Yield 55%; mp 145 ⁰C: IR (KBr) v_maks 1636. 1484, 1390, 1182, 826, 753 cm^"1; ¹H NMR

Hz. CH₂), 4.06 (2H₅ t, J=6.9 Hz, CH₂), 6.29 (IH, dd, J=2.7/7.7Hz), 7.11 (IH, dd J=2.7/7.4Hz), 7.17 (IH, dd J=L 5/8.3 Hz), 7.22-7.30 (7H, m), 7.42 (2H, d, J=8.3 Hz), 8.51 (IH, s, N=CH); ¹³C NMR (CH₃OH-^): δ ppm 38.04 (t), 58.73 (t), 108.21 (d), 111.84 (d), 128.00 (d), 129.81 (d), 130.04 (d), 130.25 (d), 130.49(d), 132.96 (s), 135.81 (s), 138.55 (s), 139.75 (d). 140.18 (d). 145.33 (d), 163.16 (s); EI-MS m/z (% relative intensity): 371 (M+2), 369 (M+), 334 (24), 105 (100), 79 (20). (C₂₀Hi₇N₃ Cl₂) C, H, N.

Example 3: 4-((2,6-dichlorobenzylidene)hydrazono)-l-(3-phenylpropyI)-l,4- dihydropyridine

Yield 52%: yellowish oil; IR (KBr) v_maks 1644, 1509, 1393, 1186. 825, 774 cm^"1; ¹H NMR (CH₃GH-^): δ ppm 2.12 (2H, quin, J=I ^'.3 Hz,- 14 CH₂), 2.68 (2H, t, J=IA Hz, CH₂). 3.86 (2H. L -/=7.3 Hz. CH₂). 6.42 (IH, d, J=6.3 Hz,), 7.18-7.33 (7H, m), 7.39-7.48 (4H, m). 8.52 (IH, s, N=CH); ¹³C NMR (CH₃OH-^: δ ppm 33.38 (t), 54.90 (t), 56.86 (t), 108.45 (d), 112.15 (d), 127.27 (d), 129.41 (d), 129.64 (d), 130.25 (d), 130.51 (d), 132.99 (s), 135.83 (s), 1 39.69 (d), 140.17 (d), 141.96 (s), 145 33 (d), 163.21 (s); EI-MS m/z (% relative intensity): 385 (M+2), 383 (M+), 348 (24), 230 (11), 91 (100), 79 (11), 51(11), 41(11). (C₂IHi₉N₃Cl₂) C, R N. Example 4: 4-((2,6-dichlorobenzylidene)hydrazono)-l-(2,6-dichIorobenzyI)-l,4- dihydropyridine

Yield 74%; yellowish oil; mp 204 ⁰C; IR (KBr) v_maks 1640, 1486, 1436, 1170, 825, 780 cm^"1; ¹H NMR (CDCl₃): δ ppm 5.14 (2H, s, CH₂-Ph;, 6.53 (IH, brs), 7.08-7.33 (8H, m), 1 Al (2H, d, J=8.6 Hz), 8.69 (IH, s, N=CH); ¹³C NMR (CDCl₃): δ ppm 53.82 (t), 107.85 (d), 1 12.31 (d), 128.81 (d), 128.95 (d), 129.17 (d), 130.16 (s), 131.02 (d), 135.03 (s), 136.71 (s), 137.56 (s), 138.62 (d), 141.15 (d), 146.49 (d), 163.05 (s); EI-MS m/z (% relative intensity): 425 (M+2), 421 (M+l), 423 (M+), 159 (100), 123 (15), 51(15), 49 (35). (Ci₉Hi₃N₃Cl₄) C, H, N.

Example 5: Lipophilieity (LogP) Determination

ihe partition coefficients were determined by means of RP HPLC using the correlation between the capacity factor and the logP values of reference compounds or by the classical shake flask method in phosphate buffer at pH 5, 6. 7 and 7,4 using UV spectroscopy.

LogP determination by means of RP HPLC: The partition coefficients of all examples were determined by RP-chromatography using methanol/phosphate buffer 70/30 as an eluent. The chromatographic systems were calibrated with solutes for which an experimental octano I/water partition coefficient was available. (Hansen, C; Sammer, ,P.G.; Taylor, JB.

