WO2005110963A1

WO2005110963A1 - (biphenyl-3-yl)-carboxylic acids and derivatives thereof and their use in therapy

Info

Publication number: WO2005110963A1
Application number: PCT/EP2005/005444
Authority: WO
Inventors: Nigel Ramsden; Francis Wilson; Alison Reid; Valerie Reader; Warren Miller; Richard John Harrison; Mihiro Sunose; Remedios Hernandez-Perni; Jeremy Major; Cyrille Boussard; Kathryn Smelt; Jess Taylor; Adeline Leformal; Andrew Cansfield; Svenja Burckhardt
Original assignee: Cellzome Ag
Priority date: 2004-05-19
Filing date: 2005-05-19
Publication date: 2005-11-24

Abstract

The present invention relates to compounds having the general formula (I) with the definitions of RI-R3 given below, and/or a salt or ester thereof. Furthermore the invention relates to the use of said compounds as a medicament, preferably for the treatment of Alzheimer's disease and their use for the modulation of g-secretase activity.

Description

(Biphenyl-3-yl)-carboxylic acids and derivatives thereof and their Use in Therapy

The present invention relates to compounds having the general formula (I) with the definitions of R_Ϊ-R₃ given below, and/or a salt or ester thereof. Furthermore, the invention relates to the use of said compounds as a medicament, preferably for the treatment of Alzheimer's disease and their use for the modulation of γ- secretase activity.

Alzheimer's disease is the most common form of age-related neurodegenerative illness. It is primarily, but not exclusively, associated with aging and presents clinically not only by progressive loss of memory, cognition, reasoning and judgement, but also by emotional instability and gradually leads to profound mental deterioration and death.

The defining pathological hallmarks of Alzheimer's disease are the presence of neurofibrillary tangles and amyloid plaques in the brain, which are also thought to play a central role in the pathogenesis of the disease.

These plaques mainly consist of peptides formed as cleavage products of the amyloid precursor protein (APP), a 695 amino acid protein, whose function so far has only been the subject of various hypotheses.

APP is processed in two steps; a first step (catalyzed by β-secretase) gives rise to a secreted peptide and a membrane-bound C99-fragment.

In the second step C99 is a substrate for a proteolytic activity, mediated by γ-secretase, resulting, inter alia, in the production of peptides in the range of 37-42 residues. The amount of the longer isoform, AB42, is selectively increased in patients carrying certain mutations in a particular protein (presenilin), and these mutations have been correlated with early-onset familial Alzheimer's disease.

Therefore, AJJ42 is believed by many to be the main culprit of the pathogenesis of Alzheimer's disease.

It has now become clear that the γ-secretase activity cannot be ascribed to a single particular protein, but is in fact associated with an assembly of different proteins comprising Aphl, Nicastrin, Presenilin and Pen-2 (reviewed by De Strooper (2003) Neuron 38, 9)

Thus, although the molecular mechanism of the cleavage-step has remained elusive until present, the γ-secretase-complex has become one of the prime targets in the search for compounds for the treatment of Alzheimer's disease.

Other hints in the search for novel treatments came from epidemiological studies, an example being the finding that the uptake of certain non-steroidal anπ^inflammatory drugs ("NSAIDs") seems to correlate with a reduced risk of developing Alzheimer's disease (Akiyama et al (2000) Neurobiol. Aging 21, 383; McGeer et al (1996) Neurology 47, 425; Rogers et al (1993) Neurology 43, 1609; Anthony et al (2004) Neurology 54, 2066; Stewart et al (1997) Neurology 48, 626; In't Veld et al (1999) Neurobiol. Aging 19, 607).

Indeed, this finding was recently supported by biochemical studies in which an effect of certain NSAIDs on γ-secretase was shown (Weggen et al (2001) Nature 414, 6860, 212; Morihara et al (2002) J. Neurochem. 4, 1009; Eriksen (2003) J. Clin. Invest. 112 , 440.

A development of further compounds showing a similar effect has been hampered so far by a lack of understanding of the molecular mechanism of the described effects. Thus, there is a strong need for novel compounds which modulate γ-secretase activity thereby opening new avenues for the treatment of Alzheimer's disease. The object of the present invention is to provide such compounds.

The object is achieved by a compound having the general formula (I)

wherein R_1? R₂ are independently of each other selected from the group consisting of H; alkyl selected from the group CH₃, C₂Hs, i-C₃H , n-C₃H₇, i-0^₉, n-G!^, 860-0 11₉, tert- C H₉; alkenyl selected from C₂H₃, i-C₃Hs, n-C₃H₅, n-C H , i-C^, sec-C,₄H₇; and wherein the alkyl and alkenyl groups can carry one or more substituents from the group: F, Cl, Br, I, CF₃ and saturated, unsaturated or aromatic 3- to 7-membered cyclic groups optionally having one or more heteroatoms from the group N, S or O, which heteroatom may be identical or different if more than one heteroatom is present, in the ring; or R_t and R₂ being part of a ring, either saturated or unsaturated, having 3 to 5 C-atoms, and which may contain in the ring one or more heteroatoms from the group N, S or O, and which heteroatom may be identical or different if more than one heteroatom is present;

and wherein R₃ is selected from the group consisting of F, Cl, Br, I, substituted and unsubstituted alkyl from the group CH₃, C₂H₅, i-C₃H₇, n-C₃H₇, i-C₄H₉, n-CφHg, sec-C^, tert-G_tHg and substituted and unsubstituted alkoxy, from the group OCH₃, OC₂H₅, Oi- C₃H₇, On-C₃H , Oi-C₄H9, On-Gflg, Osec-C₄H₉, Otert-C₄H₉ and wherein the substituents of both groups are selected from F, Cl, Br, I; preferably R₃ being selected from the group consisting of CF₃, OCF₃, F, Cl, Br, I, more preferably R₃ being selected from the group consisting of CF₃, OCF₃;

and/or a salt or ester thereof.

The term "substituted" as used herein includes both part and full substitution. Substituents can be either saturated or unsaturated.

Esters are those according to formula (I) in which H of the carboxyl group is replaced by an organic residue R₄. Suitable organic residues are known to a person skilled in the art. Preferred R_t include the following:

An unsubstituted or at least monosubstituted alkyl, preferably a C₁-C₁₀ alkyl, an alkenyl, preferably C₂-C₁o-alkenyl, an alkynyl, preferably C₃-C₁₀-alkynyl, and an unsubstituted or at least monosubstituted ring, either saturated or unsaturated, having 3 to 5 C-atoms, and which may contain in the ring one or more heteroatoms from the group N, S or O, and which heteroatom may be identical or different if more than one heteroatom is present. Said substituents being selected from the group consisting of halogen, alkyl, alkenyl, alkynyl,N, S, O, carboxyl, sulphonyl, and the like and which can be further substituted.