Comprehensive Medicinal Chemistry, 1st Ed. Vol. 6. Pergamon Press, Oxford, 1990). The capacity factors of the reference substances were correlated against with experimental octanol/water log P values, and the obtained correlation equation was used to calculate log P values of the tested compounds.

The experiments were performed on a LiChroCART^® 125-4.6 T-HPTC-Cartridge filled with LiChrospher^{^} 100, RPI8, 5 μm, endcapped (Merck, Darmstadt, Germany). The methanol (LiChrosolv^® (Merck))/buffer (70/30) mobile phase was used at a flow rate of 1.0 mL. The phosphate buffer (pH 7.4, DAB 1999) was obtained by mixing a 0.2 M potassium dihydrogen phosphate solution (1000 mL) and 0.1 M sodium hydroxide (1573 mL). 0.02 % N₉N- dimethylhexylamine was added to minimize the peak tailing. The following aromatic compounds were applied for calibration: 2-phenylethanol, benzene, dimethylaniline, toluene, biphenyl, anthracene. The analytes were dissolved in methanol at a concentration of 4 μg/niL. Experiments were run in duplicate, and peak maxima were determined at the respective absorption maximum. Capacity ratios were determined as k'= (t]-to)/to, where t] is the average retention time of the analyte and to is the average retention time of the solvent. A linear regression was performed for the log kVlogP data of the reference compounds (y 2.5935 + 1.8353; r² 0.9782), and the regression equation was used to calculate the logP of the compounds.

LogP determination by means of shake flask method: Partition coefficients of Examples 3 and 4 were determined by shake flask method using UV spectroscopy and were studied in phosphate buffer at pH 5, 6, 7 and 7.4 for each compound. The phosphate buffer was saturated with octanol prior to partitioning by adding octanol, mixing, and allowing the phases to separate overnight; in the same manner portions of octanol were saturated with the buffer. A stock solution for each compound was prepared at 10^"5 niol/L concentration in octanol saturated with phosphate buffer and the absorbance (Ao) was measured between 314 and 360 nm. A 1.0 ml portion of this solution was shaken at 37 ⁰C for 4h with 1.0 ml phosphate buffer saturated with octanol. The emulsion was allowed to stand overnight, centrifuged for 5 min (3000 U/min.). the water layer separated and again the absorption (Al) measured and the logP value was calculated as log P=log ((Ao- Ai)/ Ai).

Table 1: LogP of the exemplary compounds provided herein

Reported values for Examples 1 and 2 were determined by RP HPLC method and for Examples 3 and 4 were determined by shake flask method. Example 6t Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Inhibition

Compounds of Examples 1 to 4 were subjected to a slightly modified Ellman's test to evaluate their inhibition of AChE. (Ellman, G. L. et al.. Biochem. Pharmacol. 1961. vol. 7: 8895). The BuChE inhibition of the compounds of Examples 1 to 4 was also evaluated using a modified Ellman^'s test (Ingkaninan, K. et al., J. Chromatogr., A 2000, 872, 61 ; Hamley, LW. Angew. Chem., Int. Ed. 2007, 46, 8128).

Chemicals and Materials: Acetylcholinesterase (AChE) — E. C. 3.1.1.7., Type VI-S. from Electric Eel, 500 units, was purchased from Sigma- Aldrich (Steinheim, Germany). 5,5'- Dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's Reagent), potassium dihydrogen phosphate, potassium hydroxide, sodium hydrogen carbonate and acetylthiocholine iodide (ATC) were obtained from Fluka (Buchs, Switzerland). Deionized water prepared by means of a Milli-Q purification apparatus (Millipore^®; Eschborn, Germany) was used throughout. Spectrophotometric measurements were performed on a Varian Gary 50 UV-Vis spectrophotometer equipped with a thermostated cell holder. (Kapkova, P. et al.. Arch. Pharrrt (Wεinheim) 2003. vol. 336. 523-540). BuChE inhibitory activity was determined using BuChE (EC 3.1.1.8) from horse serum (Fallarero, A. et al., Pharmacol. Res. 2008, 58, 215).

Solutions: Phosphate buffer pH 8.0: potassium dihydrυgen phosphate (13,6Ig) was dissolved in water (IL) and adjusted with potassium hydroxide to a pH of 8.0 ± 0.1. Buffer was filtered through 0.22μm (pore size) disposable filter units (Schleicher and Schuell, Dassel, FRG) prior to use. The buffer was freshly prepared every week and kept at 4°C.