In a preferred embodiment, the invention relates to a compound having the general formula (I) wherein

R_t and R₂ being H; or Ri being H and R₂ being CH₃, C₂H₅, nC₃H₇, i-C₃H ₎ nC₄H_9; i-C Hg, sec-CφHg or tert-QHg; and wherein all the named groups can carry one or more substituents from the group C₆H₅, CF₃ or cyclopropyl; or R₁ and R₂ being CH₃; or Ri and R₂ each being a methylene group forming, together with the C-atom in α position to the carboxyl group, a cyclopropyl ring.

and wherein R₃ is selected from the group consisting of F, Cl, Br, I, CrC₄-alkyl or CrC₄- alkoxy, substituted partly or fully by F, Cl, Br, I; preferably R being selected from the group consisting of CF₃, OCF₃, F, Cl, Br, I, more preferably R₃ being selected from the group consisting of CF₃, OCF₃.

and or a salt or ester thereof.

In a more preferred embodiment, the invention relates to a compound having the general formula (I) wherein R₃ is CF or OCF₃; and the substituents Rι and R₂ have the general or preferred meanings as defined above, with the meanings having been defined as being preferred for R_\ and R₂ also being preferred in the present embodiment,

and or a salt or ester thereof.

In an even more preferred embodiment, the invention relates to a compound having the formula (II) (4'-Trifluoromethyl-biphenyl-3-yl)-acetic acid

or the compound according to the formula (III) 2-(4'-Trifluoromethyl-biphenyl-3-yι)- propionic acid

or the compound according to the formula (IV) 2-(4'-Trifluoromethoxy-biphenyl-3-yι)- propionic acid

or the compound according to the formula (V) 2-(4'-Chloro-biphenyl-3-yl)-propionic acid

or the compound according to the formula (VI) l-(4'-Trifluoromethyl-biphenyl-3-yi)- cyclopropanecarboxylic acid

or the compound according to the formula (VTf) 2-Methyl-2-(4'-trifluoromethyl-biphenyl- 3-yl)-propionic acid

or the compound according to the formula (VEII) 2-(4'-Trifluoromethyl-biphenyl-3-yl)- pentanoic acid

or the compound according to the formula (DC) 4-Methyl-2-(4'-Trifluoromethyl-biphenyl- 3-yl)- pentanoic acid

or the compound according to the formula (X) 3-Methyl-2-(4'-Trifluoromethyl-biphenyl-3- yl)- butanoic acid

or the compound according to the formula (XI) 2-(4'-Trifluoromethyl-biρhenyl-3-yl)- butanoic acid

or the compound according to the formula (XII) 3-Phenyl-2-(4'-trifluoromethyl-biphenyl- 3-yl)-propionic acid

or the compound according to the formula (XIII) 5,5,5-Trifluoro-2-(4'-trifluoromethyl- biρhenyl-3-yl)-pentanoic acid;

or the compound according to the formula (XIV) 4-Phenyl-2-(4'-trifluorometh.yl-biphenyl- 3-yl)-butyric acid;

or the compound ((XV) 3-Cyclopropyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-proρionic acid

and/or a salt or ester thereof.

Some of the compounds of the inventions and/or salts or esters thereof, will exist in different stereoisomeric forms. All of these forms are subjects of the invention. In case R- or S- enantiomers exist, the S-enantiomer is preferred.

Furthermore, the invention relates to any of the compounds according to the invention and/or a pharmaceutically acceptable salt or ester thereof for use as a medicament. Described below are exemplary salts of the compounds according to the invention which are included herein. The list of the different salts stated below is not meant to be complete and limiting.

Compounds according to the invention which contain one or more acidic groups can be used according to the invention, e.g. as their alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, e.g. emylamine, emanolamine, trie anolarnine or amino acids.

Compounds according to the invention which contain one or more basic groups, i.e. groups which can be protonated, can be used according to the invention in the form of their addition salts with inorganic or organic acids.

Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to a person skilled in the art.

The term "pharmaceutically acceptable" means approved by a regulatory agency such as the EMEA (Europe) and/or the FDA (US) and/or any other national regulatory agency for use in animals, preferably in humans.

Compounds according to the invention which contain several basic groups can simultaneously form different salts. If a compound according to the invention simultaneously contains acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines.

The respective salts of the compounds according to the invention can be obtained by customary methods which are known to the person skilled in the art, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.

Furthermore, the invention includes all salts of the compounds according to the invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts or which might be suitable for studying γ-secretase modulating activity of a compound according of the invention in any suitable manner, such as any suitable in vitro assay.

The present invention furthermore includes all solvates of the compounds according to the invention.

The present invention furthermore includes derivatives/prodrugs (including the salts thereof) of the compounds according to the invention which contain physiologically tolerable and cleavable groups and which are metabohzed in animals, preferably mammals, most preferably humans into a compound according to the invention.

The present invention furthermore includes the metabolites of the compounds according to the invention. The term "metabolites" refers to all molecules derived from any of the compounds according to the invention in a cell or organism, preferably mammal.

Preferably the term "metabolites" relates to molecules which differ from any molecule which is present in any such cell or organism under physiological conditions.

The structure of the metabolites of the compounds according to the invention will be obvious to any person skilled in the art, using the various appropriate methods.

The compounds according to general formula (I) can be prepared according to methods published in the literature or, respectively, analogous methods.

Methods for synthesis of the compounds are described e.g., in Houben-Weyl, Methoden der Organischen Chemie (Methods of Organic Chemistry), Thieme-Verlag, Stuttgart, and Organic Reactions, John Wiley & Sons, New York. Depending on the circumstances of the individual case, in order to avoid side reactions during the synthesis of a compound of the general formula (I), it can be necessary or advantageous to temporarily block functional groups by introducing protective groups and to deprotect them in a later stage of the synthesis, or to introduce functional groups in the form of precursor groups and at a later stage to convert them into the desired functional groups. Suitable synthetic strategies, protective groups and precursor groups are known to the person skilled in the art.

If desired, the compounds of the formula (I) can be purified by customary purification procedures, for example by recrystallization or chromatography. The starting materials for the preparation of the compounds of the formula (I) are commercially available or can be prepared according to or analogously to literature procedures.

Detailed protocols for the preparation of (4'-trifluoromethyl-biphenyl-3-yl)-acetic acid (i.e. the compound having the formula II); (4'-trifluoromethyl-biphenyl-3-yl)-acetic acid methyl ester; 2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid methyl ester ; 2-(4'- trifluoromethyl-biphenyl-3-yl)-propionic acid (i.e. the compound having the formula (III)), 2-(4'-trifluoromethoxy-biphenyl-3-yl)-propionic acid (i.e. the compound having the formula (IV)) ; 2-(4^l-chloro-biphenyl-3-yl)-propionic acid (i.e. the compound having the formula (V)); l-(4'-Trifluoromethyl-biphenyl-3-yl)-cyclopropanecarboxylic acid (i.e. the compound having the formula VI)); 2-Methyl-2-(4'-trifluoromethyl-biphenyl-3-yl)- propionic acid (i.e. the compound having the formula (VII); 2-(4'-Trifluoromethyl- biphenyl-3-yl)-pentanoic acid (i.e the compound having the formula (VIII)); 4-Methyl-2- (4'-Trifluoromethyl-biphenyl-3-yl)- pentanoic acid (the compound having the formula (rX)) ; 3 -Methyl-2-(4 '-Trifluoromethyl-biphenyl-3 -yl)- butanoic acid (i. e the compound having the formula (X)); 2-(4'-Trifluoromethyl-biphenyl-3-yl)-butyric acid(i.e the compound having the formula (XI)), 3-Phenyl-2-(4'-trifluoromethyl-biphenyl-3-yl)- propionic acid(i.e the compound having the formula (XH)), 5,5,5-Trifluoro-2-(4'- trifluoromethyl-biphenyl-3-yl)-pentanoic acid(i.e the compound having the formula (XIII)), 4-Phenyl-2-(4^,-trifluoromethyl-biphenyl-3-yl)-butyric acid(i.e the compound having the formula (XIV)), 3-Cyclopropyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid(i.e the compound having the formula (XV))_are given further below. These can serve as a basis for the preparation of the other compounds according to the invention by several methods well known to the person skilled in the art.

In particular the compounds according to the invention are suitable for the treatment of Alzheimer's disease.

Thus, the invention further relates to the use of a compound according to the invention and/or a pharmaceutically acceptable salt or ester thereof for use as a medicament for the treatment of Alzheimer's disease.

Details relating to said use are further disclosed below.

The compounds can be used for modulation of γ-secretase activity. Thus, the invention further relates to the use of a compound according to the invention for the modulation of γ-secretase activity.

As used herein, the term "modulation of γ-secretase activity" refers to an effect on the processing of APP by the γ-secretase-complex. Preferably it refers to an effect in which the overall rate of processing of APP remains essentially as without the application of said compounds, but in which the relative quantities of the processed products are changed, more preferably in such a way that the amount of the AJ342-peptide produced is reduced.

It has been previously shown, that the γ-secretase complex is also involved in the processing of the Notch-protein (reviewed e.g., in Schweisguth F (2004), Curr Biol 14: R129-38).

Notch is a signaling protein which plays a crucial role in developmental processes (reviewed e.g., in Schweisguth F (2004), Curr Biol 14: R129-38). Particularly with respect to the use of said compounds for the modulation of γ-secretase activity in therapy, it seems advantageous not to interfere with the Notch-processing activity of the γ-secretase activity in order to avoid putative undesired side-effects. Thus, compounds are preferred which do not show an effect on the Notch-processing activity of the γ-secretase-complex. Within the meaning of the invention, "effect on the Notch processing activity" includes both an inhibition or an activiation of the Notch-processing activity by a certain factor. A compound is defined as not having an effect on the Notch processing activity, if said factor is smaller than 20, preferably smaller than 10, more preferably smaller than 5, most preferably smaller than 2 in the respective assay as described in Shimizu et al (2000). Mol. Cell. Biol; Vol. 20: 6913-6922 at a concentration of 30 μM.

Such a modulation can be carried out, e.g. in animals such as mammals. Exemplary mammals are mice, rats, guinea pigs, monkeys, dogs, cats. The modulation can also be carried out in humans. In a particular embodiment of the invention, said modulation is performed in vitro or in cell culture.

As known to the person skilled in the art, several in vitro and cell culture assays are available.

An example for such an assay is described in WO-03/008635.

Concentrations of the various products of the cleavage (the AB-peptides) can be determined by various methods known to a person skilled in the art. Examples for such methods include determination of the peptides by mass-spectrometry or detection by antibodies.

Suitable antibodies are available for example from The Genetics Company, Inc.,

Switzerland. Further information are disclosed for example in N. Ida et al. The Journal of Biological

Chemistry, Vol. 271, No.37, pp. 22908 - 22914, 1996 and Jensen, M., et al., MoLMed. (6):

291-302,2000. Antibody-based kits are also available from Innogenetics, Belgium.

Cells which can be employed in such assays include cells which physiologically express the γ-secretase complex and cells which transiently or stably express some or all interactors of the γ-secretase complex.

Numerous available cell lines suitable for such assays are known to the skilled person.

Cells and cell lines of neuronal or glial origin are particularly suitable. Furthermore, cells and tissues of the brain as well as homogenates and membrane preparations thereof may be used.

Such assays might be carried out for example, to study the effect of the compounds according to the invention in different experimental conditions and configurations.

Furthermore, such assays might be carried out as part of functional studies on the γ- secretase complex. For example, either one or more interactors (either in their wild-type form or carrying certain mutations and/or modifications) of the γ-secretase complex of an animal, preferably a mammal, more preferably humans, might be expressed in certain cell lines and the effect of the compounds according to the invention might be studied.

Mutated forms of the interactor(s) used can either be mutated forms which have been described in certain animals, preferably mammals, more preferably humans or mutated forms which have not previously been described in said animals.

Modifications of the interactors of the γ-secretase complex include both any physiological modification of said interactors and other modifications which have been described as modifications of proteins in a biological system.

Examples of such modifications include, but are not limited to glycosylation, phosphorylation, prenylation, myristylation, farnesylation.

Furthermore, the compounds according to the invention can be used for the preparation of a medicament for the modulation of γ-secretase activity.

The invention further relates to the use of said compounds for the preparation of a medicament for the modulation of γ-secretase activity.

The activity of the γ-secretase can be modulated in different ways, i.e. resulting in different profiles of the various AB-peptides.

Uses of a compound for the preparation of a medicament for the modulation of γ-secretase activity resulting in a decrease in the relative amount of produced AJ342-protein are preferred.

Respective dosages, routes of administration, formulations etc are disclosed further below. The invention further relates to the use of the compounds according to the invention for the preparation of a medicament for the treatment of a disease associated with an elevated level of AJ542-production.

As used herein, the term "treatment" is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting, or stopping of the progression of a disease, but does not necessarily indicate a total elimination of all symptoms.

As used herein, the term "elevated level of AB42-production" refers to a condition in which the rate of production of AB42-peptide is increased due to an overall increase in the processing of APP or, preferably, it refers to a condition in which the production of the AB42 peptide is increased due to a modification of the APP-processing profile in comparison to the wild-type/non-pathological situation.

As outlined above, such an elevated AB42-level is a hallmark of patients developing or suffering from Alzheimer's disease.

In a preferred embodiment, the invention relates to the use of the compounds according to the invention for the preparation of a medicament for the treatment of Alzheimer's disease.

Further details regarding said use are disclosed further below.

Furthermore the invention relates to a composition comprising a compound according to the invention in a mixture with an inert carrier.

In a preferred embodiment, the invention relates to a composition comprising a compound according to the invention in a mixture with an inert carrier, where said inert carrier is a pharmaceutical carrier.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

Furthermore, the invention relates to a method for the preparation of a compound according to any of claims 1-4 comprising the steps of coupling phenyl acetic acid derivative, optionally said derivative being protected, with an appropriate aromatic compound and optional further functionalisation and deprotection of the thus-obtained biphenyl compound.