5.5^'-Dithiobis(2-nitrobenzoic acid) (DTNB) solution (0.01M): The solution (100ml) was prepared in water, containing DTNB (0.396g) and sodium hydrogen carbonate (0.15g). The solution was directly used or placed in the — 30⁰C freezer until needed.

Acetylthiocholine iodide (ATC) 0.075M: 0.2167g ATC were weighed and dissolved in 10ml water. This solution was stored in frozen aliquots (0.4ml) in Eppendorf-caps at ^3O⁰C. Butyrylthiocholine iodide (BTC) 0.075M: 0.237g BTC were weighed and dissolved in 10 ml water. The solution was stored in frozen aliquots (0.4ml) in Eppendorf-caps at -30⁰C.

Acetylcholinesterase (AChE)/ Butyrylcholinesterase (BChE) solution: The enzyme (500 units) was dissolved in gelatine solution (ImI, 1%) and taken into a 100ml volumetric flask, made up with water to c = 5u/ml. This stock solution was stored in frozen aliquots (0.7ml) at - 30⁰C and prior to use was thawed and diluted to a final concentration of c = 2.5u/ml.

Acetylcholinesterase /Butyrylcholinesterase activity assay: Enzyme activity was investigated using a slightly modified colorimetric method by Ellman et al. As the product of the enzymatic hydrolysis, the thiocholine, does not possess a significant chromophore for UV detection, the evaluation of enzyme activity was performed using a specific chromogenic reagent, DTNB. The measurements were carried out as follows: Stock solutions of the inhibitor compounds were prepared in 2% DMSO. The enzyme activity was determined in the presence of at least five different concentrations of an inhibitor, generally between 10^"4 and IQ^"9, in order to obtain inhibition of AChE- or BChE-activity comprised between 0 and 100%. Each concentration was assayed in triplicate. The samples were investigated immediately after preparation.

Inhibition study: Prior to use all solutions were tempered to 2O⁰C. Enzyme solution (lOOμl) and inhibitor solution (lOOμl) were added into a cuvette containing the phosphate buffer (3.0ml, 0.1M; pH= 8.0). After 5 min incubation, required aliquots of the DTNB solution (lOOμl) and of the acetylthiocholine/butyrylthiocholine iodide (20μl) were added. After rapid and immediate mixing the absorption was measured at 412 nm. As a reference, an identical solution of the enzyme without the inhibitor is processed following the same protocol. The blank reading contained 3.0ml buffer, 200μl water, lOOμl DTNB, and 20μl substrate.

The relative enzyme activity was calculated according to the equation: activity [%] = M_corr x 100 / R_corr where M_COi_r is the enzyme activity in the presence of the inhibitor, R_c0n- is the enzyme activity of the reference solution (without the inhibitor). Both of the values are corrected with blank- reading value. The results were analyzed with the program GraphPad Prism™ (GraphPad Software, San Diego, CA, USA) using non-linear regression analysis toward a sigmoid dose- response (variable slope) model. Inhibition curves were obtained for each compound by plotting the % enzyme activity in the presence of the inhibitor versus logarithm of inhibitor concentration in the assay solution. For each inhibition curve four variables were determined: IC₅₀ (the log[Dose] when response = 50% ), hill slope, the bottom plateau, the top plateau.

The IC₅₀ values of several compounds according to the invention for the inhibition of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) as determined in the AChE inhibition assay and the BChE inhibition assay are provided in Tables 2 and 3, respectively.

IC₅₀ values for AChE inhibition were initially determined to be 17.40 + 2.56 μM for the compound of Example 1, 56.70 + 9.48 μM for the compound of Example 2. 5.66 + 0.17 μM for the compound of Example 3, and 6.40 + 1.62 μM for the compound of Example 4. In a further, more elaborate determination, the AChE inhibition values as indicated in Table 2 have been obtained. Compounds as provided herein clearly inhibit AChE.

Table 2: AChE Inhibition (IC₅₀ values) of compounds provided herein

Table 3: BuChE Inhibition (IC₅0 values) of compounds provided herein

For BuChE inhibition, only two experiments were performed and, therefore, no SEM are given. Example 7: Thioflavin T Fluorescence Assay

Thioflavin was used to visualize the block of fibril formation. To assess the fibril formation inhibition of the compounds disclosed herein, a compound (DU03) known to retard the nucleation phase of amyloidogenesis was used for comparision (Kapkova. P. et al., Bioorg. Med. Chem., 2006, vol. 14:472-478).