Furthermore, the invention relates to a method for the preparation of a medicament comprising the steps of: a) preparing a compound according to the invention b) formulation of a medicament containing said compound. The compounds according to the invention and their pharmaceutically acceptable salts, optionally in combination with other pharmaceutically active compounds suitable to treat or prevent Alzheimer's disease such as Aricept (Eisai), Donepezil (Pfizer), Cognex (Warner-Lambert), Tacrine (Warner-Lambert), Axura (Merz), Memantine (Merz) or with any other of the drugs known to a person skilled in the art suitable to treat or prevent Alzheimer's disease, can be administered to animals, preferably to mammals, and in particular humans, as pharmaceuticals by themselves, in mixtures with one anther or in the form of pharmaceutical preparations.

Various delivery systems are known and can be used to administer a compound of the invention for the treatment of Alzheimer's disease/for the modulation of the γ-secretase activity, e.g., encapsulation in liposomes, microparticles, and microcapsules: If not delivered directly to the central nervous system, preferably the brain, it is advantageous to select and or modify methods of administration in such a way as to allow the pharmaceutical compound to cross the blood-brain barrier.

Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.

The compounds may be administered by any convenient route, for example by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings and may be administered together with other biologically active agents.

Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In another embodiment, the Therapeutic can be delivered in a vesicle, in particular a liposome (Langer, 1990, Science 249:1527-1533; Treat et al., 1989, In: Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler, eds., Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet another embodiment, the Therapeutic can be delivered via a controlled release system. In one embodiment, a pump may be used (Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240; Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl. J. Med. 321:574-579). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise, eds., CRC Press, Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball, eds., Wiley, New York, 1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228: 190-192; During et al., 1989, Ann. Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (e.g., Goodson, 1984, In: Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

In order to select an appropriate way of administration, the person skilled in the art will also consider routes of adrninistration which have been selected for other known Anti- Alzheimer-drugs.

For example, Aricept/Donepezil and Cognex/Tacrine (all acetylcholmesterase-inhibitors) are being taken orally, Axura/Memantine (an NMDA-receptor antagonist) has been launched both as tablets/liquid and as an i.v. -solution.

Furthermore, the skilled person in the art will take account the available data with respect to routes of administration of other members of the NSAID-family in clinical trials and other studies investigating their effect on Alzheimer's disease.

In order to select the appropriate dosage, the person skilled in the art will choose a dosage which has been shown to be not toxic in preclinical and/or clinical studies and which can be in accordance with the values given beforehand, or which may deviate from these. The precise dose to be employed in the formulation will also depend on the route of adrriinistration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

An exemplary animal model is the transgenic mouse strain "Tg2576" containing an APP695-form with the double mutation KM670/671NL. For reference see e.g., patent US5877399 and Hsiao, K et al (1996), Science 274: 99-102 and also Kawarabayahsi T (2001), J. Neurosci 21: 372-81; Frautschy et al (1998), Am J Pathol 152: 307-17, zarry et al (1997) J Neuropathol Exp Neurol 56: 965-73; Lehman et al (2003) Neurobiol Aging 24: 645-653.

Substantial data from several studies are available to the skilled person in the art which are instructive to the skilled person to select the appropriate dosage for the chosen therapeutic regimen.

Numerous studies have been published in which the effects of molecules on the γ-secretase activity are described. Exemplary studies are Lim et al, Neurobiol Aging. 2001 Nov- Dec;22(6):983-91, Lim et al, J Neurosci. 2000 Aug 1;20(15):5709-14; Weggen et al. Nature. 2001 Nov 8;414(6860):212-6; Eriksen et al. J Clin Invest. 2003 Aug;112(3):440-9; Yan et al. J Neurosci. 2003 Aug 20;23(20):7504-9; Lanz et al. Abstract. Meeting Soc f. Neurosci, 2003; Friedman et al. Abstract. Meeting Soc f. Neurosci, 2003.

General

All reactions were carried out under inert atmosphere. NMR spectra were obtained on a Bruker dpx400. LCMS was carried out on an Agilent 1100 using a zorbax SBC-18, 4.6mmxl50mm column. Column flow was lml/min and solvents used were water and acetonitrile (0.1%TFA) with an injection volume of lOul. Wavelengths were 254 and 210nm. Methods are described below:

Abbreviations

EXAMPLES:

Example 1: Preparation of (4^t-Trifluoromethyl-biphenyl-3-yl)-acetic acid (II)

To a mixture of 4-trifluoromethylphenylboronic acid (0.09g, 0.49mmol), 3- bromophenylacetic acid (O.lg, 0.46mmol) and sodium carbonate (0.15g, 1.4mmol) in water (3ml) was added a catalytic amount of palladium acetate. This mixture was irradiated in a CEM microwave at 120°C for 9 min. The mixture was then filtered through a plug of celite then acidified with HC1 (2M aq) to pH 2. The aqueous layer was then extracted with DCM (x3) and the organics were dried (MgSO ) and concentrated to afford (4'- trifluoromethyl-biphenyl-3-yl)-acetic acid as a white solid (0.1 lg, 0.39mmol) in 85% yield. 1H NMR (CDC1₃) δ 7.69 (4H, s), 7.53 (2H, m), 7.45 (1H), 7.34 (1H), 3.75 (2H, s); LCMS method (A), MH^" (279), RT = 4.1min.

Example 2: Preparation of 2-(4'-Triflttoromethyl-biphenyI-3-yI)-propionic acid (IIP

Preparation of (4'-trifluoromemyl-biphenyl-3-v0-acetic acid methyl ester

To a solution of the 4'-trifluoromethyl-biphenyl-3-yl)-acetic acid prepared as in example 1 (0.500g, 2.54mmol) in methanol (14ml) was added a catalytic amount of sulphuric acid. The reaction mixture was stirred overnight and then concentrated. The oil was diluted with EtOAc (45ml), quenched by NaHCO (sat aq) until the gas release stopped then washed with brine (x2) and dried (MgSO ). The organics were concentrated and the yellow oil was purified by flash chromatography (EtOAc: petroleum ether) to afford (4'-trifluoromethyl- biphenyl-3-yl)-acetic acid methyl ester as a colourless oil (0.55g, 2.43mmol) in 96% yield. 1H NMR (CDCls) δ 7.70 (s, 4H), 7.50 (m, 2H), 7.40 (t, 1H), 7.30 (m, 1H), 3.72 (s, 3H), 3.71 (s, 2H); LCMS method (B), (MH-) 257, RT = 12.0min.

Preparation of 2-f4'-Trifluoromemyl-biphenyl-3-yl)-propionic acid methyl ester A solution of LDA in hexanes (1.89ml, 3.4mmol, 1.8M) was added dropwise to a solution of (4'-Trifluoromethyl-biphenyl-3-yl)-acetic acid methyl ester (0.5g, 1.7mmol) in THF (8mL) at -78°C. After 30 min methyl iodide (0.32ml, 5.1mmol) in THF (2ml) was added and the mixture slowly allowed to warm to room temperature. Upon completion of reaction (3h) the mixture was quenched by the addition of sat. NE C1. This was then extracted with DCM (x3), washed with HCl (0.5N, 15ml) and the organics were dried (MgSO₄) and concentrated in vacuo to afford a brown oil. The oil was purified by flash column chromatography (EtOAc: petroleum ether) to afford 2-(4'-trifluoromethyl-biphenyl-3-yl)- propionic acid methyl ester (0.45g, 1.47mmol) in 86% yield. 1H NMR (CDC1₃) δ 7.70 (s, 4H), 7.50 (m, 2H), 7.44 (t, IH), 7.35 (dm, IH), 3.80 (q, IH), 3.70 (s, 3H), 1.56 (d, 3H); LCMS method (B), (MH^") 257, RT = 12.0min.

Preparation of 2-(4'-trifluoromethyl-biphenyl-3-yl^')-propionic acid

2-(4'-Trifluoromethyl-biphenyl-3-yl)-propionic acid methyl ester (1.15g, 3.73mmol) was dissolved in THF (11ml) and water (11ml) and cooled to 0°C. Lithium hydroxide (0.18g, 7.46mmol) was dissolved in 2ml THF and added dropwise to the solution. The mixture was allowed to stir for lh then warmed to room temperature for 16h. The mixture was acidified by addition of HCl (IM) and extracted with DCM. The organics were washed with water dried (MgSO4) and concentrated to afford 2-(4^,-trifluoromethyl-biphenyl-3-yl)- propionic acid (1.03g, 3.49mmol) in 94% yield. 1H NMR (CDC1₃) δ 7.70 (s, 4H), 7.50 (m, 2H), 7.50 (t, IH), 7.40 (d, IH), 3.80 (quartet IH), 1.50 (d, 3H); LCMS method (B), (MH-) 293, RT = 11.0min.

Example 3: Preparation of 2-(4^t-Trifluoromethoxy-biphenyl-3-yl)-propionic acid (TV)

Procedure as for Example 2 replacing 4-trifluoromethylphenylboronic acid with 4- trifluoromethoxyphenylboronic acid. 1H NMR (CDC1₃) δ 7.57 (d, 2H), 7.49-7.38 (m, 3H), 7.33-7.26 (m, 3H), 3.81 (q, IH); 1.56 (d, 3H); LCMS method (B), (MH^") 309, RT = 11. lmin. Example 4: Preparation of 2-(4'-Chloro-biphenyl-3-vI)-propionic acid (V)

Procedure as for Example 2 replacing 4-Trifluoromethylphenylboronic acid with 4- chlorophenylboronic acid. 1H NMR (CDC1₃) δ 7.57 (d, 2H), 7.49-7.38 (m, 3H), 7.33-7.26 (m, 3H), 3.81 (q, IH); 1.56 (d, 3H); LCMS method (B), (MH-) 309, RT = l l.lmin.

Example 5: Preparation of l-(4'-Trifluoromethyl-biphenvI-3-yl)- cyclopropanecarboxylic acid (VI)

(4'-Trifluoromethyl-biphenyl-3-yl)-acetic acid methyl ester as prepared in example 2 (0.085g, 0.29mmol) in DMF (1ml) was added dropwise to a mixture of 60% NaH in mineral oil (0.035g, 0.87mmol) in DMF (0.5ml) at 0°C. After 20min dibromoethane (0.03ml, 0.35mmol) was added and the mixture slowly allowed to warm to room temperature. After 16h starting material was consumed and the mixture was quenched by the addition of sat. NaHCO₃. The rnixture was then extracted with EtOAc and the organics were dried (MgSO₄) and concentrated in vacuo to afford l-(4'-trifluoromethyl-biphenyl-3- yl)-cyclopropanecarboxylic acid methyl ester. This brown oil was purified by flash column chromatography (50% EtOAc:Petroleum Ether) to afford the desired colourless oil. This oil was dissolved in THF (2ml) then water (2ml) was added followed by lithium hydroxide (0.16g, 6.8mmol). The mixture was allowed to stir for 6h at room temperature then acidified by addition of HCl (2M aq) and extracted with EtOAc. The organics were dried and concentrated to afford l-(4'-Trifluoromethyl-biphenyl-3-yl)-cyclopropanecarboxylic acid which was purified by preparative HPLC to give the desired compound (6mg,

3%yield). 'H NMR^DCls) δ 7.30-7.26 (m, 3H), 7.18-7.14 (m, IH), 7.10-7.07 (m, IH), 7.01-6.97 (m, 2H), 6.84 (s, IH), 1.33-1.30 (s, 2H), 0.93-0.90 (s, 2H). LCMS method (A), (MH-) 305, RT = 4.2min.

Example 6: Preparation of 2-Methyl-2-(4'-trifluoromethyl-biphenyl-3-yI)-propionic acid (VIP 2-(4'-Trifluoromethyl-biphenyl-3-yl)-propionic acid methyl ester as prepared in example 2 (0.1 lg, 0.35mmol) in DMF (1ml) was added dropwise to a mixture of 60% NaH in mineral oil (0.028g, 0.7mrnol) in DMF (0.5ml) at 0°C. After 20min Methyl iodide (0.066ml, 1.06mmol) was added and the mixture slowly allowed to warm to room temperature. After 3h starting material was consumed and the mixture was quenched by the addition of sat. NH₄CI. The mixture was then extracted with EtOAc and the organics were dried (MgSO₄) and concentrated in vacuo to afford a brown oil. This was purified by flash column chromatography (10:1 Petrol: EtOAc) to give the desired 2-Methyl-2-(4'-trifluoromethyl- biphenyl-3-yl)-propionic acid methyl ester. The colourless oil was then heated to 100°C in NaOH (5M aq) for lh then cooled to room temperature. A white solid precipitated and was removed by filtration and dried to afford 2-Methyl-2-(4'-trifluoromethyl-biphenyl-3- yD-propionic acid (14mg, 7% yield). 1H NMR (CDC1₃) δ 7.81-7.78 (m, 4H), 7.69-7.67 (m, IH), 7.44-7.40 (m, 2H), 7.35-7.31 ( , IH), 1.39 (s, 6H); LCMS method (A), (Mff) 307, RT = 4.3min.

Example 7: Preparation of 2-(4'-Trifluoromethyl-biphenyl-3-yl)-pentanoic acid (VHP as racemate and in form of the enantiomers

(RS) 2-(4^,-Trifluoromethyl-biphenyl-3-yl)-pentanoic acid

Procedure as for Example 2 replacing methyl iodide with bromopropane. ^!H NMR (CDC1₃) δ 7.94 (m, 8H), 3.53 (t, IH), 2.05 (m, IH), 1.71 (m, IH), 1.35 (m, 2H); 0.95 (t, 3H); LCMS method (A), (M-H^") 321, RT = 4.5min.