AB: Amyloid β-Protein (1-40) trifluoroacetate salt was obtained from Bachem (Switzerland, Lot 1012093; Lot 9004754). The peptide was stored at -20⁰C, as recommended by the manufacturer. It was dissolved in hexafiuoroisopropanol (HFIP) at 20mg/ml. This solution was kept at room temperature until the peptide was completely dissolved (30 min to Ih). The HFIP was removed under a stream of nitrogen until a clear film remained in the test tube. The residue was then dissolved in DMSO to obtain a 2 mM stock solution, which was subsequently stored frozen at -20⁰C for maximum three days. (Goldsbury, C. et al., J Struct. Biol. 2000, vol. 130: 217-231).

Solid phase synthesis of HHQKLVFF AED: The shortened peptide was synthesized by solid- phase peptide synthesis on a Milligen 9050 PepSynthesiser using standard Fmoc protocol. Synthesis was performed in DMF/DCM (60:40) with DIC/HOBt activation starting from Fmoc-Asp(OtBu)- Wang-resin. The peptide was cleaved from the resin by treatment with TFA/H2O/TIS (95/2.5/2.5). The following side chain protecting groups were used during the automated synthesis: tert -butyl ester (GIu), tert-butyloxycarbonyl (Lys) and trityl (GIn, His). The product substrate as TFA salt was purified by HPLC (H2O + 0.1% TFA; acetonitril (Merck); LiChroCART® 125-4.6 HPLC-Cartridge (LiChrospher® 100, RP 18 (5 μm, endcapped), (Merck); pressure: 143 bar; flow-rate: 1.0ml; temperature: 23°C; eluent gradient: acetonitril/water +0.1% TFA 20 - 80% in 40 min. The peptide was stored at -20⁰C. For each test a fresh solution of 2 mM in DMSO was prepared.

Thioflavin T (ThT) fibril formation assay: A 2 mM stock solution of Aβ and Aβ peptide (HHQKLVFFAED), respectively, in DMSO was diluted in buffer (25 mM NaH₂PO₄, 120 mM NaCl, 3 μM thioflavin T, 0.02% NaN₃ and a final pH of 7.4) to reach a final peptide concentration of 100 μM. (LeVine 3rd, H., Protein Sci. 1993. vol. 2, 404-410). Inhibitors were added to reach concentrations between 0.005 mM and 0.5 mM. Incubations were performed at 37°C on 96- well fluorescence rnicrotiler plates (Nunc GmbH, Wiesbaden, Germany). The negative control contained no inhibitor. The fluorescence was measured (excitation wavelength 450 nm. emission wavelength 482 ran) on a Cary Eclipse fluorescence spectrophotometer (Varian, Darmstadt, Germany).

Thioflavin T (ThT) fibril destruction assay: Compounds according to the invention were subjected to a modified test system, where fibril destruction can be measured. A 2 mM stock solution of the peptide HHQKL VFF AED in DMSO was diluted in buffer (25 mM NaH₂PO₄. 120 mM NaCl, 3 μM thioflavin T, 0.02% NaN₃ and a final pH of 7.4) to reach a final peptide concentration of 100 μM. Incubations were performed on 96-well fluorescence microtiter plates (Nunc GmbH, Wiesbaden, Germany) for 10 h. Inhibitors were added to reach concentrations between 0.005 and 0.5 mM. The fluorescence was measured (excitation wavelength 450 nm, emission wavelength 482 nm) on a Cary Eclipse fluorescence spectrophotometer (Varian, Darmstadt, Germany).

The compound of Example 3 exhibited fibril formation inhibition in the assay with Aβ40 (see Figure IA). The compounds of Examples 1 to 4 exhibited fibril formation inhibition in the assay with Aβ peptide (HHQKLVFF AED) (see Figures IB, 1C and ID). For the compounds of Examples 1 to 4, a concentration-dependent destruction of fibrils was observed, as representatively shown for the compound of Example 3 (see Figure IE).