(R)-2-(4'-trifiuoromethyl-biphenyl-3-yI)-pentanoic acid

Preparation of 2-(4'-Trifluoromethyl-biphenyl-3-yl)-acetic acid

To a mixture of 4-trifluoromethylphenylboronic acid (0.09g, 0.49mmol), 3- bromophenylacetic acid (O.lg, 0.46mmol) and sodium carbonate (0.15g, 1.4mmol) in water (3ml) was added a catalytic amount of palladium acetate. This mixture was irradiated in a CEM microwave at 120°C for 9 min. The mixture was then filtered through a plug of celite then acidified with HCl (2M aq) to pH 2. The aqueous layer was then extracted with DCM (x3) and the organics were dried (MgSO ) and concentrated to afford 2-(4'- trifluoromethyl-biphenyl-3-yl)-acetic acid as a white solid (0.1 lg, 0.39mmol) in 85% yield. 1H NMR (CDCI₃) δ 7.69 (4H, s), 7.53 (2H, m), 7.45 (IH), 7.34 (IH), 3.75 (2H, s); LCMS method (A), MH^" (279), RT = 4.1min.

(R)-2-(4^,-trifluoromethyl-biphenyl-3-yl)-pentanoic acid

2-(4'-tτifluoromethylbiphenyl-3-yl)-acetic acid underwent asymmetric alkylation by the method of Myers et al (1997) J. Am. Chem. Soc. Vol 119: 6496-6511 using R,R- pseudephedrine as a chiral auxiliary, 1-iodopropane as alkylating agent and sulphuric acid to remove the auxiliary, thus affording (R)-2-(4'-trifluoromethyl-biphenyl-3-yl)-pentanoic acid in 21% yield (3 steps) and >95% e.e. as measured by HPLC of the corresponding amide with (S)-α-memylbenzylamine. 1H NMR (CDCI₃) δ 7.94 (m, 8H), 3.53 (t, IH), 2.05 (m, IH), 1.71 (m, IH), 1.35 (m, 2H); 0.95 (t, 3H); LCMS method (A), (M-H^") 321, RT = 4.5min.

(S)-2-(4 '-trifluoromethyl-biphenyI-3-yl)-pentanoic acid

This can be prepared in an analogous fashion replacing_R,R-pseudephedrine by S,S- pseudephedrine Example 8: Preparation of 4-Methyl-2-(4'-Triflnoromethyl-biphenyl-3-yl)-pentanoic acid (IX)

Procedure as for Example 2 replacing methyl iodide with l-bromo-2-methyl-propane. 1H NMR (CDCI₃) δ 7.68 (m, 4H), 7.46 (m, 4H), 3.76 (t, IH), 2.02 (m, IH), 1.74 (m, IH), 1.54 (m, IH), 0.93 (d, 6H); LCMS method (A), (M-H-) 334.8, RT = 4.7min.

Example 9: Preparation of 2-(4'-TrifluoromethvI-biphenyl-3-yl)-3-Methyl-butanoic acid (X)

Procedure as for Example 2 replacing methyl iodide with 2-bromo-propane. 1H NMR (CDCI₃) δ 7.50 (m, 8H), 3.17 (d, IH), 2.33 (m, IH), 1.04 (d, 3H), 0.72 (d, 3H); LCMS method (A), (M-H-) 320.8, RT = 4.5min.

Example 10: Preparation of 2-(4^,-Trifluoromethyl-biphenyl-3-yl)-butyric acid (XT)

Procedure as for Example 2 replacing methyl iodide with ethyl iodide. 1H NMR (CDC1₃) δ 7.66-7.70 (m, 4H), 7.50-7.54 (m, 2H), 7.43 (t, IH), 7.37 (d, IH), 3.55 (t, IH), 2.13-2.20 (m, IH), 1.84-1.91 (m, IH), 0.95 (t, 3H); LCMS method (A), (M-H-) 320.8, RT = 4.3min.

Example 11: Preparation of 3-Phenyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid (X T)

Procedure as for Example 2 replacing methyl iodide with benzyl bromide. ^!H NMR (CDCI₃) δ 7.68 (d, 2H), 7.62 (d, 2H), 7.48-7.51 (m, 2H), 7.43 (t, IH), 7.37 (d, IH), 7.11- 7.26 (m, 5H), 3.95 (t, IH), 3.46 (dd, IH), 3.09 (dd, IH); LCMS method (B), (M-H-) 369, RT = 12.0min. Example 12: Preparation of 5_<5,5-Trifluoro-2-(4'-trifluoromethyl-biphenyl-3-yI)- pentanoic acid(XULl)

Procedure as for Example 2 replacing methyl iodide with 1,1,1 -trifluoro-4-iodo butane. 1H NMR (CDC1₃) δ 7.70 (d, 2H), 7.67 (d, 2H), 7.45-7.56 (m, 3H), 7.35 (d, IH), 3.71 (t, IH), 2.34-2.43 (m, IH), 1.99-2.18 (m, 3H); LCMS method (B), (M-H^") 375, RT = 11.7min.

Example 13: Preparation of 4-Phenyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-btttyric acid (XIV)

Procedure as for Example 2 replacing methyl iodide with 2-bromoethyl benzene. 1H NMR (CDCI₃) δ 7.66-7.70 (m, 4H), 7.52 (d, 2H), 7.45 (t, IH), 7.37 (d, IH), 7.26-7.30 (m, 2H), 7.20 (d, IH), 7.15 (d, 2H), 3.65 (t, IH), 2.63 (t, 2H), 2.43-2.53 (m, IH), 2.11-2.21 ( , IH); LCMS method (B), (M-H⁺) 383, RT = 12.5min.

Example 14: Preparation of 3-Cyclopropyl-2-(4'-trifluoromethyl-biphenyI-3-yl)- propionic acid (XV)

Procedure as for Example 2 replacing methyl iodide with bromomethyl cyclopropane. 1H NMR (CDCI₃) δ 7.66-7.70 (m, 4H), 7.56 (s, IH), 7.50 (d, IH), 7.38-7.45 (m, 2H), 3.78 (t, IH), 1.93-2.00 (m, IH), 1.78-1.85 (m, IH), 0.65-0.71 (m, IH), 0.40-0.44 (m, 2H), 0.04- 0.15 (m, 2H); LCMS method (B), (M-H⁺) 333, RT = 12.0min. Example 15: Screening of the compounds of the invention for γ-secretase-modulating activity

Screening was carried out using SKN neuroblastoma cells carrying the APP-"swedish mutation" (point mutation at 695 of APP) grown in DMEM/NUT-mix F12 (HAM) provided by Gibco (cat no. 31330-38) containing 5% Serum/Fe supplemented with 1% non-essential amino acids, 100 U/ml Pen/Strep.

Cells were grown to near confluency.