Claims

Claims

1. A pharmaceutical composition comprising a compound of the formula (I)

(I)

wherein:

L¹ is selected from a covalent bond, optionally substituted C_1-12 alkylene, optionally substituted C₂.₁₂ alkenylene or optionally substituted C_2-12 alkynylene, wherein said optionally substituted Ci_-O alkylene, said optionally substituted C_2-12 alkenylene or said optionally substituted €₂-₁₂ alkynylene is optionally interrupted by one or more groups independently selected from -0-, -S-, -CO-, -NH-, -NH-CO- or -CO-NH- , and further wherein said optionally substituted C_1-12 alkylene, said optionally substituted C_2-J2 alkenylene or said optionally substituted C_2-12 alkynylene is optionally substituted with one or more groups independently selected from hydroxyl, C ₁.₄ alkoxy, C_1-4 alkylamino, (Ci_-4 alkyl)(C_1-4 alkyl)amino or halogen;

L² is selected from a covalent bond, optionally substituted C_1-12 alkylene, optionally substituted C_2-12 alkenylene or optionally substituted C_2-12 alkynylene, wherein said optionally substituted C 142 alkylene, said optionally substituted C_2-12 alkenylene or said optionally substituted 0₂-₁₂ alkynylene is optionally interrupted by one or more groups independently selected from -O-, -S-, -CO-, -NH-, -NH-CO- or -CO-NH- , and further wherein said optionally substituted Ci_-I2 alkylene, said optionally substituted C_2-J2 alkenylene or said optionally substituted C_2-12 alkynylene is optionally substituted with one or more groups independently selected from hydroxyl, C _1-4 alkoxy, Cj_-4 alkylamino, (C_1-4 alkyl)(C_{1 -4} alkyl)amino or halogen; and A and B are each independently selected from optionally substituted aromatic 5- to 10- membered monocyclic or bicyclic ring systems, which optionally contain one or more ring heteroatoms, said heteroatoms being independently selected from N, O or S, and wherein said optionally substituted aromatic 5- to 10-membered monocyclic or bicyclic ring system is optionally substituted with one or more groups independently selected from halogen, Ci_-4 alkyl, C₂-₄ alkenyl, C_2-4 alkynyl, Cj_-4 halogenalkyl, C₂-₄ halogenalkenyl, C_2-4 halogenalkynyl, C_1-4 alkoxy, C_2-4 alkenyloxy, C_2-4 alkynyloxy, C₁. 4 halogenalkoxy, C_2-4 halogenalkenyloxy, C2-4 halogenalkynyloxy, cyano, nitro, Ci_-4 alkylthio, C2-4 alkenylthio or C₂-₄ alkynylthio;

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

2. The pharmaceutical composition of claim 1, wherein A and B are each independently selected from optionally substituted aromatic 5- or 6-membered monocyclic ring systems, which optionally contain one or more ring heteroatoms, said heteroatoms being independently selected from N, O or S.

3. The pharmaceutical composition of claim 1 or 2, wherein A is optionally substituted phenyl.

4. The pharmaceutical composition of any of claims 1 to 3, wherein B is optionally substituted phenyl.

5. The pharmaceutical composition of any of claims 1 to 4, wherein L¹ is a covalent bond.

6. The pharmaceutical composition of any of claims 1 to 5, wherein h" is optionally substituted C₁.₄ alkylene.

7. The pharmaceutical composition of any of claims 1 to 6, wherein the compound of formula (I) has one of the following formulae (i) to (iv):

(iii) (iv)

8. The pharmaceutical composition of any of claims 1 to 7 further comprising a pharmaceutically acceptable excipient.

9. The pharmaceutical composition of any of claims 1 to 8 for use in the treatment or prevention of a neurodegenerative disease/disorder.

10. A method of treating or preventing a neurodegenerative disease/disorder, the method comprising the administration of the pharmaceutical composition of any of claims 1 to 8 to a subject in need of such a treatment or prevention.

11. The pharmaceutical composition of claim 9 or the method of claim 10, wherein said neurodegenerative disease/disorder is selected from Alzheimer's disease, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis). Creutzfeldt- Jacob disease, Huntington's disease, HIV-related dementia, Lewis body dementia or motor neuropathy.

12. The pharmaceutical composition of any of claims 1 to 9 or 11 or the method of claim 10 or 11 , whereby said pharmaceutical composition is to be administered orally, topically, parenterally, intracisternally, intravaginally, rectally, pulmonarily, gastrointestinally, intrauterinally, subcutaneously, intraocularly or opthalmically.

13. The method of any of claims 10 to 12, wherein said subject is a human.