The screening was performed using the assay as described in Citron et al (1997), Nature

Medicine. Vol 3: 67-72

IC50- values of selected compounds of the invention on the γ-secretase activity. (For comparison, in addition IC50-data for R-Flurbiprofen used as a control are given in the bottom row)

Example 16: Determination of the effect the compounds according to the invention on cyclooxygenase-1 and cyclooxygenase -2 (COX-1 and COX-2)

Inhibition of Cox-1 and Cox-2 was determined using the Colorimetric Cox inhibitor screening assay provided by Cayman Chemical Company, Ann Arbor, MI, USA. (Cat. No. 760111) according to manufacturer's instructions.

Exemplary studies were carried out using 2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid, l-(4'-Trifluoromethyl-biphenyl-3-yl)-cyclopropanecarboxylic acid, 2-Methyl-2-(4'- trifluoromethyl-biphenyl-3 -yl)-propionic acid, 2-(4 '-Trifluoromethyl-biphenyl-3 -yl)- pentanoic acid, 3-Phenyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid and 4-Memyl-2-(4 '-Trifluoromethyl-biphenyl-3 -yl)- pentanoic acid.

No significant effect on Cox-1 or Cox-2 activity was observed at concentrations of up to 300uM

Example 17: Effect of alpha - substitution on CNS penetration

Dosed at lmg/kg iv in the rat, up to 15% CNS penetration was observed for 2-(4'- trifluoromethyl-biphenyl-3-yl)-pentanoic acid (example 7) (ratio of brain to plasma concentration of compound 60 min after dosing)

Protocol for measuring CNS penetration Male Sprague Dawley rats (source Charles River U.K. Ltd, Margate, Kent, U.K) (Weight range and age: Approximately 250 - 300 g (approximately 8 - 10 weeks of age)) can be used for this study.

The test material is formulated in 10% DMSO/90% hydroxypropyl β cyclodextrin (10% w/v in phosphate buffered saline)

Doses are administered as a single bolus dose into a lateral tail vein at dose volumes of 1 mL/kg

Groups of rats are dosed intravenously with the test material at a dose of 1 mg kg.

Blood samples are taken from a lateral tail vein not used for dosing. One hour after dosing a blood sample is taken as described above. Immediately following collection of this sample the chest cavity of each rat is then carefully opened and 50 - 100 mL of heparinised (100 IU/mL) saline perfused directly into the left ventricle via a butterfly cannula.

Once saline is being perfused the animal is killed by cutting the right atrium, allowing blood to flow out. Perfusion is continued until the liver and lungs are pale and the paws, eyes and lips turn whitish.

After completion of perfusion, the cranium of the rat is then opened and the brain carefully removed and washed with saline and excess saline removed with adsorbent paper. The brain is then rapidly frozen and stored at approximately -20 C pending analysis of test compound concentrations.

The level of CNS penetration is then calculated as the ratio of compound in the brain to that of the compound in plasma

Example 18: Measurement of Abeta 1-42 lowering in vivo

Abeta 1-42 lowering in vivo can be measured in a single transgenic mouse model (APPLd2) of Alzheimer's Disease, developed by Dr. Fred Van Leuven (K.U.Leuven, Belgium) and co-workers, with neuron-specific expression of a clinical mutant of the human amyloid precursor protein [V717I] (Moechars et al., 1999). The commercial rights on this model have been transferred to reMYND NV. The single transgenic mouse displays spontaneous, progressive accumulation of β-amyloid (Aβ) in brain, eventually resulting in amyloid plaques within subiculum, hippocampus and cortex. In such a model (R)-2-(4' -trifluoromethyl-biphenyl-3 -yl)-pentanoic acid dosed at 50 mg/kg po showed 20% lowering of Abeta 1-42 as compared to control animals

Protocol for measuring Abeta 1-42 lowering in vivo Aliquots of compounds can be dissolved in DMSO (volume equal to 1/10th of the final formulation volume), vortexed and further diluted (1:10) with a 10 % (w/v) hydroxypropyl β cyclodextrin (HBC, Aldrich, Ref N° 33,260-7) solution in PBS, where after they are sonicated for 20 seconds.

A single oral administration of compounds is given two hours before sacrifice. Blood is collected at sacrifice. The blood collection is performed via a heart puncture during anesthesia with a mixture of Ketalar (Ketamin), Rompun (Xylazin 2%) and Atropin (2:1:1) and collected in heparinised Eppendorf tubes. Blood is centrifuged at 4000 g for 5 minutes at 4°C.

The mice are anaesthetized with a mixture of Ketalar (Ketamin), Rompun (Xylazin 2%) and Atropin (2:1:1) and flushed trans-cardially with physiological serum at 4°C.

The brain is removed from the cranium and hindbrain and forebrain are separated with a cut in the coronal/frontal plane. The cerebellum is removed. The forebrain is divided evenly into left and right hemisphere by using a midline sagital cut. One hemisphere is immediately immersed in liquid nitrogen and stored at -70°C until homogenization for biochemical assays.

Brains are homogenized using a Potter, a glass tube (detergent free, 2 cm³) and a mechanical homogenizer (650 rpm). A volume of 6,5 x Vi brain weight of freshly prepared 20 mM Tris/HCl buffer (pH 8,5) with Proteinase Inhibitors (1 tablet per 50 ml Tris/HCl buffer, Complete™, Roche, Mannheim, Germany) is used as homogenization buffer.

Samples are transferred from -70°C into a sample holder with liquid nitrogen and each individual sample is pre- warmed by incubation on the bench for a few seconds prior to homogenization. The homogenates are collected in Beckman centrifuge tubes TLX and collected on ice prior to centrifύgation. Between two samples, the Potter and the glass tube are rinsed carefully with distilled water (AD) without detergents and dried with absorption paper.

Samples are centrifuged in a pre-cooled ultracentrifuge (Beckman, Mannheim, Germany) for 1 hour and 20 minutes at 48000 rpm (135.000 x g) at 4°C. The supernatant (soluble fraction containing secreted APP and amyloid peptides) is separated from the pellet (membrane fraction containing membrane-bound APP-fragments and plaque-associated amyloid peptides in case of aged mice).

Small reversed phase columns (Cl 8-Sep-Pack Vac 3cc cartridges, Waters, Massachusetts, MA) are mounted on a vacuum system and washed with 80% acetonitrile in 0,1% Trifluoroacetic acid (A-TFA) followed with 0,1% TFA twice. Then the samples are applied and the columns are washed successively with 5% and 25% A-TFA. Amyloid peptides are eluted with 75 % A-TFA and the eluates are collected in 2 ml tubes on ice. Eluates are freeze-dried in a speedvac concentrator (Savant, Farrningdale, NY) overnight and resolved in 240 μl of the sample diluent furnished with the ELISA kits. To quantify the amount of human Aβ-42 in the soluble fraction of the brain homogenates, commercially available Erizyme-Linked-Immunosorbent-Assay (ELISA) kits are used (h Amyloid β42 ELISA high sensitive, The Genetics Company, Zurich, Switzerland). The ELISA is performed according to the manufacturer's protocol. Briefly, the standard (a dilution of synthetic Aβl-42) and samples are prepared in a 96- well polypropylene plate without protein binding capacity (Greiner bio-one, Frickenhausen, Germany). The standard dilutions with final concentrations of 1000, 500, 250, 125, 62.5, 31.3 and 15.6 pg/ml and the samples are prepared in the sample diluent, furnished with the ELISA kit, to a final volume of 60 μl. Samples, standards and blancs (50 μl) are added to the anti-Aβ-coated polystyrol plate (capture antibody selectively recognizes the C-terminal end of the antigen) in addition with a selective anti-Aβ-antibody conjugate (biotinylated detection antibody) and incubated overnight at 4°C in order to allow formation of the antibody-Amyloid- antibody-complex. The following day, a Streptavidine-Peroxidase-Conjugate is added, followed 30 minutes later by an addition of TMB/peroxide mixture, resulting in the conversion of the substrate into a colored product. This reaction is stopped by the addition of sulfuric acid (IM) and the color intensity is measured by means of photometry with an ELISA-reader with a 450 nm filter. Quantification of the Aβ content of the samples is obtained by comparing absorbance to a standard curve made with synthetic Aβl-42.

List of abbreviations

APP amyloid precursor protein

Aβ Amyloid-β-peptide CSF cerebrospinal fluid

ELISA enzyme-linked immunosorbent assay

PBS phosphate-buffered saline

PCR polymerase chain reaction

PI protease inhibitor cocktail tablets (Complete, Roche) PSl Presenilin 1 rpm rotations per minute

RI recognition index

SB sample buffer

SEM standard error of means TBS Tris-buffered saline

TFA Trifluoroacetic acid

TMB 3,3' 5,5' -teframethylbenzidine

Claims

A compound having the general formula (I)

wherein R_ls R₂ are independently of each other selected from the group consisting of H; alkyl selected from the group CH₃, C₂H₅, i-C3H₇, n-CsH , i-C H₉, n- Hg, sec-CφH_θ, tert-C^g; alkenyl selected from C₂H₃, i-C₃H₅, n-C3H₅, n-C H , i-C₄H , sec-C₄H₇; and wherein the alkyl and alkenyl groups can carry one or more substituents from the group: F, Cl, Br, I, CF₃ and saturated, unsaturated or aromatic 3- to 7-membered cyclic groups optionally having one or more heteroatoms from the group N, S or O, which heteroatom may be identical or different if more than one heteroatom is present, in the ring; or R_\ and R₂ being part of a ring, either saturated or unsaturated, having 3 to 5 C-atoms, and which may contain in the ring one or more heteroatoms from the group N, S or O, and which heteroatom may be identical or different if more than one heteroatom is present;

and wherein R3 is selected from the group consisting of F, Cl, Br, I, substituted and unsubstituted alkyl from the group CH3, C₂H₅, i-C3H , n-C₃H₇, i-C₄H9, n-C₄H₉, sec- C H₉, tert-G_tHg and substituted and unsubstituted alkoxy, from the group OCH₃, OC₂H₅, Oi-C₃H₇, On-C₃H₇, Oi-C₄H₉, On-GjHg, Osec-C^, Otert-C^ and wherein the substituents of both groups are selected from F, Cl, Br, I; preferably R₃ being selected from the group consisting of CF₃, OCF₃, F, Cl, Br, I, more preferably R₃ being selected from the group consisting of CF₃, OCF₃. and/or a salt or ester thereof.

The compound according to claim 1, wherein Ri and R₂ being H; or Ri being H and R₂ being CH₃, C₂H₅, nC₃H₇, i-C₃H _> nC^Hg, i-C-tfϊg, sec-C-d^ or tert-C Hg; and wherein all the named groups can carry one or more substituents from the group C₆H₅, CF₃ or cyclopropyl; or R} and R₂ being CH₃; or Ri and R₂ each being a methylene group forrning, together with the C-atom in α position to the carboxyl group, a cyclopropyl ring.

3. The compound according to claim 1 or 2, wherein R₃ is selected from the group consisting of F, Cl, Br, I, C₁-C₄-alkyl or C C and/or a salt or ester thereof.

4. The compound according to any of claims 1 to 3, wherein R₃ is selected from the group consisting of CF_3; OCF₃ F, CL, Br, I, preferably from CF3 or OCF₃.

5. As a compound according to claim 1, a compound according to the formula (IT)

or the compound according to the formula (III)

or the compound according to the formula (TV)

or the compound according the formula (V)

or the compounds: (VI) l-(4'-Trifluoromethyl-biphenyl-3-yl)-cyclopropane- carboxylic acid; (VII) 2-Methyl-2-(4 '-trifluoromethyl-biphenyl-3 -yl)-propionic acid; (VIII) 2-(4'-Trifluoromethyl-biphenyl-3-yl)-pentanoic acid, in racemic form and in the form of the enantiomers; (LX) 2-(4'-Trifluoromethyl-biphenyl-3-yl)-4- Methyl-pentanoic acid; (X) 2-(4'-Trifluoromethyl-biphenyl-3-yl)-3-Methyl- butanoic acid, (XI) 2-(4'-Trifluoromethyl-biphenyl-3-yl)-butyric acid; (XII) 3- Phenyl-2-(4'-trifluoromethyl-biphenyl-3-yl)-propionic acid; (XIII) 5,5,5-Trifluoro- 2-(4'-trifluoromethyl-biphenyl-3-yl)-pentanoic acid; (XTV) 4-Phenyl-2-(4'- trifluoromethyl-biphenyl-3-yl)-butyric acid; (XV) 3-Cyclopropyl-2-(4'- trifluoromethyl-biphenyl-3 -yl)-propionic acid and/or a salt or ester thereof.

6. A medicament comprising a compound according to claim 1 and/or a pharmaceutically acceptable salt or ester thereof, optionally in admixture with an inert carrier.

7. A medicament according to claim 6 for the treatment of Alzheimer's disease.

8. A medicament according to claim 6 for the modulation of γ-secretase activity.

9. A method of modulating γ-secretase activity, which method comprises administering a compound as defined in any of claims 1 to 5 to a mammal.

10. The method according to claim 9, wherein the γ-secretase activity is associated with an elevated level of AB42-production.

11. The method according to claim 9, wherein the mammal is a human

12. A method of treating Alzheimer's disease, which method comprises administering a compound as defined in any of claims 1 to 5 to a mammal.

13. The method according to claim 12, wherein the mammal is a human

14. A method for the preparation of a compound according to any of claim 1 to 5, which method comprises the steps of coupling an optionally protected phenyl acetic acid derivative, optionally said derivative being protected, with an appropriate aromatic compound and optional further functionalisation and deprotection of the thus-obtained biphenyl compound.

15. A method for the preparation of a medicament comprising the steps of: a) preparing a compound according to any of claims 1 to 5 b) formulation of a medicament containing said compound